INTRODUCTION ABSTRACT. J. Am. Soc. Brew. Chem. 72(4): , 2014

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1 Comprisons of Brley Mlt Amylolyti Enzyme Thermostilities to Wort Osmolyte Conentrtions, Mlt Extrt, ASBC Mesures of Mlt Qulity, n Initil Enzyme Ativities 1 Stnley H. Duke, 2 Deprtment of Agronomy, University of Wisonsin, Mison, WI; Cynthi A. Henson, Unite Sttes Deprtment of Agriulture-Agriulturl Reserh Servie (USDA-ARS), Cerel Crops Reserh Unit (CCRU), Mison, WI n Deprtment of Agronomy, University of Wisonsin, Mison, WI; n Mrus A. Vinje, USDA-ARS, CCRU, Mison, WI ABSTRACT J. Am. So. Brew. Chem. 72(4): , 2014 The hypothesis tht wort osmolyte onentrtion (OC) woul orrelte muh etter thn mlt extrt (ME) with rley mylolyti enzyme thermostilities of mlts proue over severl ys of germintion ws teste. Sees of four two-row n four six-row North Amerin rley ultivrs were mlte in miromlter n smple every 24 hr throughout 6 ys of germintion. α- n β-mylses n limit extrinse were ssye efore n fter mshing t 70 C for 30 min to etermine thermostilities. Wort OC, ME, n ASBC mesures of mlt qulity were etermine for eh y of germintion. For ll ultivrs omine, over ll ys of germintion, wort β-mylse thermostilities orrelte negtively n highly signifintly with oth wort OC n ME, lthough more strongly with OC (r = 0.62, ; r = 0.46, P < 0.001, respetively). Correltions of limit extrinse thermostilities with wort OC were lso muh stronger thn with ME (r = 0.87, ; r = 0.68,, respetively). α-amylse thermostility ws either unffete y or inrese fter mshing t 70 C. These t suggest tht β- mylse n limit extrinse thermostilities eome more limiting to strh egrtion s reflete y OC thn s reflete y ME s germintion proees. β-amylse intron III lleli vrition h no effet on OC or ME in these North Amerin rley ultivrs. For ll ultivrs omine over 6 ys of germintion, the orreltions for β-mylse n limit extrinse thermostilities versus the initil tivities of α- n β-mylse n limit extrinse were signifint n negtive ([β-mylse thermostilities versus initil tivities of α-mylses: r = 0.63,, β-mylses: r = 0.68,, n limit extrinses: r = 0.54, ] n [extrinse thermostilities versus initil tivities of α-mylse: r = 0.63,, β-mylse: r = 0.68,, n limit extrinse: r = 0.54, ]). These t suggest tht seletion for high initil tivity of ny of these mylolyti enzymes woul lso selet for high thermostility of β-mylse n limit extrinse. Keywors: α-amylse, β-amylse, Enzyme thermostility, Limit extrinse, Mlt extrt, Mshing, Osmolytes RESUMEN L hipótesis e que l onentrión osmolito el mosto (OC) se orrelion muho mejor que el extrto e mlt (ME) on l enzim milolíti termoestilies e e mlt prouios urnte vrios ís e l germinión se puso prue. Semills e utro e os hilers y utro e seis hilers ultivres e e mlte nortemerinos 1 Mention of proprietry prout oes not onstitute gurntee or wrrnty of the prout y the U.S. Deprtment of Agriulture n oes not imply its pprovl to the exlusion of other suitle prouts. 2 Corresponing uthor. E-mil: shuke@wis.eu Amerin Soiety of Brewing Chemists, In. fueron en un miro-mlteo y se tomron muestrs 24 hors lo lrgo e 6 ís e germinión. Ls enzims e α- y β-milss y extrins límite, se nlizron ntes y espués e merión 70 C urnte 30 min pr eterminr termoestilies. Se eterminron ls meis e li el OC el mosto, ME, y ASBC e l mlt pr í e l germinión. Pr toos los ultivos ominos, sore toos los ís e l germinión, termoestilies e β-mils en el mosto orrelion negtiv y ltmente signifitiv on mos OC y ME, unque más fuertemente on OC (r = 0.62,, r = 0.46, P < 0.001, respetivmente). Correliones e termoestili e límite extrins on el OC el mosto ern tmién muho más fuerte que on ME (r = 0.87,, r = 0.68,, respetivmente). L termoestili e α-mils er o no feto por o umentó espués e merión 70 C. Estos tos sugieren que l termoestili e l β-mils y extrins límite se vuelven más limitnte pr l egrión el lmión omo se reflej por OC que omo se reflej por ME omo prouto e germinión. L vriión léli e l β-mils intrón III no tuvo efeto sore OC o ME en estos ultivres e e e Améri el Norte. Pr toos los ultivos ominos e más e 6 ís e germinión, ls orreliones pr β-mils y límite extrins termoestilies frente ls tivies iniiles e α- y β-mils y límite extrins estn signifitiv y negtiv ([termoestilies e β-mils frente ls tivies iniiles e α-milss: r = 0.63,, β-milss: r = 0.68,, y extrinss límite: r = 0.54, ] y [extrins termoestilies frente ls tivies iniiles e l α-mils: r = 0.63,, β-mils: r = 0.68,, y el límite extrins: r = 0.54, ]). Estos tos sugieren que l seleión pr l lt tivi iniil e ulquier e ests enzims milolítis tmién seleionr pr lt termoestili e β-mils y extrins límite. Plrs lves: α-amils, β-amils, Enzim termoestili, Extrto e mlt, Límite extrins, Merión, Osmolitos INTRODUCTION Mshing is n extremely omplex proess involving myri of physil n iohemil events tht re mostly hnging onstntly with time (9,15,31). Quntittively n qulittively, the most signifint event uring mshing is the onversion of strh to fermentle sugrs y mlt mylolyti enzymes (6,15,51). Strh omprises ~65% of the ry weight of mlt (6) n its onversion to fermentle sugrs is the primry purpose of mshing (15). Strh in msh must e geltinize efore it is highly suseptile to hyrolysis y mlt mylolyti enzymes n this requires heting (6). The het require for geltiniztion results in some, to lmost totl, enturtion of two mlt mylolyti enzymes, β-mylse n α-gluosise (31,48). Due to the importne of rley mlt mylolyti enzyme thermostility in mshing, there hve een mny stuies on the thermostility of rley β-mylse n α-gluosise (e.g., 19,35,39,41,43,48,51, 59,60,62,66,71,84). As the high tempertures use in mshing re 271

2 272 / Duke, S. H., Henson, C. A. n Vinje, M. A. not enountere in nture uring germintion, the evolution of rley grin mylolyti enzymes tht funtion well in oth germintion n mshing woul not e expete to our. Although eer hs een proue for millenni (8), it hs only een reltively reently tht mn hs intervene sientifilly to selet rley genotypes for more effiient proution of fermentle sugrs uring mshing. The use of mlt extrt (ME) s quntittive mesure of the mss of mteril in wort, whih is lrgely strh or sugrs, egn in the lte 1700s n hs een the mjor mesure of mlt qulity sine then (1,13,14,16,38,55). This unnturl seletion of rley for its utility to proue prout esire y humnkin hs resulte in rley with inrese mshing effiieny s etermine y inreses in wort ME (e.g., 76,78). The rley mlt enzymes tht re iretly responsile for enosperm strh egrtion re α-mylse, β-mylse, limit extrinse, n α-gluosise (9,48,71,82). Their omine tivities re referre to s istti power (DP), whih is often onsiere to e the seon most importnt stnr mesure of mlt qulity (30). High DP mlts re espeilly importnt when using unmlte junt in msh (5). Purifition n hrteriztion of n iniviul rley mlt mylolyti enzyme oes not neessrily give one n ppreition of its signifine in the presene of the other mylolyti enzymes euse the four enzymes t inepenently n/or intertively in the egrtion of strh (82). α- Amylse n α-gluosise n ttk ntive, non-geltinize strh grnules; however, their tivities with these strh grnules re very low n onfine to ifferent sites on strh grnules for eh enzyme (11,81 83). Inomplete hyrolysis of n queous strh mixture will ontin strhy mteril tht stins lk (mylopetin) or lue (mylose or β-limit extrin) with n ioine (KI/I 2 ) solution (24,57). β-limit extrin is the prout of limit extrinse hyrolysis of α-1 6-D-gluosii ons of mylopetin. Inomplete hyrolysis of strh uring mshing n e ue to lk of suffiient DP, insuffiient geltiniztion of strh (82), or enturtion of DP enzymes y geltiniztion temperture tht is so high tht most or ll of the mylolyti enzymes re enture (15). The rte of het enturtion of mylolyti enzymes uring mshing epens on the type of mshing. Isotherml mshing is usully hel t reltively high temperture (e.g., 65 C) for out one hour n ws stnr metho for mesuring mlt qulity y the Institute of Brewing (IoB) (55) n is now one of two reommene methos y the Europen Brewing Commission (EBC) (38). In ontrst, eotion n temperture progrmme mshing re strte t reltively low temperture (e.g., 45 C), whih is rise to firly high temperture (e.g., 63 to 70 C) y either ing k portion of the msh tht hs een oile or y slowly rising the temperture of the msh vi heting evie (7). The Congress mshing metho of the Amerin Soiety of Brewing Chemists (ASBC) n the EBC is suh metho n is stnr metho for mesuring mlt qulity (2,38). All of these methos hve perios in whih mylolyti enzymes re enture y het. Hene, regrless of the mshing metho use, equte thermostility of the mylolyti enzymes in the msh is importnt in suessful onversion of mlt strh to fermentle sugrs. β-amylse, n exomylse, hyrolyzes the α-1 4-D-gluosii ons from the nonreuing ens of mylose, mylopetin, n mltoextrins prouing mltose, n from mylopetin lso prouing limit extrin. The only mltoextrin tht it nnot egre is mltotriose (65,87). Its ility to rpily proue mltose uring mshing hs le it to e terme the shrifying enzyme (14). Brley ontins two forms of β-mylse, the enosperm speifi β-mylse, β-mylse1, n the so-lle uiquitous β-mylse, β-mylse2 (86). In rley mlt, β-mylse hs y fr the highest tivity of ll of the mylolyti enzymes (e.g., 22,27,31,40,48). The uiquitous β-mylse2 is foun in most tissues n in numerous lotions within ells (22,58,70,88). In the eveloping n mture grin of the rley enosperm, β-mylse1 is synthesize uring grin filling n is y fr the preominnt β-mylse (85,86). It is lrgely oun to insolule mteril efore germintion n must e relese y sulfhyryl regents or proteinses uring germintion to hve tivity (18,80). β-amylse usully orreltes etter with DP thn o the other mlt mylolyti enzymes (20,21,28,40,42,43,44,48,75,82), suggesting entrl role in rley enosperm strh egrtion uring mshing. Also, supporting this oservtion, mximl mlt β-mylse tivity orreltes muh etter with sugr proution thn tht of other mlt mylolyti enzymes uring mshing (32). α-amylse is n enomylse, hyrolyzing internl α-1 4-Dgluosii ons in mylopetin, mylose, n mltoextrins. As with β-mylse, the only mltoextrin tht it nnot egre is mltotriose. As n enomylse, α-mylse more rpily liquefies strh in n queous solution thn oes β-mylse with n equl tivity (23), n is referre to s the liquefying enzyme (14). α-amylse hs 10 genes from t lest two gene fmilies (46,53, 56,61,67,68) tht re lmost exlusively expresse uring grin germintion (14,64). The sene or very low tivity of α-mylse in the eveloping grin of rley n fruits of other speies (22) is ontrry to its seemingly uiquitous presene in vegettive tissues of plnt speies, where it is foun in oth intrellulr n extrellulr (poplsti) lotions (10,58,73,74). Brley α-mylse is the most thermostle of ll of the enosperm mylolyti enzymes (31,48,51). Hene, the role of α-mylse in strh n mltoextrin egrtion eomes inresingly importnt s β- mylse is het enture uring mshing. Limit extrinse (i.e., ernhing enzyme, pullulnse) hyrolyzes the α-1 6-D-gluosii ons of mylopetin n smller rnhe oligoshries. Most rley enosperm strh is mylopetin (70 80% of totl), whih hs oth α-1 6-D-gluosii n α-1 4-D-gluosii ons (5). Limit extrinse is synthesize uring oth grin evelopment n germintion (14,77,79); hene, there is no lg phse for the evelopment of tivity s with α-mylse. Only one gene for rley enosperm limit extrinse hs een ientifie (63). The thermostility of limit extrinse lies etween tht of α- n β-mylse (39,48,51) n is suffiiently high to funtion throughout mshing (39,48,81). Unlike β-mylse, limit extrinse tivity inreses uring the rmping from 45 to 70 C in Congress mshing, n usully elines in the finl isotherml perio t 70 C (31). Brley enosperm α-gluosises re exo-glyosie hyrolses tht hyrolyze α-1 1-, α-1 2-, α-1 3-, α-1 4-, n α-1 6- D-gluosii ons (55,87). As with α- n β-mylse, α-gluosise ppers to our in more thn one suellulr site with n unknown funtion in the ytosol of plnts (12,88). In the rley enosperm, α-gluosise hs the lowest tivity n thermostility of ll rley mlt mylolyti enzymes (38,55,74). Unlike α- n β-mylse, α-gluosise n utilize mltose n mltotriose s sustrtes (54). However, the three min prouts of strh egrtion uring mshing re gluose, mltose, n mltotriose (26,29), n these re the only fermentle sugrs proue from strh hyrolysis (1). The ition of high thermostility α- gluosise to msh results in the proution of higher levels of fermentle sugrs (71). The thermostilities of β-mylse n limit extrinse in mlts proue over 6 ys of germintion re highly signifintly n negtively orrelte with the proution of fermentle sugrs uring mshing (51). ME is highly positively orrelte with totl sugr proution uring mshing of mlts proue over 6 ys of germintion (26) n with eight ultivrs over the ourse of Congress mshing (29). However, in the stuy with mlts proue over 6 ys of germintion, wort osmolyte onentrtion (OC) orrelte with wort sugr proution muh etter thn ME

3 Osmolytes n Amylolyti Enzymes / 273 (26) n, in the Congress mshing stuy, OC orrelte mrginlly etter thn ME with wort sugrs with ll ultivrs omine (29). OC is mesure of osmolrity n represents the egree of egrtion of storge ompouns (primrily strh) uring mshing. OC hs een foun to e superior to ME in preiting the thermostilities of mylolyti enzymes uring mshing (48), mylolyti enzyme tivities in wort (27,31), mesures of mlt qulity (e.g., ME, wort protein, S/T protein, β-glun onentrtions, free mino is, n wort lrity) over rief perio of isotherml mshing or over n entire perio of Congress mshing or isotherml mshing (25,27,28,47,48,76), n sugr onentrtions of wort (26,29). Furthermore, OC hs een utilize to preit extrose equivlents in the egrtion of strh y α-mylse (69) n prehrvest sprouting in rley (50). In this stuy, we ompre OC n ME of mlts proue over 6 ys of germintion to their mylolyti enzyme thermostilities fter rief perio of isotherml mshing. Our hypothesis is tht OC woul e etter preitor of thermostility thn ME. This hypothesis is primrily se on the forementione stuies initing tht some mylolyti enzyme thermostilities re highly orrelte with wort sugr proution (34) n tht wort sugr proution is more highly orrelte with OC thn ME (26,29). Also, OC hs een foun to e superior preitor of thermostility in mlts proue t 5 ys of germintion n sujete to omplete mshing regime (48). Also, ny ssoition of Bmy1 intron III lleli vrition to OC n ME uring mshing ws exmine. β-amylse tivity in mlts proue over 6 ys of germintion orreltes well with wort OC (28). Mximl wort β-mylse tivity orreltes well with mximl OC vlues uring mshing, wheres wort ME oes not (31). Bmy1 intron III lleli vrition hs een ssoite with β-mylse tivity n thermostility in severl stuies (e.g., 36, 37,45), lthough we hve foun no ssoition in North Amerin germplsm (43,52). This suggests tht there woul e no ssoition etween Bmy1 intron III lleli vrition n OC or ME uring mshing. Aitionlly, mylolyti enzyme thermostilities re ompre to mesures of ASBC mlt qulity, other thn ME, n omprisons of mylolyti enzyme initil tivities to their thermostilities were me. EXPERIMENTAL Cultivrs Use n Mlting Proeure Brley mlt ws proue from eight genotypes (four tworow [Hrrington, Grnet, Merit, B1202] n four six-row [Legy, Ley, Morex, Trition {formerly B2482}]), n were the sme mlts use in previous stuies (26 28,47,51). This stuy is ontinution of those stuies. β-amylse Bmy1 intron III lleles, inluing intron size n mirostellite motifs, for ll ultivrs were lone n sequene s previously esrie (84). All sequenes for Bmy1 intron III lleles were registere with GenBnk numers for eh ultivr n ssigne numers re pulishe (Tle I, ref. 84). See smples (60 g ) were steepe to 47% moisture, germinte, n kilne in Joe White Miromlter. The steeping regime ws n lternting wet step t 15 C with n ir rest t 17 C t the following intervls: 6 hr wet, 10 hr ir, 7 hr wet, 6 hr ir, 7 hr wet, 5 hr ir t 30% ir flow, 0% reirultion. Germintion ws t 20 C for 24 hr followe y 18 C for 120 hr t 30% ir flow, 0% reirultion. Cns ontining see were turne four times every 2 hr. Every 24 hr uring the germintion regime, germinte green mlt ws remove n ple in seon Joe White Miromlter n kilne. Green mlt ws kilne s follows: 49 C for 9 hr, 54 C for 4 hr, 60 C for 2 hr, n 85 C for 3 hr with 30 min rmps etween eh temperture stge. Mshing Regime Three replitions of seven kilne sees from eh smple were groun to fine power in offee griner (Brun KSM 2B) for 10 se. Smples (150 mg) were ple in 2 ml entrifuge tue with 750 µl of 18MΩH 2 O, mixe with Vortex Genie, n hel t 70 C in wter th for 30 min with mixing every 5 min. Smples t 0 n 30 min were remove n entrifuge t 13,000 g for 2 min n frozen ( 20 C) for enzyme (0 n 30 min) n sugr ssys (30 min). Enzyme Assys Mlt smples (3.5 g ) were mille to power with Lono (Knss City, MO) urr mill. Smples were ivie into three 1.0 g replitions ple in 50 ml entrifuge tues n 5 ml of 0.5% NCL ws e n mixe for 15 min. Extrts were entrifuge t 14,000 g for 15 min n superntnts were remove n store t 20 C n use for mylolyti enzyme ssys within ~3 5 ys. Amylolyti enzymes were ssye t 0 n 30 min t 70 C. For α-mylse tivity ssys, 10 µl of smples were ilute with 990 µl of uffer (50 mm soium suinte, 3 mm CCl 2, ph 6.0). For eh mlt smple, eight 10 µl liquots of this mixture were loe into 96-well PCR plte n 10 µl of Megzyme (Bry, Ireln) Cerlph sustrte t the Megzyme reommene onentrtion ws e to eh well to strt ssys. Assys were inute t 40 C n were stoppe t 5 n 10 minutes (4 susmples for eh mlt smple for eh time point) with 150 µl 1% Trizm se. A 150 µl liquot of eh stoppe ssy ws trnsferre to 96-well flt-ottome miroplte n sorne ws re t 400 nm. Aury ws within the limits (<5% SE) for the full sle Megzyme Cerlph kit. For β-mylse tivity ssys, 20 µl of smples were ilute with 2.48 ml of uffer (100 mm soium suinte, 1 mm EDTA, TABLE I Simple Correltions (r) of Mlt Amylolyti Enzyme Ativity Thermostilities (% Ativity Remining After 30 min t 70 C) with ASBC Mesures of Mlt Qulity, Osmolyte Conentrtion of Wort, n Initil Ativity n Thermostility of Amylolyti Enzyme Ativites of Eight Brley Cultivrs Mlte Eh Dy Over Six Dys of Germintion n Mshe for 30 min t 70 C Mlt qulity mesurement α-amylse thermostiliy β-amylse thermostiliy Limit extrinse thermostility Osmolyte onentrtion (OC) r = P = r = r = Mlt extrt (ME) r = P = r = P = r = Distti power (DP) r = P = r = r = β-glun onentrtion r = P = r = r = Solule/totl protein r = P = r = P < r = ASBC α-mylse tivity r = P = r = P = r = Initil α-mylse tivity r = P = r = r = Initil β-mylse r = r = r = tivity Initil limit extrinse tivity α-amylse thermostiliy β-amylse thermostiliy P = r = P = r = r = P = r = r = P = r = Correltion oeffiients (r) were lulte for the mens of three replitions for eight ultivrs (4 two- n 4 six-row) for 6 ys of germintion (n = 48).

4 274 / Duke, S. H., Henson, C. A. n Vinje, M. A. 1 mg ml 1 BSA, ph 6.0). For eh mlt smple, eight 10 µl liquots of the ilution were loe into 96-well PCR plte n 10 µl of the originl Megzyme Betmyl kit ontining the sustrte p-nitrophenyl-α-d-mltopentosie n α-gluosise. The Megzyme reommene onentrtion of sustrte ws e to eh well to strt ssys. Assys were inute on hilling/ heting plte t 40 C n stoppe t 5 n 10 minutes (4 susmples for eh mlt smple for eh time point) with 150 µl 1% Trizm se. A 150 µl liquot ws trnsferre to 96-well fltottome miroplte n sorne ws re on Powerwve XS miroplte spetrophotometer t 400 nm. Aury ws within the limits (<5% SE) for the full sle Megzyme Betmyl kit. For limit extrinse tivity ssys unilute smples were use for y 1 of germintion n for ys 2 6 of germintion, 500 µl ws ilute with 1.5 ml H 2 O. Buffer (250 µl of 200 mm soium suinte, 3 mm CCl 2, 0.2 g per 25 ml ithiothreitol [DTT], ph 5.5) ws e to six 250 µl liquots of eh smple. DTT ws e to uffer immeitely efore use. Assys were initite y ition of Megzyme Limit-Dextrizyme tlet n were onute for 1 n 2 hr t 40 C. Assys were stoppe with 1.5 ml 1% Trizm se followe y entrifugtion t 3,500 g for 15 min. Asorne t 590 nm ws etermine with Shimzu (Kyoto, Jpn) UV-2101PC spetrophotometer. Ativities were lulte using the equtions for the Megzyme Limit- Dextrizyme kit. Thermostilities re expresse s the perentge of tivity remining t 30 min of mshing s ompre to the strting tivity (tivity t 30 min/tivity t 0 min 100). Determintions of Osmolyte Conentrtion (OC) Three replitions of seven sees from eh rley smple were groun in offee griner (Brun KSM 2B) for 10 se. Eh smple (150 mg) ws ple in 2-mL entrifuge tue with 750 µl of 18MΩ H 2 O, mixe vigorously with Vortex Genie, n hel t 70 C in wter th. Smples were mixe every 5 min for 30 min. Mixe smples were entrifuge t 13,000 g for 2 min, n 8 µl of eh replition ws ple in vpor pressure osmometer (Wesor 5100C) to mesure OC. The osmometer ws lirte with NCl Wesor stnrs t 100, 290, n 1,000 mmol kg 1. Blnks (18MΩ H 2 O) were sutrte from eh reing, n OC ws onverte into millimoles per kilogrm per grm of see. Fig. 1. Correltion n regression lines of wort α-mylse, β-mylse, n limit extrinse thermostilities versus wort osmolyte onentrtions (OCs) n mlt extrts (MEs) for omine two- n six-row ultivr mlts mshe t 70 C for 30 min [(α-mylses thermostilities versus OCs: r = 0.101, P = 0.494; versus MEs: r = , P = 0.815); (β-mylse thermostilities versus OCs: r = 0.616, ; versus MEs: r = 0.464, P = ); (limit extrinse thermostilities versus OCs: r = 0.874, ; versus MEs: r = 0.680, )]. Dt points re the mens of three replitions of four two-row n four six-row ultivrs omine (n = 48).

5 Osmolytes n Amylolyti Enzymes / 275 Determintion of Stnr Mesures of Mlt Qulity ME ws etermine for smples using the ASBC Mlt-4 proeure (2), exept tht ll weights n volumes speifie for the metho were hlve. Speifi grvity of the filtrtes ws etermine with ensitometer (Anton/Prr DAM5000). Density t were use to lulte the mount of solule mteril in the filtrte n the perent extrt from mlt. DP ws etermine on n nlyzer (Sklr SAN plus) using the utomte ferriynie ASBC Mlt-6A proeure (2). ASBC α-mylse tivity ws etermine on n nlyzer (Sklr SAN plus) y heting extrts to 73 C to intivte β-mylse n then mesuring DP using the ASBC Mlt- 6A metho (2). β-glun levels were etermine on n nlyzer (Sklr SAN plus) using the ASBC Wort-18 fluoresene flow injetion nlysis metho (2) with Clofluor s fluoresent gent. Mlt nitrogen ws mesure using Dums omustion proeure with n nlyzer (LECO FP-528). Nitrogen vlues were onverte to totl protein through multiplition y 6.25 using the ASBC Brley-7C metho (2). Solule protein levels were etermine on n nlyzer (Sklr SAN plus) using the ASBC Wort-17 UV spetrophotometer metho (2). S/T rtios were lulte from the two vlues mesure s esrie ove. Sttistil Anlysis Simple orreltion oeffiients (r) etween enzyme thermostilities n sugr onentrtions were etermine using Person s prout-moment orreltion. Mens of three replitions per y for ll two-row n six-row genotypes over 6 ys of germintion were use to etermine orreltion oeffiients for ll genotypes omine (n = 48) n two-row or six-row ultivrs (n = 24). RESULTS AND DISCUSSION Comprisons of Mlt Amylolyti Enzyme Thermostilities n Wort Osmolyte Conentrtions n Mlt Extrt A highly signifint negtive orreltion ws foun for ll ultivrs omine for β-mylse n limit extrinse thermostilities versus OC over 6 ys of germintion, whih were onsierly etter thn the orreltion with ME (Tle I, Fig. 1). α- Amylse, whih ws thermostle t 70 C, i not orrelte signifintly with OC or ME (Tle I). The orreltion for β-mylse thermostility versus OC (Tle I, Fig. 1; r = 0.616, ) ws similr to tht for its orreltion with totl wort sugrs (ref. 52, r = 0.656, ). In ontrst, the orreltion for limit extrinse thermostility versus OC (Tle I, Fig. 1; r = 0.874, ) ws onsierly etter thn its orreltion with totl wort sugrs (ref. 52, r = 0.767, ). This inites tht limit extrinse thermostility negtively ffets wort osmolrity to greter extent thn it negtively ffets sugr proution, wheres β-mylse thermostility negtively ffets osmolrity n sugr proution lmost eqully. The t presente here re prtilly ue to the pttern for thermostility of mylolyti enzymes in mlts proue over 6 ys of germintion, whih ppers to e unusul, with the highest thermostility of β-mylse n limit extrinse ourring in mlts from y 1 of germintion (51). Erly in germintion, β- mylse is lrgely oun or ltent n intive n hs no tivity when extrte without high level of sulfhyryl-reuing gent in the extrtion meium (39). We i not use sulfhyryl gent for extrtion t time zero or 30 min of mshing n we remove insolule mteril y entrifugtion; hene, we were mesuring the mylolyti tivities tht our in solution t the eginning n en of mshing. We think tht ny mtrix effet of inomplete moifition on thermostility woul not hve een oserve if it were epenent on sulfhyryl relese n/or tivtion of β-mylse. It seems more likely tht erly germintion effets on inrese mylolyti thermostilities my hve een ue to the ining of thermoprotetnt osmolytes (3,4). Also, mlts proue eh y over 6 ys of germintion n then mshe t 70 C for 30 min proue inrese wort OC in mlts from y 5 of germintion, fter whih OC remine ner the sme or eline y y 6 (47). In ontrst, wort ME inrese in mlts from ys 3 or 4 of germintion n then slowly eline in most ultivrs (47). Wort sugrs h more similr pttern to OC thn ME in mlts germinte over 6 ys, peking TABLE II Simple Correltions (r) of Mlt Amylolyti Enzyme Thermostilities (% Ativity Remining fter 30 min t 70 C) n ASBC Mesures of Mlt Qulity, Wort Osmolyte Conentrtion, n Initil Ativities of Amylolyti Enzymes for 4 Two- n 4 Six-Row Brley Cultivrs Mlte Eh Dy Over 6 Dys of Germintion Mlt qulity mesurement Osmolyte onentrtion (OC) α-amylse thermostility r = P = Mlt extrt (ME) r = P = Distti power (DP) r = P = β-glun r = onentrtion P = Solule/totl protein r = P = ASBC α-mylse r = tivity P = Initil α-mylse r = tivity P = Initil β-mylse r = tivity P = Initil limit r = extrinse tivity P = α-amylse thermostiliy β-amylse thermostiliy Two-Row Cultivrs β-amylse thermostility r = P = r = P = r = P = r = P = r = P = r = P = r = P = r = P = r = P = r = P = Limit extrinse thermostility r = r = r = P = r = r = r = r = r = r = r = P = r = P = α-amylse thermostility r = P = r = P = r = P = r = P = r = P = r = P = r = P = r = P = r = P = Six-Row Cultivrs β-amylse thermostility r = P = r = P = r = P = r = P = r = P = r = P = r = P = r = r = r = P = Limit extrinse thermostility r = r = P = r = r = r = P = r = r = r = r = r = P = r = Correltion oeffiients (r) were lulte for the mens of three replitions for four ultivrs eh, two- n six-row for 6 ys of germintion (n = 24).

6 276 / Duke, S. H., Henson, C. A. n Vinje, M. A. on ys 4 to 6, epening on ultivr (26). Hene, it ws not surprising tht wort OC orrelte positively n highly signifintly n muh etter thn ME with wort sugrs from mlts proue over 6 ys of germintion (26). All of the mlt mylolyti enzyme tivities in this stuy orrelte positively n highly signifintly with OC n muh etter thn ME, hving ptterns of inrese more similr to OC thn ME over the 6 y perio of germintion (27). A similr pttern is seen for inreses n ereses in initil α- n β-mylse n limit extrinse tivities s seen for wort OC, ut not ME (27,47). These previously oserve ptterns of OC n ME evelopment lso ontriute to orreltions oserve in this stuy. The sme ptterns for orreltions of β-mylse n limit extrinse thermostilities versus OC n ME for ll ultivrs omine (Tle I, Fig. 1) were exhiite y oth two- n six-row ultivrs seprtely (Tle II, Figs. 2 n 3). For two-row ultivrs, β-mylse n limit extrinse thermostilities orrelte negtively n highly signifintly with OC, lthough the OC orreltion with limit extrinse thermostility ws muh etter thn for β-mylse thermostility (Tle II, Fig. 2). In ontrst, the orreltions of β-mylse n limit extrinse thermostilities versus ME were muh lower thn those with OC, n with β- mylse thermostility the orreltion with ME ws not signifint t the P 0.05 level (Tle II, Fig. 2). As with ll ultivrs omine, the orreltion for β-mylse thermostility versus OC (Tle I) ws similr to tht for its orreltion with two-row totl wort sugrs (51) n the orreltion for two-row limit extrinse thermostility versus OC ws onsierly etter thn the two-row limit extrinse thermostility orreltion with totl wort sugrs (51). As with two-row ultivrs, six-row β-mylse n limit extrinse thermostilities orrelte negtively n highly signifintly with OC n the OC orreltion with limit extrinse thermostility ws muh etter thn for β-mylse thermostility (Tle II, Fig. 3). Also, s with two-row ultivrs, the orreltions of six-row β-mylse n limit extrinse thermostilities versus ME were muh lower thn those with OC (Tle II, Fig. 3). The orreltions of six-row β-mylse n limit extrinse thermostilities versus OC were muh more ner the sme s these enzyme thermostilities versus totl wort sugr proution (Tle II, ref. 51) (β-mylse thermostility versus OC = 0.647, versus totl wort sugrs = 0.686; limit extrinse thermostility versus OC = 0.865, versus totl wort sugrs = 0.808) thn two-row row β-mylse n limit extrinse thermostilities. With ll ultivrs omine, two-row, n six-row ultivrs OC orrelte muh etter with β-mylse n limit extrinse thermostilities thn ME, strongly supporting the propose hypothesis. β-amylse Intron III Alleli Vrition n Rnking of Cultivr Osmolyte Conentrtions n Mlt Extrt on Eh Dy of Germintion The ptterns of wort OC n ME evelopment in worts proue from mlts germinte over 6 ys oviously ffete the etter orreltions of OC versus mylolyti enzyme thermostili- Fig. 2. Correltion n regression lines of wort α-mylse, β-mylse, n limit extrinse thermostilities versus wort osmolyte onentrtion (OCs) n mlt extrt (MEs) for two-row ultivr mlts mshe t 70 C for 30 min [(α-mylse thermostilities versus OCs: r = , P = ; versus MEs: r = , P = 0.925); (β-mylse thermostilities versus OCs: r = 0.577, P = ; versus MEs: r = 0.403, P = ); (limit extrinse thermostilities versus OCs: r = 0.914, ; versus MEs: r = 0.719, )]. Dt points re the mens of three replitions of four tworow ultivrs omine (n = 24).

7 Osmolytes n Amylolyti Enzymes / 277 ties s ompre to those for ME. OC n ME vlues re lrgely ue to sugrs proue uring mshing (29,30,34, n refs. therein) n sugrs proue uring mshing re lrgely ue to β- mylse tivity (32,51, n refs therein). A few stuies hve shown tht Bmy1 intron III lleli vrition ffets β-mylse tivity n thermostility (e.g., 36,37). However, we n others hve shown tht in North Amerin germplsm, there is no ssoition of Bmy1 intron III lleli vrition with β-mylse tivity or thermostility (31,43,51,84). Hene, one woul not neessrily expet Bmy1 intron III lleli vrition to e ssoite with OC or ME in this stuy. However, we hve ompre OC n ME from mlts proue over 6 ys of germintion to etermine whether our ssumption is orret. Mlts from y 1 of germintion from ultivrs ontining the Bmy1. or Bmy1. intron III lleles proue worts tht were not signifintly ifferent for proution of the highest levels of ME (Bmy1. intron III llele: Grnet n Hrrington; Bmy1. intron III llele: Trition) or OC (Bmy1. intron III llele: Hrrington, Merit, Grnet; Bmy1. intron III llele: Legy) (Tle III). Worts from 2 ys of germintion were not signifintly ifferent for the highest ME from genotypes ontining either the Bmy1. intron III llele (Hrrington) or the Bmy1. III llele (Ley) (Tle III). In ontrst, Hrrington h signifintly higher wort OC thn ll other from mlts on y 2 of germintion. The reverse ws foun with mlts from y 3 of germintion, where the signifintly highest wort OC vlues were foun from ultivrs with oth the Bmy1. intron III llele (Ley, Legy, n Trition) n Bmy1. intron III llele (Grnet, Hrrington, n Merit) (Tle III). Only Hrrington (Bmy1. intron III llele) proue the signifintly highest ME on y 3. As mlts pprohe esirle egree of moifition for rewing on y 4, the signifintly highest wort OC n ME vlues were foun for ultivrs ontining either the Bmy1. intron III llele or the Bmy1. intron III lleles (ME: Bmy1. intron III llele, Hrrington; Bmy1. intron III llele, Legy n Ley) (OC: Bmy1. intron III llele, Ley, Legy; Bmy1. intron III llele, Merit, Grnet, Hrrington) (Tle III). Only one ultivr from mlts from y 5 of germintion proue ME tht ws signifintly higher thn the other ultivrs, Hrrington, whih hs the Bmy1. intron III llele. However, there were ultivrs ner the low en of ME proution tht ontine either the Bmy1. intron III (e.g., Morex) or Bmy1. intron III lleles (e.g., B1202) (Fig. 3), initing tht there is no prtiulr ssoition with high n low ME proution with the lleli vrition in Fig. 3. Correltion n regression lines of wort α-mylse, β-mylse, n limit extrinse thermostilities versus wort osmolyte onentrtion (OCs) n mlt extrt (MEs) for six-row ultivr mlts mshe t 70 C for 30 min [(α-mylse thermostilities versus OCs: r = 0.197, P = 0.355; versus MEs: r = , P = 0.951); (β-mylse thermostilities versus OCs: r = 0.647, P = ; versus MEs: r = 0.498, P = ); (limit extrinse thermostilities versus OCs: r = 0.865, ; versus MEs: r = 0.630, P = )]. Dt points re the mens of three replitions of four sixrow ultivrs omine (n = 24).

8 278 / Duke, S. H., Henson, C. A. n Vinje, M. A. question. There were no signifint ifferenes in OC proution mong ultivrs mlte on y 5 of germintion. At 6 ys of germintion, the signifintly highest wort OC vlues were proue from ultivrs with either the Bmy1. or the Bmy1. intron III lleles (Bmy1. intron III llele, Legy, Ley; Bmy1. llele, Grnet n Hrrington) (Tle III). As t 5 ys of germintion, 6-y germintion Hrrington h the signifintly highest wort ME vlue. However, s t 5 ys of germintion, the lowest ME vlues i not signifintly iffer from ultivrs with either the Bmy1. or Bmy1. intron III lleles. These t inite tht β- mylse intron III lleli vrition hs little or no influene on wort OC or ME or tht OC or ME nnot e use to preit the tivity of β-mylse. No known geneti mrker hs een ientifie to relily preit rley mlt β-mylse tivity or thermo- TABLE III Rnking of Osmolyte Conentrtion (OC) n Mlt Extrt (ME) of Brley Cultivr Mlts Proue Eh Dy Over Six Dys of Germintion. Differenes in OC n ME Vlues Within Columns Were Determine y LSD Anlysis (α Level = 0.05). Vlues for Eh Cultivr in Eh Column Followe y the Sme Letter re not Signifintly Different Dys of Germintion Dy 1 Dy 2 Dy 3 Dy 4 Dy 5 Dy 6 Rnking of Cultivr OC Hrrington Grnet Legy Ley Trition Hrrington Ley Grnet Legy Trition Ley Hrrington Grnet Legy Trition Ley Grnet Hrrington Legy Trition Legy Ley Hrrington Grnet Trition Legy Grnet Hrrington Ley Trition Mmol kg 1 g 1 1,739 1,658 1,645 1,616 1,586 1,565 1,522 1,499 2,144 2,089 2,081 2,061 2,045 1,960 1,886 1,864 2,310 2,305 2,297 2,293 2,267 2,240 2,164 2,124 2,417 2,364 2,364 2,352 2,348 2,261 2,243 2,198 2,523 2,507 2,461 2,454 2,449 2,436 2,412 2,367 2,487 2,447 2,440 2,438 2,362 2,323 2,284 2,250 P = P = Perentge inrese from y P = P = Rnking of ultivr ME Grnet Hrrington Trition Ley Legy Hrrington Ley Grnet Legy Trition Hrrington Ley Legy Grnet Trition Hrrington Legy Ley Grnet Trition Hrrington Grnet Legy Ley Trition Hrrington Grnet Ley Legy Trition P vlues > 0.05 were not onsiere signifint for eh LSD test n letters initing ifferenes re not shown. = 2-Row, = 6-row ultivrs. = β-amylse intron III llele Bmy1.. = β-amylse intron III llele Bmy1.. e Previously nme B2482. % Perentge inrese from y e e e e e e

9 Osmolytes n Amylolyti Enzymes / 279 stility n s we n others hve note, the est seletion tool urrently ville is iretly ssying for tivity or thermostility (35,62,84). However, there is n n e generte geneti vrition in Bmy1 tht will influene oth rley β-mylse tivity n thermostility (19,52,60,62,66). Comprisons of Mlt Amylolyti Enzyme Thermostilities with ASBC Mesures of Mlt Qulity Three ASBC mesures of mlt qulity were selete to ompre to mlt mylolyti enzyme thermostilities. DP ws selete euse it ppers to e the riving fore for sugr proution uring mshing (27 n refs. therein, 30) n is lrgely ue to β-mylse tivity (27 n refs. therein, 30), n mylolyti enzyme thermostility mesure in this stuy. The ASBC α- mylse tivity mesure ws selete euse its thermostility ws mesure in this stuy n euse it is the primry mylolyti enzyme stuie tht is totlly or ner totlly proue vi e novo synthesis uring rley germintion (27,64, n refs. therein). The ASBC α-mylse tivity mesurement reporte here is ifferent from ll other α-mylse tivity mesurements reporte in this pper, whih were onute with Megzyme kits. Wort β-glun onentrtions were selete euse of their very strong negtive orreltions with the tivities of mylolyti enzymes oserve in previous stuy utilizing the sme smples use in this stuy (28). Wort solule protein ivie y totl protein vlues were selete euse of their highly signifintly positive orreltions with mylolyti enzymes in the sme stuy. Tht stuy suggeste strong temporl oorintion etween strh n β-glun egrtion n protein egrtion n/or soluiliztion uring rley germintion, mlting, n mshing. Some egree of protein egrtion is t lest require in some instnes for soluiliztion of some rley mlt proteins suh s β-mylse (18), ut is ertinly not require for ll solule proteins. For mlts of ll ultivrs omine over 6 ys of germintion, the orreltion for β-mylse thermostility versus DP ws negtive n moest, ut highly signifint (Tle I, r = 0.550, ). A signifintly higher negtive orreltion for limit extrinse thermostility versus DP ws foun (Tle I, r = 0.667, ). The mgnitue of the ifferenes e- Fig. 4. Correltion n regression lines of wort α-mylse, β-mylse, n limit extrinse thermostilities versus wort initil tivities of α-mylse, β-mylse, n limit extrinse for omine two- n six-row ultivr mlts mshe t 70 C for 30 min [(α-mylse thermostilities versus initil α-mylse tivities: r = 0.103, P = 0.485; versus initil β-mylse tivities: r = , P = 0.792); versus initil limit extrinse tivities: (r = , P = 0.855)], [(β-mylse thermostilities versus initil α-mylse tivities: r = 0.628, ; versus initil β-mylse tivities: r = 0.682, ); versus initil limit extrinse tivities: (r = 0.541, )], n [(limit extrinse thermostilities versus initil α-mylse tivities: r = 0.857, ; versus initil β-mylse tivities: r = 0.720, ); versus initil limit extrinse tivities: (r = 0.910, )]. Dt points re the mens of three replitions of four two-row ultivrs n four six-row omine (n = 48).

10 280 / Duke, S. H., Henson, C. A. n Vinje, M. A. tween orreltions of β-mylse n limit extrinse with DP re similr to tht oserve with their orreltions with totl sugr proution uring mshing (52) n reflet the steeper eline in thermostility of limit extrinse s ompre to β-mylse over ys of germintion. There ws no signifint orreltion for α- mylse thermostility n DP (Tle I, Fig. 1) s expete se on the oservtion tht rley mlt α-mylses re thermostle t 70 C (51), the temperture t whih mshing ws onute in this stuy. In tht stuy, α-mylse tivity inrese uring mshing t 70 C, possily ue to the enturtion of mylse/sutilisin inhiitor (BASI) or onformtionl hnge in α- mylse tht i not llow BASI to inhiit tivity (51). BASI hs een shown to signifintly n negtively orrelte with α- mylse tivity in rley mlt (52). Comprisons of DP to β-mylse thermostility of two- n six-row ultivrs revele muh stronger signifint negtive orreltion etween the two with six-row ultivrs (r = 0.683, P = ) s ompre to two-row ultivrs (r = 0.430, P = ) (Tle II). In ontrst, orreltions of two- n sixrow ultivr totl wort sugrs n DP were very similr (51). Also, limit extrinse thermostilities orrelte muh etter with DP with six-row ultivrs (r = 0.814, P < ) s ompre to two-row ultivrs (r = 0.662, P = ), lthough oth orreltions were highly signifint n etter thn those for two- n six-row β-mylse thermostilities versus DP (51). The six-row orreltion of limit extrinse thermostilities with DP ws lmost ientil with its orreltions with totl wort sugrs (51). As with ll ultivrs omine (Tle I), orreltions of twon six-row ultivr α-mylse thermostilities versus DP were not signifint (Tle II), ue to the forementione lk of thermolility t the mshing temperture utilize. For mlts of ll ultivrs omine over 6 ys of germintion, β-mylse thermostility versus ASBC α-mylse tivity ws negtive n moest, ut highly signifint (Tle I, r = 0.525, P = ). This orreltion is very similr for β-mylse thermostility versus DP. As for the orreltion with DP, signifintly higher negtive orreltion for n β-mylse n limit extrinse thermostility versus ASBC α-mylse tivity ws foun (Tle I, r = 0.525, ; r = 0.776,, respetively). As with the orreltion the thermostilities of α- Fig. 5. Correltion n regression lines of wort α-mylse, β-mylse, n limit extrinse thermostilities versus wort initil tivities of α-mylse, β- mylse, n limit extrinse tivities for two-row ultivr mlts mshe t 70 C for 30 min. [(α-mylse thermostilities versus initil α-mylse tivities: r = , P = 0.928; versus initil β-mylse tivities: r = , P = 0.593); versus initil limit extrinse tivities: r = , P = 0.780], [(β-mylse thermostilities versus initil α-mylse tivities: r = 0.590, P = ); versus initil β-mylse tivities: (r = 0.576, P = ); versus initil limit extrinse tivities: (r = 0.474, P = )], n [(limit extrinse thermostilities versus initil α-mylse tivities: r = 0.802, ; versus initil β-mylse tivities: r = 0.827, ); versus initil limit extrinse tivities: (r = 0.927, )]. Dt points re the mens of three replitions of four two-row ultivrs omine (n = 24).

11 Osmolytes n Amylolyti Enzymes / 281 mylse versus DP, there ws no signifint orreltion for α- mylse versus ASBC α-mylse tivity. These t re very similr to those for DP versus mylolyti enzyme thermostilities. Comprisons of ASBC α-mylse tivity to β-mylse thermostility of two- n six-row ultivrs revele slightly stronger signifint negtive orreltion etween the six-row ultivrs (r = 0.548, P = ) s ompre to two-row ultivrs (r = 0.493, P = ) (Tle II), lthough neither orreltion ws prtiulrly strong. In ontrst, two- n six-row ultivr limit extrinse thermostilities versus ASBC α-mylse tivities the negtive orreltions were oth highly signifint (Tle II, r = 0.802, n r = 0.751, for twon six-row ultivrs, respetively). These orreltions were issimilr to the orreltions for DP versus limit extrinse thermostilities for two- n six-row ultivrs in tht omprison of oth types of ultivrs h highly signifint strong orreltions. As with the orreltions of ASBC α-mylse thermostilities versus DP, there ws no signifint orreltion for α-mylse versus ASBC α-mylse tivities of either two- or six-row ultivrs. Wort β-glun onentrtions signifintly n negtively orrelte with S/T, wort sugr proution, n wort mylolyti tivities with mshing of mlts proue over 6 ys of germintion (26,28). With the erese in thermostility of β-mylse n limit extrinse in wort proue from mlts proue over 6 ys of germintion (51), it ws expete tht there woul e signifint positive orreltions etween wort β-glun onentrtions n signifint negtive orreltions with S/T mesurements with the thermostilities of β-mylse n limit extrinse. This is wht ws foun for wort β-glun onentrtions n S/T mesurements versus thermostilities of β-mylse n limit extrinse for ll ultivrs omine (Tle I). For oth two- n six-row seprtely, the orreltions of wort β-glun onentrtions versus thermostilities of β-mylse were lso signifint n positive (Tle II). The orreltions of two- n six-row ulti- Fig. 6. Correltion n regression lines of wort α-mylse, β-mylse, n limit extrinse thermostilities versus wort initil tivities of α-mylse, β- mylse, n limit extrinse tivities for six-row ultivr mlts mshe t 70 C for 30 min. [(α-mylse thermostilities versus initil α-mylse tivities: r = , P = 0.779; versus initil β-mylse tivities: r = 0.120, P = 0.575); versus initil limit extrinse tivities: (r = 0.149, P = 0.487)], [(β-mylse thermostilities versus initil α-mylse tivities: r = 0.590, P = ; versus initil β-mylse tivities: (r = 0.695, P = ); versus initil limit extrinse tivities: (r = 0.775, )], n [(limit extrinse thermostilities versus initil α-mylse tivities: (r = 0.882, ); versus initil β-mylse tivities: (r = 0.763, ); versus initil limit extrinse tivities: (r = 0.907, )]. Dt points re the mens of three replitions of four two-row ultivrs omine (n = 24).

12 282 / Duke, S. H., Henson, C. A. n Vinje, M. A. vr S/T mesurements versus thermostilities of limit extrinse were highly signifint for two-row ultivrs n more moestly signifint for six-row ultivrs. Beuse of the previously isusse resons, α-mylse thermostilities i not orrelte signifintly with wort β-glun onentrtions or S/T mesurements (Tles I n II). Comprisons of Mlt Amylolyti Enzyme Thermostilities with Initil Mlt Amylolyti Ativities During Mshing For mlts of ll two- n six-row ultivrs omine over 6 ys of germintion, the orreltions for β-mylse thermostilities versus the initil tivities of α- n β-mylses n limit extrinses were highly signifint, negtive, n somewht moest (Tle I, Fig. 4, initil α-mylses: r = 0.628,, β-mylses: r = 0.682,, limit extrinses: r = 0.541, ). In ontrst, orreltions for limit extrinse thermostilities versus the initil tivities of α- n β-mylses n limit extrinses were muh stronger n highly signifint n negtive (Tle I, Fig. 4, initil α-mylses: r = 0.628,, β-mylses: r = 0,682,, limit extrinses: r = 0.541, ). As for omine ultivr orreltions for mlt α-mylse thermostilities versus initil tivities over 6 ys germintion, there were none tht were signifint. This fining is onsistent with the orreltions previously isusse n for the resons previously isusse. Comprisons of β-mylse thermostilities revele stronger signifint negtive orreltions with initil tivities of α- n β-mylses n limit extrinses with six-row ultivrs thn with two-row ultivrs (Tle II, Figs. 5 6; six-row: α-mylse tivities, r = 0.695, P = ; β-mylse tivities, r = 0.775, ; limit extrinse tivities, r = 0.763, s ompre to two-row: α-mylse tivities, r = 0.590, P = , β-mylse tivities, r = 0.576, P = ; limit extrinse tivities, r = 0.474, P = ). In ontrst, limit extrinse thermostilities of two- n six-row ultivrs versus initil tivities of α- n β-mylses n limit extrinses were ner the sme (Tle II, Figs. 5 6; six-row: α-mylse tivities, r = 0.882, ; β-mylse tivities, r = 0.763, ; limit extrinse tivities, r = 0.907, s ompre to two-row: initil α-mylse tivities (α-mylse tivities, r = 0.827, ; β-mylse tivities, r = 0.748, ; limit extrinse tivities, r = 0.927, ). As for omine ultivr n two- n six-row row ultivr orreltions for mlt α-mylse thermostilities versus initil tivities over 6 ys germintion for two- n six-row ultivrs, there were none tht were signifint (Tles I n II). These results present, to our knowlege, hitherto unnotie phenomenon initing tht high initil tivities of rley mlt α- n β-mylses n limit extrinses re ssoite with high thermostilities of β-mylses n limit extrinses. This oservtion suggests tht seletion for high initil tivity of ny of these mylolyti enzymes woul lso selet for high thermostility of β-mylse n limit extrinse. The mehnism for this reltionship is unler; however, it suggests tht some ftor or ftors other thn hnge in the genetis of ll three enzymes is involve. This oul e the proution of hemil ompoun tht enhnes tivity n thermostility. Certin osmolytes hve een shown to enhne enzyme stility n thermostility (3). An exmple of suh n osmolyte tht ws ientifie y Arkw n Timsheff (3) is proline, whih umultes in unne in plnt tissues sujete to ioti stress (33,49, n refs. therein). Free proline lso umultes in unne n fr more thn ny other mino i uring rley germintion (72). It is lso in unne n in fr greter onentrtion thn other mino is uring mshing (17,72). Even though muh of this free proline pool ppers to e trnsporte from the enosperm to the eveloping emryo uring germintion, levels of proline in the enosperm remin very high n ontinue to inrese (72). This suggests tht proline enhnement of enzyme thermostility woul e most pronoune in mlts proue ner mximum moifition. Sugrs, whih re lso known to enhne enzyme thermostility (4), lso umulte in unne uring mshing (26,32), n shoul lso enhne thermostility of ll or most proteins. We oserve the gretest thermostility in β-mylse n limit extrinse uring mshing t 70 C in mlts proue from grin germinte for only 1 or 2 ys (51) when levels of free proline re rpily inresing, ut not ner their mximl onentrtion oserve lter in germintion (72). Correltions of omine ultivr α- n β-mylse n limit extrinse thermostilities over 6 ys of germintion versus eh other s thermostilities over the sme time perio revele positive orreltion with β-mylse thermostilities versus limit extrinse thermostilities (Tle I, r = 0.665, ). The sme reltionship ws foun for two-row (r = 0.545, P = ) n six-row ultivrs (r = 0.728, ) (Tle II). α-amylse thermostilities i not signifintly orrelte with the thermostilities of ny other mylolyti enzymes. These results inite temporl reltionship etween the thermostilities of β-mylse n limit extrinse. CONCLUSIONS Wort osmolyte onentrtion (OC) is etter initor thn mlt extrt (ME) of the egrtion of strh s iretly etermine y sugr proution or s iniretly reflete y mylolyti enzymes tivities. The t presente here suggest tht β-mylse n limit extrinse thermostilities eome more limiting to strh egrtion s reflete y OC thn s reflete y ME s mlts proue on suessive ys of germintion re mshe. In generl, wort OC vlues peke with mlts proue until y 5 of germintion, wheres ME only inrese until y 4 of germintion. β- Amylse intron III lleli vrition h no effet on OC or ME on the ys where LSD nlysis ws signifint, initing tht, s in pst stuies, North Amerin rley germplsm, β-mylse thermostility n tivity re not influene y my2 intron III lleli vrition. High initil tivities of rley mlt α- n β-mylses n limit extrinses orrelte negtively n signifintly with high thermostilities of α-mylse, β-mylses, n limit extrinses. The one exeption ws for orreltions for initil tivities of α-mylse versus thermostility of α-mylse. This ws pprently ue to the thermostility of α-mylse t the 70 C mshing temperture. These t suggest tht seletion for high tivity of ny of these mylolyti enzymes woul lso selet for high thermostility of β-mylse n limit extrinse. ACKNOWLEDGMENTS Finnil support ws provie in prt y the Amerin Mlting Brley Assoition, In. n the USDA-ARS, USDA Coopertive Stte Reserh, Eution n Extension Servie, U.S. Brley Genome Projet Speil Grnt. We thnk Chrles B. Krpeleni, Joseph T. Dietrih, Mrih K. Peronto, n Roert D. Vogelzng for their expert tehnil ssistne, n Allen D. Bue n Christopher H. Mrtens for their ssistne in mlting. LITERATURE CITED 1. Alves-Jr., S. L., Hererts, R. A., Holltz, C., Miletti, L. C., n Stmuk, B. Mltose n mltotriose tive trnsport n fermenttion y Shromyes erevisie. J. Am. So. Brew. Chem. 65:99-104, Amerin Soiety of Brewing Chemists. Methos of Anlysis, 8th e. Brley-7C Protein (N 6.25) y Comustion; Mlt-4 Extrt;