CORRELATON BETWEEN THE CHEMCAL STRUCTURE AND RHEOLOGCAL PROPERTES OF GLUTEN By R. L.,(SZTTY Department f Bichemistry and Fd Technlgy, Technical University Budapest Received April 1, 1981. ntrductin The prminent poston f wheat and wheat flur, amng the raw materials f the baking industry is strngly cnnected with its unique prteins. Amngst cereals nly bread wheats - and t a lesser extent triticale - psses reserve prteins which interact with water t yield dughs having the necessary chesivity and elasticity fr making leavened bread. The reasns fr this, and the structure f gluten and its prtein cnstituents have intrigued cereal chemists since the days f Osbrne. t is evident that the factrs playing a rle in defining the rhelgical prperties f gluten are f a cmplex character. Based n the!'etical cnsidemtins and als n all the studies which have been carried ut it wuld appear that the fll"\ving tw grups f factrs are the mst imprtant: the quantity and quality (slubility, amin acid cmpsitin, mlecular mass distributin etc.) f the prtein cmpnents f the gluten cmplex, the interactins (disulphide bnds, hydrgen bnds, hydrphbic interactins, electrstatic inte!'actins etc.) between the prtein fractins. n the framewrk f the research cal'l'ied ut in ur labratry the col'l'elatin between the chemical structure and the rhelgical prperties f glut ens was investigated. The effect f amin acid cmpsitin, mlecular mass distributin f prteins and as mst imprtant, the interactins f prteins were studied. The results f this investigatin are discussed in this paper and cmpared with the cnclusins f the!' authrs. 2. ~:lateria1s and methds Glutens were prepared frm different varieties f wheats and wheat flurs (Bezstaya, Fertdi 279, San Pastre, Kavkaz, Yubileynaya, MV-4) by washing and additinal purificatin. The methd was described earlier [1,2]. 1*
4 LASZTTY, R. The rhelgical prperties f the gluten were characterized by a threeelement mdel (cmbined frm the Maxwell and Hke mdel; H/H-N). Frm the experimental data the relaxatin time and the relaxatin cnstant were calculated. The stress relaxatin was measured by a mdified Nelabrgraph instrument. The details f the measurement and calculatins were published in an earlier paper [2]. The amin acid cmpsitin f the gluten prteins was determined after hydrlysis with hydrchlric acid using an autmated amin acid analyzer (AAA 881 Mikrtechna, Praha). The prtein fractins f the gluten cmplex were separated by gel chrmatgraphy. Fr peptizatin, acetic acid slutins were used. The details f the methds were als described earlier [3a, c]. Chemically mdified glut ens (desamidated, acetylated, esterified glutens, N-ethyl maleic imide derivatives) were prepared with the methds described by LisZTTY [3], BECKWTH et al. [4], BARNAY et al. [5], HOLl\1E and BRGGS [6]. Fr splitting disulphide bnds and their re xidatin the methd f BECK WTH et al. [7, 8] was used. 3. Results and discussin 3.1. Aminracid cmpsitir. The amin acid cmpsitin f the gjutens prepared frm different wheats shws slight differences which may, in many cases, be statistically significant. Nevertheless the amin acid cmpsitin f the gluten is relatively stable and may be characterised as fllws: high glutamic acid cntent relatively high prline cntent lw amunt f basic amin acids (lysine, arginine, histidine) high degree f amidatin nearly equivalent t the aspartic and glutamic acid cntent remarkable cystein and cystine cntent. The mathematical-statistical evaluatin f the results f the verall amin acid cmpsitin and the rhelgical characteristics des nt exhibit significant crrelatin in mst cases. The ttal disulphide bnd (cystine) cntent shws a significant crrelatin with the rhelgical prperties. This is characterised by a linear crrelatin cefficient varying frm.3 t.6. A strng psitive crrelatin between the cystine cntent and wheat flur baking value was reprted earlier by WOSTMANN [9]. The lwer degree f crrelatin may be explained by the assumptin that nt nly the abslute number f disulphide hnds but als their distributin is imprtant frm the view f rhelgical
CHEl,nCAL STRUCTURE AND PROPERTES OF GLUTEN 1~....,.8 eo." G '\,. c 85 9 Degree f amidatin {%J 95 Fig. 1. Crrelatin between the degree f amidatin and the relaxatin time f gluten prperties. The prblem f the disulphide bnds ",ill be discussed in Chapter 3.3 f this paper. The crrelatin between the degree f amidatin (calculated n the basis f aspattic and glutamic acid cntent and ammnia cntent, resp.) and the Thelgical chatactel'istics is sh'w-:l in Figme 1. The results indicate that an ptimum degree f amidatin exists. The bs ervatin may pattly be explained by the wrk f BRGGS and HOLl\E [6], CUNl'<l\"GHAM et al. [1] and BECKWTH et al. [4]. They pstulated that an increase in the number f the amid gtoups inctease the pssibility f the fotmatin f secndary bnds in the gluten sttuctmewhich imptve the rhelgical pl'operties f the gluten. Hwevel', this hypthesis des nt explain the negativ effect f the highest degrees f amidatin. An intel'esting cnelatin was fund between the amunt f amin acids with hydl'phbic side chain (leucine, isleucine, prline) and relaxatin time. The cxrelatin can be descl'ihed hy a secnd-l'det equatin. Based n the :results mentined abve a ne)'! "Quality index" was pr ps eo. serving a ptelimina:ry eyaluatin f the wheat's baking value ftm the d. ta f the amin acid cmpsitin f the gluten. where C A H KA KH (1) QN =1 C.15 A-RA = cystine cntent (%) = degree f amidatin (%) = prline, leucine and isleucine cntent = (87 - A) (abslute value) = (22.5 - H) (abslute value)
6 LASZTTY. R. 3.2. Distributin f prtein fractins The readily dispersed and undispersed fractin f the gluten cmplex (in.5 ml/dm 8 acetic acid slutin) was measured and the crrelatin between the quantities f prtein fractins and relaxatin time were calculated statistically. The results are shwn in Figures 2 and 3. The results indicate that the amunt f the readily dispersible cmpnents is in negative crrelatin with the rhelgical quality. The increase in the quantity f nn-peptisable fractins has an imprving effect. These bservatins are in gd agreement with the statements f many authrs and with sme methds fr determining wheat baking value based n the swelling r dispersibility f gluten prteins in dilute rganic acids (lactic acid, acetic acid). The effect f the rati f the high mlecular mass and lw mlecular mass fractins (separated by gel chrmatgraphy) was als investigated. n "1 c ~ 1 i 5~ j 1 O+,---T---T,---,,----,,----,,;----., ---" -- 1 2 3 4 5 6 7 Readily dispersible fmctin (%J Fig. 2. Crrelatin between the quantity f readily dibpersible prtein fractins and the relaxatin time f glutens c c 6 g.;; c Fig. 3. Crrelatin between the quantity f nn-peptisable prtein fractins and the relaxatin time f glutens
CHEMCAL STRUCTURE AND PROPERTES OF GLUTEN 7 2 25 3 High mleculer m.ss rrccti:! (Cl::) 35 Fig. 4. Crrelatin between the rati f high mlecular weight prtein fractins and the relaxatin time f glutens Fig. 4 the crrelatin between the quantity f high mlecular mass prteins and the relaxatin time f the gluten is demnstrated. The crrelatin can be expressed by a secnd rder equatin and a crrespnding curve having a maximum value. The imprtance f the rati f the higher mlecular mass glutenin t the lwer mlecular mass gliadin fractin was cnfirmed by the investigatins f many authrs. Summarized results are given by KASARDA et al. [11], SMONDS [12], L.(SZTTY [13, 14]. 3.3. nteractins between the prtein fractins 3.3.1. The rle f disulphide bnds Many examples are knwn abut the imprtant rle f disulphide bnds cncerning the prtein structure and its mechanical prperties. The gluten prteins cntain relatively small quantities f cystine and cysteine, (in average 2 t 3%). Primarily their imprtance was bserved in relatin with prblems f flur imp rvers. n cnsequence, very extensive research wrk was started in this dmain, especially in the last tw decades. Nevertheless there are still many unslved prblems. T clear up the rle f disulphide bnds needs further cmprehensive investigatins. Sme useful data may be btained by studying the changes ccurring in the rhelgical prperties due t the decmpsitin f the disulphide bnds. A.mng the prcedures used in prtein chemistry t break dwn disulphide bnds, reductin seems t be particularly prmising, because f the pssibility t recnvert the thil grups frmed int disulphide bnds, after the remval f the reducing agent. Earlier tests f BECKWTH et al. [7, 8] n wheat gliadin and glutenin shwed that gliadin reductin (breakdwn f the S-S bnds) invlves n
8 LASZTTY, R. perceptible change in mlecular mass. Thugh fergrams btained by gel electrphresis shw minr deviatins in mbility, these can be explained by changes in cnfrmatin. Reduced and alkylated gliadin cntain practically n helical structures, accrding t ptical dispersin tests, whereas in active gliadin their cmputed rati is abut 15%. n the case f re xidatin in a diluted slutin, investigatins f the abve-mentined authrs shw that the native gliadin is practically recvered. Their finding has been cnfirmed by ultra centrifugal, electrphresis and rtatry dispersin tests. N data have been published abut the rhelgical prperties f the rexidized prduct. The statement accrding t which rexidatin in a mre cncentrated slutin (5%) yields prducts with higher mlecular masses and less sluble than the riginal gliadin is very interesting. Presumably intermlecular disulphide bnds are being frmed in this case. The decmpsitin f the disulphide bnds in glutenin by reductin l'esults in the disintegratin f the large gluten cmpnent mlecules. n the case f rexidatin in slutins f higher cntentratin, a prduct with prperties very clse t thse f native glutenin may be btained. Accrding t the cited authrs, the physical prperties f the prduct frmed in the curse f rexidatin arc much influenced by the rexidatin cnditins, primarly by ph and by UTea cncentratin. K numerical data n the rhelgical prperties f the glutenins prduced under different cnditins are reprted; prbably n such measurements haye been carried ut. We therefre cnducted studies n the cnditins f rexidatin fllwing glutell l'eductin and n the rhelgical prperties f the prducts btained, t pryide the necessary infrmatin. T reduce and rexidize the glutcn samples, we adpted the methd applied by BECKWTH et a1. [7,8] its rugh utlines being as fllws: 5% slutins f the gluten samples in a 6 mljdm 3 m'pa slutin,yere prcpal'ed. The l'eductin was suhscauenth- canied ut with p-merca }tethanl under nitl'gen 1..t.;! <..,.; circulatill fr 12 hurs. Part f the Teduced gluten was alkylatcd with acl'ylnitile, yielding, after dialysis and lyphilizatin, the S-cyanethyl-gluten derivative. Rexidatin was c ani ed ut in different urea cncentratins (slutins f 1 t 8 ml/dm 3 ) and at different ph values (3.5-5.5-8.5). The gluten cntent f the slutin l'anged frm t 1% since preliminary tests shwed this cncentratin range t he the mst favul'ahle f1' prducing a pl'duct similar t native gluten. Rexidatin was perfrmed by xygen cil:culatin fr 168 hurs. By the end f the rexidizing pl'ocess the prduct,vas purified by dialysis, cmpacted in a centl'ifugal appal'atus and finally free water was remved frm the agglmerating mass by hand kneading in a plyethylene bag. The samples were tested fr stress relaxatin by the methd f LASZTTY [3a, 3c].
CHEMCAL STRUCTURE AND PROPERTES OF GLUTEN 9 Table 1 summarizes the rhelgical prperties f rexidized glutens prduced under different cnditins. Table 1 Rhelgical prperties f rexidized glutens N Urea cne. (ml/dm') Th S/m' 'r (sec) Remarks 1 2 3 1 15 1 5.5 22 1 8.5 35 nt measurable 4. 5 6 2 3.5 24 2 5.5 28 2 8.5 12 3 12 nt measurable 7 8 9 4 3.5 54 4 5.5 77 4 8.5 18 68 92 15 elastic 1 11 12 6 3.5 3 -, 6 5.5 ~ 6 8.5 19 t:.: 29 18 slightly elastic 13 14 15 8 3.5 32 8 5.5 27 8 8.5 28 ~-.:);) nt 111casul'able nt l!leasurable no!.l~elastic nolwelustic 16 native gluten 59 (cntrl sample) 83 gluten f average elasticity and extensibility Data in Table 1 sh\v that the Thelgical prperties f the prdllcts btained are highly infll1enced by~ the re:y~idatin cnditir..s. Pl'OdllctS })est appraching the prperties f native gluten ale prduced with a (3 ml/dm 3 urea) slutin at ph 5.5 and with a 6 ml/dm 3 urea slutin at ph 3.5. n general, vrith 1'1'''- urea cncentratins chesive prducts,dthut elastic prperties are btained, whereas in the case f 8 ml/dm 2 urea cncentratin, the rexidized gluten is tugher, but has n apprpriate elasticity. n alcaline media (ph 8.5) n prduct with prperties similar t native gluten culd be btained at any urea cncentratin. t shuld be nted that re xidatin was essentially mre rapid in alcaline media, and in general, the rexidized prduct yielded a practically inelastic, chesive mass when mixed with water. ts prperties were largely similar t gluten extensively denaturated by heat. The present study f the rhelgical prperties distinctly shws the imprtant rle f disulphide bnds n the physical prperties f gluten. At the
1 LASZTTY, R. same time it is apparent that the abslute number f disulphide bnds alne des nt unambiguusly define these prperties. n this respect the site f the disulphide bnds is als essential. The influence f ph and urea cncentratin n the prperties f the rexidized prducts can h' explained as fllws: depending n ph and urea cncentratin, changes may ccur in the cnfrmatin f the peptide chains, in the steric psitin f the individual grups, their dissciatin cnditins, their reactivity, defining the type and site f the disulphide bnds frmed. The effects f urea, f ph and f in cncentratin n the prtein cnfrmatin are generally knwn. The disulphide bnds frmed might be either intramlecular r intermlecular bnds. The rati f inter- and intramlecular disulphide bnds is presumably very imprtant in the develpment f rhelgical prperties. T check this assumptin, the changes in the viscsity f the slutin during decm psitin by perfrmic acid f rexidized glutens btained in different ways have heen fllwed. Characteristic curves are presented in Figs 5, 6 and 7. t can distinctly be seen that the viscsity versus time graphs are different fr rexidized gluten prducts having different rhelgical prperties. The sim plest curve was btained fr a gluten rexidized in alcaline medium at a high urea cncentratin (Fig. 5). After a relatively rapid viscsity decrease, it remains at an apprximately cnstant value during further xidatin with perfrmic acid. The rexidized prduct, with prperties apprximating thse f the native gluten, fllws a different curse (Fig. 6). _,vter a rapid viscsity decrease a minimum is reached. Viscsity then rises t reach a limit value. The slutill f a rexidized gluten prduct prepared at a small urea cnccntratin at acid ph (Fig. 7) is suhject t a smaller viscsity decrease during the per frmic acid xidatin, the yiscsity graph shws a sharp minimum, and the subsequent increase in viscsity is highel' than in Fig. 6. Disulphide hnds f rexidized glutens have als heen suhjected t decmpsitin hy sulphitlysis. The character f the curves htained is clsely similar t the ahve-discussed curves. The results f hth series f experiments clearly shw the differences hetween the disulphide hnd systems f the rexidized glutens with different rhelgical prperties. The viscsity decrease ccurring in all cases at the beginning f the disintegratin f the disulphide bridges indicates that wing t the rupture f intermlecular disulphide hnds, larger prtein mlecules will hreak dwn t smaller units. The subsequent curse f the viscsity curve will depend n whether the smaller units frmed cntain intramlecular disulphide hnds at all, r t what extent, since the disintegratin t these intramlecular disulphide bnds will result in a change f the cnfrmatin f the mlecule, and the mlecule, "pening" t a certain extent, will increase viscs-
CHEMCAL STRUCTURE AND PROPERTES OF GLUTEN 11 2 Time (hurs) Fig. 5. Changes in the viscsity f a rexidized gluten slutin during the splitting f S-S bnds by perfrmic acid (Prduct C: rexidatin in 8 ml/dm 8 urea, ph 8.5) ll.o~ ri--r-"""'--'/.- >- 'lii 13 8 Ul :>.~ 15 &i 12 2 Time (hurs) Fig. 6. Changes in he viscsity f a rexidized guten slutin during the splitting f S-S bnds by perfrmic acid (Prduct B: rexidatin in 3 mljdm 3 urea, ph 5.5) 13 " 2 Time (hurs) Fig. 7.SChanges in the viscsity f a rexidized gluten slutin during the splitting f S-S bnds by perfrmic acid (Prduct A: re xidatin in 1 mljdm 3 urea, ph 3.5)
12 LASZTTY, R. ity. When disintegratin f the intramlecular disulphide bnds cmes t an end, bth the cnfrmatin f the mlecule and the viscsity will attain cnstant values. Studying the curves frm this pint f view, it may be seen that the rexidized gluten prepared in an alcaline medium at a high urea cncentratin practically cntains intermlecular disulphide bnds nly. n the prduct clse t native gluten, the number f intramlecular disulphide bnds is high and the same applies als t rexidized gluten btained at acid ph at a lw urea cncentratin. Ou principle the viscsity recvery after a minimum culd als be ascribed t new aggregates frming secndary bnds, after the breakdwn f the primary disulphide bnds. This assumptin is, hwever, imprbable, because n such increase is recrded fr the rexidized gluten prepared at alcaline ph in high urea cncentratin. A gd example fr the slwer reactin f the intramlecular disulphide bnds is presented by extensive investigatins with insulin. These studies indicated that nly ne f the three disulphide bnds present culd he reduced hy thiglyclate. Tw disulphide hnds culd he disintegrated with sdium sulphite, hut cmpletely disintegratin f all disulphid e hnds was nly pssihle in the ptesence f urea, guanidine and phenylmercuric hydrxide, resp. Mre detailed studies Tevealed that it 'was the intramlecular disulphide hnd which 'was the slwest and mst reluctant t react. Thugh there is n kn wledge ~,vailable ahut mre cmplex prteins and abut the system f disulphide hnds in gluten prteins, the cnsideratin f sme gene"a1 relatinships and 2:u.aigies all ws t presume that - at least in the case f the larger gluten prtein mlecules - the intramlecular disulphide bnds react slwer wing t steric inhibitin, and becme aecessihle nly after a certain "lsening" f the mlecul21' cnfrmatin. The hservatin fbeckwte and "'ALL (1966) is f interest: the ampermetrically determined disulphide cntent f glutenin btained hy s-ulphite bl'cakd\yll des nt e::~c-eed abut ""CV,"O thirds f present, up t a urea cncentratie f 3 ml! dm 3. The ttd detel'mined dy in a mea slutin f 6 ml/dm 3 the,rrk f BERNACKA and KACZO,\YSK [15] shuld be mentined. Based n reduetin dynamics, they divided the S-S bnds f the gluten cmplex in t fm grups (three intermlecular and ne intramlecular). Reductin and re xidatin studies f gluten indicate the imprtance f the disulphide hnds in the structme f gluten mlecules and als in its rhelg c ical prperties. Accrding t these studies high mlecular mass gluten prtein fractins cnsist f plypeptide chains cnnected by disulphide hridges. n additin t these, the number f disulphide bnds within the mlecule is als f imprtance. Dming the reductin f the gluten, all disulphide bnds will hreak dwn, and the btai'led prduct lacks the rhelgical prperties f the
CHEMCAL STRUCTURE AND PROPERTES OF GLUTEN 13 Native gluten J Reduced gluten Reductin (B - mercaptethanl) (Mix f prteins with a mlecular mass f 2-4 thusand) Rexidatin (air, xygen) High gluten cncentratin (6-1%) in 8 ml/dm3 urea slutin Lw gluten cncentratin (.1-1 'fa) in 1 ml/dm3 urea slutin Mderate gluten cncentratin@gvp (5-6 %) in 3 ml/dm3 urea slutin Slightly sluble, chesive, but nn-elastic prduct, hight mlecular mass Easily sluble, sft, sticky, nn-elastic prduct, medium mlecular mss Chesive, elastic prduct, similar t nctive gluten "Randm cil' '1T'fTf" He i x :::::r:::::. Disulphide bnd Fig. 8. Diagram f the prcess f prducing gluten pssessing rhelgical prperties as desired riginal gluten. When rexidized, the reversin is practically quantitative. Hwever, the site f the newly frmed bnds and the prprtiu between inter- and intramlecular disulphide, bnds will depend n rexidizing cnditins. This fact is reflected in the rhelgical prperties f the rexidized prduct. t fllws frm the abve that the abslute number f the disulphide bnds alcne des nt unambiguusly define the structure and the rhelgical prperties. t becmes clear why, in general, n clser crrelatin culd be established between the disulphide cntent and the rhelgical prperties. t explains als why the crrelatin is strnger in gluten f the same wheat variety: it may be assumed that in a given wheat variety the prtein bisynthesis prceeds similarly, and hence the distributin f the disulphide bnds will als be similar. The lseness f the crrelatin is likely t be attrihuted t the many ther factrs invlved in the develpment f the rhelgical prperties.
14 L.4SZTTY, R. The finding that prducts... vith differing rhelgical prperties can be btained, depending n the cnditins f re xidatin after the reductin t a given gluten, culd be f paramunt imprtance fr practice. t implies the pssibility t develp an ecnmically feasible methd f reductin and rexidatin fr breaking dwn the native glutens t units cnsisting f the fundamental plypeptide chains and subsequently, by selecting the apprpriate cnditins fr rexidatin t prduce glut ens with rhelgical prperties as required by the particular grain-prcessing technlgy in questin. The llilderlying cncept f this gluten-prcessing methd is presented in Fig. 8. EWART'S recent wrk [16, 17] cnfil:ms the imprtance f the distributin f disulphide bnds. The thery f rhelgically active SE-grups (BLOKS MA [18], JONES [19]) als stresses the rhelgical imprtance f the sites f the S-S-bnds. 3.3.2. The rle f hydrgen bnds The gluten cntain a great number f side chains frming hydrgen bnds (Table 2). This fact supprts the assumptin that the hydrgen bnds Table 2 Functinal grups in gluten prteins (HOLME 1966, POMERANZ 1968) (mml/l g prtein) Grup A.mm acids Glutenin Acidic glutamic acid 27 36 aspartic acid Basic lysine 39 52 arginine histidine tryptphane Amide glutamine 39 266 asparagine Sulphhydril and cysteine 12 12 disulphide cystine Ttal inic (acid + basic) 66 87 Ttal plar (hydrxy + amide) 381 365 Ttal nnplar 39 31
CHEMCAL STRUCTURE 41\'1) PROPERTES OF GLUTEN 15 als cntribute t the rhelgical prperties f gluten. lvlany experimental facts cnfirm the pssible rle f nn-cn valent bnds. Mst insluble glutens can be dispersed in strng urea slutins r in ther hydrgen-bnd-disrupting agents. Hydrgen bnding was als shwn t be respnsible fr aggregatin and disaggregatin phenmena f prteins during separatin by gel chrmatgraphy (JANKEWCZ and POl\ER.~NZ [2]). The cntributin f hydrgen bnds and reactive sulphur-cntaining grups f prteins t the rhelgical prperties f dugh were studied by JANKEWCZ and POMERANZ [2] by adding urea and N-ethylmaleic imide. VAKAR et al. [21] reprted that freshly washed gluten becmes sttnget and mre elastic aftet dipping in t D 2. These facts als indicate that hydrgen bnds play an imprtant rle in the gluten structure. The TOle f amid grups was studied by HOLME and BRGGS [6] and BECKWTH et al. [4]. n OUT labratoty we investigated chemically mdified glutens. The influence f desamidatin. A great number f amidated catbxy grups ate present in gluten prteins. n view f this number, high in cmparisn with ther plar grups, the Tle f amid grups in the frmatin f secndary bnds might be imprtant. We therefote determined penetratin indexes f glutens desamidated t varius degrees, and measured the viscsities f desamidated gluten disslved in acetic acid and in 8 ml/dm 3 urea slutins. Results are listed in Tables 3, 4 and 5. The data demnstrate that, cmpared t cntrl samples, desamidated glutens are f sfter cnsistence, i.e. their rhelgical ptoperties are inferir. Rhelgical Table 3 prperties f desamidated glutens Sample N. Difference in penetratin.cl P(~) Cmpared t cntrl sample degree f desamidatin (~~) 1 3 7 9 1 1 3 4 5 6 7 8 9 1 15 12 17 1 8 7 13 15 11 14 24 39 39 42 23 35-16 J~ " 5 29 39 51 55 57 21 31 35 3,1 39 17 29 37 42 42 23 29 35 37 39 21 34 45 49 ;);) 21 29 42 47 48 19 33 42 47 48 23 32 44 43 43
16 LASZTTY, R. Table 4 nstrinsic viscsity f desamidated gluten slutins in acetic acid Ser. N. (7)] dj/g degree f desamidatin 1 3 5 7 9 1 Acetic acid cncentratin 1.425.445.462.48.55.51.5 ml/dm 3.43.435.44.437.435.42 1. ml/dm 3 2.48.485.492.57.58.51.5 mljdm 3.475.48.481.475.47.469 1. ml/dm 3 3.46.475.49.52 i.5.499.5 ml/dm3.462.467.47.465!.459.458 1. ml/dm 3 4.52.52.53.536.535.537!.5 ml/dm 3!.49.51.56.58.56.51 1. ml/dm3 5.397.412.431.432.43.429.5 ml/dm 3.4.45.45.42.4.39 1. ml/dm 3 Table 5 ntrinsic viscsity f the slutins f desamidated gluten in 8 ml/dm 3 urea Ser. N. (7)] dj/g degree f desamidatin 1 ; 3 5 7 9 1 1.48.475.475.478.468.45 2.55.552.545.547.53.527 3.51.497.52.54.486.479 4.56.55.552.54.531.535 5.48.482.48.47.447.45 The differences, in terms f relative per cent penetratin increase with the degree f desamidatin, substantially at the start, and then tending twards a limit value. As far as sluhilities are cncerned, desamidated gluten is mre difficult t disslve in strng acid media (belw ph 3), but is easly disslved - in cntrast t cntrls - in ph 8. phsphate buffer. Cnsidering the substantial increase f free carbxyl grups and the acid character f the prtein frmed, this fact seems t be understandable. Based n the results f viscsity measurements it can be stated that desamidatin primarily affects the viscsities f slutins in acetic acid. Accrding t the data in Table 4, this mdificatin leads t an increase in
CHEMCAL STRUCTURE AND PROPERTES OF GLf;TE1Y 17 intrinsic viscsity, demnstrating that a change had ccurred in the cnfrmatin f the mlecules, which results in higher asymmetry f the structure. Fr an explanatin, ne might suggest that the remval f the ami d grups nl"l;lves the eliminatin f secndary, e.g. hydrgen bnds, resulting in a lser structure. Presumably, due t the dissciatin f carbxyl grups liberated within a mlecule, repulsive frces,vill be perative between grups with identical charge. This assumptin is supprted by the experimental finding that increase in viscsity is substantially less in 1. mll dm 3 acetic acid at higher ph and lwe:r dissciatin, r that n these cnditins n increase f viscsity is fund in sme cases. Prbably n the ne hand, electrstatic repulsin is weaker, and n the ther hand, new hydrgen bnds are frmed between grups at sterically favurable sites. Similar cnclusins can be drawn frm viscsity data relating t glutens and t desamidated glut ens disslved in 8 ml/dm 3 urea. A cmparisn f viscsity data f slutins in.5 ml/dm 3 acetic acid and in 8 ml/dm 3 urea reveals that as cmpared t the acetic acid slutin the increase in viscsity f nt desamidated cntrl samples is significantly higher than that f partially desamidated samples. The difference can be explained n the basis that in amidated glutens there are substantially mre hydrgen bnds present, and these are disrupted by the urea added and thus the cnfrmatins are altered. At lwer (ph/l ml/dm 3 acetic acid) desamidated gluten samples als shw viscsity data widely differing frm thse f slutins with urea. n sme cases these values are very nearly the same as thse fr the cntrl samples. This finding supprts the idea f new hydrgen bnds being frmed as mentined in the discussin f the data shwn in Table 4. Effect f esterificatin. Esterificatin is ne f the pssibilities t transfrm the free carbxyl grups. Partial cnversin f amid grups int esters is als feasible. n ur experiments the rhelgical prperties f glutens esterified with methanl r ethanl were studied tgether with the viscsity f the slutns prepared frm the derivatives with.5 ml/dm 3 acetic acid. Results arc listed in Tables 6 and 7. The data in Table 6 shw that with increasing degrees f esterificatin the rhelgical prperties f gluten deterirate, relaxatin time becmes significantly shrter. n the first stage crrespnding t the esterificatin f the free carbxyl grups present, n essential change ccurs; this suggests that the rle f free carbxyl grups in the frmatin f secndary bnds is insignificant. The viscsity data indicating a substantial decrease in intrinsic viscsity implicate mre cmpact and less asymmetric mlecules, pssibly due t the fact that alkylated prtein is highly hydrphbic, and hence its hydratin degree will be lwer. 2
18 LAsZTTY. R. Table 6 Re axatin f gluten esterified,dth methanl Relaxatin t..;ne, sec. Ser. N. extent f methylatin, m...--uljg :.3.5 1. 1.5 2. 3.! 1 82 79 6 54 48 45! 46 2 45 44 36 3 28 'r -;) 26 3 73 7 6 52 45 4 41 4 55 56 51 41 35 32 34 5 69 65 54. 48 4 36 3 Table 7 ntrinsic viscsity f slutins f gluten esterified with methanl Ser. N. [7)] d1/g extent f methylatin, mml/g.3.5 1. 1.5 2. 3.! 1.442.439.384.35.321.295.28 2.495.48.41.362.318.288.275 3.48.469.36.331.297.291.32 4.53.529.46.431.39.321.35 5.42.415.34.291.27.258.261 Effect f acylatin. n rder t study the rhelgical prperties f acylated gluten, the penetratin values f the hydrated gluten derivatives and the viscsities f their slutins in.1 ml/dm 3 acetic acid were measured. Results are summarized in Tables 8 and 9. The data reveal that the rhelgical prperties f acylated gluten are very much inferir t thse f native gluten. The decrease f chesivity suggests that primary amin grups play a substantial rle in the frmatin f intermlecular nn-cvalent bnds. Experimental results shw that n majr alteratin in viscsity takes place; after an initial small decrease the viscsity values remain practically cnstant. Thus it can be cncluded that n imprtant change f mlecular cnfrmatin ccurs, r that primary amin grups participate principally in the frmatin f intermlecular bnds.
CHEMCAL STRUCTURE AND PROPERTES OF GLUTEN 19 Table 8 Penetratin values f acylated gluten Ser. N. 1 2 3 4 5 6 7 8 1 72 58 85 7 94 45 48 73 Penetratin,.1 mm per cent f acylatin 4 8 142 18 l2 15 143 92 89 i4 152 112 135 l21 162 18 12 13 1 i 1 16 135 142 143 18 l25 117 135 ntrinsic viscsity Table 9 f the slutins f acylated gluten Ser. N. ['7][dljg per cent f acylatin 4 8 1 1!.425.396.392.388 2.48.428.42.422 3.46.432.417.418 4.52.477.472.465 5.397.368.362.365 6.485.444.427.43 7.51.482 9.469.461 8.447.417.45.396 3.3.3. Effect f hydrphbic bnds Gluten prteins cntain several amin acids with hydrphbic side chains (alanine, leucine, phenylalanine, isleucine, valine, prline). Mrever, taking int cnsideratin that the hydrphbic parts f lnger plar side chains (e.g. in the case f lysine and glutamic acid) may als interact, there can be n dubt abut the ptential pssihility f the frmatin f hydroa phbic bnds. Dugh and gluten frmatin prceed in aqueus media. Owing t the fact that an interactin f the nn-plar grups with water is "unfavurable" 2*
2 LASZTTY, R. frm the thermdynamic viewpint, the thermdynamic tendency pints twards a linkage f the nn-plar grups with each ther (with a cnsequent weakening f the interactin between these grups and water). This prblem is dealt 'with in the review by KAUZMAN [22]. n general, the frmatin f hydrphbic bnds is an endthermic prcess, i.e. the change in thermdynamic ptential is negative since the effect f the change in entrpy (T LS) exceeds that f the change in enthalpy (LlH). Up t a certain temperature limit, the strength f hydphbic bnds increases "with increasing temperature, s that hydrphbic bnds are f particular imprtance frm the viewpint f thermal stability f prteins. The slubility f gliadin in nn-plar slvents, and the influence f the latter n slubility are als indicative f the pssible imprtance f the rle f hydrphbic bnds. All this shws cnv-incingly that a study f the hydrphbic bnds is unavidably necessary fr the understanding f factrs which influence the structure and rhelgical prperties f gluten prteins. Up t nw wheat prtein research paid little attentin t this prblem. Only the bservatin might perhaps be m.entined that the"rhelgical prperties f dughs are changed already by small quantities f certain aliphatic hydrcarbns (MUELLER et al. [22], PONTE et al. [24-26]). n the present wrk indirect methds were used. We studied the effect f cmpunds able t react with the hydrphbic grups f the gluten cmplex, and can, thrugh these grups, interfere with the hydrphbic bnds e::;,:j"+;no' earlier. Effect f hydrcarbns n the rhelgical prperties f glutei These tests were carried ut as fllws. Dehydrated gluten was cntacted with water cntaining a knwn amunt f hydrcarbns. After hydratin and s'... elling, the hydrated mass was subjected t mechanical wrking until a cherent, hmgeneus material was btained. The excess slutin was remved, and the relaxatin test was perfrmed as described earlier. The experimental results are presented in Table 1. These data shw that the rhelgical prperties f gluten are affected unfavurably by the presence f higher aliphatic hydrcarbns. When tested rganleptically, gluten becmes less stretchable and crumbly. n the case f pentane and hexane, an increase in the relaxatin time and in the frce necessary t cause a defrm.atin f identical degree can be bserved, particularly with glutens f prer quality. The prnunced changes which can be detected even at the relatively lw cncentratins used, definitely indicate an interactin between the hydrcarbns and the prteins f gluten. As cncerns the character f this interactin, n the basis f thermdynamical cnsideratins ne can assume that
CHEMCAL STRUCTURE AND PROPERTES OF GLUTEN 21 Table 1 Effect f hydrcarbns n the rhelgical prperties f gluten Relaxatin time (sec) N. Cntrl Pentane Hexane Heptane! Octane i Undecane.3 ml/loo g gluten 1 12 16 11 94 82 65 2 95 95 96 82 76 72 3 88 91 82 71 66 5 4-73 72 7 72 68 53 5 69 65 63 58 55 48 6 62 65 6 55 54 45 7 58 6 62 59 51 4-5 8 53 48 49 44 4 37 9 43 44 4 38 37 35 1 38 45 4 38 4 41 a linkage is frmed between the hydrcarbns and the hydrphbic side chains f prteins. n the case f pentane and hexane, a weaker bnd is frmed, extending r rather prtecting thse hydrphbic nuclei, which - in the curse f hydratin, smtic uptake and swelling, and peptizatin - prevent the aggregates frm unlimited swelling and disintegratin. When higher hydrcarbns are added, the interactin may becme strnger due t the higher affinity, s that existing interactins between side chains may cease, i.e. existing hydrphbic bnds may be ruptured, and replaced by bnds between the added hydrcarbn and the side chains. This situatin is analgus t that assumed fr the rupture f the hydrgen bnds by urea. The tw analgus prcesses are illustrated by the fllwing scheme. '1 "./'~"- "./' N... c "- ~ CHz H CH3 CH \./" CH-CH3 CH3 / CH 3 CH3 H CH3 1 1 H N C./ \ 1\ C 11 :1 C.-/' '-...,.../ N... l.... ~ CH2 H 1 CH./' "- CH3 CH3 CH3 CH3 " CH./ 1 CH3 H CH 3 i, d./ H./' "c./ " 1
22 LAsZTrry, 11. Effect f fatty acids n the rhelgical prperties f gluten The prcedure was the same as in the preceding series f experiments; sme f the fatty acids were disslved, thers were emulsified in the aeqeus medium. The rhelgical prperties f the glutens treated under these cnditins are summarized in Table 11. The data in the table shw an interesting pattern. The changes in rhelgical prperties differ, depeding n the acid and the gluten. n the case f frmic and acetic acid, the peptizing effect predminates, which brings abut rapid deteriratin f the rhelgical prperties. Acids with increasing numbers f carbn atm up t and including valeric acid, cause an increase in relaxatin Table 11 Effect f fatty acids n the rhelgical prperties f gluten Relaxatin time (sec) N. Acid added:.1 mlfdm 3 slutin (emulsin) Cntrl : Frmic Acetic Prpinic Butyric Valeric Palmitic! Oleic Stearic 1 12 39 4 56 72 85 8 b 75 2 r 95 47 3 5 68 8 82 b 71 3 88 39 28 52 59 69 79 b 8 4 73 48 a 41 48 53 63 b 65 5 69 41 a 44 5 6 62 b 58 6 62 42 a 48 47 i 5 52 b 47 7 58 29 a 32 39 4 52 b 49 8 52 a a 3 3 42 48 b 41 9 43 a a 27 3 4 42 46 3 1 38 a a 25 28 32 32 57 28 r a = nt measurable (sticky, spreading mass) b = nt measurable (crnmbing, disintegrating mass) time, while palmitic and stearic acid lead t a slight deteriratin fthe rhelgical prperties. T explain the bserved changes it may be assumed that, similarly t pentane and hexande, fatty acids "with 3-5 carbn atms bring abut hydrphbizatin. On the ther hand, the decreasing relaxatin time bserved with higher fatty acids indicates that the interactin f mre strngly hydrphbic cmpunds with prteins may result in the rupture f existing hydrphbic bnds. Unsaturated leic acid des nt fit at all int the series. Oleic acid causes a change similar in character t a very high degree f thermal denaturatin.
CHEMCAL STRUCTURE AND PROPERTES OF GLUTEN 23 Presumably this is caused by the interactin f leic acid with a preferred side chain. Effect f hydrcarbns n the frmatin f gluten in the presence f urea n earlier wrks cncerned with the rle f hydrgen bnds, the effect f the additin f increasing amunts f urea n the rehydratin f dry gluten and n gluten frmatin has been studied. These experiments were nw repeated with the difference that varius hydrcarbns were added t the urea slutin (.3 ml per 1 g f gluten). The experimental results btained under these cnditins indicate the absence f gluten frmatin r a cnsiderable decrease in the amunt f gluten frmed already at a lwer urea cncentratin. The results are pltted in Figs 9 and 1. The bserved behaviur can be explained by assuming that a cmbined additin f urea and hydrcarbn hinders nt nly the frmatin f intermlecular hydrgen bnds but als the frmatin f the crrespnding hydrphbic bnds, as a l'esult f the interactin between hydrcarbn and nnplar side chains f the prtein. 1~~-----------------------------------------, ----- Urea slutin --..."..-- Urea. ctane (.3 mll Urea cntent (ml!t!t) Fig. 9, Effect f ctane n the frmatin f gluten in the presence f urea }JO~ i ~"~ i 't\~ \1 \ \ \ \ \ \ ~, " " "l>..... ---- Urea slutin ---6--- Ure-c.-..-heptane \.3 rr,{) O+-------.-----~._~~--._------r_----~ 1 2 }. 5 6 Urea cncentratin (ml/litl Fig. 1. Effect f heptane n the frmatin f gluten in the presence f urea
24 LASZTTY, R. Summary The general statement is that tw main grups f factrs influence the rhelgical prperties f the gluten the quality and quantity f the prtein fractins in the gluten cmplex (amin acid cmpsitin, mlecular mass and cnfrmatin etc.) - the interactins between the different prtein cmpnents f the gluten cmplex (disulphide bnds, hydrgen bnds, hydrphbic interactins etc.) n the framewrk f the research cnducted in ur labratry the influence f the fllwing factrs was investigated:. amin acid cmpsitin - rati f lw and high mlecular mass fractins - interactins betwee~ prtein cmpnents. We fund that the cystine cntent, the degree f amidatin and the cntent f amin acids,~ith hydrphbic chains have a significant effect n the rhelgical prperties. The lwer mlecular mass fractins determined by gel chrmatgraphy, gel electrphresis r peptizatin were fund t impair the rhelgical quality f the gluten cmplex. The quantity and distributin f disulphide bnds play a very imprtant rle in the determinatin f the rhelgical prperties. The distributin f the disulphide bnds can be changed by reductin and rexidatin f the gluten cmplex. The investigatin f chemically mdified glutens shwed that in frming intermlecular hydrgen bnds, ami d and primary amin grups play the main rle. References 1. L.~SZTTY, R.: Elelmiszenizsg. Kzl. 6 (196) 2. L.~SZTTY, R.: Acta Chim. Hung. 53, 169 (1967) 3a LASZTTY, R.: Acta Chim. Hung. 62 (1), (1969) 3b LASZTTY, R.: Die Nahrung 13, 131 (1969) 3e LASZTTY, R.: Die Nahrung 14, 569 (197) 4. BECKWTH, A. C.-WALL, T. S.-DlLER, R..: Arch. Bichem. Biphys. 13, 319 (1963) 5. BARNEY, T. E.-POLLOCK, H. B.-BLZE, C. C.: Cereal Chem. 42 (3), 215 (1965) 6. HOLlE, J.-BRGGs, D. R.: Cereal Chem. 36, 321 (1959) 7. BECKWTH, A. C.-WALL, J. S.-JORDA:N, R. W.: Archiv. Bichem. Biphys. 112, 16 (1965) 8. BECKWTH, A. C.-WALL, J. S.: Bichem. Biphys. Acta 13, 155 (1966) 9. WOSTMANN, B.: Cereal Chem. 27, 391 (195) _ 1. CU:Nl'HNGL.\.M, D. K.-GEDDES, W. F.-ANDERs:.'i, T. A.: Cereal Chem. 32 (2), 91 (1955) 11. KASARDA, D. D.-BERNARDN, T. E.-NllO, C. C.: Wheat Prteins. n: Advances in Cereal Sciences and Technlgy (Ed. by PO)ERA:NZ Y.) A.A.C.C. nc. St. Paul lilinn. p. 16. (1976) 12. SMMONDS, D. H.: Structure, Cmpsitin and Bichemistry f Cereal Grains. n: Cereals '18: Better Nutritin fr the Wrld's lllillins (Ed. by POMERA:NZ, Y.) Publ. A.A.C.C. nc. Winnipeg-St. Paul, 1979, p. 15-137 13. L.~SZTTY, R.: A siker kemiai szerkezete es relgiai sajatsagai kztti sszefiiggesek (Crrelatin between the Chemical Structure and Rhelgical Prperties f Gluten). n: A kemia ujabb eredmenyei (Recent Results in Chemistry: Ed. by Csakvary B.). Vl. 11. Akademiai Kiad6, Budapest, 1972. p. 81-134 14. LASZTTY, R.: Cereal Prteins. lliezgazdasagi Kiad6, Budapest 1981. 15. BER:NACKA-MELESTK, T.-KACZKOWSK, T.: Die Nahrung 19 (9/1), 769 (1975) 16. EWART, T. A. D.: J. Sci. Fd. Agric. 29, 551 (1978) 17. EWART, T. A. D.: J. Sci. Fd. A"ric. 3, 482-492 (1979) 18. BLOKSMA, A. H.: Cereal Chem. 49, 14 (1972) 19. JONES, 1. K.-PHLPS, T. W.-BRD, F. T. R.: J. Fd. Sci. Agric. 28, 1 (1974) 2. JAl'<lUEWCZ, M.-POMERANZ, Y.: J. Sci. Fd. Agric. 16, 644 (1965). Cereal Chem. 42, 37 (1965) 21. VAKAR, A. B.-PUMPYA:NSK, A. YA.-SEMENOVA, L. V.: Prikl. Bikhim. Mikrbil. 1, 25 (1965)
CHEMCAL STRUCTURE AND PROPERTES OF GLUTE 25 22. KA.uzMANN, W.: Sme Factrs in the nterpretatin f Prtein Denaturatin. n: Advances f Prtein Chemistry (Ed. by Anfinsen C. B.-Ansn, M. L.-Bailey, K.-Edsall, 1. T.). Vl. 14. 1959, p. 1-64 23. MUELLER, H. G.-HLYNKA, 1.-KUZNA, F. D.: Cereal Chem. 42, 33 (1965) 24. PONTE, 1. G.-TTCOMB, S. T.-COTTON, R. H.: Cereal. Chem. 41 (4), 23 (1964) 25. PONTE, T. G.-TTCOMB, S. T.-DESTEFANS, V. A.-CTTN, R. H.: Cereal Chem. 43, 475 (1966) 26. PONTE, T. G.-DESTEFA..'\l"S, V. A.-TTCOMB, S. T.-COT:rON, R. H.: Cereal Chem. 44, 211 (1967) 27. POMERA.l,"Z, Y.: Relatin between Chemical Cmpsitin and Bread-Making Ptentialities f Wheat Flur. n: Advances f Fd Research, V. 16. 1968, p. 335-455 (Ed. by C. O. Chichester, E.1\1. 1\rak, G. F. Stewart) Prf. Dr. Radmir L(SZTTY H-1521 Budapest