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1 Molecules 2009, 14, ; doi: /molecules OPEN ACCESS molecules ISSN Article Structurl Chnges of Mlt Proteins During Boiling Bei Jin 1, Lin Li 1, *, Guo-Qin Liu 1,2, Bing Li 1, Yu-Kui Zhu 1 nd Lio-Ning Lio Deprtment of Food Science nd Technology, South Chin University of Technology, Gungzhou , P.R. Chin Gungzhou Zhujing Brewery Group Compny, Gungzhou , P.R. Chin * Author to whom correspondence should be ddressed; E-mil: felinli@scut.edu.cn; Tel.: ; Fx: Received: 23 December 2009; in revised form: 3 Mrch 2009 / Accepted: 4 Mrch 2009 / Published: 9 Mrch 2009 Abstrct: Chnges in the physicochemicl properties nd structure of proteins derived from two mlt vrieties (Budin nd Gungmi) during wort boiling were investigted by differentil scnning clorimetry, SDS-PAGE, two-dimensionl electrophoresis, gel filtrtion chromtogrphy nd circulr dichroism spectroscopy. The results showed tht both protein content nd mino cid composition chnged only slightly during boiling, nd tht boiling might cuse grdul unfolding of protein structures, s indicted by the decrese in surfce hydrophobicity nd free sulfhydryl content nd enthlpy vlue, s well s reduced α-helix contents nd mrkedly incresed rndom coil contents. It ws lso found tht mjor component of both worts ws boiling-resistnt protein with moleculr mss of 40 kd, nd tht ccording to the two-dimensionl electrophoresis nd SE-HPLC nlyses, smll mount of soluble ggregtes might be formed vi hydrophobic interctions. It ws thus concluded tht chnges of protein structure cused by boiling tht might influence beer qulity re lrgely independent of mlt vriety. Keywords: Mlt vriety; Structure; Wort boiling; Protein unfolding; Two-dimensionl electrophoresis.

2 Molecules 2009, Introduction Mlted brley is the mjor source of beer components. The mlt qulity of given brley vriety is determined by its genetic bckground nd the physicl conditions during growth, hrvest nd storge [1]. A comprtive study of mlt vrieties indicted tht in poor ones there were lrger mounts of ggregte forms [2], nd this ggregted frction hs been found to hve negtive effect on beer qulity [3]. It ws lso reported tht the rte of decrese of totl hordein during mlting differed cross vrieties [4]. Quntittive differences were observed between the protein frctions of Scrlett nd Prestige mlt vrieties, resulting in quntittive differences in the two worts protein profiles [5]. All these results seem to confirm tht both the content nd the distribution of proteins in mlt depend on the mlt vriety nd determine beer qulity. Proteins re essentil to the qulity of mlt nd beer. During the mlting nd brewing processes, series of chnges to the brley proteins occur, including glyction by Millrd rections during the mlting, cyltion during the mshing nd structurl unfolding during the brewing [6]. Moreover, two-dimensionl electrophoresis nd mss spectrometry hve been combined to highlight some brley proteins tht could resist the het tretments during the mlting nd brewing processes. Most beer proteins in the kd size rnge [7], which minly originte from brley proteins, re products of the proteolytic nd chemicl modifictions occurring during the brewing [8]. As expected, from brley to mlt nd further to beer, most of the het-stble proteins re disulfide-rich ones [9]. In prticulr, three mjor components hve been identified in beer, nmely polypeptide with moleculr mss of 40 kd, known s Protein Z [8,10,11], 9.7 kd polypeptide known s LTP1, which is responsible for fom stbility [12], nd group of hordein-derived polypeptides (with sizes rnging from 10 kd to 30 kd) tht re involved in hze formtion [13]. Both LTP1 nd Z4 re tolernt of high tempertures nd resistnt to proteolysis, which contribute to their resilience nd survivl through the brewing process [10,14]. As reported by Levis nd Young, wort ws boiled in wort boiling kettle to inctivte enzymes, remove undesirble flvor components, sterilize the wort, isomerize hop α-cids, nd to precipitte hze-forming proteins nd polyphenols [15]. It ws observed tht n increse of the bck-pressure on the wort in n externl boiler system, might increse the men boiling tempertures up to 103~110 C, which ccelerted protein cogultion, wort dimethyl sulfide stripping, nd the isomeriztion of hop α-cids. Consequently, the boiling time reduced by 30%-40% [16]. The wort boiling temperture during the brewing process is importnt for the LTP1 content nd formtion of the finl product (beer); higher wort boiling temperture (bout 102 C), resulting from the low ltitude t se level, reduces the LTP1 level of beer to 2~3 μg/ml, wheres lower wort boiling tempertures (bout 96 C), resulting from higher ltitudes, leds to LTP1 level of 17~35 μg/ml. In ntive brley seed, LTP1 gives poor foming properties. However, it exhibits fom-promoting form fter unfolding during wort boiling. It ws found tht unfolding would occur in the wort boiling process [6] nd glyction might prevent from precipittion due to unfolding during the boiling process. Both glyction nd denturtion increse the mphiphilicity of LTP1 polypeptides nd contribute to better dsorption t ir-wter interfces of beer fom. [12,14]. Bech et l. [17] found tht brley LTP1 with moleculr mss of kd ws converted to fom-ctive form with moleculr mss of 9.6 to 9.99 kd during wort boiling. Vg et l. [18] lso reported polypeptide of 17 kd which ws rendered fom-ctive during the mshing nd

3 Molecules 2009, the boiling in similr mnner s reported with LTP1 [12]. All these results confirmed tht the boiling process influence beer finl composition. Gungmi, stndrd mlt vriety, is widely used in Chin for beer production. Budin, nother vriety of mlt originlly from Austrli, is now being incresingly employed in Chin for its good brewing qulity. The physico-chemicl chrcteriztion of finl mlts revel tht Budin smples presented lower quntities of totl protein nd free α-mino nitrogen thn Gungmi smples (11.84% nd 185 for Gungmi nd 11.08% nd 132 for Budin, respectively). It should be pointed out tht the totl protein quntity of mlt is criticl for the brewing process [36]. Budin nd Gungmi re new vrieties of gret commercil interest in the beer production mrket. Although these two kinds of mlts re both used to produce beer in south Chin, gret differences in beer qulity exist. In order to seek the resons for these differences in beer qulity, the whole brewing process is being investigted. As the only difference mong beers is the difference in the composition of the wort during the boiling, this boiling must be importnt [7]. Furthermore, informtion bout the chnges in physicochemicl properties nd structure of wort proteins during the boiling process is still scrce. Therefore, this study sought to investigte the effects of boiling process on some physicochemicl properties nd structure of wort proteins, nd revel the differences in composition nd structure of Budin nd Gungmi worts during the boiling process. Results nd Discussion Protein nd mino cid nlysis As the chnges of the quntity of protein nd certin mino cids during the wort boiling process were relted to the qulity nd stbility of the finl product, this pper sought to exmine the chnges in the content nd composition of proteins nd mino cids during the wort boiling process tht hve not been previously investigted in depth. According to the protein contents of the wort before nd fter boiling (Tble 1), stedy decline in the protein content during the wort boiling process ws observed, which mtches the results reported by Gorinstein et l. [19], but differs from those of Osmn et l. [20] who reported similr protein contents before nd fter boiling. Tble 1. Chnges of protein contents of the worts during the boiling step. Protein content (g/l) Budin wort Gungmi wort before boiling fter boiling before boiling fter boiling Ech vlue is the men of duplicte mesurements. Moreover, the decline in protein content during the wort boiling ws more pronounced for Budin thn for Gungmi wort, reveling greter effect of boiling on the proteins of the former. As shown in Tble 2, fter boiling the mino cids content of Budin wort decresed, while tht of Gungmi wort chnged only slightly, nd tht glutmic cid nd proline were the two mjor components of both Budin nd Gungmi, suggesting tht hordein is possibly the mjor component in the wort proteins with the two vrieties. Moreover, the two vrieties hd similr levels of ll essentil mino cids

4 Molecules 2009, except leucine nd histmine. Furthermore, little difference in wort protein nd mino cid contents between the two vrieties ws observed. Tble 2. Chnges in mino cid contents of the two wort vrieties during boiling. Amino Acids Budin Gungmi contents (mg/100g) before boiling fter boiling before boiling fter boiling Asp 2, , , , Glu 11, , , , Ser 2, , , , Gly 2, , , , His 1, , , , Arg 2, , , , Thr 1, , , , Al 1, , , , Pro 4, , , , Tyr 1, , , , Vl 2, , , , Met Cys Ile 1, , , , Leu 2, , , , Phe 1, , , , Lys 1, , Totl content 40, , , , Ech vlue ws the men of duplicte mesurements. Emission fluorescence spectroscopy nlysis nd surfce hydrophobicity (H 0 ) nd SH contents Protein fluorescence origintes from the tryptophn/tyrosine residues present in the beer protein [23]. The boiling process cused n obvious decrese in fluorescence intensity (Figure 1), indicting therml-induced unfolding nd ssocition/ggregtion of exposed hydrophobic groups. As shown in Figure 2, there were lso significnt decreses in surfce hydrophobicity (H o ) vlues during the wort boiling. These results re consistent with the dt of fluorescence intensity of the two worts (Figure 1). Surfce hydrophobicity (ANS binding) of proteins my decrese when the proteins unfold s result of the heting nd re then ggregted through hydrophobic interctions, thus reducing the number of ANS binding sites. The surfce hydrophobicity ws expected to decrese becuse it is enthlpiclly fvourble [24]. On the other hnd, The ANS-binding sites of the wort proteins were possibly rerrnged through unfolding during the boiling nd ANS did not bind there nd thereby led to decrese in surfce hydrophobicity. Moreover, the boiling-induced chnges in surfce hydrophobicity vlues (H o ) were slightly pronounced in the Budin vriety. As result, we conclude tht boiling led to prtil unfolding of wort protein nd the formtion of smll mounts of soluble ggregtes vi hydrophobic interctions.

5 Molecules 2009, Figure 1. Emission fluorescence intensity spectr of wort proteins before nd fter boiling with the two vrieties (using ANS s fluorescence probe). Emission fluorescence intensity (A.U.) Budin wort before boiling Budin wort fter boiling Gungmi wort before boiling Gungmi wort fter boiling Wvelenth (nm) Figure 2. Surfce hydrophobicity (H 0 ) of wort proteins (before nd fter boiling) with the two vrieties in 10 mmol/l phosphte buffer (ph 7.0). Different chrcters (-d) on the top of columns represent significnt difference t p<0.05 level during the boiling Gungmi Budin b Surfce hydrophobicity (H o ) c d 0 wort before boiling wort fter boiling Figure 3 shows tht the totl SH contents of wort proteins of the two vrieties decresed fter boiling. The results further confirmed the occurrence of boiling-induced protein unfolding [6] nd subsequent ggregtion/re-ssocition of unfolded proteins. The boiling-induced chnges in totl SH contents were more pronounced in the Budin worts, compred with the Gungmi ones, in ccordnce with the surfce hydrophobicity results. As demonstrted by Perrocheu et l. [9], there ws no reoxidtion of cysteine in the 2D electrophoresis, becuse the protein sulfhydryls were lkylted immeditely fter boiling. It ws thus resonbly concluded tht the wort boiling process gretly ffected the SH content nd ggregtion of those unfolded wort proteins would become prominent due to hydrophobic interctions, s illustrted by SDS-PAGE pttern (Figure 4).

6 Molecules 2009, Figure 3. Totl SH contents of wort proteins (before nd fter boiling) with the two vrieties. Results re mens nd stndrd devitions of replictes. Different chrcters (-d) on the top of column indicte the significnt differences (p<0.05) between wort proteins with the two vrieties during the boiling. A 16 Gungmi Budin 14 Totl SH content (mol/g protein) b c d 0 wort before boiling wort fter boiling DSC therml chrcteristics The therml properties of the wort proteins with the two vrieties were evluted by mens of DSC, nd the relted DSC chrcteristics re summrized in Tble 3. Wort proteins in the two vrieties before boiling showed wek DSC endothermic response (dt not shown) suggesting lrge mount of components in wort proteins were unfolded nd dentured. The DSC thermogrm usully shows the protein denturtion s n endothermic pek, which is net vlue of endothermic nd exothermic processes occurring in highly coopertive mnner [25]. Tble 3. DSC chrcteristics of wort smples with the two vrieties. Budin wort boiled before Budin wort boiled fter Gungmi wort boiled before Gungmi wort boiled fter T o ( C) T d ( C) ΔH (J/g) ΔT 1/2 ( C) Ech vlue ws the men of duplicte mesurements. The scn rte ws 5 C per minute. The wort smples were dispersed t bout 20% (w/v) in 50 mmol/l phosphte buffer (ph 7.0). T o, on-set temperture of denturtion; T d, therml denturtion temperture; ΔH, enthlpy chnges of endotherm nd ΔT 1/2, width t hlf pek height of endothermic pek. Vlues were expressed s the men nd stndrd devitions of triplicte mesurements.

7 Molecules 2009, The results showed tht the two wort proteins before boiling were of similr DSC ptterns. The denturtion temperture (T d ) cn be used s mesure of therml stbility of proteins. However, there ws slight decrese in therml denturtion temperture (T d ) of the two kinds of wort proteins due to the presence of highly dentured protein in wort, implying tht the proportion of undentured protein or ordered structure found in wort between the two vrieties were decresed upon boiling. The boiling process cused slight decrese in the enthlpy chnge (ΔH) of the endothermic pek of the two wort protein components (Tble 3). The enthlpy vlue (ΔH) is correlted with the content of ordered secondry structure of protein [26], s indicted by the proportion of undentured protein. Therml tretments could disrupt the chemicl forces tht mintin the structurl integrity of protein molecules, such s hydrophobic nd ionic interctions, hydrogen bonds nd disulfide bonds, resulting in protein denturtion. Rupture of hydrogen bonds is considered n endothermic rection which could increse the net endothermic contribution wheres the brekup of hydrophobic interctions nd ggregtion re exothermic rections which could lower the net endothermic contribution resulting in decrese in ΔH [27]. Thus, the boiling-induced unfolding of undentured protein in worts nd the exothermic rections of ggregtion s well s the brekup of hydrophobic interctions were more prominent since prtilly unfolded protein would require less het energy to denture completely thn ntive protein. As listed in Tble 3, the ΔH vlue of Gungmi wort ws higher thn tht of Budin wort, which indicted tht former hd much more ordered structure. On the other hnd, s for the width t hlf-pek height of the mjor endothermic pek (ΔT 1/2 ), which is n index of the coopertivity of the trnsition from ntive to dentured stte [28], it slightly incresed fter boiling, thus reveling the loss of coopertivity in the denturtion process nd the wekening of protein-protein interctions. Furthermore, similr DSC profiles were observed in wort protein before nd fter boiling for the two vrieties, suggesting tht little effect of vriety difference on the chnges of protein during boiling ws obtined in this study. Electrophoretic seprtion To elucidte the chnge of protein composition during wort boiling, SDS-PAGE ws performed respectively under both reducing (dding mercptoethnol to smple buffer) nd non-reducing conditions (without mercptoethnol). In the presence of reducing gent β-mercptoethnol (2-ME), the ptterns were similr between wort before boiling nd fter boiling of the two vriety. An intense bnd of pproximte 40 kd ws observed in the worts before nd fter boiling between the two vrieties, correspondent to the protein Z, indicting tht protein Z ws mjor component in wort. This result confirmed tht protein Z could resist to het denturtion nd exist in beer [8,29]. Other bnds were observed, including ~66 kd, kd nd kd in the Budin wort before boiling, while bnd of 116 kd ws lso observed in Gungmi wort before boiling (Figure 4). In ccordnce with the study of Kordilik-Bogck nd Ambrozik [30], the 66 kd bnd ws detected in the two vrieties in this study. However, the bnd intensities of kd nd ~35 kd in Budin wort evidently decresed fter boiling, while not only did the bnd intensities of kd nd ~35 kd decrese in Gungmi wort, but bnd t 116 kd lso disppered fter boiling. The worts of both vrieties displyed similr protein profiles fter boiling under non-reducing conditions. The mjor protein bnd ws lso found t moleculr mss of 40 kd. Moreover, the bnds of kd nd kd were detected in the non-reducing SDS-PAGE ptterns of both worts before boiling. In

8 Molecules 2009, ddition, the Gungmi wort before boiling contined high moleculr weight bnds of kd, while the Budin wort included ~66 kd, ~60 kd nd kd ones, indicting the presence of disulphide bonds within their structures. After boiling, the bnd intensities of kd obviously decresed in the Gungmi wort under non-reducing conditions, while the bnd intensities of kd obviously decresed nd the two bnds of ~66 kd nd ~60 kd disppered in Budin wort. The bove results indicted tht prt of high moleculr weight proteins dissocited into smller ones due to reduction or dentured with the presence of hops nd led to their precipittion nd removl, nd tht some low moleculr weight protein formed high moleculr weight protein vi hydrophobic interctions or might be cylted nd glycted through Millrd rections with reducing sugr becuse the Millrd rection still hppens during the wort boiling to produce protein glyction [31] nd further form precipittes with the hops. However, there ws no evidence to support the contention tht high moleculr weight proteins were removed in noticeble quntities by het denturtion during boiling, bsed on SDS-PAGE results. In this study it ppered tht only smll mounts were removed, in greement with some recent reports [37-39], s similr protein ptterns were observed in the two wort vrieties before boiling nd fter boiling under reducing nd non-reducing conditions, therefore little effect of vriety difference on the protein chnges during boiling ws found in this study. Figure 4. SDS-PAGE of wort proteins under reducing nd non-reducing conditions: 1. Mrker (kd); 2. Budin wort before boiling (with 2-ME); 3. Budin wort fter boiling (with 2-ME); 4. Gungmi wort before boiling (with 2-ME); 5. Gungmi wort fter boiling (with 2-ME); 6. Budin wort before boiling (without 2-ME); 7. Budin wort fter boiling (without 2-ME); 8. Gungmi wort before boiling (without 2-ME); 9. Gungmi wort fter boiling (without2-me). In order to chrcterize exctly the composition of wort proteins, these were nlyzed using 2-D nlysis. However, the 2-D ptterns of Gungmi wort proteins were poorly resolved due to the high sugr content, thereby only Budin wort proteins were investigted in this study. The observed chnges in the protein ptterns displyed in the silver-stined gels could be relted to the events occurring in boiling. Figure 5 shows the 2-D gels of Budin wort protein before nd fter boiling. As expected, cler differences could not be extrcted from the 2-D mps. Approximtely 120 well-defined spots

9 Molecules 2009, could be detected on ech wort gel in the pi rnge of 3-10 using the 2-D gel nlysis softwre Melnie. It ws noteworthy tht the protein profiles of the wort proteins before nd fter boiling were quite similr. Moreover, the wort before boiling gel displyed much higher number of spots in the pi rnge of 5-8, mss of kd (Figure 5A), in ccordnce with the study of Gorinstein [19]. It ws observed tht significnt decrese in the number of protein spots in the rnge of nd kd nd in the pi rnge of 5-8 for wort protein fter boiling, in ccordnt with SDS-PAGE results. However, slight increse in the number of protein spots in the rnge of kd nd in the pi rnge of 4-6 for wort protein fter boiling ws detected in 2-D ptterns, indicting tht the formtion of smll mount of soluble ggregtes vi hydrophobic interctions. As shown in Figure 5, lrge, intense stining spot ws observed t round n isoelectric point (pi) of 4-6 nd moleculr mss of kd in ll of the 2-D gels. Presumbly this gel region included protein Z, which hs coincident chrcteristics with this region. Bsed on the study of Perrocheu [9], it ws speculted tht protein Z (40 kd), LTP1 (10 kd) nd α-mylse inhibitor (12-16 kd) could resist het denturtion during boiling nd exist in beer. However, further reserch is required to elucidte the reltionship of wort protein composition nd beer qulity using immunologicl method. The improved understnding of the impct of boiling process on ech mlt protein will potentilly provide further scope for optimizing both mlt chrcteristics nd beer qulity. Figure 5. 2-D gel protein ptterns of wort protein before boiling (A) nd fter boiling (B). Proteins were seprted in the first dimension on IPG strip pi 3-10 nd in the second dimension on 15% crylmide SDS gel. The proteins were silver stined. (Protein Mrkers: 116, 90, 75, 50, 37, 25, 20, 15 nd 10 kd) A Gel filtrtion chromtogrphy nlysis of wort proteins B The SEC elution profiles of the wort proteins with the two vrieties during the boiling were investigted, using 50 mmol/l cette buffer (ph 5.8) contining 0.02% NN 3 s the eluting solvent. During the boiling, Sephcryl S-200 High Resolution gel filtrtion chromtogrphy frctioned the wort protein into incompletely resolved peks. As shown in Figure 6, the proteins showed similr profile ptterns. For ech mlt vriety, the chromtogrms were divided into three prts nd there were three components in wort proteins during the boiling. However, the obtined SE-HPLC curves were different from those reported by Osmn et l. [20] with eight peks of vrying mgnitudes nd degree of seprtion due to different mlt vriety nd gel filtrtion chromtogrphy column used. After

10 Molecules 2009, boiling, the re of the intermedite-size pek (pek II) incresed nd the pek position moved bckwrds concomitntly with decrese in the low moleculr mss pek (pek III), while the shpe nd position of pek I remined unchnged. This ws ttributed to the fct tht the smller proteins formed medium nd high moleculr weight proteins vi hydrophobic interctions or oxidtion nd further form precipittes with hops t the sme time the components of the intermedite-size pek (pek II) dissocite into smll-size ones. The quntittive nlysis of the proteins recovered in the frctions, using the Brdford Coomssie blue dye binding ssy (Figure 7), reveled tht the protein contents in pek II nd III decresed while tht of pek I slightly incresed fter boiling, reveling the formtion of smll mount of soluble ggregtes. Figure 6. Gel-filtrtion protein profile of Budin nd Gungmi wort before nd fter boiling. 2 Budin wort before boiling Budin wort fter boiling Gungmi wort before boiling Gungmi wort fter boiling 280nm ABS Tube Number Furthermore, it is pertinent to dd tht the pek II frction ws coloured (yellow), while the other pek frctions showed limpidity or were slightly yellow, due to the combintion of phenolic nd polyphenolic groups with sugrs nd proteins through Millrd rections [20]. Figure 7. Protein contents of the peks I, II nd III from Gungmi (A) nd Budin (B) worts before nd fter boiling. A Gungmi wort before boiling Gungmi wort fter boiling protein content (mg/ml) b b c c pek1 pek2 pek3

11 Molecules 2009, Figure 7. Cont. B Budin wort before boiling Budin wort fter boiling protein content (mg/ml) b b c c pek1 pek2 pek3 Some of the mterils from the peks of SE-HPLC were collected nd electrophoresed in the SDS-PAGE system (dt not shown here), but Commssie Blue regent stining of the gels filed to detect ny permnent bnds fter the destining of pek III. No ttempts were mde to use other regents t this stge. Pek II in the two kinds of worts represented the protein with the moleculr mss of 40 kd nd the corresponding bnd intensity reduced fter boiling. A finter bnd of ~ 66 kd ws detected in pek I nd disppered fter boiling. Moreover, the sme electrophoretogrm ppered in the two kinds of worts. This evidence confirmed the bove SDS-PAGE nlysis tht 40 kd protein could resist het denturtion nd exist in the finl product (beer) nd tht prt of the high nd middle moleculr weight protein might form smll mounts of soluble ggregtes fter boiling. Futhermore, little effect of vriety difference on the chnges of protein during boiling ws lso found due to very similr gel filtrtion chromtogrm profiles. CD nlysis The influence of boiling on the secondry structure of wort protein between the two vrieties were evluted by fr-uv CD nlysis. The CD spectr for wort protein before nd fter boiling were very similr between the two vrieties (Figure 8), indicting tht the effect of vriety difference on the chnges of protein during boiling ws insignificnt. Figure 8. The CD spectr for wort protein before nd fter boiling between the two vrieties Budin wort before boiling Budin wort fter boiling Gungmi wort before boiling Gungmi wort fter boiling 0 Ellipticity [deg.cm 2 /dmol] Wvelength (nm)

12 Molecules 2009, The CD spectr for wort protein before between the two vrieties showed similr strong negtive bnd round 216 nm, which ws chrcteristic of β-sheet structure. However, boiling significntly reduced the bnd intensity t 216 nm, which ws indictive of rndom structure nd confirmed tht boiling effectively disrupted the α-helix secondry structure. The α-helix, β-sheet, nd rndom coil content of wort protein before nd fter boiling were estimted for the two vrieties (Tble 4). The results showed tht α-helix contents were reduced, β-sheet nd β-turn contents were slightly incresed, nd rndom coil contents were mrkedly incresed for both vrieties fter boiling, indicting prtil loss of the α-helix structure nd thereby prtil unfolding of the wort protein nd tht some retined their secondry structures throughout the brewing process. Moreover, the dt confirmed the results of DSC indicting tht unfolding strted on mshing nd ws chieved on boiling. Tble 4. Comprison of secondry structure contents of wort protein before nd fter boiling between the two vrieties. Budin wort Gungmi wort before boiling fter boiling before boiling fter boiling α- helix (%) β- sheet (%) β- turn (%) Rndom coil (%) Ech vlue ws the men of duplicte mesurements. Conclusions In conclusion, the study hs confirmed tht the boiling gretly influences the physicochemicl properties nd structure of wort proteins. Boiling results in decrese in protein nd mino cid contents. Boiling lso results in the decrese in surfce hydrophobicity nd free sulfhydryl content nd enthlpy vlue, indicting protein unfolding nd denturtion, s well s the formtion of smll mount of soluble ggregtes vi hydrophobic interctions during the boiling. Two-dimensionl electrophoresis nd SE-HPLC nlysis revel tht mjor protein component 40kD in wort proteins is ble to resist het denturtion, while confirming wort protein denturtion nd the formtion of smll mount of soluble ggregtes vi hydrophobic interctions. Results from CD nlysis show tht boiling disrupts the secondry structure to prtil unfolding. Additionlly, this study hs shown tht the effect of wort boiling on protein properties is independent of mlt vriety. Further studies should be crried out to revel the conformtionl chnges in protein structure during the boiling nd to correlte the chnges of protein during the wort boiling with beer qulity.

13 Molecules 2009, Experimentl Worts nd preprtion of wort protein Worts were produced from Budin nd Gungmi mlts on n industril scle. 100% of the mlt used in ech cse ws from single brley cultivr. Similr mshing nd boiling procedures were used. The wort smples of Budin nd Gungmi before nd fter boiling were provided by Zhujing Brewery Group Co., Ltd., P.R. Chin. Wort proteins before nd fter boiling were extrcted by dding solid mmonium sulfte to the wort to chieve reltive sturtion of 80%. The precipitte formed overnight t 4 C ws collected by centrifugtion (7000 g) for 10 min, then the superntnt ws discrded nd the pellet ws resuspended in wter. Afterwrds, the mixture ws homogenized to ensure the pellet to be completely broken up. The smples were dilyzed nd lyophilized before storge t 4 C until use. The protein contents of the products were determined by mens of the micro-kjeldhl method (N 6.25). Mesurements were performed in duplicte. Anlysis of mino cids The lyophilized protein ws hydrolyzed in seled tube for 24 h with 6 mol/l HCl t 110 C, then the mino cid composition ws nlyzed by high-performnce ion-exchnge chromtogrphy nd post-column derivtiztion using o-phthlldehyde nd sodium hypochlorite using Wters mino cid nlyzer equipped with PICO TAG column. The determintion ws crried out t 38 C, with detection wvelength of 254 nm, nd flow rte of 1.0 ml/min. Tryptophn ws not determined. Mesurement of surfce hydrophobicity (H o ) H o ws determined using ANS, ccording to protocol of Kto nd Nki [32]. Fluorescence intensity (FI) ws mesured t wvelengths of 390 nm (excittion) nd 470 nm (emission) using F-4500 spectrofluorometer t mbient temperture, with constnt excittion nd emission slit of 5 nm. The initil slope of the FI versus protein concentrtion plot ws clculted by mens of liner regression nlysis nd used s n index of H o. Mesurements were performed in triplicte. Mesurement of free sulfhydryl (SH) content The mount of free sulfhydryl of lyophilized wort protein ws mesured with colorimetric method, s modified by Chn nd Wssermn [33]. Smples ( 15 mg) were suspended in rection buffer A, consisting of 8 mol/l ure, 3 mmol/l EDTA, 1% SDS, nd 0.2 mol/l Tris-HCl (ph 8.0, 1.5 ml). Smples were vortexed for 30 sec nd plced on constnt gittion shker t room temperture. After 1 hr, buffer B (0.15 ml, 4 mg DTNB dissolved in 1 ml of 0.2 mol/l Tris-HCl) ws dded to ech smple, nd shking continued for nother 1 hr. Smples were then centrifuged t 13,600 g for 10 min t room temperture, nd the bsorbnce of the superntnt ws red t 412 nm ginst blnk consisting of 1.5 ml of buffer A nd buffer B. Ech smple ws determined in triplicte.

14 Molecules 2009, Differentil Scnning Clorimetry The therml denturtion of wort proteins were exmined using TA Q100-DSC therml nlyzer (TA Instruments, New Cstle, DE), ccording to the procedure of Meng nd M [34], with some modifictions. Approximtely mg protein smples were ccurtely weight into luminum liquid pns, nd 10 μl 50 mmol/l phosphte buffer (ph 7.0) ws dded. The pns were hermeticlly seled nd heted from 20 to 160 C t rte of 5 C/min. A seled empty pn ws used s reference. Pek or denturtion temperture (T d ) of different protein components nd denturtion enthlpy chnge (ΔH) nd width t hlf pek height of endothermic peks (ΔT 1/2 ) were computed from the thermogrms by the Universl Anlysis 2000, version 4.1D softwre (TA Instruments-Wters LLC, USA). All experiments were conducted in triplicte. In ll cses, the seled pns contining wort protein smples nd buffers were equilibrted t 25 C for more thn 6 h. Gel filtrtion chromtogrphy Gel filtrtion chromtogrphy ws performed by using Wters 650E Advnced Protein Purifiction System nd Sephcryl S-200 High Resolution column (GE Helthcre UK Limited, Φ 2.6 cm 60 cm). Wort protein liquots contining mg of protein were pplied to the column. Elution rte ws 0.8 ml/min using 50 mmol/l cette buffer (ph 5.8) contining 0.02% NN 3 in deionized wter. Frctions of 6 ml were collected nd the bsorbnce t 280 nm ws mesured. The column ws wshed fter ech smple with 0.1 mol/l NOH, distilled wter nd re-equilibrted before loding the next smple. Electrophoretic nlyses Sodium dodecylsulfte-polycrylmide gel electrophoresis (SDS-PAGE) ws done s described by Lemmli [35] on discontinuous buffer system with resolving gels (totl crylmide [T] 15%, cross-linking [C] 2.7%) nd stcking gel (T 4%, C 2.7%) by using Mini-Proten II electrophoresis unit (Bio-Rd). Smples were dissolved in reducing smple buffer (5 mol/l ure, 4% SDS, Tris buffer ph 8.0, contining 1% 2-mercptoethnol) nd non-reducing smple buffer (without 2-mercptoethnol). Seprtions were crried out t 15 ma in the stcking gel, nd t 25 ma in the resolving gel. The gel ws stined with Coomssie Brillint Blue R-250 in 50% trichlorocetic cid nd destined in 7% cetic cid [methnol/cetic/wter, 227:37:236 (v/v/v)]. For 2-D electrophoresis the freeze-dried protein smples were completely dissolved in 8 M ure, 2% w/v CHAPS, 2% v/v IPG buffer ph 3-10 (Amershm Biosciences, Uppsl, Sweden), 0.01% bromophenol blue, 20 mm DTT, nd were centrifuged t 12,000 rpm for 10 min. IEF ws performed using Phrmci Biotech IPG phor electrophoresis system (Amershm Biosciences). Prior to second dimension development, the IPG strips were equilibrted for 10 min in equilibrtion buffer (50 mm Tris/HCl ph 8.8, 6M ure, 30% v/v glycerol, 2% w/v SDS, 0.01% bromophenol blue) contining 10 mg/ml DTT, followed by 10 min in equilibrtion buffer contining 25 mg/ml iodocetmide. The second dimension ws run on 15% crylmide gels on verticl system. Gels were stined with silver nitrte [21, 22].

15 Molecules 2009, Circulr Dichroism Spectroscopy The secondry structure of proteins ws determined by CD t 25 C in the fr UV (from 190 to 250 nm) using CD6 Jobin-Yvon dichrogrph. Proteins were solubilized in finl concentrtion of 0.5 mg/ml. A qurtz cell of 0.2 nm pth length ws used. Dt were expressed s men-residue ellipticity. Secondry structures were determined using the CONTIN softwre. Sttisticl nlysis An nlysis of vrince ws performed on the dt, nd lest significnt difference test with confidence intervl of 95% ws used to compre the mens. Acknowledgments This study is prt of the reserch projects of NSFC (seril number: ). The uthors lso grtefully cknowledge the technicl support from Zhujing Brewery Group Compny, Gungzhou, P.R. Chin. References nd Notes 1. Ostergrd, O.; Melchior, S.; Roepstorff, P.; Svensson, B. Initil proteome nlysis of mture brley seeds nd mlt. Proteomics 2002, 2, Jnes, P.W.; Skerritt, J.H. High performnce liquid chromtogrphy of brley proteins: reltive quntities of hordein frctions correlte with mlt extrct. J. Inst. Brew. 1993, 99, Poyri, S.; Mikol, M.; Strohm-Sontg, T.; Norj-Kukovirt, A.; Home, S. The formtion nd hydrolysis of brley mlt gel-protein under different mshing conditions. J. Inst. Brew. 2006,108, 2, Mrchylo, B.A.; Krucrr, L.E.; Htcher, D. High performnce liquid chromtogrphy nd eletrophoretic nlysis of hordein during mlting for two brley vrieties of contrsting mlting qulity. J. Cerel Chem. 1986, 63, 3, Silv, F.; Nogueir, L.C.; Gonclves, C.; Ferreir, A.A.; Ferreir, I. M.P.L.V.O.;Teixeir, N. Electrophoretic nd HPLC methods for comprtive study of the protein frctions of mlts, worts, nd beers produced from Scrlett nd Prestige brley (Hordeum vulgre L.) vrieties. Food Chem. 2008, 106, Perrocheu, L.; Bkn, B.; Boivin, P.; Mrion, D. Stbility of brley nd mlt lipid trnsfer protein 1(LTP1) towrd heting nd reducing gents: Reltionships with the brewing process. J. Agric. Food Chem. 2006, 54, Leiper, K.A.; Stewrt, G.G.; Mckeown, I.P. Beer polypeptides nd silic gel Prt I. Polypeptides involved in hze formtion. J. Inst. Brew. 2003, 109, 1, Curioni, A.; Pressi, G.; Furegon, L.; Peruffo, A.D.B. Mjor proteins of beer nd their precursors in brley: Electrophoretic nd immunologicl studies. J. Agric. Food. Chem. 1995, 43,

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