Effect of Microwave Radiation Pretreatment of Rice Flour on Gluten-Free Breadmaking and Molecular Size of β-Glucans in the Fortified Breads

E ec o mic owa e adia ion p e- ea men o ice lou on glu en- ee b eadmaking and molecula size o β-glucans in he o i ied b eads * Sand a Pé ez-Qui cea, Felicidad Rondaa*, A hina Laza idoub, Cos as G. Biliade isb. Food Biop ocess Technology. 2017, 10(8), 1412-1421 DOI: 10.1007/s11947-017-1910-7. E ec o mic owa e adia ion p e- ea men o ice lou on glu en- ee b eadmaking and molecula size o β-glucans in he o i ied b eads Sand a Pe ez-Qui ce1, Felicidad Ronda1*, A hina Laza idou2, Cos as G. Biliade is2 1Depa men o Ag icul u e and Fo es y Enginee ing, Food Technology, College o Ag icul u al and Fo es y Enginee ing, Uni e si y o Valladolid, A . Mad id, 44, 34004 Palencia, Spain. 2Depa men o Food Science and Technology, School o Ag icul u e, A is o le Uni e si y o Thessaloniki, P.O. Box 235, Thessaloniki, G eece, 541 24. * Co esponding au ho : [email p o ec ed]; Tel: +34979108339; Fax: +34979108200 Abs ac Ce eal β-glucan concen a es can be used in glu en- ee b eads o imp o e dough handling p ope ies and quali y o inal p oduc s as well as o enhance hei nu i ional alue; howe e , he p esence o endogenous β-glucanases in ice lou , in combina ion wi h p olonged mixing, e men a ion and p oo ing ime, can cause a subs an ial educ ion in β-glucan molecula weigh , a ec ing de imen ally hei e icacy o bioac i i y. In his s udy, mic owa e (MIWA) hea ing was applied o he ice lou s be o e b eadmaking a di e en lou wa e con en s (13-25%) and ea men imes (0- 4min) o educe β-glucanase ac i i y. Glu en- ee b eads made om he MIWA ea ed ice lou s we e o i ied wi h oa β-glucan concen a e o enhance hei nu i ional p o ile. The molecula weigh o added β-glucan in he inal p oduc s inc eased wi h inc easing bo h lou wa e con en and ime o MIWA ea men , e lec ing he magni ude o esidual β-glucanase ac i i y in he lou . P e ea men wi h MIWA adia ion o 4 min o he ice lou empe ed a 25% mois u e esul ed in negligible esidual β-glucanase ac i i y and p ese ed o a g ea ex en he molecula weigh o β-glucans in he en iched b eads. End-p oduc quali y was no a ec ed by lou MIWA p e ea men , and e en a sligh ly highe loa speci ic olume was no ed o b eads made om he MIWA - ea ed lou s (4min MIWA a 25% mois u e con en ) compa ed o ha o un ea ed lou . These indings can con ibu e o he imp o emen o nu i ional alue o ice-based glu en- ee b eads o celiac consume s as well as o any β-glucan con aining yeas -lea ened bake y p oduc wi hou al e ing i s senso ial a ibu es. Addi ional s udies a e s ill equi ed o u he e alua ion o he e ec o mo e in ense mic owa e ea men on ice lou and i s applica ion on b eadmaking. Keywo ds: β-glucan molecula weigh ; β-Glucanase inac i a ion; glu en- ee b ead; mic owa e ea men ; ice lou 1. In oduc ion Celiac disease (CD) is a gene ically linked au oimmune diso de a ec ing he gas oin es inal sys em and cha ac e ized by li e-long in ole ance o he inges ion o glu en. CD has a di e se clinical pic u e anging om angible symp oms such as nu ien s malabso p ion, dia hea, weigh loss, and abdominal discom o o ague symp oms such as i on and olic acid de iciency, a h algia, a igue, and os eopo osis (Sollid & Lundin, 2009). The demand o glu en- ee (GF) p oduc s is inc easing as a esul o he inc eased numbe s o CD diagnosed pa ien s. Ma ke ends ha e encou aged ex ensi e esea ch o he de elopmen o glu en- ee p oduc s, especially b eads and o he bake y i ems (Houben e al. 2012; Foschia e al. 2016). Gene ally, dough and b ead de elopmen wi hou glu en in ol es he use o di e se ing edien s and addi i es wi h he aim o imi a ing he iscoelas ic p ope ies o glu en and he eby ob aining high quali y p oduc s (Demi kesen e al. 2014, Hage and A end , 2013, Scia ini e al. 2010). Hyd ocolloids a e one g oup o polysaccha ides which can ul il his need. They a e used in glu en- ee b eads o imp o e he dough handling p ope ies and enhance he quali y a ibu es ( olume, ex u e, mois u e e en ion, e c.) and shel -li e o b ead (Ahmad e al. 2016). Mo eo e , some hyd ocolloids, belonging o he die a y ibe s g oup, a e used in dough o mula ions o enhance he nu i ional alue o glu en- ee b eads. Nowadays, a wide ange o non-s a ch polysaccha ides, including ce eal β-glucan (BG), a e hough o exe se e al nu i i e and physiological e ec s in he human diges i e ack. The US Food and D ug Adminis a ion (USFDA) (2005) and ecen ly, he Eu opean Food Sa e y Au ho i y (EFSA) ha e app o ed heal h claims acco ding o which ba ley and oa β-glucan inges ion, wi h a daily consump ion o 3g o β-glucan soluble ibe , leads o he educ ion o blood plasma choles e ol le els, which is a majo isk ac o o he de elopmen o co ona y hea disease (EFSA 2011a). O he heal h claims o oa and ba ley β-glucans we e also app o ed by EFSA conce ning he educ ion in pos -p andial glycemic esponses, a ecommended doses o abou 4g o β-glucans pe 30g o a ailable ca bohyd a es in b ead and pas a p oduc s (EFSA 2011b), and he inc ease o aecal bulk (EFSA 2011c); he la e claim can be used o oods con aining ba ley o oa g ain ibe o a leas 6g/100g p oduc o 3g/100 kcal. Ou p e ious wo ks ha e demons a ed he po en ial o baking ice-based glu en- ee (GF) b eads en iched wi h comme cial BG concen a es o ul ill he EFSA heal h claim equi emen s as well as o p o ide p oduc s wi h accep able quali y (Pe ez- Qui ce e al. 2014; Ronda e al. 2015). Howe e , a educ ion o he BG molecula weigh in ice-based GF b eads compa ed wi h ha o he ini ial concen a es used as ing edien s in he o mula ion mix u e has been no ed p obably implying he p esence o endogenous β-glucanase ac i i y in ice lou (Hage e al. 2011; Ronda e al. 2015). I seems o be necessa y o minimize BG depolyme iza ion du ing ood p ocessing in o de o e ain he ull physiological impac o β-glucans in o mula ed p oduc s, since is ela ed o he iscosi y o β-glucan aqueous dispe sions; he la e is a unc ion o molecula weigh and concen a ion o he polysaccha ide (Tosh, 2008; Wood, 2007; Wole e e al. 2010). The ac i i y o endogenous lou β-glucanases, in combina ion wi h he long con ac ime du ing mixing, e men a ion and p oo ing, can cause a subs an ial educ ion in β-glucan molecula weigh du ing p oduc ion o β-glucan con aining yeas -lea ened baked p oduc s (Aman e al. 2004; Ande sson e al. 2004; 2009; Laza idou e al. 2014; Ronda e . al. 2015; T ogh e al. 2004). The inac i a ion o lou enzymes and he use o ela i ely sho p ocessing ime ha e been p oposed as e ec i e means o minimize β-glucan deg ada ion (Ande sson e al. 2004; Laza idou e al. 2014; Mo ia ey e al. 2010; Va andous e al. 2012). Some me hods p e iously used o he β-glucanase inac i a ion we e au ocla ing, scalding, o en hea ing, mic owa e hea ing and e hanol e luxing (Laza idou e al. 2014; Riede e al. 2015; Mo ia ey e al. 2010; Pe ez-Qui ce e al. 2016). Among hese op ions, he p ocess wi h he g ea es po en ial on he inac i a ion o endogenous β-glucanase ice lou seemed o be mic owa e hea ing since i has been p e iously ound ha a ea men o jus 4 min a e empe ing o he lou o 25% mois u e con en esul ed in enzyme inac i a ion (Pe ez-Qui ce e al. 2016); mo eo e , he deg ee o c ys allini y o he s a ch was una ec ed and he side e ec s o such ea men on lou pas ing and he mal p ope ies we e a he negligible. Laza idou e al. (2014) and Pe ez-Qui ce e al. (2016) ha e ecen ly e alua ed he β-glucanase inac i a ion including he lou empe ing up o ce ain mois u e le el be o e he he mal ea men s as a c i ical pa ame e o he su icien enzyme inac i a ion. Howe e , main aining he molecula weigh o β-glucans in ice-based b eads when hey a e o i ied wi h hese polysaccha ides has no been ye e i ied. Fu he mo e, he e ec o he mic owa e hea ing on quali y a ibu es o he glu en- ee b eads made wi h he mic owa e- ea ed ice lou s is s ill unknown. The e o e, in he p esen s udy glu en- ee b eads o i ied wi h a high molecula weigh β-glucan concen a e we e made om ice lou s p e ea ed by mic owa e (MIWA) adia ion a di e en imes and mois u e le els aiming a he e en ion o β- glucan molecula size in b eads and hence a maximizing hei physiological unc ionali y. Fu he o he amoun and molecula weigh o β-glucans ound in he end-p oduc s, he e ec s o ice lou hea ea men on physical p ope ies o lou s and glu en- ee b eads we e also explo ed. 2. Ma e ials and me hods 2.1. Ma e ials Fi e samples o ice lou , a ying in wa e con en and ime o mic owa e hea ing we e examined in his wo k. Rice lou om an Indica a ie y was supplied by He ba Ricemills SLU (Ta agona, Spain), ha ing 13.12% mois u e, 79.1% s a ch, 0.46% ash, 7.5% p o ein and 0.49% a . The pa icle size dis ibu ion o he lou was 6% > 150 µm, 150 µm > 63.2% > 100 µm and 30.8% < 100 µm acco ding o da a p o ided by he manu ac u e . Di e en combina ions o lou wa e con en and ime o MIWA ea men ha could lead o i e almos equally-spaced esidual β-glucanase ac i i ies, co esponding o abou 0, 25, 50, 75 and 100% β-glucanase inac i a ion, ha e been es ed. These ea men condi ions a e shown in Table 1 and we e adop ed acco ding o he indings om a p e ious wo k (Pe ez-Qui ce e al. 2016). A high molecula weigh oa (1→3) (1→4)-β-D-glucan concen a e a ailable on he ma ke (P omoa ™) was supplied by Bio elop AB (Kims ad, Sweden) and was u he pu i ied. The p oxima e composi ion o his comme cial concen a e as p o ided by he supplie was: 6% mois u e, 54-56% ca bohyd a es (dex ins), <4.5% p o ein, 1-3 % ash and 0.5-1 % a ; β-glucan con en 33-36%. The pu i ica ion p o ocol in ol ed aqueous ex ac ion o he polysaccha ide (50oC x 1 h) using an aqueous slu y o he P omoa ™ lou (1:40 solids:liquid) ollowed by cen i uga ion (2400 g x 30 min). The supe na an was diges ed (37oC x 3 h) by po cine panc eas α- amylase (100.000U/g lou , Megazyme In e na ional L d, B ay, I eland), concen a ed (90oC x 2 h o ½ o he olume) and he β-glucan was p ecipi a ed wi h wo olumes o e hanol (4oC x 24 h). Finally, he polysaccha ide was e-suspended in 2-p opanol (4oC x 24 h), il e ed and d ied (50oC x 18 h) o ob ain a high molecula weigh β-glucan p epa a ion (HMW-BG). As a esul o his pu i ica ion scheme, he P omoa ™ was concen a ed om 33 o 72 % β -glucan con en (HMW-BG) as assessed by he mixed-linkage (1→3), (1→4) β-D-glucan assay ki pu chased om Megazyme. The ing edien s used in he b eadmaking p ocess, like sal , suga and sun lowe oil we e ob ained om he local ma ke , whe eas he hyd oxy-p opyl-me hyl-cellulose (HPMC) 4 KM p epa a ion was a gi om Dow Chemical (Midland, EEUU). 2.2 Me hods 2.2.1 Mic owa e ea men o ice lou samples Rice lou s we e hea ed in a Panasonic In e e NN-GD566M (Osaka, Japan) mic owa e o en ollowing he me hod p e iously de eloped by Pe ez-Qui ce e al. (2016), acco ding o which samples o hyd a ed lou s (50.0 g) we e in oduced and he me ically closed in o polyamide and polyp opylene bags and subsequen ly hea - ea ed wi h mic owa e powe (900 W) applied in cycles o 20 seconds in e als combined wi h down imes o 1 min. Se e al ba ches om each ea men we e p ocessed and hen mixed in o de o ob ain he equi ed amoun o lou equi ed o he b eadmaking. A pa icula a en ion was paid o achie e a uni o m empe a u e and cons an humidi y du ing he MIWA p ocess. The mois u e con en s o he lou be o e and a e he mic owa e ea men we e de e mined ollowing he AACC 44-19 me hod, and he wa e equi ed o adjus i o a ce ain alue was calcula ed as in a p e ious wo k (Pe ez-Qui ce e al. 2016); he empe ing p ocedu e o lou s be o e MIWA ea men was also desc ibed in he la e s udy. 2.2.2 β-Glucanase ac i i y de e mina ion β-Glucanase ac i i y in con ol (un ea ed) and hea ea ed ice lou s was assessed by measu ing he a e o dec ease in speci ic iscosi y o a dilu e solu ion o a pu e β-glucan p epa a ion, ollowing addi ion o lou ex ac s, acco ding o a me hod desc ibed in ou p e ious wo ks (Laza idou e al. 2014; Pe ez-Qui ce e al. 2016); lou ex ac s (1:10 w/w ice lou :wa e ) we e ob ained by aqueous ex ac ion (25oC x 25 min) and hey we e mixed wi h an aqueous solu ion (0.1% w/ ) o a high molecula weigh (2x106) β-glucan p epa a ion (pu i y 95%). The mix u e was hen ans e ed in o an Ubbelohde glass capilla y iscome e (UBBEL04NC, K 0.01, ange 2-10 cS , b and Pa agon Scien i ic L d, Wi al, UK) and he speci ic iscosi y (ηsp = (η-η0)/η0, whe e η and η0 is he iscosi y o abo e mix u e and wa e , espec i ely) was measu ed o e 1h pe iod a 20±0.1 ºC e e y 5 min in e als. The ηsp da a e sus ime we e i ed o a linea eg ession model and he β-glucanase ac i i y in ice lou s was calcula ed om he slope o he i ed line and exp essed as he dec ease in speci ic iscosi y pe hou o he pu e β-glucan solu ion upon addi ion o he lou ex ac s. Residual β-glucanase ac i i y o each ea ed lou sample as well as enzyme ac i i y o he un ea ed lou we e analyzed a leas in iplica e. 2.2.3 Pas ing p ope ies o ice lou aqueous dispe sions Pas ing p ope ies we e s udied in he mic owa e- ea ed and con ol lou s by he Rapid Visco Analyze (RVA-4, Newpo Scien i ic P . L d., Aus alia) using he ICC S anda d me hod 162. The pas ing empe a u e, peak iscosi y, holding s eng h o ough iscosi y, as well as b eakdown, inal and se back iscosi y we e calcula ed om he pas ing cu e using he The mocline .2.2 so wa e. Viscoamylog aphy o aqueous lou dispe sions (3 g o 14% mois u e basis lou in 28 g o o al weigh ) was ca ied ou in iplica e. 2.2.4 B eadmaking p ocess A s aigh dough p ocess was pe o med using he ollowing o mula on a 100 g ice lou basis: 92% wa e , 6% oil, 5% suc ose, 2% HPMC, 1.8% sal and 3% d ied yeas . GF dough-making was achie ed by blending he solid ing edien s i s in a ki chen-aid p o essional mixe (Model 5KPM50, Ki chen Aid, S . Joseph, MI, USA) o 2 min a speed 2. Then, he liquid ing edien s (oil and wa e a 20 ± 2 °C) we e added and mixed o 8 min a speed 4. The dough (200 g) was placed in o an aluminum pan and p oo ed a 30 °C and 90% ela i e humidi y o 50 min. Subsequen ly, baking was ca ied ou in a S eba Dahlen o en (F is ad, Sweden) a 170°C o 20 min wi h s eam injec ion o 7s a he beginning o he p ocess. A e baking, b eads we e emo ed om he pan and s o ed o one hou a oom empe a u e be o e u he analysis. B eads om MIWA ea ed ice lou s en iched wi h he HMW-BG p epa a ion we e also made a 3.9 % o pu e β-glucan o i ica ion le el ollowing he same b eadmaking p ocess. 2.2.5 Con en and molecula weigh de e mina ion o β-glucan in b eads The con en and molecula weigh o he added BG in en iched b eads we e de e mined o e alua e any change du ing he b eadmaking p ocess due o endogenous β-glucanase ac i i y o ice lou . The concen a ion o BG in b ead was de e mined using he mixed-linkage (1→3)(1→4)β-D-glucan assay ki o Megazyme. Fo molecula weigh e alua ion, he β-glucans we e i s ly ex ac ed om he o i ied b eads acco ding o an isola ion p o ocol desc ibed in de ails elsewhe e (Laza idou e al. 2014); his includes an aqueous diges ion wi h Te mamyl 120 L (1% / , No ozymes), ollowed by a suspension wi h Fulle ’s ea h o p o ein emo al, hyd olysis wi h xylanase (100U/100g b ead, Megazyme), exhaus i e dialysis, concen a ion and epea ed p ecipi a ions wi h e hanol. The con en o β- glucan concen a es de i ed om he b ead c umbs we e 55-80% as assessed by he espec i e assay ki o Megazyme. The appa en peak molecula weigh (Mp) o he ex ac ed-BG om b eads was es ima ed ollowing he me hod desc ibed in de ail by Laza idou e al. (2004; 2014) using a high pe o mance size exclusion ch oma og aphy sys em (HPSEC) sys em, which consis ed o a single pump (Ma a hon IV, Rigas Labs, Thessaloniki, G eece), a gua d TSKPWH column and wo SEC columns in se ies, 7.5x300 mm TSK G6000 PW and 7.5x600 mm TSK G5000 PW (Tosoh Bioscience GmbH, S u ga , Ge many), and a e ac i e index (RI) de ec o (ERC-7515A, ERC- Inc. Nishiaoki, Kawaguchi-Ci y, Japan). 2.2.6 E alua ion o b ead quali y Fo loa speci ic olume de e mina ion, b eads we e weighed a e emo al om he pan and cooling down and he loa olume was measu ed in ou eplica es using a Volscan p o ile 300 analyse (S able Mic osys ems, Su ey, UK). Tex u e pa ame e s o he b ead c umb we e e alua ed in quad uplica e samples wi h a TA- XT2 ex u e analyse (S able Mic osys ems, Su ey, UK) using he so wa e “Tex u e Expe ”; o his analysis an aluminum 20 mm diame e cylind ical p obe was employed o submi c umb specimens o a wo-cycle comp ession es (Tex u e P o ile Analysis, TPA) a 1 mm/s speed es , wi h a 30 s delay be ween i s and second comp ession, and a a de o ma ion le el up o 50%. This es was ca ied ou a 20 ± 3 ºC on b ead slices, wi h 20 mm hickness, aken om he cen e o each loa . Ha dness (N), chewiness (N), cohesi eness, sp inginess and esilience o he b ead c umb we e calcula ed om he TPA cu es. B ead c us colou was measu ed wi h a Minol a spec opho ome e CN-508i (Minol a, Co.LTD, Japan); esul s we e ob ained in he CIE L*a*b* and CIE L*C*h coo dina es using he D65 s anda d illuminan , and he 2º s anda d obse e (In e na ional Commission on Illumina ion, CIE). Colou de e mina ions we e made 4 x 4 imes; i.e. colou pa ame e s we e measu ed in ou di e en b ead loa es a ou di e en poin s o hei c us . 2.2.7 S a is ical analysis The S a g aphics Cen u ion .6 (Bi s eam, Camb idge, MN, USA) was used o ANOVA analysis, and signi ican di e ences (p<0.05) be ween samples we e iden i ied by he LSD (Leas Signi ican Di e ence) es . Figu e 1. E ec o lou mic owa e ea men on he ex e nal appea ance and in e nal c umb s uc u es o glu en- ee ice-based b eads. Mic owa e (MIWA) ea men Ex e nal appea ance C umb s uc u e 1 2 3 4 5 3.4 E ec o β-glucanase inac i a ion o lou on molecula weigh o β-glucan isola ed om in o i ied glu en- ee b eads The β-glucan con en in he inal p oduc s o i ied wi h he β-glucan isola e was simila o ha expec ed om he amoun o polysaccha ide added o he dough o mula ion (Table 4); a sligh decline in he BG con en in b eads was no ed only in p oduc s made by lou s wi h high β-glucanase ac i i y (MIWA ea men 1 and 2) (Tables 1 and 4). The le el (3.9% o pu e β-glucan on ice lou basis) o HMW-BG added o he glu en- ee o mula ions can mee he heal h claim equi emen s o US Food and D ug Adminis a ion (FDA, 2005) and EFSA (EFSA, 2011) o he educ ion o se um choles e ol and can be accomplished by a daily in ake o ~ 170 – 214 g p oduc which on a e age, co esponds o ou se ings o 50 g o he GF o i ied wi h β-glucan b eads. Table 4. β-Glucan (BG) con en added o dough and measu ed in b ead and b ead in ake o ul il he EFSA claim (choles e ol educ ion). Mic owa e (MIWA) ea men BG added o he dough (% d y ma e ) BG con en measu ed in b ead (% d y ma e ) B ead mois u e con en (%) BG con en (% in b ead) B ead in ake o ul il he EFSA claim (g) 1 3.2 2.8 (± 0.12)a 49.3 (± 1,08)a 1.4 214 2 3.2 3.0 (± 0.16)ab 49.2 (± 0,92)a 1.5 200 3 3.2 3.2 (± 0.16)ab 48.7 (± 1,07)a 1.7 181 4 3.2 3.4 (± 0.22)b 48.0 (± 0,90)a 1.8 169 5 3.2 3.1 (± 0.09)ab 50.4 (± 1,10)a 1.5 197 Mean alues wi h di e en le e s o he same pa ame e imply signi ican di e ences be ween means a p<0.05. The molecula weigh dis ibu ions o he HMW-BG p epa a ion added o he glu en ee o mula ions as well as he β-glucan concen a es de i ed om b eads made om ice lou s submi ed o di e en mic owa e p e- ea men s we e analysed by a HPLC- SEC-RI sys em o e alua e any changes in molecula weigh o he polysaccha ide du ing b eadmaking (Figu e 2); he Mp alues o hese p epa a ions we e es ima ed om he peak ac ion o he elu ing peaks o he polysaccha ides using a calib a ion cu e made wi h β-glucan s anda ds. A la ge po ion o he elu ing peak and he peak ac ion o he HMW-BG sample we e elu ed in he oid olume o he size exclusion columns, i.e. he β-glucan added o he glu en- ee doughs had a Mp > 9 x 105 Da. Howe e , o he β-glucans isola ed om he con ol b ead made om he un ea ed ice lou he e was a la ge educ ion in Mp, as he main elu ing peak was ~ 0.23 x 105 Da, p esumably due o polysaccha ide deg ada ion by endogenous β-glucanases o he ice lou du ing he b eadmaking p ocess (Table 1). Simila ly, se e al esea che s ha e p e iously no ed a conside able educ ion o β-glucan molecula weigh du ing p oduc ion o yeas -lea ened bake y p oduc s om oa b an as well as ye and ba ley lou which was a ibu ed o endogenous β-glucanase ac i i y (Aman e al. 2004; Ande sson e al. 2004; 2008; 2009; Laza idou e al. 2014; T ogh e al. 2004); appa en ly, he molecula weigh o β-glucans in he inal p oduc s dec eases wi h inc easing mixing and dough e men a ion ime. Recen ly, an endogenous β-glucanase ac i i y was also ound in ice lou (Pe ez-Qui ce e al. 2016) which can cause se e e educ ion o oa and ba ley β-glucans du ing b eadmaking when hese polysaccha ides a e added as concen a es o ice-based glu en- ee b ead o mula ions (Ronda e al. 2015). Tempe ing o ice lou up o 16% mois u e con en ollowed by 1 min mic owa e hea ing esul ed in b ead wi h highe Mp (0.61 x 105 Da) compa ed o ha made om he un ea ed lou (Figu e 2), p esumably due o a pa ial dec ease in β-glucanase ac i i y as e idenced om he da a in Table 1. A mino ac ion o β-glucans in bo h b eads om he con ol and his ea ed (MIWA ea men 1) lou p ese ed hei ini ial molecula size, since small peaks ep esen ing 3% and 6% o he ch oma og aph a ea, espec i ely, had Mp>9 x 105 Da (Figu e 2, slan ed do ed a ow). Wi h inc easing ime o MIWA ea men up o 4 min o he lou empe ed a 16% mois u e he e was a g adual inc ease o β-glucan molecula weigh as shown by he shi ing o polysaccha ide elu ing peaks o lowe e en ion imes and inc ease o Mp up o 4.37 x 105 Da (Figu e 2); his obse a ion is consis en wi h he decline in β-glucanase ac i i y o he espec i e ea ed lou s (Table 1). The elu ing p o ile o β-glucans in b ead p epa ed om he lou hea ed wi h MIWA o 2 min showed wo main peaks wi h Mp alues o 1.41 and 0.23 x 105 Da which ep esen 61 and 39% o he o al ch oma og aph a ea, espec i ely (Figu e 2). Simila bimodal molecula weigh dis ibu ions o β-glucans isola ed om oa , ye and ba ley b eads ha e been p e iously epo ed and a ibu ed also o he endogenous β-glucanase ac ion in he lou s (Aman e al. 2004; Ande sson e al. 2004; 2009). Figu e 2. HPSEC elu ion p o iles de ec ed by RI and appa en peak molecula weigh , Mp, (slan ed solid a ows) o he elu ing peaks o he en iched in high molecula weigh oa β-glucan concen a e (HMW- BG) added o he glu en- ee o mula ions and o he β-glucan ex ac ed om he c umbs o he o i ied b eads made by he mic owa e (MIWA) ea ed lou s; MIWA ea men condi ions (mois u e con en and hea ing ime) o he ice lou s a e also gi en on he espec i e elu ion cu es. The slan ed do ed a ows show he Mp o mino elu ing peaks o he molecula weigh o he ac ion o shoulde s. The e ical a ows indica e he elu ion ime o he peak ac ion o six (1→3) (1→4) β-D-glucan s anda ds (Mp: 15, 33, 83, 186, 340 and 466 x 103 Da) used o plo ing o he s anda d cu e (inse ); (1→3) (1→4) β-D-glucan s anda d wi h Mp 941 x 103 Da is elu ed a he oid olume o he columns. All Mp alues showed on igu e is exp essed in Dal ons (Da). Wi h inc ease o lou mois u e le el o 25% be o e MIWA ea men he e was a u he inc ease o he molecula weigh o β-glucan in b ead, as indica ed by he highe Mp alue, 5.01 x 105 Da, compa ed o ha om lou wi h 16% mois u e le el ea ed wi h MIWA o he same ime, 4 min (Figu e 2). In con as , o he MIWA ea men 5, i seemed ha he β-glucans main ained o a conside able ex en hei ini ial molecula size, since a la ge po ion o he elu ed polysaccha ides we e o high molecula weigh (>9 x 105 Da) and appea ed as a shoulde o he main peak elu ed in he oid olume o he ch oma og aph (slan ed do ed a ow). This ac is in acco dance wi h he β- glucanase ac i i y o he espec i e ea ed lou used o b eadmaking which appea ed o be negligible, i.e. appa en ly, non-de ec able ac i i y by he employed iscome ic me hod (Table 1). As ound p e iously, inc eased le els o lou mois u e a e c ucial o adequa e β-glucanase inac i a ion, mos likely due o he d op o dena u a ion empe a u e o he enzyme wi h inc easing wa e con en (Laza idou e al. 2014; Pe ez- Qui ce e al. 2016). Pe ez-Qui ce e al. (2016) ha e ecen ly epo ed ha esidual ac i i y o endogenous β-glucanase in ice lou dec eased wi h inc easing ime o mic owa e hea ing and mois u e le el o he lou ; o ins ance, an inc ease o ini ial wa e con en up o 19 and 25% in ice lou ollowing by mic owa e ea men o 8 and 4 min, espec i ely, esul ed in non-measu able β-glucanase ac i i y using he same iscome ic me hod. In he p esen s udy, he sligh d op o β-glucan molecula weigh du ing p oduc ion o b eads made by he MIWA ea men 5 lou (wi h no appa en β- glucanase ac i i y) could be a ibu ed ei he o mino esidual enzyme ac i i y, non- de ec able by he iscome ic me hod o by oxida i e deg ada ion eac ions in ol ing o β-glucans; appa en ly, he e is e idence o hyd oxyl adical media ed depolyme isa ion o β-glucans ha could occu in ce eal baked p oduc s (Ki ela e al., 2011). 4. Conclusions Mic owa e p e ea men o hyd a ed ice lou s used as a base ma e ial o p oduce glu en- ee b eads, o i ied wi h β-glucans, do no cause educ ion in he molecula weigh o he bioac i e polysaccha ides upon baking, p esumably due o inac i a ion o he endogeneous ice lou β-glucanases. The β-glucan molecula weigh in he inal p oduc inc eased wi h ime o mic owa e hea ing and ini ial mois u e con en o he lou in acco dance wi h he magni ude o he esidual endogenous β-glucanase ac i i y ound in he ea ed lou s. A sligh inc ease in loa olume was also obse ed o he b eads made om he ice lou ea ed o he longes ime, while no p ac ically impo an changes in he pas ing p ope ies o he lou as well as in ex u e and colo o he inal p oduc s we e no ed as a esul o lou mic owa e ea men . The indings o he p esen s udy could con ibu e o imp o ing he quali y and bioac i i y o ice-based glu en- ee baked p oduc s, con aining ce eal β-glucan concen a es, o b oaden he ood i em choices o celiac consume s. 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