Inac i a ion o endogenous ice lou β-glucanase by mic owa e
adia ion and impac on physico-chemical p ope ies o he ea ed
lou
Sand a Pe ez-Qui ce1, Felicidad Ronda1*, Ca men Melend e1, 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.
Abs ac
The appa en educ ion o β-glucan (BG) molecula weigh in ice based glu en- ee
(GF) b eads o i ied wi h ce eal BG concen a es e eals he p esence o β-
glucanase ac i i y in ice lou . Inac i a ion o endogenous β-glucanase in ice lou
hus seems o be necessa y s ep when de eloping GF b eads en iched wi h BG o
high molecula -weigh . The aim o his wo k was o s udy he he mal inac i a ion o
endogenous β-glucanase in ice lou by means o mic owa e (MW) p ocessing; ice
lou s p econdi ioned a ou di e en mois u e le els (13%, 16%, 19%, 25%) we e
ea ed by MW adia ion a 900 W and i e MW ea men imes ( anging om 40s
o 8 min, applied s epwise a 20s in e als). The e ec s o mic owa es on s a ch
c ys allini y, pas ing and he mal p ope ies o MW- ea ed ice lou s we e also
explo ed. The β-glucanase ac i i y in ice lou s was assessed by he a e o dec ease
in speci ic iscosi y o a dilu e solu ion o a pu i ied β-glucan p epa a ion, upon
addi ion o lou ex ac s. MW p o ed o be a use ul al e na i e o he mal
inac i a ion o endogenous β-glucanase in ice lou s when applied o mois ened
samples. The inac i a ion p ocess ollowed a i s o de kine ic esponse and he
appa en a e cons an o he mal inac i a ion inc eased exponen ially wi h he
mois u e con en o he lou , M, acco ding o he equa ion 0.0146·exp (0.212·M)
(R2 = 0.97). The MW ime equi ed o comple e β-glucanase inac i a ion was only 4
min when he ini ial lou mois u e inc eased o 25%. Following MW ea men , he
s a ch c ys allini y was una ec ed (p>0.05) and he side e ec s o he ea men on
lou pas ing and he mal p ope ies we e a he negligible.
Keywo ds: β-Glucanase inac i a ion; mic owa e ea men , pas ing p ope ies, ice
lou ; he mal p ope ies; X-Ray di ac ome y
1. In oduc ion
The (1-3) (1-4) β-D-glucans (BG) a e majo componen s o cell walls in he s a chy
endospe m and he aleu one laye o comme cially impo an ce eals, mos ly oa and
ba ley and o a lesse ex en ye and whea (Laza idou and Biliade is 2007). These
polysaccha ides a e classi ied as soluble die a y ib e wi h well ecognized
nu i ional implica ions as speci ied in se e al heal h claims au ho ized by many
egula o y au ho i ies a ound he globe. The US Food and D ug Adminis a ion
(USFDA) (2005) has app o ed a heal h claim o he educ ion o co ona y hea
disease isk wi h a daily consump ion o 3g o β-glucan soluble ibe om whole
g ain ba ley o oa and d y milled ba ley/oa g ain p oduc s as pa o a low sa u a ed
a and low choles e ol die . Recen ly, he Eu opean Food Sa e y Au ho i y (EFSA)
has also au ho ized a heal h claim, acco ding o which ba ley β-glucan inges ion
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); he ecommended
daily in ake is 3g o oa β-glucan as pa o a balanced die . 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 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 a leas 6g/100g p oduc o 3g/100 kcal.
On he o he hand, he demand o glu en- ee p oduc s s eadily inc eases. Al hough
se e al glu en ee (GF) p oduc s a e nowadays a ailable on he ma ke , baked
p oduc s wi h glu en- ee ing edien s a e gene ally o poo nu i ional and senso ial
quali y and exhibi undesi able physicochemical p ope ies; i.e. hey con ain low
amoun s o ib e, i amins and o he essen ial nu ien s, which exe a wo sening
e ec on he al eady nu i ionally unbalanced die o celiac disease (CD) su e e s
(Thomson 2009). Despi e he cu en end o de elopmen o ood p oduc s wi h
imp o ed nu i ional quali y, he GF p oduc s o en ecei e only ma ginal a en ion
om a o mula ion poin o iew. The en ichmen o GF b eads and o he bake y
i ems (cookies, pas a p oduc s, e c.) wi h BG, holds a special in e es among he
ulne able popula ion o celiac pa ien s which encoun e s a signi ican incidence a e
o o he associa ed ch onic diseases, such as obesi y and diabe es due o highe a
and calo ic in ensi y die s (C onin and Shanahan 1997).
Rice lou is he mos sui able ing edien o GF bake y o mula ions due o i s bland
as e, whi e colou , diges ibili y and hypoalle genic p ope ies. O he a ibu es such
as he low con en o p o ein and sodium as well as he p esence o easily diges ed
ca bohyd a es a e addi ional bene i s (Rosell e al. 2014). Fu he mo e, in ice lou ,
he a io o albumin-globulin-p olamin-glu elin is a he unique among he ce eals,
e ealing a high concen a ion in glu elins and low in p olamins. As wi h o he
ce eals, ice p o eins a e de icien in he essen ial amino acid lysine, bu as a
consequence o he espec i e a io o p o ein ac ions, ice has highe con en o
lysine han o he ce eals, and his is sha ed by oa s (Rosell e al. 2014).
P e ious wo ks ha e demons a ed he po en ial o baking ice-based 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). In hese s udies, al hough he inal con en o BG in he
b ead was no a ec ed, he molecula weigh o BG was no ably educed compa ed
wi h he ini ial concen a e used as ing edien in he o mula ion mix u e (Hage e al.
2011; Ronda e al. 2015).
The abili y o β-glucans o dec ease se um choles e ol le els and mode a e he
glycemic esponses is o en linked o he po en ial o hese polysaccha ides o
enhance he iscosi y o he in es inal con en s (Tosh e al. 2008; Wole e e al.
2010). Since he iscosi y o a β-glucan solu ion is a unc ion o i s molecula weigh
and polysaccha ide concen a ion, he in ensi y o MW ea men and he amoun o
wa e -ex ac able β-glucans in a gi en ood p oduc can in luence he ex en o hei
physiological e ec (B umme e al. 2012; Laza idou and Biliade is 2007; Tosh
2013; Tosh e al. 2008; Wole e e al. 2010). In o de o e ain he ull physiological
impac o β-glucan in o mula ed p oduc s i is he e o e c ucial o minimize i s
depolyme iza ion (hyd olysis) du ing ood p ocessing and s o age. This is a
challenging p oblem du ing p oduc ion o β-glucan en iched b ead, since he ac i i y
o endogenous lou β-glucanases, in combina ion wi h he long con ac ime du ing
mixing o ing edien s, e men a ion and p oo ing, can cause a subs an ial educ ion in
β-glucan molecula weigh (Aman e al. 2004; Ande sson e al. 2004; T ogh e al.
2004).
The ole o enzymes om whea lou in β-glucan deg ada ion du ing dough
handling has been demons a ed by Mo ia ey e al (2010), who showed ha e hanol
e luxed whea lou ( esul ing in enzyme inac i a ion) ga e lowe β-glucan
deg ada ion and highe ex ac iscosi ies om doughs p epa ed wi h added ba ley β-
glucan concen a e. The addi ion o yeas , on he o he hand, did no seem o a ec β-
glucan deg ada ion (Ande sson e al. 2004; Mo ia ey e al. 2010) no did di e ences
in e men a ion empe a u e o he wa e addi ion (Ande sson e al. 2004). Ande sson
e al. (2004) also sugges ed ha he baking p ocess i sel does no esul in u he
deg ada ion o he polysaccha ide, whe eas he endogenous β-glucanase ac i i y in
lou (whea o ba ley) and eac ion ime in he dough sys em (du ing mixing,
e men a ion and p oo ing) a e he mos impo an de e minan s o β-glucan
deg ada ion du ing p oduc ion o β-glucan en iched b eads (Ande sson e al. 2004;
Mo ia ey e al. 2010). Inac i a ion o lou enzymes and he use o ela i ely sho
p ocessing imes ha e been p oposed as e ec i e means o minimize β-glucan
deg ada ion (Ande sson e al. 2004; Mo ia ey e al. 2010; Va andous e al. 2012).
Howe e , adop ing long mixing and e men a ion-p oo ing egimes is o en c ucial
o quali y pa ame e s o he inal p oduc s, i.e. imp o ed loa olume, c umb
po osi y and ex u e. Thus he educ ion o e men a ion ime o a oid β-glucan
deg ada ion does no appea as a easible-p ac ical app oach. Consequen ly, he
des uc ion o β-glucanase ac i i y in aw lou ma e ials seems o be a necessa y and
con enien s ep o de elop b eads en iched wi h BG o high molecula weigh .
Endogenous β-glucanase ac i i y has no been demons a ed in ice lou so a . In
spi e o he low BG con en o ice lou , ha would make someone o an icipa e a
a he low β-glucanase ac i i y, an appa en educ ion o he BG molecula weigh in
ice based GF b eads, o i ied wi h ba ley and oa BG concen a es, has poin ed
owa ds he p esence o β-glucanase ac i i y in ice lou (Ronda e al. 2015; Hage
e al. 2011).
The aim o his wo k was o s udy he inac i a ion o β-glucanase enzymes in ice
lou by means o mic owa e ea men s (MW). Some o he me hods p e iously
used o inac i a e β-glucanase ac i i y a e au ocla ing, scalding, o en hea ing and
e hanol e luxing (Laza idou e al. 2014; Riede e al. 2015; Mo ia ey e al. 2010).
Howe e , he e is no li e a u e in o ma ion on he use o mic owa e hea ing o
inac i a ion o β-glucanase enzymes. The high e iciency and he ela i ely sho
ea men s adop ed in MW p ocessing, in compa ison o o he con en ional hea ing
p ocedu es, led us o explo e his al e na i e me hod o he mal inac i a ion o he
endogenous ice lou β-glucanase. Only ew wo ks, wi h p omising esul s, ha e
been epo ed ill now on lou s o whole ce eal g ain s abiliza ion by MW (Jiaxun-
Tao e al. 1993). In ice p ocessing MW has been applied o con ol he g ow h o
pes s and mildew (Zhao e al. 2007) as well as o inac i a e lipases and lipoxygenase
enzymes in o de o inc ease i s s abili y du ing s o age (Chang and El-Dash 1998;
Zhong e al. 2013).
In o he hea ea men s o lou s (e.g. au ocla ing), hyd a ion has been iden i ied as
a c i ical pa ame e o success ul β-glucanase inac i a ion in ba ley lou s
(Laza idou e al. 2014); only in lou s wi h he highes mois u e con en he e was a
ull des uc ion o his enzyme. Taken his in o accoun and he ac ha he p inciple
o mic owa e hea ing is mainly based on wa e molecules pola iza ion, a s udy o he
e ec o ice lou mois u e con en on β-glucanase inac i a ion kine ics by MW
seemed o be necessa y. Mo eo e , o limi s a ch gela iniza ion du ing he he mal
p ocessing s age he mois u e con en o he lou was kep below 30% (Biliade is e
al. 1980; Maache-Rezzoug e al. 2008; Biliade is 2009). The e ec o MW hea ing
unde hese condi ions on s a ch c ys allini y, pas ing and he mal p ope ies o
mic owa es- ea ed ice lou s was also es ed.

2. Ma e ials and me hods
2.1. Ma e ials
Six een di e en ice lou samples, a ying in mois u e con en and hyd o he mal
ea men using MW, we e examined in his wo k as speci ied in Table 1. 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) (da a p o ided by he manu ac u e ).
2.2. Me hods
Se e al p elimina y es s we e conduc ed in o de o es ablish he wo king condi ions
o mic owa e ea men . A pa icula a en ion was paid o achie e a uni o m
empe a u e and cons an wa e con en du ing he p ocess. The ini ial mois u e
con en o he ice lou was measu ed ollowing he AACC 49.19 me hod, and he
wa e needed o adjus i o he selec ed alue was calcula ed. Flou s we e sp ayed
wi h he app op ia e amoun o wa e while hey we e mixed in a Ki chen-Aid
(Model 5KPM50, Ki chen Aid, S . Joseph, MI, USA) mixe o 10 min. The samples
we e hen allowed o s and 24 h a 4±2 ºC in o de o equilib a e he mois u e. Fou
di e en wa e con en s (13%, 16%, 19%, 25%) and i e mic owa e ea men imes,
(1, 2, 4 and 8 min o mois u es o 13 o 19%, and 40 s, 1, 2, 4 min o 25%
mois u e) we e es ed. The β-glucanase ac i i y o un ea ed lou , was aken as he
alue co esponding o ze o ime condi ions.
2.2.1. Mic owa e T ea men o Flou Samples
Rice lou s we e hea ed in a Panasonic In e e NN-GD566M (Osaka, Japan)
mic owa e o en. The equency o mic owa e adia ion was 2450 MHz. Samples o
hyd a ed lou s (50.0 g) we e in oduced in o (polyamide + polyp opylene) bags o
20 x 30 cm (NOP101, C yo ac, Sealed ai , NY, USA) and he me ically closed by
hea -sealing (Magne a 300 MG model, b and Audio Elek o, Holland) in o de o
main ain he mois u e con en cons an du ing he MW ea men . All samples we e
sp ead ou o o m a hin laye inside he bag (laye hickness  1 mm) o ensu e a
uni o m mois u e and empe a u e dis ibu ion in he lou du ing ea men . The
mic owa es dish, whe e he sample was placed, o a ed du ing he ea men o
assu e a good dis ibu ion o MW ene gy in he sample. The mic owa es powe ,
900W, was applied in cycles o 20 seconds in e als combined wi h down imes o 1
min. Unde hese condi ions he sample bags wi hs ood he wa e apo p essu e
wi hou b eaking du ing he ea men . Be ween s ops he packed lou s we e
manually mixed by mo ing and u ning he bags o acili a e a common empe a u e
wi hin he sample. Samples we e subsequen ly le o cool o 30 min and hen we e
s o ed a 4 °C un il u he analyses. 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 49.19 me hod .
Wa e losses did no exceed 1%, which implies a good he me ic sealing o he bags.
P e ious es s ha e showed a ma ked loss o wa e in samples p ocessed in non-
he me ically closed con aine s; i.e. samples in open con aine s ea ed o 6 min in
he mic owa e o en exhibi ed mois u e losses om 13% o 5%. Some expe imen s
desc ibed in li e a u e also used open o non-he me ically closed con aine s (glass
beake s sealed wi h a pe o a ed polye hylene ilm), bu he mois u e e en ion in he
samples was no epo ed. As mois u e shows a ma ked e ec on hea -induced
inac i a ion o enzymes in plan ma e ials, i s con ol seems o ex eme impo ance
in o de o ob ain conclusi e esul s; he e o e, sealing o he bags was adop ed in all
expe imen al ials o he p esen s udy. The empe a u e e olu ion o e ime o he
mic owa e ea men was moni o ed wi h an in a ed he mome e Tes o 826-T2
(Lenzki ch, Ge many) in he shu down pe iods o he ea men cycles. All
mic owa e ea men s we e ca ied ou a leas in duplica e.
2.2.2. β-Glucanase Ac i i y De e mina ion
The β-glucanase ac i i y in con ol and hyd o he mally 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 he
me hod epo ed in a p e ious wo k (Laza idou e al. 2014). The lou ex ac s we e
ob ained by aqueous ex ac ion ( lou : wa e 1:10) unde s i ing a 25 ºC o 30 min
and subsequen cen i uga ion a 2500 g o 20 min. An aliquo (6 ml) o he esul an
supe na an was added o 36 ml o he 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 o lou
ex ac wi h he β-glucan solu ion 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 = (-o)/o, whe e o is he
iscosi y o wa e ) 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. The p inciple o his p ocedu e is based on he linea
ela ionship be ween he in insic iscosi y and he molecula weigh o some
polyme s in solu ion which, in u n, ollows an in e se linea esponse
(depolyme iza ion) wi h he eac ion ime when he polyme , i.e. β-glucan, is
subjec ed o a andom depolyme iza ion by he ac ion o β-glucanase (Riede e al.
2015). A low polyme concen a ions speci ic iscosi y can be used ins ead o
in insic iscosi y (Hje de e al. 1994). Residual β-glucanase ac i i y o each ea ed
lou sample was analyzed a leas in iplica e.
2.2.3 β-Glucanase Inac i a ion Kine ics
The β-glucanase ac i i y alues ob ained in duplica e om hea ea ed lou s unde
he six een ea men condi ions, plus he un ea ed lou ac i i y alue, we e i ed
agains he lou mois u e and he ime o mic owa e ea men using a nonlinea
mul i a ia e eg ession model. The indi idual e ec o he ime o mic owa es
applica ion on β-glucanase inac i a ion kine ics a each le el o mois u e con en o
he lou was desc ibed by a i s o de kine ic model, acco ding o he equa ion, A =
A0 exp (-k· ), whe e A is he β-glucanase ac i i y, is he ime o he mic owa e
ea men , A0 is a cons an ha ep esen s he ini ial ac i i y o he un ea ed lou (
= 0); and k (min-1) is he appa en a e o enzyme inac i a ion, ep esen ing he
educ ion o enzyme ac i i y pe uni ime. The lou mois u e con en also had a
ma ked e ec on he a e cons an k which a ied exponen ially wi h i .
2.2.5. Pas ing P ope ies o Flou s
Pas ing p ope ies we e s udied by using he Rapid Visco Analyze (RVA-4,
Newpo Scien i ic P . L d., Aus alia) using ICC S anda d me hod 162. The pas ing
empe a u e (PT), peak ime (when peak iscosi y occu ed) (VT), peak iscosi y
(PV), holding s eng h o ough iscosi y (TV), b eakdown (BD), inal iscosi y
(FV) and se back ( inal iscosi y minus peak iscosi y) (SB) we e calcula ed om
he pas ing cu e using The mocline .2.2 so wa e. Viscoamylog aphy o aqueous
lou dispe sions (3 g o lou 14% mois u e basis o 28 g o al weigh wi h dis illed
wa e ) was ca ied ou in iplica e.
2.2.6. X- ay Di ac ion
Samples we e analyzed using a B uke D8 Disco e A25 di ac ome e (B uke
AXS, Rhein elden, Ge many) equipped wi h a coppe ube ope a ing a 40 kV and
40mA, using he CuKa adia ion o 0.154-nm wa eleng h. Di ac og ams we e
ob ained by scanning om 5° o 40° (2θ) a a a e o 1.2°/min, a s ep size o 0.02°, a
di e gence sli wid h a iable (DS) o 5mm and a sca e sli wid h (SS) o 2.92° and
a nickel il e 0.02 o exclude he Kβ adia ion.
2.2.7. Di e en ial Scanning Calo ime y
The mal cha ac e is ics o lou s we e de e mined using a di e en ial scanning
calo ime e (DSC-822e, Me le Toledo, SAE). Flou samples we e weighed in o
aluminum pans (40 L) and dis illed wa e was added using a mic opipe e o make
70% mois u e con en o he aqueous lou dispe sions o a oid he e ec s o wa e
sca ci y on he he mal p o ile (non-equilib ium mel ing) o g anula s a ch
(Biliade is e al. 1980; Biliade is, 2009). Flou weigh s we e abou 8 mg. The
samples we e scanned om 0ºC o 110 ºC a 5 ºC/min using an emp y pan as
e e ence. S a ch e og ada ion was e alua ed in he samples which ha e been
p e iously gela inized in he DSC o en and s o ed in he pans a (4 ± 2) ºC o 7
days; he s aled samples we e e-scanned using he same hea ing p o ocol as o
gela iniza ion. The en halpy (H) alues, exp essed in J/g, based on d y- lou basis,
he onse and endse empe a u es (To and Te) and he peak empe a u e (Tp) we e
es ablished in bo h scans, o gela iniza ion and e og ada ion. Samples we e un in
duplica e.
2.2.8. S a is ical Analysis
S a g aphics Cen u ion .6 (Bi s eam, Camb idge, MN, USA) was used o
mul i a ia e non-linea eg ession. STATISTICA package (Tulsa, OK, EEUU) .6,
allowed pe o mance o MANOVA analysis, and LSD (Leas Signi ican Di e ence)
es was used o e alua e signi ican di e ences (p<0.05) be ween samples.
Some s udies ha e been conduc ed on he e ec o MW on ce eals, mainly s a ches.
Lewandowicz e al. (2000) ound an inc ease in gela iniza ion empe a u e and a
dec ease in solubili y o mic owa ed maize and whea s a ches. S e enson e al.
(2005) also epo ed an inc ease o gela iniza ion empe a u e and a dec ease o pas e
iscosi y o mic owa ed maize s a ch. Ande son and Gu aya (2006) and Luo e al.
(2006) in es iga ed, espec i ely, he e ec o mic owa e on ice and maize s a ches
wi h di e en p opo ions o amylose/amylopec in. These au ho s epo ed
ea angemen s o he molecula s uc u e upon mic owa e hea ing ha could
explain he signi ican changes in iscosi y p ope ies o bo h waxy and non-waxy
s a ches. Pink o á e al. (2003) ha e obse ed ha he peak iscosi y o ice lou
dec eased as empe a u e and mic owa e powe le el applied o ice g ain inc eased.
On he o he hand, Fan e al. (2012) epo ed ha mic owa e i adia ion had no e ec
on he op ical and he mal p ope ies o ice s a ch du ing gela iniza ion compa ed o
con en ional hea ing. Many s udies ha e been ca ied ou on he s uc u al and
physical p ope ies o s a ches a e hea -mois u e (HMT) ea men (Za a eze and
Dias 2011). HMT p omo es in ense changes in s a ches, hus signi ican ly al e ing
hei pas ing p o ile as e idenced by inc eased pas ing empe a u e and dec eased
peak iscosi y, inal iscosi y, and b eakdown (Wa cha a ewinkul e al. 2009).
Acco ding o hese esea che s, he changes in hea - ea ed s a ch’s pas ing p ope ies
a e due o associa ions be ween he chains in he amo phous egions o he g anule as
well o changes in c ys allini y du ing he hyd o he mal ea men . Such s uc u al
modi ica ions a e in ensi ied as he mois u e con en in he HMT inc eases (Olayinka
e al. 2008). As he o ces o he in a-g anula chain in e ac ions a e s eng hened,
he s a ch equi es mo e hea o s uc u al disin eg a ion and pas e o ma ion
(Olayinka e al. 2008). A high pas ing empe a u e hus indica es ha mo e o ces
and c oss-links exis wi hin he s a ch g anules (Olayinka e al. 2008). Lan e al.
(2008) ha e shown ha he e og ada ion p ocess is in luenced by he amoun o
leached amylose, g anule size, and he p esence o igid, non- agmen ed swollen
g anules. Chung e al. (2009) also ound ha HMT educes amylose leaching om
s a ch g anules and ha his educ ion is mo e signi ican in s a ches con aining high
le els o amylose. They ha e epo ed ha HMT p omo es addi ional amylose–
amylose and/o amylopec in–amylopec in chain in e ac ions, which es ic amylose
leaching and dec ease e og ada ion e en s. A ecen s udy by Roman e al. (2015)
epo s on he e ec s o sho ime (0.5 o 4 min) MW ea men s o co n lou s a

30% mois u e. They ound opposi e e ec s on pas ing p ope ies depending on he
MW ea men ime. T ea ed samples showed inc eased iscosi y du ing he hea ing-
cooling cycle when a e y sho ime o mic owa e ea men was applied (0.5 and 1
min). Ins ead, longe ea men s (4 min) induced he opposi e e ec , and a dec ease
in he iscosi y was no ed. A dec ease o he maximum (peak) iscosi y was also
obse ed by Pink o á e al. (2003) by inc easing empe a u e o mic owa e
ea men o he ice g ain and inc easing powe ou pu a a mois u e con en o 30
%. Roman e al. (2015) ha e examined he e ec o MW on lou s uc u e. They
ound a mo e disagg ega ed s uc u e and a less compac ma ix, wi h s a ch g anules
being mo e naked and sligh ly swollen. Luo e al. (2006) obse ed e en mo e ma ked
changes on s a ch g anule s uc u e when mois ened (30 %) maize s a ch was ea ed
o 20 min wi h mic owa es a 1 W/g. They ha e no ed some b eakage, c acks and
po es on he su ace o he s a ch g anules. These obse a ions can be a ibu ed o
chain segmen al ans e s and in e nal ea angemen s in he g anula ma e ial,
acili a ed by wa e plas iciza ion. The absence o such e ec s in ou MW ea ed
lou s could be due o he sho e ea men imes employed (lowe powe ) as well as
he use o comple ely ai igh bags, whe e la ge wa e ans e s wi hin he sample
we e p obably hinde ed by he apo p essu e aised in he bag headspace. Figu e 3
shows he di e en pas ing p ope ies o wo aliquo s o 30% mois ened- ice lou
ea ed wi h he same MW hea ing p ocedu e used in all he expe imen s ca ied ou
in his s udy, and in an opened beake . Al hough u he s udies should be ca ied ou
o ully un a el he di e en e ec s hea -mois u e ea men s ha e on he s a ch
g anula ma e ial using MW, he no able di e ences in pas ing p ope ies shown in
Fig. 3 clea ly indica e ha wa e plays an impo an ole in s a ch modi ica ions
du ing MW ea men s. Mo eo e , i is impo an o no e ha he lou ea ed in he
close bag main ained i s o iginal whi e colo compa ed o ha ea ed in he open
beake o he same ime which showed a isible da ke -b own colo .

Figu e3:Pas ingcha ac e is icso (9.2gd y lou /100g)aqueousdispe sionso  ice lou s;
con olandmic owa ehea ‐ ea eda 30%mois u econ en  o 4min.Viscosi yp o ile o 
na i e(),andmic owa e‐ ea edsamplesinhe me icbags()andopenbeake ().
3.4. X- ay Di ac ion
The di ac og ams ob ained om he mic owa e- ea ed and he un ea ed 25%
mois ened- ice lou s (Fig. 4) showed ha all samples main ained he A- ype
c ys allini y, ypical o many ce eal s a ches (Imbe y, e al. 1991; Zobel 1964) wi h
peaks cen e ed a app oxima ely 15, 17.1, 18 and 23 (2), o bo h na i e sample
and all mic owa e ea ed ice lou s. Indeed, he A- ype c ys alline pa e n was
conse ed in he MW ea ed lou s wi h 25%, o any p ocessing ime (0.67, 1, 2 and
4 min), and no signi ican di e ences in he ela i e c ys allini y alue compa ed o
he na i e lou (s a ch) we e obse ed. Luo e al. (2006) and Roman e al. (2015)
epo ed an inc ease in X- ay in ensi ies o mic owa e ea ed maize s a ch and
lou , espec i ely. I has been pos ula ed ha mic owa e adia ion can lead o
o ma ion o addi ional double helical s uc u es wi hin he s a ch c ys alli es, leading
o a highe molecula o de han ha in na i e s a ch (Luo e al. 2006). The
mo emen o double helices migh be ela ed o he apo iza ion o wa e molecules
ha elease posi ions o iginally occupied by wa e molecules, allowing o mo e
0
20
40
60
80
100
120
0
500
1000
1500
2000
2500
3000
3500
4000
0 200 400 600 800
Tempe a u e(ºC)
Viscosi y(cp)
Time(min)
Con ol
He me ic
bag
Openbeake
compac o de ed c ys alline a ays (Luo e al. 2006). The use o he me ically sealed
bags in ou s udy p o ided a small posi i e p essu e inside he bags and p e en ed
he loss o wa e , and his in u n migh ha e esul ed in limi ed molecula
eo ganiza ion wi hin he s a ch g anules. Di e en e ec s o HMT on s a ch
c ys allini y ha e been no ed by o he au ho s, howe e , o mo e p olonged he mal
ea men s. Jyo hi e al. (2010) obse ed an inc ease in c ys allini y o swee po a o
and a ow oo s a ches and a sligh dec ease in he c ys allini y o cassa a s a ch,
a e he h ee s a ches we e hea -mois u e ea ed a 120ºC o 14 h a 20% mois u e.
Simila esul s o ou indings we e ob ained by Maache-Rezzoug e al. (2008).
These au ho s did no ind any signi ican di e ence in he ela i e c ys allini y when
applied a con olled p essu e d op (DIC) hyd o he mal ea men a 1 ba o p essu e
(100 ºC, 14-15% mois u e) o any p ocessing ime (10 – 60 min) o maize s a ch,
whe eas hey ha e no ed a signi ican educ ion in ela i e c ys allini y when applied
ea men s a 2 and 3 ba o p essu e (122 and 135ºC, espec i ely); in he la e case,
he A- ype c ys alline pa e n was p og essi ely changing o a V- ype c ys alline
pa e n, implying he o ma ion o amylose-lipid complexes in he hea - ea ed s a ch
g anules. O e all, he indings o he p esen wo k clea ly indica e ha i is possible
o apply MW ea men s o comple e inac i a ion o β-glucanase wi hou modi ying
s a ch s uc u e, e en a he highes mois u e con en o 25%. Main aining a cons an
wa e con en o he lou and he lou empe a u e below 100 °C, h oughou he
mic owa e ea men a e p obably he mos impo an pa ame e s in his espec .

Figu e 4: X- ay di ac ion pa e ns o un ea ed ice lou (con ol) and mic owa ed
(di e en ea men imes) ice lou s a mois u e con en 25%. .
0
10000
20000
30000
40000
50000
60000
10 15 20 25 30 35 40
Di ac ionangle(2)
con ol
0.67min
1min
2min
4min
3.5. Impac o MW on The mal P ope ies
The esul s o X- ay di ac ome y we e u he con i med by di e en ial scanning
calo ime y. The he mal p ope ies o un ea ed (con ol) and mic owa e- ea ed
25% mois ened- ice lou s a e summa ized in Table 3. In he ange o empe a u es
es ed, he aqueous dispe sions o lou s exhibi ed wo endo he mic peaks,
co esponding o he gela iniza ion o s a ch and he dissocia ion o amylose-lipid
complexes (Biliade is 2009). A e se en-day s o age (in he DSC pans) a 4ºC o he
gela inized samples a second scan also showed wo endo he mic ansi ions. The
peak co esponding o he mel ing o he ec ys allized amylopec in ha appea ed a
lowe empe a u es han he gela iniza ion peak o na i e s a ch, and he amylose-
lipid complex ansi ion ound in he i s scan ha appea ed a he same empe a u e
and had he same en halpy alue. As can be seen, he e ec o mic owa es on s a ch
gela iniza ion, amylopec in e og ada ion and amylose-lipid complex empe a u es
and en halpies was p ac ically negligible, e en hough he samples es ed by DSC
we e hose o he highly mois ened- lou s. The only signi ican e ec (p<0.01) was
ob ained in he gela iniza ion peak empe a u e (weak annealing, Biliade is 2009),
al hough he quan i a i e impo ance o his change was a he mino . O he au ho s
ha e also epo ed an inc ease in he gela iniza ion peak empe a u es o mic owa e
ea ed 30% mois ened-maize lou s (Roman e al. 2015) when MW ene gy was
applied o 2, 4 min; he inc eases obse ed by hese au ho s we e 3°C, while ha
o he p esen wo k was less han 1°C. Lewandowicz e al. (2000) also obse ed ises
in he gela iniza ion peak empe a u es o 30% mois ened-s a ches ea ed by MW
o 60 min a 0.5 W/g, ha a ied om 13ºC o whea o 6ºC o waxy co n s a ch,
in compa ison o hei un ea ed coun e pa s. Mo eo e , hese au ho s ha e epo ed
an impo an dec ease, ~72%, in he appa en gela iniza ion en halpy alue o whea
s a ch ha was only ~ 50% o co n, and insigni ican o waxy co n compa ed wi h
he un ea ed s a ches. O he au ho s also ound a educ ion in s a ch gela iniza ion
en halpy o MW ea ed co n s a ches (Luo e al. 2006; S e enson e al. 2005). In
con as , Roman e al. (2015) ha e epo ed an almos double gela iniza ion en halpy
o all MW- ea ed co n lou samples wi h espec o he na i e one. Consequen ly,
he ex en o MW hea ing e ec s on he mal beha io o g anula s a ches depends
on he s a ch sou ce, he amylose and mois u e con en , as well as he in ensi y and
ime o he applied mic owa e ea men (Za a eze and Dias 2011).
Table 3. The mal p ope ies o aqueous dispe sions (70 % wa e ) o lou ea ed by mic owa es (a 25% wa e con en ).


T ea men
ime
(min)
Hgel
(J/g db)
To-gel
(ºC)
Tp-gel
(ºC)
Te-gel
(ºC)
R
(ºC)
Ham-lip
(J/g db)
Tp-am-
lip
(º C)
H e
(J/g db)
Tp- e
(ºC)
0 7.7 a 68.0 a 74.16 a 89.6 a 12.4 a 1.22 bc 100.7
a
4.7 bc 52.7 a
0.67 7.5 a 68.3 ab 74.30 b 90.2 a 12.0 a 1.09 ab 100.0
a
4.2 a 54.5 ab
1 8.0 a 68.1 a 74.25 ab 91.2 a 12.2 a 1.32 c 100.2
a
4.4 ab 55.2 b
2 7.7 a 68.4 ab 74.47 c 91.5 a 12.2 a 1.06 ab 101.3
a
4.7 bc 53.3 ab
4 7.9 a 68.9 b 74.71 d 91.5 a 11.6 a 0.97 a 101.7
a
4.8 c 52.8 a

Hgel: En halpy alue associa ed wi h gela iniza ion; To-gel and Te-gel: onse and endse empe a u es o he gela iniza ion peak; Tp-gel, Tp- e , Tp-amil:
Tpeak o gela iniza ion, e og ada ion and amylose-lipid complex dissocia ion peaks, espec i ely; R= 2·(Tp-To) ; Hamyl-lipid: En halpy alue o
he dissocia ion o he amylose-lipid complex; H e : Mel ing en halpy o he ec ys alized amylopec in a e s o age o he gela inized sample a
4ºC o 7 days. Each alue is he a e age o duplica e measu emen s.
Wi hin he da a se o each pa ame e , di e en le e s in he mean alues o he espec i e columns imply signi ican di e ences be ween means
a p < 0.05.

4. Conclusions
Mic owa e ea men is a use ul al e na i e o inac i a ion o he endogenous β-
glucanase in ice lou s when applied o mois ened lou samples. The enzyme
inac i a ion p ocess ollows a i s o de kine ic esponse (R2 = 0.97). The cons an a e
o he he mal inac i a ion by MW inc eased exponen ially wi h he mois u e con en o
he lou , so ha , he mic owa e ea men ime equi ed o comple e β-glucanase
inac i a ion was only 4 min when he ini ial lou mois u e was aised o 25%.
Following he MW ea men , he c ys allini y o he s a ch was una ec ed and he side
e ec s o he ea men on lou pas ing and he mal p ope ies we e a he negligible.
Acknowledgmen s
The au ho s g a e ully acknowledge he inancial suppo o he Spanish Ins i u ions
Minis e io de Economía y Compe i i idad and he Eu opean Regional De elopmen
Fund (FEDER) (P ojec s AGL2012-35088 and AGL2015-63849-C2-2-R).
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Scien i ic opinion on he subs an ia ion o heal h claims ela ed o be a-glucans om
oa s and ba ley and main enance o no mal blood LDL-choles e ol concen a ions
(ID 1236, 1299), inc ease in sa ie y leading o a educ ion in ene gy in ake (ID
851, 852), educ ion o pos -p andial glycaemic esponses (ID 821, 824), and
“diges i e unc ion” (ID 850) pu suan o a icle 13(1) o Regula ion (EC) No
1924/2006, 2207. (2011).