Remo al o a inose and s achyose om soy-based be e ages using soluble
and immobilized MelA
α
-D-galac osidase om Lac iplan ibacillus
plan a um WCFS1
Leand o Al es dos San os
a,d
, Paloma Delgado-Fe n´
andez
b
, Ana Mu˜
noz-Lab ado
b
,
Diego Ma in-Gu ie ez
a
, E elyn C. Rome o
b
, F anzo y Cua an
a
, And ea Ga cia-Al a ez
a
,
Elena Ga cía-Cal o
a
, Blanca de las Ri as
c
, Rosa io Mu˜
noz
c
, Nie es Co zo
b
, F. Ja ie Mo eno
b
,
Cesa Ma eo
a,*
a
Depa amen o de Bioc´
a alisis, Ins i u o de Ca ´
alisis y Pe oleoquímica, CSIC, Ma ie Cu ie 2, 28049, Mad id, Spain
b
Depa amen o de Bioac i idad y An´
alisis de Alimen os, Ins i u o de In es igaci´
on en Ciencias de la, Alimen aci´
on, CIAL (CSIC-UAM), Nicol´
as Cab e a 9, 28049,
Mad id, Spain
c
Ins i u o de Ciencia y Tecnología de Alimen os y Nu ici´
on, ICTAN (CSIC), Juan de la Cie a 3, 28006, Mad id, Spain
d
Depa men o Chemis y, Fede al Uni e si y o Pa an´
a, Poly echnic Cen e , P.O. Box 19032, Cu i iba, 81531-980, Pa an´
a, B azil
ARTICLE INFO
Keywo ds:
Soy-based be e ages
α
-galac osidase
Galac ose hyd olysis
Docking
Enzyme immobiliza ion
ABSTRACT
The hyd olysis o
α
-galac ose-ca bohyd a es in soy-based be e ages using he enzyme MelA
α
-D-galac osidase
(Lp_3485) om Lac iplan ibacillus plan a um WCFS1 is p esen ed. MelA is a highly uns able ime ic enzyme
whose ac i i y and s abili y a e s ongly dependen on he dilu ion and pola i y o he eac ion medium.
Conside ing his, he enzyme was immobilized and s abilized using di e en immobiliza ion p o ocols. Pos -
immobiliza ion echniques consis ing o c oss-linking wi h di e en poly unc ional polyme s we e used. The
op imal de i a i e was used o hyd olyse di e en oligosaccha ides o he a inose amily ( a inose and s a-
chyose) p esen in soy d ink p epa a ions. Rema kably, MelA showed speci ic ac i i y and high hyd oly ic e -
iciency owa ds RFOs in soy-based be e ages a a ela i ely low empe a u e (20 ◦C). The e o e, hese esul s
sugges ha his enzyme could be p e e en ially used in he ea ly s age o soybean p ocessing o emo e RFOs
om soy-based be e ages and soy-de i ed p oduc s, he eby imp o ing hei nu i ional alue.
1. In oduc ion
Soybean (Glycine max (L) Me ) is a highly aluable ood sou ce,
p o iding essen ial p o eins, ib e, mic onu ien s and phy ochemicals
ha play an impo an ole in he die s o di e en egions o he wo ld
(Fehily, 2003; Guo e al., 2022). Soybeans a e also no able o hei
ca bohyd a e composi ion, which accoun s o a signi ican p opo ion
(30%) o hei o al con en . Wi hin hese ca bohyd a es, soluble suga s
make up abou 15% o he seed composi ion. The p edominan suga s
ound in soybean seeds a e suc ose, which can ange om 40% o 70% o
he suga con en , ollowed by a inose wi h 5%–15% and s achyose
wi h 12%–35% (Hou e al., 2009).
Soybeans can be used o make a ious p oduc s such as b ead, bis-
cui s, cakes, chocola e, e c (Amigo-Bena en , Sil ´
an, Mo eno, Villamiel,
& delCas illo, 2008). Soy p oduc s can also be used as a mea subs i u e
in ege a ian die s due o hei high p o ein p o ile and balanced amino
acid pa e n (Bha ia, Singh, Ba a, & Singh, 2020). In pa icula ,
soy-based be e ages s and ou as a nu i ious be e age and a iable
al e na i e o mammalian milk. In addi ion, soy d inks a e lac ose- ee,
making hem an ideal choice o people wi h lac ose in ole ance
(Ka olia, Liu, Li, & Koppa apu, 2019).
Howe e , soy-based be e ages con ain an inu i ional ac o s
including ypsin inhibi o s, phy ic acid, lec ins and soluble bu indi-
ges ible ca bohyd a es. Among he oligosaccha ides o he a inose
amily (RFOs) such as a inose, s achyose, e bascose and ajugose a e
conside ed majo an inu ien s (Padalka e al., 2023). S uc u ally,
hese ca bohyd a es a e
α
-galac osyl (
α
-1,6) de i a i es o suc ose, wi h
a inose, s achyose, e bascose and ajugose con aining one, wo, h ee
and ou galac ose uni s, espec i ely, linked o suc ose. These
α
-galac-
osides a e unable o be diges ed by humans and o he monogas ic
* Co esponding au ho .
E-mail add ess: [email p o ec ed] (C. Ma eo).
Con en s lis s a ailable a ScienceDi ec
LWT
jou nal homepage: www.else ie .com/loca e/lw
h ps://doi.o g/10.1016/j.lw .2024.116864
Recei ed 1 Ap il 2024; Recei ed in e ised o m 1 Augus 2024; Accep ed 2 Oc obe 2024
LWT - Food Science and Technology 210 (2024) 116864
A ailable online 3 Oc obe 2024
0023-6438/© 2024 The Au ho (s). Published by Else ie L d. This is an open access a icle unde he CC BY license (
h p://c ea i ecommons.o g/licenses/by/4.0/ ).
animals due o he absence o panc ea ic
α
-D-galac osidase, which is
equi ed o hyd olyse he
α
-1-6 linkages. As a esul , RFOs emain un-
diges ed in he small in es ine and pass h ough wi hou being abso bed.
Subsequen ly, in he la ge in es ine, gas-p oducing bac e ia such as
Clos idium sp. me abolize he RFOs ia anae obic e men a ion, p o-
ducing excess ca bon dioxide, hyd ogen, aces o sho -chain a y acids
(SCFAs) and me hane. These la us accumula ion lead o symp oms such
as in es inal discom o , la ulence and dia hoea in suscep ible in-
di iduals, which limi s he consump ion o oods wi h high concen a-
ions o RFOs (Connes e al., 2004; Mu uyemungu, Singh, Liu, & Rose,
2023; Sanyal, Kuma , Pa anayak, Ka , & Bishi, 2023). The e o e,
add essing he emo al o RFOs om soy d inks is key o po en ially
imp o ing hei nu i ional alue and inc easing hei consump ion.
Cu en indus ial app oaches o emo ing RFOs om soy-based
be e ages include he use o enzymes, speci ically
α
-D-galac osidases.
These enzymes a e widely used in many indus ial p ocesses o soy-
de i ed p oduc s (Falkoski e al., 2006; Çelem &
¨
Onal, 2022).
α
-D-ga-
lac osidases (
α
-D-galac oside galac ohyd olase, EC 3.2.1.22) a e exo-
glucosidases ha ca alyse he hyd olysis o
α
-1,6-linked e minal
non- educing
α
-D-galac osyl esidues om RFOs such as a inose, s a-
chyose and e bascose, as well as glycoconjuga es, glycop o eins, gal-
ac omanans and galac olipids using e en ion mechanisms (Jang e al.,
2019; Li, Loman, Co man, & Ju, 2017; Wang e al., 2014). In addi ion,
α
-D-galac osidase clea es he
α
-1,6 linkage be ween galac osyl and
glucosyl uni s om melibiose, hence i is also known as melibiase
(Bha ia e al., 2020; Delgado-Fe nandez e al., 2020).
α
-D-galac osidases
a e widely dis ibu ed in plan s, animals and mic oo ganisms ( ungi and
bac e ia) and due o he inc easing indus ial demand o enzymes o
emo e RFOs, many
α
-D-galac osidases ha e been s udied (Bha ia e al.,
2020; Wang e al., 2019; Çelem &
¨
Onal, 2022).
The g owing demands o indus ial p ocesses and he need o
simpli y downs eam p ocessing and ope a ional s abili y, ha e
ende ed immobilized enzymes highly desi able. Mo e speci ically, he
use o soluble
α
-D-galac osidases as ca alys s o indus ial-scale e-
ac ions is no easible because i is no possible o eco e he enzyme a
he end o he p ocess. The e o e, enzyme immobiliza ion has eme ged
as an a ac i e opic o enable enzyme euse and con inuous ope a ion
(Geng e al., 2022; Magh aby, El-Shabasy, Ib ahim, & Azzazy, 2023).
Va ious me hods o enzyme immobiliza ion ha e been desc ibed, bu
hei e ec i eness a ies depending on he na u e o he enzymes,
pa icula ly hei e ia y and qua e na y s uc u es in he case o mul-
ime ic enzymes (Ma eo e al., 2020). In he case o mul ime ic enzymes,
s abili y depends on he immobiliza ion o all subuni s o p e en hei
dissocia ion in he eac ion medium. The e o e, he e is a g owing en-
dency o de elop immobiliza ion me hods ha in ol e all subuni s.
Howe e , geome ic cons ain s (e.g, e ahed al enzymes o enzymes
wi h many subuni s) o en make his app oach in easible. The e o e,
pos -immobiliza ion echniques using polyme s o poly unc ional com-
pounds capable o c oss-linking he non-immobilized subuni s o he
suppo become necessa y (Fe nandez-La uen e, 2009; Fe n´
andez-La-
uen e e al., 1999).
α
-D-galac osidases om di e en sou ces ha e been
immobilized on di e en suppo s and success ully used o he emo al
o RFOs om soy d inks (Ka olia e al., 2019).
Recen ly, an e icien hyd olysis (84%) o RFOS ( a inose and s a-
chyose) was achie ed using MelA
α
-D-galac osidase om Lac iplan iba-
cillus plan a um WCFS1, demons a ing ha MelA has a high capaci y o
hyd olyse indiges ible RFOS in simple bu e ed solu ions
(Delgado-Fe nandez e al., 2020). In his wo k, once he enzyme ac i i y
o MelA
α
-D-galac osidase was op imized, di e en immobiliza ion
suppo s we e p epa ed o un a el he e iciency o he enzyme immo-
biliza ion p io o hei applica ion. This p ocess was ollowed by a
chemical cha ac e iza ion in o de o depic he esul ing oligosaccha-
ides a e hyd olyza ion. The e o e, he main objec i e o his wo k
aims o in es iga e he abili y o immobilized MelA
α
-D-galac osidase
(Lp_3485) om L. plan a um WCFS1 in compa ison wi h he soluble
o m, o hyd olyse ca bohyd a es as RFOs p esen in eal ood ma ices
as a echnological s a egy o soy-based be e ages.
2. Ma e ials and me hods
In all cases, expe imen s we e pe o med a leas in duplica e and he
mean e o was ne e g ea han 5%.
2.1. MelA
α
-D-galac osidase ac i i y es
The enzyme
α
-D-galac osidase (MeIA) om Lac obacillus plan a um
WCFS1 was pu i ied and cha ac e ized as desc ibed (Cu iel, de las Ri as,
Manche˜
no, & Mu˜
noz, 2011). The enzyma ic ac i i y was e alua ed by
measu ing he p-ni ophenola e (pNP) eleased om a solu ion o
p-ni ophenyl-
α
-D-galac opy anoside (pNPG). The measu emen was
pe o med con inuously by kine ics using a JASCO V-730 spec opho-
ome e . A 5 mM solu ion o pNPG in 50 mM sodium phospha e a pH 7
was used. The eleased p-NP was measu ed a 420 nm and 25 ◦C. One
enzyma ic uni is de ined as he amoun o MeIA equi ed o p oduce 1
μ
mol o p-NP pe minu e unde he assay condi ions (
ε
=9310
M
−1
cm
−1
).
2.2. P epa a ion o he di e en aga ose-based suppo s
The di e en suppo s used o he immobiliza ion o he enzyme
we e de i ed om aga ose p e iously de i a ized wi h epoxide sup-
po s. These epoxide g oups we e subsequen ly con e ed in o di e en
eac i e g oups as desc ibed below.
2.2.1. Ac i a ion o aga ose wi h epoxide g oups
1 g o aga ose (4BCL; ABT, Spain) was suspended in 4.4 mL o wa e
and mixed wi h 0.328 g o NaOH, 0.02 g o NaBH
4
, 1.6 mL o ace one
and inally 1.1 mL o epichlo ohyd in. This mix u e was s i ed a 25 ◦C
o 16 h. Finally, he suppo was washed wi h plen y o wa e and d ied
by acuum il a ion.
2.2.2. Ac i a ion o epoxy aga ose wi h o he unc ional g oups
Aga ose-CHO (glyoxyl-aga ose): 1 g o epoxy aga ose was hyd o-
lyzed wi h 10 mL o 0.5 M aqueous HCl solu ion o 90 min. A e his
ime, he suppo s we e washed wi h wa e and d ied. A solu ion con-
sis ing o 0.1 mL o 0.2 M NaIO
4
and 9 mL o wa e was hen added. This
suspension was allowed o eac o 90 min a 25 ◦C, hen washed wi h
wa e and d ied.
T ie hylamine aga ose (TEA): 1 g o aga ose epoxide was mixed wi h
10 mL o a solu ion consis ing o 5 mL ace one, 1.4 mL TEA and 3.6 mL
wa e . This suspension was s i ed a 25 ◦C o a leas 24 h. Finally, i
was washed wi h wa e and d ied unde acuum.
Sodium iminodiace a e (IDA). To 1 g o aga ose epoxide, 10 mL o a
0.5 M IDA solu ion a pH 11 was added. This suspension was allowed o
eac o a leas 24 h wi h s i ing, a e which he suppo was washed
and d ied.
Aga ose-IDA/Zn. To 1 g o aga ose-IDA suppo , 10 mL o an aqueous
solu ion o ZnCl
2
(0.2 M) was added. A e 30 min o eac ion, he
suppo was washed wi h wa e and d ied o la e use.
Ac i a ion o he di e en bi unc ional suppo s used: In all cases, he
de i a ized aga ose epoxide suppo s (IDA, TEA, Zn) we e oxidized ac-
co ding o he ollowing p o ocol: 0.1 mL o a 0.1 M aqueous NaIO
4
solu ion and 9 mL o wa e we e added o 1 g o each suppo and
allowed o eac o 90 min. A e his ime, hey we e washed wi h
wa e and d ied unde acuum.
Aga ose-e hylenediamine (EDA) wi h glu a aldehyde: 1 g o aga ose-
CHO was amina ed by eac ion wi h 10 mL o a 2 M aqueous solu ion o
e hylenediamine a pH 10 o 2 h. A e his eac ion ime, 10 mg o
NaBH
4
was added. A e ano he 2 h, he suppo was washed wi h 1 M
NaCl and plen y o wa e . Finally, he suppo was suspended in a
mix u e consis ing o 1.7 mL o 0.2 M phospha e bu e a pH 7 and 1.1
mL o comme cial 25% glu a aldehyde solu ion. The suspension was
L. Al es dos San os e al.
LWT 210 (2024) 116864
2
allowed o s and o 16 h a 25 ◦C wi h gen le agi a ion and hen washed
wi h plen y o wa e (Be anco e al., 2006).
The o he eagen s used in he de elopmen o he immobiliza ion
p ocedu e we e pu chased om Me ck (S enheim, Ge many).
2.3. Immobiliza ion o MeIA on di e en suppo s
Di e en bu e s we e used depending on he suppo used o
immobiliza ion: 5 mM sodium phospha e and 50% glyce ol o he
aga ose-TEA and aga ose-IDA ac i a ed suppo s, and 25 mM sodium
phospha e wi h 150 mM NaCl and 15% glyce ol o he o he suppo s.
Then, 1.0 g o he di e en suppo s we e suspended in 10 mL o MelA
enzyme solu ion (100 U g-1 suppo ) and gen ly shaken a 4 ◦C o
di e en imes. Immobiliza ion was conside ed comple e when no ac-
i i y was de ec ed in he supe na an .
The immobiliza ion e iciency (IE, %) was calcula ed as (Equa ion
(1)):
IE =Ai−A
Ai
×100% (1)
whe e Ai is he ini ial enzyma ic ac i i y (U) o he enzyme and A is he
enzyma ic ac i i y (U) emaining in he supe na an a he end o he
immobiliza ion p ocess.
The eco e ed ac i i y (R, %) was calcula ed as (Equa ion (2)):
R=A0
AT
×100% (2)
whe e A
0
is he quo ien be ween he eal ac i i y (U g-1 suppo ) o he
immobilized p epa a ion and AT is he heo e ical ac i i y (U g-1 sup-
po ) o he immobilized p epa a ion. A he end o he immobiliza ion
p ocess, he enzyme immobilized on suppo s wi h aldehyde g oups was
educed wi h 1 mg mL-1 NaBH4 o 30 min a 4 ◦C and hen washed wi h
he immobiliza ion bu e p e iously cooled o 4 ◦C. In he case o he
glu a aldehyde suppo , i was ea ed wi h 5 mL o 1M glycine a pH 7
and 4 ◦C o 1 h o block he eac i e g oups and hen washed wi h he
immobiliza ion bu e .
2.4. Pos -immobiliza ion ea men
1 g o he p epa a ion immobilized on glu a aldehyde-ac i a ed
suppo s was suspended in 5 mL o pH 5.7 bu e con aining 15%
glyce ol and 5 mg/mL o he di e en polyme s (aspa ic dex an and
glycine dex an) in he p esence o 10 mM N-(3-dime hylaminop opyl)-
N
′
-e hylca bodiimide hyd ochlo ide (EDC). The suspension was allowed
o s and o 90 min a 4 ◦C wi h gen le shaking and hen washed wi h 25
mM phospha e bu e , pH 7, con aining 150 mM NaCl and 15% glyce ol.
2.5. Soyd ink based p epa a ion (sample L)
The be e age was p epa ed as was desc ibed by (Mulimani and
Ramalingam, 1995) using comme cially a ailable soybeans. The soy-
beans we e g ound and de a ed wi h n-hexane (1:1; / ). This de a ed
meal was suspended in 10 ol o dis illed wa e and boiled o 10 min.
Insoluble esidues we e emo ed by cen i uga ion a 2000×g o 5 min.
2.6. T ea men o soy-based be e ages MelA
α
-D galac osidase o emo e
RFOs
Eigh b ands o soy-based be e ages, labelled as SA, SL, P, VS, SCa, A,
K, and SSA, we e pu chased om local supe ma ke s whose selec ion
c i e ia included hei ma ke ep esen a ion, p oduc di e si y and
accessibili y. Table 1 shows he ca bohyd a e composi ion o he
di e en soy-based be e ages and he lab-p epa ed soy-milk. All sam-
ples we e s o ed a e ige a ion empe a u e and analyzed in duplica e
be o e he expi y da e.
Hyd olysis o RFOS p esen in comme cial soy-based be e ages
(Table S1) and he labo a o y p epa ed soy d ink sample we e pe -
o med in ba ch eac ions using soluble and immobilized enzyme in
ba ch eac ions. B ie ly, 5 U o ee o immobilized MelA
α
-D-galac o-
sidase we e added o 0.460 mL aliquo s o soy-based be e ages and
incuba ed a 22 ◦C and pH 6.8 o 7 h unde agi a ion. Aliquo s we e
wi hd awn pe iodically 0, 1, 3, 5 and 7 h and kep in a boiling wa e ba h
o 10 min o s op he enzyme eac ion.
2.7. De e mina ion o pH and d y ma e (DM)
The pH o he samples was measu ed a 22 ◦C using an MP225 pH
me e wi h a glass elec ode (Me le Toledo GmbH, Schwe zenbach,
Swi ze land).
The d y ma e (DM) was de e mined in he soy-based be e age
p e iously p epa ed in he labo a o y (L) as desc ibed in sec ion 2.5. The
samples we e hea ed o 15 h and he d y esidue was weighed a e
cooling in a desicca o . The DM alue (%) was abou 6.8 o he soy
be e ages.
2.8. De e mina ion o ca bohyd a es by gas ch oma og aphy- lame
ioniza ion de ec o (GC-FID)
Ca bohyd a es we e ex ac ed acco ding o he me hod o Pa il,
P a een Kuma , Mulimani, Vee anagouda, & Lee, 2010 (Pa il e al.,
2010) wi h some modi ica ions. Fi s , an op imiza ion o he ex ac ion
p ocess o comme cial soy-based be e ages was pe o med using wo
di e en alcoholic sol en s: e hanol and me hanol. Rega ding he
quan i a i e esul s o ca bohyd a es and hei coe icien o a ia ion,
me hanol ex ac ion p o ided mo e accu a e esul s o he Nu i ion
Fac s panel o p oduc labels (Table S1) and coe icien s o a ia ion
<10% compa ed o e hanol. Once he ex ac ion sol en was op imized,
aliquo s (3 mL) o soy-based be e age samples we e mixed wi h 7 mL o
me hanol. The mix u e was cen i uged a 3200 g o 15 min a 40 ◦C.
The di e en ca bohyd a es we e analyzed as ime hylsilyla ed oxime
(TMSO) de i a i es, p epa ed as p e iously desc ibed (Ga cia Banos,
Olano, & Co zo, 2000). 0.4 mL o in e nal s anda d (IS) solu ion (0.5 mg
mL-1 phenyl-β-glucoside) was added o di e en samples con aining
be ween 2.5 and 5 mg o suga s. These mix u es we e d ied in a o a y
e apo a o a 38–40 ◦C. Suga oximes we e o med by adding 250
μ
L o
hyd oxylamine chlo ide (2.5%) in py idine and hea ing he mix u e a
70 ◦C o 30 min, hen silyla ed wi h hexame hyldisilazane (250
μ
L) and
TFA (25
μ
L) and kep a 50 ◦C o 30 min. Reac ion mix u es we e
Table 1
– Composi ion in suc ose and a inooligosaccha ides (RFOs; a inose and s a-
chyose) ound in comme cial UHT soy-based be e ages (n =2).
Soy-based be e ages
labelling
Suga con en (mg/100 mL)
Suc ose Ra inose S achyose To al
RFOs
SA 120.18 ±
6.71
2.28 ±0.27 11.30 ±
0.184
13.58
SL 82.13 ±
4.26
3.17 ±031 14.11 ±1.71 17.28
P 392 ±
36.33
90.2 ±1.64 373 ±8.51 463.2
VS 478.18 ±
21.01
66.22 ±
3.95
383.01 ±
35.22
449.23
SCa 362.76 ±
1.87
10.64 ±
0.14
13.72 ±0.44 23.36
A 648.5 ±
6.37
119 .14 287.2 ±0.71 406.34
K 578.57 ±
67.27
173.47 ±
21.56
375.40 ±
56.64
548.87
L 340.9 ±
32.37
103.94 ±
10.20
436.44 ±
45.42
540.38
SSA (Wi hou added
suga s)
19.23 ±
1.86
2.51 ±0.21 10.71 ±0.67 13.22
L. Al es dos San os e al.
LWT 210 (2024) 116864
3
cen i uged a 6708 g o 1 min a oom empe a u e. Supe na an s we e
injec ed o s o ed a 4 ◦C p io o analysis. GC-FID analysis o TMSO
de i a i es was pe o med on an Agilen Technologies 7820A gas
ch oma og aph (Wilming on, DE, USA) using a DB-5HT used silica
capilla y column, c oss-linked and bonded phase (5%
phenyl-me hylpolysiloxane; 30 m ×0.25 mm i.d., 0.10
μ
m ilm hick-
ness) (J&W Scien i ic, Folson, CA, USA). The ini ial o en empe a u e
was 120 ◦C, which was inc eased o 380 ◦C a a a e o 1 ◦C/min and held
o 80 min. Injec o and de ec o empe a u es we e 280 ◦C and 370 ◦C,
espec i ely. Injec ions we e pe o med in spli mode (1:20 o 1:5 in he
case o diges ed samples) using ni ogen a a low a e o 1 mL/min.
Quan i ica ion o each ca bohyd a e was pe o med by calib a ion
using phenyl-β-glucoside (0.5 mg mL-1) as an in e nal s anda d. Mix-
u es o s anda d solu ions o glucose, galac ose, uc ose, melibiose,
a inose, and s achyose (Sigma Ald ich; S enheim, Ge many) we e
p epa ed in he expec ed concen a ion ange o calcula e esponse
ac o s o each s anda d ca bohyd a e. All analyses we e pe o med in
duplica e. Da a acquisi ion was e alua ed using Agilen ChemS a ion
so wa e (Wilming on, DE, USA).
3. Resul s and discussion
3.1. Ca bohyd a e composi ion o he soy-based be e ages
GC-FID analysis was ca ied ou in o de o cha ac e ize ei he he
comme cial soy-based samples and he lab-p epa ed soy-based sample
s udied in his manusc ip . Mo eo e , his analy ical me hodology was
used as a sc eening o he eac ion p oduc s o e ime in o de o depic
he enzyme ac i i y e iciency and eco e y o ei he he soluble enzyme
o m o wi hin hei immobiliza ion suppo s.
Fig. 1 shows he ch oma og ams o wo comme cial soy-based be -
e ages, samples K and SSA, wi h di e en ca bohyd a e con en s. As can
be seen, he quan i iable ca bohyd a es we e suc ose, a inose and
s achyose. Suc ose was he main ca bohyd a e ound in sample K,
Fig. 1. GC-FID ch oma og ams o wo comme cial soy-based be e ages, a) sample K, b) sample SSA (wi hou added suga ).
L. Al es dos San os e al.
LWT 210 (2024) 116864
4
ollowed by s achyose and a inose. In gene al, his pa e n was ound
in all analyzed soy be e ages (Table 1). Simila ly, high le els o suc ose
we e ound in A, VS, P and SCa, whe eas i was e y low in sample SSA
(Fig. 1) in acco dance o hei comme cial label (Table S1). The la e
was labelled as ha ing no added suga s, so his soy-based d ink may
e lec he p opo ion o ca bohyd a es o iginally p esen in soybeans.
Conside ing RFOs, s achyose was he dominan ca bohyd a e in all
samples. and he o al amoun o RFOs anged om 13.22 o 548.87 mg/
100 mL. Rada-Mendoza and co-wo ke s (Rada-Mendoza, Villamiel,
Ramí ez, Usu iaga, & Mon illa, 2022) analyzed h ee samples o
soy-based be e ages and he ange o RFOs was wi hin he alues e-
po ed in ou wo k (i.e., be ween 30 and 274 mg/100 mL).
While some o he soy-based be e ages samples exhibi ela i ely
low RFO alues (samples SA, SL, SCa and SSA; 13.22–23.36 mg/100
mL), he samples namely: P, VS, A, K and L, s ill show high quan i a i e
alues o 406.34–548.87 mg/100 mL acco ding o he ca bohyd a e
composi ion in Table 1. The RFO concen a ions o he la es samples
may lead o la ulence and in es inal issues since humans and mono-
gas ic animals lack he enzyme esponsible o hei hyd olysis in he
small in es ine (Sanyal e al., 2023). The e o e, enzyma ically emo ing
hese oligosaccha ides is key o allowing he elabo a ion o soy-based
p oduc s wi h enhanced nu i ional alue while educing hei an i-
nu i ional and la ulence-causing compounds (Elango e al., 2022).
3.2. S udy o soluble MelA
α
-D-galac osidase
Since he pu pose o his s udy was o sugges an enzyme hyd o-
lyza ion o an inu i ional ac o s o soy-based be e ages such as
α
-ga-
lac osides, he s udy o soluble MelA enzyme was ca ied ou . The
α
-D-
galac osidase enzyme om L. plan a um WCFS1 is an enzyme wi h a
ime ic s uc u e. I is composed o 80 kDa monome s wi h a o al mass
o 240.5 kDa (Panwa , Shubhashini, Chaudha i, P ashan h, & Kapoo ,
2020). This enzyme ga e a hyd oly ic ac i i y o 484 U/mg when he
syn he ic subs a e o-NPG was used. Howe e , he enzyme ac i i y was
highly a iable depending on he eac ion medium in which i was used.
The e o e, he enzyme ac i i y was measu ed a e dilu ion in media
wi h di e en bu e s and in he p esence o di e en addi i es, as
shown in Table 2.
This a iabili y in ca aly ic ac i i y is cha ac e ized by he mul i-
me ic na u e o he enzyme, wi h loss o ca aly ic ac i i y in media o
lowe pola i y. The p esence o an addi i e such as glyce ol had a e y
posi i e e ec on he s abili y o he enzyme s uc u e. The op imal
condi ions, bo h o he p oduc ion o he enzyme and o i s subsequen
use o immobiliza ion, we e dilu ion in phospha e bu e wi h a con-
cen a ion anging om 25 o 150 mM and 15% glyce ol.
3.3. Immobiliza ion o MelA
α
-D-galac osidase
The u iliza ion o enzyme immobiliza ion on o di e en suppo s
se es he pu pose o s abilizing he enzyme while p ese ing i s enzy-
ma ic ac i i y. Ac i a ed aga ose suppo s wi h di e en unc ional
g oups, mono- and he e o- unc ional, we e used o immobiliza ion o
he enzyme. Table 3 p o ides he esul s o he me ics calcula ed
ega ding he IE (%) - Eq. (1) and he R (%) - Eq. (2). Immobiliza ion is
achie ed by he eac ion o he suppo wi h di e en eac i e g oups on
he amino acids o he enzyme and he e o e by di e en egions o he
su ace whe e hese amino acids a e mo e abundan . The use o di e en
suppo s can p oduce ca alys s wi h di e en p ope ies om hose o
he soluble enzyme in e ms o ac i i y, s abili y and selec i i y (Ma eo,
Palomo, Fe nandez-Lo en e, Guisan, & Fe nandez-La uen e, 2007).
The bes suppo s o immobiliza ion we e hose ac i a ed wi h
g oups capable o immobilizing he enzyme by anion exchange mech-
anisms (TEA-CHO and EDA-glu a aldehyde) and hose ac i a ed wi h
me al g oups. In he case o me als, his is no mal conside ing ha he
enzyme is labelled wi h 6 his idine esidues pe subuni . The ac i i ies
ob ained e ained almos unchanged he ini ial ac i i y o e ed, excep
in he case o suppo s ac i a ed wi h Cu
2+
, whe e hal o he ac i i y
o e ed was los a e he immobiliza ion p ocess. The high ac i i y
eco e ed in he i s s age o he immobiliza ion indica es ha he e
would be no mass ans e limi a ions. This is consis en wi h no ha ing
ca alys s wi h he maximum possible suppo load.
The s abili y o he mos ac i e de i a i es was also s udied in
Table 2
– Ac i i y o he soluble MelA in di e en bu e s. Dilu ion (1:5). 100 % ac i i y was conside ed as he undilu ed enzyme (n =3).
Composi ion o he bu e Rela i e ac i i y (%)
Sal Concen a ion (mM) Sal Concen a ion (mM) Addi i e % –
Phospha e 50 NaCl 20 – – 1.27
Phospha e 50 NaCl 75 – – 19.3
Phospha e 50 NaCl 150 – – 50
Phospha e 50 NaCl 300 – – 53
Phospha e 50 NaCl 500 – – 40
Phospha e 50 NaCl 150 Glice ol 5 % 84
Phospha e 50 NaCl 150 Glice ol 10 % 85
Phospha e 50 NaCl 150 Glice ol 15 % 98
Phospha e 50 NaCl 20 Glice ol 15 % 95
Phospha e 25 – – Glice ol 15 % 95
Phospha e 25 – – Glice ol 15 % 68
Phospha e 25 – – Glice ol 25 % 68
Phospha e 25 – – Glice ol 50 % 78
Phospha e 5 – – PEG 15 % 2
Phospha e 5 – – PEG 25 % 5
Phospha e 5 – – PEG 50 % 50
MOPS 50 NaCl 20 – – 30
MOPS 50 NaCl 75 – – 56
MOPS 50 NaCl 150 – – 75
MOPS 50 NaCl 300 – – 78
Table 3
Immobiliza ion o MelA
α
-D-galac osidase om L. plan a um WCFS1 on
di e en aga ose ac i a ed suppo s. The o e ed ac i i y was 75 U pe g am o
suppo (n =3).
Suppo Immobilized enzyme (%) Reco e ed ac i i y (%)
TEA 100 100
IDA 10 38
Glyoxyl 10 0
IDA (Cu) 80 49
IDA (Zn) 100 100
IDA – glu a aldehyde 0 0
EDA – glu a aldehyde 100 100
IDA (Zn) – glu a aldehyde 100 100
L. Al es dos San os e al.
LWT 210 (2024) 116864
5
compa ison wi h ha o he enzyme in soluble o m. The di e en
p epa a ions we e incuba ed a 29 ◦C. Unde hese condi ions, he hal -
li e o he soluble enzyme was less han 30 min (Fig. 2). The mos s able
de i a i es we e hose immobilized on suppo s ac i a ed wi h EDA-
glu a aldehyde. Howe e , he low s abiliza ion ac o s (a ound 2- old
inc ease) sugges ha he qua e na y s uc u e o he enzyme was no
ully s abilized. This is o be expec ed as i is a mul ime ic enzyme and i
is some imes di icul o all subuni s o be co alen ly bound o he
suppo su ace.
Wi h his in mind, a pos -immobiliza ion ea men was ca ied ou .
This ea men consis ed o c oss-linking he immobilized ca alys s on
he op imal suppo s wi h di e en poly unc ional polyme s capable o
co alen ly in e ac ing wi h he di e en amino acids p esen on he
p o ein su ace. This ype o ea men is able o p omo e he co alen
immobiliza ion o all he subuni s p esen in di e en mul ime ic p o-
eins. The conse ed ac i i y was 35% and 15% when he de i a i es
we e c oss-linked wi h dex an-aspa ic acid and dex an-glycine,
espec i ely. The di e en c oss-linked de i a i es we e incuba ed a
di e en empe a u es and hei ac i i y was compa ed bo h wi h ha o
he soluble enzyme and wi h ha o he non-polyme ized de i a i e.
A e incuba ion a 29 ◦C, he ac i i y o he c oss-linked de i a i es
emained unchanged o 24 h. Inc easing he empe a u e up o 35 ◦C
p omo ed he inac i a ion o he de i a i e c oss-linked wi h dex an-
glycine polyme s, whe eas hose c oss-linked wi h dex an-aspa ic
acid e ained 85% o hei ini ial ac i i y a e 24 h a his empe a-
u e (Fig. 3).
3.4. Elimina ion o RFOs in soy-based be e ages by ee and immobilized
MelA
α
-D-galac osidase
The capabili y o MelA
α
-D-galac osidase om L. plan a um WCFS1
o hyd olyse single bu e ed solu ions o RFOS has al eady been
demons a ed (Delgado-Fe nandez e al., 2020), as well as he e icien
capaci y o syn hesize
α
-GOS (Delgado-Fe nandez e al., 2021). How-
e e , as a new ba ch o enzyme was used in his wo k, he hyd oly ic
capaci y o he enzyme was again es ed on a inose. This isaccha ide
was clea ed a he melibiose moie y, eleasing suc ose and galac ose,
achie ing 82% deg ada ion a e 24 h o eac ion.
A e es ing he hyd oly ic ac i i y o he enzyme, he ollowing
assays we e pe o med o s udy he hyd olysis o RFOS p esen in he
soy-milk be e age using soluble MelA
α
-D-galac osidase om
L. plan a um WCFS1. Conside ing he composi ion o di e en soy-based
be e ages, sample L showed he highes con en o RFOs whose alue is
impo an o compa a i e pu poses a e enzyme hyd olysis o hese
an inu i ional ac o s (Table 1). The pH analysis o he samples was
c ucial p io o conduc ing he eac ions, as he pH needed o be op i-
mized o ei he he enzyma ic ac i i y o MelA and o each
immobiliza ion suppo . The ca bohyd a e e olu ion esul ing om he
hyd olysis o he RFOs in he soy-based be e age sample ea ed wi h
he ee enzyme o m is shown in Fig. 4. In he un ea ed soy-based
be e age, he quan i iable ca bohyd a es we e suc ose, a inose, s a-
chyose and mino monosaccha ides such as uc ose, glucose and
galac ose.
Du ing he incuba ion imes s udied, comple e disappea ance o
s achyose and a inose was obse ed a e 1 and 3 h o ea men wi h
ee soluble MelA, espec i ely, oge he wi h a concomi an inc ease in
suc ose and elease o galac ose as a esul o hyd olysis o hese oli-
gosaccha ides, whe eas glucose (≈20 mg/100 mL) and uc ose (≈10
mg/100 mL) le els emained ai ly s able o e he cou se o he eac ion
ime. S achyose was comple ely hyd olyzed a e 1 h o eac ion, while
a inose unde wen 53% deg ada ion, eaching almos comple e
deg ada ion (96%) a e 3 h o eac ion and no ace could be de ec ed
a e 5 h o eac ion. A simila hyd olysis pa e n was obse ed o all soy
be e ages desc ibed in Table 1.
Many
α
-D-galac osidases ha e been in es iga ed o he emo al o
RFOs om soy-based be e ages. Go e and cowo ke s (Go e, Umalka ,
Khan, & Khi e, 2004) success ully hyd olyzed a inose and s achyose
p esen in soy be e ages a 65 ◦C wi hin 2 h o ea men using a he -
mos able
α
-D-galac osidase om Bacillus s ea o he mophilus (NCIM 51
46). P ashan h and Mulimani (P ashan h & Mulimani, 2005) achie ed
93% deg ada ion o RFOs in a soy-based be e age a 50 ◦C a e 12 h o
eac ion using a ungal
α
-D-galac osidase om Aspe gillus o yzae. How-
e e , ea men o soy d ink wi h an
α
-D-galac osidase isola ed om
Bacillus mega e ium VHM1 esul ed in comple e hyd olysis o RFOs om
Fig. 2. The mal s abili y o he di e en MelA
α
-D-galac osidase p epa a ions.
Samples we e incuba ed a 29 ◦C. (n =3) ◆: IDA-Cu; ▴: soluble enzyme; Δ:
IDA-Zn; ●: IDA-Cu; □: IDA-Zn-glu a aldehyde; ■: EDA-glu a aldehyde.
Fig. 3. The mal s abili y o di e en MelA
α
-D-galac osidase p epa a ions (n =
3). ◆: soluble enzyme; ■: immobilized on glu a aldehyde suppo s; ●:
immobilized on EDA-glu a aldehyde suppo s and ea ed wi h dex an-glycine;
▴: immobilized on EDA-glu a aldehyde suppo s and ea ed wi h dex an-
aspa ic acid.
Fig. 4. Reac ion cou se o L soy-based be e age ea ed wi h ee MelA (n =2).
Δ: Ra inose; ■: S achyose; ●: Suc ose;
○: F uc ose; ◆: Glucose; ▴: Galac ose.
L. Al es dos San os e al.
LWT 210 (2024) 116864
6
soy-based be e age wi hin 1.5 h o incuba ion a 55 ◦C and pH 7 (Pa il
e al., 2010). T ea men o soy-based be e age wi h an
α
-D-galac osidase
om Bacillus mega e ium 3–7 esul ed in he comple e emo al o a i-
nose and s achyose a e 4 h o eac ion (Huang e al., 2018). Using a
ecombinan
α
-D-galac osidase om I pex lac eus exp essed in Pichia
pas o is o hyd olyse RFOs in soy d ink, Jang and cowo ke s (Jang e al.,
2019) achie ed almos comple e hyd olysis o a inose and s achyose a
60 ◦C in 30 min. In all cases, he op imum empe a u e o
α
-D-ga-
lac osidases o hyd olyse he RFOs was conside ably highe han ha
used in ou wo k o MelA ac i i y. In he p esen s udy, he enzyme is
ac i e a 20 ◦C, which is o g ea in e es om he poin o iew o i s
applica ion in indus y. Ou esul s a e in ag eemen wi h hose ob ained
by Wang e al. (Wang e al., 2020) who, using an
α
-D-galac osidase gene
om Aspe gillus o yzae o e exp essed in Pichia pas o is, achie ed 97.8%
hyd olysis o RFOs a e 3 h o incuba ion a 25 ◦C and wi h he highe
enzyme concen a ion s udied. Ou esul s con i m p e ious indings
indica ing a g ea e p e e en ial ac i i y o
α
-D-galac osidases owa ds
s achyose han a inose as subs a e.
Conce ning he hyd olysis o RFOs wi h immobiliza ion o MelA
α
-D-
galac osidase, expe imen s we e pe o med a he same condi ions as o
he soluble o ms. The beha io o he immobilized enzyme was e y
simila o ha o he soluble one, eleasing galac ose and suc ose
h oughou he eac ion. As in he soy-based d ink ea ed wi h ee
soluble MelA, s achyose was comple ely hyd olyzed a e 1 h o eac ion,
and a his ime a inose was 78% hyd olyzed, eaching comple e
deg ada ion (100%) a e 3 h o eac ion. In his case, a inose deg a-
da ion was as e han in he soluble enzyme assays. I was ound a 79%
and 66% educ ion o RFOs by ee and immobilized
α
-D-galac osidase
om Gibbe ella ujiku oi, espec i ely, om soy d ink a e 3 h o incu-
ba ion (Thippeswamy & Mulimani, 2002). Using he soluble and
immobilized
α
-D-galac osidase om Aspe gillus o yzae, P ashan h and
Mulimani (P ashan h & Mulimani, 2005) achie ed 93% and 81%
deg ada ion o RFOs, espec i ely, in soy d ink a 50 ◦C a e 12 h.
Ka olia e al., 2019; Ka olia e al., 2019) s udied he elimina ion o
RFOs om soy-based be e ages using he soluble and immobilized
α
-D-galac osidase om Aspe gillus o yzae o e calcium-algina e and
chi osan. A e 4 h o incuba ion, he ee enzyme hyd olyzed only 30%
o he RFOs, whe eas he algina e-immobilized and
chi osan-immobilized enzyme hyd olyzed 97.6% and 93.7% o he
RFOs, espec i ely.
α
-D-galac osidase cloned om Bac e oides he aio-
aomic on and exp essed in Esche ichia coli (iB _3292) immobilized on
algina e suppo s esul ed in he consump ion o 98.9% o he RFOs
p esen in soy d ink a e 48 h o incuba ion (Shin e al., 2020).
The esul s ob ained in he p esen s udy show ha ei he in ee o
immobilized o m,
α
-D-galac osidase om L. plan a um WCFS1 p o-
duced a high pe cen age o hyd olysis o RFOs in soy-based be e age in a
sho pe iod o ime and unde mild empe a u e condi ions (20 ◦C).
The e o e, he immobilized enzyme could be used on an indus ial scale
o hyd olyse indiges ible RFOs in soy-based be e ages, making he
p ocess cheape .
3.5. Reusabili y o he immobilized
α
-D-galac osidase
One o he majo limi a ions associa ed wi h he indus ial applica-
ion o ee enzymes is hei high cos and ope a ional ins abili y.
The e o e, om an economic and sus ainable poin o iew, he he -
e ogeneous bioca alys mus be eused o imp o e he p oduc i i y o
he p ocess. Fo his pu pose, he ca alys was eused in successi e e-
ac ion cycles and i s hyd oly ic capaci y was e alua ed. A e 10 e-
cycles, he deg ee o hyd olysis o bo h s achyose and a inose was
s able, abo e 80% and 98%, espec i ely (Fig. 5). These esul s align
wi h hose epo ed by Çelem and ¨
Onal (2022) and Falkoski e al. (2006)
Fig. 5. E ec o he mul iple use o immobilized, on suppo o aga ose-EDA-glu a aldehyde, MelA
α
-D-galac osidase om L. plan a um WCFS1 in di e en eac ion
cycles (n =1). The eac ion was pe o med using (0.450 mL) o a comme cial soy-based be e age, 5 U/g o immobilized enzyme, a 25 ◦C. The ini ial concen a ion o
each ca bohyd a e was, espec i ely, 14.61 and 23.51 mg/100 mL, o a inose and s achyose.
L. Al es dos San os e al.
LWT 210 (2024) 116864
7
o non-immobilized
α
-galac osidases, whe e he deg ee o hyd olysis
o s achyose and a inose anges om 78% o 100%. This demons a es
he high e iciency o he a ious immobiliza ion suppo s used in his
s udy and highligh s he po en ial o enzyme eusabili y.
4. Conclusions
Conside ing he an inu i ional ac o s o soy-based be e ages and
he impo ance o emo ing he esponsible RFOs o his e ec , he
indus y conside s a ious me hodologies o he ea men o hese
seeds including ei he con en ional o non-con en ional p ocedu es. To
he bes o ou knowledge, his is he i s s udy on he ca bohyd a e
composi ion o soy-based be e ages on he Spanish ma ke and he
applica ion o MelA
α
-D-galac osidase om L. plan a um WCFS1 o he
emo al o RFOs. The ac i i y is s ongly dependen on he enzyme
concen a ion and he eac ion medium. Fo his eason, his wo k also
p oposes a ious suppo s o immobilizing he enzyme in o de o
s abilize he enzyme o i s use in he eac ions. MelA
α
-D-galac osidase
om L. plan a um WCFS1 showed speci ic ac i i y and high hyd oly ic
e iciency owa ds RFOs in soy-based be e ages a a ela i ely low
empe a u e (20 ◦C). The e o e, hese esul s sugges ha his enzyme
could be p e e en ially used in he ea ly s age o soybean p ocessing o
emo e RFOs om soy-based be e ages and soy-de i ed p oduc s,
he eby imp o ing hei nu i ional alue. Compa ed o o he
α
-D-ga-
lac osidases, MelA om L. plan a um WCFS1 has g ea indus ial po-
en ial o he emo al o RFOs om soy d inks and ela ed oods.
Aga ose enzyme immobilized ca alys s a e e y use ul especially o
s i ed ank eac o s, and conside ing ha he eac ions would be ca ied
ou a oom empe a u e (20 ◦C), as well as he low ca alys olume/
eac ion olume a io, he e would be no majo hea ans e p oblems.
Despi e hese good p ope ies, he limi a ion o hese ca alys s would
be hei use a high empe a u es due o he high ins abili y o he sol-
uble s a ing enzyme.
CRediT au ho ship con ibu ion s a emen
Leand o Al es dos San os: So wa e, Me hodology, In es iga ion,
Fo mal analysis. Paloma Delgado-Fe n´
andez: In es iga ion, Fo mal
analysis. Ana Mu˜
noz-Lab ado : Fo mal analysis. Diego Ma in-
Gu ie ez: In es iga ion. E elyn C. Rome o: In es iga ion. F anzo y
Cua an: In es iga ion. And ea Ga cia-Al a ez: In es iga ion. Elena
Ga cía-Cal o: In es iga ion. Blanca de las Ri as: Resou ces. Rosa io
Mu˜
noz: Resou ces. Nie es Co zo: W i ing – o iginal d a , Supe ision.
F. Ja ie Mo eno: W i ing – e iew & edi ing, Supe ision. Cesa
Ma eo: W i ing – e iew & edi ing, Valida ion, Supe ision,
Concep ualiza ion.
Decla a ion o compe ing in e es
The au ho s decla e ha hey ha e no known compe ing inancial
in e es s o pe sonal ela ionships ha could ha e appea ed o in luence
he wo k epo ed in his pape .
Da a a ailabili y
Da a will be made a ailable on eques .
Acknowledgemen s
This wo k has been inanced by he Spanish Minis ies o Economy,
Indus y and Compe i i eness (P ojec s AGL2017-84614-C2-1-R and
AGL2017-84614-C2-2-R; PID2021-123862OB-I00. Paloma Delgado-
Fe n´
andez hanks o Minis y o Science, Inno a ion and Uni e si ies o
Spain o p o iding FPI p edoc o al ellowship. The au ho s also wish o
acknowledge he unding om Eu opean Commission, p ojec “Twin-
ning o in ensi ied enzyma ic p ocesses o p oduc ion o p ebio ic-
con aining unc ional ood and bioac i e cosme ics “g an no.
101060130, HORIZON-WIDERA-2021-ACCESS-02-01. Resea ch schol-
a ship was g an ed o Leand o Al es dos San os by CAPES - Coo denaç˜
ao
de Ape eiçoamen o de Pessoal de Ní el Supe io . (Finance code
88887.750039/2022-00).
Appendix A. Supplemen a y da a
Supplemen a y da a o his a icle can be ound online a h ps://doi.
o g/10.1016/j.lw .2024.116864.
Re e ences
Amigo-Bena en , M., Sil ´
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