Developing and improving enzyme-driven technologies to synthesise emerging prebiotics

G een Chemis y
TUTORIAL REVIEW
Ci e his: G een Chem., 2025, 27,
8777
Recei ed 8 h Ap il 2025,
Accep ed 12 h May 2025
DOI: 10.1039/d5gc01723h
sc.li/g eenchem
De eloping and imp o ing enzyme-d i en
echnologies o syn hesise eme ging p ebio ics
Noelia Losada-Ga cia,
b
Milica Simo ić,
c
Ma ija Ćo o ić,
c
Ana Mili oje ić,
c
Nikola Nikače ić,
c
Cesa Ma eo,*
a
Dejan Bezb adica*
c
and Jose M. Palomo *
a
Eme ging p ebio ics, mainly oligosaccha ides and phenolic compounds, a e gaining inc easing a en ion
in he scien ific communi y owing o hei heal h benefi s and b oad indus ial po en ial. P ebio ics a e
widely used in oods, cosme ic o mula ions and die a y supplemen s. Eme ging p ebio ics offe
addi ional ad an ages as hey can be de i ed om low-cos , enewable ma e ials and p oduced sus ain-
ably, in line wi h he p inciples o a ci cula economy. G een echnological app oaches, in eg a ing expe -
ise om diffe en scien ific disciplines, will be essen ial o de elop efficien and en i onmen ally iendly
me hods o he p oduc ion o eme ging p ebio ic-en iched p oduc s. This e iew p o ides a comp ehen-
si e o e iew o he ad ances in his field, highligh ing he ad an ages and op imisa ion o enzyme-based
ca alysis. Insigh s in o how enzymes enhance he con ol o oligosaccha ide p oduc ion by enabling he
selec i e syn hesis o egioisome s wi h desi ed chain leng hs and modifica ion o phenolic p ebio ics a e
p o ided. Fu he mo e, diffe en echnologies o imp o e bioca alys s o con ibu e o he no el biop o-
cess in ensifica ion s a egies applicable o eme ging p ebio ic p ocessing a e elucida ed.
G een ounda ion
1. We discussed g eene me hods o eplace complex and edious chemical p ocesses in he syn hesis o eme ging oligosaccha ides wi h p ebio ic ac i i ies
ia ca aly ic con e sion using enzymes. This app oach allows hei syn hesis in one-s ep in aqueous media and a low o mode a e empe a u es.
2. P ebio ics ha e a c i ical effec as heal h-p omo ing agen s, wi h a global ma ke es ima ed o be $13 billion by 2030. Thei ole is based on hei bene icial
effec s on he hos , s imula ing he g ow h o “good”bac e ia and inhibi ing “bad”bac e ia. They ha e been e alua ed as nondiges ible ood ing edien s and,
mo e ecen ly, o he ea men o skin p oblems.
3. Al hough some p omising ca alys s ha e been de eloped o he syn hesis o p ebio ics, he use o enzymes as ca alys s offe s signi ican ad an ages owing
o hei selec i i y and speci ici y, wi h mo e sus ainable and efficien syn hesis app oaches. This e iew will discuss me hodologies o imp o e he inal bio-
ca aly ic p ocess.
1. In oduc ion
P ebio ics a e s able chemical molecules, mainly ca bohyd a es,
which ha e c i ical effec s as heal h p omo ing agen s.
1
They
bene icially affec he hos by selec i ely s imula ing he g ow h
and/o ac i i y o one o a limi ed numbe o bene icial bac e ia
in hei colon, hus imp o ing he hos heal h and, in he ideal
case, inhibi ing pa hogenic bac e ia.
2
Cu en a ge s o p ebio-
ics ha e now expanded beyond he ypical p obio ic gene a
Lac obacillus and Bi idobac e ium o a wide ange o mic obial
esponde s.
3
Simila o p obio ics, hese include candida e
heal h-p omo ing gene a such as Rosebu ia spp., Eubac e ium
spp., Akke mansia spp., Ch is ensensella spp., P opionibac e ium
spp., and Faecalibac e ium spp. A signi ican heal h-p omo ing
bene i o hese gene a is hei capaci y o p oduce sho -
chain a y acids (SCFAs), which ha e been demons a ed o
egula e a a ie y o unc ions wi hin he gas oin es inal
sys em, including he unc ions o gu epi helial cells and
mucus ba ie , immuni y, in lamma ion, glucose and lipid
me abolisms, ene gy expendi u e, and sa ie y.
4
Pa icula ly,
hey ha e been ex ensi ely e alua ed as nondiges ible ood
ing edien s,
2,3,5,6
bu ecen ly, hei ole in he ea men o
illnesses such as pso iasis, a opic de ma i is, skin heal h,
colon cance , COVID, bone heal h and calcium abso p ion
has been iden i ied.
7–11
Thus, hey ha e been highligh ed o
hei ele ance in he a ea o p ebio ics.
The global p ebio ics ma ke , es ima ed a $6 billion in
2022, is o ecas ed o each a e ised size o $13.8 billion by
2030, g owing a a CAGR (compound annual g ow h a e) o
a
Ins i u o de Ca álisis y Pe oleoquímica (ICP), CSIC, C/Ma ie Cu ie 2,
28049 Mad id, Spain. E-mail: josem[email p o ec ed], [email p o ec ed]
b
BioISI—Biosys ems and In eg a i e Sciences Ins i u e, Facul y o Sciences, Uni e si y
o Lisboa, Campo G ande, C8, 1749-016 Lisboa, Po ugal
c
Facul y o Technology and Me allu gy, Uni e si y o Belg ade, Ka negije a 4,
11000 Belg ade, Se bia. E-mail: [email p o ec ed].ac. s
This jou nal is © The Royal Socie y o Chemis y 2025 G een Chem.,2025,27, 8777–8803 | 8777
Open Access A icle. Published on 14 May 2025. Downloaded on 11/19/2025 9:38:26 PM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
View Jou nal
| View Issue
11% o e he analysis pe iod o 2022–2030. Conside ing he
ongoing pos -pandemic eco e y, he g ow h o he galac o-
oligosaccha ide (GOS) segmen has been adjus ed o a e ised
CAGR o 12.1% o e he nex 8 yea s.
11
In ecen imes, a ange o compounds classi ied as p ebio-
ics has expanded beyond he “es ablished p ebio ics”(inulin
and galac o- and uc o-oligosaccha ides (GOS and FOS,
espec i ely)) o “eme ging p ebio ics”, comp ising new ca bo-
hyd a e-based molecules
12–14
such as xylooligosaccha ides
(XOS),
15–20
isomal ooligosaccha ides (IMO),
21,22
pec in oligo-
saccha ides (POS),
23–25
mannooligosaccha ides (MOS),
26
chi -
ooligosaccha ides (CHOS),
27
lac osuc ose, affinose, epilac ose,
and glucomannans.
12
Fu he mo e, as he la es de ini ion o p ebio ics includes
skin mic obio a as he a ge o hei ac i i y,
3
non-ca bo-
hyd a e subs ances, such as polyphenols and o he phenolic
compounds, ha e been iden i ied as candida es o exe p e-
bio ic effec s on he hos .
3,12,28
Since hey a e de i ed om
plan -based ma e ials o by-p oduc s gene a ed du ing ood
and be e age p ocessing, hey align well wi h cu en ma ke
ends and he p inciples o a ci cula economy. The e o e,
esea ch on eme ging p ebio ics, including hei syn hesis
and/o ex ac ion and modi ica ion, in i o models o con i -
ma ion o hei p ebio ic ac i i y, g een p ocessing and inco -
po a ion in ood p oduc s, is a mul idisciplina y esea ch
opic. Owing o he impo ance o his opic, he numbe o
publica ions has inc eased o e he las 20 yea s. In ac , he e
has been an exponen ial inc ease in he numbe o publi-
ca ions on his opic o e he las 10 yea s (Fig. 1).
Eme ging p ebio ics a e a an ea lie de elopmen s age
han es ablished p ebio ics, acing challenges in hei disco -
e y, es ing, and applica ion. Ensu ing esis ance o gas ic
diges ion is he key o p ebio ics o each he colon and be e -
men ed by gu mic obio a, which depends on hei molecula
s uc u e. Fo ins ance, long-chained IMO is mo e esis an o
deg ada ion in he uppe GI ac , making i mo e po en han
sho -chained IMO.
29
Selec i ely s imula ing bene icial bac e ia, such as
Bi idobac e ium and Lac obacillus, is also c ucial, gi en ha
diffe en p ebio ics can impac he mic obial composi ion
diffe en ly. Fo example, XOS was ound o s imula e
Bi idobac e ia g ow h mo e effec i ely han FOS in a s.
30
Howe e , alida ing he efficacy o p ebio ics is challenging
due o a ia ions in mic obiomes and me abolic esponses,
al hough new e alua ion me hods such as dynamic in i o
diges ion and ecal e men a ion expe imen s a e helping.
Despi e hese challenges, ongoing esea ch is unco e ing no el
p ebio ics ha show esis ance o diges ion and p omo e
heal h bene i s, bo h in i o and in i o.
31
One o he mos in e es ing aspec s is he p epa a ion o oligo-
saccha ides, which can be p oduced ei he ia he op–down
app oach (hyd olysis o he pa en polysaccha ides o oligosac-
cha ides) o bo om-up app oach (syn hesis o oligosaccha ides
om simple suga s) (Fig. 2). The i s app oach makes i possible
o p oduce hese molecules om diffe en sou ces by hyd olysis,
especially om was e om he ag i- ood o ma ine indus y, ligno-
cellulosic was e,
32
seaweeds,
33
and c us acean was e
34
(Fig. 2). In
hese cases, he p oduc ion o ca bohyd a es in ol es ea men
wi h s ong acids, whe eas he use o enzymes allows hei p epa-
a ion in aqueous media unde mild condi ions, and he e o e a
highe deg ee o pu i y wi h espec o o he by-p oduc s.
The enzyma ic hyd olysis o polysaccha ides se es a dual
pu pose in he p oduc ion o eme ging p ebio ics. I no only
acili a es oligosaccha ide gene a ion bu also ac s as a ool o
b eaking down he cell wall in plan s, enhancing he ex ac ion
o phenolic compounds, ano he class o eme ging p ebio ics,
h ough a g een me hod commonly known as enzyme-aided o
enzyme-assis ed ex ac ion.
35
Fig. 1 Numbe o publica ions pe yea on p ebio ics. Sou ce: Scopus.
Tu o ial Re iew G een Chemis y
8778 |G een Chem.,2025,27, 8777–8803 This jou nal is © The Royal Socie y o Chemis y 2025
Open Access A icle. Published on 14 May 2025. Downloaded on 11/19/2025 9:38:26 PM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
The second app oach, a syn he ic me hod, s a s om
single building blocks (Fig. 2), whe e diffe en chemical glyco-
syla ion eac ions (using me al ca alys s, base, e c.) ha e been
desc ibed.
36
The e sa ili y o chemical p ocesses allows he
selec i e p oduc ion o a ious molecules such as di-, i- and
e a-saccha ides, bu makes he p oduc ion o la ge com-
pounds mo e difficul .
35,36
In his sense, he use o enzymes can o e come his
d awback,
37–42
p oducing diffe en egioisome s o a gi en
chain leng h wi h high selec i i y and acili a ing he o -
ma ion o la ge oligome s mo e easily (Fig. 2).
To da e, mos o hese compounds on he ma ke a e based
on a mix u e and hei p ebio ic efficacy has been e alua ed in
a gene al way, ob iously conside ing he ques ion o p ice-pe -
o mance a io.
The e o e, se e al issues such as inal o e all yield o p o-
duc s, was e p oduc ion, numbe o syn he ic s eps (based on
p o ec ion and pu i ica ion s eps) can be imp o ed using
enzymes as ca alys s (Fig. 2). The ex ao dina y selec i i y and
speci ici y o enzymes (pa icula ly, glycosidic bond enzymes)
allow hese molecules o be ob ained in one o a ew syn hesis
s eps in aqueous media a mode a e empe a u e, also wi h
high e sa ili y, enabling he same enzyme o be used in
se e al p ocesses. One example is shown in Fig. 2 ega ding
he syn hesis o a hexame -oligosaccha ide.
43
The p oduc was
syn hesized in 94% o e all yield using glycosyl ans e ases (in
ou enzyma ic s eps by using co ac o - ecycling
43
), whe eas
he chemical app oach equi ed mul iple glycosyla ion s eps
(wi h a high numbe o p e ious syn hesized building blocks
p epa ed), gi ing a inal o e all yield o 2.6%.
45
Mo e ecen ly, syn hesis s a egies ha e also been de eloped
whe e enzyma ic and chemical s eps a e combined o ob ain
ailo -made oligosaccha ides.
42,44,45
Thus, a la ge numbe o enzyma ic glycosyla ion p ocesses
has been desc ibed, in which, in addi ion o aking ad an age
o hei excellen na u al p ope ies, s a egies ha e been
applied o imp o e hem by inc easing hei s abili y unde he
eac ion condi ions o hei e sa ili y in ecognizing o he
simila subs a es, o example h ough p o ein enginee ing,
o by imp o ing hei p ope ies and ecyclabili y, hus imp o -
Fig. 2 Ad an ages o he enzyma ic s a egy in he g een syn hesis o p ebio ic oligosaccha ides. (a) Top-down app oach syn hesis, (b) bo om-up
app oach syn hesis, (c) compa ison be ween enzyma ic and chemical app oaches, and (d) example o he syn hesis o a ailo -made bioac i e oligo-
saccha ide using enzyma ic e sus chemical app oach.
G een Chemis y Tu o ial Re iew
This jou nal is © The Royal Socie y o Chemis y 2025 G een Chem.,2025,27, 8777–8803 | 8779
Open Access A icle. Published on 14 May 2025. Downloaded on 11/19/2025 9:38:26 PM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
ing he economic sus ainabili y o he p ocess, which is o
indus ial in e es .
This e iew discusses he majo ad ances in he enzyma ic
syn hesis, modi ica ion and ex ac ion o a ious ypes o
eme ging p ebio ics, as well as con ibu ions in he a ea o
p ope y imp o emen p ocessing and equipmen h ough new
in ensi ied echnologies o he ood and cosme ics indus ies.
2. Enzyme-d i en de elopmen o
eme ging p ebio ics
2.1. Eme ging oligosaccha ide p ebio ics
Cu en ly, GOS, FOS and lac ulose a e he ca bohyd a es wi h
p ebio ic ac i i y con i med by clinical s udies. Howe e , wi h
he expanded de ini ion o p ebio ics, he e is a g owing
demand o he iden i ica ion, p oduc ion and e alua ion o
new p ebio ic ca bohyd a es ha can affec he b oade ange
o bene icial mic oo ganisms and a ge ed hos s.
46
These com-
pounds, such as XOS, POS, MOS, IMO and CHOS (Fig. 3), a e
known as “eme ging”p ebio ics, gi en ha e idence o hei
esis ance o gas ic diges ion and selec i e g ow h s imula ion
o bene icial bac e ia is s ill lacking. These compounds
p esen a g ea scien i ic and echnological challenge due o
hei s uc u al di e si y, a ising om hei complex na u al
subs a es. The la es achie emen s and end owa ds sus ain-
able ans o ma ion o diffe en bioma e ials in his ield will
be p esen ed nex .
XOS a e non-diges ible oligome s consis ing o 2 o10
linked xylose uni s by β-1,4 linkages (Fig. 3a). Na u ally, XOS
a e p esen in small amoun s in ege ables, ui s, honey and
dai y p oduc s. XOS a e conside ed o be compe i i e eme ging
p ebio ics, ideal o inco po a ion in o a ious p oduc s in he
ood and eed sec o s, especially conside ing hei excellen
applica ion p ope ies such as high s abili y in a wide ange o
empe a u es (up o 100 °C) and pH (2.5–8), good swee ening
powe and low calo ic alue.
12
Depending on he xylan sou ces
used o he p oduc ion o XOS, he s uc u es o XOS a e
diffe en in e ms o deg ee o polyme iza ion (DP), mono-
me ic uni s, and ypes o linkages. Gene ally, XOS a e mix u es
o oligosaccha ides o med by xylose linked h ough β-(1 →4)-
linkages.
47
Se e al examples o he syn hesis o XOS om
suga monome s ha e been desc ibed. The enzyme
β-xylosidase is capable o syn hesising a ious alkyl β-xylosides
h ough ansxylosyla ion p ocesses.
48
The β-xylosidase om
A. nige IFO 6662 has s ong ansxylosyl ac i i y and has been
epo ed o p oduce a no el non- educing disaccha ides. The
syn hesis o XOS om β-(1 →4)-xylobiose in he p esence o
D-mannose by ans-xylosyla ion was epo ed, esul ing in he
p oduc ion o wo xylosylmannoses and non- educing XOS
49
(Fig. 4a).
Howe e , in mos cases, XOS a e p oduced using comme -
cially a ailable xylan, bu o enable hei economically iable
p oduc ion, signi ican emphasis has been placed nowadays
on he use o lignocellulosic biomass, which is an abundan
(up o 34% w/w), and mo e impo an ly, cheap sou ce o
xylan.
50
Lignocellulosic ma e ials can be hyd olyzed o XOS
using a combina ion o he mal and chemical p e- ea men s
o deg ade hei complex s uc u e, mainly composed o cell-
ulose, hemicellulose and lignin, ollowed by enzyma ic hyd o-
lysis, as shown in Fig. 4b. To da e, nume ous lignocellulosic
ma e ials (whea and ice s aw, suga cane bagasse, co ncob,
beech and bi ch wood) ha e been s udied o XOS p oduc ion,
wi h a ying efficiencies depending on he amoun and ype o
xylan p esen . Namely, xylan is a biopolyme wi h a β-1,4-
linked xylose backbone wi h α-D-glucopy anu onic acids and/o
L-a abino u anose esidues, and acco dingly can be classi ied
in o one o ou main g oups (homoxylan, glucu onoxylan, a a-
binoxylan, a abinoglucu onoxylan and glucu onoa abinoxylan)
based on he p esence and dis ibu ion o subs i uen s.
26
Fig. 3 Typical s uc u e o eme ging p ebio ics, emphasizing he monome ic uni o each one.
Tu o ial Re iew G een Chemis y
8780 |G een Chem.,2025,27, 8777–8803 This jou nal is © The Royal Socie y o Chemis y 2025
Open Access A icle. Published on 14 May 2025. Downloaded on 11/19/2025 9:38:26 PM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
The e o e, diffe en me hods o XOS p oduc ion and he g ea
di e si y o po en ial xylan subs a es esul in a wide spec um
o diffe en XOS s uc u es wi h diffe en subs i uen s on he
xylose backbone and deg ees o polyme iza ion, which conse-
quen ly ha e a g ea impac on hei p ebio ic and o he unc-
ional p ope ies.
Fo example, Viei a e al.
51
demons a ed ha he ligno-
cellulosic by-p oduc o palm p ocessing (Bac is gasipaes
Kun h) can se e as an excellen subs a e o XOS p oduc ion.
A e mild alkali p e ea men o peach palm was e (inne
shea h and peel), XOS we e ob ained by enzyma ic hyd olysis
using comme cial xylanase om Aspe gillus o yzae wi h XOS
yields om he xylan inne shea h and xylan peel o 50.1% and
48.8%, espec i ely. The ob ained XOS showed excep ional
an ioxidan capaci y, signi ican ly highe han ha ob ained
om comme cial xylan. In he las ew decades, se e al p o-
cesses o XOS p oduc ion ha e been e alua ed, bu his is s ill
an e ol ing ield, whe e he bes me hods, ca alys s and sub-
s a es a e no comple ely clea . The e o e, special e alua ion
o he s uc u e and con en o xylan is o u mos impo ance
o he de elopmen o iable XOS syn hesis p ocesses,
especially in e ms o enzyme selec ion. Endo-1,4-β-xylanases
(EC 3.2.1.8) a e he main enzymes esponsible o he hyd o-
lysis o xylan o XOS. Gi en hei di e si y, some o hem wo k
well on unsubs i u ed xylan, while o he s a e mo e suscep ible
o subs i u ed xylan. Ne e heless, he use o auxilia y enzymes
o emo e xylan subs i uen s such as α-glucu onidases (EC
3.2.1.139), α-a abino u anosidases (EC 3.2.1.55) and ace yl
es e ases (EC 3.1.1.72) has been in es iga ed. An example o
enzyme syne gism is he deg ada ion o oa spel xylan using
α-L-a abino u anosidases om Aspe gillus ho ai, p oduced and
pu i ied om a medium con aining ci us pulp and o ange
peel, and a p e iously desc ibed pu i ied endoxylanase om
he same mic oo ganism. The expe imen s we e ca ied ou a
40 °C using 2% (w/ ) oa spel xylan solu ion in ammonium
ace a e buffe (pH 4.5), and he enzymes we e used indi idu-
ally o in sequen ial eac ions. The esul s showed ha he
p io ac ion o he α-L-a abino u anosidases, which emo ed
he side a abinose subs i uen s and made he main chain
mo e accessible o xylanase ac ion, esul ed in a wo- old
inc ease in he hyd olysis yields achie ed.
52
Howe e , i should
be no ed ha some xylanases show a p e e ence o subs i u ed
xylans, whe e auxilia y enzymes should be included in he
p ocess o gene a e unsubs i u ed XOS and allow hei be e
u iliza ion by a ge mic oo ganisms.
53
Fo example, Zhou
e al. ca ied ou he hyd olysis o ha dwood xylan (gluco onox-
ylan) using The mo oga ma i ima xylanase (XynB) and
α-glucu onidase (AguA) co-exp essed in Esche ichia coli ia
dual-p omo e and bicis onic cons uc s o educe he enzyme
cos s. The α-glucu onidase enabled he emo al o 4-O-me hyl-
D-glucu onic acid esidues om he b anched XOS, esul ing
in an inc ease in he an ioxidan capaci y o he XOS mix u es
p oduced wi h bo h XynB and AguA. In addi ion, hese unsub-
s i u ed XOS a e belie ed o signi ican ly accele a e he g ow h
o some bac e ia om Bi idobac e ium sp.
53
Thus, xylanases a e
excellen ools o ailo p ebio ic oligosaccha ides wi h he
in en ion o s imula ing diffe en ypes o bac e ia om
diffe en niches, in cong uence wi h he newly es ablished
de ini ion o p ebio ics.
54
Recen ly, o achie e be e XOS p o-
duc ion, emendous effo s ha e been in es ed in he de elop-
men o new enzymes wi h imp o ed ac i i y and s abili y by
inc easing he XOS/xylose a io.
Also, selec ed mic oo ganisms ha e been cul i a ed o
p oduce ex acellula xylanases ha would he ea e hyd olyze
xylan o XOS. A new s ain, Bacillus sub ilis KCX006, was ound
o cons i u i ely p oduce endo-xylanase and xylan deb anching
enzymes wi hou β-xylosidase ac i i y in he p esence o ligno-
cellulosic biomass.
55
The e o e, his mic oo ganism was used
o he simul aneous p oduc ion o xylanase and XOS om
lignocellulosic biomass (whea b an, ice b an, ice husk and
suga cane bagasse (SB)) h ough solid-s a e e men a ion. To
p o ide ni ogen sou ces o he g ow h o Bacillus, o ganic and
ino ganic ni ogen sou ces oge he wi h diffe en oil-cakes
(g ound nu oil-cake, sun lowe oil-cake, cas o oil-cake, and
co on seed cake) we e used. Among he a ious subs a es,
whea b an and g oundnu oil-cake suppo ed he highes xyla-
nase and XOS p oduc ion, which unde he op imized con-
di ions yielded 3102 IU g
−1
and 48 mg g
−1
, espec i ely.
Fig. 4 Syn hesis o XOS. (a) Syn hesis using β-xylosidase and (b) enzyme hyd olysis o xylan.
G een Chemis y Tu o ial Re iew
This jou nal is © The Royal Socie y o Chemis y 2025 G een Chem.,2025,27, 8777–8803 | 8781
Open Access A icle. Published on 14 May 2025. Downloaded on 11/19/2025 9:38:26 PM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online

POS (Fig. 3b) ep esen he mos di e se g oup o unc ion-
ally ac i e ca bohyd a es wi hin he g oup o eme ging p ebio-
ics. Nume ous s udies ha e shown he diffe en physiological
ac i i ies o POS mix u es, such as p ebio ic, an ibac e ial,
an icance and an ioxidan p ope ies.
56
The main eason o
his di e si y is he complexi y o he s uc u e o hei mole-
cule o o igin, pec in. This he e opolysaccha ide, which is he
majo cons i uen o he cell wall in highe plan s, consis s o
ou main s uc u al componen s, homogalac u onan (HG),
hamnogalac u onan-I (RG-I), hamnogalac u onan-II (RG-II)
and xylogalac u onan (XG).
57
The mos abundan pec in sub-
s uc u e (mo e han 65%) is homogalac u onan (HG), which
ep esen s a sequence o galac u onic acid esidues wi h α-1,4-
linkages, occasionally es e i ied wi h me hyl (C6) o ace yl
g oups (O2 and O3). The es o he molecule is comp ised o
diffe en monosaccha ides ( hamnose, a abinose, galac ose,
and xylose) and a ious linkages.
56
POS can be p oduced om
pec in ia se e al me hods such as enzyma ic and acid hyd o-
lysis, hyd o he mal ea men me hod o a combina ion o
acid/enzyma ic/hyd o he mal ea men . The choice o
employed me hods is p ima ily dependen o he pec in
sou ce; howe e , he applica ion o enzymes showed he g ea -
es po en ial (Fig. 5).
To da e, pec in om a ious sou ces has been u ilized o
he p oduc ion o POS, especially gi en ha a ious ag icul-
u al by-p oduc s (apple pomace, suga bee pulp, ci us was e,
be y pomace, e c.) ha e been ecognized as cheap and abun-
dan sou ces.
56,57
The enzyma ic hyd olysis p ocedu e is highly
complex and equi es he syne gis ic ac ion o diffe en g oups
o enzymes including hyd olases, es e ases and lyases.
58
The
enzymes esponsible o pec in deg ada ion can be majo ly
di ided in o HG deg ading (polygalac u onase, EC 3.2.1.15,
pec a e lyase EC 4.2.2.2, and pec in lyase EC 4.2.2.10, pec in
me hyl es e ase EC 3.1.1.11, and pec in ace yl es e ase EC
3.1.1.6) and hamnogalac u onan-I (RG-I) deg ading enzymes
(α-a abino u anosidase EC 3.2.1.55, endoa abinase EC
3.2.1.99, β-galac osidase EC 3.2.1.23, endogalac anase EC
3.2.1.89, e uloyl EC 3.1.1.73 and p-couma oyl es e ases EC
3.1.1.B10).
Al hough he e a e examples o newly isola ed enzymes ha
a e u ilized o he syn hesis o POS,
57
mos o he publica ions
p esen ed he esul s o comme cial hyd oly ic enzyme mix-
u es de i ed om Aspe gillus sp.
56
Fo ins ance, Saba e and
co-wo ke s used he comme cial enzyme p epa a ion
Viscozyme® L, mul i-enzyme complex om Aspe gillus aculea-
us, o he hyd olysis o a ichoke pec in in o POS. The enzy-
ma ic p ocess was op imized using an expe imen al design
and u he analyzed by he applica ion o a i icial neu al ne -
wo ks, yielding 65.9% pec in con e sion o POS unde he
op imal condi ions. The s uc u al elucida ion o he ob ained
POS e ealed he p esence o oligosaccha ides DP2–DP6 ( om
dime o oligome s wi h 6 uni s o monome s), which exhibi-
ed s ong adical sca enging ac i i ies.
59
The same enzyme
was u ilized o he con e sion o pec in om indus ial byp o-
duc s, such as lemon peels, suga bee pulp
60
and onion
skin,
61
o o m a unc ionally ac i e POS. The c ude pec ic
ex ac om onion skins, mos ly made o homogalac u onan
wi h e y sca ce hamnogalac u onan egions, ob ained by
sodium hexame aphospha e (SHMP) ex ac ion was p ocessed
u ilizing a con inuous c oss low memb ane bio eac o . The
enzyma ic hyd olysis and in si u memb ane sepa a ion we e
combined o ob ain high yields o ailo -made POS because
he memb ane allows he con inuous emo al o p oduc s o
he a ge ed DP simul aneously, p o ec ing he POS om
u he hyd olysis and monosaccha ide o ma ion, and hence
a oiding enzyme inhibi ion by eac ion p oduc s. The eac ion
esul ed in high POS yields (a ound 60%) p o ided ha
enzyme and subs a e concen a ions we e 41.4 U mL
−1
and
50 g L
−1
, espec i ely. Unde hese condi ions, he highes POS
olume ic p oduc i i y (22.0 g L
−1
h
−1
), as well as he lowes
POS/monosaccha ide a io (4.5 g g
−1
) we e achie ed. Also, i
mus be no ed ha a s able p oduc ion was achie ed o he
whole eac ion pe iod and sho POS (DP2, DP3, and DP4)
we e obse ed as he main eac ion p oduc s.
61
In addi ion o he dominan p ocesses, which include he
u iliza ion o hyd olases, lyases a e o en applied o he syn-
hesis o POS by clea ing he α-1,4-glycosidic bond o he sub-
s a e molecules ia ans β-elimina ion eac ions.
62
A ecen
Fig. 5 Enzyma ic hyd olysis o pec in.
Tu o ial Re iew G een Chemis y
8782 |G een Chem.,2025,27, 8777–8803 This jou nal is © The Royal Socie y o Chemis y 2025
Open Access A icle. Published on 14 May 2025. Downloaded on 11/19/2025 9:38:26 PM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
example o cold-ac i e pec a e lyases a ac ed pa icula a en-
ion due o hei abili y o e ain high ca aly ic efficiency a
lowe empe a u es, simul aneously enabling ene gy sa ing,
cos educ ion and po en ially he p ese a ion o physiochem-
ical p ope ies o he ea ed p oduc s. A new cold- ole an
pec a e lyase (E PelPL1) gene om Echinicola osea was cloned,
and he ea e he e ologously exp essed in Esche ichia coli bac-
e ia. The enzyme was pu i ied by high-affini y Ni-cha ged
esin FF (Ni–NTA Sepha ose) and he molecula mass o
50 kDa was obse ed. This enzyme exhibi ed high ca aly ic
ac i i y a a low empe a u e (4 °C), al hough i exhibi ed
op imal ac i i y a 35 °C and pH 8.0 in he p esence o 1 mM
o Ca
2+
. I was ound ha wide ange o oligosaccha ides
(DP2–6) was p oduced du ing he eac ion cou se wi h he p e-
dominance o DP2–3.
62
MOS (Fig. 3c) a e a g oup o non-diges ible oligosaccha -
ides, which a e comp ised o 2–10 mannose uni s linked by
wo ypes o glycosidic bonds (α-1,6 and β-1,4). Acco dingly,
MOS can be u he di ided in o wo majo g oups, α- and
β-MOS, mos ly based on hei subs a e sou ce. Small amoun s
o na u ally occu ing MOS can be ex ac ed om he s uc-
u al and s o age pa s o plan s; howe e , he g ea es amoun
o MOS is nowadays gene a ed by he hyd oly ic clea age o
diffe en na u ally occu ing mannans (Fig. 6). Al hough
α-MOS p oduc s om yeas cell wall (α-1,6-mannan) hyd olysis
ha e been well es ablished as a eed addi i e in he ag icul u e
indus y,
63
β-MOS can be de i ed om mannans, which ep-
esen one o he majo cons i uen s (a ound 50%) o hemi-
cellulose in a a ie y o plan s, p ima ily so wood, plan seeds
and legumes. Also, ce ain ag icul u al by-p oduc s ( o
example cop a meal and palm ke nel meal)
64
and was e
ma e ials such as spen coffee g ounds
65
ha e been p o en o
be a g ea mannan sou ce (up o 60% mannans). Plan
mannans may appea as homo- (linea mannan) o he e o-
polyme s (galac omannan, glucomannan and galac ogluco-
mannan) o mannose depending on hei sou ce, and he e-
o e β-MOS a e p ima ily gene a ed h ough he hyd oly ic
ac i i y o β-mannanase (EC 3.2.1.78), leading o he gene-
a ion o β-MOS wi h a ying deg ees o polyme iza ion (DP).
Diffe en β-mannanases showed diffe en p e e ences in e ms
o po en ial subs a es and po en ial p oduc s, and hus he
achie ed yields and DP o he ob ained MOS p o ed o be
highly dependen on he β-mannanase u ilized. Fo example,
using β-mannanase om Aspe gillus o yzae (ManAo) unde he
same condi ions, MOS we e ob ained om a ious ag icul u al
byp oduc s (locus bean gum (LBG), gua gum (GG), konjac
gum (KG), palm ke nel cake (PKC) and cop a meal (CM)) in
wide ange o yields 9–56%,
46
clea ly showing speci ici y
owa ds pa icula subs a es o mo e speci ically ypes o
mannans wi hin hese subs a es. Magengelele e al. examined
he po en ial o ecombinan Aspe gillus nige endo-mannanase
(Man26A) exp essed in Saccha omyces ce e isiae Y294 o he
con e sion o h ee subs a es, i o y nu linea mannan and
wo galac omannan subs a es wi h a ying amoun s o galac-
osyl subs i u ions (GG and LBG).
66
The enzyme exhibi ed high
subs a e speci ici y owa ds locus bean gum and i o y nu
mannan wi h he majo p oduc s DP 2–4, while i s speci ici y
owa ds gua gum was a he low and hese eac ions gene -
a ed MOS o highe DP. Howe e , when conside ing he quan-
i y o ob ained MOS, he esul s showed a disc epancy, gi en
ha highe yields we e achie ed when using galac omannans
(4.91 mg mL
−1
o al educing suga s o gua gum and
3.89 mg mL
−1
o al educing suga s o locus bean gum) as
subs a es han linea mannan (2.24 mg mL
−1
o al educing
suga s) du ing ex ended pe iods o mannan hyd olysis. Unlike
hese enzymes, some β-mannanases show an ob ious p e e -
ence owa ds unsubs i u ed egions o linea mannan.
The e o e he u iliza ion o diffe en auxilia y enzymes such as
β-glucosidase (EC 3.2.1.21), β-mannosidase (EC3.2.1.25), as
main-chain mannan-deg ading enzymes, and α-galac osidase
Fig. 6 Hyd olysis o mannans.
G een Chemis y Tu o ial Re iew
This jou nal is © The Royal Socie y o Chemis y 2025 G een Chem.,2025,27,8777–8803 | 8783
Open Access A icle. Published on 14 May 2025. Downloaded on 11/19/2025 9:38:26 PM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
(EC 3.2.1.22), and ace yl mannan es e ase (EC 3.1.1.6), which
can enable he emo al o he side-chain subs i uen s and
b eak down he complex s uc u e o subs i u ed mannans,
should be conside ed.
67
Yang e al. showed ha he hyd olysis
efficiency o he β-mannanase om T ichode ma eesei is
g ea ly affec ed by he side chain o galac oses, which in o-
duces s e ic hind ance o enzymes. They examined he po en-
ial o α-galac osidase o enhance he hyd olysis o galac o-
mannan om Sesbania seeds (26% w/w), by adding he
enzyme sepa a ely ( i s ly ea ed wi h α-galac osidase),
sequen ially, and simul aneously. Finally, enzyma ic hyd olysis
by he simul aneous addi ion o α-galac osidase signi ican ly
imp o ed he ob ained MOS yields ( om 17% o 31%).
68
Howe e , i mus be no ed he e ha besides he enzyme and
subs a e selec ion, ca e ul op imiza ion o he p ocess con-
di ions should be pe o med o achie e sa is ac o y p oduc
yields, and some o hese examples a e p esen ed in se e al
e iew pape s.
64,67
Besides he achie ed yields, he de e mi-
na ion o he DP o he ob ained p oduc s plays an impo an
pa in he de elopmen o he MOS syn hesis p ocess,
gi en ha hei p ebio ic unc ion, as well as hei an ioxidan ,
an i-in lamma o y, c yop o ec an , an i-s ess and an i-
diabe ic po en ial is highly dependen on his p ope y.
A un a anamook e al. used gu he mic obio a model mic o-
o ganism Lac obacillus eu e i o examine he p ebio ic po en-
ial o syn hesized β-MOS.
69
Thei s udy showed ha medium-
leng h MOS (DP 4 and DP 5) exhibi ed he highes p ebio ic
po en ial, gi en ha highe MOS we e poo ly u ilized, whe eas
he excessi e hyd olysis o mannans esul ed in he loss o
selec i i y owa d bene icial bac e ia. The e o e, in e ms
o ob aining he mos p omising MOS p ebio ic mix u e
and inc easing he selec i i y owa d he p oduc ion o
medium-leng h MOS, diffe en s a egies ha e been p oposed,
such as changing he sou ces o mannan o op imizing
he enzyma ic hyd olysis condi ions and manipula ion wi h
β-mannanase.
66–68
In he wo k by A un a anamook e al.,
69
hey imp o ed he speci ici y o β-mannanase om Aspe gillus
nige (ManF3) owa d he desi ed p oduc size h ough
a ional-based enzyme enginee ing. Namely, hey eplaced
y osine (Ty 42 and Ty 132) in he enzyme ac i e si e wi h
glycine, which being smalle amino acid, enabled he o -
ma ion o an ex ended subs a e-binding si e, and conse-
quen ly inc eased he possibili y o highe molecula weigh
MOS o bind o he enzyme. Finally, his mu a ion esul ed in
addi ional space o he enzyme o accommoda e la ge hyd o-
lysis p oduc s. Mu a ions o y osine in o glycine esul ed wi h
enhanced yields o medium-chain MOS (DP4 and DP5);
howe e , hese mu a ions had educed hyd ophobic in e -
ac ions wi hin he enzyme molecule, and hus nega i ely
affec ed he mal and con o ma ional s abili y o he mu an
enzymes. This is why i was impossible o ob ain a double
mu an enzyme despi e i being success ully cons uc ed.
To da e, nume ous β-mannanases ha e been es ed o he
syn hesis o MOS, p e alen ly om gene a Aspe gillus and
Bacillus.
67
Howe e , ecen s udies ha e b ough o ou knowl-
edge a p e iously sca cely explo ed g oup o β-mannanoly ic
enzymes o igina ing om he gu mic obio a. Fo example,
Bha acha ya e al.
70
explo ed he po en ial o a cell-su ace
exposed β-mannanase (BoMan26B) om he abundan gu bac-
e ium Bac e oides o a us o he syn hesis o MOS om galac-
omannan, which is abundan in legumes, and ace yl-galac o-
glucomannan, abundan in so woods. The eac ion yielded
pa ially ace yla ed linea and galac osyl-con aining β-MOS
(MOS/GMOS) wi h an app oxima e deg ee o polyme iza ion
(DP) be ween 2 and 6, which was e ealed using a newly de el-
oped high- esolu ion anion-exchange ch oma og aphy p o-
cedu e. The abundance o MOS de i ed om galac omannan
ollowed he o de o p e alen ly DP 5, DP 6 = DP 2 and DP 4,
and inally DP 3, while he p o ile o MOS de i ed om ace yl-
galac oglucomannan was sligh ly diffe en , i.e., p e alen ly DP
5, ollowed by DP 4 and DP 2 = DP 3, and inally DP 6. Unde
he op imal condi ions, he yields o he ob ained oligosac-
cha ides we e 33% (w/w) and 30% (w/w), espec i ely. The p e-
bio ic po en ial o he ob ained oligosaccha ides was con-
i med by means o measu ing he p oduc ion o sho -chain
a y acids using he human gu bac e ia Bi idobac e ium ado-
lescen is ATCC 15703 and Rosebu ia hominis DSMZ 6839 as
ace a e and bu y a e p oduce s, espec i ely.
70
IMO (Fig. 3d) ha e been de ined as a mix u e o oligosac-
cha ides composed o se e al (usually 2–10) glucose uni s
linked by α-(l →4) and α-(1 →6) glycosidic bonds. They a e
mainly comp ised o isomal ose, panose, isomal o iose, iso-
mal o e aose, isopanose, and highe b anched oligosaccha -
ides. Nowadays, a g ea deal o IMO is syn hesized h oughou
diffe en chemical and enzyma ic p ocesses, bu hey can be
ound in honey and soy-based e men ed p oduc s, such as
miso and soy sauce, in small quan i ies. Besides hei con o-
e sial p ebio ic ac i i y due o he exis ence o eadily diges i-
ble α-l,4 linkages, which places hem in he eme ging p ebio-
ics ca ego y, hey a e cha ac e ized by high empe a u e and
pH s abili y, low iscosi y, and low wa e ac i i y. These p o-
pe ies enabled he app o al o a ious heal h claims, making
hem in e es ing o applica ion in he ood and eed sec o s.
71
IMO a e p ima ily syn hesized ia he enzyma ic con e sion o
s a ch om na u al and sus ainable plan sou ces (Fig. 7).
Ce eal c ops (whea , ice, ba ley, and co n), pulses, and ube s
(cassa a and po a o) a e conside ed he majo sou ces o
s a ch o p oduce IMO.
72
This p ocess in ol es he mul ime ic
enzyma ic ac ion o α-amylase (EC 3.2.1.1) and β-amylase (EC
3.2.1.2), which hyd olyze he long and in e nal s a ch
b anches, oge he wi h he s a ch deb anching pullulanase
(EC 3.2.1.41) and ansglycosyla ing α-glucosidase (EC
3.2.1.20), as shown a Fig. 7. Du ing he i s phase, he mo-
ole an α-amylase lique ies he s a ch ia andom clea age o
he α-1,4 linkages, and he ea e α-amylase oge he wi h
β-amylase, and pullulanase enables s a ch con e sion o mal-
ooligosaccha ides. The subsequen eac ion is ca alyzed by
α-glucosidase, an enzyme wi h p ima y hyd oly ic unc ion
ha ac s on he non- educing e minal o α-glucosides, bu
al e na i ely can ans e a glucosyl esidue o ano he glucose,
mal ose, isomal ose o isomal o iose molecule ia he o -
ma ion o α-1,6 linkages. S udies ha e shown ha his s ep is
Tu o ial Re iew G een Chemis y
8784 |G een Chem.,2025,27,8777–8803 This jou nal is © The Royal Socie y o Chemis y 2025
Open Access A icle. Published on 14 May 2025. Downloaded on 11/19/2025 9:38:26 PM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
c ucial in he syn hesis o IMO, and ha he igh choice o
he α-glucosidase and eac ion condi ions may g ea ly in lu-
ence he unc ional ac i i y o ob ained p oduc s h ough an
inc emen in he panose and isomal ose sha e in oligosaccha -
ides.
72
Fo example, i has been de e mined ha he simul-
aneous p ocesses o saccha i ica ion and ansglycosyla ion
a e mo e likely o p oduce an inc ease in he yield and p o-
duc i i y o IMO om s a ch compa ed o he con en ional
p ocess, esul ing in lowe glucose accumula ion h ough
g ea e ansglycosyla ion. Fo ins ance, Duong Hong e al.
de eloped a simple wo-s ep p ocedu e o he p oduc ion o
IMO using swee po a o s a ch as a low-cos subs a e.
73
Fi s ly,
hey examined he effec o se e al comme cial α-amylase p ep-
a a ions (Spezyme X a, Liquozyme SC DS, Spezyme Alpha,
and Te mamyl SC DS) on he p ocess o s a ch lique ac ion
(25% w/ ) a e 30 min wi h aim o ind he mos sui able
α-amylase based on he ob ained mix u e o oligosaccha ides.
The a ge ed oligosaccha ide mix u e in he i s s ep was DP
2–6, gi en ha i was p esumed ha i would sho en he sim-
ul aneous saccha i ica ion and ansglycosyla ion ime
equi ed o he maximum concen a ion o u u e IMO wi h
DP be ween 2 and 4. Acco dingly, he Spezyme X a p epa-
a ion (1.0 CU g
−1
) was chosen o he lique ac ion eac ion
gi en ha he highes concen a ion o desi ed oligosaccha -
ides (49.24% w/w) wi hou any gene a ion o ee glucose and
low gene a ion o s a ch esidue (1.98% w/w) was achie ed.
The ea e , ba ley β-amylase, pullulanase M2 om Bacillus
licheni o mis, and ansglucosidase om Aspe gillus nige we e
used o simul aneous saccha i ica ion and ansglycosyla ion
upon he de ailed op imiza ion o he enzyme dosage and eac-
ion condi ions. Finally, β-amylase (3 U g
−1
), pullulanase (0.8 U
g
−1
), and ansglucosidase (10 U g
−1
) we e applied, leading o
he gene a ion o 68.85 g L
−1
o IMO a o al eac ion ime
h ee imes sho e han in p e ious s udies.
73
Ano he app oach owa d he de elopmen o IMO ich in
panose in ol es he isola ion o a new α-glucosidase enzyme
p epa a ion possessing ad anced kine ic cha ac e is ics, p i-
ma ily inc easing he a io o ansglycosyla ion o hyd oly ic
ac i i y. Kuma e al. isola ed o he i s ime α-glucosidase
om a non-nige Aspe gillus isola e wi h high ansglycosyla-
ion po en ial.
74
The new soil isola e was iden i ied as
Aspe gillus neonige . A e pu i ica ion using a DEAE
Sepha ose-CL6B column, α-glucosidase was ound o possess
he molecula mass o 145 kDa and some s uc u al simi-
la i ies wi h he comme cially a ailable α-glucosidase om
Aspe gillus nige , al hough hei gene sequences a e conside -
ably diffe en . When compa ing hei ac i i ies, i mus be
no ed ha he mal ose consump ion p o iles o bo h enzymes
we e simila , al hough he ini ial a e o consump ion was
sligh ly slowe in case o he new enzyme. Likewise, he hyd o-
lysis a e o mal ose o glucose is lowe compa ed o he com-
me cial enzyme, which is qui e impo an gi en ha he low
gene a ion o undesi able by-p oduc can be expec ed.
Addi ionally, he new enzyme showed highe po en ial o he
syn hesis o panose in compa ison o he comme cial enzyme,
as well as a educ ion in he seconda y hyd olysis o panose
a e.
74
The e o e, i can be concluded ha his enzyme shows
g ea po en ial o he syn hesis o IMO. Success ul mal ose
con e sion in o desi able IMO (panose, isomal ose and iso-
mal o iose) can be also achie ed using Saccha omyces ce e i-
siae cells wi h α-glucosidase ac i i y.
73
Namely, he aglA gene
ha encodes α-glucosidase om Aspe gillus nige , known o
i s ansglycosyla ing ac i i y, was exp essed in Saccha omyces
ce e isiae in a manne ha yeas cells can be used di ec ly as
Fig. 7 Hyd olysis o s a ch using diffe en enzymes.
G een Chemis y Tu o ial Re iew
This jou nal is © The Royal Socie y o Chemis y 2025 G een Chem.,2025,27,8777–8803 | 8785
Open Access A icle. Published on 14 May 2025. Downloaded on 11/19/2025 9:38:26 PM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
His, espec i ely) exhibi ed a supe io pe o mance in XOS syn-
hesis. A e 14 h, he concen a ion o XOS eached 10.6 mg
mL
−1
, which was abou 1.6- old highe yield han ha o wild-
ype XynLC9. Addi ionally, bo h mu an s showed enhanced
he mos abili y.
146
The second app oach is ocused on e adica ing he hyd o-
ly ic capaci y o he enzymes by an unique amino acid modi i-
ca ion in he p o ein s uc u e o ge a syn hase, which is he
so-called glycosyn hase. This concep is based on he exchange
o he ca aly ic esidue (usually an aspa a e o glu ama e)
esponsible o p omo ing nucleophilic a ack on he subs i-
u ed anome ic ca bon (Fig. 13) by a non- unc ional esidue
(usually alanine o glycine). As a esul , hey become hyd oly i-
cally incompe en .
147,148
Then, he enzyma ic abili y o he
enzyme goes h ough he ans e o an ac i a ed glycosyl
dono ( ypically a glycosyl luo ide in anome ic posi ion) o a
sui able accep o o ca alyze he o ma ion o a glycosidic
bond in high yields.
147
This syn he ic po en ial has an eno mous impac in
he syn hesis o oligosaccha ides.
149
As p ebio ic compounds,
complex human milk oligosaccha ides a e gaining inc easing
a en ion.
150,151
Mic oo ganisms enginee ed o he syn hesis
o oligosaccha ides om nu ien s a e conside ed he mos
p omising sys ems o p ocess de elopmen .
152
The e o e, K.
Schmölze e al.
153
de eloped a glucosyn hase ( a ian D746E)
om Bi idobac e ium bi idum β-N-ace ylhexosaminidase
JCM1254 h ough he β-glycosyla ion o ac i a ed N-ace yl-D-
glucosaminyl dono s by 1,2-oxazoline (Fig. 14a). This ep-
esen s an impo an syn he ic s a egy owa ds oligosaccha -
ides.
154
The au ho s emphasized enzyma ic chemoselec i i y
gi en ha i is decisi e in he highly efficien glycosyla ion o
lac ose (∼90%) o NAG-oxa lac ose, gi ing ise o lac o-N- iose
II (LNT II), a cen al building block o human milk oligosac-
cha ides (HMOs). In con as , he wild- ype enzyme hyd olyzed
bo h he NAG-oxa dono and he isaccha ide p oduc wi h
signi ican ly highe ac i i y han glycosyn hase. This makes
he wild- ype enzyme qui e unsui able o syn he ic appli-
ca ion. The syn hesis in ol ed he use o chemically p epa ed
NAG-oxa in 40% yield om N-ace yl-D-glucosamine (GlcNAc).
Using equi alen amoun s o NAG-oxa and lac ose a hei
solubili y limi (600 mM), LNT II was ob ained (515 mM;
281 mg mL
−1
;∼90% yield; ≤1 h eac ion ime), which could be
immedia ely eco e ed om he bioca aly ic eac ion wi h 85%
pu i y. These p ocess efficiency me ics e eal he ema kable
po en ial o glycosyn hase o chemical p ocess applica ion
and highligh i as supe io o al e na i e syn he ic op ions o
isaccha ide p oduc ion.
BbhI is an exo-ac ing β-N-ace ylhexosaminidase and
belongs o he GH-20 amily o glycoside hyd olases. Enzymes
o he GH-20 amily use he pa icipa ion o he neighbo ing
2-ace amido g oup o he subs a e in ca alysis. The enzyma ic
eac ion is p omo ed by a highly conse ed iad o esidues
(Glu, Asp, and Ty ; Fig. 14b) and p oceeds h ough an in e -
media e 1,2-oxazolinium ion. A close-up o he ac i e si e o a
modeled s uc u e o BbhI is shown in Fig. 14c. Based on e i-
dence o endo-β-N-ace ylglycosaminidases om he GH-18
and GH-85
155,156
amilies, a p omising design o he BbhI gly-
cosyn hase was o subs i u e he esidues (Asp746 and Ty 827)
in ol ed in he s abiliza ion o he oxazolinium in e media e.
The s uc u e model (Fig. 14c) co obo a es he e idence o
he sequence alignmen (Fig. 14b) by sugges ing ha Asp746
and Ty 827 a e posi ionally conse ed a he BbhI ac i e si e.
Wi hin he GH-20 amily, he e is limi ed p ecedence in he
de elopmen o glycosyn hases. The D313A a ian o
β-hexosaminidase om S ep omyces plica us (GH-20) and
Ty 470 (Phe, His, and Asn) a ian s o β-hexosaminidase om
Tala omyces la us
157
(GH-20) e lec concep ually simila
enzyme design s a egies o ha applied o BbhI. Fou si e-
di ec ed a ian s (D746E, D746A, D746Q, and Y827F) o BbhI
we e p epa ed (Fig. 14d). The Asp746 a ian s in ol e he loss
o elec os a ic s abiliza ion (D746A) o he in e media e, a
likely s e ic con lic in he subs a e/in e media e posi ioning
(D746E), o bo h (D746Q).
The Y827F a ian in ol es he emo al o a hyd ogen bond
o subs a e binding and ca alysis.
158
Howe e , al hough
hese BbhI a ian s a e p edic ed o ha e low ac i i y o LNT II
hyd olysis, hey can u ilize he 1,2-oxazoline o N-ace yl-D-
glucosamine (NAG-oxa) as a dono o he β-1,3-glycosyla ion
o lac ose.
3.2. Chemical modi ica ion o p o ein su ace: con olling
selec i i y in he syn hesis o sho -oligosaccha ides
Selec i i y ep esen s a key cha ac e is ic in enzyma ic syn-
hesis. S uc u al modi ica ions can in luence he capaci y o
ecognize one egioisome o a pa icula ca bohyd a e o e
ano he . Thus, modi ica ions by in oducing chemical mole-
cules on he p o ein su ace ha e been success ully de eloped
o enhance his ecen ly.
159–163
One example demons a ed he
selec i i y o le ansuc ose in con olling he polyme size.
159
The au ho s pe o med a chemical modi ica ion on he y o-
Fig. 13 ans-Glycosyla ion p ocess ca alyzed by β-glycosyn hases.
Tu o ial Re iew G een Chemis y
8792 |G een Chem.,2025,27, 8777–8803 This jou nal is © The Royal Socie y o Chemis y 2025
Open Access A icle. Published on 14 May 2025. Downloaded on 11/19/2025 9:38:26 PM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online

sine o le ansuc ase om B. mega e ium, which p e e en ially
happened a esidues Y196 and Y247 (Fig. 15). The modi i-
ca ion was based on a luminol de i a i e con aining an alkyne
g oup, which ep esen s an o hogonal g oup. Then, egio-
selec i i y monosaccha ides we e inco po a ed on he p o ein
ia click chemis y eac ion using azide-anome ic ac i a ed
glucose. This modi ica ion allowed he egioselec i i y o he
enzyme o be al e ed, and he bioconjuga e could p oduce
mainly la ge polyme s (Fig. 15).
Ano he new al e na i e in he p oduc ion o new ca bo-
hyd a e molecules is he in si u cascade me allo-enzyma ic
eac ion. Employing his app oach, a new ype o a i icial
me alloenzyme con aining wo ac i e si es, one om he
enzyme an ano he a i icial si e c ea ed by he enzyme-
induced in si u gene a ion o coppe nanopa icles (CuNPs),
has been de eloped.
162,163
The g een p ocess o he p o-
duc ion o new bioca alys s allowed he p oduc ion o a p e-
ious lipase suppo ed on g aphene shee s, enabling he selec-
i e o ma ion o CuNPs on he enzyme su ace exclusi ely,
whe e he p ocess in ol ed he coo dina ion o he me al wi h
he amino acids o he enzyme in aqueous media. This CuNP-
modi ied lipase conse ed he excellen enzyma ic egio-
selec i i y agains monodeace yla ion o pe ace yla ed glucal a
C-3 and combined he oxida i e capaci y o CuNPs o he syn-
hesis o disaccha ides ia he o ma ion o an epoxy in e -
media e (Fig. 16). This chemical modi ica ion enabled he
p epa a ion o e y obus bioac i e ca alys s wi h excellen
ecyclabili y.
162,163
3.3. Immobiliza ion echnology o ob ain oligosaccha ides
o indus ial applica ion
The use o immobilized enzymes in chemical p ocesses offe s
se e al ad an ages o e p ocesses ca alyzed by soluble
enzymes. One o he mos signi ican ad an ages is he abili y
o euse he ca alys du ing diffe en eac ion cycles. This elim-
ina es he need o pu i ica ion p ocesses caused by he
mix u e o he soluble enzyme and diffe en componen s o
he eac ion in he case o homogeneous ca alysis p ocesses.
164
Fig. 14 (A) ans-Glycosyla ion by β-glycosyn hases and β-N-ace ylhexosaminidases. (B) Pa ial sequence alignmen o p oka yo ic and euka yo ic
GH-20 β-N-ace ylhexosaminidases displaying esidues c i ical o subs a e-assis ed ca alysis. C i ical amino acids (g een) and mu a ion si es
(a ows). (C) Close-up iew o he modeled ac i e si e o BbhI. The homology model (shown in blue). Key ac i e-si e esidues o BbhI used o mu a-
genesis a e d awn as s icks (Asp746; D746, pola izing esidue; Ty 827; Y827, s abiliza ion o eac ion in e media e by hyd ogen bonding). The LNB-
hiazoline bound in lac o-N-biosidase is shown wi h g een-colo ed ca bon a oms. (D) Compa ison o enzymes ega ding he maximum yield o LNT
II (black ba s) and he selec i i y pa ame e RTH (whi e ba s). Copy igh 2019, he Ame ican Chemical Socie y.
G een Chemis y Tu o ial Re iew
This jou nal is © The Royal Socie y o Chemis y 2025 G een Chem.,2025,27, 8777–8803 | 8793
Open Access A icle. Published on 14 May 2025. Downloaded on 11/19/2025 9:38:26 PM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
In addi ion o hese ad an ages, o he bene i s ha e been
desc ibed, such as he abili y o use immobiliza ion p ocesses
o enhance a ious enzyme p ope ies, including ac i i y, s abi-
li y and selec i i y.
165–168
In he ield o oligosaccha ide p o-
duc ion, enzymes a e o en imp o ed o enhance hei p o-
pe ies and enable he euse o he ca alys s in successi e eac-
ion cycles. In his case, immobiliza ion has been shown o
imp o e he p ope ies o enzymes, as demons a ed by he
immobiliza ion o xylanase om Aspe gillus nige on po ous
aga ose suppo s using co alen a achmen me hods. The
de i a i es esul ing om his p ocess we e 1100- imes mo e
s able han he soluble enzyme. This allowed he p oduc ion o
60% o oligosaccha ides anging om X2 o X6 xylose uni s.
169
Polygalac u onase om Aspe gillus aculea us was
immobilized by en apmen in a sol-gel sys em, esul ing in
a highly ac i e de i a i e (94.6% o i s ini ial ac i i y) and
imp o ed s abili y. The immobilized enzyme e ained 57%
o i s ca aly ic ac i i y a e incuba ion a 55 °C o 2 h,
compa ed o only 17% o he ee enzyme. Fu he mo e,
he an ibac e ial ac i i y o he ob ained POS using s an-
da d me hods was con i med o POS in he ange o
DP2−DP4. This immobiliza ion me hod allowed he euse
o he enzyme, wi h 65% ac i i y e ained a e 6 ba ch
eac ions.
170
β-Mannanase om a Konjac glucomannan p epa a ion was
immobilized by en apmen in algina e gels. The enzyme
ex ac , wi h a pu i y o 95%, e ained 68.3% o i s ca aly ic
ac i i y a e immobiliza ion. The immobilized ca alys s we e
mo e s able agains ex eme pH and high empe a u es, wi h
he op imal pH and empe a u e o 6 and 75 °C, espec i ely.
These ca alys s could be eused in he p oduc ion o MOS o 8
cycles, e aining 70.3% o hei ca aly ic ac i i y. In he oligo-
saccha ide p oduc ion assay, MOS was gene a ed a a concen-
a ion o 8 mg mL
−1
, consis ing o 5 mg mL
−1
o mannobiose
and 3 mg mL
−1
o manno iose.
171
To make he p ocess o MOS syn hesis mo e cos -effec i e
and economically iable by enhancing he s abili y o he
enzyme, Su yawanshi e al. ca ied ou LBG hyd olysis u ilizing
an immobilized p epa a ion om he newly isola ed
Aspe gillus quad ilinea us RSNK-1.
172
Namely, he mix u e o
hemicellulo y ic enzymes comp ised o endo-β-mannanase,
endo-β-xylanase, β-xylosidase, β-glucosidase and α-galac osidase
gene a ed by SSF e men a ion on low-cos cop a meal was co-
alen ly immobilized on aluminum oxide pelle s (AOP) a e glu-
a aldehyde 1% ( / ) ac i a ion. A e op imiza ion o he immo-
Fig. 15 Polyme -elonga ion specifici y ia chemical modifica ion o le ansuc ase om B. mega e ium. Figu e adap ed om e . 159 wi h pe mission
om RSC Publishing, Copy igh 2018.
Fig. 16 Syn hesis o new disaccha ides using bioconjuga e CuNP-
enzyme hyb id. Adap ed om e . 162 wi h pe mission om he
Ame ican Chemical Socie y, Copy igh 2023.
Tu o ial Re iew G een Chemis y
8794 |G een Chem.,2025,27, 8777–8803 This jou nal is © The Royal Socie y o Chemis y 2025
Open Access A icle. Published on 14 May 2025. Downloaded on 11/19/2025 9:38:26 PM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
biliza ion p ocedu e using he s a is ical me hod o esponse
su ace me hodology, MOS p oduc ion was pe o med in a
column bio eac o . The eac ion inished in 20 min, and he e-
a e he ob ained immobilized p epa a ion (Man-AOP) was suc-
cess ully ecycled 10 imes wi hou signi ican loss in i s ac i i y.
The a e age MOS gene a ion wi hin he 10 successi e cycles was
0.95 mg pe cycle (1.50, 5.15 and 2.84 mg mL
−1
o DP4, DP3
and DP2 MOS, espec i ely).
Chalane e al.
173
desc ibed an endodex anase sys em
(D8144) om Penicillium sp. immobilized on an epoxy-ac i-
a ed monoli hic Con ec i e In e ac ion Media (CIM®) disk o
p oduce on-line IMO om Dex an T40. The sys em e ained
mo e han 80% o i s esidual ac i i y a e 5000 column
olumes, demons a ing he high s abili y o he immobilized
endodex anases.
Hooda co-immobilized chi inase and glucosaminidase on
polyu e hane nanopa icles coa ed wi h zinc oxide.
174
The co-
immobilized ca alys demons a ed g ea e ac i i y unde he
op imal pH and empe a u e condi ions and highe s abili y
agains empe a u e han soluble enzymes. A e incuba ing
he soluble enzymes a 75 °C, 75% loss in ac i i y was
obse ed, while ha in he case o he co-immobilized ca alys
was only 40%. Fu he mo e, he ca alys p ope ies we e
imp o ed, enabling i s euse o a leas 50 eac ion cycles,
wi h a 50% loss in ca aly ic efficiency.
Ano he in e es ing example o immobiliza ion was
desc ibed by Ruzic e al.,
175
employing he same a ian , i.e.,
Bi idobac e ium bi idum β-N-ace yl hexosaminidase a ian
D746E. Speci ically, glucosyn hase immobilized on Cu
2+
-
aga ose beads (4%) (∼30 mg g
−1
) packed in a ixed bed (1 mL)
Fig. 17 (a) Asp746 o BbhI acili a es he o ma ion o he oxazoline in e media e in he enzyma ic eac ion. (b) Con e sion o GlcNAc–oxa in o LNT II.
(c) Recyclabili y p ocess using immobilized D746E glycosyn hase.
G een Chemis y Tu o ial Re iew
This jou nal is © The Royal Socie y o Chemis y 2025 G een Chem.,2025,27, 8777–8803 | 8795
Open Access A icle. Published on 14 May 2025. Downloaded on 11/19/2025 9:38:26 PM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
was u ilized o he s able con inuous p oduc ion o LNT II
(145–200 mM) wi h a quan i a i e yield o dono subs a e
(Fig. 17a). Wild- ype β-N-ace yl-hexosaminidase used unde
exac ly he same condi ions mainly gene a ed he hyd olysis
p oduc D-glucosamine (∼85%). By allowing a sho esidence
ime (2 min), which is difficul o es ablish o mixed-po
eac o ypes, he glycosyn hase low eac o achie ed he
effec i e uncoupling o LNT II o ma ion (∼80–100 mM min
−1
)
om slowe side eac ions (decomposi ion o dono subs a e
and enzyma ic hyd olysis o LNT II) o he op imal syn he ic
efficiency (Fig. 17b and c). Thus, his s udy p o ides a s ong
case o he applica ion o low chemis y p inciples in gluco-
syn hase eac ions, and he eby e eals he impo an syne gy
be ween enzymes and eac ion enginee ing o he bioca aly ic
syn hesis o oligosaccha ides.
3.4. P ocess in ensi ica ion in oligosaccha ides syn hesis
P ocess in ensi ica ion (PI) concep s and me hods can deli e
signi ican bene i s in he p oduc ion o biochemicals and
g een chemicals. The mos p omising PI concep s o he syn-
hesis o enzyma ic biop oduc s a e ansi ioning om ba ch
o con inuous p ocessing in no el ypes o eac o s (e.g. mic o/
milli eac o s, oscilla o y low eac o s, spi al eac o s, and
o a ing disk eac o s), in eg a ion o eac ion and sepa a ion
in mul i unc ional uni s (e.g. memb ane eac o s and ch oma-
og aphic eac o s) and u iliza ion o al e na i e ene gy
sou ces (e.g. mic owa es and sonica ion). The applica ion o
he abo e-men ioned me hods in eac o and p ocess design
can esul in signi ican imp o emen s in p oduc i i y, highe
desi ed p oduc selec i i y, educ ion o eac o olume and
o he ixed cos s, lowe ene gy and u ili ies cos s, be e
p ocess ope abili y and con ol, and educed was e gene a ion.
Recen e iews
176–178
shed ligh on he e sa ile and ample
possibili ies o PI in biochemical applica ions, p oposing he
new e m biop ocess in ensi ica ion (BPI/BI).
Rega ding he syn hesis o eme ging p ebio ics, he
esea ch and u iliza ion o PI a e s ill in hei in ancy.
Expec edly, mo e examples in he li e a u e can be ound o
es ablished p ebio ics, e.g. galac o-oligosaccha ides (GOS) and
uc o-oligosaccha ides (FOS). Fig. 18 p esen s he p ocess
in ensi ica ion me hods and de ices used o he syn hesis o
p ebio ics, including he main esul s achie ed (Fig. 18). To
ans e om ba ch o con inuous p ocessing, he classical
ubula eac o s wi h a ixed bed o immobilized enzymes ha e
been in es iga ed (Fig. 18a). Shin e al.
179
used a con inuous
ixed-bed eac o wi h β-galac osidase adso bed on chi osan
pa icles and ob ained a good GOS yield o 55% wi h s able
ope a ion o 15 days. Wa me dam e al.
180
epo ed ha hey
achie ed he same GOS yield in a ixed-bed bio eac o as in a
compa able ba ch bio eac o , bu he olume ic p oduc i i y
inc eased by 6 imes in he con inuous eac o . Lo enzoni
e al.
181
used bo h ixed-bed and luidized-bed eac o s o he
con inuous p oduc ion o FOS. They epo ed simila FOS
yields (54–59%) in bo h eac o s, al hough he immobilized
enzymes we e mo e s able in he ixed-bed (no changes in
ac i i y o 40 days). Zambelli e al.
182
showed o FOS syn-
hesis ha he eac ion ime o 72 h in ba ch p ocessing can
be educed o 10 h in a ixed-bed eac o , esul ing in a much
highe olume ic p oduc i i y. Howe e , he d awbacks o
ixed-bed eac o s include a ela i ely high p essu e d op and
possible mass ans e limi a ions, bo h ex e nal due o he
low eloci ies used (long esidence imes needed) and in e nal
due o in e pa icle diffusion effec s (in he case o po ous pa -
icles wi h immobilized enzymes) (Fig. 18b). These nega i e
effec s can be somewha educed in luidized-bed eac o s.
Memb ane eac o s ha e also demons a ed po en ial o
p ocess imp o emen , gi en ha he in si u sepa a ion o com-
ponen s can lead o highe con e sions o subs a e and yields
o he p ebio ic p oduc s. Pe zelbaue e al.
183
used a mem-
b ane eac o o GOS syn hesis and achie ed a GOS concen-
a ion 3- o 4- imes highe han in ba ch mode unde he
same condi ions. Có do a e al.
184
designed and op imized an
ul a il a ion memb ane bio eac o . By using high concen-
a ions o lac ose, he amoun o gene a ed GOS pe uni
mass o ca alys inc eased by 2.44- imes compa ed o he ba ch
sys ems. Po a z e al.
185
in es iga ed he p oduc ion o GOS
by immobilizing he enzyme β-galac osidase on a me hac ylic
mac opo ous monoli h, which was used as he memb ane
eac o . In his eac o , he luid lows unhinde ed h ough he
po es, which educes he mass ans e limi a ions ( ha occu
when con en ional suppo s a e used). The esul s showed
ha du ing con inuous p oduc ion, he GOS yield was up o
60% highe compa ed o he ba ch p oduc ion. These eac o s
a e sui able o indus ial p oduc ion due o he simple
p ocess scale-up achie ed by adding monoli hs. Sen e al.
186
compa ed a o a y disk memb ane bio eac o (Fig. 18d) wi h a
ba ch eac o in e ms o GOS p oduc ion. Thei esea ch
showed ha he p oduc yield and pu i y we e highe in he
o a ing disk memb ane bio eac o , wi h yields 80.2% and
77%, espec i ely. When compa ing p oduc pu i y, he
ob ained pu i y was wice as high when using he o a y disk
memb ane bio eac o (67.4%) han he ba ch eac o ollowed
by memb ane il a ion (32.4%). Howe e , he disad an ages
o memb ane eac o s a e he use o high p essu es, which can
educe he ac i i y o enzymes by damaging hei ac i e cen e s
and possibili y o memb ane clogging and ouling, all edu-
cing he yield and p oduc i i y.
Fu he mo e, newe ypes o eac o s wi h special cons uc-
ions and ope a ion ha e been p oposed o he syn hesis o
p ebio ics. These eac o s a e oscilla o y low eac o s wi h
baffles (OBR) (Fig. 18e), which exhibi e y good mixing due o
he induced and s uc u ed o ices appea ing a low ne low
a es (needed o longe esidence imes). Sla nić
187
showed
ha OBRs can be efficien ly used o GOS syn hesis, bo h wi h
ee- lowing and immobilized enzymes, ob aining highe p o-
duc i i y han in he ba ch eac o . The shea s esses in OBRs
a e lowe han in he classical mixing uni s (wi h p opelle s),
and hus he enzymes main ain hei s abili y.
187
Recen ly,
Todiće al.
188,189
p oposed a modi ied OBR, eplacing he
s aigh ube wi h a cu ed one in a helical oscilla o y baffled
eac o (HOBR, Fig. 18 ), which displayed e en be e mixing
due o addi ional Dean o ices. P a ilo iće al.
190
con i med
Tu o ial Re iew G een Chemis y
8796 |G een Chem.,2025,27, 8777–8803 This jou nal is © The Royal Socie y o Chemis y 2025
Open Access A icle. Published on 14 May 2025. Downloaded on 11/19/2025 9:38:26 PM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
ha GOS can be p oduced in 3D-p in ed SOBR wi h a 3- old
inc ease in olume ic p oduc i i y and highe selec i i y pe
enzyme consumed in compa ison o he ba ch eac o . This
wo k
189–191
also demons a ed how modeling, op imiza ion
and 3D p in ing can aid he apid de elopmen o in ensi ied
enzyma ic p ocesses and no el eac o s o he syn hesis o
p ebio ics.
I is easonable o assume ha many o he abo e-p esen ed
PI me hods can also be applied o he syn hesis o eme ging
p ebio ics wi h compa able success. One should no e ha he
p oduc ion o eme ging p ebio ics is mo e demanding, gi en
ha i commonly in ol es complex-s uc u e subs a es and
mul iphase p ocessing. Thus, some o he abo e-men ioned
concep s would no be sui able o would need conside able
modi ica ions. Ne e heless, se e al esea ch effo s ha e been
epo ed on he in ensi ica ion o eme ging p ebio ics syn-
hesis, e.g. XOS and POS. A low- ype mic o eac o (150 ×
300 µm, 18.7 µL) was used o in ensi y and inc ease he
efficiency o enzyma ic XOS p oduc ion om beechwood xylan
using pu i ied endoxylanase om The momyces lanuginosus.
The xylan hyd olysis pe o mance was compa ed wi h he
same eac ion pe o med unde ba ch condi ions (100 pm).
The expe imen al esul s showed ha XOS syn hesis was sig-
ni ican ly imp o ed compa ed o he ba ch eac o , namely a
yield o o e 80% was achie ed in a esidence ime o less han
one minu e. The enzyma ic xylan hyd olysis pe o mance and
xylan o XOS con e sion we e h ee imes highe unde he
op imized low condi ions in compa ison o he examined
ba ch p ocess.
192
Howe e , he disad an ages o mic o- eac o s
a e ela ed o hei small-size channels, which a e no sui able
o solid-liquid lows (o en equi ed o he syn hesis o eme -
ging p ebio ics), as well as high ixed cos s pe uni o low a e
(cos s o indus ial le el capaci y).
Ano he con inuous p ocess was designed o he p o-
duc ion o POS om suga bee pulp in a c oss low con inu-
ous enzyme memb ane eac o , achie ing s able p oduc ion
o 28.5 h unde he op imized condi ions, wi h an a e age
POS yield o 82.9% (w/w) combined wi h a olume ic p o-
duc i i y o 17.5 g L
−1
h
−1
and a speci ic p oduc i i y o 8.0 ±
1.0 g g
−1
Eh
−1
. This wo k demons a ed he s able con inuous
p oduc ion o POS om suga bee pulp using a eac o mo e
sui able o upscaling.
193
Fig. 18 P ocess in ensifica ion s a egies o he syn hesis o p ebio ics. (a) Fixed bed eac o s, (b) Fluidized bed eac o s, (c) Memb ane eac o s, (d)
Ro a ing memb ane eac o s, (e) Oscila o y buffle eac o z, ( ) Helical oscilla o y baffled eac o s, (g) Table con aining he p ocesses iden ifica ion
esul s in p ebio ic molecules p oduc ion. Figu es we e adap ed om e . 179, 181, 183, 186–189.
G een Chemis y Tu o ial Re iew
This jou nal is © The Royal Socie y o Chemis y 2025 G een Chem.,2025,27, 8777–8803 | 8797
Open Access A icle. Published on 14 May 2025. Downloaded on 11/19/2025 9:38:26 PM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online

4. Conclusions and ou look
Eme ging p ebio ics a e compounds wi h high impac , no
only h ough he gene al bene icial effec o p ebio icson
human heal h and well-being, which is e y impo an
nowadays due o he inc easing occu ence o diseases
ela ed o ood in ake and seden a y li es yles, bu also
h ough hei con ibu ion o a g een ansi ion gi en ha
hey a e compounds ha can be p oduced by g een bio ech-
nological p ocesses. These p ebio ics, p ima ily oligosac-
cha ides and polyphenols, a e eco- iendly because hey a e
sus ainably sou ced, gi en ha hey can be de i ed om aw
ma e ials such as indus ial by-p oduc s o was e (e.g.,
meals, pomaces, bagasse, and biomass) h ough ex ac ion
o hyd olysis om polysaccha ide cons i uen s. This e iew
p esen ed he mos no able chemical compounds iden i ied
as eme ging p ebio ics, oge he wi h e idence o hei p e-
bio ic ac i i y. Efficien enzyma ic p ocesses a e essen ial o
he op imal use o aw ma e ials and ine- uning he physio-
logical ac i i y o p ebio ics. These g een ca alys s a e
employed o p oduce p ebio ic oligosaccha ides and
enhance he ex ac ion and s uc u al modi ica ion o poly-
phenolic p ebio ics. Consequen ly, his e iew highligh ed
he signi icance o enzyma ic glycosyla ion and glycolysis in
he p oduc ion o eme ging p ebio ics and desc ibed he
mos ele an enzymes used in hese p ocesses. Howe e ,
he high cos o enzymes o en poses a signi ican obs acle
in he de elopmen o new enzyme-based g een echno-
logies, and hence his e iew p o ided a comp ehensi e
o e iew o he cu en effo s o maximize he bioca aly ic
po en ial o enzymes using enginee ing ools such as
p o ein enginee ing, immobiliza ion echnology, and
p ocess in ensi ica ion. The sha e o eme ging p ebio ics in
he apidly g owing p ebio ic ma ke is expec ed o inc eases
om 4.7% o 12.9% o e he nex decade, and hence he
join applica ion o he diffe en ypes o scien i ic expe ise
p esen ed in his e iew is necessa y o comme cialize p o-
cesses ha mee ma ke demands, while also adhe ing o
he g een ansi ion egula ions o indus y. Fu he mo e,
li e cycle assessmen (LCA) s udies can be conside ed o
he quan i ica ion o he impac s o p oduc s and p ocesses
on hei whole li e cycle, adop ing a holis ic app oach.
194,195
This analysis can acili a e he iden i ica ion o he main
en i onmen al ho spo s o he p oduc ion p ocess, he com-
pa ison o he diffe en p ocess al e na i es, and he sugges-
ion o eco-design op ions o he imp o emen o hese p o-
cesses. Mo eo e , hese indings can be e-used in o he
LCA s udies including eme ging p ebio ics in he p o-
duc ion sys em.
Au ho con ibu ions
All au ho s e ised and edi ed he manusc ip . All au ho s
ha e ead and ag eed o he published e sion o he
manusc ip .
Da a a ailabili y
No p ima y esea ch esul s, so wa e o code ha e been
included, and no new da a we e gene a ed o analysed as pa
o his e iew.
Conflic s o in e es
The au ho s decla e no con lic o in e es , inancial o
o he wise.
Acknowledgemen s
The au ho s hank he suppo om he Spanish Na ional
Resea ch Council (CSIC) and Minis y o Science, Inno a ion
and Uni e si y o Spain. Se bian pa o esea ch is inanced by
he axpaye s o he Republic o Se bia (Con ac No. 451-03-
136/2025-03/200135 o Minis y o Technological De elopmen
and Inno a ions o he Republic o Se bia and p og amme
IDEAS, p ojec P In P Enzy, no. 7750109) and ca ied ou
unde condi ions o legal and inancial ep ession exe ed by
he Minis y o Educa ion on he eaching s affo Uni e si y o
Belg ade. The au ho s acknowledge he unding om
Eu opean Commission, p ojec “Twinning o in ensi ied enzy-
ma 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. N. L-G. hanks he
unding by he Eu opean Union (BioNanoAc , GA 101153145).
Views and opinions exp essed a e howe e hose o he au ho (s)
only and do no necessa ily e lec hose o he Eu opean
Union o Eu opean Resea ch Execu i e Agency (REA). Nei he
he Eu opean Union no he REA can be held esponsible o
hem. We acknowledge he suppo o he publica ion ee by
he CSIC Open Access Publica ion Suppo Ini ia i e h ough
i s Uni o In o ma ion Resou ces o Resea ch (URICI).
Re e ences
1 R. Todd, M. Klee ebezem, M. V. Kopp and M. Rescigno,
Na . Re . Immunol., 2012, 12, 728–734.
2 G. R. Gibson and M. B. Robe oid, J. Nu ., 1995, 125,
1401–1412.
3 G. R. Gibson, R. Hu kins, M. E. Sande s, S. L. P esco ,
R. A. Reime , S. J. Salminen, K. Sco , C. S an on,
K. S. Swanson, P. D. Cani, K. Ve beke and G. Reid, Na .
Re . Gas oen e ol. Hepa ol., 2017, 14, 491–502.
4 E. E. Blaak, E. E. Can o a, S. Theis, G. F os , A. K. G oen,
G. Mi hieux, A. Nau a, K. Sco , B. S ahl, J. an Ha sselaa ,
R. an Tol, E. E. Vaughan and K. Ve beke, Bene ic.
Mic obes, 2020, 11, 411–455.
5 K. Swennen, C. M. Cou in and J. A. Delcou , C i . Re .
Food Sci. Nu ., 2006, 46, 459–471.
6 R. Y. Wu, P. Mää änen, S. Nappe , E. Sc u en, B. Li,
Y. Koike, K. C. Johnson-Hen y, A. Pie o, L. Rossi,
Tu o ial Re iew G een Chemis y
8798 |G een Chem.,2025,27, 8777–8803 This jou nal is © The Royal Socie y o Chemis y 2025
Open Access A icle. Published on 14 May 2025. Downloaded on 11/19/2025 9:38:26 PM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
S. R. Bo s, M. G. Su e e and P. M. She man, Mic obiome,
2017, 5, 135.
7 V. Lolou and M. I. Panayio idis, Fe men a ion, 2019, 5, 41.
8 Y. H. Lee, N. K. Ve ma and T. Thanabalu, J. Func . Foods,
2021, 78, 104352.
9 P. Mish a, V. M. Badiyani, S. Jain, S. Sub amanian,
S. V. Maha aj, A. Kuma and B. N. Singh, Cance Rep.,
2023, 6, e1870.
10 D. Xa ie -San os, M. Padilha, G. A. Fabiano, G. Vinde ola,
A. G. C uz, K. Si ie i and A. E. Cos a An unes, T ends Food
Sci. Technol., 2022, 120, 174–192.
11 P ebio ics: Global S a egic Business Repo , Region: Global
Indus y Analys s, Inc., ID: 1206755, 2023.
12 B. B. Ca doso, C. Amo im, S. C. Sil é io and
L. R. Rod igues, Ad . Food Nu . Res., 2021, 95,41–95.
13 Y. Nakazawa, M. Kageyama, M. Ma suzawa, T. Z. Liang,
K. Kobayashi, H. Shimizu, K. Maeda, M. Masuhi o,
S. Mo ouchi, S. Kumano, N. Tanaka, K. Ku amochi,
H. Nakai, H. Taguchi and M. Nakajima, Commun. Biol.,
2025, 8, 66.
14 I. Igna o a, A. A so , P. Pe o a and K. Pe o , Molecules,
2025, 30, 803.
15 M. Ma ins, P. F. A ila, C. C. P. de And ade and
R. Goldbeck, Bioca al. Ag ic. Bio echnol., 2020, 28, 101747.
16 M. Ma ins, M. F. Sil a, T. M. Dinama co and
R. Goldbeck, P ocess Biochem., 2022, 122, 331–340.
17 P. Ka apodis and P. Ch is akopoulo, LWT, 2008, 41, 1239–
1243.
18 L. Khaleghipou , J. A. Lina es-Pas en, H. Rashedi,
S. O. R. Siada , A. Jasilionis, S. Al-Hamimi, R. R. R. Sa da i
and E. N. Ka lsson, Bio echnol. Bio uels, 2021, 14, 153.
19 B. Han, J. Gao, X. Han, H. Deng, T. Wu, C. Li, J. Zhan,
W. Huang and Y. You, Food Res. In ., 2022, 162, 112019.
20 G. P ecup, J. Venus, M. Heie mann, R. Schneide ,
I. D. Pop and D. C. Vodna , Polyme s, 2022, 14, 1336.
21 C. Be ocal, H. Chico, E. Ca anza and R. Vega, Biochem.
Eng. J., 2021, 171, 108003.
22 S. Renga ajan and R. Palani el, P ocess Biochem., 2020, 98,
93.
23 J. Kang, X. Sun, S. Yu, Z. Wang, J. Zhang, Y. Zhao, S. Wang
and Q. Guo, In . J. Biol. Mac omol., 2024, 281, 136302.
24 D. O ego, M. L. Oli a es-Teno io, L. V. Hoyos, C. Al a ez-
Vasco, B. K. Cebe io and N. Caicedo, LWT, 2024, 207,
116681.
25 Y. Wandee, D. U apap, P. Mischnick and
V. Rungsa d hong, Food Chem., 2021, 348, 129078.
26 J. Chen, J. Yin, H. Xie, W. Lu, H. Wang, J. Zhao and J. Zhu,
Food Func ., 2024, 15, 3810–3823.
27 S. A. Ismail, H. S. El-Sayed and B. Fayed, Ca bohyd .
Polym., 2020, 234, 115941.
28 M. Cunningham, G. Vinde ola, D. Cha alampopoulos,
S. Lebee , M. E. Sande s and R. G imaldi, T ends Food Sci.
Technol., 2021, 112, 495.
29 J. Kaulpiboon, P. Rudeekul ham ong, S. Wa anasa i a pa,
K. I oand and P. Pongsawasdi, J. Mol. Ca al. B:Enzym.,
2015, 120, 127.
30 C.-K. Hsu, J.-W. Liao, Y.-C. Chung, C.-P. Hsieh and
Y.-C. Chan, J. Nu ., 2004, 134, 1523–1528.
31 J. C. Gomes de Alenca , G. T. da Sil a Pin o, K. F. C. Sil a,
J. M. S. San os, M. D. Hubinge , J. L. Bicas,
M. R. Ma os ica, C. L. de Oli ei a Pe kowicz and
B. N. Paulino, T ends Food Sci. Technol., 2025, 155,
104808.
32 R. Saini, A. K. Pa el, J. K. Saini, C.-W. Chen, S. Va jani,
R. R. Singhania and C. D. Dong, Bioenginee ed, 2022, 13,
2139–2172.
33 P. K. Pe umal, C.-Y. Huang, C.-W. Chen, G. S. Anisha,
R. R. Singhania, C.-D. Dong and A. K. Pa el, Bioenginee ed,
2023, 14, 2252659.
34 H. Ami i, M. Aghbashlo, M. Sha ma, J. Gaffey,
L. Manning, S. M. M. Bas i, J. F. Kennedy, V. K. Gup a and
M. Taba abaei, Na . Food, 2022, 3, 822–828.
35 C. J. C aw o d and P. H. Seebe ge , Chem. Soc. Re ., 2023,
52, 7773–7801.
36 O. Gligo , A. Mocan, C. Moldo an, M. Loca elli, G. C işan
and I. C. F. R. Fe ei a, T ends Food Sci. Technol., 2019, 88,
302–315.
37 M. Hue a, A. San Ma ín, B. A ancibia, F. A. Co nejo,
F. A enas, A. Illanes, C. Gue e o and C. Ve a, Food
Biop od. P ocess., 2024, 147, 474–482.
38 C. Ve a, A. Illanes and C. Gue e o, Cu . Opin. Food Sci.,
2021, 37, 160–170.
39 M. Filice, J. M. Guisan, M. Te eni and J. M. Palomo, Na .
P o oc., 2012, 7, 1783–1796.
40 H.-W. Tseng, H.-K. Tseng, K.-E. Ooi, C.-E. You,
H.-K. Wang, W.-H. Kuo, C.-K. Ni, Y. Manabe and C.-C. Lin,
JACS Au, 2024, 4, 4496–4506.
41 J. Zhen, H. Xu, J. Fang and X. Zhang, Ca bohyd . Polym.,
2022, 291, 119564.
42 M. Filice and J. M. Palomo, RSC Ad ., 2012, 2, 1729–1742.
43 T.-I. Tsai, H.-Y. Lee, S.-H. Chang, C.-H. Wang, Y.-C. Tu,
Y.-C. Lin, D.-R. Hwang, C.-Y. Wu and C.-H. Wong, J. Am.
Chem. Soc., 2013, 135, 14831–14839.
44 S. S. Mandal, G. Liao and Z. Guo, RSC Ad ., 2015, 5,
23311–23319.
45 R. Zhong, L. Gao, Z. Chen, S. Yuan, X. Chen and C. Zhao,
Food Chem.: X, 2021, 12, 100152.
46 L. San ibáñez, C. Hen íquez, R. Co o-Tejeda, S. Be nal,
B. A mijo and O. Salaza , Ca bohyd . Polym., 2021, 251,
117118.
47 A. A. Aacha y and S. G. P apulla, J. Ag ic. Food Chem.,
2008, 56, 3981–3988.
48 H. Shinoyama, Y. Kamiyama and T. Yasui, Ag ic. Biol.
Chem., 1988, 52, 2197–2202.
49 H. Kizawa, H. Shinoyama and T. Yasui, Ag ic. Biol. Chem.,
1991, 55, 671–678.
50 A. Palaniappan, U. An ony and M. N. Emmambux, T ends
Food Sci. Technol., 2021, 111, 506–519.
51 T. F. Viei a, R. C. G. Co êa, R. d. F. P. M. Mo ei a,
R. A. Pe al a, E. A. de Lima, C. V. Helm, J. A. A. Ga cia,
A. B ach and R. M. Pe al a, Was e Biomass Valo iza ion,
2021, 12, 6727–6740.
G een Chemis y Tu o ial Re iew
This jou nal is © The Royal Socie y o Chemis y 2025 G een Chem.,2025,27, 8777–8803 | 8799
Open Access A icle. Published on 14 May 2025. Downloaded on 11/19/2025 9:38:26 PM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
52 C. C. Te one, J. M. F. Nascimen o, C. R. F. Te asan,
M. B ienzo and E. C. Ca mona, Bioca al. Ag ic. Bio echnol.,
2020, 23, 101460.
53 T. Zhou, Y. Xue, F. Ren and Y. Dong, J. Ca bohyd . Chem.,
2018, 37, 210–224.
54 E. N. Ka lsson, E. Schmi z, J. A. Lina es-Pas én and
P. Adle c eu z, Appl. Mic obiol. Bio echnol., 2018, 102,
9081–9088.
55 S. S. Reddy and C. K ishnan, P ep. Biochem. Bio echnol.,
2016, 46,49–55.
56 R. P. Singh and J. M. R. Tingi ika i, Bioca al. Ag ic.
Bio echnol., 2021, 31, 101910.
57 N. Babba , W. Dejonghe, M. Ga i, S. S o za and K. Els ,
C i . Re . Bio echnol., 2016, 36, 594–606.
58 J.-L. Xia and P.-J. Li, Pec ic Enzymes, in Encyclopedia o
Food Chemis y, ed. L. Mel on, F. Shahidi and P. Va elis,
Academic P ess, Ox o d, 2019, pp. 270–276.
59 C. Saba e , A. Blanco-Do al, A. Mon illa and N. Co zo,
Food Hyd ocolloids, 2021, 110, 106161.
60 B. Gómez, C. Peláez, M. C. Ma ínez-Cues a, J. C. Pa ajó,
J. L. Alonso and T. Requena, LWT, 2019, 109,17–25.
61 S. Baldassa e, N. Babba , S. Van Roy, W. Dejonghe,
M. Maesen, S. S o za and K. Els , Food Chem., 2018, 267,
101–110.
62 L. Zheng, Z. Guo, S. Cao and B. Zhu, Bio esou .
Biop ocess., 2021, 8, 121.
63 M. Faus ino, J. Du ão, C. F. Pe ei a, M. E. Pin ado and
A. P. Ca alho, Ca bohyd . Polym., 2021, 272, 118467.
64 N. Hlalukana, M. Magengelele, S. Malgas and
B. I. Ple schke, Foods, 2021, 10, 2010.
65 C. Wongsi ide chai, V. Jonja oen, T. Sawangwan,
T. Cha oen a and S. Chan o n, LWT, 2021, 148, 111717.
66 M. Magengelele, N. Hlalukana, S. Malgas, S. H. Rose,
W. H. an Zyl and B. I. Ple schke, Enzyme Mic ob. Technol.,
2021, 150, 109893.
67 U. K. Jana and N. Kango, In . J. Biol. Mac omol., 2020, 149,
931–940.
68 L. Yang, G. Shi, Y. Tao, C. Lai, X. Li, M. Zhou and Q. Yong,
Appl. Biochem. Bio echnol., 2021, 193, 405–416.
69 N. A un a anamook, R. Wansuks i, T. Uengwe wani and
V. Champ eda, J. Biosci. Bioeng., 2020, 130, 443–449.
70 A. Bha acha ya, M. Wiemann and H. S ålb and, LWT,
2021, 151, 112215.
71 A. S. Hima , S. Gau am, J. P. C. Ga cia, A. X. Vid io-
Sahagún, Z. Liu, D. B essle and T. Vasan han, Bioac .
Ca bohyd . Die . Fib e, 2021, 26, 100275.
72 C. Be ocal, H. Chico, E. Ca anza and R. Vega, Biochem.
Eng. J., 2021, 171, 108003.
73 Q. D. Hong, T. H. Minh, H. G. N. Thi, T. H. N. Thi,
N. H. Nguyen, T. L. N. Thi, T. T. Vu and H. N. Luong,
J. Food Qual., 2021, 2021, 1987219.
74 S. Kuma , A. Basu, K. A. Anu-Appaiah, B. S. Gnanesh
Kuma and S. Mu u i, J. Appl. Mic obiol., 2020, 129, 1644–
1656.
75 J. L. Shamshina, P. Be on and R. D. Roge s, ACS
Sus ainable Chem. Eng., 2019, 7, 6444–6457.
76 C. P. Souza, B. C. Almeida, R. R. Colwell and I. N. Ri e a,
Ma . Bio echnol., 2011, 13, 823–830.
77 H. Wang, B. Li, F. Ding and T. Ma, P og. O g. Coa ., 2020,
149, 11.
78 M. Dohendou, K. Pakzad, Z. Neza a , M. Nas ollahzadeh
and M. G. Dekamin, In . J. Biol. Mac omol., 2021, 192,
771–819.
79 Z. Fan, Y. Qin, S. Liu, R. Xing, H. Yu, X. Chen, K. Li, R. Li,
X. Wang and P. Li, Ca bohyd . Polym., 2019, 224, 115155.
80 A. Maia, Y. C. Glo ia, K. Fuchs, T.-H. Chang, P. Engels,
M. Zhou, T. Hinnen hal, E. Rusch, C. Gou e angeas and
A. N. R. Webe , J. Leukocy e Biol., 2023, 114, 180–186.
81 X. Liu, Y. Zhang, Z. Liu and X. Xie, OncoTa ge s The .,
2019, 12, 7581.
82 H. Zhen, Q. Yan, Y. Liu, Y. Li, S. Yang and Z. Jiang, Food
Sci. Hum. Wellness, 2022, 11, 999–1009.
83 M. Guo, X. Wei, S. Chen, J. Xiao and D. Huang, In . J. Biol.
Mac omol., 2022, 209, 631–641.
84 M. Yamabhai, M. Khamphio, T. T. Min, C. N. Soem,
N. C. Cuong, W. R. Ap ilia, K. Luesukp ase ,
K. Tee ani aya a n, A. Maneedaeng, T. R. Tu eng,
S. B. Lo en zen, S. An onsen, P. Ji p ase wong and
V. G. H. Eijsink, Ca bohyd . Polym., 2024, 324, 121546.
85 G. R. Gibson, R. Hu kins, M. E. Sande s, S. L. P esco ,
R. A. Reime , S. J. Salminen, K. Sco , C. S an on,
K. S. Swanson, P. D. Cani, K. Ve beke and G. Reid, Na .
Re . Gas oen e ol. Hepa ol., 2017, 14, 491–502.
86 A. And eu, M. Ćo o ić, C. Ga cia-Sanz, A. S. San os,
A. Mili oje ić, C. O ega-Nie o, C. Ma eo, D. Bezb adica
and J. M. Palomo, Ca alys s, 2023, 13, 1359.
87 F. Nazza o, F. F a ianni, V. De Feo, A. Ba is elli, A. G. Da
C uz and R. Coppola, Ad . Food Nu . Res., 2020, 94,35–89.
88 M. C. Rod íguez-Daza, E. C. Pulido-Ma eos, J. Lupien-
Meilleu , D. Guyonne , Y. Desja dins and D. Roy, F on .
Nu ., 2021, 8, 689456.
89 A. Bačić, J. Ga ilo ićand M. Rajilić-S ojano ić,A h.
Fa m., 2023, 73, 535–553.
90 M. Fidélix, D. Milenko ic, K. Si ie i and T. Cesa , Food
Func ., 2020, 11, 1599–1610.
91 R. C. Ba nes, H. Kim, C. Fang, W. Benne , M. Nemec,
M. A. Si en, J. S. Suchodolski, N. Deu z, R. A. B i on and
S. U. Me ens-Talco , Mol. Nu . Food Res., 2019, 63,
1800512.
92 A. L. Molan, Z. Liu and M. K uge , Wo ld J. Mic obiol.
Bio echnol., 2010, 26, 1735–1743.
93 A. L. Molan, Z. Liu and G. Plimme , Phy o he . Res., 2014,
28, 416–422.
94 S. N. Hes e , A. Mas aloudis, R. G ay, J. M. An ony,
M. E ans and S. M. Wood, J. Nu . Me abol., 2018, 2018,
7497260.
95 M. C. Rod íguez-Daza, M. Roquim, S. Dudonné, G. Pilon,
E. Le y, A. Ma e e, D. Roy and Y. Desja dins, F on .
Mic obiol., 2020, 11, 2032.
96 D. E. Roopchand, R. N. Ca mody, P. Kuhn, K. Moskal,
P. Rojas-Sil a, P. J. Tu nbaugh and I. Raskin, Diabe es,
2015, 64, 2847–2858.
Tu o ial Re iew G een Chemis y
8800 |G een Chem.,2025,27,8777–8803 This jou nal is © The Royal Socie y o Chemis y 2025
Open Access A icle. Published on 14 May 2025. Downloaded on 11/19/2025 9:38:26 PM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
97 J. Li, T. Wu, N. Li, X. Wang, G. Chen and X. Lyu, Food
Func ., 2019, 10, 333–343.
98 Y. Liu, L. Luo, Y. Luo, J. Zhang, X. Wang, K. Sun and
L. Zeng, J. Ag ic. Food Chem., 2020, 68, 6368–6380.
99 S. Wes all, N. Lomis and S. P akash, A i . Cells,
Nanomed., Bio echnol., 2018, 46, 441–455.
100 Y. Fuji a, H. Tsuno and J. Nakayama, PLoS One, 2017, 12,
e0169240.
101 Z. Wang, K. L. Lam, J. Hu, S. Ge, A. Zhou, B. Zheng,
S. Zeng and S. Lin, Food Sci. Nu ., 2019, 7, 579–588.
102 J. Liu, W. Hao, Z. He, E. Kwek, Y. Zhao, H. Zhu, N. Liang,
K. Y. Ma, L. Lei and W. S. He, Food Func ., 2019, 10, 2847–
2860.
103 M. Xie, G. Chen, P. Wan, Z. Dai, X. Zeng and Y. Sun,
J. Ag ic. Food Chem., 2018, 67, 171–183.
104 E. B. Mojze , M. K. H nčič,M.Ške ge , Ž. Knez and
U. B en, Molecules, 2016, 21, 901.
105 K. Amee , H. M. Shahbaz and J. H. Kwon, Comp . Re .
Food Sci. Food Sa ., 2017, 16, 295–315.
106 Y. Hu, B. Yan, Z. S. Chen, L. Wang, W. Tang and
C. Huang, J. Renewable Ma e ., 2022, 10, 1471–1490.
107 U. E xebe ia, N. A ias, N. Boque, M. T. Maca ulla,
M. P. Po illo, F. I. Milag o and J. A. Ma inez, Bene ic.
Mic obes, 2015, 1,97–111.
108 D. Po as, E. Nis al, S. Ma ínez-Fló ez, S. Pisone o-
Vaque o, J. L. Olcoz, R. Jo e , J. González-Gallego,
M. V. Ga cía-Media illa and S. Sánchez-Campos, F ee
Radicals Biol. Med., 2017, 102, 188–202.
109 J. Xie, W. Song, X. Liang, Q. Zhang, Y. Shi, W. Liu and
X. Shi, Biomed. Pha maco he ., 2020, 127, 110147.
110 S. Liu, Y. T. Loo, Y. Zhang and K. Ng, Food Chem., 2024,
434, 137508.
111 S. Lindahl, J. Liu, S. Khan, E. N. Ka lsson and C. Tu ne ,
Anal. Chim. Ac a, 2013, 785,50–59.
112 I. S. Choi, E. J. Cho, J.-H. Moon and H. J. Bae, Food Chem.,
2015, 188, 537–542.
113 L. Wang, Y. Wu, Y. Liu and Z. Wu, Molecules, 2017, 22,
1648.
114 P. Mikšo sky, C. Ko npoin ne , Z. Pa andeh,
M. Goessinge , K. Bica-Sch öde and H. Halbwi h,
ChemSusChem, 2024, 17, e202301094.
115 Z. Liu, Z. Ma, H. Zhang, B. S. Summah, H. Liu, D. An,
Q. Zhan, W. Lai, Q. Zeng and H. Ren, Biomed.
Pha maco he ., 2019, 120, 109482.
116 Y. Li, Z. Xie, T. Gao, L. Li, Y. Chen, D. Xiao, W. Liu, B. Zou,
B. Lu and X. Tian, Food Func ., 2019, 10, 4046–4061.
117 H. Z. Zheng, I. W. Hwang and S. K. Chung, J. Zhejiang
Uni ., Sci., B, 2009, 10, 912–919.
118 S. Chamo o, A. Vi e os, I. Al a ez, E. Vega and A. B enes,
Food Chem., 2012, 133, 308–314.
119 M.-R. Meini, I. Cabezudo, C. E. Bosche i and
D. Romanini, Food Chem., 2019, 283, 257–264.
120 A. Takagaki and F. Nanjo, Biol. Pha m. Bull., 2015, 38,
325–330.
121 A. Takagaki and F. Nanjo, Biosci., Bio echnol., Biochem.,
2016, 80, 199–202.
122 P. Dey, B. D. Olms ead, G. Y. Sasaki, Y. Vodo o z, Z. Yu
and R. S. B uno, J. Nu . Biochem., 2020, 84, 108455.
123 R. Pacheco-O daz, A. Wall-Med ano, M. G. Goñi,
G. Ramos-Clamon -Mon o , J. F. Ayala-Za ala and
G. González-Aguila , Le . Appl. Mic obiol., 2018, 66,25–31.
124 K. N. Ba uah, S. Singha, P. Mukhe jee and R. V. Uppalu i,
Biomass Con e s. Bio e in., 2024, 14, 24407–24425.
125 Y.-H. Hong, E. Y. Jung, Y. Pa k, K.-S. Shin, T. Y. Kim,
K.-W. Yu, U. J. Chang and H. J. Suh, Biosci., Bio echnol.,
Biochem., 2013, 77,22–29.
126 V. Ba es in, G. Macedo and V. De F ei as, Food Chem.,
2008, 108, 228–233.
127 A. Liang, W. Leona d, J. T. Beasley, Z. Fang, P. Zhang and
C. S. Ranadhee a, C i . Re . Food Sci. Nu ., 2024, 64,
7563–7588.
128 Y. Zhu, H. Sun, S. He, Q. Lou, M. Yu, M. Tang and L. Tu,
PLoS One, 2018, 13, e0195754.
129 X. Liu, K. K. Hu and V. S. Ha i os, Food Chem., 2024, 435,
137562.
130 W. Yue and F. Han, Food Chem.: X, 2022, 16, 100501.
131 L. Zhang, G. Fan, M. A. Khan, Z. Yan and T. Be a, Food
Chem., 2020, 323, 126714.
132 P. Amulya and R. ul Islam, Food Chem., 2023, 18, 100643.
133 L. Pan, H. Ye, X. Pi, W. Liu, Z. Wang, Y. Zhang and
J. Zheng, F on . Mic obiol., 2023, 14, 1092729.
134 M. Muelle , B. Za l, A. Schle i zko, M. S enzl,
H. Vie ns ein and F. M. Unge , Biop ocess Biosys . Eng.,
2018, 41, 221–228.
135 A. E. Paha ik, C. P. Pa le , N. Chung, D. A. Todd,
E. I. Rod iguez, M. J. Van Dyke, N. B. Cech and
A. R. Ho swill, Cell Hos Mic obe, 2017, 22, 746–756.
136 K. Bie and B. Schi ek, Exp. De ma ol., 2021, 30, 1442–
1452.
137 A. S acy and Y. Belkaid, Science, 2019, 363, 227–228.
138 S. Di Lodo ico, F. Gaspa i, E. Di Campli, P. Di Fe mo,
S. D’E cole, L. Cellini and M. Di Giulio, Mic oo ganisms,
2020, 9, 37.
139 C. Le Bou go , C. Meunie , E. Gaio, V. Mu a ,
M. Michele o, E. Tedesco and F. Bene i, Sci. Rep., 2022,
12, 9702.
140 A. P. I anko ić, A. Mili oje ić,M.Ćo o ić, M. Simo ić,
K. Banjanac, P. Jansen, A. Vukoičić, E. Van den Bogaa d
and D. Bezb adica, Chem. Biol. Technol. Ag ic., 2023, 10,
125.
141 A. P. I anko ić,M.Ćo o ić, A. Mili oje ić, M. Simo ić,
K. Banjanac, M. Veljko ićand D. Bezb adica, In . J. F ui
Sci., 2024, 24,85–101.
142 D. Gwiazdowska, K. Juś, J. Jasnowska-Małecka and
K. Kluczyńska, Ac a Biochim. Pol., 2015, 62, 895–901.
143 A. Duda-Chodak, J. Physiol. Pha macol., 2012, 63, 497–503.
144 Z. Zhang, X. Peng, S. Li, N. Zhang, Y. Wang and H. Wei,
PLoS One, 2014, 9, e90531.
145 Spo ligh on p o ein s uc u e design, Na . Bio echnol.,
2024, 42, 157.
146 L. Wang, K. Cao, M. M. Ped oso, B. Wu, Z. Gao, B. He and
G. Schenk, J. Biol. Chem., 2021, 297, 101262.
G een Chemis y Tu o ial Re iew
This jou nal is © The Royal Socie y o Chemis y 2025 G een Chem.,2025,27, 8777–8803 | 8801
Open Access A icle. Published on 14 May 2025. Downloaded on 11/19/2025 9:38:26 PM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online