In i o p o ein diges ion and mine al accessibili y o edible ilamen ous
ungi cul i a ed on wine y and dis ille y by-p oduc s
Luziana Hoxha
a,b
, Ricky Wang
b,*
, Mohammad J. Tahe zadeh
b
, Ing id Undeland
c
a
Depa men o Ag onomy, Food, Na u al Resou ces, Animals and En i onmen , Uni e si y o Pado a, 35020, Legna o, PD, I aly
b
Swedish Cen e o Resou ce Reco e y, Uni e si y o Bo ås, 50190, Bo ås, Sweden
c
Food and Nu i ion Science, Depa men o Li e Sciences, Chalme s Uni e si y o Technology, 412 96, Go henbu g, Sweden
ARTICLE INFO
Keywo ds:
Wine y and dis ille y by-p oduc s
Filamen ous ungi
Mine al accessibili y
P o ein diges ibili y
In i o diges ion
ABSTRACT
Edible ilamen ous ungi (mycop o ein) o e a sus ainable p o ein sou ce ha suppo s he upcycling o ag i- ood
indus y sides eams, s imula ing ci cula p oduc ion sys ems. This s udy e alua ed he nu i ional quali y,
including p o ein diges ibili y and mine al accessibili y o h ee edible ungal species Neu ospo a in e media (NI),
Aspe gillus o yzae (AO), and Rhizopus o yzae (RO), cul i a ed on wine y by-p oduc s (g ape ma c, wine lees, and
inasse) and on syn he ic glucose medium as a con ol. P o ein con en , amino acid p o ile, essen ial mine als, in
i o p o ein deg ee o hyd olysis (DH%), and mine al accessibili y we e assessed. One impo an hypo hesis
explo ed was whe he en iched ungal biomass polyphenol le els would nega i ely in luence p o ein diges ibili y
and mine al accessibili y. Wine lees suppo ed he highes biomass p o ein con en (27.2–30.6 % d y weigh ,
dw), ollowed by g ape ma c, and inasse. The amino acid p o ile e ealed ha essen ial amino acids comp ised
40.88–51.69 % o he o al p o ein, wi h lysine (8.43–14.18 %) and leucine (7.62–9.95 %) being he mos
abundan . No ably, RO g own in g ape ma c accumula ed highe polyphenol le el compa ed o NI and AO, up o
96 mg gallic acid equi alen /g dw. A e in i o diges ion, NI and AO e ealed highe p o ein diges ibili y han
RO (38–80 % s 9–53 % DH), and all ungal species cul i a ed in wine lees—pa icula ly RO—exhibi ed he
highes le els o accessible i on and zinc G ape ma c-g own RO showed signi ican ly educed p o ein di-
ges ibili y and mine al accessibili y. These indings p esen a p omising ou e o p oduce mycop o ein, while
lowe ing he wine and dis ille y sec o oo p in . Polyphenol le els should be op imized o a oid hampe ing
p o ein diges ibili y and mine al accessibili y.
1. In oduc ion
Add essing hunge , malnu i ion, and clima e change simul a-
neously aligns wi h Uni ed Na ions Sus ainable De elopmen Goal 2, 12,
and 13, which emphasize a die a y shi owa d sus ainable p o ein
sou ces p oduced in a esponsible manne wi h minimal en i onmen al
impac . Achie ing his will equi e maximizing esou ce e iciency such
as minimizing was e h oughou he ood alue chain. In his con ex ,
edible ilamen ous ungi ha e eme ged as p omising d i e s among
nume ous di e en al e na i e p o ein sou ces (Shahid e al., 2024).
They no only hos a aluable nu i ional p o ile cha ac e ized by high
le els o essen ial amino acids and die a y ibe s such as β-glucan, bu
a e also capable o u ilizing ag i- ood indus y sides eams as eeds ocks,
he eby p omo ing ci cula i y in ood p oduc ion sys ems (Ng e al.,
2024).
The wine y and dis ille y indus ies play a p ominen ole in he
Eu opean Union ag i- ood sec o . They gene a e subs an ial amoun s o
bo h solid and liquid o ganic and ino ganic esidues, collec i ely known
as “oenological by-p oduc s,” amoun ing o app oxima ely 20 million
ons annually wo ldwide (Hoxha, Tahe zadeh, & Ma angon, 2025). The
mos abundan oenological by-p oduc is g ape ma c, comp ising 30 %
(w/w) o p ocessed g apes du ing winemaking and con ain up o 60 % o
solid by-p oduc s (Nanni e al., 2021). Ano he oenological by-p oduc
includes p e-dis illa ion wine lees, collec ed a e alcoholic e men a-
ion, and he pos -dis illa ion wine lees o inasse, collec ed a e he
dis illa ion p ocess. These by-p oduc s, ich in polyphenols, p esen bo h
en i onmen al challenges and oppo uni ies o sus ainable alo iza ion
in ood, eed, bioene gy, cosme ics and many sec o s (Hoxha, Tahe za-
deh, & Ma angon, 2025).
The e is cu en ly an inc eased end in o he de elopmen and
* Co esponding au ho
E-mail add ess: [email p o ec ed] (R. Wang).
Con en s lis s a ailable a ScienceDi ec
Food Bioscience
jou nal homepage: www.else ie .com/loca e/ bio
h ps://doi.o g/10.1016/j. bio.2025.107711
Recei ed 1 Augus 2025; Recei ed in e ised o m 1 Oc obe 2025; Accep ed 3 Oc obe 2025
Food Bioscience 73 (2025) 107711
A ailable online 3 Oc obe 2025
2212-4292/© 2025 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/ ).
comme cializa ion o mycop o ein-based oods (Lübeck & Lübeck,
2022). The mycelial biomass o he ungal species Fusa ium enena um is
used as he main ing edien in comme cial mea al e na i es Quo n™
(K. Li, Yu, e al., 2023). Se e al o he species o ilamen ous ungi ha e
also been widely used o ood indus y applica ions such as soy sauce,
and sake ( ice wine) based on Aspe gillus o yzae (I o & Ma suyama,
2021), oncom based on Neu ospo a in e media (Maini Rekdal e al.,
2024); and empeh p oduc ion based on Rhizopus o yzae and Rhizopus
oligospo us (Ma ín-Migu´
elez e al., 2025). These ilamen ous ungi a e
also in e es ing om he pe spec i e o ood sys em ci cula i y since
hey can u ilize di e se ag i- ood indus y sides eams, including ish
p ocessing (Sa e al., 2021), pea p ocessing (Souza Filho e al., 2018),
dai y (Mahboubi e al., 2017), and o he ag icul u al sides eams (Maini
Rekdal e al., 2024) and a he same ime building up a high c ude
p o ein con en , ypically anging om 20 o 45 % dw.
An essen ial aspec dic a ing he nu i ional alue o ungi-based
oods is howe e he quali y o he p o eins being accumula ed,
including hei amino acid p o ile and diges ibili y. Fu he o his, he
accessibili y o essen ial mine als has eme ged c ucial when eplacing e.
g. ed mea as a main p o ein sou ce, wi h mo e en i onmen ally sound
p o ein sou ces (La unde-Dada e al., 2023). Wine y and dis ille y
by-p oduc s also con ain polyphenols (Chiappe o e al., 2023), which
may be u he accumula ed by ilamen ous ungi du ing e men a ion.
Polyphenols a e known o hei an ioxidan p ope ies, which ha e
bo h ood s abilizing e ec s (Lei e al., 2024) and po en ial heal h
bene i s (Rana e al., 2022). Howe e , hey can also in e ac wi h p o-
eins and mine als, leading o p ecipi a ion, which may educe nu ien s
bioa ailabili y (Lund, 2021; Thaku e al., 2019).
The s anda dized INFOGEST in i o ood diges ion p o ocol p o ides
a consis en app oach o e alua ing mac onu ien diges ibili y and
mic onu ien accessibili y. Exis ing in i o s udies add essing he p o-
ein diges ibili y o edible ilamen ous ungi ha e examined Fusa ium
enena um and species o Aspe gillus o yzae, Neu ospo a in e media, and
Rhizopus sp. (A i¨
ens e al., 2021; Colosimo e al., 2020; Wang e al.,
2023). Using he same model, we ha e p e iously assessed mine al
accessibili y o Rhizopus oligospo us (Wang e al., 2024). The INFOGEST
in i o me hod has also been success ully used o e alua e p o ein
diges ion o se e al o he mic obial biomasses such as bac e ia and yeas
(No dlund e al., 2024).
To he bes o he au ho s’ knowledge, no p e ious s udy has
in es iga ed he p o ein diges ibili y and mine al accessibili y o ila-
men ous ungi cul i a ed on wine y and dis ille y by-p oduc s. In he
p esen s udy, h ee species o edible ilamen ous ungi, Aspe gillus
o yzae, Neu ospo a in e media, and Rhizopus o yzae we e cul i a ed on
g ape ma c, p e-dis illa ion wine lees, and inasse, alongside a syn he ic
medium consis ing o glucose and yeas ex ac s as a con ol. Following
ha es , he ungal biomasses we e subjec ed o p o ein, essen ial amino
acids, essen ial mine als and polyphenol quan i ica ion, as well as in
i o diges ibili y and accessibili y assessmen . We hypo hesized ha
bo h he composi ion o he cul i a ion subs a es and he ungal species
would in luence he nu i ional quali y and polyphenol con en o he
ungal biomass. In ela ion o polyphenols, i was also hypo hesized ha
a highe accumula ion would hampe he ungal p o ein diges ibili y
and mine al accessibili y. This s udy p o ides new insigh s bo h in o he
nu i ional po en ial o ilamen ous ungi cul i a ed on wine y and
dis ille y by-p oduc s, highligh ing hei polyphenols, p o eins, and
mine als, and i s e ec s on nu ien diges ibili y and accessibili y.
2. Ma e ials and me hods
2.1. Chemicals
All chemicals we e o analy ical g ade (Sigma-Ald ich, Sweden).
Ni ic acid (68 % ace me al g ade) was pu chased om Fische Sci-
en i ic. Ul apu e wa e 18.2 MΩ-cm (Milli-Q IQ7000) was used o he
amino acid and mine al analysis. Pepsin om po cine gas ic mucosa
(P6887), bile bo ine, and panc ea in om po cine panc eas (8x USP,
P7545) we e pu chased om Sigma-Ald ich, Sweden.
2.2. Wine y and dis ille y by-p oduc s
In his s udy, he wine y and dis ille y by-p oduc s we e collec ed
be ween No embe and Decembe 2023 om he Acqua i e dis ille y
loca ed in Vazzola, Vene o, I aly. The g ape a ie ies used in he p o-
duc ion o hese by-p oduc s we e sou ced exclusi ely om he Vene o
egion. The g ape ma c (GM) consis ed o abou 80 % skins om he
P osecco/Gle a g ape a ie y, along wi h skins om o he whi e and ed
g ape a ie ies. GM was collec ed a e p essing and dis illa ion, ol-
lowed by mechanical seed emo al, d ying, and milling as desc ibed by
(Hoxha, Lenna sson, & Tahe zadeh, 2025; Hoxha, Tahe zadeh, &
Ma angon, 2025). The esul ing GM was acuum-packaged and s o ed a
4 ◦C du ing anspo and h oughou he s udy un il use.
P e-dis illa ion wine lees (WL) was collec ed du ing he cla i ica ion,
sedimen a ion, and acking s eps o P osecco wine p oduc ion, p io o
dis illa ion. The wine lees used in his s udy consis ed o a blend o bo h
g oss and ine lees, wi h an app oxima e composi ion o 60 % liquid and
40 % solids ac ions. The WL was s o ed a 4 ◦C du ing anspo and
h oughou he s udy un il use in maximum one mon h.
Vinasse o pos -dis illa ion wine lees (VIN) was gene a ed om he
dis illa ion o wine lees ha we e collec ed om mul iple wine ies and
blended in he dis ille y’s s o age anks. These wine lees we e s o ed o
2–3 weeks p io o dis illa ion. Vinasse was collec ed immedia ely a e
dis illa ion and s o ed a 4 ◦C un il u he use in maximum six mon hs.
Fo he u he analysis, GM was analyzed in i s o iginal o m. WL and
VIN we e eeze-d ied o ob ain solid samples.
2.3. Mic oo ganisms
Th ee edible ilamen ous ungal s ains we e sou ced om he
Wes e dijk Fungal Biodi e si y Ins i u e (U ech , The Ne he lands) o
his s udy. These included wo Ascomyce es—Aspe gillus o yzae a .
o yzae (CBS 819.72) and Neu ospo a in e media (CBS 131.92)—and one
Zygomyce e, Rhizopus o yzae a . delema (CBS 145940). The s ains
we e main ained on Po a o Dex ose Aga (PDA) composed o 4 g/L
po a o ex ac , 20 g/L glucose, and 15 g/L aga . Spo e suspensions we e
p epa ed by adding 20 mL o s e ile Milli-Q wa e o each PDA pla e,
ollowed by gen ly eleasing he spo es using a s e ile L-shaped plas ic
sp eade . The esul ing spo e-con aining suspension was hen collec ed
o use in subsequen ungal cul i a ions.
2.4. Fungal cul i a ions in 4 L bubble column eac o s
G ape ma c (GM) was hyd o he mally p e ea ed by p epa ing 4 %
(w/ ) solu ions and au ocla ing a 121 ◦C o 20 min. A e cooling o
oom empe a u e, he suspension was cen i uged and il e ed o ob ain
he GM liquo . P e-dis illa ion wine lees (WL) was cen i uged (4500 g
o 15 min a 4 ◦C) and he liquid ac ion was collec ed and used as
cul i a ion medium dilu ed 1:1 ( / ) wi h wa e . Vinasse (VIN) was
dilu ed wi h wa e in o a concen a ion o 10 % ( / ) and hus was used
as a suspension mix u e o ungal cul i a ions. No addi ional nu ien
supplemen a ion was added o he ungal cul i a ions. In addi ion,
ungi we e cul i a ed in a syn he ic glucose media (SYN), which was
p epa ed using 15 g/L glucose and 5 g/L yeas ex ac , se ing as a
con ol.
Bubble column bio eac o s (Belach Bio eknik, Sweden) we e illed
wi h 3 L o ei he 4 % g ape ma c liquo , 50 % dilu ed wine lees liquid
ac ion, 10 % dilu ed inasse, and syn he ic glucose media. They we e
s e ilized a 121 ◦C o 20 min, hen cooled a oom empe a u e and
inocula ed wi h 20 mL/L o spo e suspension. Cul i a ions we e con-
duc ed a 35 ◦C and pH 5, con olled wi h ei he 2 M NaOH o 2 M H
2
SO
4
acco ding o Hoxha, Lenna sson, and Tahe zadeh (2025). An i oam
BIOSPUMEX™ 200K (PMC Ou ie SAS, F ance) was added o con ol
L. Hoxha e al.
Food Bioscience 73 (2025) 107711
2
he excessi e oams (only o GM e men a ions). The cul i a ion las ed
48 h wi h a cons an ae a ion a e o 1 ol o ai pe olume o liquid pe
minu e. Biomass was ha es ed h ough a ki chen sie e 1 mm
2
. Ha -
es ed biomass was washed and d ied in a d ying o en a 70 ◦C o e -
nigh . D ied samples we e hen milled using a ball mill (Re sch,
Ge many) and subsequen ly subjec ed o mine al analysis, amino acid
analysis, polyphenol de e mina ion, and in i o gas oin es inal diges-
ion. Fungal cul i a ion was pe o med in duplica es (n =2) o WL, and
single eplica es (n =1) o GM, VIN, and SYN.
2.5. S a ic in i o gas oin es inal diges ion
The in i o gas oin es inal diges ion was pe o med ollowing he
INFOGEST 2.0 p o ocol (B odko b e al., 2019), including s anda dized
elec oly e composi ions o simula ed sali a y luid (SSF), simula ed
gas ic luid (SGF), and simula ed in es inal luid (SIF), and s anda dized
enzyme ac i i y measu emen s. Pepsin and panc ea in ac i i ies we e
measu ed a 2867 U/mg and 5.1 U/mg, espec i ely. Bo ine bile was
measu ed o con ain bile sal s a 1.55 mmol/mg. Mino modi ica ions, as
desc ibed by Wang e al. (2023), included a educ ion in panc ea in
enzyme and bile concen a ion.
B ie ly, 100 mg d ied ungal biomass was placed in a ube, and 1 mL
o wa e was added. While o al diges ion was omi ed, 1 mL o SSF
(wi hou sali a y amylase) was added. Gas ic diges ion was ini ia ed by
adding 2 mL o SGF con aining pepsin, wi h he inal concen a ion
adjus ed o 2000 U/mL in a o al gas ic olume o 4 mL. The mix u e
was incuba ed a pH 3 o 2 h. The in es inal phase was ini ia ed by
adding 4 mL o SIF con aining panc ea in and bile, adjus ed o each a
inal concen a ion o 10 U/mL and 1 mmol/L in he o al olume,
espec i ely. The pH was adjus ed o 7, and he mix u e was incuba ed
o ano he 2 h. Diges ion was s opped by adding 800
μ
L o soybean
ypsin-chymo ypsin/Bowman–Bi k inhibi o (0.05 g/L). All in-
cuba ions we e ca ied ou a 37 ◦C wi h gen le mixing (7 pm) using a
S ua Ro a o SB3 (UK). Blank diges ions we e ca ied ou by ollowing
he same p ocedu e as abo e, bu wi hou adding he d ied ungal
biomass. The in es inal diges s we e hen ozen and s o ed a −80 ◦C.
Each sample was diges ed in iplica e.
2.6. Analy ical me hods
2.6.1. Ca bon- o-ni ogen (C/N) a io
Solid subs a es, namely GM, eeze-d ied WL, and VIN we e
analyzed o hei ca bon- o-ni ogen (C/N) a io using a FlashSma ™
Elemen al Analyze (The mo Scien i ic, USA).
2.6.2. Amino acid p o iling
Amino acids we e analyzed ollowing he p ocedu e ou lined by
T igo e al. (2021). B ie ly, 50 mg o d ied ungal biomass sample was
combined wi h 8 mL o 6 M hyd ochlo ic acid (HCl). A e pu ging ai
om he ubes and eplacing i wi h ni ogen, he samples unde wen
hyd olysis a 110 ◦C o 24 h on a hea ing block. A e hyd olysis, 0.5 mL
aliquo o he hyd olysa e was d ied by lushing wi h ai , hen esus-
pended in 5 mL 0.2 M ace ic acid and il e ed using a 0.22
μ
m sy inge
il e p io o analysis wi h liquid ch oma og aphy–mass spec ome y
(LC-MS) as p e iously epo ed by T igo e al. (2021).
Two mic oli e s o each sample we e analyzed using an LC/MS sys-
em (Agilen 1100 HPLC and 6120B Single Quad upole MS), equipped
wi h a Phenomenex C18 column (250
μ
m ×4.6
μ
m ×3
μ
m).
Ch oma og am da a we e p ocessed using MassHun e Quan i a i e
Analysis so wa e ( e sion B.09.00, Agilen Technologies). Due o acid
hyd olysis, yp ophan could no be quan i ied, and aspa agine and
glu amine we e quan i ied alongside aspa ic acid and glu amic acid,
espec i ely. Resul s a e shown in e ms o o al amino acids (% dw) and
essen ial amino acids (% o o al amino acids).
2.6.3. Deg ee o hyd olysis
The quan i ica ion o he deg ee o p o ein hyd olysis (DH%) o he
sample o he in es inal diges s was accomplished u ilizing he o-
ph halaldehyde eagen (OPA) o measu e he p ima y amines. The OPA
eagen was eshly p epa ed by mixing 10 ml o OPA s ock solu ion
(0.05M in e hanol), 10 mL o n-acyl-L-cys eine (0.057 M in wa e ), 5 mL
SDS 20 %, and 75 mL 100 mM bo a e bu e . Aliquo o he in es inal
diges s was cen i uged a 2000 pm o 5 min. The supe na an was
dilu ed and hen added (100
μ
L) o he OPA eagen (1000
μ
L), incu-
ba ed a oom empe a u e o 10 min, and measu ed a 335 nm using a
UV–Vis spec opho ome e (Lib a, Bich om). Sample abso bance ead-
ings we e compa ed wi h he L-se ine s anda d cu e (0.15–0.8 mM).
The deg ee o hyd olysis was calcula ed by Equa ion (1):
Deg ee o hyd olysis (%) = hsample −hdiges ion blank
h o al (sample)
x100 Eq. 1
whe e h is he measu ed alue o o al p ima y amines (mmol se ine
equi alen s) by he OPA me hod, and h
o al
is he maximum numbe o
p ima y amines in each sample (mmol amino acid) ob ained h ough he
amino acid analysis.
2.6.4. Mine al analysis
Mine als, including phospho us (P), zinc (Zn), coppe (Cu), magne-
sium (Mg), and i on (Fe) we e quan i ied ollowing he p o ocol
desc ibed by Wang e al. (2024). Fo all solid samples, namely GM,
eeze-d ied WL and VIN, and o en d ied ungal biomass, 100 mg
samples we e ini ially ea ed wi h 1 mL o 68 % HNO
3
a 110 ◦C o 2 h.
Subsequen ly, an addi ional 1 mL o 68 % HNO
3
and 0.5 mL o 30 %
H
2
O
2
we e added, and he mix u e was hea ed o ano he 2 h a 110 ◦C.
A e diges ion, he samples we e ans e ed in o olume ic lasks o a
inal olume o 10 mL wi h Milli-Q wa e and il e ed h ough a 0.22
μ
m
PES il e . Mine al quan i ica ion was ca ied ou using Mic owa e
Plasma A omic Emission Spec oscopy (MP-AES 4200, Agilen Tech-
nologies). Analysis was conduc ed a wa eleng hs o 213.6 nm o P,
213.8 nm o Zn, 324.7 nm o Cu, 285.2 nm o Mg, 371.9 nm o Fe,
and 422.7 nm o Ca. S anda ds we e p epa ed using s anda d mix u es
(Agilen Technologies) anging om 0.05 ppm o 2 ppm o each mine al
in 2 % HNO
3
. All measu emen s we e conduc ed in iplica es.
To assess mine al accessibili y, il e ed diges s we e acidi ied o 2 %
HNO
3
and analyzed unde he same condi ions as desc ibed o he
ungal biomass. S anda d cu es o mine als anging om 0.05 o 2
ppm we e simila ly p epa ed using simula ed diges ion luid (a 1:2:4
mix u e o SSF, SGF, and SIF), and acidi ied o 2 % HNO
3
.
Mine al accessibili y was calcula ed using Equa ion (2):
2.6.5. To al phenolic con en
Fo he ungal biomass, ex ac s we e p epa ed ollowing he p o-
cedu e desc ibed by Qin e al. (2013), wi h mino modi ica ions. B ie ly,
100 mg o ungal biomass was mixed wi h 8 mL o an e hanol:wa e :HCl
Mine al accessibili y (%) = o al mine al in il e ed diges − o al mine al in il e ed blank diges
o al mine al in he 100 mg sample x100 Eq. 2
L. Hoxha e al.
Food Bioscience 73 (2025) 107711
3
solu ion (80:19.9:0.1, / / ) and o exed o 1 min. The mix u e was
incuba ed o 4 h a 37 ◦C and gen ly agi a ed a 7 pm using a o a o
(SB3, S ua ). A e incuba ion, he samples we e cen i uged a 3000×g
o 1 min, and he supe na an was il e ed using 0.22
μ
m il e s.
To al polyphenol con en was de e mined acco ding o he me hod
by Single on and Rossi (1965). Ex ac s we e combined wi h
Folin–Ciocal eu eagen (1:10 / ) and a 7.5 % Na
2
CO
3
solu ion in a
a io o 1:5:4. The mix u es we e incuba ed a oom empe a u e o 30
min, cen i uged a 18,900×g o 2 min, and abso bance measu ed a
765 nm using a UV–Vis spec opho ome e (Lib a, Bich om). Polyphenol
concen a ions we e calcula ed based on a gallic acid s anda d cu e
(7.5–250 ppm,
2
>0.99) and exp essed in millig ams o gallic acid
equi alen s (GAE) pe g am o d y ungal biomass. All analyses we e
pe o med in iplica e.
2.7. S a is ical analysis
S a is ical analysis was pe o med using Mini ab® 21.1.1 so wa e.
One way ANOVA was applied ollowed by Tukey’s pos hoc o pai wise
compa ison a a signi icance le el o 5 %. Di e en g oups o supe -
sc ip s a e used in ables o indica e he s a is ically signi ican pai wise
compa isons a a h eshold p- alue <0.05. Resul s in ables and igu es
a e p esen ed as mean alues ±s anda d de ia ion. Resul s in ables and
igu es a e p esen ed as mean alues ±s anda d de ia ion (n =3),
excep o cul i a ion in wine lees (n =5)
3. Resul s and discussion
This s udy aimed o e alua e he nu i ional quali y, in e ms o
p o ein con en , essen ial amino acid p o iles, mine al con en , p o ein
diges ibili y, and mine al accessibili y—o ilamen ous ungi cul i a ed
on wine y by-p oduc s. Th ee species o ilamen ous ungi; Neu ospo a
in e media (NI), Aspe gillus o yzae (AO), and Rhizopus o yzae (RO) we e
cul i a ed on h ee di e en wine y and dis ille y by-p oduc , namely
wine lees (WL), g ape ma c (GM), and inasse (VIN), and a syn he ic
glucose media (SYN) as a con ol. I was hypo hesized ha a ia ions in
he composi ion o cul i a ion medium and ungal s ains would a ec
he ungal biomass’s nu i ional quali y and o al polyphenol con en ,
wi h he la e po en ially a ec ing p o ein diges ibili y and mine al
accessibili y.
3.1. P o ein con en and essen ial amino acid p o ile o ungal biomass
The p o ein con en o ungal biomass was measu ed as o al con en
o amino acids, and esul s a e p esen ed in Table 1. When cul i a ed on
glucose media, he p o ein con en anged om 18.37 % o 21.5 % dw,
while when cul i a ed on wine lees esul ed in a signi ican ly highe
p o ein con en , anging om 27.25 % o 30.6 % dw. In con as , when
g ape ma c was used as he cul i a ion media, he p o ein con en was
signi ican ly lowe compa ed o wine lees and glucose media ac oss all
ungal species: NI a 16.1 %, AO a 10.98 %, and RO a 11.8 % dw.
Addi ionally, when inasse was used as he cul i a ion medium, no able
species-speci ic di e ences we e obse ed. NI showed he highes p o-
ein con en a 25.1 %, ollowed by AO a 18.89 %, while RO had he
lowes con en a 9.25 % dw.
The p o ein con en o N. in e media, A. o yzae, and R. o yzae in his
s udy was conside ably lowe compa ed o ou p e ious s udy (Wang
e al., 2023) ha epo ed 37.9 % o N. in e media, 45.1 % o A. o yzae,
and 32.1 % o R. o yzae. This di e ence is p ima ily a ibu ed o
cul i a ion media composi ion and cul i a ion pe iod. While he p e i-
ous s udy used 30 g/L glucose and 24-h cul i a ion pe iod, he p esen
s udy comp ised cul i a ion in 15 g/L glucose o 48 h. C ude p o ein
con en o ilamen ous ungi cul i a ed in syn he ic glucose media a e
ela i ely high be ween 42 and 56 %. The disc epancy be ween c ude
Table 1
P o ein con en ( o al amino acids) and essen ial amino acids p o ile o ilamen ous ungi N.in e media (NI), A.o yzae(AO), and R.o yzae (RO) cul i a ed on syn he ic
glucose media (SYN), p e-dis illa ion wine lees (WL), g ape ma c (GM), and inasse (VIN).
Subs a e Fungi
species
To al amino acids
(% dw)
Amino acid p o ile (% o al AA)
Lysine His idine Th eonine Valine Me hionine Isoleucine Leucine Phenylalanine Essen ial
AA
SYN NI 19.6 ±0.68
de
9.74 ±
0.1
b
2.87 ±
0.07
abcd
6.28 ±0.1
a
5.83 ±
0.08
e
1.31 ±0.03
4.62 ±
0.22
g
7.71 ±
0.23
cd
4.49 ±0.06
d
42.85 ±
0.51
de
AO 18.4 ±0.69
de
8.9 ±
0.23
b
2.67 ±
0.05
abcd
6.22 ±
0.02
abc
6.77 ±
0.37
abc
1.48 ±
0.08
e
5.2 ±
0.04
cde
8.29 ±
0.12
bcd
5.1 ±0.05
abcd
44.62 ±
0.79
cd
RO 21.5 ±1.22
cd
11.33 ±
0.03
ab
3.52 ±
0.04
a
6.22 ±
0.28
abc
6.31 ±
0.06
cde
1.51 ±
0.04
de
5.21 ±
0.23
cde
7.87 ±
0.16
cd
5.04 ±
0.07
abcd
47.01 ±
0.74
bc
WL NI 28.9 ±1.75
ab
9.92 ±
0.57
b
2.59 ±
0.28
bcd
6 ±0.83
abc
6.21 ±
0.15
de
1.74 ±
0.15
abcd
4.83 ±
0.14
e g
9.29 ±
0.79
ab
5.02 ±
0.26
abcd
45.61 ±
2.25
cd
AO 27.3 ±2.55
ab
9.69 ±
0.73
b
2.5 ±
0.38
bcd
5.76 ±
0.09
abc
7.04 ±
0.17
a
1.84 ±
0.06
ab
5.4 ±
0.16
bcd
9.35 ±
0.21
ab
5.28 ±0.11
abc
46.87 ±
0.95
bc
RO 30.6 ±2.49
a
14.18 ±
3.51
a
3.23 ±
0.5
ab
5.33 ±
0.14
c
6.9 ±
0.35
ab
1.9 ±0.18
a
5.83 ±
0.34
ab
8.92
±1
abc
5.39 ±0.45
ab
51.69 ±
1.55
a
GM NI 16.1 ±0.32
e
9.19 ±
0.07
b
1.95 ±
0.38
d
6.05 ±
0.29
abc
6.82 ±
0.02
abc
1.73 ±
0.05
abcde
5.32 ±
0.17
bcde
9.05 ±
0.07
abc
4.82 ±0.12
bcd
44.93 ±
0.34
cd
AO 10.9 ±0.78
g
8.43±0
b
1.95 ±
0.24
d
6.35 ±
0.28
a
7.35 ±
0.17
a
1.51 ±
0.08
de
5.63 ±
0.15
abc
9.95 ±
0.29
a
5.34 ±0.15
abc
46.5 ±
0.85
bc
RO 11.9 ±0.32
g
9.59 ±
0.23
b
2.27 ±
0.36
cd
6.26 ±
0.15
ab
7.28 ±
0.22
a
1.82 ±
0.08
abc
6.21 ±
0.34
a
9.91 ±
0.25
a
5.49 ±0.29
a
48.83 ±
1.08
ab
VIN NI 25.2 ±4.24
bc
8.55 ±
0.25
b
2.78 ±
0.06
abcd
5.68 ±
0.13
abc
5.63 ±
0.13
1.6 ±
0.03
bcde
4.43 ±
0.03
g
7.62 ±
0.15
d
4.59 ±0.04
d
40.88 ±
0.49
e
AO 18.89 ±0.9
de
11.39 ±
0.35
ab
2.57 ±
0.65
abcd
5.31 ±
0.03
bc
6.09 ±
0.18
de
1.58 ±
0.07
cde
4.92 ±
0.18
de g
7.82 ±
0.33
cd
4.79 ±0.09
cd
44.47 ±
0.61
cd
RO 9.25 ±0.7
g
8.77 ±
0.28
b
3.06 ±
0.11
abc
5.57 ±
0.38
abc
6.44 ±
0.08
bcd
1.51 ±
0.07
de
5.51 ±
0.16
bcd
7.88 ±
0.11
cd
5.24 ±0.14
abc
43.97 ±
0.63
cde
Adul amino acid equi emen (%p o ein)* 4.5 1.5 2.3 3.9 1.6 3.0 5.9 3.8
Da a a e exp essed as mean alues ±s anda d de ia ion. Fo all media n =3, excep wine lees n =5.
Adul amino acid equi emen is based on FAO/WHO ecommenda ion (FAO, 2007).
Di e en le e s in he same columns deno e s a is ically signi ican di e ences (p <0.05) among samples.
SYN: syn he ic glucose media, WL: p e-dis illa ion wine lees, GM: g ape ma c, VIN: inasse.
NI: Neu ospo a in e media, AO: Aspe gillus o yzae, RO: Rhizopus o yzae.
L. Hoxha e al.
Food Bioscience 73 (2025) 107711
4
p o ein con en and o al amino acids is mos likely due o he p esence
o non-p o ein ni ogen such as RNA, and chi in and chi osan in ungal
cell wall.
The signi ican di e ences in he p o ein con en obse ed o ungi
cul i a ed on di e en wine y and dis ille y by-p oduc s a e mos likely
d i en by se e al easons. One o he eason could be he cul i a ion
media di e s in he ca bon- o-ni ogen (C/N) a ios. The ungal biomass
N. in e media showed he highes p o ein con en when cul i a ed on
wine lees (C/N a io: 8.43), ollowed by inasse (C/N a io: 15.38) and
g ape ma c (C/N a io: 21.36). I is well es ablished ha he ca bon,
ni ogen, and phospho us con en o ilamen ous ungi is highly in lu-
enced by he cul i a ion media (Zhang & Else , 2017). P e ious s udy
using oa as cul i a ion medium showed a educ ion in p o ein con en
o Rhizopus oligospo us om 39 % in glucose media, wi h C/N a io o
7.54 o p o ein con en 21 % in oa -based media wi h C/N a io o 23.2
(Wang e al., 2024).
When cul i a ed on inasse, R. o yzae exhibi ed signi ican ly lowe
p o ein con en compa ed o N. in e media. This is likely due o he
mixed ungal biomass en angled wi h he un e men ed solid pa icle o
ungus R.o yzae. Vinasse also con ain la ge amoun s o da k colo pig-
men s; melanoidin, hyd oxyme hyl u u al, and u u al (Pan &
Adholeya, 2007). These p oduc s o he Mailla d eac ion and suga
ca ameliza ion a e known o inhibi ungal g ow h (Ka imi e al., 2005).
In ou expe imen , he inasse was equi ed o be dilu ed en- old o
accommoda e ungal g ow h. I is likely ha A. o yzae and N. in e media
ha e highe ole ance o hese inhibi o y compounds (Nai e al., 2018)
and capable o u ilizing he solid ac ion o inasse, hus achie ing mo e
ungal g ow h and highe p o ein con en .
The h ee ungal species cul i a ed on wine y and dis ille y by-
p oduc s exhibi ed high lysine con en , anging om 8.43 % o 14.18
% o he o al amino acids dw. The highes lysine con en was achie ed
by R. o yzae when cul i a ed on wine lees. This high lysine le el o
ilamen ous ungi is a ibu ed o he ungi abili y o syn hesize lysine
ia he
α
-aminoadipa e pa hway (Xu e al., 2006). His idine le els in
ungi cul i a ed on g ape ma c (1.95–2.27 %) we e signi ican ly lowe
han hose in ungi g own on syn he ic media (2.67–3.62 %) and wine
lees (2.50–3.23 %). In con as , ungi g own on g ape ma c (9.05–9.95
%) was oge he wi h ungi g own on wine lees (8.92–9.29 %) signi i-
can ly highe in leucine compa ed o ungi cul i a ed on syn he ic media
(7.71–8.29 %) and inasse (7.62–7.88 %).
Based on he ecommended daily p o ein in ake o 0.66 g pe kilo-
g am o body weigh , he essen ial amino acid equi emen s o adul
humans, as ecommended by FAO/WHO (FAO, 2007), a e: his idine (15
mg/g p o ein), isoleucine (30 mg/g), leucine (59 mg/g), lysine (45
mg/g), me hionine (16 mg/g), phenylalanine and y osine (38 mg/g),
h eonine (23 mg/g), yp ophan (6 mg/g), and aline (39 mg/g). The
ilamen ous ungi cul i a ed on wine y and dis ille y by-p oduc s had
p o ein con en s anging om 9 % o 30 % dw, which is lowe han ha
o con en ional animal-based p o ein sou ces such as bee , chicken, and
ish a abou 50–90 % dw (Wang e al., 2023). Howe e , he amino acid
p o iles o he ungal biomass ul ill FAO/WHO essen ial amino acid
equi emen s, excep o me hionine, which only me o su passed he
equi emen when he ungi we e cul i a ed on wine lees and g ape
ma c. Among he essen ial amino acids, lysine and leucine a e pa icu-
la ly impo an om a nu i ional s andpoin . Lysine is he limi ing
amino acid in mos ce eal g ains, and is a key amino acid in p o ein
syn hesis, collagen o ma ion, and nu ien abso p ion, while leucine
plays a key ole in s imula ing muscle p o ein syn hesis (Young & Pel-
le , 1990). Gi en i s well-balanced amino acid p o ile, he ungal
biomass s udied in his wo k could se e as a aluable al e na i e p o-
ein sou ce, especially as a complemen o plan -based die s.
3.2. Essen ial mine al con en o ungal biomass
The essen ial mine al con en o he ungal biomass o he h ee
edibles ilamen ous ungi species cul i a ed on h ee di e en wine y
and dis ille y by-p oduc s we e quan i ied and a e p esen ed in Table 2.
The e we e no signi ican di e ences be ween he h ee ungal species
cul i a ed in syn he ic glucose media wi h espec o phospha e (P)
con en ; 984–1148 mg/100 g dw. Howe e , cul i a ion using wine lees
signi ican ly inc eased he phospho us con en in NI and RO, up o 2514
mg/100 g dw. When g ape ma c was used, RO had signi ican ly highe
phospho us con en compa ed o NI and AO, while cul i a ion using
inasse, NI exhibi ed he highes phospho us con en .
The adequa e in ake in adul s o phospho us is 520 mg/day and he
ole able uppe limi in ake pe day is 3000 mg (Blomho e al., 2023).
Fungi cul i a ed on wine lees and inasse, as well as RO g own on g ape
ma c, had signi ican ly highe phospho us le els, up o 2514 mg/100g
dw. Excess phospho us in ake can nega i ely impac bone and kidney
heal h (Blomho e al., 2023). The e o e, addi ional ea men s, such as
hea ea men (V doljak e al., 2015) should be conside ed o educe
phospho us con en in ha es ed ungal biomass.
I on (Fe) con en o ungal biomass cul i a ed in syn he ic media
anged om 3.3 o 5.2 mg/100 g dw. Cul i a ion in wine lees signi i-
can ly inc eased i on concen a ions ac oss all ungal species, pa icu-
la ly in RO (117.3 mg/100 g dw) compa ed o NI (57.7 mg/100 g dw)
and AO (45.7 mg/100 g dw). Cul i a ion in g ape ma c also esul ed in
highe i on con en s, wi h RO again ha ing he highes con en (85.9
mg/100 g dw), ollowed by NI and AO. Fungal biomass cul i a ed on
inasse showed in e media e i on con en , lowe han hose cul i a ed
Table 2
Con en o essen ial mine als o ilamen ous ungi N.in e media (NI), A.o yzae(AO), and R.o yzae (RO) cul i a ed on syn he ic glucose media (SYN), p e-dis illa ion
wine lees (WL), g ape ma c (GM), and inasse (VIN).
Subs a e Fungi species Mine al con en o ungal biomass (mg/100 g)
P Ca Mg Fe Zn Cu
SYN NI 984.8 ±28.8
41.5 ±1.25
72.3 ±1.44
4.8 ±0.12
e
10 ±0.36
de
1.4 ±0.21
e
AO 990.6 ±6.05
6.5 ±0.43
104.1 ±0.57
de
3.3 ±0.03
e
8 ±0.03
e
0.3±0
RO 1148.7 ±69.7
e
43.1 ±0.36
e
72.1 ±3.3
e
5.2 ±0.26
e
11.2 ±0.43
de
0.5 ±0.01
WL NI 2513.5 ±75.2
a
215.5 ±7.75
cd
224.9 ±8.87
a
57.7 ±2.43
c
46.8 ±1.1
b
7.4 ±0.14
c
AO 1558.3 ±91.6
cde
98.9 ±3.23
e
124.6 ±5.78
cd
45.7 ±2.32
c
9.8 ±0.78
de
10.3 ±0.27
ab
RO 2333.5 ±63.3
a
296.3 ±1.96
bc
240.8 ±3.64
a
117.3 ±1.36
a
55.9 ±0.62
a
11.6 ±0.17
a
GM NI 1189.4 ±245.3
e
213.3 ±28.11
cd
116.3 ±19.89
d
49.5±2
c
18.3 ±4.28
c
2.5 ±0.09
de
AO 1128.4 ±47.5
148 ±2.4
de
131.8 ±3.64
bcd
37.6 ±0.72
cd
3.7 ±0.16
1.7 ±0.1
de
RO 1845.6 ±256.3
bc
716.5 ±99.8
a
157 ±13.24
b
85.9 ±21.18
b
19.8 ±4.23
c
3.2 ±1.32
d
VIN NI 2246.1 ±22.3
ab
383.6 ±3.12
b
152.6 ±2.56
bc
14.3 ±0.3
de
21.8 ±0.1
c
2.9 ±0.12
de
AO 1646.3 ±17
cd
393.3 ±1.88
b
155.9 ±1.25
bc
17.5 ±1.49
de
16.5 ±0.1
cd
2.6 ±0.02
de
RO 1314.7 ±253.19
de
272 ±19.22
bc
163.8 ±20.03
b
34.2 ±7.39
cd
20.9 ±2.62
c
8.5 ±1.59
bc
Da a a e exp essed as mean alues ±s anda d de ia ion. Fo all media n =3, excep wine lees n =5.
Di e en le e s in he same columns deno e s a is ically signi ican di e ences (p <0.05) among samples.
SYN: syn he ic glucose media, WL: p e-dis illa ion wine lees, GM: g ape ma c, VIN: inasse.
NI: Neu ospo a in e media, AO: Aspe gillus o yzae, RO: Rhizopus o yzae.
L. Hoxha e al.
Food Bioscience 73 (2025) 107711
5
in wine lees and g ape ma c, ye highe han hose in syn he ic glucose
media, wi h RO (34.2 mg/100 g dw) showing he highes i on con en .
Based on he mine al con en o he subs a e, we es ima ed i on
concen a ion in all he cul i a ion media a : 10 ppm o wine lees, 9.5
ppm o g ape ma c, 1.6 ppm o inasse, and 1.1 ppm o glucose media
(Table S3). The i on con en o ungi hus appea ed being dependen on
he cul i a ion medium, wi h ungi cul i a ed on wine lees ha ing he
highes i on con en while he lowes le el was ound o ungi cul i-
a ed on glucose media.
In ungi, i on se es as a c i ical co ac o in a ious essen ial en-
zymes. Among he ungal species examined, RO consis en ly exhibi ed
he highes i on con en , pa icula ly when cul i a ed using wine lees
and g ape ma c. The ilamen ous ungi om Rhizopus genus ha e unique
biological capabili y o s o e excess i on in e i in-like p o eins
(Canessa & La ondo, 2013). Fe i ins e ec i ely seques e la ge
amoun s o i on, he eby mi iga ing po en ial oxici y, including damage
om he Fen on eac ion associa ed wi h low molecula weigh i on
ions. Fu he esea ch is equi ed in ol ing di ec measu emen s o
e i in p o ein abundance o alida e hese indings.
Rega ding calcium (Ca) con en , NI and RO cul i a ed in syn he ic
media con ained 41.5 and 43.1 mg/100 g dw, espec i ely, whe eas AO
exhibi ed signi ican ly lowe calcium con en a 6.5 mg/100 g dw.
Cul i a ion in wine lees, g ape ma c, and inasse ma kedly inc eased he
calcium con en in all ungal biomasses compa ed o syn he ic media,
wi h alues anging om 98 o 716 mg/100 g dw. Fungi biomass wi h
he highes calcium con en was obse ed in RO g own on g ape ma c.
We es ima ed he calcium concen a ion o he cul i a ion media a 52,
342 and 18 ppm o wine lees, g ape ma c, and inasse, espec i ely
(Table S2). These esul s e lec ed highe calcium concen a ion in he
media could yield ungal biomass wi h highe calcium con en .
The zinc (Zn) con en o h ee species o ungi biomass g own in
syn he ic glucose media anged om 8.0 o 11.2 mg/100 g dw. When
cul i a ed in wine lees, NI (46.8 mg/100 g dw) and RO (55.9 mg/100 g
dw) showed much highe zinc le els, while AO emained low a 9.8 mg/
100 g dw. A simila pa e n was obse ed wi h g ape ma c: NI and RO
had highe zinc con en s (18.3 and 19.8 mg/100 g dw, espec i ely),
whe eas AO was again lowe (3.7 mg/100 g dw). In con as , cul i a ion
in inasse esul ed in compa able zinc le els ac oss all h ee ungal
species, anging om 16.5 o 21.8 mg/100 g dw.
The ungus A. o yzae consis en ly exhibi ed lowe calcium and zinc
accumula ion compa ed o N. in e media and R. o yzae. Unde excess
mine al condi ions, ungi can main ain zinc and calcium homeos asis
h ough wo main mechanisms: e lux anspo ac oss he plasma
memb ane, which emo es excess mine als, and in acellula s o age in
acuoles (Lange & Pei e , 2020; Robinson e al., 2021). The lowe zinc
and calcium le els obse ed in A. o yzae sugges ha his species may
p ima ily egula e homeos asis h ough e lux anspo . In con as ,
N. in e media and R. o yzae may ely mo e on acuola s o age. These
hypo heses equi e u he in es iga ion, including s udies wi h de ined
media o examine he e ec s o zinc concen a ion on he g ow h and
mine al accumula ion o hese species.
3.3. To al polyphenol con en o ungal biomass
Filamen ous ungi cul i a ed in syn he ic glucose media exhibi ed
mode a e polyphenol le els (17.1–23.1 mg GAE/g biomass dw), while
hose g own on wine lees showed lowe concen a ions (7.18–12.36 mg
GAE/g biomass dw), wi h no signi ican di e ences among ungal spe-
cies (Fig. 1). In compa ison, cul i a ion on g ape ma c led o signi i-
can ly highe polyphenol con en . RO showed he highes le el (96.42
mg GAE/g biomass dw), while NI and AO displayed mode a e alues
(26.38 and 28.92 mg GAE/g biomass dw, espec i ely). Fungal biomass
cul i a ed on inasse had ela i ely low polyphenol con en
(10.25–12.57 mg GAE/g dw), simila o wine lees, again wi hou no able
species-speci ic di e ences.
Compa ed o he Ascomyce e ungi N. in e media and A. o yzae, he
Zygomyce e ungi R. o yzae exhibi ed signi ican ly g ea e polyphenol
accumula ion when cul i a ed in g ape ma c. This is could be due o
di e ences in cell wall composi ion. R. o yzae con ains chi osan in i s
cell wall (S ensson e al., 2021), which con ains ee amino g oups om
glucosamine ha can in e ac wi h and o m complexes wi h poly-
phenols (Popa e al., 2000; Sun e al., 2014; Yi e al., 2021). Ano he
hypo hesis is ha ilamen ous ungi accumula e only speci ic poly-
phenols, and his accumula ion depends on he ungal species. Addi-
ionally, wine lees, inasse, and g ape ma c di e signi ican ly in hei
polyphenol p o iles (da a is no shown). Howe e , u he s udies a e
equi ed o in es iga e he unde lying mechanisms and o explain why
R. o yzae beha es in his manne .
P e ious s udies suppo he capaci y o ilamen ous ungi o g ow in
polyphenol- ich media, ole a ing he inhibi o y p ope ies o poly-
phenol on ce ain mic oo ganism (Wikanda i e al., 2015). While
Aspe gillus and Neu ospo a biomass yields emain una ec ed by high
polyphenol concen a ions (2.4–240 mg/L), Rhizopus oligospo us—an-
o he Zygomyce e—has shown a h ee- old inc ease in biomass yield
when cul i a ed in media con aining 240 mg/L ellagic acid compa ed o
2.4 mg/L (Bulkan, Si a esmi, e al., 2022; Bulkan, Yudhan i, e al.,
2022). This sugges s ha ce ain ilamen ous ungi no only ole a e, bu
also may bene i om polyphenol- ich subs a es.
Polyphenols a e known o hei an ioxidan p ope ies, which can
enhance he s o age s abili y o p oduc s by p e en ing lipid oxida ion.
Fo example, a ecen s udy showed ha using lingonbe y p ess cake in
combina ion wi h he ing es aw ma e ials du ing p o ein ex ac ion
imp o ed he p oduc ’s s o age s abili y in a dose-dependen manne
(Zhang e al., 2024). Since Rhizopus o yzae can accumula e polyphenols,
his highligh s i s po en ial o use in alo izing indus ial sides eams
ha a e ich in bo h polyphenols and polyunsa u a ed a y acids, such
as sides eams om oli e o apeseed p ocessing.
3.4. P o ein deg ee o hyd olysis o ungal biomass ollowing in i o
diges ion
In he p esen s udy, p o ein diges ibili y was assessed a e in i o
diges ion by measu ing he p o ein deg ee o hyd olysis. The deg ee o
hyd olysis (DH%) indica es he ex en o p o ein hyd olysis in a sample,
de e mined by compa ing he numbe o p ima y amines in he diges ed
sample o he o al amino acids. Fig. 2 depic s he o al DH% be o e and
a e in es inal diges ion o he h ee species o ungal biomass cul i a ed
on syn he ic glucose media and h ee di e en wine y and dis ille y by-
p oduc s (g ape ma c, wine lees and inasse).
When g own on glucose media, all h ee ungal species Rhizopus
Fig. 1. Polyphenol con en (mg GAE/d dw biomass) o ilamen ous ungi N.
in e media, A.o yzae, and R.o yzae cul i a ed on syn he ic glucose media (SYN),
p e-dis illa ion wine lees (WL), g ape ma c (GM), and inasse (VIN). Da a a e
exp essed as mean alues ±s anda d de ia ion depic ed as e o ba s. Fo all
media n =3,excep wine lees, n =5. Values ha do no sha e he same sub-
sc ip s deno e s a is ically signi ican di e ences (p <0.05) among samples.
L. Hoxha e al.
Food Bioscience 73 (2025) 107711
6
o yzae, Neu ospo a in e media, and Aspe gillus o yzae exhibi ed simila ly
high o al DH alues a he end o he diges ion, anging om 84 % o 95
%, wi h no signi ican di e ences among species. Cul i a ion on wine
lees esul ed in a mode a e bu non-signi ican educ ion in o al DH%,
wi h alues be ween 72 % and 82 % ac oss all ungal species. Using
g ape ma c, a dis inc pa e n eme ged: while N. in e media and A. o yzae
main ained high o al DH% le els (86 % and 101 %, espec i ely),
R. o yzae showed a d ama ic decline o jus 20.4 %, indica ing a sub-
s an ial inhibi ion o p o ein hyd olysis in his ungus species. On
inasse, o al DH% alues o N. in e media and A. o yzae emained
compa able a 78 %, whe eas R. o yzae again exhibi ed a lowe o al DH
a 56.6 % compa ed o wine lees.
As shown in Fig. 2a, he e we e high ini ial DH% in he di e en
ilamen ous ungi p io o diges ion, e lec ing p ima y amines eleased
in o he aqueous phase be o e he addi ion o diges i e enzymes. This
may e lec he p esence o non-p o ein ni ogen sou ces (e.g., RNA),
sho e pep ides, o ee amino acids wi hin he ungal cy oplasm.
The ne p o ein diges ibili y (calcula ed as o al DH% – ini ial DH%)
esul ing om gas oin es inal diges ion anged om 38 o 80 % o
N. in e media and A. o yzae. Pai wise compa isons showed ha
N. in e media cul i a ed on g ape ma c had signi ican ly highe di-
ges ibili y han when cul i a ed on glucose medium bu was no
signi ican ly di e en om N. in e media cul i a ed on wine lees. In
con as , A. o yzae cul i a ed on g ape ma c exhibi ed signi ican ly
highe diges ibili y han when cul i a ed on ei he glucose medium and
wine lees. Fo R. o yzae, inasse esul ed in highes ne p o ein di-
ges ibili y among ou subs a es, a abou 53 %, while diges ibili y was
s ill e y low (<10 %) when using g ape ma c as cul i a ion media.
In compa ison o con en ional p o ein sou ces, he diges ibili y
alues obse ed o ungal biomass a e wi hin he ange epo ed o
animal-based p o eins such as chicken (62 %), bee (60 %), and salmon
(67 %) unde simila in i o condi ions (Wang e al., 2023). Among
al e na i e sou ces, algae p o ein ypically shows a DH o a ound 30 %
(Ve sp ee e al., 2021), while edible mush ooms each app oxima ely
40 % (X. Li, Yu, e al., 2023). Ce eal-de i ed p o ein concen a es om
oa , whea , and ba ley exhibi DH alues anging om 50 o 60 % (Gong
e al., 2022), and comme cial plan -based bu ge s o mula ed om pea
p o ein ha e epo ed DH alues be ween 50 and 58 % (Cu oneo e al.,
2023). These compa isons sugges ha ungal p o eins cul i a ed on
wine y sides eams can achie e diges ibili y le els compa able o bo h
adi ional and al e na i e p o ein sou ces.
The no ably low DH% obse ed in R. o yzae cul i a ed on g ape ma c
suppo s ou hypo hesis ha a high polyphenolic con en in he ungal
biomass may hinde he in i o p o ein diges ibili y. Phenolic com-
pounds can in e e e wi h p o ein hyd olysis by in e ac ing wi h bo h
p o ein subs a es and diges i e p o eases. Thei binding o p o-
eins—p ima ily h ough hyd ophobic in e ac ions—can educe acces-
sibili y o p o eases ha p e e en ially clea e a hyd ophobic esidues
(Ci ko ic Velicko ic & S anic-Vucinic, 2018). While he speci ic in-
e ac ions be ween g ape-de i ed polyphenols and ungal p o eins ha e
no ye been in es iga ed, he inhibi o y e ec s o g ape polyphenols on
diges i e enzymes a e well documen ed. Fo example, p ocyanidins can
inhibi ypsin ac i i y depending on hei deg ee o polyme iza ion, and
bo h es e a ol and an hocyanins ha e been shown o inhibi pepsin
ac i i y (Ci ko ic Velicko ic & S anic-Vucinic, 2018). Iden i ica ion o
he speci ic polyphenols accumula ing in he ungal biomass is subjec
o a u u e s udy.
The Ascomyce e ungi, pa icula ly N. in e media, main ained ela-
i ely high p o ein con en and p o ein diges ibili y ac oss all wine y
and dis ille y by-p oduc s explo ed. This highligh s i s supe io i y as a
nu i ious al e na i e p o ein sou ce among he h ee ungal species
s udied. In con as , he high polyphenol con en o R. o yzae comes wi h
he challenge o lowe p o ein diges ibili y when cul i a ed on g ape
ma c. The e o e, u he s udies a e needed o imp o e he p o ein di-
ges ibili y o R. o yzae, especially when g own on g ape ma c.
3.5. Mine al accessibili y o ungal biomass
Mine al accessibili y desc ibes he ex en o which essen ial mine als
in solid oods a e eleased and solubilized a e gas oin es inal diges-
ion. In his s udy, he insoluble po ion o he in i o diges ed samples
was sepa a ed om he soluble ac ion using a 0.2
μ
m il e . The
accessibili y o i on (Fe) and zinc (Zn) was de e mined by measu ing he
pe cen age o each mine al eleased in o he soluble ac ion ela i e o
he o al con en in he o iginal solid sample (Figs. 3a and 4a).
Filamen ous ungi g own on syn he ic glucose media showed he
highes mine al accessibili y o e all. I on accessibili y eached 100 %
o NI and AO, while RO showed a lowe alue o a ound 52 %. When
wine lees was used as he cul i a ion media, Fe accessibili y dec eased
ac oss all ungal species, anging om 24 o 34 %. The mos d ama ic
educ ion in Fe accessibili y was obse ed in ungi g own on g ape ma c,
whe e alues d opped below 9 % o NI and AO, and became negligible
o RO. A simila pa e n eme ged wi h inasse, whe e NI and AO
e ained mode a e Fe accessibili y (23–33 %), bu RO again exhibi ed
nea -ze o le els.
Zinc accessibili y ollowed a simila end. Th ee species o ungi
Fig. 2. P o ein deg ee o hyd olysis (DH%) (a) o al DH%, including ini ial DH
%, and (b) ne DH% o he ilamen ous ungi N.in e media (NI), A.o yzae (AO),
and R.o yzae (RO) cul i a ed on syn he ic glucose media (SYN), p e-dis illa ion
wine lees (WL), g ape ma c (GM), and inasse (VIN). “Ini ial DH” shows he DH
% p esen in samples p io o he diges ion. “ne DH” shows he DH% ob ained
in he in es inal s ep, induced by pepsin and panc ea in a e gas oin es inal
diges ion. Da a a e exp essed as mean alues ±s anda d de ia ion depic ed as
e o ba s. Fo all media n =3,excep wine lees, n =5. Values ha do no sha e
he same subsc ip s deno e s a is ically signi ican di e ences (p <0.05)
among samples.
L. Hoxha e al.
Food Bioscience 73 (2025) 107711
7
cul i a ed on wine lees esul ed in Zn accessibili y a ound 22–26 %, a
mode a e educ ion compa ed o syn he ic media, anging om 39 % o
77 %. G ape ma c cul i a ion led o he lowes alues, wi h NI unde 10
%, while AO and RO e en showed nega i e alue, indica ing an i-
nu ien p ope ies o zinc. In inasse-g own ungal biomass, NI and AO
main ained mode a e Zn accessibili y (25–27 %); howe e , RO
emained low (app oxima ely 2.85 %).
I is impo an o no e ha p esen ing mine al accessibili y in ela i e
e ms (i.e., as a pe cen age o o al mine al con en in he biomass), can
be misleading when compa ing biomass wi h e y di e en mine al
con en . The o al accessible mine als a e in i o diges ion o ungi a e
p esen ed in Figs. 3b and 4b. Al hough ungi g own on syn he ic media
showed he highes ela i e i on accessibili y, he absolu e amoun o
i on eleased in o he soluble phase om a 100 mg ungal sample was
signi ican ly lowe , anging om 2.7 o 4.9
μ
g Fe. In con as , ungi
cul i a ed on wine lees, eleased signi ican ly highe o al amoun o
i on (10–40
μ
g Fe). Despi e o he signi ican ly lowe accessibili y pe -
cen age alue compa ed o syn he ic media, bo h Ascomyce es ungi, NI
and AO, cul i a ed on g ape ma c and inasse e ealed simila o al i on
elease (3.3–3.6 and 3.3–5.9
μ
g Fe, espec i ely). Howe e , RO showed
negligible alues when cul i a ed on g ape ma c and inasse. The same
pa e n was also exhibi ed o zinc excep o AO, which ga e ela i ely
low amoun s o accessible zinc when cul i a ed bo h on wine lees and
g ape ma c.
These esul s suppo ou hypo hesis ha he accessibili y o essen ial
mine als om ilamen ous ungi could be a ec ed by bo h he ungal
species and he ype o wine y and dis ille y by-p oduc ; pa icula ly he
po en ial o he ungi o accumula e an inu ien s om he subs a e.
Thus, ha ungal biomass cul i a ed on syn he ic glucose media
exhibi ed he highes ela i e i on and zinc accessibili y may be a ib-
u ed bo h o he lowe o al mine al con en in hese samples and he
absence o an inu ien s. In con as , ilamen ous ungi cul i a ed on
wine y and dis ille y by-p oduc s, such as wine lees and g ape ma c,
accumula ed subs an ially highe mine al le els, e lec ing he le els
p esen in hese by-p oduc s and likely he ungi abili y o s o e excess
i on and zinc in acellula ly (Robinson e al., 2021). We hypo hesized
ha mine als s o ed in ese e o ms a e less bioaccessible han hose
inco po a ed in o unc ional oles such as enzyme co ac o s, howe e
u he esea ch is needed o con i m his hypo hesis.
The subs an ially educed mine al accessibili y obse ed in R. o yzae
cul i a ed on g ape ma c suppo s he hypo hesis ha accumula ed
polyphenols in he ungal biomass nega i ely a ec mine al solubili y.
Fig. 3. (a) I on accessibili y (%), and (b) o al accessible i on (
μ
g pe 100 mg
dw) o he di e en ilamen ous ungi N.in e media (NI), A.o yzae (AO), and R.
o yzae (RO) cul i a ed on syn he ic glucose media (SYN), p e-dis illa ion wine
lees (WL), g ape ma c (GM), and inasse (VIN). Da a a e exp essed as mean
alues ±s anda d de ia ion depic ed as e o ba s. Fo all media n =3,excep
wine lees, n =5. Values ha do no sha e he same subsc ip s deno e s a is i-
cally signi ican di e ences (p <0.05) among samples.
Fig. 4. (a) Zinc accessibili y (%), and (b) o al accessible zinc (
μ
g pe 100 mg
dw) o he di e en ilamen ous ungi N.in e media (NI), A.o yzae (AO), and R.
o yzae (RO) cul i a ed on syn he ic glucose media (SYN), p e-dis illa ion wine
lees (WL), g ape ma c (GM), and inasse (VIN). Da a a e exp essed as mean
alues ±s anda d de ia ion depic ed as e o ba s. Fo all media n =3,excep
wine lees, n =5. Values ha do no sha e he same subsc ip s deno e s a is i-
cally signi ican di e ences (p <0.05) among samples.
L. Hoxha e al.
Food Bioscience 73 (2025) 107711
8
Phenolic compounds a e well known o impai i on bioa ailabili y by
o ming insoluble i on(III)-phenol complexes in he gas oin es inal
ac (Saini e al., 2016). Addi ionally, phenolic compounds ha e been
shown o in e ac wi h zinc, wi h he s eng h o binding a ying
depending on he speci ic phenolic s uc u e (Cle geaud e al., 2016;
Duan e al., 2023).
An in e es ing end was obse ed wi h ungi cul i a ed on inasse.
While NI and AO main ained mode a e le els o i on and zinc accessi-
bili y, RO showed a ma ked educ ion. One plausible explana ion is he
men ioned p esence o melanoidins—complex Mailla d eac ion p od-
uc s known o bind i on and po en ially educe i s bioa ailabili y (Pan
& Adholeya, 2007). The lack o inhibi o y e ec in NI and AO is unclea ,
howe e , i migh be due o ha NI and AO accumula ed less o he
melanoidins compa ed o RO.
In his s udy, mine al accessibili y was assessed by measu ing he
p opo ion o soluble mine als in he in es inal phase de i ed om in
i o diges ion. Fu he wo k is equi ed o e alua e he bioa ailabili y o
mine als om ilamen ous ungi g own on wine y and dis ille y by-
p oduc s ha include he in es inal abso p ion, ei he h ough in i o
s udies using Caco-2 in es inal cell models o ul ima ely in in i o ials.
4. Conclusion
In his s udy, he con en o p o ein, mine als and polyphenols was
e alua ed along wi h essen ial amino acid p o ile, p o ein diges ibili y
and mine al accessibili y o h ee edible ilamen ous ungi species,
Neu ospo a in e media, Aspe gillus o yzae, and Rhizopus o yzae, g own on
h ee wine y and dis ille y by-p oduc s: g ape ma c, wine lees, and
inasse. I was demons a ed ha he nu i ional quali y o edible ila-
men ous ungi was s ongly a ec ed by he cul i a ion subs a e and
ungal species, e lec ing di e ences in nu ien composi ion and up ake
capaci y. Fungi g own on wine lees exhibi ed highe p o ein con en
compa ed o hose cul i a ed on g ape ma c and inasse. The mine al
con en was s ongly a ec ed by he species and cul i a ion medium.
No ably, R. o yzae accumula ed mo e i on han he o he species, and
wine lees as a subs a e ga e he highes i on le els. Among he key
ac o s, polyphenols eme ged as pa icula ly in luen ial due o hei
po en ial o in e ac wi h p o eins and mine als, he eby hampe ing
p o ein diges ibili y and mine al accessibili y. The highes polyphenol
con en was obse ed o R. o yzae cul i a ed on he g ape ma c. The
ungi N. in e media and A. o yzae displayed consis en ly high p o ein
diges ibili y ac oss all media, whe eas R. o yzae had educed di-
ges ibili y when g own on g ape ma c, mos likely due o accumula ed
polyphenols om he cul i a ion media. Mo eo e , all ungi cul i a ed
in g ape ma c exhibi ed a no able educ ion in Fe and Zn accessibili y.
These indings unde sco e he po en ial o op imize he nu i ional alue
o ungal biomass h ough subs a e selec ion, demons a ing ha win-
e y and dis ille y by-p oduc s a e iable media o cul i a ing ungi as
unc ional mycop o ein ing edien s.
CRediT au ho ship con ibu ion s a emen
Luziana Hoxha: W i ing – e iew & edi ing, P ojec adminis a ion,
Me hodology, Funding acquisi ion, Fo mal analysis, Da a cu a ion,
Concep ualiza ion. Ricky Wang: W i ing – o iginal d a , Me hodology,
Fo mal analysis, Da a cu a ion, Concep ualiza ion. Mohammad J.
Tahe zadeh: W i ing – e iew & edi ing, Supe ision, Funding acqui-
si ion, Concep ualiza ion. Ing id Undeland: W i ing – e iew & edi ing,
Concep ualiza ion.
E hical app o al
No applicable.
Funding
This esea ch was unded by he Eu opean Commission, Ho izon
Eu ope Resea ch and Inno a ion P og amme, Ma ie Skłodowska-Cu ie
G an Ag eemen No. 101105437, p ojec Biono FOOD, and Swedish
Resea ch Council FORMAS G an No. 2023-02018.
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 .
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. bio.2025.107711.
Da a a ailabili y
Da a will be made a ailable on eques .
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