Solid-state fermentation with mushroom mycelium elevates plant protein quality and umami taste

17 Sol id- s tat e fun g al f e r m en t at i on ( SSFF ) o ff ers a low-t ec h , low - e ne r gy , and mini mal pr oc e ssing me t h od 18 t o e nhanc e the pr o t e in co n tent a nd qua lity of f ood s. Thi s s tu dy evalua te s the pot e n ti al of S SFF 19 e x ecu t e d with m y c elium of edi ble b a sidi om ycet e s t o impr ov e b o t h nu triti onal an d s e n s o ry qua li tie s 20 of br ow n ric e, br e w er ’ s spe n t g r ain ( BS G ), and lupin. The c o n v e n ti ona l tempe h fung us , R h iz o pus 21 m i cr os po r us v a r . o l ig os po r us , w as us e d as c on tr o l . D if f er en t s u b s t r a t e -f u n g us c om b in a t i on s v ar i e d in 22 impa ct on fla vour a nd pr o t ein quali t y . SS FF impr oved the pr otein qual ity and uma mi t as te of br ow n 23 rice a nd m ainly i mpr o v ed i n umami t a ste of lupin, whi l e f e rme n ta ti on o f BS G w ith ba s i diom y c e t e s 24 even decrea se d pr o t ei n quali ty . Ba s i dio m y c et ou s S SFF produc ts e xhi bi t e d high er umami po t e n ti al , 25 wi t h equivalent umami conc entr a t i on s (EU C) r e achi ng 15 9 g M S G -e q/ 10 0 g D W , sur pa ssing t h e 26 v a lues f ound f or R. micro sp oru s -f e r me nt ed p r oduc t s. In t e rms of s ub str ate s , t he p r o t ei n content 27 inc r ea se d mo s t in b r ow n r i ce f e rme n ta t ions, whil e th e EUC a nd pr o t e i n qu ali ty inc r e a s e d mo st i n 28 lupi n. P r o t ei n q u ality and c ont e n t inc r e as ed more in ba sidiomy ce t e s, indic at ed by the up t o 35 .1% 29 inc r ea se in t he pr o tein dig e stibili ty c orre ct ed a mino ac id sc o r e o f th e limiting a mino ac id lys i n e and a 30 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted September 24, 2025. ; https://doi.org/10.1101/2025.09.24.678313doi: bioRxiv preprint 36.4 % rise in utiliz abl e amino aci ds. B as i di om y c et ou s SS FF t hu s o ff e r s a pr o mising a ppr oach t o 31 upgr a de low - q ual ity p lant pr o tein s int o mor e pa l at able a nd nu t ri tiou s food s. 32 Key wo rd s : Ba sidiomy ce t e s , mu shr oom -p r oducing fungi, m yceli um, s oli d - s t ate f er men tation , pr o tei n 33 qual ity , um ami, py r a z in e s 34 1 Introduction 35 In de v elope d n a tio ns a hig her por t io n o f t h e pr o t ein int ake need s to come from non-anim a l pr otein s 36 f or s u st ain abi lity , he a lth , and e thica l r e a s on s. Higher c on sumptio n o f ( r ed ) me a t is a s soci at ed w i th 37 i.a . highe r ca r diovascul ar di se as e i ncide n ce, h igher gr e e nhou se g as emis sio ns, an d def o r e sta ti on (Ki m 38 et al. , 2019; Po or e & Nemec ek, 20 18) . D es pi t e t h e f ac t th at medi an p r o t ein i nt akes g en er al ly e xcee d 39 r equi r eme nts in de v elo p ed nation s, ve g an a nd v ege t aria n di ets le ad t o a hig her inc idence o f 40 ina dequ at e pr o t e i n int a k e s (Broek ema et al. , 202 0; Kim et a l., 2 019). Addi t io n ally , in developing 41 na ti on s a sub s t a n t i al porti on of t h e pr o t e in int ak e c om es fr om pla n t sou r c e s, lik e c er e als a nd r oo t s. 42 The s t apl e f ood gr oup c ereal s c o nt ri bu t e t o 30 % of the tot a l pr o t ein int ak e (FA O , 2024). Th e s e s taple 43 f ood s are char ac teriz e d by a low pr ot ei n qua lity (Sá & Hou se , 2024), a nd t hi s, and thi s lead s t o a n 44 inc r ea se i n the pr ev a l ence o f p r o tei n ina dequa cy ( Mo ug han, 202 1) . The r ef o r e, in de v elop ing a s w el l 45 as in dev eloped n at i on s , a h ighe r pr opo rt i on o f high-q ual ity pla n t protein in th e die t could al levi at e 46 the se i s s ue s. 47 A method t o i ncr e a se t he pro t e in qu alit y of ce r e al s a n d legume s i s solid - stat e f ung al f erme nt a t i on 48 (SS FF) ( Villa c r é s & R os ell, 20 21; Z wi nk els et al., 2 023). This minima l pr oces s i ng method can impr ov e 49 t h e d i g e s t i b i l i t y , a s w e l l a s i n c r e a s e t h e a m o u n t o f u t i l i z a b l e l y s i n e , w h i c h i s c o m m o n l y t h e l i m i t i n g 50 ami no ac id in the se food s . SS FF h as a particul a r advant a g e over o the r pr oc e ssing method s a s i t 51 minim ally im pacts othe r b e n e ficia l nu tr i e n t s, such a s fibre s a nd miner al s , and d oe s not r e qui r e t h e 52 addi t i on of sa lt o r o the r pre ser v ative s . Mor eov er , low -inc ome popul a ti on s o ften ha ve li t tl e a cce ss t o 53 hea lth y a nd su st a inabl e pr o tein al t e r na tive s (Lum sden e t al ., 2024 ). The low ener gy a nd 54 t ec hnol ogical r equi r em e n t s of SS FF ma k e it a tec hnique th a t can b e w idely a ppli ed i n man y pa rt s o f 55 the wo r l d. Cur r e n tly , SSFF r elie s ma i nly o n a limi t e d s e t o f fungi: muc or om y c ete s suc h as Rh i z op us s p. 56 (tempeh ), and a s com y c e t e s inc luding As p er g il l us s p . ( k o j i ) , Neur o spor a i n term ed ia (oncom ), a nd 57 Pe n i c i l l i u m sp . (mould -rip ened ch ee se s ) (Allwood e t al ., 2 021; Finnigan, 2 011; Han et al . , 2004 ; 58 St ar z yn sk a- Jani s z e w sk a e t al. , 2017; W ol k ers – R ooij ack ers e t a l., 20 18; Y in et al ., 2 020). 59 Be side s Muc or om y co t a a nd A s com y co t a , t he fungal kingdom harb ours one mo re ph ylum tha t h a s 60 potential , p artic ul arly for food f e r me n t atio n. T he ph y lum Ba s i diom y co t a i s c o mpose d o f r oughl y 61 40,0 00 s p ecie s , of w hich a r o und 2,000 p r odu ce ed ibl e fr uiting bod ie s (mus hr oo ms) ( H e et al ., 202 2) . 62 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted September 24, 2025. ; https://doi.org/10.1101/2025.09.24.678313doi: bioRxiv preprint The se mushroom s hav e a long hist o ry of con sumption and a r e de sired f or t heir umami t aste. Thi s 63 mak es t h em i n tere s t i ng op tion s for me a t al t e r n at i ves (Ma u, 2005 ; Mill er e t al . , 2014) a s uma mi ta st e 64 is a k ey f a ctor in a l t e r n ative pr o tein acc e pt abil ity a nd su st ainabl e e ating (Sch midt & Mou rit se n, 202 2; 65 Y a maguc hi & Ninomiya, 2000) . How ever , t he s e mu shrooms ge nerally hav e a lo w e r pr o t ei n quali t y 66 and c o n ten t , maki ng t hem le s s id eal to mee t die t a ry pr o tei n requireme nts ( W al lis et al ., 201 2 ; 67 Z w ink e ls , van Oor s c h ot, et al. , 202 5). N o n ethel es s, the my ce lium of the se s p e cie s c a n s til l be a goo d 68 potential s our ce o f diet a ry pr o t ein . A r ec ent s tudy ind ica ted th a t th e m y c elium of bas i diom y c e t e s 69 pos se ss e s both a high c ont e n t o f uma mi-activ e c ompoun d s , as well a s a high pr otein c o nt e nt an d 70 qual ity , ou tper f or mi ng the c onv e n ti o nal fungi us ed i n s olid- st a t e f erm ent a t io n f or tempe h 71 pr oduc t i on (Z wink els , v an Oors c h ot, e t a l., 202 5). S econ dly , basid iom y c et e s ar e w ell -known t o gr ow 72 on lign ocell ulo s i c m a terial , commonly f ound in f ood i ndu s try side - s tream s, maki ng t h em a 73 potential ly su s t ai nabl e solu tion f or pr o t e in pr oduc t ion (Ilić e t al ., 20 23) . Thirdly , t he m yceliu m o f t h e 74 basidi omy ce t e P l e u ro tu s os t r ea tu s d i d n o t c o n t a i n k n o w n m y c o t o x i n s . M o r e o v e r , f o u r p e p t i d e t o x i n s 75 w e r e l ow er in m y c elium compa r ed to the f ruiting b od y (v an Dam e t al. , 2024 ). A s tr ong i ndica t i on 76 that m ycel ium o f e dible mu s h r oom -produc ing f u ngi i s sa f e for human c on su mption. L a stly , t h e 77 m yceliu m o f f ive ba sidi om y c et e sp ecie s w a s f ound t o g r ow on pla n t sub s tr ate s sui t abl e f o r huma n 78 con s umptio n, where it r e duced t h e an ti -nut ritio nal f a c t or ph ytic ac id mor e ef f e ctive ly than R. 79 m i cr os po r us (Z w ink el s , Oo rs c hot, e t al ., 2025). Th e f ou r abov e -me n tion ed fac t ors supp ort t h e 80 potential o f ba sidi om ycet ou s m ycel ium i n S SFF a s a hea l th y , s u s tain abl e, and t a s ty novel p r o t ei n 81 sou r ce. 82 How ever t o date, r e se ar ch on ba s i di om y c et e s h a s pr e domina n tly f ocu sed on mu shroom pr o duc t io n , 83 enz yme p r oducti on, wa st e - s tr e am v al o r i sa ti on for f eed pr o duc tion, or pure bioma ss pr oduc t i on 84 thr ough s u bme r g ed f e r me nt a tion (Ahl b orn et al ., 201 9; Bac h e t al., 2017; da Silveir a & Badial e -85 Furlong , 2009; Hensk e e t al., 2 018; Step ha n et al., 2018; S ur uga et al. , 2020; van Dam et a l., 2024 ).I n 86 cont r a st, r e s ear ch o n SSFF of f o od-g r a de sub s t r at e s with ba s idi om y c e t ou s m y ce lia a nd their eff ec t o n 87 pr otein qu ali ty a nd fla v our i s s ca r ce. 88 The r ef o r e , thi s s tu dy ai ms t o ev a lu at e th e p o t e nt i al o f S SFF f o od s produce d w i th ba s i diom y c etou s 89 m ycelia as no v el protein sou r ce s f o r hu man nutri tion by a s s e ssing th eir p r o t ein cont e n t and quali t y 90 (via pr o t ein dige st ib ili ty c or r ec t ed am in o a cid s c ore ( P DC AA S ) ), al ong side t h e p r e senc e of uma mi-91 ac tiv e m olecul e s , and r oa sted me at-lik e ar oma s. T e mpeh ma de wi t h R h i z o pus m i c r o s po r us v a r . 92 o l i gos por us on th e sam e s u b s trat e s s e rv e s a s a ref erence . 93 T o pr odu ce a pr o t ein sou r c e fi t f or hum a n con sumption t hr o ugh SSFF , w ith out requi r i ng addi tiona l 94 pr oce ssing techni que s, pl a n t sub st r ate s f i t f or h uma n c on sumption a r e r e qui r e d. There f o r e , t h r ee 95 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted September 24, 2025. ; https://doi.org/10.1101/2025.09.24.678313doi: bioRxiv preprint sub s t r at e s are sel ec t e d t h at r e pr e s ent fo od g r o up s u sed in f o o d f e rmenta tio n. Fir stly , br own rice i s a 96 ce r e al an d s t ap le f ood i n man y par ts o f t he w orld . It i s hig h in st a r ch a nd low in prot ei n, but du e t o it s 97 hig h c onsumpti on contr i bu t i ng t o a sub s t a nt i al por tion o f diet a ry pr o t ein int ak e i n s ome area s (FA O, 98 2023 ). L upin i s a l e gume wi t h a hig h p rot ei n an d fib r e co n tent (Mar tín e z -Vill al u eng a et al. , 200 6) . 99 Furthe rmore, it se rve s a s a su st a in able a lt e rnativ e t o soy , d ue to its ni tr ogen fixation an d po s si bili t y 100 of cultivation in t e mperat e r e gion s (Pa l mer o et al ., 2 022). La s tly , br ewer ’ s spe n t g r a in ( BS G) is a n 101 indu s t ry s i de - s tream o f bee r br ew ing , made fr om ma lted barley . It ha s a low s ta r ch c o nt e n t , due t o 102 the mal ting a nd ma shing pr oc e ss a nd i s hig h i n fi br e a nd mod er ately hi gh in prot e i n (B r ig gs e t al . , 103 1981 ). 104 2 Materials & Methods 105 2.1 Substrates, species, and solid-state fermentation 106 Sub s tr ate s a nd fun g al speci e s u sed , as well as t h e f e r me nt ati on pr o ce s s ar e de scrib ed in Zwink els, 107 Oor sc hot, et al. (2025) . In bri e f , ba s m a ti br own r i ce ( Oryz a s ativ a) , br e w er ’ s s p ent gr ain (BSG , 108 Hordeu m v ulg are ) , and w hit e lupin ( L upinu s alb u s ) w e r e f erm ent ed w ith t he ba s i diom y c ete s 109 Stroph aria rug o so -a nnul ata , Sc hiz op h y llum c omm une , Volv ar i ella vol v ace a , Ple urot us p ul m onari u s , 110 and Py cnop or u s ci nna bari nu s . For c omp a r i son t o c onventiona lly u sed fungi f or f er m enta ti on, the se 111 s u bs tr a t es w e r e al so f er m e n t e d w i t h Rhi z o pu s mi c r os po r us v a r . o l ig os po r us , c o m m o nl y us e d i n 112 t emp eh pr o duc tion. 113 Sub s tr ate s were so ak ed o v e rnight a t pH 4.0, dr aine d , r i n s ed , a nd au t ocl a ved i n a Micr obo x 114 poly pr opylene f er m e n tation c o n t a ine r with an a i r fil ter s trip. T h e s ub s trat e s w ere inoc ulated w it h a 115 m yceliu m s u spen si on in the c a se of basidi o m y ce t e s or a s pore su s p en s i o n in the ca se o f R. 116 m i cr os po r us . Co n tai ners w ere inc ubated at t he op timal g r owth t emp er atu r e o f th e fungi (de t ermin e d 117 in Z w ink els , Oo rs c hot , et al . (2025 ), w hi ch was 33 °C f or P . c inna ba r i n u s a n d V. v olv acea , 30° C f o r P. 118 pulm onari u s and S. c omm u ne , 2 7°C f or S. rugo s o- an nula t a and 28° C for R. micro sp oru s . 119 Fe r me n t a tion time f or ba s idi om y c et e s was eig h t da ys and f or R . mic r o spor us was 48 hour s. 120 2.2 Sample pr eparation 121 A ft er f e r m en t a t i o n , t h e f e r m en t ed c ak e w a s c u t in t o 8 id e n t i c al s l i ce s an d p i e ces f r o m al l s i d es of t he 122 sampl e wer e t a k en, f r o z e n w ith liqui d nitr ogen an d g r ou nd t o a fine powd er u s ing a spic e g r i nde r 123 (K rups F203 , S olingen , Germany) . Ho mog eniz ed sampl es we r e i mmedi a tel y s t o r ed in a u r in e 124 cont ain er ( VW R in t e r na t i onal , ML1 5) a t - 2 0 °C unt i l fu r th er ana lysi s . D ry ma tter c on t e nt was 125 calcul at e d f r om s a mple s ( 1 .0 g) d ried t o a con s t a nt w eight in a moi st ure a nalys er MA100 (Sa r t oriu s , 126 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted September 24, 2025. ; https://doi.org/10.1101/2025.09.24.678313doi: bioRxiv preprint German y) a t 105°C , f o llowin g the pr oce dur e s de scrib ed by the A s socia t io n of O f f ic ial Agricul t u r a l 127 Chemi s ts (AO AC , 2 005). 128 2.3 Quantification of umami-acti ve and volatile metabolites 129 2.3.1 F ree amino acids 130 Fr ee a mino ac id c o nce n tra ti on s in homo geniz ed s a mpl es w e r e de t e rmin ed by f ol lo wing the m etho d 131 desc ribed by S c ott et al . (2021 ). 132 2.3.2 ‘5-nucleotides 133 5’-nuc le otid es we r e ext r acted and m ea sur e d ac cor din g t o the met hod fr om S ei f ar et a l . (200 9 ). 134 Homog en iz e d sample s (100 mg) w ere dispe rs e d i n 5 mL mi lliQ wat er in a 1 5 m L cen trifug a l t ub e . 135 T u b e s w e r e m i x e d b y v o r t e x i n g a n d h e a t e d f o r 2 m i n a t 1 0 0 ° C i n a s h a k i n g w a t e r b a t h . T h e h e a t e d 136 tube s were v o rt e xed f o r 30 s e c ond s and cooled to r oom t e mpera tu r e. Sam ple s wer e cen t ri fuge d ( 5 137 min, 10.0 00 g ) and the s up e r n a t ant wa s fil t e r ed u s i ng a Co st a r S pin-X centri fu g e fil t er (0 .22 µm , 138 ce llulo se ac e t ate) . S ub sequ ently , 2 00 µL w as tr a n s f er r ed t o a pla st i c au t o sampl er vial , ca pped a n d 139 st o r e d a t -20 °C until a n alysi s. S epa r a t i on, de tection a nd qua n t ific ation o f 5’-nucl e otid es wa s 140 perf o rmed u sing u lt r a p er forma nce l iqu id c hr om at ogr aph y (UPLC ) with a n U l t iMa t e 3000 UPL C 141 s yst em (D i o ne x, Id stein, Germ an y ) e quip ped w ith an Acc Q-T a g Ult r a BE H C 18 c ol u mn (150 mm x 2. 1 142 m m , 1 . 7 µ m ) ( W a t e r s , M i l f o r d , M A , U S A ) , a n d a B E H C 18 gua r d column (5 m m x 2 .1 m m, 1 .7 µm; 143 W at ers , Mil f o r d , M A, USA) . The c ol umn t e mp er ature w a s s et 25 °C and th e mo bile ph a s e flow r a t e 144 w a s ma i nt aine d at 0 .2 ml/min. E lue n t A w a s compo sed of 10 mM d ibu tyl amm on ium ac etat e (D B A A, 145 pH 6.7) (TCI che mica l s , T okyo , Japa n ) and el ue n t B wa s compo sed o f 10 mM DB A A (pH 6 .7 ) 146 cont aining 84 % ace t on i t rile (S up elco , Be l l ef o n t e , P A , U SA) . T he se p ar a t ion gr adi e n t w as 0 – 2 m in 147 99.9 % A, 2 – 12 min st e adil y dec rea sing to 70% A, 12 – 15 min 7 0% A, 15 – 17 min 0% A, 17 – 27 mi n 148 99.9 % A. One mic r o li t er o f s ample w a s inje ct ed f o r a nalysi s and c ompo und s we r e de t e c t ed by UV 149 mea s u r em ents at 25 0 nm. Co nc entr ati ons we r e c al cul at e d ba s ed on a ca libra tio n c ur v e of 5’ -150 ade no s in e monopho sph at e (Th e r mo Fi she r), 5 ’-guan o sine monoph o sph a te (T he r mo F i sher), 5’ -151 inosin e mon oph o s ph ate (Th ermo Fi she r) , and 5’ -xan t ho sine mon oph o s ph a t e ( Bi o -C onnect , Hui s s e n , 152 The N e t h e r la nd s) (0 . 78 – 25 mg /L). 153 2.3.3 Equi v alent umami concentration 154 The equivalent uma mi c onc entra tion (EU C) e x pre s s e s t h e s y ner gi stic e f f e ct of um am i fr e e amino aci d 155 and 5’ -nuc leo tide s . Ba sed on th e rel at i v e umami con centra tion o f th e se com pound s, t h e EU C o f 156 MSG -eq pe r c eiv e d w a s calc ul a ted (E q u ati on 1;Y amag uchi e t al. , 1 971). 157 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted September 24, 2025. ; https://doi.org/10.1101/2025.09.24.678313doi: bioRxiv preprint /g1831/g1847/g1829 /g3404 /g3533 /g1853 /g3036 /g1854 /g3036 /g3397 1218 /g4672 /g3533/g1853 /g3036 /g1854 /g3036 /g4673/g4672 /g3533/g1853 /g3037 /g1854 /g3037 /g4673 # /g4666 1 /g4667 Where a i (g / 1 00 g DW) i s th e c onc e n tr atio n o f th e r e s pe c tiv e umam i a mino a cid (As p o r G lu ); a j 158 (g /100 g DW) i s th e c o nce n tra ti on of th e r e s pe ctive u mami 5’-nuc leo tide (5-iz) -M P , 5-iz)- GMP , 5-iz)-XM P , 159 or 5-iz)- AM P); b i i s t h e r e l a t i v e u m a m i c o n c e n t r a t i o n ( R U C ) f o r u m a m i a m i n o a c i d s t o M S G ; a n d b j i s 160 the RU C f or umam i 5-iz) -nuc leo tide s t o 5-iz) - IMP (Y amagu chi e t al. , 19 71). 161 2.3.4 V olatile org anic compounds 162 V ol a t i le o r g anic c ompo un ds ( V O C s ) , wi th a f oc u s on a lkyl pyr azines , we r e de t e r mined by head spac e 163 solid -pha s e mic r o extr acti on ga s c hr om a togr aphy mas s sp ectr ome try (HS SP ME G C- MS) (Sco t t et al . , 164 2021 ). In bri ef , 1.0 g r a m ho mogeniz ed s a mpl es w ere w eig hed in a 5 mL g as c hr omat ogr ap h y vial . 165 Raw sample s were c a pp ed an d s t ored a t -2 0 ° C u n til ana lysi s . He a ted s a mple s wer e c ov e r e d w it h 166 alu minium f oil a nd he at ed in a co n v ec t i on oven at 150 °C f o r 5 min, after wh i ch t he y wer e al so 167 capped and st o r ed at -20 °C . V OC s w ere e x tr a c t ed and de t e c t ed ac c o r ding t o th e method describ e d 168 by Sc ot t e t a l . (2021 ) , with th e mo difi c a t i o n that a 1: 5 s pl it mod e wa s u s e d. 169 2.4 Protein quality 170 2.4.1 In vitro protein digestibility 171 In v itro p r o t e i n d i g e s t i b i l i t y ( I V P D ) w a s d e t e r m i n e d u s i n g t h e M e g a z y m e t e s t k i t K - P D C A A S b y 172 f ollow ing the manuf ac tur e r ’ s pr oc edu r e wi t h s ome adap t atio n s (M eg az ym e, 201 9). I n bri ef , 173 appr o x imat ely 500 mg homog en iz ed t e s t , bla nk, co n tr ol and st a nda r d s a mple wa s w eig h ed into a 5 0 174 mL cen tri fug a l tube . T o t h e s e tube s 19 m L HCl (0.06 M) wa s add ed, a nd the tub es were inc ubat e d i n 175 a hot ai r sh ak ing inc ubat or (37 °C, 30 min, 300 RPM). Thi s w a s foll ow ed by a firs t dige s t i on s t ep w it h 176 the ad ditio n o f p epsin (60 min , 37 °C, 300 RPM ). S ubseq ue n tly , s am pl e s w e r e ne u tr al iz e d t o pH 7.4 by 177 addi t i on of 2 mL 1 .0 M T ri s bu ff er . Th e n, sample s w e r e s ub jec t e d t o a secon d dige s tion st e p by 178 addi t i on o f 200 µL tr y psin / c h ymotryp sin mix tur e and i ncubatio n f o r 4 h a t 37°C at 300 RPM. Th is wa s 179 f ollowed by h e a ting t h e sampl es i n a boilin g wat er b ath f o r 10 min. P r o t ei n s w e r e p r e cipi tat e d 180 ov e r n ight a t 4° C by addi t i on of 1 mL T CA solut ion (40% ). T ube s w e r e c entri fuge d (10 min , 15,000 g ) 181 and the s upe r na t a n t s w e r e dilu t e d 10-fol d a nd 20 - f o ld i n a ce t ate bu ff er (50 mM, pH 5. 5). Amin e 182 concentra tion wa s me a sured again st a L -g ly c ine calibra ti on cur v e t hr o ug h c ol o r i m etr ic de t e rmina t io n 183 of t e st , blank and s t a ndard sample s at 570 nm aft er the additi on of ninh y drin ( 2 %) and in cuba tion at 184 70°C f or 35 min . I V PD w as calc ul at ed from t h e p r im ary amin e co ncentra tion of t e st sampl e s fi tt e d 185 against s t and a r d s a mple s with k nown in v i vo pr o t ein d i g e st i bility value s. 186 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted September 24, 2025. ; https://doi.org/10.1101/2025.09.24.678313doi: bioRxiv preprint 2.4.2 Amino acid composition 187 The pr oce s s i ng , e x t r a cti on, a nd qua n ti fic a tio n of a mino ac id s in the sampl e s a r e de scribe d i n 188 Z w ink e ls , O ors c hot, et al. (2025) . The p r oc edure s gui ded by ISO13904 (ISO, 20 05b) and ISO1390 3 189 (ISO , 20 05a ) we r e f ollo wed. L upin p r oduc t s w ere defa tt ed b ef ore h y d r ol ys i s . 190 2.4.3 Protein quality and utilizable amino acid content 191 Pr o t ein qu a lity wa s determine d ba sed on t h e pr o t ein di ge s tibi lity co r r ec t e d amino acid s co r e 192 (P DC AAS ), a s de scrib ed by (FA O / WHO , 1 991). So me ad aptat i o n s were made t o m a k e the sco r e mo r e 193 in li ne with the dig e s tible i ndi spen sa ble amin o acid sc o r e ( DIA AS), whi ch w a s rec ommended by t h e 194 FA O (2013 ) . The ada ptation s were: (1) th e inc lu s i on of non -limiting amin o a cid s i n the sc o r e, a nd (2 ) 195 not tr un c a ting s c o r e s abov e 1. 0 . T he a mino ac id s co r e (AAS ) wa s calcu l a ted u s i ng E quation 2. The 196 r ef erenc e protein w a s ba sed on th e di et a ry r e f e r ence i n tak e o f a pre- scho ol chil d (0.5– 3 y) , a s 197 pr e scrib ed b y the FA O (2 013). 198 /g1827/g1827/g1845 /g3404 /g3040/g3034 /g3042/g3033 /g3036/g3041/g3031/g3036/g3046/g3043/g3032/g3041/g3046/g3028/g3029/g3039/g3032 /g3028/g3040/g3036/g3041/g3042 /g3028/g3030/g3036/g3031 /g3036/g3041 /g2869 /g3034 /g3042/g3033 /g3047/g3042/g3047/g3028/g3039 /g3028/g3040/g3036/g3041/g3042 /g3028/g3030/g3036/g3031/g3046 /g3040/g3034 /g3042/g3033 /g3046/g3028/g3040/g3032 /g3028/g3040/g3036/g3041/g3042 /g3028/g3030/g3036/g3031 /g3036/g3041 /g2869 /g3034 /g3042/g3033 /g3045/g3032/g3033/g3032/g3045/g3032/g3041/g3030/g3032 /g3043/g3045/g3042/g3047/g3032/g3036/g3041 ( 2 ) 199 The P DCA AS wa s c al cul at e d by multiply ing the IV P D by t he AA S o f t h e limiting a mino ac id (E qua t io n 200 3) 201 /g1842/g1830/g1829/g1827/g1827/g1845 /g3404 /g1835/g1848/g1842/g1830 /g1499 /g1827/g1827/g1845 ( 3 ) 202 The utili z a b le amin o ac id cont e n t (U A A) w as c a lcul a t e d b y multiplyi ng t he trun cat ed P D C AAS w it h 203 the t o t al amino a cid c ont e n t . 204 2.5 Data analysis 205 D a t a g e ne r a t ed b y U PL C a n d H S S P M E GC -M S w er e an a ly s ed in C hr o m e leo n 7 . 3.1 ( T h e r m o S cie n t i f i c, 206 MA, USA). G C-MS pe ak int egra tion wa s p erf o r m ed u sing th e I CI S alg or i t hm an d th e NIST main li br a r y 207 w a s u s e d f o r i d e n t i f i c a t i o n b y m a t c h i n g m a s s s p e c t r a l p r o f i l e s w i t h t h e p r o f i l e s i n N I S T . O n e 208 quant i fy ing pe ak (g e n er ally t h e high e st m/z p e ak pe r comp ound) wa s u s e d per c omp ound for 209 quant i fi cation, whil e two c onfi r mi ng p ea ks w e r e us ed f or c onfirm a ti on. Da t a was anal yse d, a nd 210 figur e s w ere pr oduc ed u sing R s tudi o softwar e v . 4 .0.2 (R S tudi o®, Bo ston, M A, U SA ). All v a lu e s 211 pr e se nt ed are mean s o f bi ologi cal tri plic a t e s ± st a ndard devia t i on, unle s s s t a t ed ot h e r w i s e . 212 Si gnifica n t di ff e r ence s that a r e ind ic a ted with lett e r s we r e de t e rmined u sing On e-W a y AN O V A w ith 213 T u k ey pos t h o c t e st. Sig ni fica nce l ev el ( p -value ) w a s set a t 0.05 . 214 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted September 24, 2025. ; https://doi.org/10.1101/2025.09.24.678313doi: bioRxiv preprint 3 Results 215 3.1 T aste & aroma 216 The e ff ect o f so lid- sa t e f erme n tation by no v el ba s idi om y c etou s fung i on umami ta s te compoun ds of 217 ce r e al s a nd leg ume s w a s c o mp ared to th e c onv e n ti ona l f o od fungu s R . m i cr os por u s , u s ed in t empe h 218 pr oduc t i on . The e quiv a le n t um ami c onc e n tr atio n (EU C) was calcul at ed a s a me a s u r e for uma mi t a st e 219 of t he food produc t ( Fi g ur e 1 A) . The un f e r me nt e d s ub s trat e s , BS G, br o wn ric e, a nd lupin, h ad a low 220 EUC o f 0.007 , 0 .028 a nd 1.52 g MSG - e q /10 0 g DW , r e spec tively , plac ing t hem in u mami level 4 (0 -1 0 221 g MSG -eq /100 g DW), ac c o r ding t o Mau (2 005) . Ferme nt at i on inc r ea s ed t he EU C in a ll sa mple s, bu t 222 the e x tend o f thi s i nc r ea s e wa s d e pen de n t on t he i n t e r a c tion be twe en sub s tra te an d f ung al spec i es . 223 EUC inc r e a se d t o l ev e l 3 (10 -100 g MSG- eq/100 g DW) in P . pu lm o na r i us f erm ented BSG and br ow n 224 rice , R. m i cr os p or u s -lupi n and P . c in na barin us - b r o w n r i c e . T h e h i g h e s t E U C w a s o b t a i n e d i n P. 225 ci nnab arinu s -lupin (158.7 g MSG -eq /10 0 g DW), r ea ching le v el 2. G e ner ally , the hig he s t EUC wa s 226 obser ved i n lupin, t h e n br own ric e , f oll o w ed b y BSG. 227 The umami t a ste in f ood i s d eriv ed f r om a s yne r gi s tic e ff ec t of um am i fr e e amin o a cids (FAA) and 5 ’ -228 nuc leotid e s. In un f e rmented sub s tr at e s the c onc e n tration o f um ami FAA was slig ht l y l ow e r t h a n 229 neutr al , bitt er , or sweet t a sting FAA ( Fig ur e 1 B). Howe v er , t he c onc entr atio ns o f all FAA wer e 230 insu ffici e n t t o pa ss the ta st e th r e shold . Ferme n tation incr e as ed t h e co ncentra tio n of F AA in al l 231 inst anc e s . However , ma inly um ami FAA s urpa ssed the t a st e thre s hol d, r e achi ng dos e-over -th r e s ho l d 232 (D O T ) up to 60 in P . c in na ba r i n us-lupin . Umami FAA s e x hibi t ed a higher DO T c om pared t o ot he r 233 t a sting FAA, p rimarily du e t o their low taste th r e s hol d . L a s tly , th e co n t rib utio n of 5’ -nuc leo tide s to 234 the EUC is depi c t ed in Figur e 1C. Sim ilarl y t o F AA, 5’-nuc leo tide s w e r e only pr e se n t in m inor amoun t s 235 in t h e unf e rmented pr oduct s . In most f erme nt ation s, 5’ -g uano sin e mon opho s p hate (GM P ) 236 cont ribu t e d mo s t t o the EU C o f t h e f erm ent ed pr oduc ts, ex ce pt f or V. vo lv ac ea f er m e n t e d b r o w n r i c e 237 and R. micro spor u s f e r m ented lupin, wher e 5’ -i n os i ne mon opho sph at e (IM P) w as th e highe st 238 cont ribu t or . O ver a ll, pr oduc t i on of 5’ -nu cle otide s w a s highe r in b a s i diom y c e t ou s f e r m e nta tion s th a n 239 in R. m icros por us . Particu l arly , P . c inna barin u s and P . pulm on a r i u s p r oduced t hes e c ompound s i n 240 con s ide r a b le amou n t s. B a s idi om y c etes se emed t o f avour br ow n r ic e f o r 5’- nuc leotid e pr oduc tion , 241 whi le R. m icro spor u s pr odu ce d mor e in l upin . 242 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted September 24, 2025. ; https://doi.org/10.1101/2025.09.24.678313doi: bioRxiv preprint 24 3 Figure 1. A) Equi v ale n t u m am i con c entra ti on (EUC, g MSG-eq/ 1 00 g DW ) o f u nfermente d and fer m ented lup in 24 4 (or a n ge), br own rice (g r een) and br e w er’s sp en t g r ain (BSG, pu rple) (me an ± s tandar d devi a tio n). B) R ela ti onship 24 5 betw e e n log 10 ( D o s e o ve r t h r es h o l d) an d l o g 10 ( conce n tr a tio n) of f r ee a m i no acids w i th bitter (r ed), ne u tra l ( g r ee n ), 24 6 s w e e t ( b l u e ) , o r u m a m i ( p u r p l e ) t a s t e i n u n f e r m e n t e d a n d f e r m e n t e d s u b s t r a t e s . C l o s e d p o i n t s r e p r e s e n t i n d i v i d u a l 24 7 a mino a cid s , w h ile o p e n poin ts r e pr e s ent th e ave r a g e of all amino a cids in a certa in ta ste c ategory . R oun d po ints ar e 24 8 below the tas te thr e s h old, whi le triangular p oin ts a r e abov e the tas te thres h old. C) C ontributi on of 5’-nucle o ti des to 24 9 t h e e q ui v a l e n t u m a m i c o n c e n t r a t i on o f un f e rm e n t e d a nd f e r m e nt e d s ub s t r a t e s ( E qu a t io n 1 ) . C o lo u r s r e pr e s e n t 5’ -25 0 a de no sine m o nop h osph a te (AM P , r ed ), 5 ’-gua n osine m o no pho s p ha te (GMP , ol i ve g r een), 5’-inosine m o no pho s p ha te 25 1 (IM P , g r een), 5’-x anthano s in e m o nop hosph a te (XMP , blue) , and t he sum of a ll nucleoti d e s con tributions (pu rple) . 25 2 Alkyl pyr azines a r e he t e r oc y c lic nitr ogen ous a r omatic compound s , a nd t h e ir pre senc e is a ssoc i at ed 25 3 with meaty a nd r o a s t ed a r oma s ( R en e t al., 2 024) . Level s o f th e se com pound s i n unf e r me n ted and 25 4 f e r m en t e d s u bs t r at e s , e i t he r r a w o r af t er h e a t t r eat m en t w er e d e t er m i n e d b y H S S P M E G C - M S. 25 5 Alkyl pyr azines w e r e o nly detec t ed at t r ac e level s in un f e rmented sub str a t e s. F erme n tation wit h 25 6 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted September 24, 2025. ; https://doi.org/10.1101/2025.09.24.678313doi: bioRxiv preprint basidi omy ce t e s p artic ul arly pr oduc ed al ky lpyr azines in BS G (u p to 5.6 *10 5 count s * min ) and in l upi n 257 f erme n ted w ith R. mic r o s por us (6.9*10 5 count s*min) . The mos t ab unda n t com po und w a s ei the r 2 ,5 -258 dime t h y lpyr azine or t etr ame th yl pyr azine. H e ating increa se d t he al kylpy r az i ne concen t ra tion in R. 259 m i cr os po r us f erm ent ed sub st r ate s (up t o 7.0*10 5 count s * min) , whi le cha ng e s we r e le ss pr on ounce d 260 in basidiom yce t e s f erme nt atio n s . The d iv ersity of pyr azine -de riv a tiv e s did inc r e a s e wi t h he a ting , 261 fr om maxima lly fiv e in the r aw s u b s tr at e ( i n P . c innab arinu s f er me n t e d B SG ) t o t w e lv e d if f er en t 262 compound s in th e he a ted sub s trat e s (i n R. micro spor us f erme nt ed lupi n ). 263 264 Figure 2. Al ky lp y r a zin e areas (coun ts * min) pe r g r am of d ry w eight un f er me nted o r fer m ented substra te. Sample s 265 w ere m e a sure d either unt r eated ( R a w) or aft er he a t trea t me nt (H e ated). Co lo urs r e p r e s en t compou nds a n d a r e 266 e x p la in e d in the figu r e legend. 267 3.2 Protein quality 268 In a ddition t o ta st e and a r om a, the eff ec t of f erm enta tion by basidi om ycet e s on nutriti ona l quali ty of 269 t h e s u bs t r a t es w as a s sess e d. Pr o t e in q ua l i t y w as d et e r m i ned b as ed o n t h e in v it r o p r o t e i n 270 dige s ti bi lity a nd ami no ac id c ompo si tio n of the f e rme n ted pr oduc t s. ( Fi gu r e 3). In un f e r m en t e d 271 br own r ic e, the p r o t ei n dig e stibili t y w as 82. 3% and f erme nt ation a l t e r e d the pr o tein dige s t i b ility w ith 272 les s th an 1.7 per ce n tage p oi n t s. How ever , i n BS G , f erm ent ation s ig nifi cantly r e duced the pr otei n 273 dige s ti bi lity fr om 88.1% , to as low a s 80. 0% ( p < 0 . 0 5 ) , w h e r e a s i n l u p i n f e r m e n t e d w i t h P. 274 ci nnab arinu s di g e stibili ty s ig nifi c a ntly i ncr ea sed with 7 .5 per c ent age poin t s , r e ac hing 99.4% ( p < 275 0.05 ) . O verall, t he eff ec t of f e rmenta ti on on p r o t ein dige s tib ili ty wa s ba s ed on t h e inter ac t i o n 276 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted September 24, 2025. ; https://doi.org/10.1101/2025.09.24.678313doi: bioRxiv preprint betwe en sub s tr ate an d fung al s p ec ie s. This w a s il lus tra ted by the eff ec t o f P . c innaba r i n u s on pr o t e i n 277 dige s ti bi lity , whic h did no t al ter in b r ow n rice , reduc ed the di g e st i bili ty i n BSG, a nd inc r e a s e d 278 dige s ti bi lity in lupin. 279 280 Figure 3. In vitro p r otein dige stibi lit y (%) o f unfe rmented a n d fe r m ented s u bstr a te s . Di ff e rent le tters ind ica te 281 sig n ifica n t di ffer ences b e t w e en s p e cie s w ith in a su bstr ate (T uke y HS D , p < 0. 0 5) . 282 Pr o t ein quality in un f erme n ted and f e rmen t e d sub s tr at e s w a s de t ermin ed ba sed o n th e pr o tei n 283 dige s ti bi lity co rr e cted amino acid s c o r e (P DC AAS ) o f all indi sp en sable ami no ac ids ( Fig u r e 4 ). I n 284 unf e rmented br ow n ric e, lysin e wa s the limi t in g amino ac id, with a P D CAAS of 0 .62. Ferme n tat i o n 285 inc r ea se d the P D CAAS be twe en 2.6 and 18.6 %, up t o 0 .73. In a ddition t o ly s ine , t he PD CA AS simila rly 286 inc r ea se d con s i stently in i sole ucine an d t hreon ine . Fur t he r more, in BS G , lysi ne w a s th e limiting 287 ami no ac id with a P DC AAS o f 0.7 8. Ferm en t a t i on o f BS G al t e r e d th e P DC AAS d ep e nding on t h e fung a l 288 speci e s . Ferme nt at i on w ith P . p ul mon ar ius a nd R. mic r o sporu s h a d r el a t i v ely mino r eff ec t on t he 289 PD CAA S, whil e S. ru go so- an nula t a f erm ent ation inc r e a se d th e P D CAAS of l ysin e by 14.8% t o 0 .90 . 290 Fe r me n t a tion w ith P . c innab arin u s s ub st a n tial ly i ncr e a sed and d ecrea se d t he pr otein dig e stibili t y 291 cor rec ted amino acid r a t i o ( PDCA AR) o f c er ta in indis pen sabl e a mino ac id s. O n th e ot he r ha nd, lupi n, 292 in it s un f erm ent ed st at e , wa s l imiti ng in sulphu r a mino ac ids, lysine , an d t ryp t op ha n, with a P DC A AS 293 of t ryp t oph a n at 0.86 . Ferme n ta ti on impr ov ed P DCA AS. P a rticula r l y , in P . cin nabarin us f e r m e n t e d 294 lupi n, the P DC AAR o f s ulp h ur am ino aci ds inc r e a s e d by 35.1% , l y sine by 17.8 %, and trypt oph an by 295 52.7 %. T his f e rmenta ti on i ncr ea se d t he PDC AAS by such an e x t ent that i t c an be consid ered a 296 c o m p l e t e p r o t e i n , w i t h a P D C A A S o f 1 . 0 3 ( l y s i n e ) . S i m i l a r t o t h e i n c r e a s e s i n P D C A A S , f e r m e n t a t i o n 297 signifi ca n tly r ai sed t h e pr opor tion o f ind is p e n s ab le a mino acid s acr o s s all s ub s tr at e s by 0 .2–3.4% 298 (Supp lem ent a ry Fig ur e 1 ), in dic a ting that m ycel ium pr ov ide s a more bal anc ed amino aci d 299 compo s i t i on than pla n ts . 300 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted September 24, 2025. ; https://doi.org/10.1101/2025.09.24.678313doi: bioRxiv preprint 301 Figure 4. Pr ote in qu a l i ty o f f ermented sub s tr at e s. Pr otein q ua lit y is r e p r e se nted b y the pr otein dige stib ility c orr e cte d 302 am in o ac i d sco r e ( P DCAAS) of indi s p e n sable a m in o ac i ds (c o lou r ed). P o ints r e p r e se n t the PDCA A S pe r a m i no acid (± 303 sta n dar d deviation ) , whi le bar s repre s e n t the r e lati ve chan g e in PDCAAS f r om the unfermen ted substr ate (%). The 304 PDCAA S of the amino ac id s in the unfermented su bstr ate i s gi v en in sup plem enta r y figu r e 2 a n d writte n in te xt f o r th e 305 limitin g amino a cid. Ar om atic amino a cid s (phen yla l a ni ne a n d ty r osine) w ere no t meas u r ed. 306 The inc r ea s ed P D C AAS o f f erm ent ed s u bs t r a t e s indi cate s th a t fung a l f erm entati on en hance s thei r 307 pr otein qu a lity . Ho w e v er , a s de scrib ed b y Z wink els, O o r sch ot, et al . (2 025), fungal f er m e nt ation c a n 308 also al t e r the pr o t e i n c ont e n t o f t h e substr a t e t hr o ug h the br e a k dow n of ami no acids via 309 dea mination (decrea se ), or thr o ugh loss o f dry w eig ht (inc r e a se ). This w a s in cluded thr o ugh t h e 310 calcul ation of utili z abl e amino aci d c ont e nt (U AA ), indic a ting t he t o t a l amino aci d c ont e n t th at can be 311 absor bed a nd util iz e d by the bo dy . T he cha ng e in U A A due to f erm enta tion wa s plott ed a g ain s t t he 312 EUC ( Fig u r e 5 ) . The U AA of un f e rme n ted br o wn rice , BS G, and lu pin we r e 5.7 , 14 . 6, and 31.2 g /100 g 313 DW . In b r ow n rice , the U A A incr e a sed by 15.3 t o 36 .4% , due t o the combin ed inc r e a s e in PD CAA S a nd 314 inc r ea se in t otal amino ac ids c ont e n t ( Z w ink e ls , Oor sch o t , et al. , 2025). Co n trast i ngly , in BSG t h e 315 ch ang e i n U AA vari ed f r om 6.1% incr e a se t o 46 .0% dec r ea s e, due t o the d e gr adation o f a mino ac id s , 316 partic ula rly pr e se n t in BS G f erm ent e d with P . c innab arin u s a nd P . p ulmo nariu s . In l upin, f erme n ta t io n 317 inc r ea se d th e U A A up t o 6 .1%. T hi s i ncre as e in U AA wa s ac c ompa nied by t h e large s t inc r ea se i n EUC 318 t o 158 .7 g MS G- eq/100 g DW . 319 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted September 24, 2025. ; https://doi.org/10.1101/2025.09.24.678313doi: bioRxiv preprint 320 Figure 5. E ff e ct o f f er m ent ation on the util iz able a m i no a cid c o ntent (%) a n d equiv ale n t uma mi co nce ntr ation (EUC, g 321 M S G-eq/100 g DW ) . T o c o m p a re substr ates, the change in the uti liza bl e a mino ac i d con te n t compa r ed to the 322 u n fer m en t ed s u b st ra t e i s giv en . P D CA AS o f t h e s am p l e i s i n d i c at e d by t h e s i z e o f t h e po i nt . Co lo u r o f t h e p o i n t 323 in dica tes the subst r at e. The shape of the po int i ndicate s th e a mino a cid w i th th e l ow er P DCAAS . A r o m atic amino ac i ds 324 ( p he n y l a l a ni n e a n d t y r o s i ne ) w er e no t m e as u r e d. 325 326 4 Discussion 327 This st u dy e x plor ed t he impa ct of sol id - s t ate f erme nt a t i on with basidi omy ce t e s o n the t a st e , ar oma , 328 and pr ot e in qua lity of pla n t -ba s ed f oo ds. Th e impa ct o f ba s idi om y c et e s w a s al s o c ompa r ed t o 329 f erme n ta ti on w ith R. m ic r os p o r us , r ep r e se n ting a fungu s tha t i s c onv e n ti onal ly use d f o r solid - st at e 330 f ood f e r me n ta ti on (i .e . t empeh m akin g ) . Wi t h th e s e a s se ssm ent s we a im ed t o un ders t an d t h e 331 potential o f s oli d - s ta te f e rmen t at i on wit h m yceli um of ba sid iom ycet e s to pr od uce n ov el hi gh -quali t y 332 pr otein sour c e s f or huma n c on sumption . 333 Umami t a ste i s a c ritical fact o r in f ood likeabili t y an d n ov el f oo d acc eptanc e, w i t h umami b eing call e d 334 a ma jor driv e r o f su s t ai nabl e f o od c on su mption (S chmidt & M ouri t s en, 2 022 ). Th e equivale n t umam i 335 concentra tion (EUC) , ba se d on t he s yn er gi s tic eff ect s o f um a mi FAA an d 5’ -n ucleo t i d es, clo sely 336 cor rela te s with umami t a ste pe r c eptio n (Zhu et a l., 2022 ). W hi le m eat i s a we ll-k nown s our c e of 337 umami , mushr o oms a lso ha v e a p artic ul a rly s t r ong uma mi ta st e ( Mau, 200 5) . 338 In our s tudy ba s id iom y c ete s pr oduc ed h ighe r EUC v a lue s tha n R. m ic r o s p o r u s a c r o s s a l l s u b s t r a t e s , 339 r eac hing up to 159 g MSG-eq /100 g DW . The s ub st a n ti al EUC v al ue s in ba sidio m y ce t o u s S SFF 340 pr oduc ts i s in line with pr eviou s w ork on id entic a l fungal s p ec ie s (Zwink el s, van Oorscho t, et al . , 341 2025 ). I n t h i s s tudy the E U C of pure bas i di om y c et ou s m yceli a surpa ss ed tha t o f R. m i cr os po r us 342 m yceliu m and thei r cor r e s pond ing mush rooms, r each i ng up t o 300 g M S G - eq / 100 g DW . Th ereby , t he 343 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted September 24, 2025. ; https://doi.org/10.1101/2025.09.24.678313doi: bioRxiv preprint EUC o f SS FF pr oduc t r e ach ed hal f that o f the highe s t EUC v a lue of pure m yceli um but o utp erf o r me d 344 all mush r oo m s . T h ese r e s ult s sug g e st t hat th e EUC o f op timi z ed s oli d -st ate f e rmen t e d p r oduc ts c oul d 345 sur p a s s the u mami i nt e n s ity typic ally as s oc i at ed w ith mu shroom s , highli gh ti ng t he po t e n tial o f 346 basidi omy ce t e s for c r e ating a ppeal in g umami t a s t e in f o od through soli d- s tat e f e rmen t a t i on . 347 Int e r e stingly , the EU C o f pu r e m yceli um of a fung a l s p e cie s did n ot c orr e spond w ith th e EUC of thei r 348 f erme n ted pr oduc t produc ed with that f u ng al sp eci e s . In stead, th e in ter ac t i on be t we en the fung a l 349 speci e s and th e s ub str ate a pp ears t o b e the k ey f ac t or . Thi s i n ter ac t i on s ug g e s t s th a t high fu ng a l 350 pr oteoly tic ac t iv ity c o mbined w it h a hig h pr otein co n tent i s li k ely r e qui r ed t o ac hi ev e a high umami 351 free amino ac id c ont e n t . Moreov e r , this i n ter acti on sug g e st s th a t t h r oug h sub s tr a t e -spec ie s 352 sel ec t i on , the umami ta st e o f the s e f erm ent ed produc ts c ou ld b e fur t h e r op t i miz e d. T ak en t og e t he r , 353 the se finding s und ersc o r e the po tenti al of solid - sta te f e rmenta ti on of pl ant-b as ed f o od w ith 354 basidi omy ce t e s t o off er a ric h u mami t a s te. This i s a k ey f ac t or in inc r e a sing t he pa lat abili t y a nd 355 pr omoting t h e c on sumpti on o f t he s e su s t ainab le pr o tein s our ce s . 356 Alky lpyr azines a r e impo rtant c ompou nd s in t h e ar om a pr o file o f r o a st ed me a t , mu s hroom s, a n d 357 f erme n ted f o o ds l ik e n a t t o , and soy sauc e (Moon e t al . , 2006; Rocch i et al., 2024; Z hang e t a l., 201 8) . 358 Alky lpyr azines ar e nit r o g e nou s cycli c c ompound s, w hich t h r oug h alk yl a tion gr e a tly reduce thei r 359 odour t h r e sho ld, typic ally pr oduci ng r o a s ted, nu t ty , and m e a ty aroma s (Suppl ement a ry t abl e 1) . 360 T h ey a r e g e n e r a l l y f o r me d i n a M a i l l ar d r ea c t i o n oc c ur r in g a t l o w w a t e r a ct i v i t y wi t h a m i n o a c id s ( i. e . 361 ala nine, gl y ci ne, th r e oni ne, or lysine ) an d r educ ing s u g ars (i. e . gluco s e , fruc t o se , and rhamnos e) a s 362 r eac t a n t s ( A da ms & Kimpe , 2009 ; Amr a n i-Hemaimi et al., 1 995) . 363 2,5-di meth ypy r a z ine and t etr a meth ylp y r a z ine w e r e the mo s t ab unda n t c ompound s in r aw , 364 f erme n ted B S G and lupi n . He ating inc r ea sed th e abu ndance and / or div ersi ty of th e a lkylp yr az i ne s u p 365 t o t w el v e compound s . This i s in line with the alk ylpy r a z in e s f oun d in pur e m yceli u m of the se spec ie s 366 (Z w ink e ls , v a n Oo rs c hot, et al. , 2025) . How e v er , S SFF s a mpl es c o nt a ined a higher dive rs ity o f 367 alk ylpy r a z in es in t he r aw p r odu c t t ha n p ur e m y c elium did. M or eov er , h eat e d m ycel ium pr oduce d a 368 hig her divers i ty of alk ylpyr azine s t ha n he at e d SSF F pr od uc t s . Fu rthe rmore, in pur e m ycel ium, the 369 hig he s t abund an ce of al kylpy r az i ne s w a s f ou nd in basidi omy ce t e s , whil e in SS FF s a mple s, 370 alk ylpy r a z in es w ere mo s t a bunda nt in R. mic ro s p oru s fe r m e nt e d s u b s t ra te s . I n te re s ti n g l y , th e 371 compo s i t i on o f th e s ub str at e seem s to hav e a la r ge im pact o n th e di vers i t y and abund a nce of 372 alk ylpy r a z in es. T here for e , alk ylpyr a z ine a bundanc e in th e m ycel ium is lik el y not a good pr e dictor f o r 373 alk ylpy r a z in e f ormat i on i n S SFF . Mor e over , Soh ail et a l. (2022) and Z w ink el s et al. (2025) r ep or t e d 374 that he at e d be ef cont ai ns t he highe st di v e r sity of a lkyl pyr azine s, highe r than chi c k en, my ce lium, an d 375 mushr o oms, w ith twelve py r az i ne de riv a tiv es un i que t o thi s me a t type. In li n e w ith t h e s e find ing s, 376 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted September 24, 2025. ; https://doi.org/10.1101/2025.09.24.678313doi: bioRxiv preprint sev eral of the s e unique compound s were also detec t ed in our SSFF pr oduct s. N ot abl y , 3-eth yl - 2 ,5 -377 dime t h y lpyr azine, id entifi ed by Soh a il et al. ( 2 022) a s a di stinc t i ve a r oma c ompou nd in ch ick en, be ef , 378 and pork, wa s p r e se n t in th e f erm ent ed s ample s . 379 The IV P Ds of u n f er m e nt ed b r own r ic e (8 2% ) and l upin (92% ) w e r e slig ht l y hig her t h an t ho s e r epo rt e d 380 in lit era tu r e f o r wh it e ric e ( 8 1%) and lu pin (82%) (Vill ac r é s & R os ell, 202 1; Zwink els e t al ., 202 3). 381 Contr arily , th e I VP D o f unf erme nt e d BS G (88 %) w a s s li ghtly lower t h a n r e p ort e d in ba r l ey (90-91% ) 382 (Z w ink e ls et al. , 2 023) . The eff e ct o f S SFF on IVPD vari es acros s th e lit erature. In c r ea sing IV PD ha ve 383 bee n r ep o rt ed i n f e r me nt atio ns w ith P . o s t r eat u s , R. m i cr os p or us , L e n t in u la e do d e s , and A s p e r g i l l u s 384 oryz ae ( Clark e t al ., 2022; E spin o sa -P á ez et al ., 201 7, 2021; T odor ov et al ., 202 4; Villac r é s & R o sell , 385 2021 ). How e v er , f erme nt ation s with Rh iz op us sp. w ere f o und t o dec r e a se th e I VP D (d a Si lv eir a & 386 Badi ale -Fu rlong , 2009; Ranjan e t a l. , 201 9 ; Z wi nk els et a l ., 2023 ). In thi s study , s i milar t o the eff ec t o f 387 f erme n ta ti on on t a ste and ar oma, th e eff ec t I VP D was dep ende n t on s ub s trat e - s pe ci e s in ter acti on . 388 I V P D d i d n o t c h a n g e s i g n i f i c a n t l y i n b r o w n r i c e , w h i l e d i g e s t i b i l i t y d e c r e a s e d i n B S G a n d i n c r e as e d in 389 lupi n f erme nt ed w ith P . c i nn aba r i nu s . 390 Although SS FF al t ered t h e IV PD s ig nifi ca n tly , the i ncrea sed PDC A AS o f f erme nt ed sub s tr a t e we r e 391 lar gely du e t o cha nge s in the a mino ac id compo s i t i on . P ar ticul arly th e in cr ea s e in l y sin e, in t e n ou t o f 392 e l ev e n f e r m en t a t i o ns , r a is e d t he PD CA A S. Thes e r es u lts a r e in li n e w i t h e a r l ier SS FF b y R. 393 m i cr os po r us an d A. o r y z a e on ba r l ey and whit e ric e (Z w ink e ls e t a l., 20 23 ). This inc r e a se in ly s in e i s o f 394 partic ula r import a nce be c au s e lys in e i s t he main l imiting ami no acid i n plan t-b as ed diets ( L e inone n 395 et al. , 2019) . Alth ough b a s i diom y c ete s could i ncr ea s e the protein qua li ty of s p ec ific s ub str ate s mor e 396 than R. m ic r o s po r u s , fungal specie s fr om both ph yl a impr ov e t he p r o t e in quali ty of p la n t s ub s trat e s . 397 Nev er thel e ss , a s ha s bee n de s c r i b ed i n Z wink els, Oors c ho t, et al. (2025), readi ly av ail able c a r bo n 398 sou r ce s in th e sub s trat e ( e. g . s t ar c h ) cou ld imp act the p r o t ein c ont e n t o f the f e rme nt ed product, by 399 w e igh t lo s s d ue to their con sumption a n d c on vers i on t o CO 2 . Th ere fore, t o a s s e s s t he t o t a l quali t y of 400 the pr o tein sour c e , both p r o t ein qu al ity a nd pr o t ein c ont e n t n eed t o be evalu at e d . 401 For thi s rea son, the u t i liz abl e amino ac i d c ont e nt (U A A ) w a s c a lcul at e d, whic h evalua t e s bo th t he 402 pr otein qu a lity a s well a s the total ami n o acid co nt e n t ( Figu r e 5) . T he t ot al a m i n o a ci d c on t e n t was 403 use d in st e ad o f the c r u de pr o t ei n c o nt e n t, b e c au se in f erme n ted p r o duct s, method s t o d etec t 404 nitr ogen -ba s ed crude pr o t e in, ma y no t a cc ur a tely e s tima t e pr o t e in co nt e n t beca u s e deamin at i on ca n 405 inc r ea se n on- amin o nitrog en l evel s, l e ading t o an ove r e st i m a tion o f pr o tein c ont e n t (Zwink el s, 406 Oor sc hot, e t a l., 2025 ). The c hang e i n U AA fr om un f erme nt ed sub s tr a t e s enc ompas s chang es ari s in g 407 fr om i) c hange s in pr o t e in qu ality , ii ) c h ang e s in conc e n tr at i on of amin o ac id s , du e t o lo s s of d r y 408 w e igh t , and iii ) lo s s of am ino aci d s by dea mination . Thi s i s illu s tr a t e d by the f oll owi ng e x a mples: i) 409 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted September 24, 2025. ; https://doi.org/10.1101/2025.09.24.678313doi: bioRxiv preprint f erme n ta ti on o f lup in wi th P . c i nn aba r inu s i n c r e a s e d t h e p r o t e i n q u a l i t y b u t s l i g h t l y d e c r e a s e d t o t a l 410 ami no a cids c o nt ent (Suppl eme n tary t a ble 2 & S uppleme nt a r y fi gure 3); i i) f e rme n t a tion of br ow n 411 rice with S. comm une increa sed t o t al a m ino ac id c on t e n t w ith minima l cha n g e in pr otein q uality; ii i ) 412 f erme n ta ti on o f BS G w ith P . ci n n aba r inus d e c r e a s e d t h e t o t a l a m i n o a c i d c o n t e n t , d u e t o c a t a b o l i s m 413 of amin o acid s, counter actin g th e incr ea s e i n pr o t e in quali t y r e s ulti ng in an over all dec r ea s e in U AA. 414 The se r e sul ts indi c ate that both an inc re ase in pr ot e in c ontent and pr o t ein qu ali ty can be obt ain e d 415 wi t h SSFF by basidiom ycete s wi t h t h e c orrec t sub s tr a t e - s pec i es f ormul a ti on. A pr e r equi si t e f or 416 succ e ssful imp r ov e me n t of the nutri tion al q uality app ea rs t o b e th e p r e s e nc e o f a readil y a v a ilabl e 417 carbon s our ce . Th i s pr eve nts t h e util i z a t io n o f amin o ac ids a s an a ltern a tiv e c a rbon s o ur c e, whil e 418 inc r ea sing the t o t a l amino ac id c o nt e nt due t o we igh t l o ss in f o rm o f non -pr o t e in c ompo und s (e. g . 419 carboh y drat e s ). Simil a r ob ser va tion s w er e made by S t o ff e l e t al . (20 19 ), w ho s ho wed th a t 420 f erme n ta ti on of BSG a nd g r a pe bagas se by the basi diomy ce t e s Pl euro tu s al bid u s, A gar i cu s 421 fu sc o suc c ine a , an d Ag aricus bl az ei h a d varying eff ect s on t h e pr o t ei n a nd amino a cid co n tent. They 422 obser ved th at when carboh y d r at e s w e re met a b oliz ed, a s se en wi t h P. a l b i d u s i n BSG, both p r o t e i n 423 and amino acid c ont e nt incr ea sed. H o wev e r , wh en carboh y drat e s we r e eit he r a bs e nt (in gr ape 424 bagas s e ) or could no t be utili z ed (by A garicu s s pe ci e s), t o t a l a mino acid cont e n t de crea sed , ev e n 425 though crude p r o t ein c o nt e n t still in cr e a s ed. 426 Fi nally , ba sidiom ycetou s s pe cie s impr oved th e t o t al UAA c ontent o f t he ce r e al ( br own rice ) a nd t h e 427 leg ume (lupi n) mo r e eff ectively than R. microsp oru s , by increa sing th e A AS o f l y sin e and the t o t a l 428 concentra tion of am in o aci d s through a lo ss o f d r y weig h t. T hi s is p articu l arly rel evan t s in ce c e r e al s 429 c u r r e n t l y p r o v i d e a p p r o x i m a t e l y 3 0 % o f t h e p r o t e i n i n h u m a n d i e t s , w i t h m a i z e , w h e a t , a n d r i c e a s 430 the domina n t c r op s (FA O , 20 24). Thi s r a t i o i s e x pected t o ri s e with an inc r e a s e i n plan t -ba s ed diet s 431 (Le inone n e t al., 2 019 ). How e v er , c er e als typi call y hav e a high c alo r i e -to-p r o t ein r at i o a nd a r e 432 defic ient in lysine (S á & H ou s e , 2 024). There f o r e , th e c ombined eff ec t o f inc r e a sing t he PD CA AS o f 433 lys in e and el ev ation o f t he t o t a l ami no acid c ont e n t, du e to r emov a l of non-ni tr o g en cal oric 434 nutrie n t s, signifi cantly enha nce s t he po t e n ti a l of ce r e al s t o c ontribu t e t o mee ting di et ary p r otei n 435 r equi r eme nts . 436 In c onc lu si on, s ol id - s tat e f erme nt ation with edibl e ba s idi om y c et e s c o uld i ncr e as e in the utilizabl e 437 ami no acid con tent a s well a s th e umami t aste, par tic ularly in P . cinn abari nu s f er m en t a t io ns . Th es e 438 r e sult s imply th a t s ol id - s ta te f erm enta tio n with edibl e b a s i di om y c et e s co u ld pr odu ce nov e l 439 altern at ive p r o t ein -en rich ed food s or in gr edi e n t s w it h i mpr ov e d nutri tional valu e an d um ami t a st e , 440 whi ch a r e impor t ant d r ive r s f or al t e rn a tive pr o t ein a d option ( Onwez en et al. , 2021; Sc hmidt & 441 Mourit sen , 2022 ). Fu rt h e r mo r e , the al t e r ed t exture, d efin ed by the t r a n s f or ma tion fr om loo se 442 k e r ne l s t o a soli d pa t t y , t r a n s f o rms the per ce pti on o f the f o od. The r eby , t he s e novel cer e al -b a s e d 443 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted September 24, 2025. ; https://doi.org/10.1101/2025.09.24.678313doi: bioRxiv preprint t emp eh-l ik e pr oduc ts cou ld bett e r s er v e a s t he pr o t ei n sou r ce o f the me al . H owev e r , mo r e 444 r efin eme nts in sub str at e -spec ie s formul ation a r e r e quired to optimiz e th e f e rmen t e d f ood produc t i n 445 t er m s of p r ot e in q u a l it y , t as t e , an d t e xt ur e. Fi n al l y , l e g is la t i v e h u r d les m i g ht ha v e t o be t a k e n as s u ch 446 pr oduc ts lik ely f all u nd er th e EU Nov el Fo od r egu l a tion (201 5/ 22 83 ) (Moli tori s ová et a l., 202 5 ). 447 CRediT authorship contribution statement 448 Jasper Zwink els : C o nceptu al iz at i on , M ethodolog y , Inve s t i ga tion , Formal ana l y si s, D a t a cura ti on , 449 V al idation, W riting – origina l d r a f t . Os c ar van M a s trigt: C onc eptua liz a t i on, M e t h odology , W r iting – 450 r evi ew & edi ting , S uper vi sion . E ddy J. Sm id: Conc ep tuali z a ti on , Me thodol o gy , W riting – r e view & 451 editin g , Supe rv i s i on, Fund i ng ac quisi tion, Pr ojec t admin i s tra ti on. 452 Funding statement 453 This pr o ject w a s suppo r t ed b y the Go o d Food I n s ti tu t e t hr oug h th e Al t e r n a tive pr o t e in re sea r c h 454 gr an t u nd er c o n trac t numbe r 22-FM -NL - F C A-1 -31 0 . 455 Declaration of competing interest 456 The autho r s d ec la r e th at they ha v e n o know n c omp eting fin anc ial int e r e s t s or p e r s on a l r e lation ship s 457 that could have a pp eared t o in f l u ence th e w ork r e p ort ed in t hi s paper . 458

459 W e would lik e to thank S te f van Oo r scho t f or hi s co n tr i b ut i on t o the pr od uctio n a nd ana lysi s of s ol id -460 st ate f erme nt ed BS G. Addi tio nally , th e ex plor a tory s t ud i e s of P hili p van K oolw ijk, Ran X u, a nd Ales s ia 461 de Cha u d we r e v e ry valuabl e f o r thi s pa per . W e gr ate fully ack nowle dge D r . R e b ecca R oc chi f o r her 462 inspira ti on in th e gr aph ical d e s ig n. La stl y , we w ou ld lik e t o th ank D r . Nikk ie v a n der Wi el en, Mich e l 463 Br eu er and D r . Leo n d e Jon g e f o r th eir ad vi ce a nd perf o rming the amino a cid e xtr a ction and a nalysi s . 464 Data availability 465 Dat a w ill be mad e av ai lable o n r equ e st. 466 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted September 24, 2025. ; https://doi.org/10.1101/2025.09.24.678313doi: bioRxiv preprint

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No reuse allowed without permission. The copyright holder for this preprintthis version posted September 24, 2025. ; https://doi.org/10.1101/2025.09.24.678313doi: bioRxiv preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted September 24, 2025. ; https://doi.org/10.1101/2025.09.24.678313doi: bioRxiv preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted September 24, 2025. ; https://doi.org/10.1101/2025.09.24.678313doi: bioRxiv preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted September 24, 2025. ; https://doi.org/10.1101/2025.09.24.678313doi: bioRxiv preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted September 24, 2025. ; https://doi.org/10.1101/2025.09.24.678313doi: bioRxiv preprint