1
Hyd o he mal ea men o ches nu shells (Cas anea sa i a) o p oduce oligosaccha ides 1
and an ioxidan compounds 2
Bea iz Gullón1, Gemma Eibes*1, Izaskun Dá ila2, Ma ía Te esa Mo ei a1, Jalel Labidi2, Pa icia 3
Gullón2
4
1Depa men o Chemical Enginee ing, Ins i u e o Technology, Uni e sidade de San iago de Compos ela, 5
15782 San iago de Compos ela, Spain 6
2Chemical and En i onmen al Enginee ing Depa men , Uni e si y o Basque Coun y, 20018 San 7
Sebas ián, Spain 8
*E-mail add ess: [email protected] 9
Abs ac 10
Hyd o he mal ea men is an en i onmen ally iendly echnology ha allows he solubilisa ion 11
o hemicellulosic oligosaccha ides wi h po en ial o hei use as p ebio ics. The pu pose o his 12
s udy was o solubilize oligosaccha ides and an ioxidan compounds om ches nu shells by a 13
hyd o he mal p ocessing. The highes con en o oligosaccha ides (18.3 g/L), wi h a ela i ely 14
low le el o monosaccha ides (2.4 g/L) and deg ada ion p oduc s (0.5 g/L) was ob ained a 180 15
ºC (se e i y o 3.08). In addi ion, he liquo s p esen ed a high con en o phenolic and la onoid 16
compounds wi h good an ioxidan p ope ies. The GC-MS e ealed ha he mos abundan 17
phenolic compound was py ogallol (13.2%). The molecula weigh dis ibu ion o he 18
solubiliza ion p oduc s showed ha a 26.5% p esen ed an appa en Mw o 6077 g/mol and a 19
73.5% p esen ed an appa en Mw o 586 g/mol wi h a high polydispe si y index. MALDI-TOF, 20
FTIR, and TGA analyses e ealed s uc u al in o ma ion o hese compounds and hei he mal 21
s abili y. 22
23
Keywo ds: au ohyd olysis, oligosaccha ides, an ioxidan ac i i y, s uc u al cha ac e iza ion, 24
ches nu shells 25
26
27
28
2
1. In oduc ion 29
Changes in he li es yle o he i s wo ld socie y a e b inging abou a shi in ea ing 30
habi s and ends in ood manu ac u e and consump ion, which ha e an impac on heal h, 31
en i onmen and socie y (Cencic & Chingwa u, 2010; Isanga, & Zhang, 2007). In his con ex , 32
he demand o as and eady-made ood is cons an ly inc easing. Opposi e o his end, 33
consume s a e inc easingly a en i e o ood sa e y, quali y and heal h- ela ed issues (Cencic & 34
Chingwa u, 2010). In his sense, he g owing awa eness o he ela ionship be ween heal h and 35
nu i ion has os e ed he sea ch o and isola ion o bioac i e subs ances as a way o coun e ac 36
unbalanced die s (An o & Ðo de ic, 2017; Cencic & Chingwa u, 2010). In pa icula , he e is 37
a g owing in e es in he sea ch o new sou ces o ob ain bioac i e compounds. 38
In his pe spec i e, unde alued by-p oduc s gene a ed om he indus ial p ocessing o 39
ag o-indus ial eeds ocks can po en ially be exploi ed as an inexpensi e and enewable sou ce 40
o bio-compounds (Mandelli e al., 2014). The e o e, he u iliza ion o ag o-indus ial by-41
p oduc s needs o ind sui able app oaches wi h a double bene icial e ec : 1) was e 42
managemen ; 2) alo iza ion o added alue p oduc s (Mo ana e al., 2017). 43
Among he p ocessing indus y, in ecen decades, he ches nu (Cas anea sa i a) 44
indus y has g own signi ican ly in Eu ope, especially o he p oduc ion o ma on glace and 45
ches nu lou , he la e used as ing edien in glu en- ee die s (Vella, La a a, La Ca a, & 46
Mo ana, 2017). Du ing he ches nu peeling p ocess, he ches nu shell is emo ed, a ac ion 47
ha ep esen s be ween 10 and 15% o he weigh o he whole ches nu (Vázquez, Mosque a, 48
F ei e, An o ena, & González-Ál a ez, 2012). Ches nu shells a e lignocellulosic ma e ials 49
comp ised o majo cons i uen s: cellulose, hemicelluloses and lignin (González-López, Mou e, 50
Domínguez, & Pa ajó, 2012), wi h di e en ypes o unc ional g oups which can be binding 51
si es o ion exchange and complexa ion eac ions (Vázquez e al., 2012). 52
Nowadays, ches nu shells a e cu en ly used as uel, bu hei ex ac s ha e been 53
epo ed o ha e an ioxidan ac i i y due o hei high polyphenolic con en (Vázquez e al. 54
2008). In addi ion, he mode a e amoun o hemicelluloses (González-López e al., 2012) can be 55
a ema kable subs a e o ob aining non diges ible oligosaccha ides (NDO) desc ibed as 56
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po en ial p ebio ic subs a es, bu o ou bes knowledge, hei exploi a ion o his applica ion 57
has no ye been conside ed. 58
A p ebio ic is “a selec i ely e men ed ing edien , o a ibe ha allows o speci ic 59
changes in bo h he composi ion and/o ac i i y o he gas oin es inal mic obio a, con e ing 60
bene i s on he well-being and heal h o hos ” (Robe oid e al., 2010). Mo eo e , p ebio ics 61
p omo e o he indi ec e ec s such as immunological, an i-in lamma o y, an icance , 62
an ialle gic ac ion and also imp o e in es inal unc ion and bioa ailabili y o calcium (Aacha y 63
& P apulla, 2011; Aze edo-Ca alho, de Oli a Ne o, da Sil a, & Pas o e, 2013). Among he 64
di e en oligosaccha ides, xylooligosacha ides (XOS) p o ide addi ional bene i s beyond 65
p ebio ic p ope ies: mode a e deg ee o swee ness, e ec on s a ch e og ada ion, s abili y o 66
pH and empe a u e and imp o ed nu i ional and senso y p ope ies o ood (Vo agen, 1998). 67
All o hese p ope ies make xylooligosaccha ides sui able compounds o be inco po a ed in o 68
ood (Ayyappan e al. 2016). 69
The p oduc ion o NDO om ag icul u al esidues has ecei ed much a en ion due o 70
hei high a ailabili y and low cos . Be ween he di e en echniques ha could be used o 71
ob ain NDO, hyd o he mal ea men , also called au ohyd olysis o liquid ho wa e , is a low 72
cos and en i onmen ally iendly echnology ha allows he solubiliza ion o compounds o be 73
used as p ebio ics (Qui ain, Sa o, Daimon, & Fujie, 2003), a oiding he use o chemicals. 74
Oligosaccha ides ob ained by hyd o he mal ea men om di e en aw ma e ials has been 75
exploi ed in ecen yea s. Gullón e al. (2008) and Dá ila, Go dobil, Labidi, and Gullón (2016) 76
ob ained xylooligosaccha ides om ice husks and ine shoo s by an au ohyd olysis ea men , 77
espec i ely, while Gullón, Yáñez, Alonso, and Pa ajó (2010), Ros o e al. (2014) and Rico, 78
Gullón, Alonso, Pa ajó and Yáñez (2018) epo ed he p oduc ion o oligosaccha ides om ye 79
s aw, maize pe ica p o peanu shells, espec i ely. The liquo s om he hyd o he mal p ocess, 80
in addi ion o he p esence o oligosaccha ides, ha e also been epo ed o con ain compounds 81
wi h an ioxidan ac i i y. Gullón e al. (2017) s udied he in luence o he se e i y o he 82
hyd o he mal ea men on he an ioxidan ac i i y o au ohyd olysis liquo s om ine shoo s. 83
Rico e al. (2018) also s udied he an ioxidan p ope ies o he solubilized p oduc s om peanu 84
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shells, while Mou e, Conde, Falqué, Domínguez, and Pa ajó (2014) e alua ed he pu i ied 85
ex ac s o au ohyd olysis liquo s o ches nu bu s. 86
The goal o his wo k was he solubiliza ion o oligosaccha ides and an ioxidan 87
compounds om ches nu shells by a hyd o he mal p ocessing, since i has no been exploi ed 88
ye . The ches nu shells we e hyd o he mally p ocessed unde di e en se e i ies o de e mine 89
he condi ions ha allowed maximum solubiliza ion o oligosaccha ides. The e ec o ea men 90
se e i y on he composi ion o he liquid and solid phases esul ing om hyd o he mal 91
ea men s, as well as he an ioxidan ac i i y o au ohyd olysis liquo s we e analyzed. 92
Solubilized hemicellulose unde op imal au ohyd olysis condi ions we e cha ac e ized 93
by echniques such as FTIR, TGA, HPSEC and MALDI-TOF. The GC-MS analysis o e hyl 94
ace a e ex ac s allowed he iden i ica ion o compounds de i ed om suga and lignin o 95
ex ac ion-de i ed subs ances ha p o ide an ioxidan ac i i y o he liquo . This app oach 96
could be he i s s age o a new in eg a ed bio e ine y o ches nu shells ha aims a a sui able 97
e alo iza ion s a egy o his unexploi ed was e biomass. 98
2. Ma e ials and me hods 99
2.1. Raw ma e ial and chemicals 100
The ches nu shells used in his wo k we e supplied by a ches nu p ocessing plan 101
(Cue as & Cia S.A., San Cib ao das Viñas, Spain) a e being ob ained by a slow s eam peeling 102
p ocess. The collec ed shells we e ai d ied, milled and sie ed o achie e a pa icle size o less 103
han 0.4 mm. The milled shells we e mixed o acqui e a single ba ch and a oid aliquo 104
a ia ions. The lo was s o ed a oom empe a u e in a da k and d y place un il i s u he use. 105
Sulphu ic acid (95-97%), glucose (≥99.5%), xylose (≥99%), a abinose (≥99%), u u al 106
(99%), hyd oxyme hyl u u al (≥98%), e hyl ace a e (99.8%), gallic acid (≥98%), u in (95%), 107
olox (6-hyd oxy-2,5,7,8- e ame hylch oman-2-ca boxylic acid), Folin-Ciocal eu eagen , 108
ABTS (2,2'-azino-di(3-e hylbenzo hiazoline-6-susl onic acid; 98%), TPTZ (2,4,6- i(2-py idyl)-109
S- iazine; 99%), DPPH (2,2-diphenyl-1-pic ylhyd azyl; 97%), sodium ca bona e, sodium 110
ace a e 3-hyd a e, po assium pe sul a e, ace ic acid, hyd ochlo ic acid and i on(III) chlo ide 111
5
hexahyd a e we e ob ained om Sigma-Ald ich (Ba celona, Spain), pullulan polysaccha ides 112
(≥98%) we e pu chased om Va ian (England). 113
2.2. Cha ac e iza ion o he aw ma e ial 114
Milled ches nu shells we e subjec ed o mois u e (TAPPI T264- om-88), ash (TAPPI 115
T244-om-93) and e hanol- oluene ex ac i es (TAPPI T204 cm-97) de e mina ion. The ches nu 116
shells wi hou ex ac i es we e milled and sie ed o ob ain a pa icle size o less han 0.25 mm 117
in o de o subjec hem o a quan i a i e acid hyd olysis wi h 72% (w/w) H2SO4 (TAPPI T-249-118
cm-09) o he de e mina ion o he hemicellulosic, glucan and lignin con en . The solid phases 119
eco e ed by il a ion a e o en-d ying we e conside ed as Klason lignin, while he liquid 120
phases we e analyzed by High Pe o mance Liquid Ch oma og aphy (HPLC) o he 121
de e mina ion o monosaccha ides (glucose, xylose and a abinose), galac u onic acid and ace ic 122
acid and deg ada ion p oduc s: u u al (F) and hyd oxyme hyl u u al (HMF). A Jasco LC Ne 123
II/ADC ch oma og aph equipped wi h a e ac i e index de ec o was used o hese analyses. 124
20 µL o he samples we e elu ed wi h a low a e o 0.6 mL/min o H2SO4 0.005 M h ough an 125
Aminex HPX-87H 300 x 7.8 mm (Bio-Rad Labo a o ies, USA) column a 50 ºC. All analyses 126
we e pe o med in duplica e. 127
2.3. Hyd o he mal p ocessing o he ches nu shells 128
The au ohyd olysis ea men s o he milled ches nu shells we e ca ied ou in non-129
iso he mal egimen a di e en empe a u es ( om 170-220 ºC). This esidue was mixed wi h 130
wa e in a liquid/solid a io o 8 kg/kg (o en d ied basis) in a 1.5 L s ainless s eel eac o using a 131
Pa PID con olle o con ol empe a u e. Once he eac o was cooled down, he liquid and 132
solid phases we e sepa a ed by il a ion, he liquid phases being s o ed a 4 ºC un il hei la e 133
use and he solid phases d ied a oom empe a u e a e being washed. 134
Wi h he pu pose o acili a ing he compa ison o wo king condi ions, he se e i y (S0) 135
o ea men s was de e mined. This pa ame e , which is de ined as he loga i hm o he se e i y 136
ac o (R0), conside s he e ec caused by ime and empe a u e h oughou he non-iso he mal 137
6
ea men , including bo h he hea ing and cooling pe iod. This pa ame e is exp essed by he 138
ollowing equa ion (Rico e al., 2018): 139
𝑆𝑆
0
=𝑙𝑙𝑙𝑙𝑙𝑙𝑅𝑅
0
=𝑙𝑙𝑙𝑙𝑙𝑙�𝑅𝑅
0𝐻𝐻𝐻𝐻𝐻𝐻𝐻𝐻𝐻𝐻𝐻𝐻𝑙𝑙
+𝑅𝑅
0𝐶𝐶𝑙𝑙𝑙𝑙𝑙𝑙𝐻𝐻𝐻𝐻𝑙𝑙
�=𝑙𝑙𝑙𝑙𝑙𝑙�� 𝐻𝐻
�𝑇𝑇(𝐻𝐻)−𝑇𝑇𝑅𝑅𝐻𝐻𝑅𝑅
𝜔𝜔�
𝑑𝑑𝐻𝐻+� 𝐻𝐻
�𝑇𝑇´(𝐻𝐻)−𝑇𝑇𝑅𝑅𝐻𝐻𝑅𝑅
𝜔𝜔�
𝑑𝑑𝐻𝐻
𝐻𝐻𝐹𝐹
𝑇𝑇𝑇𝑇𝐻𝐻𝑇𝑇
𝑇𝑇𝑇𝑇𝐻𝐻𝑇𝑇
0
�
[1]
whe e Max is he ime (min) equi ed o achie e he maximum empe a u e o each 140
hyd o he mal ea men (TMax, ºC); F is he ime (min) o he en i e hea ing–cooling cycles; T( ) 141
and T’( ) (ºC) a e he empe a u e p o iles in he hea ing and cooling p ocesses, espec i ely, 142
and ɷ and TRe a e pa ame e s whose alues ha e been epo ed in he li e a u e (ω = 14.75 ºC; 143
TRe = 100 ºC). 144
2.4. Chemical cha ac e iza ion o he spen solids om hyd o he mal ea men 145
The spen solids om he hyd o he mal ea men s a e ai -d ying we e subjec ed o 146
g a ime ic and mois u e analyses o de e mine he solid yield and he solubiliza ion o he aw 147
ma e ial. The composi ion o he p e- ea ed solids was analyzed by a quan i a i e acid 148
hyd olysis, as desc ibed o he aw ma e ial in Sec ion 2.2. 149
2.5. Chemical cha ac e iza ion o he liquid phases o hyd o he mal ea men 150
The con en o monosaccha ides (glucose, xylose and a abinose), ace ic and 151
galac u onic acids and deg ada ion p oduc s ( u u al and hyd oxyme hyl u u al) in he liquid 152
phase was analyzed by HPLC as desc ibed in Sec ion 2.2. In addi ion, aliquo s o each ea men 153
we e subjec ed o pos -hyd olysis acid ea men (4% H2SO4 a 121 ºC o 30 min) and he 154
eac ion p oduc s we e quan i ied by HPLC using he same me hodology as desc ibed in Sec ion 155
2.2. The inc ease in he concen a ions o monosaccha ides, galac u onic and ace ic obse ed 156
du ing he quan i a i e pos -hyd olysis makes i possible o de e mine he concen a ion o 157
oligome s and hei deg ee o subs i u ion by ace yl and galac u onic g oups. Oligosaccha ides 158
(OS) we e exp essed as monosaccha ide equi alen s. 159
An aliquo o he a ious liquo s was o en-d ied a 105±2 °C un il cons an weigh , in 160
o de o de e mine he con en o non- ola ile compounds (NVC). The quan i ica ion o non-161
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ola ile compounds ha we e no saccha ides (called o he non- ola ile compounds, ONVC) 162
p esen in he au ohyd olysis liquo s was ca ied ou by he di e ence be ween NVC and 163
saccha ides (conside ing monosaccha ides, OS and OS subs i uen s) (Gullón e al., 2010). 164
Apa om he componen s de e mined abo e, he acid soluble lignin (ASL) solubilized 165
by he hyd o he mal ea men was quan i ied (TAPPI UM 250 um-83 me hod). An aliquo o 166
he au ohyd olysis liquo s ob ained a di e en empe a u es was dilu ed wi h H2SO4 1 M un il 167
he abso bance measu ed a 205 nm wi h an UVmini-1240 spec opho ome e (Shimadzu 168
Co po a ion) was be ween 0.1 and 0.8. 169
[2] 170
Whe e Abs250nm is he abso p ion ( ela i e o 1 M H2SO4 a 205 nm), DF is he dilu ion 171
ac o , VF is he olume o he hyd o he mally liquo , ε is he abso p i i y o lignin a his 172
wa eleng h (110 L/g·cm), DMi is he weigh o he aw ma e ial used in he au ohyd olysis (g as 173
100% d y ma e ) and l (cm) is he leng h o solu ion he ligh passes h ough. 174
2.6. To al phenolic con en (TPC) and o al la onoid con en (TFC) de e mina ion 175
The liquo s o ches nu shells we e e alua ed o o al phenolic con en (TPC) using he 176
Folin-Ciocal eau me hod (Single on and Rossi, 1965) and exp essed as g o gallic acid 177
equi alen s (GAE)/L o au ohyd olysis liquo s. The o al la onoid con en (TFC) o liquo s 178
was quan i ied by he colo ime ic me hod o aluminum chlo ide desc ibed by Blasa e al. 179
(2005). TFC was eco ded in g o u in equi alen s (RE)/L o au ohyd olysis liquo s. All 180
measu emen s we e made in iplica e. 181
2.7. An ioxidan ac i i y 182
An ioxidan ac i i y was e alua ed using h ee complemen a y me hods: DPPH (α,α-183
Diphenyl-β-pic ylhyd azyl adical sca enging assay), ABTS (2,2-azino-bis-3-184
e hylbenzo hiazoline-6-sulphonic acid) and FRAP ( e ic educing an ioxidan powe ) acco ding 185
o he p ocedu es desc ibed in he li e a u e by Gullón e al. (2017). Fo all an ioxidan ac i i y 186
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assays, T olox was used as s anda d and esul s we e exp essed as g o T olox equi alen s 187
(TE)/L o au ohyd olysis liquo s as mean o h ee eplica es. 188
2.8. Chemical and s uc u al cha ac e iza ion o he solubiliza ion p oduc s om 189
hemicelluloses o ches nu shells 190
Solubilized oligosaccha ides a he op imal empe a u e o au ohyd olysis we e eeze-191
d ied and subjec ed o di e en analy ical echniques o ob ain de ailed in o ma ion on hei 192
chemical and s uc u al cha ac e is ics. 193
2.8.1. Fou ie ans o m in a ed spec oscopy (FTIR) 194
The chemical g oups and bonding a angemen o cons i uen s p esen in he 195
oligosaccha ides we e de e mined by FTIR in a Pe kinElme Spec um Two FT-IR spec ome e 196
wo king in ansmission mode wi h a esolu ion o 4 cm-1 and accumula ing a o al o 8 scans. 197
2.8.2. The mog a ime ic analysis (TGA) 198
The mal s abili y was e alua ed by a TGA/SDTA RSI 851 Me le Toledo analyze . 199
Be ween 3 and 5 mg o eeze-d ied liquo s we e es ed unde ni ogen a mosphe e a a hea ing 200
a e o 10 ºC/min om 25 ºC o 800 ºC. 201
2.8.3. Molecula weigh dis ibu ion analysis 202
The molecula weigh dis ibu ion o oligosaccha ides was es ima ed by High 203
Pe o mance Size Exclusion Ch oma og aphy (HPSEC) using a Jasco LC Ne II/ADC 204
ch oma og aph equipped wi h a e ac i e index (RI) de ec o was used. 40 µL o he sample 205
was elu ed wi h a low a e o 0.6 mL/min o 0.005 N H2SO4 a 40 ºC h ough a Va ian Polyme 206
Labo a o ies Aquagel-OH mixed-H 8 µm column. The HPSEC calib a ion was ca ied ou wi h 207
pullulan polysaccha ides wi h di e en molecula weigh s (be ween 180 and 805000 Da). 208
209
210
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2.8.4. Ma ix assis ed lase deso p ion/ioniza ion- ime o ligh mass spec oscopy (MALDI-211
TOF MS) 212
The absolu e masses o OS we e de e mined by MALDI-TOF MS using an Ul a lex III 213
TOF/TOF mass spec ome e equipped wi h a Sma beam® lase (B uke Dal onics). 214
Measu emen s we e pe o med in e lec on ope a ing mode and posi i e pola i y. The 215
accele a ion ol age was se a 25 kV and a o al o 1200 lase sho s pe spo we e au oma ically 216
acqui ed. Sample p epa a ion was ca ied acco ding o he p ocedu e desc ibed in Gullón e al. 217
(2014) using 2,5-dihyd oxybenzoic acid (DHB) as ma ix. 218
2.8.5. Quali a i e Analysis o he E hyl Ace a e Soluble F ac ion 219
The non-saccha ide componen s p esen in he liquo ob ained unde op imum 220
condi ions we e analyzed by ca ying a liquid-liquid ex ac ion wi h e hyl ace a e (EAc). A 221
single ex ac ion s age was ca ied ou by s i ing a mix u e o he liquo wi h EAc using a 222
liquo -sol en a io o 1:3 ( / ) o 15 min. The immiscible phases we e sepa a ed by 223
decan a ion, wi h he o ganic phase acuum e apo a ed a 40 ºC o elimina e he sol en and he 224
dissol ed ola ile compounds. These ex ac s we e dissol ed in EAc and analyzed by Gas 225
Ch oma og aphy-Mass Spec ome y (GC-MS) using an Agilen Technologies 7890A gas 226
ch oma og aph (GC) coupled o an Agilen Technologies 5975C mass spec ome e (MS). 1 µL 227
o he sample was in oduced in he GC in spli mode and using a low o 1 mL/min o He i was 228
passed h ough a column o 30 m x 0.25 mm x 0.25 µm hickness HP-5MS (5% 229
phenylme hylpolysiloxane). The sepa a ion me hod used was he one epo ed by Gullón e al. 230
(2017). The iden i ica ion o he compounds was ca ied ou by compa ing hei mass spec a 231
wi h hose o he Na ional Ins i u e o S anda ds (NIST) lib a y da abase and wi h compounds 232
epo ed in he li e a u e. Mola peak a eas we e calcula ed o compounds wi h a peak a ea 233
g ea e han 0.4% and o de e mine he ela i e abundance o he compounds, he sum o he 234
mola peak a ea was no malized o 100%. 235
236
237
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Figu e 1. E ec o hyd o he mal ea men empe a u e on he composi ion o liquo s: (a)
oligosaccha ides (b) monosaccha ides and (c) deg ada ion compounds.
a)
b)
c)
17
Ano he aspec e alua ed in his wo k was he an ioxidan po en ial o he liquo s om
ches nu shells. Table 3 shows he cha ac e iza ion o he di e en au ohyd olysis liquo s in
e ms o an ioxidan phenolic compounds. As o o al phenolic con en (TPC), i eached a
maximum concen a ion a 180 ºC (4.69 g GAE/L, co esponding o 3.9 g GAE/100 g ches nu
shell), coinciding wi h he maximum XOS yield, and hen s a ed o dec ease, p obably due o
deg ada ion eac ions. A ew esea ch wo ks ha e desc ibed he g adual inc ease in phenolic
con en wi h empe a u e du ing hyd o he mal ea men s and was ela ed o he elease o
phenolic compounds linked o oligosaccha ides and he pa ial depolyme iza ion o lignin
(Conde e al., 2011; Gullón e al., 2017).
The TPC alues epo ed he e show ha ches nu shells a e a ich sou ce o phenolic
compounds. In ac , he ex ac ion o his esidue wi h a 2.5% Na2SO3 solu ion esul ed in a o al
phenolic con en o 13.4 g GAE/100 g o en-d ied shells (Vázquez e al., 2008). This di e ence
in he esul s o bo h s udies could indica e ha some o he phenolic compounds om ches nu
shells could be deg aded a he au ohyd olysis empe a u es e alua ed. Ne e heless, he
phenolic con en de e mined in he liquo was subs an ially highe han hose ob ained by
au ohyd olysis ea men o di e en ma e ials. Fo example, Jesus e al. (2017) epo ed a
maximum o 2.09 g GAE/L in he au ohyd olysis liquo o ine p uning esidue (S0=4.13), and
Gullón e al. (2017) eached a maximum TPC o 2.25 g GAE/100 g ine shoo s a highe
empe a u e (T=215 ºC). Conde e al. (2011) e alua ed he phenolic con en o au ohyd olysis
liquo s o i e di e en lignocellulosic was es, and only hose o ches nu bu s liquo s had a
highe phenolic con en (4.4 g GAE/100 g ches nu bu a 240 ºC) han ha achie ed in he
p esen s udy.
The same end was obse ed o he o al la onoid con en (TFC), wi h he maximum
also a 180 ºC (4.82 g RE/L, co esponding o 4.0 g RE/100 g ches nu shell). The la onoid
con en in his liquo was 3.6 imes highe han ha epo ed o he au ohyd olysis liquo s o
ine shoo s (1.1 g RE/100 g, 215 ºC; Gullón e al. 2017). In e es ingly, he end in an ioxidan
ac i i y depended on he me hodology used. Mo eo e , he DPPH me hod indica ed ha he
18
an ioxidan ac i i y o he liquo emained almos cons an in he ange 170 ºC o 200 ºC, and a
he highes empe a u es, ac i i y d opped sligh ly o 84% o he ini ial ac i i y (4.6 g TE/L).
The ABTS me hod, on he o he hand, showed a con inuous dec ease o he an ioxidan ac i i y
wi h he au ohyd olysis empe a u e, leading o 49% o he ini ial ac i i y in he ha she
condi ions. The an ioxidan capaci y measu ed by he FRAP assay showed ha liquo s
gene a ed a 170-180 ºC exhibi ed simila an ioxidan ac i i y, and a highe empe a u es,
dec eased o a inal ela i e alue o 69%. Con a y o hese obse a ions, Gullón e al. (2017)
obse ed a s eady inc ease o he an ioxidan ac i i y o he ine shoo s au ohyd olysis liquo s
wi h empe a u e, consis en wi h he ise o he TPC le els. Howe e , he maximum
an ioxidan ac i i ies epo ed in ha wo k we e in he ange 2.9-4.4 imes lowe han hose
achie ed he e (1.05, 4.45 and 2.68 g TE/100 g ine shoo s, o DPPH, ABTS and FRAP assays,
espec i ely).
Since he p incipal objec i e o he p esen esea ch was o ob ain a liquo wi h a high
con en o bo h XOS and phenolic an ioxidan s, he op imum empe a u e ha allowed his
objec i e o be accomplished was 180 ºC. A his empe a u e, he o al oligosaccha ide con en
was 18.3 g/L, monosaccha ides accoun ed o 2.4 g/L, ace ic acid as he only deg ada ion
p oduc was p esen a 0.5 g/L and he o al phenolic con en was 4.7 g GAE/L. In his sense, he
mass a io o oligosaccha ide o monosaccha ide was 7.6, which is mo e a o able han he
alue o 5 de e mined o peanu shells (Rico e al., 2018).
19
Table 3. E ec o empe a u e o hyd o he mal ea men on he TPC, TFC and an ioxidan
ac i i y (analyzed by he DPPH, ABTS and FRAP me hods) o au ohyd olysis liquo s. TPC:
o al phenolic con en ; TFC: o al la onoid con en ; GAE: gallic acid equi alen s; RE: u in
equi alen s; TE: T olox equi alen s.
Tempe a u e
(ºC)
TPC
(g GAE/L)
TFC
(g RE/L)
DPPH
(g TE/L)
ABTS
(g TE/L)
FRAP
(g TE/L)
170
4.49 ± 0.08
4.55 ± 0.24
5.60 ± 0.09
16.27 ± 0.09
9.36 ± 0.32
175
4.43 ± 0.04
4.68 ± 0.09
5.50 ± 0.14
15.66 ± 0.14
9.44 ± 0.21
180
4.69 ± 0.01
4.82 ± 0.07
5.35 ± 0.02
15.17 ± 0.25
9.33 ± 0.33
185
4.17 ± 0.19
4.15 ± 0.18
5.14 ± 0.16
13.99 ± 0.36
8.94 ± 0.05
190
3.88 ± 0.12
3.42 ± 0.06
5.38 ± 0.03
13.43 ± 0.23
8.47 ± 0.05
200
3.25 ± 0.08
2.66 ± 0.03
5.31 ± 0.04
9.89 ± 0.21
7.29 ± 0.16
210
2.89 ± 0.03
2.51 ± 0.04
4.92 ± 0.11
8.49 ± 0.26
6.90 ±0.21
215
2.77 ± 0.05
2.44 ± 0.14
4.63 ± 0.15
8.05 ± 0.18
6.72 ±0.13
220
2.57 ± 0.05
2.44 ± 0.03
4.64 ± 0.05
7.80 ± 0.12
6.31 ±0.14
Values a e he mean o wo hyd o he mal ea men s ± s anda d de ia ions
Figu e 2 shows he mass balance o he hyd o he mal p e ea men pe o med a 180
ºC. F om 100 kg o d ied ches nu shells, 15.11 kg o subs i u ed oligosaccha ides (sum o GOS,
XOS and A OS), 1.87 kg o monosaccha ides and 0.29 kg o an ioxidan compounds can be
ob ained. In addi ion, 72.8 kg o spen solid (en iched in cellulose and lignin), ha can be used
as subs a e o a a ie y o ma ke able chemicals, we e also eco e ed.
20
Figu e 2. Ma e ial balances o he p oduc s ob ained om ches nu shells a he eac ion
empe a u e o 180 ºC
Abb e ia ions: NVC, non- ola ile compounds; ONVC, o he non- ola ile compounds; S0,
se e i y
3.3. S uc u al cha ac e iza ion o he solubiliza ion p oduc s om ches nu shells
hemicellulose
A b oade s udy o he s uc u al cha ac e is ics o oligosaccha ides was ca ied ou o
assess he po en iali y o he wa e -soluble compounds as nu aceu icals. The au ohyd olysis
liquo om he expe imen ca ied ou a 180 ºC (S0=3.08) was analyzed by HPSEC, MALDI
TOF, FTIR, and TGA o ob ain s uc u al and he mal beha io in o ma ion o hese
compounds. This liquo was ob ained unde he au ohyd olysis condi ions which led o he
maximum concen a ion o oligosaccha ides and an ioxidan compounds.
3.3.1. De e mina ion o he molecula weigh dis ibu ion o solubilized oligosaccha ides
The molecula weigh o he oligosaccha ides is an impo an cha ac e is ic ha
in luences hei biological p ope ies (Gullón e al., 2014). In his sense, Gullón e al. (2008)
AUTOHYDROLYSIS
(S0=3.08)
Wa e
(789 kg)
Ches nu shells
(111 kg o ma e ial,
equi alen o 100 kg o d ied ches nu shells)
Fil a ion
Spen solid
(72.8 kg)
Liquo s
(827.2 kg)
Wa e and ola ile eac ion p oduc s: 804.98 kg
NVC: 22.22 kg
Monosaccha ides: 1.87 kg
Subs i u ed oligosaccha ides: 15.11 kg
An ioxidan compounds: 0.29 kg
ONVC: 4.95 kg
21
sugges ed ha xylooligosaccha ides wi h a deg ee o polyme iza ion (DP) g ea e han 4 p esen
po en ial p ebio ic applica ions. In his wo k, he molecula weigh dis ibu ion o he
oligosaccha ides con ained in he au ohyd olysis liquo s was analyzed by HPSEC
(Supplemen a y Figu e S1). Values we e calcula ed based on equi alen RI signals o pullulan
s anda ds o known Mw. The oligosaccha ides could be di ided in o wo ac ions: a mino
p opo ion (26.5%) wi h highe appa en molecula weigh (Mw) o 6077 g/mol (DP: 46.03) and
he majo ac ion (73.5%) wi h a lowe appa en Mw o 586 g/mol (DP: 4.43), which can be
ega ded as low molecula weigh oligome s (Tunc and Van Heiningen, 2011).
The appa en Mw o he solubilized oligosaccha ides om ches nu shells (S0=3.08) is
lowe han he one o he oligosaccha ides om ine shoo s (Dá ila e al. 2016). Wang e al.
(2016) also ob ained oligosaccha ides wi h highe appa en Mw (8430 g/mol) du ing he
au ohyd olysis o apeseed s aw ca ied ou wi h an S0 o 3.26. Du ing he au ohyd olysis
ea men , depolyme iza ion o solubilized hemicellulosic oligosaccha ides o high molecula
weigh in o oligome s o low molecula weigh akes place, so he di e ence o he Mw o he
oligosaccha ides is a consequence o he s uc u e o he aw ma e ial. I should be no ed ha
he high polydispe si y index (10.25) o he ches nu shells oligosaccha ides is due o he high
di e ence o he Mw o he wo ac ions. As epo ed in he li e a u e, oligosaccha ides wi h
di e en a e age deg ees o polyme iza ion may p esen di e en p ebio ic p ope ies.
Acco ding o Sanchez e al. (2009), sho e -chain-leng h molecules we e p ima ily e men ed in
he p oximal colon, whe eas longe molecules (DP: 29) eached he dis al colon. Hence,
oligosaccha ides wi h high deg ees o polyme iza ion should be conside ed o p e en o al
e men a ion du ing ansi h ough p e ious compa men s (Sanchez e al., 2009).
3.3.2. Ma ix Assis ed Lase Deso p ion/Ioniza ion Time o Fligh Mass Spec ome y
Fo mo e in o ma ion on he s uc u al ea u es o he solubilized oligosaccha ides a he
op imum empe a u e, a MALDI-TOF MS analysis was ca ied ou (Supplemen a y Figu e
S2). Table 4 p esen s he mass signals in MALDI-TOF spec a, along wi h he main s uc u es
iden i ied ( he speci ic compounds we e de ec ed as sodium and po assium adduc s). The
22
MALDI-TOF-MS spec a p o ile showed he p esence o di e en ypes o oligosaccha ides
ha consis ed p edominan ly o chains o pen oses and hexoses. The iden i ied pen aoligome s
(which co espond o xylose esidues acco ding he composi ion analysis o he liquo s
men ioned abo e) showed a ich subs i u ion pa e n, mainly by ace yl and me hylglucu onosyl
g oups, wi h DP in he ange o 2-12. I is impo an o no e ha pa o he XOS may be
subs i u ed wi h a abinose uni s. Howe e , a abinose subs i u es canno be de ec ed by MALDI-
TOF-MS as bo h xylose and a abinose ha e he same molecula weigh . As o hexose
oligome s, hese included a se ies o unsubs i u ed o ace yla ed oligome s wi h DP anging
om 4 o 12. The p esence o hese oligome s can be explained by he depolyme iza ion o
cellulose and he deg ada ion o glucan (Wang e al., 2016). The subs i u ion pa e n o
oligosaccha ides a ec s hei p ebio ic po en ial as con i med in he li e a u e. In his sense,
subs i u ed oligosaccha ides can display a di e en e men a ion beha io and bioac i i y
po en ial (Kabel, Ko enoe en, Schols & Vo agen, 2002).
The s uc u al cha ac e is ics o oligome ic compounds ob ained om di e en aw
ma e ials by hyd o he mal ea men ha e been ex ensi ely conside ed in he li e a u e (Rico e
al., 2018; Ruiz e al., 2017; Gullón e al., 2014). Howe e , his is he i s ime ha a de ailed
s uc u al cha ac e iza ion o oligosaccha ides om ches nu shells is epo ed.
23
Table 4. MALDI-TOF esul s o he au ohyd olysis liquo s a 180 º C and sugges ed s uc u es.
m/z
S uc u e
m/z
S uc u e
627.19
Pen 4AcK
1239.38
Hex6Ac5K
669.19
Pen 4Ac2K
1265.4
Pen 8Ac4Na
711.11
Pen 4Ac3K
1281.39
Pen 7Ac3MeU Na
727.10
Pen 3Ac2MeU K
1287.39
Pen 8MeU Na
741.16
Pen 2Ac2U 2Na
1329.44
Pen 8AcMeU Na
743.18
Pen 5AcNa
1337.47
Hex8Na
745.15
Pen 4U Na
1353.46
Hex8K
747.18
Hex4AcK
1371.44
Pen 8Ac2MeU Na
759.2
Pen 4MeU Na
1395.45
Hex8AcK
761.19
Pen 4U K
1397.44
Pen 9Ac4Na
785.18
Pen 5Ac2Na
1413.46
Pen 9Ac4K
787.18
Pen 4AcU Na
1419.45
Pen 9MeU Na
789.16
Pen 3U 2Na
1429.45
Pen 7Ac2MeU 2Na
849.24
Pen 6K
1455.47
Pen 8Ac4MeU Na
867.25
Hex5K
1461.47
Pen 9AcMeU Na
975.27
Pen 6Ac3K
1487.51
Pen 10Ac3Na
1011.29
Hex4Pen 2AcK
1497.49
Pen 8Ac5MeU Na
1013.32
Hex6Na
1499.54
Hex9Na
1029.32
Hex6K
1515.54
Hex9K
1049.30
Pen 7Ac2Na
1529.51
Pen 10Ac4Na
1065.32
Pen 7Ac2K
1545.52
Pen 9Ac3MeU Na
1091.35
Pen 7Ac3Na
1587.53
Pen 9Ac4MeU Na
1107.32
Pen 7Ac3K
1593.54
Pen 10AcMeU Na
1113.33
Hex6Ac2K
1635.56
Pen 10Ac2MeU Na
1123.30
Pen 5AcMeU 2Na
1677.59
Pen 10Ac3MeU Na
1131.33
Pen 4Ac5U 2Na
1719.58
Pen 10Ac4MeU Na
1133.34
Pen 7Ac4Na
1725.57
Pen 11AcMeU Na
1139.35
Hex6Ac3Na
1767.59
Pen 11Ac2MeU Na
1175.39
Hex7Na
1793.62
Pen 12Ac4Na
1181.36
Hex6Ac4Na
1809.62
Pen 11Ac3MeU Na
1191.38
Hex7K
1839.67
Pen 10Ac3U 2Na
1197.36
Hex6Ac4K
2001.73
Hex12K
1233.37
Hex7AcK
(Pen =pen ose; Hex=hexose; Ac=Ace yl g oup; MeU , O-me hyl-u onic acid; U : u onic acid)
24
3.3.3. FTIR Analysis
FTIR spec oscopy was used o de e mine he speci ic abso p ion bands o he wa e -
soluble compounds p esen in he au ohyd olysis liquo ob ained a 180 ºC. The FTIR spec a
(Supplemen a y Figu e S3) o he au ohyd olysis liquo o ches nu shells p esen ed ypical
bands epo ed o hemicellulosic oligosaccha ides (Jiang e al., 2014 and S ä d e al., 2015;
Dá ila e al., 2016; Rico e al., 2018). The bands a 1021.69 and 895 cm-1 co espond o he C-
O, C-C s e ching ib a ion o o he C-OH bending ib a ion obse ed in xylan and o he β-(1-
4) glycosidic bonds be ween suga s espec i ely. Acco ding o Peng e al. (2009), he bands
loca ed a 1364.70 cm-1 and 1243.16 cm-1 also could be a ibu ed o he s uc u e o
hemicelluloses. The p esence o a abinosyl side chains in he suga s uc u e is con i med by he
band a 1144.51 cm-1, while he band obse ed a 1719.50 cm-1 indica ed he p esence o ace yl
acids linked o he backbone o oligosaccha ides (Dá ila e al. 2016). The wide band a 3305.22
cm-1 is asc ibed o he O-H s e ching o hyd oxyl g oups and he band obse ed a 2927.84 cm-1
co esponds o he s e ching ib a ions o C-H bonds. The band loca ed a 1609.68 cm-1 could
be a ibu ed o wa e bound o he suga chain, bu i could also co espond o sy ingyl de i ed
linked o oligosaccha ides.
3.3.4. The mal g a ime ic analysis (TGA)
The he mal s abili y o he solubilized oligosaccha ides unde he op imum empe a u e
o au ohyd olysis was de e mined by he mog a ime ic analysis, as depic ed in Figu es 3a and
3b. By combining he in o ma ion om hese wo cu es, i can be obse ed ha h ee di e en
deg ada ion p ocesses occu ed. The lyophilized liquo showed an ini ial weigh loss o 10.38%
below 150 ºC. This e en was associa ed o wo deg ada ion p ocesses as can be seen in he
de i a i e he mog a ime ic (DTG) cu e a 105 and 135 ºC, which could co espond o he
e apo a ion o he abso bed wa e . The g ea es weigh loss (43.33%) o he lyophilized liquo s
ook place be ween 150 and 400 ºC, being wo p ocesses o maximum deg ada ion a 239 and
304 ºC. This second s age could be a ibu ed o he decomposi ion o oligosaccha ides, as
25
Demi bas (2000) epo ed ha be ween 150 and 350 °C, he dehyd a ion and decomposi ion o
he glycosyl uni s ook place. These da a show ha solubilized oligosaccha ides om ches nu
shells can wi hs and high empe a u es, sugges ing hei s abili y in p ocesses such as cooking,
pas eu iza ion, and s e iliza ion in ood and d ug indus ies.
0
10
20
30
40
50
60
70
80
90
100
25 125 225 325 425 525 625 725
Weigh (%)
Tempe a u e (°C)
Figu e 3. The mog a ime ic analysis (TGA) (a) de i a i e he mog a ime ic (b) cu es o
au ohyd olysis liquo s ob ained a 180 ºC
25 125 225 325 425 525 625 725
Tempe a u e (°C)
b)
a)
32
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34
Supplemen a y Da a
Hyd o he mal ea men o ches nu shells (Cas anea sa i a) o p oduce oligosaccha ides and
an ioxidan compounds
Bea iz Gullón1, Gemma Eibes*1, Izaskun Dá ila2, Ma ía Te esa Mo ei a1, Jalel Labidi2, Pa icia
Gullón2
1Depa men o Chemical Enginee ing, Ins i u e o Technology, Uni e sidade de San iago de
Compos ela, 15782 San iago de Compos ela, Spain
2Chemical and En i onmen al Enginee ing Depa men , Uni e si y o Basque Coun y, 20018
San Sebas ián, Spain
*E-mail add ess: [email protected]
1
10
100
1000
10000
100000
1000000
10000000
0
50000
100000
150000
200000
250000
11 13 15 17 19 21 23 25
Log Molecula weigh
IR esponse (In ensi y)
Time (min)
180 ºC
Mw
805000
393000
210000
113000
21700
11300
6000
667
180
Figu e S1. Molecula weigh dis ibu ion o he oligosaccha ides con ained in he
au ohyd olysis liquo o ches nu shells a 180 °C.
35
Figu e S2. MALDI-TOF mass spec a o he oligosaccha ides con ained in he au ohyd olysis liquo o ches nu shells a 180 °C.
36
3305.22 cm-1
2927.84 cm-1
1719.50 cm-1
1609.68 cm-1
1364.70 cm-1
1243.16 cm-1
1144.51 cm-1
1021.69 cm-1
895.41 cm-1
1
2
Figu e S3. FTIR spec a o au ohyd olysis liquo s om ches nu shells a 180 ºC. 3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
37
0
100000
200000
300000
400000
500000
600000
700000
800000
3 8 13 18 23 28 33
In ensi y
Re en ion ime (min)
Fu u al
5-me hyl u u al
2- u oi acid
Ca chol
5-hyd oxyme hyl u u al
Py ogallol
3,4-dihyd oxybenzaldehyde
No iden i ied
P o oca echuic acid
No iden i ied
No iden i ied
22
23
Figu e S4. GC-MS ch oma og am o ace yl soluble ex ac s om au ohyd olysis liquo s om 24
ches nu shells a 180 ºC. 25
26
27
