Reactions of Soy Flour and Soy Protein by Non-Volatile Aldehydes Generation by Speciﬁc Oxidation

polymers

Article Reactions of Soy Flour and Soy Protein by Non-Volatile Aldehydes Generation by Speciﬁc Oxidation

Charles R. Frihart 1, Antonio Pizzi 2,3,* , Xuedong Xi 2 and Linda F. Lorenz 1

1 Forest Products Laboratory, USDA, 1 Giﬀord Pinchot Ave., Madison, WI 53726, USA 2 LERMAB-ENSTIB, University of Lorraine, 27 rue Philippe Seguin, 88000 Epinal, France 3 Department of Physics, King Abdulaziz University, Al Ehtifalat St, Jeddah 21589, Saudi Arabia * Correspondence: [email protected]; Tel.: +33-623126940

Received: 30 August 2019; Accepted: 7 September 2019; Published: 10 September 2019

Abstract: Soy protein isolate (SPI) and insoluble soy flour polymeric carbohydrates have been reacted with sodium periodate for the specific oxidation of vicinal –OH groups to investigate the reactions involved in this approach to soy flour adhesives. The reactions have been shown to generate carbohydrate oligomer fractions presenting one, two or multiple aldehyde groups. With the exception of the small molecular weight heptanedial, the smaller molecular weight aldehydes generated from mono- and disaccharides by the same reaction do not appear to form from the insoluble soy flour carbohydrates, or have already reacted. The reaction of periodate with soy protein isolate has been shown to generate some aldehydes too. When the mix of SPI and soy insoluble carbohydrates is treated with periodate, the majority of the observed aldehyde carrying species appear to be higher molecular weight carbohydrate oligomer fractions.

Keywords: soyflour; soy protein isolate; insoluble carbohydrates; periodateoxidation; aldehydesgeneration; condensation reactions; soy adhesives

1. Introduction Recently, to meet new environmental standards, a number of different approaches to the use of natural materials have been used to prepare wood adhesives without the use of formaldehyde. The use of carbohydrate oxidants is a long well-known practice for a number of different applications. Thus, a number of different oxidation systems have been used to generate aldehydes from polymeric carbohydrate, the literature on this being rather abundant. These include sulfoxide-carbodiimide and related methods [1], nitroxyl radical mediated aqueous oxidation [2], special salts oxidation [3] and more recently nitro-oxidation methods [4,5], this latter being well suited for nanocellulose generation. Among these, the periodate specific oxidation of oligomeric carbohydrates is a well-known system. Thus, such a system is reported from the older periodate oxidation literature on aldehyde generation [6–8] to the more recent literature on nanocellulose generation by such a system followed by ozonization [9]. Among the more recent applications, the use of specific oxidants of monomeric or oligomeric carbohydrates in soy flour have shown particularly interesting results, allowing not only the protein fraction but also the carbohydrate fraction to participate positively in the preparation of wood panel adhesives [10–12]. In this approach, soy flour adhesives have been successfully prepared by treating soy flour with either potassium permanganate or sodium periodate [12]. Sodium periodate is a specific oxidant for carbohydrates reacting with adjacent vicinal hydroxyl groups to form dialdehydes [6,12] according to the reaction:

Polymers 2019, 11, 1478; doi:10.3390/polym11091478 www.mdpi.com/journal/polymers Polymers 2019, 11, 1478 2 of 18 Polymers 20192019,, 1111,, xx FORFOR PEERPEER REVIEWREVIEW 22 ofof 1919 Polymers 2019, 11, x FOR PEER REVIEW 2 of 19

O O _ O O M _ H22O OOM O O M H2O OOM I II I II OO OO MM HO OH HO OH O O HO II OHOH HOHO OHOH MIO OO O O MIOMIO44 O OO H H H H H H H HH H H R R R 1 R M = H, Na RR11 1 RR222 R R R1 1 R2 22 M =M H, = NaH, Na R11 R222

WhileWhile this thisthis reaction reaction reaction andand and outcome outcome areare better arebetter better known known known for forfor carbohydrate carbohydratecarbohydrate for carbohydrate monomers monomersmonomers monomers [6,13] [6,13][6,13] and andand [dimers6,13 dimersdimers] and [13]dimers[13][13] suchsuch such [13 as]asas such glucoseglucoseglucose as glucose andandand sucrose,sucrose, and sucrose, equivalentequivalentequivalent equivalent re rereactionsactions reactions are are known are known known also also alsofor for forhigher higher higher carbohydrate carbohydrate carbohydrate oligomersoligomers up up to to cellulosecellulosecellulose itself [12,14,15]. [[12,14,15].12,14,15]. In In the the case case of of cellulose cellulosecellulose and andand long longlong carbohydrate carbohydratecarbohydrate oligomers oligomersoligomers alone,alone, the the reactions reactions that that havehave been shown shown to to occur occur are are the the condensation condensation condensation of of the the aldehydes aldehydes aldehydes formed formed with with otherother carbohydrate carbohydrate chains chainschains to to yield yield crosslinking,crosslinking, crosslinking, leading leading leading to to tosolid solid solid panels panels panels in inthe in the the case case case of celluloseof of cellulose cellulose [15]. [15]. [ 15]. ByBy increasing increasing the the levellevel ofof oxidationoxidation withwith further further periodate periodate in in the the presence presencepresence of ofofan ananaldehyde-reactive aldehyde-reactivealdehyde-reactive speciesspeciesspecies such such such as asas a aa soy soysoy protein proteinprotein [12], [[12],12], aa a flavonoidflavonoid ﬂavonoidflavonoid tannin tannin tannin [13] [13] [[13]13 or] or or other other other reactive reactive reactive species, species, species, including including lignin, lignin,lignin, cross-linking can also occur, leading to feasible wood adhesives [12,13].27 cross-linkingcross-linking can can alsoalso occur,occur, leadingleadingleading tototo feasiblefeasiblefeasible woodwoodwood adhesivesadhesivesadhesives [ [12,13].27[12,13].2712,13].

Further scission FurtherFurtherFurther aldehydes scissionscission FurtherFurther aldehydesaldehydes NaIO4 OH NaIO4 OH OH 4 OHO OHOHO O O O OH OH O O O O OO O O O OH HO OH HO OH OH O OO O O O NaIO O HOHO OHOH HO O Carbohydrates O 4 O O O O O O crosslinkingCarbohydrates O O NaIO44 O O Carbohydrates O O crosslinkingcrosslinking HO OH O O O O O O HO OH O O O O O with or without HO OH O with orNaIO without4 with or without Aldehydes reaction HO OH NaIO NaIO44 with other species Crosslinking Aldehydes reaction with other species Crosslinking The oxidation with the periodate ion, resulting in a 1,2–glycol scission, is one of the most widely usedThe reactions oxidation in carbohydrate with the periodate chemistry. ion, Theresulting mild reaction in a 1,2–glycol and the scission, aqueous issolvent one of conditions the most most widely widelyfor usedperiodate reactions oxidation in carbohydrate are particularly chemistry. apt for The use wimild th water-soluble reaction reaction and and carbohydrates.the the aqueous aqueous solvent solvent The development conditions conditions for for periodateand wide oxidation application are of particularlyparticularlythe reaction aptareapt due forfor useuseto its withwi highthth water-solublewater-soluble degree of selectivity carbohydrates.carbohydrates. [6,14,15]. The The development development and wide Aldehydes application are well of theknown reaction for reacting areare duedue with toto its itsthe highhigh amino degreedegree group ofof on selectivityselectivity proteins [10–12]. [[6,14,15].6,14,15 The]. aldehydes generated from the carbohydrate fraction of soy flour appear to react with the active sites of the soy Aldehydes are well known for reacting withwith the amino group on proteins [10–12]. [10–12]. The The aldehydes aldehydes protein, leading to cross-linking and yielding adhesives bonding plywood panels that are very generated from from the the carbohydrate carbohydrate fraction fraction of of soy soy flour flour appear appear to toreact react with with the theactive active sites sites of the of soy the encouraging for shear bond strengths, especially under wet testing conditions [12]. Equally, some of protein,soy protein, leading leading to cross-linking to cross-linking and and yielding yielding adhesives adhesives bonding bonding plywood plywood panels panels that that are are very the aldehydes generated by the periodate treatment of glucose and sucrose also react with a encouraging forfor shearshear bond bond strengths, strengths, especially especially under under wet wet testing testing conditions conditions [12 [12].]. Equally, Equally, some some of the of polyflavonoid tannin, yielding equally encouraging plywood bond strengths [13]. thethe aldehydesaldehydes generatedgenerated byby thethe periodateperiodate treatmenttreatment ofof glucoseglucose andand sucrosesucrose alsoalso reactreact withwith aa aldehydesIn the generated case of Na by theperiodate periodate acting treatment on glucos of glucosee at 120 and °C sucrosefor 1 hour, also reacta number with aof polyflavonoid different polyflavonoid tannin, yielding equally encouragingcouraging plywoodplywood bondbond strengthsstrengths [13].[13]. tannin,aldehydes yielding have equallybeen shown encouraging to be generated, plywood for bond example:28 strengths [13]. InIn thethethe casecasecase of ofof Na NaNa periodate periodateperiodate acting actingacting on onon glucose glucosglucos ate 120 at 120◦C for °C 1 for h, a1 number hour, a of number different of aldehydes different aldehydes have been shown to be generated, for example:28 aldehydeshave been shownhave been to be shown generated, to be generated, forCHO example: for example:28OH OH CHO CHOH OHCCHCHCHO CHO CHO OH OH CHO CHO glyoxalCHO CHOH dihydroxysuccinaldehydeOHCCHCHCHO CHO hydroxy malonicCHO glyoxal dihydroxysuccinaldehyde aldehyde hydroxyhydroxy malonicmalonic However, other aldehydes, not onlyaldehyde from the oxidative cleavage of the carbohydrates but also via different mechanisms following the cleavage, have been shown to form [13]. The first class of theseHowever, are given otherother by aldehydes,thealdehydes, recombination not not only only of from from the the dialdehydesthe oxidative oxidative cleavageobtained cleavage offrom of the the carbohydratesthe carbohydrates cleavage by but aldolbut also also via viadicondensationff erentdifferent mechanisms mechanisms [13]. Aldol following condensationfollowing the cleavage, the can cleavage, occur have un been haveder shownacid been or shown toalkaline form to [ 13conditions form]. The [13]. first [16]. The class Thus, first of these classdue are of thesegiventhese byarearethe givengiven recombination byby thethe recombinationrecombination of the dialdehydes ofof thethe obtained dialdehydesdialdehydes from theobtainedobtained cleavage fromfrom by aldolthethe cleavagecleavage condensation byby aldolaldol [13]. condensationAldolcondensation condensation [13].[13]. AldolAldol can occur condensationcondensation under acid cancan or occuroccur alkaline ununder conditions acid or [alkaline16]. Thus, conditions due possibly [16]. toThus, the aciddue

Polymers 2019, 11, x FOR PEER REVIEW 3 of 19 Polymers 2019,, 11, x 1478 FOR PEER REVIEW 3 3of of 19 18 Polymers 2019, 11, x FOR PEER REVIEW 3 of 19 possiblyPolymers 2019 to the, 11, acidx FOR environment PEER REVIEW induced by periodate, the aldol condensation reaction starting3 of 19 from possiblythe glyoxal to theformed acid isenvironment for example:29 induced by periodate, the aldol condensation reaction starting from possiblypossibly to to the the acid acid environment environment inducedduced by by periodate, periodate, the the aldol aldol condensation condensation reaction reaction starting starting from from theenvironment glyoxal formed induced is for by periodate,example:29 the aldol condensation reaction starting from the glyoxal formed is thethe glyoxal glyoxal formed formed isis forfor example:29example:29 for example: O O OH OH O O OH OH OHCO CH + OHCOO CH OHCCHCHOHOH OH OH CHO OHC CH + OHC CH OHCCHCHCHO OHCOHC CH + OHCOHC CH CH OHCCHCHOHCCHCHCHOCHO

Additionally, the following species have been found to be formed via aldol condensation [13]:30 Additionally, the following species have been found to be formed via aldol condensation [13]:30 Additionally,Additionally,Additionally, the the following following species species havehave been been foun foun founddd to to tobe be be formed formed formed via via via aldol aldol aldol condensation condensation condensation [13]:30 [13]:30 [13 ]: OH OH OH OH OH OH OH OH OH OHCCHCHOHOH OH OHCH OHOHCH CHO OHCCHCHOHOH OH OHOHOHCHOHOHCHOHOHCH CHO OH OH OH OH OH OH OH OH OH OHCCHCHOHCCHCHCH CHCH CHOCHO OHCCHCHOHCCHCHCHCHCHCHCHCHCHOCHO OHCCHCHCH CH CHO OHCCHCHCH CH CH CHO OH OH OH OH OH OH OH OH OH OH OHCOH CH OHCH OHC CHO OHCOHOH CHOHOHCHOHOHCHOHOHC CHO OH OH OH OH OH OH OH OHCOHC CH CH CCCHOCHOCHO OHC CH CH CH C CHO OHC CH CH C CHO OHC CH CH CH CCHOCHO CHO OHC CH CH CH C CHO CHO CHOCHO CHO CHO A third type of reaction has also been shown to occur due to water elimination between two A third type of reaction has also been shown to occur due to water elimination between two aldehydeA third groups type [13].of reaction Aldehydes has also in water been are shown in general to occur present due to as waterhemiacetals, elimination these between being due two to aldehydeA third groups type [13].of reaction Aldehydes has inalso water been are shown in general to occur present due as to hemiacetals, water elimination these being between due to two aldehydethe reaction groups of an [[13].13 ].aldehyde AldehydesAldehydes with inin water wateran alcohol are are in in general orgeneral water present present [16]. asThis as hemiacetals, hemiacetals, is the case, these these for being beingexample, due due to the toof aldehydethe reaction groups of an[13]. aldehyde Aldehydes with in anwater alcohol are inor general water present[16]. This as ishemiacetals, the case, forthese example, being dueof to theformaldehydereaction reaction of an of aldehydeforming an aldehyde hemiformals with an with alcohol inan water oralcohol water in the or [16 preparation ].water This is[16]. the of This case, formaldehyde-based foris the example, case, offor formaldehyde resinsexample, [17].31 of theformaldehyde reaction of forming an aldehyde hemiformals with inan water alcohol in the or preparation water [16]. of formaldehyde-basedThis is the case, for resins example, [17].31 of formaldehydeforming hemiformals forming in hemiformals water in the in preparation water in the of preparation formaldehyde-based of formaldehyde-based resins [17]. resins [17].31 formaldehyde forming hemiformals in water in the preparation of formaldehyde-based resins [17].31 elimin elimin H2O H2eliminO OHC CHO + OHC CHO OHC CH2OH + HOH2CCHOelimin OHC CH2 OCH2 CHO OHC CHO + OHC CHO OHC CH2OH + HOH2CCHO H2O OHC CH2 OCH2 CHO H2O OHC CH OCHCHO OHC CHO + OHC CHO OHC CH2OH + HOH2CCHO OHC CH2 OCH2 CHO OHC CHO + OHC CHO OHC CH2OH + HOH2CCHO 2 2 FromFrom this this reaction reaction of elimination, species species such such as as follows follows were were formed formed [3].32 [[3].323]. From this reaction of elimination, species such as follows were formed [3].32 From this reaction of elimination, species such as follows were formed [3].32 OHOH OHOH OHOH OHOHOHOH OH OH OH OH OH OHCOHCOH CH CH CHCH22 OCH2 CHOHCH CHOCHO OHCOHC CHOH CHCHCH2 OCH2 OCH2 CH2 OHCHCHOHCHCHOCHO 4 4 44 44 OHC CH4 4 CH OCHCH CH CHO OHC CH CH2 OCH2 CH CHO OHC CH CH2 OCH2 CH CH CHO OHC CH 4CH2 OCH2 CH 4CHO 4 2 2 4 4 4 4 4

CHCH2 CHOHCHOH CHO CHO 2 4 4 OH OH OCH CHOH CHO OH OH CHO 2 CHOH4 CHO 2 4 OHC CHOH CH2 OCH2 CHOH CHO CHO OHCOH CH CH2 OCH2 OHCH4 OCH CHO 44 4 OHC CH CH2 OCH2 CH CH CHO OHC CH 4 CH2 OCH2 CH 4CH CHO 4 4 CompoundsCompounds obtainedobtained byby thethe combination combinationcombination of of the the th three threeree reactions reactions reactions outlined outlined outlined above above above have have have been been been shown shown shown to to occur even at higher molecular weights, but aldehyde groups are always present, thus maintaining tooccur occurCompounds even even at at higher higher obtained molecular molecular by the weights, weights,combination but but aldehyde aldehydeof the th groupsree groups reactions are are always alwaysoutlined present, present, above thus havethus maintaining maintainingbeen shown the the Compoundsreaction capacity obtained of theseby the compoundscombination with of the any three available reactions reactive outlined sites above of haveother been present shown tothereaction occur reaction even capacity atcapacity higher of these molecularof compounds these compoundsweights, with anybut aldehyde availablewith any reactivegroups available are sites alwaysreactive of other present, presentsites ofthus compounds other maintaining present [13]. tocompounds occur even [13].at higher The reactivitymolecular of weights, the higher but molecular aldehyde weightgroups aldehydesare always is present, likely to thus be relativelymaintaining thecompoundsThe reaction reactivity [13].capacity of theThe higher reactivityof these molecular ofcompounds the weighthigher withmolecular aldehydes any weightavailable is likely aldehydes toreactive be relatively issites likely lowerof toother be than relatively present that of thelower reaction than that capacity of more of active, these lower compounds molecular with weight any aldehydes. available It reactivemust also sitesbe noted of otherthat all present the compoundslowermore active,than that [13]. lower of The more molecular reactivity active, weight lower of the aldehydes. molecular higher molecular Itweight must alsoaldehydes. weight be noted aldehydes It thatmust all alsois the likely be aldehydes noted to be that relatively involved all the compoundsaldehydes involved [13]. The in reactivity subsequent of re theactions higher also molecular have a very weight low volatility, aldehydes eliminating is likely the to possibility be relatively loweraldehydesin subsequent than involved that reactions of more in subsequent alsoactive, have lower are veryactions molecular low also volatility, haveweight a eliminatingvery aldehydes. low volatility, theIt must possibility eliminating also be of noted VOC the that emission.possibility all the lowerof VOC than emission. that of more active, lower molecular weight aldehydes. It must also be noted that all the aldehydesof VOCHowever, emission. involved in addition in subsequent to the background reactions also given have above a very and low the volatility, reactions eliminating indicated, it the has possibility also been aldehydesHowever, involved in addition in subsequent to the background reactions also given have abov a every and lowthe reactions volatility, indicated, eliminating it has the also possibility been ofdeduced VOCHowever, emission. from in applied addition bonding to the background results [3] that: given (i) abov the reactionse and the reactions do appear indicated, to occur it with has also soy flour,been ofdeduced VOC emission. from applied bonding results [3] that: (i) the reactions do appear to occur with soy flour, deducedthusHowever, on insoluble from appliedin addition polymeric bonding to the carbohydrates backgroundresults [3] that: and given soluble(i) abovthe reactionse sucrose and the and doreactions appear its alpha-galactosyl indicated, to occur withit has derivatives soy also flour, been thusHowever, on insoluble in addition polymeric to thecarbohydrates background and given soluble abov sucrosee and the and reactions its alpha-galactosyl indicated, it derivatives has also been deducedthusreacting, on insoluble afterfrom selectiveapplied polymeric oxidation,bonding carbohydrates results with its [3] proteic that:and soluble(i) part; the and reactions sucrose (ii) even anddo in appearits the alpha-galactosyl absence to occur of carbohydrates with derivatives soy flour, in deducedreacting, from after appliedselective bondingoxidation, results with its [3] proteic that: (i)part; th eand reactions (ii) even do in appear the absence to occur of carbohydrates with soy flour, thusreacting,soyprotein on insoluble after isolates, selective polymeric the oxidation, protein carbohydrates itself with is its able, proteic and alone, soluble part; to cross-link and sucrose (ii) even onceand inits treated the alpha-galactosyl absence with sodium of carbohydrates derivatives periodate, thusin soyon insolubleprotein isolates, polymeric the carbohydratesprotein itself is and able, soluble alone, sucrose to cross-link and its once alpha-galactosyl treated with derivativessodium reacting,inyielding soy protein encouragingafter selective isolates, plywood oxidation, the protein results. with itself Theits proteic researchis able, part; alone, work and presented to(ii) cross-link even herein the once is absence then treated aimed of carbohydrateswith to investigate sodium reacting,periodate, after yielding selective encouraging oxidation, plywood with its results. proteic Th part;e research and (ii) work even presented in the absence here is ofthen carbohydrates aimed to inperiodate,(i)investigate whatsoy protein aldehydes yielding (i) whatisolates, areencouraging aldehydes formed the protein via are plywood the formed itself action results. isvia of able, periodatethe Th actionalone,e research onof to theperiodate cross-link work soy flourpresented on once insolublethe soytreated here flour carbohydrates is thenwith insoluble aimed sodium to in soy protein isolates, the protein itself is able, alone, to cross-link once treated with sodium periodate,investigateexplaincarbohydrates the yielding already(i) whatto explain experiencedencouraging aldehydes the already cross-linkingareplywood formedexperienced results. via [12 ]cross-the ofTh periodate-treatedeaction linkingresearch of [12] periodatework of periodate-treated presented soy on flour; the (ii)here soy what issoyflour then occursflour; insolubleaimed (ii) in the to periodate, yielding encouraging plywood results. The research work presented here is then aimed to investigatecarbohydratesreactionswhat occurs of (i) thein towhat the cleaved explain reactions aldehydes polymericthe of already the are cleaved carbohydratesformed experienced polymeri via the cross-c with carbohydratesactionlinking the of soy periodate[12] protein with of periodate-treated the inon soy soy the protein flour; soy andflourin soy even insolubleflour; more (ii) investigate (i) what aldehydes are formed via the action of periodate on the soy flour insoluble carbohydrateswhatimportantly, occurs in (iii) tothe whatexplain reactions happens the of already the when cleaved experienced the soy polymeri protein cross-c isolate carbohydrateslinking is treated [12] of with with periodate-treated the periodate, soy protein in the soy in absencesoy flour; flour; (ii) of carbohydrates to explain the already experienced cross-linking [12] of periodate-treated soy flour; (ii) whatcarbohydrates, occurs in the to makereactions it cross-link. of the cleaved polymeric carbohydrates with the soy protein in soy flour; what occurs in the reactions of the cleaved polymeric carbohydrates with the soy protein in soy flour;

Polymers 2019, 11, x FOR PEER REVIEW 4 of 19 and even more importantly, (iii) what happens when the soy protein isolate is treated with periodate, in the absence of carbohydrates, to make it cross-link. Polymers 2019, 11, 1478 4 of 18 2. Materials and Methods

2.1.2. Materials Preparation and of MethodsSamples for Analysis 2.1. PreparationIn the purification of Samples of forthe Analysis soy protein isolate the first step involves dispersing the native flour in water at 10 percent solids, adjusting the pH to 8, centrifuging out the insoluble carbohydrates and In the purification of the soy protein isolate the first step involves dispersing the native flour in then adjusting the pH of the insoluble carbohydrates to 6.5 [12]. (a) 10 g of isolated insoluble soy flour water at 10 percent solids, adjusting the pH to 8, centrifuging out the insoluble carbohydrates and carbohydrates, (b) 10 g of soy protein isolate alone, and (c) 10 g of samples of a NaIO4-treated mix of then adjusting the pH of the insoluble carbohydrates to 6.5 [12]. (a) 10 g of isolated insoluble soy soy protein isolate + insoluble soy flour carbohydrates + sucrose in relative proportions by a weight flour carbohydrates, (b) 10 g of soy protein isolate alone, and (c) 10 g of samples of a NaIO4-treated of 50:25:25 were all three treated with 1.5 g NaIO4. Controls of 10 g of soy protein isolate + 5% mix of soy protein isolate + insoluble soy flour carbohydrates + sucrose in relative proportions by glutaraldehyde and 1% glutaraldehyde by weight were also prepared under the same conditions. a weight of 50:25:25 were all three treated with 1.5 g NaIO . Controls of 10 g of soy protein isolate The mixtures were placed in an oven at 120 °C for 1 hour, then4 cooled. + 5% glutaraldehyde and 1% glutaraldehyde by weight were also prepared under the same conditions. The mixtures were placed in an oven at 120 C for 1 h, then cooled. 2.2. Matrix Assisted Laser Desorption Ionization◦ (MALDI-TOF) Mass Spectrometry 2.2. MatrixAll samples Assisted for Laser the Desorptionmatrix assisted Ionization laser (MALDI-TOF) desorption ionization Mass Spectrometry time-of-flight (MALDI-TOF) analysisAll were samples prepared for the by matrix first dissolving assisted laserthe (a) desorption 5 mg samples ionization of NaIO time-of-flight4-treated insoluble (MALDI-TOF) soy flour carbohydrates, (b) 5 mg samples of NaIO4-treated soy protein flour, or (c) 5 mg samples of NaIO4- analysis were prepared by first dissolving the (a) 5 mg samples of NaIO4-treated insoluble soy flour treated soy protein isolate alone, in 1 mL of a 50:50 v/v acetone/water solution. Then, 10 mg of this carbohydrates, (b) 5 mg samples of NaIO4-treated soy protein flour, or (c) 5 mg samples of NaIO4-treated solutionsoy protein was isolate added alone, to 10 in µL 1 mLof a of2,5–dihydroxy a 50:50 v/v acetone benzoic/water acid solution. (DHB) matrix. Then, 10 The mg locations of this solution dedicated was toadded the samples to 10 µL on of the a 2,5–dihydroxy analysis plaque benzoic were acidfirst (DHB)covered matrix. with 2 The µL locationsof a NaCl dedicatedsolution 0.1 to theM in samples 2:1 v/v methanol/water,on the analysis plaque and pre-dried. were first coveredThen, 1 with µL 2ofµ Lthe of sample a NaCl solutionsolution 0.1 was M inplaced 2:1 v /vonmethanol its dedicated/water, location,and pre-dried. and the Then, plaque 1 µL ofwas the dried sample again. solution MALDI-TOF was placed spectra on its dedicatedwere obtained location, using and an the Axima- plaque Performancewas dried again. mass MALDI-TOF spectrometer spectra from wereShimadzu obtained Biot usingech (Kratos an Axima-Performance Analytical Shimadzu mass Europe spectrometer Ltd., Manchester,from Shimadzu UK) Biotech using a (Kratos linear po Analyticallarity-positive Shimadzu tuning Europe mode. Ltd.,The measurements Manchester, UK) were using carried a linear out makingpolarity-positive 1000 profiles tuning per mode. sample The with measurements 2 shots accumulated were carried per profile. out making The 1000spectrum profiles precision per sample is of +with 1 Da. 2 shots accumulated per profile. The spectrum precision is of 1 Da. ± 3. Results Results and and Discussion Discussion In the the case case of of the the periodate periodate treatment treatment of ofthe the insoluble insoluble polymeric polymeric carbohyd carbohydratesrates of soy of soyflour, flour, the cleavagethe cleavage of C–C of C–C bonds bonds with with vicina vicinall –OH –OH groups groups does does yield yield aldehydes, aldehydes, but but the the peaks peaks of of the the smaller smaller aldehydes, suchsuch asas glyoxal,glyoxal, hydroxymalonic hydroxymalonic dialdehyde dialdehyde and and dihydroxy dihydroxy succinaldehyde succinaldehyde as obtainedas obtained for forglucose glucose [13 ][13] and and in some in some cases cases for sucrose, for sucrose, are not are observed, not observed, either either because because they arethey not are formed not formed (most (mostlikely likely due to due the to polymeric the polymeric nature nature of the of insoluble the insoluble carbohydrates), carbohydrates), because because they arethey lost are due lost to due their to theirvolatile volatile nature, nature, or because or because they have they already have reactedalready in reacted some manner. in some One manner. peak already One peak observed already for observedglucose, given for glucose, by the recombinationgiven by the recombination of small dialdehydes of small species dialdehydes by aldol specie condensation,s by aldol condensation, is nonetheless ispresent. nonetheless This sole present. peak, This also sole present peak, in also glucose presen at 231t in Daglucose (calc. at 231Da), 231 Da including (calc. 231Da), the Na including+ enhancer, the Na+appears enhancer, to belong appears to the to action belong of to 15% the sodiumaction of periodate 15% sodium at 120 periodate◦C on the atinsoluble 120 °C on carbohydrates. the insoluble carbohydrates.It has already been It has reported already forbeen monomeric reported for carbohydrates monomeric [carbohydrates13], and has the [13], following and has structure: the following structure:33 OH OH OH OH OH OHCCHCHCH CH CH CHO

This peak, corresponding to a pentahydroxy heptanedial, is very marked when the insoluble carbohydrates are treated with periodate at 120 °C, while it is almost absent, showing just traces, carbohydrates are treated with periodate at 120 ◦C, while it is almost absent, showing just traces, when the insoluble carbohydrates alone are heated for the same period at 120 °C. It is absent at 20 °C. when the insoluble carbohydrates alone are heated for the same period at 120 ◦C. It is absent at 20 ◦C. The otherother aldehydes, aldehydes, found found when when glucose glucose and sucrose and sucrose are treated are withtreated periodate with asperiodate a consequence as a consequenceof the other reactionsof the other outlined reactions in theoutlined introduction, in the introduction, are not found are for not soy found ﬂour for insoluble soy flour polymeric insoluble polymericcarbohydrates. carbohydrates. Diﬀerent aldehydes Different ofaldehydes much greater of mu molecularch greater weight molecular are instead weight formedare instead (Figure formed1a–c and Table1). Table1 lists all the species formed, whether they carry aldehyde groups or not.

Polymers 2019, 11, 1478 5 of 18 Polymers 2019, 11, x FOR PEER REVIEW 5 of 18

(a)

(b)

(c)

FigureFigure 1.1. MALDIMALDI ToFToF spectraspectra of insoluble soy soy flour flour ca carbohydratesrbohydrates after after a treatment a treatment with with sodium sodium periodate at 120 °C for 1 hour. (a) 100 Da to 500 Da range. (b) 500 Da to 1000 Da range. (c) 1000 Da to periodate at 120 ◦C for 1 h. (a) 100 Da to 500 Da range. (b) 500 Da to 1000 Da range. (c) 1000 Da to 15001500 Da Da range.range.

Polymers 2019,, 11,, xx FORFOR PEERPEER REVIEWREVIEW 6 of 19

Polymers 2019, 11, x FOR PEER REVIEW 6 of 19 PolymersFigure 2019 1., 11MALDI, x FOR PEERToF spectraREVIEW of insoluble soy flour carbohydratesrbohydrates afterafter aa treatmenttreatment withwith sodiumsodium6 of 19 periodate at 120 °C for 1 hour. (a) 100 Da to 500 Da range. (b) 500 Da to 1000 Da range. (c) 1000 Da to Figureperiodate 1. MALDIat 120 °C ToF for spectra1 hour. (ofa) insoluble100 Da to soy500 Daflour range. carbohydrates (b) 500 Da toafter 1000 a treatmentDa range. (withc) 1000 sodium Da to 1500Figure Da range. 1. MALDI ToF spectra of insoluble soy flour carbohydrates after a treatment with sodium periodate1500 Da range. at 120 °C for 1 hour. (a) 100 Da to 500 Da range. (b) 500 Da to 1000 Da range. (c) 1000 Da to Polymers periodate2019, 11, 1478 at 120 °C for 1 hour. (a) 100 Da to 500 Da range. (b) 500 Da to 1000 Da range. (c) 1000 Da to 6 of 18 15001500 Da Da range. range. InIn thethe insolubleinsoluble carbohydratescarbohydrates treatedtreated withwith sodiumsodium peroxideperoxide andand inin thethe mixmix proteinsproteins ++ carbohydrates + sucrose + periodate at 120 °C, there are a number of peaks that do not appear in the In In the the insoluble insoluble carbohydrates carbohydrates treated treated with with sodium peroxideperoxide andand inin the the mix mix proteins proteins + + carbohydrates control at 120 °C without periodate (Supplementary Material). These are listed in Table carbohydratescarbohydrates ++ sucrose+sucrose sucrose + +periodate periodateperiodate at at 120 at120 120 °C,°C, therethereC, there are a are numbernumber a number of of peaks peaks of peaksthat that do do that not not appear do appear not in appear inthe the 1, which lists all the species formed that carry◦ aldehyde groups or not. Among these, several of the carbohydrates1, which lists all control the species at 120 °Cformed without that periodate carry alde (Shydeupplementary groups or Material). not. Among These these, are listed several in Tableof the in thecarbohydrates carbohydrates control control at 120 at °C 120 without◦C without periodate periodate (Supplementary (Supplementary Material). Material). These are These listed are in listedTable in species formed after the periodate treatment are noteworthy,teworthy, asas theythey presentpresent oneone oror moremore aldehydealdehyde Table1, which1, which1, which lists lists all lists all the allthe species thespecies species formed formed formed that that that carrycarry carry aldealde aldehydehyde groups groups oror not. not. or Among not. Among Among these, these, these, several several several of ofthe the of groups. These are the peaks at 768 Da, 791 Da, 930 Da, 975 Da, 1031 Da, 1046 Da, 1053 Da, 1092 Da, speciesthespecies species formed formed formed after after after the the theperiodate periodate periodate treatment treatment treatment areare are no noteworthy,teworthy, asas as theythey they present present present one one one or or ormore more more aldehyde aldehyde aldehyde 1135 Da, 1153 Da, 1176 Da, 1192 Da, 1198 Da, 1214 Da, 1230 Da, 1254 Da, 1362 Da, and 1416 Da. All groups.groups. These These These are are are the the the peaks peaks at at at 768 768 Da, Da, 791 791 Da, Da, 930 930 Da, 975 975 Da,Da, 10311031 Da, Da, 1046 1046 Da, Da, Da, 1053 1053 1053 Da, Da, Da, 1092 1092 1092 Da, Da, Da, present at least one aldehyde group. Among all of thesethese therethere areare monoaldehydemonoaldehyde speciesspecies obtainedobtained byby 11351135 Da, Da, 1153 1153 1153 Da, Da, Da, 1176 1176 1176 Da, Da,Da, 1192 1192 Da, Da, Da, 1198 1198 1198 Da,Da, Da, 12141214 1214 Da, Da, 1230 1230 Da,Da, Da, 12541254 1254 Da, Da, 1362 Da, 1362 1362Da, Da, and Da,and 1416 and1416 Da. 1416 Da. All Da.All differentpresent cleavage at least one sites aldehyde of one group.glucose, Among such allas ofthethe these speciesspecies there representedrepresented are monoaldehyde byby thethe speciespeakspeaks atatobtained 768768 Da,Da, by 791791 Allpresent present at least at least one onealdehyde aldehyde group. group. Among Among all of all these of these there there are aremonoaldehyde monoaldehyde species species obtained obtained by Da,different 1254 Da cleavage and 1416 sites Da:34 of one 35 glucose, such as the species represented by the peaks at 768 Da, 791 differentby diﬀerent cleavage cleavage sites sites of one of oneglucose, glucose, such such as the as thespecies species represented represented by the by peaks the peaks at 768 at Da, 768 791 Da, Da,Da, 1254 1254 Da Da and and 1416 1416 Da:34 Da:34 35 35 791 Da, 1254 Da and 1416 Da: OH OH OH OH OH OH HO OH O OH O OH O O HO O O O OH OH O OH CHO OH OH OH OH OH OH CHO HO OH O OH O OH O OH OHO OH HO O O O O O O OH OHO OH CHO OH OH OH CHO O O O OH OH OH HO OH O OHOH O OH OH O OH OH OH OH OH OH HO OH OH768 Da HO OH OH768 Da 768 Da 768 Da

OH OH OH OH OH HO OHO OHO OHO OH O OH CHO HO OHO OHO OHO OH O CHO HO O O O O OH CHO OHO OHO OHO OHO OH HO O O O O O O O O OH CHO OHO OHO OHO OH O OH OH OH OH O O O O OH OH OH OH OH OHOH OH OH OH OH OH OH 791 Da OH OH OH791 DaDa OH 791 Da ThereThereThere areare are speciesspecies species presenting presenting presenting two two two aldehyde aldehydealdehyde groups groups due duedue to toto the the the speciﬁc specific specific oxidation oxidation oxidation of of twoof two two glucosesglucoses glucoses by by the periodate, such as those represented by the peaks at 1031 Da and 1053 Da:36 thebyby the periodate,There the periodate, periodate, are suchspecies such such as those presentingas as th those representedose represented represented two aldehyde by theby the peaks groups peakspeaks at due 1031 atat 1031 1031to Da the Da andDa specific and and 1053 1053 1053 Da:oxidation Da:36 Da:36 of two glucoses by the periodate, such as those represented by the peaks at 1031 Da and 1053 Da:36 OHOH OHOH OHOH OHOH OHOH OH OH OH OH OH OHCOHC O O OO OO OO OO O O CHO OH OH OH OHCHOCHO OH OH OH OH OHOH OHO OHO OHO OHO OHOH OHOH OHOH OHC O OO O OO O OO O OO O O CHO OHOH OH OH OH OH OH OH OH OH OH OHOHO OH OHO OH OHO OH OH O OHOH OH OH OH OH OH OH 1031 Da OH OH OH1031 Da OH 1031 Da Finally,Finally,Finally, speciesspecies species presenting presenting presenting a a multitudea multitude multitude of ofof aldehyde aldehydede groups, groups,groups, such such such as as as several several several glucoseglucose glucose pyranpyran pyran ringsrings rings in thein inthe carbohydrate the carbohydrate carbohydrate oligomer, oligomer, oligomer, have have have been been been cleaved cleavedcleaved by by periodate. periodate.periodate. TwoTwo Two examples examples of of of thesethese these are areare the thethe species speciesspecies in theFinally, carbohydrate species oligomer,presenting have a multitude been cleaved of aldehy by periodate.de groups, Two such examples as several of glucosethese are pyran the species rings representedrepresented byby by thethe the peakspeaks peaks atat at 12141214 1214 DaDa Da andand and 1230 12301230Da: Da:37 38 inrepresented the carbohydrate by the peaksoligomer, at 1214 have Da been and cleaved 1230 Da:37 by periodate. 38 Two examples of these are the species represented by the peaks at 1214 Da and 1230 Da:37 38 OH OH O HOO OH OH OH OH HO OH OH OH OH O OH O OH OH HO O O OH OH OH HO O O HO O O O OH O OHO HOHO O OH HO OH O HO OH O OH O O O O O O OH OH O HO OO HO O O OHO O O O O O O HO HO O O O OH O O OH HO O O HO OH HO OO OH O O O O HO O O OH HO OO O OH O HO O HO O OHO OH OH OH HO O O HO O O HOO OH OH O HO O HO O OH O HO O OH O HO OH Polymers 2019, 11, x FORHO PEER REVIEW 1214 Da 7 of 19 O 1214HO Da HO O OH 1214 Da O HO OH OH O O OH 1214 DaOH OH OH HO O O O OH O O O O HO O OH O O O O HO HO O OH O OH O HO HO O OH O HO HO O O 1230 Da

Both species confirm the reactions brought about on cellulose and other carbohydrate oligomers by previous work [6,7]. In the structures in Table 1 for the 1214 Da and 1230 Da species, the open and oxidized forms of glucose can be placed anywhere and alternate in any manner in relation to the non- oxidized glucose closed forms. Thus, the above structures and their structures in Table 1 represent just one of the possible isomers. The 1230 Da peak is separated by 16 Da from the 1214 Da peak and by 32 Da from the 1198 Da peak. This means that these two species have respectively 1 and 2 –O- atoms less than the 1230 Da one. These can have been lost in the MALDI analysis; carbohydrate chains are known to do this [18], but it could also be due to similar but slightly different species. The marked 1198 Da peak could be a case of 6 open units linked together: 176 × 6 = 1032 + 162 units =1194 Da. It is the nearest to 1198 Da. One could also imagine that it could be a peak obtained by reactions of the small aldehydes such as glyoxal. The reactions of an aldehyde with an alcohol yields reversible hemiacetals under acid conditions. However, better and more accurate explanations are possible. Effectively, the group of peaks 1198 Da, 1214 Da, and 1230 Da are of interest due to their relative height in the high molecular weights part of the spectra in Figure 1. Of these, the 1230 Da and 1214 Da ones are interesting due to their multiple aldehyde groups. The 1198 Da species appears to be the same as the 1214 Da or the 1230 Da ones, having lost just one or two –OH groups in the MALDI analysis. There are some peculiarities too. The peak at 1092 Da is also present in the SPI treated at 120 °C without periodate. Thus, one cannot be sure that the interpretation as an aldehyde carrying carbohydrate oligomer is correct, and a doubt must logically persist. Monoaldehydes of higher molecular weights are also noted, such as the one at 1416 Da:39

OH OH OH OH OH OH OH OH OH OH HO O O O O O O O O CHO

O O O O O O OH OH OH OH OH OH OH OH OH OH HO OH HO OH OH OH OH 1416 Da

This indicates that even higher molecular weight oligomers containing aldehyde groups are likely to be generated the longer the periodate treatment is. All of the above information indicates that the specific oxidation of the periodate appears to be able to also occur with polymeric carbohydrates, also generating aldehyde groups in high molecular weight fragments of carbohydrate chains. Consequently, they are these high molecular weight carbohydrate oligomers carrying periodate-generated aldehyde groups and the heptanedial aldehyde, which contribute to the applied bonding results with the proteic part of soy flour already reported [12]. In particular, it must be noted that species presenting a number of active aldehyde groups such as those represented by the 1230 Da and 1214 Da peaks can be a centre of tridimensional networking by a reaction with the protein, but also (as already demonstrated) by a reaction with other present carbohydrate chains [14,15]. Another interesting curiosity are the peaks at 975 Da (Calc. 976 Da) and 1135 Da (Calc. 1137 Da). The species represented by these two peaks are associated with 2 Na+, the 975 Da corresponding to the 930 Da peak + 2xNa+, and the 1135 Da peak corresponding to 1092 Da + 2xNa+. The 959 Da peak is the same as the 975 Da but has lost one –OH group in the MALDI analysis. The species containing two Na+ in the MALDI analysis are relatively unusual but are not rare and have also been reported in the previous literature [19].

Polymers 2019, 11, x FOR PEER REVIEW 7 of 19

OH OH O O OH OH HO O O O OH OH O O O O OH HO O OH O O O O HO HO O OH O OH HO O O HO OH Polymers 2019, 11, 1478 O HO HO O7 of 18 O 1230 Da Both species confirm the reactions brought about on cellulose and other carbohydrate oligomers by previousBoth species work confirm [6,7]. In the the reactions structures brought in Table about1 foron cellulose the 1214 and Da other and 1230 carbohydrate Da species, oligomers the open andby previous oxidized work forms [6,7]. of glucose In the st canructures be placed in Table anywhere 1 for the and 1214 alternate Da and 1230 in any Da manner species, inthe relation open and to the non-oxidizedoxidized forms glucose of glucose closed can forms. be placed Thus, anywhere the above and structures alternate andin any their manner structures in relation in Table to 1the represent non- justoxidized one of glucose the possible closed isomers. forms. Thus, The 1230 the Daabove peak stru isctures separated and bytheir 16 structures Da from the in 1214Table Da 1 represent peak and by 32just Da one from of the the possible 1198 Da isomers. peak. This The means 1230 Da that peak these is separated two species by have16 Da respectively from the 1214 1 andDa peak 2 –O-atoms and lessby 32 than Da thefrom 1230 the Da 1198 one. Da These peak. canThis have means been that lost these in the two MALDI species analysis; have respectively carbohydrate 1 and chains 2 –O- are atoms less than the 1230 Da one. These can have been lost in the MALDI analysis; carbohydrate chains known to do this [18], but it could also be due to similar but slightly different species. are known to do this [18], but it could also be due to similar but slightly different species. The marked 1198 Da peak could be a case of 6 open units linked together: 176 6 = 1032 + 162 The marked 1198 Da peak could be a case of 6 open units linked together: 176 × 6× = 1032 + 162 units =1194 Da. It is the nearest to 1198 Da. One could also imagine that it could be a peak obtained by units =1194 Da. It is the nearest to 1198 Da. One could also imagine that it could be a peak obtained reactions of the small aldehydes such as glyoxal. The reactions of an aldehyde with an alcohol yields by reactions of the small aldehydes such as glyoxal. The reactions of an aldehyde with an alcohol reversible hemiacetals under acid conditions. However, better and more accurate explanations are yields reversible hemiacetals under acid conditions. However, better and more accurate explanations possible. Effectively, the group of peaks 1198 Da, 1214 Da, and 1230 Da are of interest due to their are possible. Effectively, the group of peaks 1198 Da, 1214 Da, and 1230 Da are of interest due to their relativerelative heightheight in in the the high high molecular molecular weights weights part part of the of thespectra spectra in Figure in Figure 1. Of1 these,. Of these, the 1230 the Da 1230 and Da and1214 1214 Da ones Da ones are interesting are interesting due dueto their to their multiple multiple aldehyde aldehyde groups. groups. The 1198 The Da 1198 species Da species appears appears to tobe be the the same same as the as 1214 the 1214 Da or Da the or 1230 the Da 1230 ones, Da ha ones,ving havinglost just lostone or just two one –OH or twogroups –OH in the groups MALDI in the MALDIanalysis. analysis. ThereThere are some some peculiarities peculiarities too. too. The Thepeak peak at 1092 at Da 1092 is also Da ispresent also presentin the SPI in treated the SPI at treated120 °C at 120without◦C without periodate. periodate. Thus, Thus,one cannot one cannot be sure be surethat thatthe theinterpretation interpretation as an as analdehyde aldehyde carrying carrying carbohydratecarbohydrate oligomeroligomer is correct, an andd a a doubt doubt must must logically logically persist. persist. MonoaldehydesMonoaldehydes of higher molecular molecular weights weights are are also also noted, noted, such such as asthe the one one at at1416 1416 Da:39 Da:

OH OH OH OH OH OH OH OH OH OH HO O O O O O O O O CHO

O O O O O O OH OH OH OH OH OH OH OH OH OH HO OH HO OH OH OH OH 1416 Da

ThisThis indicatesindicates thatthat even even higher higher molecular molecular weight weight oligomers oligomers containing containing aldehyde aldehyde groups groups are are likely tolikely be generated to be generated the longer the longer the periodate the periodate treatment treatment is. is. AllAll ofof thethe aboveabove information indicates indicates that that the the sp speciﬁcecific oxidation oxidation of ofthe the periodate periodate appears appears to be to be ableable toto alsoalso occuroccur with polymeric carbohydrates, carbohydrates, al alsoso generating generating aldehyde aldehyde groups groups in high in high molecular molecular weightweight fragmentsfragments of carbohydrate chains. chains. Conseq Consequently,uently, they they are are these these high high molecular molecular weight weight carbohydratecarbohydrate oligomersoligomers carrying carrying periodate-generated periodate-generated aldehyde aldehy groupsde groups and theand heptanedial the heptanedial aldehyde, whichaldehyde, contribute which tocontribute the applied to the bonding applied results bonding with results the proteic with the part proteic of soy part ﬂour of already soy flour reported already [ 12]. reported [12]. In particular, it must be noted that species presenting a number of active aldehyde In particular, it must be noted that species presenting a number of active aldehyde groups such as groups such as those represented by the 1230 Da and 1214 Da peaks can be a centre of tridimensional those represented by the 1230 Da and 1214 Da peaks can be a centre of tridimensional networking networking by a reaction with the protein, but also (as already demonstrated) by a reaction with other by a reaction with the protein, but also (as already demonstrated) by a reaction with other present present carbohydrate chains [14,15]. carbohydrate chains [14,15]. Another interesting curiosity are the peaks at 975 Da (Calc. 976 Da) and 1135 Da (Calc. 1137 Da). Another interesting curiosity are the peaks at 975 Da (Calc. 976 Da) and 1135 Da (Calc. 1137 Da). The species represented by these two peaks are associated with 2 Na+, the 975 Da corresponding to + Thethe species930 Da peak represented + 2xNa+ by, and these the two 1135 peaks Da peak are associatedcorresponding with to 2 Na1092, Da the + 975 2xNa Da+. corresponding The 959 Da peak to the + + 930is the Da same peak as+ the2xNa 975 ,Da and but the has 1135 lost Da one peak –OH corresponding group in the MALDI to 1092 analysis. Da + 2xNa The species. The 959 containing Da peak is thetwo same Na+ asin the MALDI 975 Da but analysis has lost are one relatively –OH group unusual in the but MALDI are not analysis.rare and have The species also been containing reported two + Nain thein previous the MALDI literature analysis [19]. are relatively unusual but are not rare and have also been reported in the previous literature [19].

Polymers 2019, 11, x FOR PEER REVIEW 8 of 19

Polymers 2019, 11, x FOR PEER REVIEW 1 8 of 19 Polymers 2019, 11, x FOR PEER REVIEW 8 of 19 OH OH OH1 OH OH Polymers 2019, 11, x FOR PEER REVIEW 8 of 19 OHCCHCHCH1 CH CH CHO Polymers 2019, 11, 1478 OH OH OH OH OH 8 of 18 Polymers 2019, 11, x FOR PEER REVIEW 8 of 19 OH OH OH1 OH OH OHCCHCHCH2 CH CH CHO OHCCHCHCH CH CH CHO PolymersTable 2019 1., 11The, x FOR proposed PEER REVIEW oligomer speciesOH forOH theOH1 MALDI OH ToFOH spectra peaks in Figure1a–c for the8 of 19 reaction at 120 C for 1 h of sodium periodateOH with2 insolubleOH soy ﬂour carbohydrates. Note: the links ◦ OHCCHCHHO CHO CH CH OHCHO Polymersbetween 2019, 11 the, x glucoseFOR PEER units REVIEW are alpha-glucosidic. OH OH TheyOH12 areOH representedOH as being beta glucosidic to shorten8 of 19 the table. OHO OHO OHCCHCHHO OH CHO CHOH CH OHCHO OHOH OH OH12 OHOHOH HO O + OH OH231O Da (with OH Na )O OHCCHCHOHOH CH2 CHOHOHCH CHO HO OH OHO OHO OH OOH OH OH OH OH OH3 OHCCHCHOHO CH2 CHOHOCH CHO HOOH OHO OH OH OH + OH365 Da (calc. 365OH Da) (with Na OH) HO O 3 O OH OHOHO OH2 OHO HO OH O3 OH OH OHO OHO OHO HO O O OH OHOHOHO OH OH OHO OH HOOH OOH3 OHOH HO OHO OHO OH OHO OHO OH O OHOHOH O OH OHOH3 O OHOH HO O OHO O OHO O OH OH527 Da (calc.OH 527 Da) (with NaOH+) OH OH4 OH OHOH OHOH OH HOOHO OHO 3 O OHO O OH OH OH OH 4 OH OH OH OH OHOHOOH OHOH3 OOH OH OHO HO HO O OH O O OH4 O OOH OH O CHO OH OH OH OH OHOHOOHO OHOHOOHO OH OHO OH OH OH HO OHO O O O 4 O O OH OH O OH OH OH OH OH OH OH OH 768OH Da (calc.766 Da)OH (with Na+) CHO HO O O O O HO O OHOHO O OH OH 4 O O OH OHO OHOHOH OHOHCHO OH OH OH OHOH OHOH OHOH HO O O O O O O OH OHO OH OH OH OH CHO HO OHOH OH OH 45 OH OH OH OH OH OH OH OH OH OH HOHO O OHO O OHO O O O OH OH OH CHO OH OH OH OH HO O 54O O OH OH OH OH HO OH OHOHO OH OHO OH OH OH HO O O O 5 O O OH OH OH CHO OHO OHO OHO + OH OHOHO OH HO OH 689O DaOH (calc. 689O Da)OH (with NaO ) OH OH HO OHO OHOHO OH OHO OH OH OH HO OHO OHO 5 OH O OH OHO HOOH OHO OHO OHO OH O OH O OH O O CHO OH OH OH OH HO O OHOHO O OH OHO OOHO OHOHO OH OH HO O 5O O OH OHOHOH OH OHOH OH6OH OH OH OH HO OHOHOHO OHOH OHO OH OHO OH OHO HO OH O OH 5O OH O OH OH 768 Da (calc. 7686 Da) (no Na+), OH OHOH OH OHO OH OHO OH+ OH O HO O OHO 791 Da (calc.OOH 7916 Da) (withOHO Na ) OH O CHO HO OH O OH 5O OH O OH OH OH OHOOH OH OHO OH OHO OH OHO OH HO O OHO OOHO 6 OHOO OHO O OH CHO OHHO OHO OHO O OH OH OHOH OHOH OHOH OHOH HO O O O O CHO OH OH OH OH OH OHO OHO O OHO 6 O OHO O O OH OHOHOH OH OHOH OH OHOH OH OHOH OH HO O O O O O O O O CHO OHOH OH OH OH OH OH OH OH OH OH67 OH OH OH OH OH OH OH HOOHO OHO O OHO O OHO + O O CHO OH OH930 Da (calc. 928 OH Da) (with Na OH) 6 OH 7 OH OH OH OH OHOHO OH OHO OH OHO OH OHO HOOH OOHO OH O OH O CHO HO OHO OHO OH7 O OH O O OH OHOH OHCHO OH OH OH OH OH HOOHO OHO O OHO 7 O OHO O O OH OH CHOOH Polymers 2019, 11,OH x FORO OH PEER REVIEWOHO OH OHO OHOHO OH O OH OH O OH 9 of 19 HO O O O CHO OHOH OHOHOH OH OHOH HO OO OO OO OO OHO OH OH OH7OH OH OHOHOHOH OH OHCHO OHO OH OHO OH OH O OH8 O OH OH OH OH OH OH OHO OHO OHO+ O OH OHO OH HO O O 930O Da (calc. 930 Da)O (no Na ) CHO OHOH OH OHOH 7OH OHOH OH OH OH OH OH OH OH OH OH OH OHOHOHO OHOHOHO OH OH OHO OH OHOHO O OH O HOHO OO OO O O O O CHOCHO OH OH OH 7 OH OH OH OH OH OH OH OH OH OHOHOO OHOHOO OH OHOO OHO O O O O HO O O O CHO OHOHOH OH OHOH OHOH OHOH OH OH OHOH OHOH OH OH OH OH OH OH OH OHHO O OHO O OHO O OHO O OHO O OH OH OH OH CHO OH OH OH OH OHO OHO OH O O OH OH OH 9 OH OH OH OH OH OH OH OH OH OH OH OH OHC O O O O O O CHO

OH OH O O O O O OH OH OH OH OH

OH OH OH OH OH

OH OH OH OH OH OH OH OH OHC O O O O O O CHO OH OH OH OH O O O O OH OH OH OH OH OH OH OH

OH OH OH OH OH OH HO O O O O O O CHO

O O O O O O OH OH OH OH OH OH HO HO OH OH OH OH

HO OH OH OH OH OH OH O O O OH O OH O O CHO O O O OH O O OH OH OH OH OH OH OH OH OH OH OH OH

OH OH OH OH OH OH OH HO O O O O O O CHO

O O O O O O OH OH OH OH OH OH OH OH OH OH OH OH OH

Polymers 2019, 11, x FOR PEER REVIEW 9 of 19

Polymers 2019, 11, x FOR PEER REVIEW 8 9 of 19

Polymers 2019, 11, x FOR PEER REVIEW 8 OH 9 of 19 OH OH OH OH OH HO O O O O O CHO Polymers 2019, 11, x FOR PEER REVIEW 8 OH 9 of 19 OH OH OH OH OH OH HO O O O O O O O O O O CHO OH OH OH OH OH Polymers 2019, 11, x FOR PEER REVIEW 8 OH 9 of 19 OH OH OH OH OH OH HOOHO OHO O OHO O OHO O OHO O O CHO OH OH OH OH OH 8 OH OH OH OH OH OH OH HOOHO OHO O OHO O OHO O OHO O O CHO OH OH OH 9 OH OH OH OH OH OH OH OH OH HOOHO OHO O OHO O OHO O OHO O O CHO OH OH OH 9 OH OH OH Polymers 2019, 11, 1478 OH OH OH OH OH 9 of 18 OHC O O O O O O OH CHO OHO OHO OHO OHO OH O OH 9 OH OH OH OH OH OH OH OH OHO OHO OHO OHO OHO OHC O O O O O O CHO OH OHOH OH OHTable OH 1.OHCont. OH OH OH OH 9 OH OH OH OHOHO OHOHO OHOHO OH OHO OH+ OHO OHC O OH 975O DaOH (calc. 976O Da) OH(= 930 DaO but withOH 2xNaO ) OH O CHO + OH 1031 Da (calc. 10329 Da) (no Na ) OH OH OH OHOHO OH1053OHO Da (calc.OH 1054OHO Da) OH(with Na OH+O) OH OHO OHC O OH O OHO OH10 O OHO OH O CHO OH OH OH OHOH OHOH OHOH OH OH OH OH OH OHC OHO O O O O 10O O O O OH OHO O OH CHO OH OHOH OH OH OHOH OH OH OHC O O O O O O OH CHOOH OHO OHO OHO OHO OH O OH OH 10 OHOH OHOH OH OH OH OHO OH OHO OH OHO OH OHO OH OHC O OHO OHO OHO OH O O CHO OH OH OH OH OH OHOH OH OH OH10 OH OH OHOH OH OHOH OHO OHO OHO OHO+ OHC O OHO 1031 OH Da (calc.O 1029 OH Da)O (with OH Na ) O O CHO OH OH OH OH OH10 OH OHOH OHOH OH OH OHO OHO 11 OHO OHO OHC O OHO OHO OHO OH O O CHO OH OH OH OH OH OH OHOH OHOH OH OH OH OHO OH OHO OH 11 OHO OH OHO OH OH HOOHC O O OHO O OHO OHO O OH O O O O CHOO CHO OH OH OH OH OH OH OH OH OHO O OHO O OHO 11 O OHO O OHO OHO HO O OHO OH OHO OH O O O OH OH 1046 DaOH (calc. 1043 Da)OH (with Na+) OH OH CHO HO OH OH OH OH OH HO OH OHOH OHOH OHOH OH OH HO O O O O O 11O O O O O O O OH OH OH OH OH OH CHO HO OHO HO OHO OHOHO OH11 OHO OHOHO OH OHO HO OH O OH O OH 12O OH O OH O OH O CHO HO OHOHO OHO OHOHO OHOHO OHOHO OH OHO HOHO OH O OHO OH 12O O OHO OH OHO CHO OH O OH O OH O OH OH O +OH OH OH HO 1092 Da (calc.1089 Da) (with Na ) O O OHO O OHO OHO OHO OH O CHO HO OHO 12 OH OH OH OH O OHOHO O OHOOH OHOOH OHO OH OH OH OH OH O O OH OH OH HO OH OH O O HO OH OH OH OH CHO OH OHO HO OHO OHO OHO OHO OH12 OH OH OH OH OH O OHO O OHO OH OH OH OH OH OH OH O O CHO OH OHO 12 HO OHO OHO OHO OHO OHO OH O OH OH OH OH OH OH O 13OH O OH OH OH OH OH OH O O 1092 Da (calc.1092 Da) (no Na+) CHO HOOH OHO OH OH OH OH OH O OHO O OHO OHOHO OH OHO OH OH OH O 13 O OH OH OHOH OH OH OH OHOH OHOH O OH O HO O O O O O O CHOCHO OH O OH O OH O OH OHO OH O OH OH OHOH OH OH 13OH OH OH Polymers 2019, OH11O, x FOR PEER OHREVIEWO OHO OHO OHO OHO 10 of 19 HOOH OH O OH O OH O OH O OH O OH O CHO OH OH OH OH OH 13 OH PolymersOH 2019OH, 11O, x FOR OH PEEROH REVIEWO OH OHO OH14OH O OHOHO OH OHO 10 of 19 HO OH O OH O OH O OH O OH O OH O CHO 1135 Da (calc. 1136 Da) (= 1092 Da but with 2x Na+) 13 + OHOH OHOHO OH OHOHO OH1153OHO Da (calc.OH 115314 OH O Da) OH (no Na OH) O OH OHO HO OH OOH OH O OH O OH O OH + O OH O CHO OH 1176 Da (calc. 1176 Da) (with Na ) O O OH O OH O OH O OH O OH OHOH OHOH OH OH OH OH OH OH HO OHO OHO OHO OHO OHO OHO HO OOHO O O O O O CHO OH OH OH OHO OH O OH O OH O OHO O O OH O OH O OH O OH O OH O OH OH OH OH OH OH OH OH OHHO O OHHO O OHO OHO OHO OH O O HO HO HO HO HO OH OH OH O OH O OH O OH O OHO O OH OH OH OH OH OH OH OHHO OH OH OH OH HO HO HO HO HO 15 1192 Da (calc. 1192 Da) (with Na+)

15 OH OH OH OH OH OH OH OH HO O O O O O O O CHO OHOH OHOH OHO OHO OHO OHO OHO OHO HO OH O OH O OH O OHO OH O OH O O CHO OHO OHO OHO OHO OHO OH O OH OH OH OH OH OH OH OH OH OH OH OH OH OH 16

16 OH O HOOH OH OH OH O O O OH OH HO O HO OH HOO OH OH O OH O O O O O O O O OH OH HO O HO O O OH O O HO O OH O O O HO O OH OH HO O O O O O HO HO O OH HO O OH O O HOO O HO HO O OH OH O HO HO O OH O HO 17

OH OH 17 O O OH OH OH OH HO O O O OH OH OH O O O O HO OO O OH OH O O O HO OH OH OH O HOO HO OO OH O O OH HO OO O OH O O HOO OH HO HO O OH O O HOO OO O O HO HO O OH O OH HO O O HO OH O HO HO O 18 O

18 OH OH OH OH OH OH OH OH OH HO O O O O O O O CHO OH OHOH OHOH OH OHO OHO OHO OHO OHO OHO HO OH O OH O OH O OH O OH O OH O O CHO HO HO OH OH OH O O OHO OHO OHO OH O OH OH OH OH OH OH HO HO OH OH19 OH OH

OH 19 OH OH O O OH O OH O OH O OH O OH O OH O OH CHO HO OHO OH OHO O O O O O O O OH O OH O OH O OH O OH O OH O OH O OH OH OH OH OH OH CHO HO HO O HO HO HO HO HO OH OH O O O O O O O OH OH OH OH OH OH OH HO HO HO HO HO HO OH

Polymers 2019, 11, x FOR PEER REVIEW 10 of 19 Polymers 2019, 11, x FOR PEER REVIEW 10 of 19 Polymers 2019, 11, x FOR PEER REVIEW 14 10 of 19 14 OH 14 OH OH OH Polymers 2019, 11, x FOR PEERO REVIEW O OH O OH O OH O OH O OH OH 10 of 19 OH OH OH HO O O OH O OH O OH O OH O OH OH OHOHO O OH OHO OHO OHO OHO OHO HO O O OH O OH O 14OH O OH O OH O OH OH OH O O O O O O HO HO OH OH O O HO OH HOOH HOOH HOOH HO OH HO O O O O O OH OH HO OH HOOH HOOH HOOH HO OH OH HO OH O HO O OH HOO 15 OH HOO OH HOO OH O HO OH OH HO O OH O O 15 O O O O OH OH OH OH OH OHOH OH HO 15 OH OH HOOH HOOH HOOH HO OH HO HO O O O O O O OH OH O CHO OH OH OH OH OH OH HO O O O O O O O O O O O O O OHOH OHOH CHO OHOH OHOH OHOH OHOH OHOHOH OH HO O O O O O O O15 O O O O O OH OH O CHO OHOH OHOH OHOH OHOH OH OH OH OH O O O O O O OH OH OHOH OHOH OHOH OHOH OH OH OH OH OH OH OH OH OH OH OH OH HO O O O O O O O CHO OH OH OH OH16 OH OH Polymers 2019, 11,O 1478 O O O16 O O OH OH 10 of 18 OH OH OH OH OH OH OH OH OH OH OH16 OH OH OHO Table 1. Cont. HOO OH OH OH HOOH OH 16 HO O OHO OHO OH OH + O HO 1214 Da (calc. 1212 Da) (no Na ) OH O O O O OH O OHO HOHO O OH OHO O O HO OH O OH O O O O OH HO O O O OH O O OH O OH O O HO OO O HO O HO O OH OH O OH HO O OHO OH O OO OHO OH O HO O OH O OH HO OH O HO O HOO OH O OOH OH OH HO O O HO HO O HO O O OH O O HO O O OH HO O OH HO O O HOO HO O OH O OH O HO O O O O HO17O OH HO O OH O O OHO O HO HO O 17 OH OH O HO O OH OH HO OH O HO O O OH 17 OH OH OH OH OH HO O O O + O O 1230OH Da (calc. 1231OH Da) (with Na ) OH O O OHO OHO HO O O O HO OH O OH 17 O O OHO OH O O O O O HO OH O OHOH O OH HO OO HO O O O O HO OH O HO OHO OHOH O O HO OO HO OH O O OH OH HO OH O HO HO OHO O O O HO OO O O OH HO OH OH O HO O O O O HOO HO OO HO OH O OH OH O O HO O OH O O O OH HO O O OH O O O HO O HO O HO O OH O OH 18 O O HO HO O O HO HO O OH O O OH HO O O HO 18 OH O + HO HO O 1254 Da (calc. 1253 Da) (with Na ) OH OHO OH OH OH OH OH OH OH OH HO O O O 18 O O O OH OH O CHO OH OH OH OH OH OH HO O O O O O O OH OH O OH O O O O 18 O O CHO OH OH OH OH OH OH OH O O O O O OHOH OHOH OH HO O OHOH HO OHOH OHOH OHOH OHOH OH OH HO O OHO OHO OHO OHO O O OH CHO HO OH OH HO OH OH OHOH OHOH OHOH OH OH OH OH OH HO O O O O O O O O O O O O O CHO OH OH OH OH OH OH + 1297 Da = glucose octamer19 missing one –OH, (no Na ) OH OH OH HO O O O O O O OH HO OH OHOH1362 Da OH (calc.OH 136419 OH Da)OH (with Na OH+) OH HO OH HO OH OH OH OH OH OH OH O O OH O OH 19O OH O OH O OH O OH O OH CHO OH OH OH OH OH OH OH PolymersHO 2019, 11, Ox FORO PEER REVIEWO O 19O O O O O11 of 19 OH O O O O O O O CHO HO OH OH OH O O OH OH OH OH OH OH OH HO O O O O O O OH OHOH HO O O OHOH HOO OHOH HOO OHOH HOO OH OH HOO OH OH OH O OH OH O OHHO 20 OHHO OH OH OH OH OH OH CHO HO O O HO1416O Da (Calc. HOO 1415 Da) HOO HOO OHO O OH O O CHO HO O O O O O O O OH OH OH O OH O OH O OH O OH O OHO OH OH OHO OH HO OH OH OH OH OH OH OH HO O O OHO OHO OHO OHO OHO OH O HO HO HO HO HO HO OH CHO HO HO HO HO HO OH O O O O O O OH OH OH OH OH OH OH OH OH OH HO OH HO OH OH OH OH

It is more interesting to examine the behavior of the soy protein in the soy protein isolate (SPI) whenIt treated is more with interesting sodium periodate.to examine Notwithstanding the behavior of the that soy the protein amino acidsin the and soy the protein peptides isolate involved (SPI) whenin these treated reactions with are sodium always periodate. rather di ffiNotwithstandingcult to identify, thethat task the isamino facilitated acids by and the the fact peptides that the involvedrelative abundance in these reactions of each are amino always acid rather in the difficult soy protein to identify, is known the [task20,21 is]. faci Thelitated results by clearly the fact show that thethat relative the protein abundance is also aofff ectedeach amino by a periodate acid in the treatment. soy protein Thus, is known on observing [20,21]. the The peaks results of theclearly soy showprotein that isolate the protein+ 15% is peroxide also affected present by ina periodate the MALDI treatment. analysis Thus, as well on as observing of the mix the protein peaks of+ soythe soyinsol. protein carbohydrates isolate + 15%+ sucrose peroxide+ periodate,present in the there MA areLDI peaks analysis clearly as well coming as of from the mix the protein + itself. soy insol.This also carbohydrates shows the formation+ sucrose + of periodate, aldehydes there via are the peaks periodate clearly oxidation coming offrom some the sites protein of the itself. protein This alsoitself, shows thus explainingthe formation why of the aldehydes soy protein via isolatethe period aloneate+ oxidationperoxide of also some yields sites good of the bonding protein results itself, withoutthus explaining the presence why the of carbohydratessoy protein isolate [12]. alone The main + peroxide species also formed yields that good is comingbonding from results the without protein theare apresence series of of monoaldehydes carbohydrates but[12] also. The one main dialdehyde, species formed as shown that in is Table coming1 and from Figure the2 proteina–c, the are rest a seriesbeing diofffi monoaldehydescult to assign. The but question also one that dialdehyde, must be answered as shown is betweenin Table which 1 and groups Figure does 2a,b,c, the the specific rest beingoxidation difficult cleavage to assign. occur The in the question protein that primary mustskeleton, be answered as vicinal is between –OH carrying which groups carbons does are notthe specificpresent. oxidation The only possibilitycleavage occur is that in thethe peptidicprotein primary bond itself skeleton, is cleaved, as vicinal as its C–OH=O doublecarrying bond carbons is in are not present. The only possibility is that the peptidic bond itself is cleaved, as its C=O double bond is in reality delocalized with the vicinal –NH, the electronegativity being somewhat similar to that of the vicinal –OH groups.40

O O

N N H H

(a)

Polymers 2019, 11, x FOR PEER REVIEW 10 of 18 Polymers 2019, 11, x FOR PEER REVIEW 11 of 19 OH OH OH O O OH O OH O OH O OH O OH O OH O 20 CHO HO O OH O O O O O O O OH OH OH OH OH OH OH OH OH OH OH HO OH OH OH OH OH OH HO O OHO O HOO HOO HOO HO O OH O CHO O O 1416 ODa (Calc. 1415O Da) O O OH OH OH OH OH OH OH OH OH OH OH HO OH OH OH OH OH HO OH OHOH OHOH OHOH OH OH HO O O O O O O O O CHO

O O O O O O OH OH OH OH OH OH OH OH OH OH HO OH HO OH OH OH OH It is more interesting to examine the behavior of the soy protein in the soy protein isolate (SPI) when treated with sodium periodate. Notwithstanding that the amino acids and the peptides involvedIt is more in these interesting reactions to areexamine always the rather behavior difficult of the to identify, soy protein the taskin the is facisoylitated protein by isolate the fact (SPI) that whenthe relativetreated abundancewith sodium of eachperiodate. amino Notwithstandingacid in the soy protein that theis known amino [20,21]. acids Theand resultsthe peptides clearly involvedshow that in these the protein reactions is also are alwaysaffected rather by a periodatedifficult to treatment. identify, theThus, task on is observing facilitated the by peaksthe fact of that the thesoy relative protein abundance isolate + 15% of eachperoxide amino present acid in the MAsoyLDI protein analysis is known as well [20,21]. as of the The mix results protein clearly + soy showinsol. that carbohydrates the protein is+ sucrosealso affected + periodate, by a periodate there are peakstreatment. clearly Thus, coming on observingfrom the protein the peaks itself. of This the soyalso protein shows isolate the formation + 15% peroxide of aldehydes present via in the the period MALDIate analysisoxidation as of well some as sitesof the of mix the protein +itself, soy insol.thus carbohydrates explaining why + sucrose the soy protein+ periodate, isolate there alone are + peaksperoxide clearly also comingyields good from bonding the protein results itself. without This alsothe shows presence the formationof carbohydrates of aldehydes [12] . The via mainthe period speciesate formed oxidation that of is some coming sites from of the the protein protein itself, are a thusseries explaining of monoaldehydes why the soy butprotein also isolateone dialdehyde, alone + peroxide as shown also in yields Table good 1 and bonding Figure results 2a,b,c, withoutthe rest Polymersthebeing 2019presence, difficult11, 1478 of carbohydrates to assign. The [12] question . The mainthat mustspec iesbe formedanswered that is isbetween coming which from thegroups protein does are the a 11 of 18 seriesspecific of monoaldehydes oxidation cleavage but occuralso one in the dialdehyde, protein primary as shown skeleton, in Table as vicinal1 and Figure–OH carrying 2a,b,c, thecarbons rest beingare notdifficult present. to Theassign. only The possibility question is that themust peptid be answeredic bond itself is betweenis cleaved, which as its C=Ogroups double does bond the realityspecificis delocalized in reality oxidation delocalized with cleavage the with vicinal occur the vicinalin –NH, the protein–NH, the electronegativity the primary electronegativity skeleton, being asbeing vicinal somewhat somewhat –OH carrying similar to carbons to that that of of the vicinalarethe not –OH vicinal present. groups. –OH The groups.40 only possibility is that the peptidic bond itself is cleaved, as its C=O double bond is in reality delocalized with the vicinal –NH, the electronegativity being somewhat similar to that of the vicinal –OH groups.O O O O N N NH N H H H

(a)

Polymers 2019, 11, x FOR PEER REVIEW 11 of 18 (a)

(b)

Data: 9.LSPI-1200001.H8[c] 2 May 2019 14:27 Cal: Ref 2 May 2019 14:06 Shimadzu Biotech Axima Performance 2.9.3.20110624: Mode Linear, Power: 64, P.Ext. @ 2300 (bin 78)

%Int. 1.5 mV[sum= 298 mV] Profiles 1-200 Smooth Av 50 -Baseline 150

1036.6 100

90 1091.5

80 1459.6

50 1212.3 40

0 1000 1100 1200 1300 1400 1500 1600 1700 18001[c] m/z (c)

FigureFigure 2. The2. The MALDI MALDI ToF ToF spectra spectra of of thethe soysoy protein isol isolateate after after a atreatment treatment with with sodium sodium periodate periodate at at 120 °C for 1 hour. (a) 100 Da to 500 Da range. (b) 500 Da to 1000 Da range. (c) 1000 Da to 1500 Da 120 ◦C for 1 h. (a) 100 Da to 500 Da range. (b) 500 Da to 1000 Da range. (c) 1000 Da to 1500 Da range. range.

The location of the formed aldehyde groups seems to support such an idea. Table 2 lists the structures of the species identified according to the peaks in Figures 2 and 3. Among these, several monoaldehydes are present, namely those represented by the peaks at 377 Da, 408 Da, 455 Da and the dialdehyde at 659–660 Da, namely a serine–[alanine]5–glutamic acid dialdehyde + Na+ with the following structure: O HO H O HO OHC N N N N N N CHO Na+ H O HO H O HO - + COO Na There could be several other aldehydes, but it is difficult to identify from the MALDI peaks the structures formed in the case of a protein.

Table 2. The proposed oligomer species for the MALDI ToF spectra peaks in Figure 2a,b,c and Figure 3a,b for the reaction at 120 °C for 1 hour of sodium periodate with the soy protein isolate.

377 Da = [alanine]4-aspartic acid monoaldehyde without Na+ 409 Da (calc. 409 Da) = alanine pentamer monoaldehyde with Na+

Polymers 2019, 11, x FOR PEER REVIEW 12 of 19

(b)

Data: 9.LSPI-1200001.H8[c] 2 May 2019 14:27 Cal: Ref 2 May 2019 14:06 Shimadzu Biotech Axima Performance 2.9.3.20110624: Mode Linear, Power: 64, P.Ext. @ 2300 (bin 78)

%Int. 1.5 mV[sum= 298 mV] Profiles 1-200 Smooth Av 50 -Baseline 150

1036.6 100

90 1091.5

80 1459.6

50 1212.3 40

0 1000 1100 1200 1300 1400 1500 1600 1700 18001[c] m/z (c)

Figure 2. The MALDI ToF spectra of the soy protein isolate after a treatment with sodium periodate at 120 °C for 1 hour. (a) 100 Da to 500 Da range. (b) 500 Da to 1000 Da range. (c) 1000 Da to 1500 Da range. Polymers 2019, 11, 1478 12 of 18

The location of the formed aldehyde groups seems to support such an idea. Table 2 lists the structuresThe location of the species of the formed identified aldehyde according groups to seems the peaks to support in Figures such an 2 idea.and 3. Table Among2 lists these, the structures several monoaldehydesof the species identified are present, according namely to the those peaks represen in Figurested2 by and the3. Amongpeaks at these, 377 Da, several 408 monoaldehydesDa, 455 Da and theare dialdehyde present, namely at 659–660 those representedDa, namely a by serine–[alan the peaks atine]5–glutamic 377 Da, 408 Da, acid 455 dialdehyde Da and the + dialdehydeNa+ with the at following659–660 Da, structure:41 namely a serine–[alanine]5–glutamic acid dialdehyde + Na+ with the following structure:

O HO H O HO OHC N N N N N N CHO Na+ H O HO H O HO

COO- Na+ Polymers 2019, 11, x FOR PEER REVIEW 13 of 19 Polymers 2019, 11, x FOR PEER REVIEW 13 of 19 ThereThere could could be be several several other other aldehydes, aldehydes, but but it it is is difficult diﬃcult to to identify identify from from the the MALDI MALDI peaks peaks the the Table 2. The proposed oligomer species for the MALDI ToF spectra peaks in Figure 2a,b,c and Figure structuresPolymersstructuresTable 2019 formed formed2., 11 The, x FOR proposed in in PEER the the case caseREVIEW oligomer of of aa protein. protein.species for the MALDI ToF spectra peaks in Figure 2a,b,c and Figure13 of 19 Polymers3a,b 2019 for, the11, xreaction FOR PEER at 120REVIEW °C for 1 hour of sodium periodate with the soy protein isolate. 13 of 19 3a,b for the reaction at 120 °C for 1 hour of sodium periodate with the soy protein isolate. TableTable 2.2. TheThe proposed proposed oligomeroligomer speciesspecies forfor thethe MALDIMALDI21 ToFToToFF spectra spectraspectra peaks peakspeaks in inin Figures FigureFigure 22a,b,c2a,b,ca–c and andand3a,b FigureFigure for 3a,b for the reaction at 120 °C for 1 hour of sodium21 periodate with the soy protein isolate. the3a,b reaction for the reaction at 120 ◦C at for 120 1 h°C of for sodium 1 hour periodate of sodium with periodate the soy with protein the isolate.soy protein isolate. H O H O H O 21 H O + 377 Da = [alanine]4-aspartic acid21 monoaldehyde without Na N N + 409 DaOHC (calc. 409 Da)N= alanineN pentamerN monoaldehydeN COOHwith Na OHC H O N H O N COOH O H O H O H O H O H O H NN NN OHCOHC NN NN COOHCOOH O H 22 O H O H 22 O H But it could also be (but less likely) O HO H + O O 409 Da = tyrosine-triptophaneHO2222 + NaH (dimer)O OHC455 Da (calc. 457 Da) = Nalanine hexamer monoaldehydeN without Na+ OHC N N N N N COOH OO N HOHON HH OO N COOH H O HO H OHCOHC H O NN HONN H NN NN NN COOHCOOH H O 23HO H H O 23HO H + OR455 Da (calc. 457 Da) = [alanine]2-[serine]3 monoaldehyde + Na OH OH HO 23OH OH HO 23 HH OO HH OO N N HO N OHN OH OHCOHCHO NN OHNN OHCOOHCOOH HH OO HH OO OO HH OO HH NN NN 659–660 DaOHCOHC= phenylalanine-triptophan-leucine-histidineNN NN +COOHCOOHNa+ (tetramer) 659–660 Da = phenylalanine-triptophan-histidine-asparticO H 24 O H acid + Na+ (tetramer) O H 24 O H + 659–660 Da = serine-[alanine]5-glutamic acid dialdehyde + Na 24 OO HOHO 24 HH OO HOHO OHC N N N OHC N N N + NN NN NN CHOCHO NaNa+ OO HOHO HH OO HOHO HH OO HOHO HH OO OHCHO N N N + OHCHO N N N N N N CHO Na + N N +N CHO Na - Na + H O HOCOOCOO- NaH O HO H O HO H O 959 Da =HOphenylalanine-triptophan-leucine-histidine-phenylalanine-arginine + Na+ (hexamer) + + 959 Da = phenylalanine-triptophan-histidine-aspartic COO acid-phenylalanine-arginine-- Na + + Na (hexamer) COO Na

Data: 3-mix-120blank2500001.C10[c] 30 Apr 2019 16:40 Cal: REF 30 Apr 2019 15:39 ShimadzuData: 3-mix-120blank2500001.C10[c] Biotech Axima Performance 30 2.9.3.20110624: Apr 2019 16:40 Mode Cal: REFLinear, 30 Power:Apr 2019 85, 15:39 Blanked, P.Ext. @ 2300 (bin 78) Shimadzu Biotech Axima Performance 2.9.3.20110624: Mode Linear, Power: 85, Blanked, P.Ext. @ 2300 (bin 78) %Int. 181 mV[sum= 36183 mV] Profiles 1-200 Smooth Av 50 -Baseline 150 %Int. 181 mV[sum= 36183 mV] Profiles 1-200 Smooth Av 50 -Baseline 150

Data: 3-mix-120blank2500001.C10[c] 30 Apr 2019 16:40 Cal: REF 30 Apr365.0 2019 15:39 ShimadzuData:100 3-mix-120blank2500001.C10[c] Biotech Axima Performance 30 2.9.3.20110624: Apr 2019 16:40 Mode Cal: REFLinear, 30 Power:Apr365.0 2019 85, 15:39 Blanked, P.Ext. @ 2300 (bin 78) Shimadzu100 Biotech Axima Performance 2.9.3.20110624: Mode Linear, Power: 85, Blanked, P.Ext. @ 2300 (bin 78) %Int.90 181 mV[sum= 36183 mV] Profiles 1-200 Smooth Av 50 -Baseline 150 %Int.90 181 mV[sum= 36183 mV] Profiles 1-200 Smooth Av 50 -Baseline 150 80 365.0 10080 365.0 100 361.0 70 361.0 9070 90 60 8060 80 50 361.0 7050 361.0 70 40 6040 60 30 5030 273.1 50 273.1 377.0 20 377.0 4020 360.0 40 177.1 360.0 393.0 10 177.1 311.1 393.0 3010 269.0 30 273.1 311.1 357.0 409.0 453.1 0 269.0273.1 357.0 409.0 453.1 0 377.0 20 150 200 250 300 350 377.0 400 450 5001[c] 20 1[c] 150 200 250 300 m/z 350360.0 400 450 500 177.1 m/z 360.0 393.0 10 311.1 10 177.1 393.0 269.0 311.1 357.0 409.0 453.1 0 269.0 357.0 409.0 453.1 0 1[c] (a) 150 200 250 300 350 400 450 500 1[c] (a) 150 200 250 300 m/z 350 400 450 500 m/z (a)(a)

Polymers 2019, 11, x FOR PEER REVIEW 12 of 18

H O H O N N OHC N N COOH O H O H But it could also be (but less likely) 409 Da = tyrosine-triptophane + Na+ (dimer) 455 Da (calc. 457 Da) = alanine hexamer monoaldehyde without Na+ O HO H O OHC N N N N N COOH

H O HO H OR 455 Da (calc. 457 Da) = [alanine]2-[serine]3 monoaldehyde + Na+ HO OH OH H O H O N N OHC N N COOH O H O H 659–660 Da = phenylalanine-triptophan-leucine-histidine + Na+ (tetramer) 659–660 Da = phenylalanine-triptophan-histidine-aspartic acid + Na+ (tetramer) 659–660 Da = serine-[alanine]5-glutamic acid dialdehyde + Na+ O HO H O HO OHC N N N N N N CHO Na+ H O HO H O HO

COO- Na+ 959 Da = phenylalanine-triptophan-leucine-histidine-phenylalanine-arginine + Na+ (hexamer) Polymers 2019959, 11Da, 1478 = phenylalanine-triptophan-histidine-aspartic acid-phenylalanine-arginine + Na+ 13 of 18 (hexamer)

Data: 3-mix-120blank2500001.C10[c] 30 Apr 2019 16:40 Cal: REF 30 Apr 2019 15:39 Shimadzu Biotech Axima Performance 2.9.3.20110624: Mode Linear, Power: 85, Blanked, P.Ext. @ 2300 (bin 78)

%Int. 181 mV[sum= 36183 mV] Profiles 1-200 Smooth Av 50 -Baseline 150

365.0 100

80 361.0 70

30 273.1 377.0 20 360.0 177.1 393.0 10 311.1 269.0 357.0 409.0 453.1 0 150 200 250 300 350 400 450 5001[c] m/z Polymers 2019, 11, x FOR PEER REVIEW 13 of 18 (a)

(b)

(c)

FigureFigure 3.3. TheThe MALDI ToF ToF spectra spectra of of the the reaction reaction of of th thee mix mix by by a weight a weight of of50% 50% soy soy protein protein + 25%+ 25% sucrose + 25% insoluble soy carbohydrates + 15% NaIO4 at 120 ℃ for 1 hour. (a) 150 Da to 500 Da sucrose + 25% insoluble soy carbohydrates + 15% NaIO4 at 120 °C for 1 h. (a) 150 Da to 500 Da range. (brange.) 500 Da(b) to500 900 Da Dato 900 range. Da range. (c) 900 (c Da) 900 to Da 1500 to Da1500 range. Da range.

ItIt is is of of interestinterest toto observe the the peaks peaks present present in in the the reaction reaction of ofthe the reconstituted reconstituted mix mix SPI SPI + sucrose+ sucrose ++soy soy insolubleinsoluble carbohydratescarbohydrates ++ NaIONaIO4 4inin Figure Figure 3a,b,c.3a–c. Among Among all all the the present present peaks, peaks, the the higher higher molecularmolecular weight weight (hence (hence lessless reactive)reactive) aldehydesaldehydes generatedgenerated byby sucrosesucrose oxidation are at 365 Da, 393 Da, andDa, 523and Da.523 Da. There There are are peaks peaks from from the the protein-generatedprotein-generated aldehydes, aldehydes, namely namely at at377 377 Da, Da, 409 409Da, Da, 453 Da, and 659 Da, and peaks from protein fragments without aldehyde groups, such as at 959 Da. The generated aldehydes by oxidation of the insoluble soy carbohydrates are represented by the peaks at 689 Da, 1036 Da, 1093 Da, 1135 Da, 1212 Da, and 1361 Da and the non-aldehyde fragment at 1297 Da. The DHB matrix enhancer are represented by the peaks at 312 Da, 361 Da, 536 Da, 550 Da and 698 Da. The question is now about which peaks that are present are due to the reaction of an aldehyde, for which it does not matter how they were generated, and which are due to a reaction with amino acids. First of all, it is clear that at a high molecular weight range, many species that are formed will be out of the possible range. Second, the majority of species generated by a reaction with the amino acids of aldehyde-generated species will be found in the middle of the examined molecular weight range. Thus, the co-reacted species are those represented by the peaks that do not belong to any of the single components at 555 Da, 559 Da, 585 Da, 619 Da, 729 Da, 780 Da, 819 Da, 849 Da 890 Da, 1389 Da and 1459 Da. These are due to the reaction of some generated aldehydes with some amino acids, and their assignments are given in Table 3. The more interesting interpretable peak is the one at 585 Da. This is obtained by the reaction of the sucrose-generated tri-aldehyde at 177 Da.

Polymers 2019, 11, 1478 14 of 18

453 Da, and 659 Da, and peaks from protein fragments without aldehyde groups, such as at 959 Da. The generated aldehydes by oxidation of the insoluble soy carbohydrates are represented by the peaks at 689 Da, 1036 Da, 1093 Da, 1135 Da, 1212 Da, and 1361 Da and the non-aldehyde fragment at 1297 Da. The DHB matrix enhancer are represented by the peaks at 312 Da, 361 Da, 536 Da, 550 Da and 698 Da. The question is now about which peaks that are present are due to the reaction of an aldehyde, for which it does not matter how they were generated, and which are due to a reaction with amino acids. First of all, it is clear that at a high molecular weight range, many species that are formed will be out of the possible range. Second, the majority of species generated by a reaction with the amino acids of aldehyde-generated species will be found in the middle of the examined molecular weight range. Thus, the co-reacted species are those represented by the peaks that do not belong to any of the single components at 555 Da, 559 Da, 585 Da, 619 Da, 729 Da, 780 Da, 819 Da, 849 Da 890 Da, 1389 Da and 1459 Da. These are due to the reaction of some generated aldehydes with some amino acids, and their assignmentsPolymers 2019,, 11 are,, xx FORFOR given PEERPEER in REVIEW TableREVIEW3. The more interesting interpretable peak is the one at 585 Da. This15 of is19 obtained by the reaction of the sucrose-generated tri-aldehyde at 177 Da. While this peak is also present inWhile SPI alonethis peak representing is also present just an aminoin SPI acid,alone it representing is also a branched just an aldehyde amino generatedacid, it is fromalso a sucrose branched via thealdehyde aldol condensationgenerated from of sucrose simpler via sucrose-generated the aldol condensation aldehydes. of simpler Thus, sucrose-generated the 585 Da peak is aldehydes. due to the reactionThus, the of 585 three Da moleculespeak is due of to the the most reaction abundant of three SPI molecules amino acid, of namelythe most glutamic abundant acid, SPI with:amino acid, namely glutamic acid, with:42 OH OH OH OHC CH CH C CHO CHO CHO

To yieldyield thethe twotwo alternativealternative structures at 585 Da, one is derived directly by the reaction of three molecules ofof glutamic glutamic acid acid with with the the 177 177 Da tri-aldehydeDa tri-aldehyde and theand second the second one is generatedone is generated via the reactionvia the ofreaction three glutamicof three acidglutamic residues acid with residues glyoxal with and glyoxal a hydroxyl and malonica hydroxyl aldehyde malonic directly aldehyde generated directly by thegenerated speciﬁc by oxidation the specific cleavage oxidatio byn the cleavage periodate: by the periodate:43

COOH

COOH HN COOH HO CH HO CH COOH HC OH H H OH OH HN COOH HO CH OH COOH N C CH CH CHOH COOH N HC CH HN COOH OH CH OH COOH N OH HN COOH COOH COOH COOH

The same peak at 585 Da can alsoalso bebe assignedassigned toto anan oligomeroligomer of:of: Lysine-CHOH-CHOH-Lysine-CHOH-CHOH-CHOH-Lysine As amino acidsLysine-CHOH-CHOH-Lysine-CHOH-CHOH-CHOH-Lysine presenting at least two amino groups can form linear oligomers, the peaks at 554–555 Da, 619 Da, 728 Da, 780 Da, 819 Da, and 849–850 Da can be interpreted as linear oligomers of the reactionAs amino of (mainly) acids presenting lysine and at arginine, least two wi aminoth glyoxal groups and can hydroxy form linear malonic oligomers, aldehyde the generated peaks at 554–555by periodate-specific Da, 619 Da, 728 oxidation Da, 780 from Da, 819 simple Da, andsugars 849–850 such Daas glucose can be interpreted [3] and sucrose, as linear as is oligomers indeed the of thecase reaction for one ofof (mainly)the assignments lysine and for arginine, the species with at glyoxal585 Da and(Table hydroxy 3). malonic aldehyde generated by periodate-speciﬁc oxidation from simple sugars such as glucose [3] and sucrose, as is indeed the case for one of the assignments for the species at 585 Da (Table3).

Polymers 2019, 11, 1478 15 of 18 Polymers 2019, 11, x FOR PEER REVIEW 16 of 19

TableTable 3. 3.Some Some possible possible assignments assignments from the MALDI MALDI ToF ToF spectra spectra in in Figure Figure 3a,b,c3a–c of of the the reaction reaction of of SPISPI+ sucrose+ sucrose+ +insoluble insoluble soysoy carbohydratescarbohydrates + NaIO4NaIO4 for for peaks peaks not not belonging belonging to toany any of ofthe the single single periodate-treatedperiodate-treated constituent constituent components. components. 554–555 Da, no Na+ 554–555 Da, no Na+ Lysine–CHOH–CHOH–Lysine–CHOH–CHOH–Lysine25Lysine–CHOH–CHOH–Lysine–CHOH–CHOH–Lysine 585 Da, no Na+, two possible species from glutamic acid COOH

COOH HN COOH HO CH COOH HC OH H H OH OH HN COOH HO CH OH COOH N C CH CH CHOH COOH N HC CH HN COOH OH CH OH OH HN COOH COOH COOH COOH and/or one from Lysine Lysine–CHOH–CHOH–Lysine–CHOH–CHOH–CHOH–Lysineand/or one from Lysine Lysine–CHOH–CHOH–Lysine–CHOH–CHOH–CHOH–Lysine619 Da, no Na+ Lysine–CHOH–CHOH–CHOH–Lysine–CHOH–CHOH–CHOH–Lysine 619728 Da, Da, no NaNa++ Lysine–CHOH–CHOH–CHOH–Lysine–CHOH–CHOH–CHOH–LysineLysine–CHOH–CHOH–Lysine–CHOH–CHOH–Lysine–CHOH–CHOH–Proline 728780 Da, Da, no NaNa++ Lysine–CHOH–CHOH–Lysine–CHOH–CHOH–Lysine–CHOH–CHOH–ProlineLysine–CHOH–CHOH–Lysine–CHOH–CHOH–Lysine–CHOH–CHOH–Phenylalanine 819 Da, no Na+ 780 Da, no Na+ Arginine–CHOH–CHOH–Arginine–CHOH–CHOH–Arginine–CHOH–CHOH–Cysteine Lysine–CHOH–CHOH–Lysine–CHOH–CHOH–Lysine–CHOH–CHOH–Phenylalanine849–850 Da, no Na+ Lysine–CHOH–CHOH–CHOH–Lysine–CHOH–CHOH–CHOH–Lysine–CHOH–CHOH–CHOH–Lysine819 Da, no Na+ Arginine–CHOH–CHOH–Arginine–CHOH–CHOH–Arginine–CHOH–CHOH–Cysteineand/or Lysine–CHOH–CHOH–CHOH–Lysine–CHOH–CHOH–CHOH–Lysine–CHOH–CHOH–CHOH–glutamic849–850 Da, no Na+ acid Lysine–CHOH–CHOH–CHOH–Lysine–CHOH–CHOH–CHOH–Lysine–CHOH–CHOH–985 Da, no Na+ Lysine–(–CHOH)3–Lysine–(–CHOH)CHOH–Lysine3–Lysine–(–CHOH) 3–Lysine CHOH–CHOH–Glycine 1388–1389and/or Da Arginine–(–CHOH)2–Arginine–(–CHOH)2–Arginine–(–CHOH)2–Arginine–(–CHOH)2–Arginine–(–CHOH)Lysine–CHOH–CHOH–CHOH–Lysine–CHOH–CHOH–CHOH–Lysine–CHOH–CHOH– 2–Tryptophan Where the structure is notCHOH–glutamic necessarily linear andacid can be better expressed as [Arginine–(–CHOH)985 Da, no2–] Na+5–Tryptophan Lysine–(–CHOH)3–Lysine–(–CHOH)3–Lysine–(–CHOH)1459–1460 Da 3–Lysine CHOH–CHOH–Glycine [Arginine–(–CHOH)1388–13892–]5–Histidine–(–CHOH) Da 4–CHO or [Arginine–(–CHOH) –] –Histidine–(–CHOH) –Alanine Arginine–(–CHOH)2–Arginine–(–CHOH)2–Arginine–(–CHOH)2–Arginine–(–CHOH)2–2 5 2 Arginine–(–CHOH)2–Tryptophan 4. ConclusionsWhere the structure is not necessarily linear and can be better expressed as [Arginine–(–CHOH)2–]5–Tryptophan To summarize, in the spectra of the reaction of SPI + sucrose + insoluble carbohydrates + NaIO4, the following peaks ascribed to the action of1459–1460 periodate Da on SPI and insoluble soy ﬂour carbohydrates [Arginine–(–CHOH)2–]5–Histidine–(–CHOH)4–CHO are present: 1053 Da, 1092–1093 Da, 1135 Da, 1153 Da, 1198 Da, 1212–1213 Da, 1230 Da, 1254 Da, or [Arginine–(–CHOH)2–]5–Histidine–(–CHOH)2–Alanine 1361–1362 Da and 1416 Da coming from the insoluble carbohydrates, and 377 Da, 408–409 Da, 453–455 Da and 659–660 Da coming from the SPI. This shows that some of the carbohydrate aldehyde 4. Conclusions species are no longer present when the SPI is present; hence, they have reacted with the SPI. Furthermore, the co-reactedTo summarize, species in are the thosespectra represented of the reaction by of the SPI peaks + sucrose that + do insoluble not belong carbohydrates to any of + the NaIO single4, components,the following at peaks 555 Da, ascribed 559 Da, to 585 the Da,action 619 of Da, peri 729odate Da, on 780 SPI Da, and 819 insoluble Da, 849 soy Da flour 890 Da,carbohydrates 1389 Da and 1459are Da. present: 1053 Da, 1092–1093 Da, 1135 Da, 1153 Da, 1198 Da, 1212–1213 Da, 1230 Da, 1254 Da, 1361– 1362 Da and 1416 Da coming from the insoluble carbohydrates, and 377 Da, 408–409 Da, 453–455 Da

Polymers 2019, 11, 1478 16 of 18

The peak at 231 Da represents just traces (almost non-existent) in the insoluble carbohydrates at 120 ◦C without periodate, but it is a huge peak in the insoluble carbohydrates with periodate at 120 ◦C. It does not seem to appear in the SPI + sucrose + insoluble carbohydrates + NaIO4. It could be that with the protein present, the aldehyde groups of the species 231 Da have reacted very readily with SPI. It is one of the best indications that the carbohydrates have not only formed an aldehyde (double here) but also that this reacts with the SPI. The following peaks coming from the periodate cleavage of the protein are present: 377 Da, 408–409 Da, 455 Da and 659–660 Da. Of these, the 408–409 Da, 455 Da and 659–660 Da could present an aldehyde function due to the cleavage but could also be interpreted otherwise. However, they are most likely carrying aldehyde groups, as without periodate these peaks do not seem to appear. However, the peaks at 408–409 Da and 453–455 Da also appear, although small, in the case of the carbohydrates at 120 ◦C without periodate, and in the SPI alone at 120 ◦C without periodate; thus, their interpretation in favour of aldehyde formation is not at all certain. Thus, one cannot be sure at all that they come from the protein. The ﬁnal conclusions for the reactions at 120 ◦C with and without periodate are then as follows:

1. There are clear indications that aldehydes are formed from the insoluble carbohydrates by periodate specific oxidation and cleavage. There are carbohydrate oligomers presenting single, double and multiple aldehyde groups coming from the cleavage of the soy flour insoluble carbohydrates. 2. There is only one, diﬀerent, low molecular weight aldehyde at 231 Da, which is in considerable proportion when the insoluble carbohydrates are treated with periodate but only in traces when treated at 120 ◦C without periodate. This peak disappears completely when the SPI is present, indicating that the aldehyde (a dialdehyde) has reacted with the SPI. 3. The peak at 231 Da is a dialdehyde derived by the aldol condensation of fragments issued by the degradation of soluble and insoluble carbohydrates. 4. There are oligomeric peptides coming from the cleavage of the protein by periodate. However, only one species (at 659–660 Da) could (we stress could) be ascribed to an oligomeric aldehyde, as other interpretations, such as peptide oligomers without an aldehyde group, are also possible. 5. Thus, the clear indications are that the carbohydrates do form aldehydes that co-react with the SPI and crosslink it. 6. There is at least one indication that aldehyde groups are formed in the cleavage of the protein, but these are not clear and could also be interpreted otherwise.

7. In the SPI + sucrose + insoluble carbohydrates + NaIO4, several aldehydes are formed, as presented in the Introduction and already reported in the literature [5].

8. In the SPI + sucrose + insoluble carbohydrates + NaIO4, mainly aldehydes generated from sucrose by periodate-speciﬁc oxidation react with amino acids, showing that the cross-linking of the protein oligomers by these aldehydes does indeed occur. This entails that at a higher temperature or longer reaction times, the other aldehydes generated from the insoluble carbohydrates and from the protein are also likely to react and contribute to cross-linking. 9. Finally, it must be noted that the peaks at 177 Da, 360–361 Da, 536 Da, 550 Da, 698–699 Da, 717 Da, and 874 Da belong to the DHB enhancer used in the MALDI analysis.

What are the future prospects of this type of approach? The first interest is that an almost classical reaction and cross-linking system based on aldehydes and a protein are generated, but with the generated aldehydes being non-volatile and non-toxic. The second interest is that all parts of soy flour, much cheaper than soy protein isolates, can be directly used for adhesives and can participate in cross-linking. Third, while one can use soy flour, nothing impedes the use of a separate mix of oligomeric and or monomeric carbohydrates to generate aldehydes via periodate specific oxidation and then adding this to a soy protein isolate, another protein isolate (such as gluten for example), or other aldehyde-reacting materials. In this respect, periodate oxidized carbohydrates could be used Polymers 2019, 11, 1478 17 of 18 as hardeners for a variety of different natural materials, such as tannins, lignin, humins, etc., or possibly even mixtures of these.

Supplementary Materials: The following are available in the Supplementary Material online at http://www.mdpi. com/2073-4360/11/9/1478/s1, Figure S1: MALDI Tof Spectra of insoluble carbohydrates treated at 120 ◦C without periodate: (a) 150 Da to 500 da range, (b) 500 Da to 900 Da range, and (c) 900 Da to 1200 Da range. Figure S2: sucrose + 15% NaIO4 at 120 ◦C for 1 h: (a) 50 Da to 1000 Da range, (b) 50 Da to 500 Da range, (c) 300 Da to 500 Da range, (d) 500 Da to 800 Da range, and (e) 800 Da to 1600 Da range. Author Contributions: C.R.F., A.P. and L.F.L. conceived, planned and designed the experiments, X.X. did the reactions and performed the MALDI analysis, A.P. analyzed the data, interpreted the MALDI analysis, coordinated the work and wrote the paper. Acknowledgments: The LERMAB of the University of Lorraine is supported by a grant overseen by the French National Research Agency (ANR) as part of the Laboratory of Excellence (Labex) ARBRE, which is herewith thanked by the second and third author. The third author thanks the China Scholarship Council for the study bursary granted to him. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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