Chitin and Cellulose Processing in Low-Temperature Electron Beam Plasma †

molecules

Article Chitin and Cellulose Processing in Low-Temperature Electron Beam Plasma †

Tatiana Vasilieva 1,*, Dmitry Chuhchin 2, Sergey Lopatin 3, Valery Varlamov 3, Andrey Sigarev 1 and Michael Vasiliev 1

1 Moscow Institute of Physics and Technology, Institutsky per., 9, Dolgoprudny, 141700 Moscow, Russia; [email protected] (A.S.); [email protected] (M.V.) 2 Core Facility Center “Arktika”, Northern (Arctic) Federal University, Severnaya Dvina Emb., 17, 163002 Arkhangelsk, Russia; [email protected] 3 Federal State Institution, Federal Research Centre, Fundamentals of Biotechnology of RAS, Institute of Bioengineering, 60 let Oktjabrja pr-t, 7/1, 117312 Moscow, Russia; [email protected] (S.L.); [email protected] (V.V.) * Correspondence: [email protected]; Tel.: +7-495-408-4744 † Presented at 13th International conference of the European Chitin Society/8th Symposium of the Iberoamerican Chitin Society.

Received: 3 September 2017; Accepted: 27 October 2017; Published: 6 November 2017

Abstract: Polysaccharide processing by means of low-temperature Electron Beam Plasma (EBP) is a promising alternative to the time-consuming and environmentally hazardous chemical hydrolysis in oligosaccharide production. The present paper considers mechanisms of the EBP-stimulated destruction of crab shell chitin, cellulose sulfate, and microcrystalline cellulose, as well as characterization of the produced oligosaccharides. The polysaccharide powders were treated in oxygen EBP for 1–20 min at 40 ◦C in a mixing reactor placed in the zone of the EBP generation. The chemical structure and molecular mass of the oligosaccharides were analyzed by size exclusion and the reversed phase chromatography, FTIR-spectroscopy, XRD-, and NMR-techniques. The EBP action on original polysaccharides reduces their crystallinity index and polymerization degree. Water-soluble products with lower molecular weight chitooligosaccharides (weight-average molecular mass, Mw = 1000–2000 Da and polydispersity index 2.2) and cellulose oligosaccharides with polymerization degrees 3–10 were obtained. The 1H-NMR analysis revealed 25–40% deacetylation of the EBP-treated chitin and FTIR-spectroscopy detected an increase of carbonyl- and carboxyl-groups in the oligosaccharides produced. Possible reactions of β-1,4-glycosidic bonds’ destruction due to active oxygen species and high-energy electrons are given.

Keywords: chitin; cellulose; oligosaccharides; low-temperature plasma; electron-beam plasma; plasma chemistry

1. Introduction Two natural renewable biopolymers, namely cellulose (linear chain of several hundreds to many thousands of β-1,4-linked D-glucose units) and chitin (linear heterocopolymer of β-1,4-linked 2-amino-2-deoxy-D-glucopyranose and 2-acet-amido-2-deoxy-D-glucopyranose units), are the most abundant polysaccharides on the earth. Cellulose, chitin, and chitosan derivatives are very promising for technological and industrial applications such as agriculture, microbiology, food processing, medicine, and the pulp and paper subsector [1–6]. Though the polymers possess unique properties (high biocompatibility with living tissues, biodegradability, ability to the complexation, low toxicity, etc.) they have limited applications in many industrial ﬁelds because of their insolubility in most solvents. For example, water-soluble low

Molecules 2017, 22, 1908; doi:10.3390/molecules22111908 www.mdpi.com/journal/molecules Molecules 2017, 22, 1908 2 of 15 Molecules 2017, 22, 1908 2 of 15 molecular weight chitooligosaccharides (COS) (less than 10 kDa) are usually required in medicine, pharmaceutics,molecular weight and chitooligosaccharides agriculture [5–10]. Low-molecular (COS) (less than weight 10 kDa) cellulose are usually fragments required are inprospective medicine, pharmaceutics,substrates for microbiology and agriculture since [5 –they10]. Low-molecularcan be converted weight into cellulosemonosaccharides fragments and are a prospectivenumber of products,substrates fermentable for microbiology sugars sincefor bio-ethanol they can besynthesis converted via microbia into monosaccharidesl fermentation among and a numberthem [11]. of products,Simple fermentable and relatively sugars low-cost for bio-ethanol chemical synthesis hydrolys viais microbialin concentrated fermentation acids amongor alkalis them at [high11]. temperaturesSimple and is arelatively conventional low-cost method. chemical However, hydrolysis these techniques in concentrated usually acids take or several alkalis hours at high or eventemperatures days and is a conventionalthe processing method. equipment However, is thesedamaged techniques due to usually corrosion take severaland neutralization hours or even proceduresdays and the are processing required equipment[12–15]. Moreover, is damaged toxic due waste to corrosion and environmental and neutralization contamination procedures are inherentare required in polysaccharide [12–15]. Moreover, chemical toxic processing. waste and Another environmental problem with contamination the chemical are methods inherent is the in polysaccharideformation of products chemical with processing. modified Another chemical problem structures. with For the instance, chemical the methods chemical is the degradation formation of productschitin can with result modiﬁed in the chemicalformation structures. of deacetylat For instance,ed COS thebut chemicalfor further degradation (bio)chemical of chitin reactions can result the stablein the formationN-acetyl groups of deacetylated available COS in the but chitin for further structure (bio)chemical are usually reactions required the [12,13]. stable N-acetyl groups availableEnzymes in the are chitin often structure preferable are to usually inorganic required compounds [12,13]. with catalytic capacity because they are environmentallyEnzymes are sustainable. often preferable Howe tover, inorganic the enzymatic compounds hydrolysis with catalytic of cellulose capacity is often because incomplete, they are andenvironmentally the products sustainable.formed have However,a high polydispersity the enzymatic index hydrolysis [16]. of cellulose is often incomplete, and theThus, products the efforts formed towards have athe high development polydispersity of new index economically [16]. feasible and environmentally friendlyThus, methods the efforts for towardspolysaccharide the development degradation of newappear economically to be reasonable. feasible The and plasma-chemical environmentally technologiesfriendly methods using for non-equilibrium, polysaccharide low-temperature degradation appear plasmas to be could reasonable. be a promising The plasma-chemical alternative to thetechnologies hydrolysis using methods non-equilibrium, mentioned low-temperatureabove. Our previous plasmas papers could have be a promisingshown that alternative electron- tobeam the plasmahydrolysis (EBP) methods can be mentioned applied for above. the effective Our previous and controllable papers have destruction shown that electron-beamof chitosan, and plasma high (EBP)yields canof water-soluble be applied for COS the can effective be attained and controllableby optimizing destruction the EBP-treatment of chitosan, procedure and high [17]. yields of water-solubleThe aims COSof the can present be attained study bywere optimizing as follows: the EBP-treatment procedure [17]. The aims of the present study were as follows:  To study the EBP-processing of chitin and cellulose and to reveal the time dependence for the • ToEBP study-stimulated the EBP-processing formation of water-soluble of chitin and celluloselow molecular and to weight reveal products the time dependence(LMWP). for the  ToEBP-stimulated characterize the formation chemical of structure water-soluble and molecular low molecular mass weight of the produced products (LMWP).LMWP. • To characterizesuggest possible the chemical mechanisms structure of EBP and action molecular on polysaccharides. mass of the produced LMWP. • To suggest possible mechanisms of EBP action on polysaccharides. 2. Results 2. Results 2.1. The EBPProcessing of Polysaccharides 2.1. The EBPProcessing of Polysaccharides Figure 1 illustrates the design and operation of the electron-beam plasma-chemical reactor (EBPR).Figure The1 illustrates EBPR, its the operation design and modes, operation and of thethe electron-beamoptimization plasma-chemicalof the biomaterial reactor treatment (EBPR). Theregimens, EBPR, as its well operation as the modes,EBP properties, and the optimization were described of the in biomaterialdetail in [18,19]. treatment regimens, as well as the EBP properties, were described in detail in [18,19].

Figure 1. Electron-beam plasma-chemical reactor and scheme of the treatment procedure: Figure 1. Electron-beam plasma-chemical reactor and scheme of the treatment procedure: 1—electron-beam gun; 2—high vacuum chamber; 3—electron beam; 4—injection window; 1—electron-beam gun; 2—high vacuum chamber; 3—electron beam; 4—injection window; 5—electromagnetic scanning system; 6—working chamber; 7—EBP cloud; 8—mixing device with 5—electromagnetic scanning system; 6—working chamber; 7—EBP cloud; 8—mixing device with polysaccharide powder to be treated. polysaccharide powder to be treated.

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Briefly,Briefly, thethe focusedfocused EB EB 3 generated3 generated by by the the electron-beam electron-beam gun 1gun that 1is that located is located in the highin the vacuum high vacuumchamber chamber 2 is injected 2 is intoinjected the working into the chamber working 6 chamber filled with 6 thefilled plasma-generating with the plasma-generating gas through gas the throughinjection the window injection 4. In window passing 4. through In passing the gasthrough the EB the is scatteredgas the EB in is elastic scattere collisionsd in elastic and thecollisions energy andof fast the electrons energy graduallyof fast electrons diminishes gradually in various diminish inelastices in interactions various inelastic with the interactions medium (ionization, with the mediumexcitation, (ionization, dissociation). excitation, As a result, dissociation). EBP cloud 7As is a generated, result, EBP all plasmacloud 7 parameters is generated, being all functions plasma parametersof the x, y and beingz coordinates functions of (z theis the x, y axis and of z thecoordinates EB injection). (z is the axis of the EB injection). TheThe electromagnetic electromagnetic scanning scanning system system 5 5 placed placed inside inside the the working working chamber chamber near near the the injection injection windowwindow is is able able to to deflect deflect the the injected injected EB EB axis axis in inthe the x andx and y directionsy directions and, and, therefore, therefore, to control to control the spatialthe spatial distribution distribution of the ofplasma the plasma particles particles throughout throughout the plasma the bulk. plasma The bulk.working The chamber working is preliminarilychamber is preliminarily evacuated evacuatedto pressure to pressure~1 Pa ~1and Pa then and thenfilled filled with with the the plasma plasma generating generating media—chemicallymedia—chemically pure pure oxygen oxygen (Sigma-Aldrich, (Sigma-Aldrich, Munich, Germany) up to 670 Pa. TheThe polysaccharide polysaccharide powders powders were were processed processed in in a a special special mixing mixing re reactoractor 8. 8. The The device device (Figure (Figure 2a)2a) containscontains a a cylindrical cylindrical quartz quartz vessel vessel 1a 1a with with internal internal ribs ribs 3a; 3a; it it is is equipped equipped with with a a stepper stepper motor motor rotatingrotating thethe vesselvessel in in various various modes modes (continuous, (continuous, intermittent, intermittent, reverse, reverse, etc.). Whenetc.). When the vessel the rotates,vessel rotates,the powder the powder material material to be treated to be (4a)treated is mixing. (4a) is mixing. The device The is device placed is inside placed the inside EBPR the working EBPR workingchamber chamber filled with filled the plasma with the generating plasma gasgenerating at required gas pressure at required and thepressure EB 2a isand injected the EB through 2a is injectedthe open through end of the reactor.open end As of a the result reactor. the aerosol As a result reaction the zoneaerosol 5a reaction is formed zone inside 5a theis formed vessel. insideThe treatment the vessel. time The duration treatment (τ) of time the polysaccharidesduration (τ) of the varied polysaccharides within the range varied 1–20 within min. Tothe prevent range ◦ 1–20the polysaccharides’ min. To prevent thermal the polysaccharides’ destruction, they thermal were destru processedction, at they material were temperature processed atT smaterial= 40 C. temperatureThe sample temperatureTs = 40 °C. The during sample the temperature treatment was during monitored the treatment by an optical was monitored IR-pyrometer by an Optris optical LS IR-pyrometer(Optris GmbH, Optris Portsmouth, LS (Optri NH,s USA)GmbH, or byPortsmouth, a miniature NH, thermo-sensor USA) or by inserted a miniature into the thermo-sensor reaction zone. insertedThe material into the temperature reaction zone. was controlledThe material by varyingtemperature the EB was current. controlled by varying the EB current.

(a) (b)

FigureFigure 2. 2. PolysaccharidePolysaccharide powders powders processing processing in in the the mixing mixing device: device: ( (aa)) Design Design of of the the mixing mixing device device forfor the the EBP-treatment EBP-treatment of of polysacch polysaccharidearide powders: powders: 1a—cylin 1a—cylindricaldrical quartz quartz vessel; vessel; 2a—the 2a—the EBP EBP cloud; cloud; 3a—internal3a—internal partitions; 4a—polysaccharides powder;powder; 5a—aerosol5a—aerosol reaction reaction zone. zone. ( b()b Chitin) Chitin powder powder in inthe the EBP EBP reaction reaction zone zone photo. photo.

2.2.2.2. The The Characterization Characterization of of the the EBP-Treated EBP-Treated Polysaccharides

2.2.1.2.2.1. Molecular Molecular Mass Mass Characterization Characterization τ TheThe originaloriginal polysaccharidespolysaccharides were were water-insoluble water-insoluble and theand EBP-treatment the EBP-treatment ( = 1–10 (τ min)= 1–10 increased min) increasedtheir solubility their in solubility water and in some water other and solvents some other due to solvents the decrease due ofto polymerization the decrease of degree polymerization and the low degreemolecular and weight the low products molecular (LMWP) weight formation. products Table (LMWP)1 illustrates formation. the solubility Table 1 changes illustrates of the the EBP-treated solubility changescellulose of sulfate the EBP-treated in distilled watercellulose and sulfate 5% NaOH. in distilled The yield water of soluble and 5% cellulose NaOH. LMWP The yield increased of soluble with the EBP-treatment time duration and reached 77.4% (for water-soluble LMWP) after a 10-min treatment. cellulose LMWP increased with the EBP-treatment time duration and reached 77.4% (for EBP-processing for 2 min significantly reduced the polymerization degree of the non-soluble fraction. water-soluble LMWP) after a 10-min treatment. EBP-processing for 2 min significantly reduced the polymerization degree of the non-soluble fraction.

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TableTable 1.1. TheThe solubilitysolubility changeschanges ofof thethe EBP-treatedEBP-treated cellucelluloselose sulfatesulfate inin distilleddistilled waterwater andand 5%5% NaOH.NaOH. Table 1. The solubility changes of the EBP-treated cellulose sulfate in distilled water and 5% NaOH. TimeTime ofof YieldYield ofof YieldYield ofof Products,Products, YieldYield ofof PolymerizationPolymerization DegreeDegree EBP-Processing, Water-Soluble Soluble in 5% Non-Soluble of Non-Soluble EBP-Processing, Water-SolubleYield of SolubleYield of in Products, 5% Non-SolubleYield of of Non-SolublePolymerization Time of 1 minmin Products,Products,Water-Soluble %% NaOH,NaOH,Soluble %% in 5% ResidueResidueNon-Soluble 1,, %% ResidueResidueDegree of EBP-Processing, min 1 00 Products,1.81.8 % NaOH,6.86.8 % Residue93.293.2 ,% Non-Soluble 960 960 Residue 022 22.722.7 1.8 45.345.3 6.8 54.754.7 93.2 460 460 960 10210 77.477.4 22.7 100100 45.3 00 54.7 - - 460 1 10 77.4 100 0 - 1 Non-solubleNon-soluble residueresidue correspondscorresponds toto productsproducts thatthat dodo notnot dissolvedissolve inin eithereither 5%5% NaOHNaOH oror distilleddistilled water.water. 1 Non-soluble residue corresponds to products that do not dissolve in either 5% NaOH or distilled water. TheThe mixturemixture ofof productsproducts withwith polymerizationpolymerization degreesdegrees varyingvarying fromfrom 1010 andand higherhigher toto glucoseglucose monomersmonomersThe mixture werewere offoundfound products inin thethe with extractsextracts polymerization ofof thethe EBP-treatedEBP-treated degrees varying cellulosecellulose from 10byby and thethe higher chromatographychromatography to glucose + monomerstechnique,technique, werethethe ion-exclusionion-exclusion found in the column extractscolumn Rezex ofRezex the EBP-treatedRSO-OligosaccharideRSO-Oligosaccharide cellulose by AgAg the+ (4%)(4%) chromatography (Phenomenex,(Phenomenex, technique, Torrance,Torrance, theCA,CA, ion-exclusion USA)USA) beingbeing used.used. column TheThe Rezex water-solublewater-soluble RSO-Oligosaccharide LMWPLMWP havehave Ag polymerizationpolymerization+ (4%) (Phenomenex, degreesdegrees Torrance,mostlymostly inin CA, thethe USA)rangerange beingofof 3–10.3–10. used. ProductsProducts The water-soluble withwith lowerlower (up(up LMWP toto glucoseglucose have polymerizationmomonomers)nomers) andand degrees higherhigher molecularmolecular mostly in theweightsweights range werewere of 3–10. alsoalso Productsobservedobserved with (Figure(Figure lower 3).3). AtAt (up first,first, to glucose thethe concentrconcentr monomers)ationation ofof and LMWPLMWP higher dropsdrops molecular withwith thethe weights decreasedecrease were inin polymerizationpolymerization also observed (Figuredegreedegree3 down).down At ﬁrst, toto 66 the andand concentration thenthen risesrises again.again. of LMWP TheThe moleculamolecula drops withrr weightweight the decrease distributiondistribution in polymerization ofof thethe formedformed degree productsproducts down isis topresentedpresented 6 and then inin risesFigureFigure again. 4.4. TheThe The LMWPLMWP molecular formationformation weight withwith distribution predominatingpredominating of the formedtetramertetramer products waswas alsoalso is confirmedconfirmed presented by inby 13 Figure13C-NMRC-NMR4. The analysis.analysis. LMWP formation with predominating tetramer was also conﬁrmed by 13C-NMR analysis.

FigureFigureFigure 3. 3.3. The TheThe ligand-exchange ligand-exchangeligand-exchange chromatogram chromatogramchromatogram of ofof the thethe water-soluble wawater-solubleter-soluble cellulose cellulosecellulose LMWP LMWPLMWP obtained obtainedobtained due duedue to toto thethethe EBP-processing:EBP-processing:EBP-processing: 1—glucose;1—glucose;1—glucose; 2–10—LMWP2–10—LMWP2–10—LMWP withwithwith variousvariousvarious polymerizationpolymerizationpolymerization degreesdegreesdegrees (the(the(the numbersnumbersnumbers correspondcorrespondcorrespond to toto the thethe degree degreedegree of ofof polymerization); polymerization);polymerization); RI—refractive RI—refractiveRI—refractive index. index.index.

FigureFigureFigure 4. 4.4. The TheThe molecularmolecularmolecular weightweightweight distributiondistributiondistribution ofof thethethe productsprodproductsucts formedformedformed inin thethethe cellulosececellulosellulose EBP-processing:EBP-processing: 1—original cellulose; 2—cellulose treated in the EBP for 2 min; 3—cellulose treated in the EBP for 10 min. 1—original1—original cellulose;cellulose; 2—cellulose2—cellulose treatedtreated inin thethe EBPEBP forfor 22 min;min; 3—cellulose3—cellulose treatedtreated inin thethe EBPEBP forfor 1010 min.min.

Molecules 2017, 22, 1908 5 of 15 Molecules 2017, 22, 1908 5 of 15 The exclusion chromatography of the EBP-treated chitin revealed formation of low-molecular weight chitooligosaccharides (COS) with weight-average molecular mass, Mw = 1000–2000 Da and polydispersityThe exclusion index chromatography 2.2. of the EBP-treated chitin revealed formation of low-molecular weightSince chitooligosaccharides the formation of COS (COS) with with Mw weight-average = 800–2000 Da molecularand polymerization mass, Mw degree= 1000–2000 varying Da from and polydispersitydimers to heptamers index 2.2.during the EBP-stimulated destruction of chitosan has been proved before [17], the sameSince mechanism the formation of the of EBP-stimulated COS with Mw =proce 800–2000ssing for Da all and polysaccharides polymerization can degree be supposed. varying from dimers to heptamers during the EBP-stimulated destruction of chitosan has been proved before [17], the2.2.2. same Main mechanism Features of of the the Polysaccharides EBP-stimulated EBP processing-Processing for all polysaccharides can be supposed.

2.2.2.Our Main present Features experiments of the Polysaccharides revealed the EBP-Processing dependence of the water-soluble LMWP yield on the treatment time and some other inherent features in chitin and cellulose EBP-processing were also found: Our present experiments revealed the dependence of the water-soluble LMWP yield on the treatment At optimized time and some treatment other inherent conditions features (plasma in chitin generating and cellulose media EBP-processing pressure, wereelectron also found:beam characteristics, the mixing device design)only 2 min were required to obtain 85% yield of • At optimized treatment conditions (plasma generating media pressure, electron beam low-molecular weight COS from original chitin powder [17]. The maximal yield of cellulose characteristics, the mixing device design)only 2 min were required to obtain 85% yield of water-soluble LMWP (77.4%) was obtained after 10 min (Table 1). low-molecular weight COS from original chitin powder [17]. The maximal yield of cellulose  The threshold relationship between LMWP yield and the duration of the EBP-treatment. At water-soluble LMWP (77.4%) was obtained after 10 min (Table1). optimized treatment conditions, chitin and cellulose destruction stopped when the processing • Thetime thresholdreached 7 and relationship 15 min, respectively. between LMWP yield and the duration of the EBP-treatment. At optimized treatment conditions, chitin and cellulose destruction stopped when the processing 2.2.3.time Chemical reached Structure 7 and 15 Characterization min, respectively. 2.2.3.A Chemical 30% decrease Structure of the Characterization orderly phase in treated cellulose samples with respect to that of the original substance was found, while the content of amorphous fraction was increased. The cellulose A 30% decrease of the orderly phase in treated cellulose samples with respect to that of the crystallinity index (CI) was determined by the XRD technique. The amorphicity was found to original substance was found, while the content of amorphous fraction was increased. The cellulose increase after plasma-chemical treatment: the CI of modified cellulose was 76.4%, whereas the CI of crystallinity index (CI) was determined by the XRD technique. The amorphicity was found to increase the untreated substance was 86.4%. The same trend was shown for chitin. Preliminary experiments after plasma-chemical treatment: the CI of modiﬁed cellulose was 76.4%, whereas the CI of the revealed the loss of CI in the following ranges: from 61.1% to 65.9% (original chitin) up to untreated substance was 86.4%. The same trend was shown for chitin. Preliminary experiments 50.9%–55.2% after the EBP-treatment in oxygen-containing media. revealed the loss of CI in the following ranges: from 61.1% to 65.9% (original chitin) up to 50.9–55.2% The low-temperature EBP is a source of several active oxygen species (O, O•, singlet oxygen, after the EBP-treatment in oxygen-containing media. OH•, etc.) (Figure 5) that are produced in plasma chemical processes in very high concentrations The low-temperature EBP is a source of several active oxygen species (O, O•, singlet oxygen, (up to 1010–1011 cm−3) [18]. These particles can be responsible for both the destruction of OH•, etc.) (Figure5) that are produced in plasma chemical processes in very high concentrations (up to polysaccharides and their oxidation. To reveal the oxidation changes in the polysaccharide 1010–1011 cm−3)[18]. These particles can be responsible for both the destruction of polysaccharides and theirmolecules oxidation. due Toto revealthe treatment the oxidation in the changes oxygen, in theFTIR polysaccharide-spectroscopy molecules was applied. due toThe the IR treatment-spectra −1 inanalysis the oxygen, of cellulose FTIR-spectroscopy showed intense was absorbance applied. The at 1720–1750 IR-spectra cm analysis (Figure of cellulose 6) that can showed be attributed intense absorbanceto the significant at 1720–1750 increase cm of− carbonyl1 (Figure 6C=O) that groups can be [20]. attributed to the signiﬁcant increase of carbonyl C=O groups [20].

Figure 5. The optical spectrum of oxygen EBP in visible and NIR ranges; the peaks attributed to the Figure 5.The optical spectrum of oxygen EBP in visible and NIR ranges; the peaks attributed to the different oxygen species are in accordance with [21,22]. different oxygen species are in accordance with [21,22].

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(a)

(b)

FigureFigure 6. The IR-spectroscopy of cellulose: ( (aa)) The The different different IR-spectra IR-spectra of of the the cellulose cellulose before before (1, (1, A 1)) andand after after the EBP-treatment (2, A 2)) in in oxygen oxygen media; media; ( (bb)) the the ratio ratio of of the the IR-spectra IR-spectra (A (A22/A/A1).1 ).

ChemicalChemical changes changes in in the structure of nitrogen-containingnitrogen-containing polysaccharides were studied using thethe EBP-treated EBP-treated chitosan chitosan films. ﬁlms. Figure Figure 6 6shows shows the the differences differences in inthe the chitosan chitosan IR-spectra IR-spectra within within the −1 wavethe wave number number range range 1500–2000 1500–2000 cm−1 cm. It is .important It is important that in that the in spectra the spectra of the of treated the treated chitosan chitosan (red −1 curves(red curves in Figure in Figure 7) the7) weak the weak band band at 1735 at 1735 cm−1 cm correspondingcorresponding to the to stretching the stretching vibrations vibrations of C=O of groupsC=O groups in carboxyl in carboxyl groups groups (–COOH) (–COOH) is observed is observed.. The The selective selective peak peak intensity intensity (the (the difference −1 betweenbetween the absorbance values at the maximum and at thethe baselinebaseline level)level) forfor thethe bandband atat 16501650 cmcm−1 (corresponding(corresponding to to C=O C=O stretching stretching vibrations vibrations of of Amide Amide I I groups) groups) in in the the spectrum for for the treated chitosanchitosan isis approximatelyapproximately 20% 20% higher higher than than that inthat the in absorbance the absorbance spectrum spectrum of the original of the substance. original −1 substance.Also there Also is a smallthere decreaseis a small in decrease the integral in the intensity integral ofintensity the bands of the at bands 1422, 1155,at 1422, and 1155, 896 cmand 896in −1 −1 cmthe−1 range in the 1250–500 range 1250–500 cm . cm Note−1. Note that thethat bandsthe bands at 1155 at 1155 and and 896 896 cm cm−correspond1 correspond to toβ β-1,4-glycosidic-1,4-glycosidic bondsbonds [23–25]. [23–25]. So So the the analysis analysis of of the the IR IR absorption absorption spectra spectra of of the the original original and and treated treated chitin showed thatthat the the EBP-treatment resulted in some increase of oxygen-containingoxygen-containing carbonyl C=O and and carboxyl –COOH groups andand partialpartial destructiondestruction ofof thethe ββ-1,4-glycosidic-1,4-glycosidic bonds.bonds. ToTo reveal the EBP effect changes on the chemical structure in chitin rings and to detect their possible oxidation,oxidation, thethe monomericmonomeric substrate substrateN -acetyl-N-acetyl-D-glucosamineD-glucosamine was was treated treated in oxygenin oxygen EBP EBP and andanalyzed analyzed by IR-spectroscopy. by IR-spectroscopy. The technique The techniqu did note detectdid not any detect oxidative any changesoxidative in changes the EBP-treated in the EBP-treatedN-acetyl-D-glucosamine. N-acetyl-D-glucosamine. TheThe reverse-phase chromatography chromatography (within (within the the se sensitivitynsitivity of of the technique) showed showed that that the retentionretention time time of of water-soluble COS COS produced produced in in th thee EBP-treatment of of chitin and and retention time of modelmodel mixture mixture of of the the individual individual N-acetylglucosamineN-acetylglucosamine oligomers oligomers was wasapproximately approximately the same the [17]. same More [17]. 1 sensitiveMore sensitive 1H-NMRH-NMR analysis analysis revealed revealed 25–40% 25–40% deacetylation deacetylation of the EBP-processed of the EBP-processed chitin depending chitin depending on the duration of the treatment (for τ = 5–10 min). The typical 1H-NMR spectrum of the EBP-treated chitin is shown in Figure 8 (τ = 15 min) and the example of the DD calculations is given below:

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Molecules 2017, 22, 1908 7 of 15 1 onMolecules the duration 2017, 22, 1908 of the treatment (for τ = 5–10 min). The typical H-NMR spectrum of the EBP-treated7 of 15 chitin is shown in Figure8( τ = 15DA/DD min) and= (10.2498/3)/(19.76/6) the example of the = DD 1.037, calculations is given below: DA/DD = DA(10.2498/3)/(19.76/6) = 50.9 mol %, = 1.037, (1) DA/DD = (10.2498/3)/(19.76/6) = 1.037, DDDA == 49.150.9 molmol %%, (1) DA = 50.9 mol %, (1) DD = 49.1 mol % (all comments and explanations are given DDin Section = 49.1 mol4). % (all comments and explanations are given in Section 4). (all comments and explanations are given in Section4).

Figure 7. Partial absorbance spectra of the chitosan film before (solid black curve) and after (dashed FigureredFigure curve) 7. 7.Partial Partialthe EBP-treatment absorbance absorbance spectra spectrain oxygen of of the themedia chitosan chitosan in the ﬁlm film range before before 1180–2000 (solid (solid black cmblack−1. curve) curve) and and after after (dashed (dashed −1 redred curve) curve) the the EBP-treatment EBP-treatment in in oxygen oxygen media media in in the the range range 1180–2000 1180–2000 cm cm−1..

1 FigureFigure 8.8. 1H-NMR spectrum of the chitin treated in the oxygen for 15 min. Figure 8. 1H-NMR spectrum of the chitin treated in the oxygen for 15 min. 3.3. Discussion 3. Discussion 3.1.3.1. Possible Mechanisms of the EBP-Action onon PolysaccharidesPolysaccharides 3.1. ThePossibleThe following Mechanisms EBP of factorsthe EBP-Action can affect on Polysaccharides the poly polysaccharidessaccharides structure duringduring their plasma chemical processing: chemicalThe processing:following EBP factors can affect the polysaccharides structure during their plasma chemical Chemically processing: active heavy particles of the EBP: excited molecules and atoms, ions, radicals  (activeChemically oxygen active species, heavy for example);particles of the EBP: excited molecules and atoms, ions, radicals  The(active fast oxygenelectrons species, of the forpartially example); degraded EB that bombard the polysaccharide material;  The fast electrons of the partially degraded EB that bombard the polysaccharide material;

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Molecules• Chemically 2017, 22, 1908 active heavy particles of the EBP: excited molecules and atoms, ions, radicals (active8 of 15 oxygen species, for example); The secondary electrons of moderate energies (up to 0.01–1 keV) produced in the EBP due to • The fast electrons of the partially degraded EB that bombard the polysaccharide material; ionization of the molecules of the plasma generating media; • The secondary electrons of moderate energies (up to 0.01–1 keV) produced in the EBP due to The EBP-radiation, especially UV one and X-ray (bremsstrahlung);  ionization of the molecules of the plasma generating media;  Possible heating by direct electron bombardment and due to heat transfer between the plasma • The EBP-radiation, especially UV one and X-ray (bremsstrahlung); cloud and the sample. • Possible heating by direct electron bombardment and due to heat transfer between the plasma Eachcloud of and these the factors sample. is able to cause transformations of polymeric molecules, but the integral effect of the polysaccharides macromolecule modification is likely to be due to their joint action, i.e., Each of these factors is able to cause transformations of polymeric molecules, but the integral synergism is expected to take place. effect of the polysaccharides macromolecule modiﬁcation is likely to be due to their joint action, The possible reaction of the fast electrons with polysaccharide chains results in the destruction i.e., synergism is expected to take place. of β-1,4-glycosidic bonds and dehydration with levoglucosan formation (Figure 9). The same The possible reaction of the fast electrons with polysaccharide chains results in the destruction of conversion occurs during the pyrolysis of cellulose and other carbohydrates in a vacuum [26]. The β-1,4-glycosidic bonds and dehydration with levoglucosan formation (Figure9). The same conversion increase of the radiation dose can stimulate further dehydration and production of low molecular occurs during the pyrolysis of cellulose and other carbohydrates in a vacuum [26]. The increase weight organic compounds such as furfural and 5-hydroxymethylfurfural, furfuryl alcohol, of the radiation dose can stimulate further dehydration and production of low molecular weight enolactones, and various aromatic structures. These substances are markers of deep polysaccharide organic compounds such as furfural and 5-hydroxymethylfurfural, furfuryl alcohol, enolactones, damage and their carbonization should be avoided. Under our experimental conditions these and various aromatic structures. These substances are markers of deep polysaccharide damage and organic compounds were not detected in the EBP-treated samples. Thus the radiation and thermal their carbonization should be avoided. Under our experimental conditions these organic compounds polysaccharides damage due to high energy electrons has been minimized by proper selection of were not detected in the EBP-treated samples. Thus the radiation and thermal polysaccharides damage plasma generating gas pressure and the EB scanning mode [27,28]. due to high energy electrons has been minimized by proper selection of plasma generating gas pressure In our previous studies it was proven that plasma chemical processes predominate [27,28]. and the EB scanning mode [27,28]. Chemically active oxygen particles break the β-1,4-glycosidic bound and decrease the In our previous studies it was proven that plasma chemical processes predominate [27,28]. polysaccharides’ molecular weight. These particles can abstract hydrogen atoms at all ring C–H Chemically active oxygen particles break the β-1,4-glycosidic bound and decrease the polysaccharides’ bonds in carbohydrates except C-2 of N-acetyl hexosamine [29–31]. Figure 10 illustrates a possible molecular weight. These particles can abstract hydrogen atoms at all ring C–H bonds in carbohydrates degradation mechanism of chitin during its treatment in the EBP of water vapor [32]. The reaction except C-2 of N-acetyl hexosamine [29–31]. Figure 10 illustrates a possible degradation mechanism involves the abstraction of a hydrogen atom with the generation of carbon-center radicals. Then the of chitin during its treatment in the EBP of water vapor [32]. The reaction involves the abstraction radicals at carbons that form glycosidic bonds undergo a β-scission reaction, resulting in the of a hydrogen atom with the generation of carbon-center radicals. Then the radicals at carbons that breakdown of polysaccharide chains [29,30,33]. The oxidation changes in the polysaccharides form glycosidic bonds undergo a β-scission reaction, resulting in the breakdown of polysaccharide molecules with increase of carbonyl C=O and carboxyl –COOH groups after the treatment in the chains [29,30,33]. The oxidation changes in the polysaccharides molecules with increase of carbonyl EBP of oxygen also support this hypothesis. C=O and carboxyl –COOH groups after the treatment in the EBP of oxygen also support this hypothesis.

FigureFigure 9. 9. TheThe scheme scheme of of cellulose cellulose degradation degradation under under action action of of high-energy high-energy electrons electrons in in the the EBP. EBP.

Molecules 2017, 22, 1908 9 of 15 Molecules 2017, 22, 1908 9 of 15 Molecules 2017, 22, 1908 9 of 15

Figure 10. The scheme of chitin degradation under hydroxyl radical action in the EBP: the attack of FigureOHFigure radicals 10. 10.The The on schemethe scheme chitin of of –NH chitin chitin2 groups degradation degradation [32]. under under hydroxyl hydroxyl radical radical action action in in the the EBP: EBP: the the attack attack of of

OHOH radicals radicals on on the the chitin chitin –NH –NH2 2groups groups [ 32[32].]. The second possible site of the OH radical action is the chitin N-acetyl group. This conversion was ThefoundThe second second for both possible possible amino site siteacid of of derivatives the the OH OH radical radical like actionN action-acetyl is is themethionine the chitin chitinN N -acetyl[34]-acetyl and group. group. glycosaaminoglycans This This conversion conversion was(e.g.,was found hyaluronan)found for for both both [35,36] amino amino under acid acid derivatives oxygen-containingderivatives like likeN N-acetyl-acetyl radicals methionine methionine and some [ 34[34] other] and and active glycosaaminoglycans glycosaaminoglycans oxygen species. (e.g.,The(e.g., mechanism hyaluronan) hyaluronan) presented [ 35[35,36],36] under underin Figure oxygen-containing oxygen-containing 11 assumes the radicals radicals C-4 carbon-centered and and some some other other radicals active active oxygen oxygenformation species.species. and ThesubsequentThe mechanism mechanism scission presented presented reaction in ofin Figure theseFigure radicals11 11 assumes assumes leading the the to C-4 C-4the carbon-centered cleavagecarbon-centered of β-1,4-glycosidic radicals radicals formation formation bonds. and and subsequentsubsequent scission scission reaction reaction of of these these radicals radicals leading leading to to the the cleavage cleavage of ofβ -1,4-glycosidicβ-1,4-glycosidic bonds. bonds.

Figure 11. The scheme of chitin degradation under hydroxyl radical action in the EBP: the attack of

OH radicals on the chitin –NH2 groups. Figure 11. The scheme of chitin degradation under hydroxyl radical action in the EBP: the attack of OHFigure radicals 11. The on the scheme chitin of –NH chitin2 groups. degradation under hydroxyl radical action in the EBP: the attack of OH radicals on the chitin –NH2 groups.

Molecules 2017, 22, 1908 10 of 15

Simultaneously with the destruction of β-1,4-glycosidic bonds and the stimulation of oxidation reactions heavy plasma radicals and ions etch the polysaccharides crystals resulting in their CI loss and the amorphicity increase. The mechanisms responsible for the plasma-stimulated etching of organic polymers are well-known and described in details in [37–39]. These mechanisms, which are shown to be common for various polymers, explain the same CI decrease values and trends for both chitin and cellulose.

3.2. The Comparison of the EBP-Stimulated Polysaccharides Destruction with Respect to Acid-Catalyzed Hydrolysis The comparison of the EBP-stimulated polysaccharides destruction with respect to conversional acid-catalyzed hydrolysis is given in Table2. In contrast to conventionally used hydrolysis techniques the EBP-stimulated processing is rapid single-stage, and environmental friendly procedure. Often the stable N-acetyl group presented in chitin structure is required for further (bio)chemical modiﬁcations of chitooligosaccharides. The chemical hydrolysis of chitin resulted in the formation of signiﬁcantly deacetylated LMWP whereas the intensive deacetylation of chitin oligomers does not occur during the EBP-treatment.

Table 2. The comparison of the EBP-stimulated polysaccharides destruction with respect to acid-catalyzed chemical hydrolysis.

Criteria Chemical Hydrolysis EBP-Processing Treatment time Several hours or days Minutes (τ = 2–10) Number of stages Multi-stage Single-stage Destruction of β-1,4-glycosidic bonds; Destruction of β-1,4-glycosidic bounds; Destruction of amorphous parts of Destruction of both amorphous and biopolymer predominates; Speciﬁcity and Efﬁciency crystalline parts of biopolymer occurs; Both oxidation changes of produced Oxidation changes of oligosaccharides oligosaccharides and deep oxidation of (formation of C=O and –COOH groups). original biopolymers are possible. Deacetylated low molecular weight The DD of produced chitin Chitin deacetylation derivatives of chitin are produced. oligosaccharides 25–40%. Reacetylation is needed Cellulose fragments with polymerization LMWP from dimers to heptamers Molecular mass of products degree 200 or monomers are produced [40] are produced. Low yields of LMWP and large amounts of LMWP yields Up to 80–85% monomeric units [40,41]. Highly concentrated acidic or alkaline Environmentally friendly: hazardous Ecological safety solutions are needed. Toxic wastes are byproducts and toxic wastes are produced. High energy consumption. not generated.

The advantages of EBP-treatment with respect to gas discharge plasmas have been discussed in our previous papers [17–19]. Furthermore, the effective controllable destruction of polysaccharides occurs due to the EBP-hydrolysis, whereas the treatment in gas discharge plasmas mostly results in biomaterial oxidation and an improvement of its hydrophilic properties [42–44].

4. Materials and Methods

High molecular weight crab shell chitin (viscosity-average molecular weight, Mν = 1000 kDa) obtained from red king crab Paralithodes camtschaticus, cellulose sulfate (molecular weight 160 kDa), and microcrystalline cellulose with molecular weight of 55 kDa were used as the original substances for the further EBP-treatment. The chitin was of high purity and free of any other biopolymers/inorganic content and pigments (content of proteins and other impurities <0.5%). The substance was obtained from Bioprogress Co., (Shchelkovo, Russia) and certiﬁed by the manufacturer. All substances were water-insoluble white powders. Molecules 2017, 22, 1908 11 of 15

For solubility measurements, 100 ± 0.1 mg of the preliminary dried sample (ms) were placed into a tube and 1.5 mL of distilled water or 5% NaOH (Sigma-Aldrich, Munich, Germany) was added to the sample. The resulting mixture was incubated for 24 h at room temperature under periodic mixing. After the incubation the mixture was centrifuged for 5 min and 1 mL of centrifugate was taken and dried. The mass of the dry residue (mdr) was measured with an accuracy ±0.1 mg. The sample solubility was calculated as the (mdr/ms) × 100% ratio. Molecular masses (weight-average Mw, number-average Mn, and z-average mass Mz) of the chitin EBPtreatment products were determined by the size exclusion chromatography on a LC-20 Prominence HPLC system (Shimadzu, Tokyo, Japan) equipped with refractometric detector RID-10A. The chromatographic column was Ultrahydrogel 500 7.8 × 300 mm (Waters, Milford, MA, USA). Other analysis conditions were as follows:the mobile phase—buffer containing 0.05 M acetic acid and 0.15 M ammonium acetate (pH = 5.1); the flow rate—0.5 mL/min; temperature—30 ◦C; volume of the injected sample—40 µL; duration of the analysis—25 min. Dextrans (Sigma, St. Louis, MO, USA) were used as standard samples for mass scale calibration. Individual N-acetylglucosamine oligomers were determined by the reverse phase chromatography using the Asahipak NH2P-LF (Shodex, Tokyo, Japan) chromatographic column. The column was calibrated with a model mixture of N-acetylglucosamine oligomers with the polymerization degree n = 1–6. The size exclusion chromatography of cellulose materials was performed using the chromatograph Staier (Akvilon, Moscow, Russia) and the chromatographic column Phenomenex BioSep-Sec-S-3000 (Phenomenex, Torrance, CA, USA) with the efficiency of 30,000 theoretical plates. The analysis conditions were as follows:the elutriating agent—0.1 M phosphate buffer (pH 6.86) containing 0.05% ◦ NaN3; the elution rate—1 mL/min; temperature—30 C; UV-detector with the wavelength 280 nm. The correlation between elution time (t) and molecular mass of cellulose oligosaccharides (M) is described by Equation (2): ln(M) = −0.193t2 + 1.91t + 9, (2) where (t = 6–11 min); the validity coefficient of approximation R2 = 0.99. After t = 11 min the column operates by the reversed-phase mechanism. The ligand-exchange high-performance chromatography of cellulose products was performed at 30 ◦C under the following conditions: ion-exclusion column Rezex RSO-Oligosaccharide Ag+ (4%) (Phenomenex, Torrance, CA, USA) with the efficiency of 12,000 theoretical plates; the elutriating agent—H2O; the elution rate—600 mcl/min. Differential refractometric detection (Water, Milford, CT, USA) was applied. Solutions from the original and the EBP-treated chitosan (Mν = 500 kDa with the degree of deacetylation 85–98% and polydispersity index 1.5) were prepared by dissolving them in 1% acetic acid and the distilled water respectively. The solutions were cast and dried at 37 ◦C to form thin films 8.0 ± 1.5 µm thick. The obtained films were then analyzed by Fourier transform infrared (FT-IR) spectroscopy. In particular, the film thickness was estimated by analyzing a baseline modulation period in a transmittance spectrum of the chitosan film. IR spectral measurements in a wavenumber (ν) range 500–5000 cm−1 with a resolution of 4 cm−1 were carried out by a FT-IR spectrometer Perkin-Elmer Spectrum 100 (Perkin-Elmer, Boston, MA, USA). A transmittance spectrum of the studied chitosan or cellulose film, T(ν), was measured at normal incidence of IR radiation on the film surface as a ratio:

T(ν) = T1(ν)/T0(ν), (3) where T0(ν) and T1(ν) are the registered transmission spectra of the spectrometer channel for an empty sample holder and for a sample holder with the ﬁlm, respectively. To get acceptable signal-to-noise Molecules 2017, 22, 1908 12 of 15 ratio 16 scans were statistically averaged. The transmittance spectrum was converted to obtain the absorbance spectrum of the ﬁlm, A(ν), through Equation (4):

A(ν) = −log10(T1(ν)/T0(ν)). (4)

MoleculesThe 2017 cellulose, 22, 1908 crystallinity index was determined by means of X-ray diffraction analysis (XRD)12 of 15 Molecules 2017, 22, 1908 12 of 15 using the method developed by Segal and coworkers [45]. standard 5-mm probe from 150 mg of sample dissolved in 1.0 mL D2O after 10,000 scans. The 1H- and 13C-NMR spectra of the EBP-produced cellulose and chitin oligosaccharides were 2 13standardC-NMR 5-mmspectra probe of the from EBP-treated 150 mg of cellulosesample dissolvedwere obtained in 1.0 mLunder D Ototal after proton 10,000 ◦ decoupledscans. The recorded13 on a Bruker Avance 500 MHz instrument (Bruker, Billerica, MA, USA) at 25 C using a C-NMR spectra of the EBP-treated→ cellulose were obtained under total proton decoupled standardconditions 5-mm and probe the model from 150 β(1 mg4)-tetramer of sample dissolved (Figure in12) 1.0 was mL DusedO after as a 10,000 reference scans. substance. The 13C-NMR The conditions and the model β(1→4)-tetramer (Figure 12) was used2 as a reference substance. The spectraaverage of degree the EBP-treated of the EBP-treated cellulose were chitin obtained acetylation under (DA, total mol proton %) decoupledand deacetylation conditions (DD, and mol the average degree of the EBP-treated chitin acetylation (DA, mol %) and deacetylation (DD, mol model%)wereβ determined(1→4)-tetramer according (Figure to 12 Equation) was used (5) [46]: as a reference substance. The average degree of the %)were determined according to Equation (5) [46]: EBP-treated chitin acetylation (DA, mol %) and deacetylation (DD, mol %)were determined according DD (mol %) = {1 − [1/3ICH3/(1/6IH2–H6)]}100, (5) to Equation (5) [46]: DD (mol %) = {1 − [1/3ICH3/(1/6IH2–H6)]}100, (5) where ICH3—the integral intensity of CH3 residue and IH2–H6—the sum of the integral intensities of DD (mol %) = {1 − [1/3ICH3 /(1/6IH2-H6 )]}100, (5) where ICH3—the integral intensity of CH3 residue and IH2–H6—the sum of the integral intensities of IH2, IH3, IH4, IH5, IH6 and IH6’ protons (Figure 13). where ICH3 —the integral intensity of CH3 residue and IH2–H6 —the sum of the integral intensities of IH2, IH3, IH4, IH5, IH6 and IH6’ protons (Figure 13). IH2 ,IH3 ,IH4 ,IH5 ,IH6 and IH6 ’ protons (Figure 13).

FigureFigure 12.12. TheThe structurestructure ofof thethe modelmodelβ β(1(1→→4)-tetramer.4)-tetramer. Figure 12. The structure of the model β(1→4)-tetramer.

Figure 13. The scheme of the protons in the chitin monomer. Figure 13. The scheme of the protons in the chitin monomer. Figure 13. The scheme of the protons in the chitin monomer. 5.5. ConclusionsConclusions 5. Conclusions 1.1. The possibility of the EBP-stimulatedEBP-stimulated degradationdegradation of chitin andand cellulosecellulose andand formationformation ofof thethe 1. water-solubleThe possibility oligosaccharidesoligosaccharides of the EBP-stimulated waswas provedproved degradatio experimentally.experimentally.n of chitin and cellulose and formation of the 2.2. Chitooligosaccharideswater-soluble oligosaccharides with weight-average was proved experimentally. molecularmolecular mass 1000–20001000–2000 DaDa andand polydispersionpolydispersion 2. indexChitooligosaccharides 2.2 and cellulosecellulose with oligosaccharidesoligosaccharides weight-average withwith mo polymerizationpolecularlymerization mass 1000–2000 degreesdegrees 3–103–10 Da werewere and formedpolydispersionformed duedue toto theindex destructiondestruction 2.2 and cellulose of ofβ-1,4-glycosidic β-1,4-glycosidic oligosaccharides bonds bonds duringwith during po thelymerization EBP-treatment. the EBP-treatment. degrees The 3–10 EBP-processing The were EBP-processing formed resulted due to inresultedthe 25–40% destruction in deacetylation 25–40% of β -1,4-glycosidic ofdeacetylation original chitin bondsof and original partialduring oxidation chitinthe EBP-treatment. and of the partial produced Theoxidation oligosaccharides. EBP-processing of the 3. Atproducedresulted optimized oligosaccharides.in 25–40% treatment deacetylation conditions (plasmaof original generating chitin mediaand partial pressure, oxidation electron of beam the 3. characteristics,Atproduced optimized oligosaccharides. designtreatment of the conditions mixing device) (plasma 85% yieldgenerating of low-molecular media pressure, weight COSelectron and 77.4%beam 3. yieldcharacteristics,At optimized of cellulose designtreatment oligosaccharides of the conditions mixing were device) obtained(plasma 85% yield ingenerating only of 2low-molecular and media 10 min, pressure, respectively. weight COSelectron and 77.4%beam 4. Bothyieldcharacteristics, theof cellulose fast electrons design oligosaccharides andof the the mixing active were device) oxygen obtained 85% particles yieldin only producedof 2 low-molecular and 10 in min, the respectively. EBP weight are responsibleCOS and 77.4% for 4. theBothyield destruction the of cellulosefast electrons of oligosaccharidesβ-1,4-glycosidic and the active bondswere oxygen obtained in the pa originalrticles in only produced polysaccharides. 2 and 10 in min, the respectively.EBP By properare responsible selection for of 4. treatmenttheBoth destruction the fast conditions, electrons of β-1,4-glycosidic the and polysaccharide the active bonds oxygen damage in the pa originalrticles due to produced high-energypolysaccharides. in electronsthe EBP By properare can responsible be selection minimized forof andtreatmentthe destruction the plasma conditions, chemicalof β-1,4-glycosidic the processes polysaccharide bonds predominate. in thedama originalge due polysaccharides. to high-energy By properelectrons selection can be of minimizedtreatment andconditions, the plasma the chemical polysaccharide processes dama predominate.ge due to high-energy electrons can be Acknowledgments:minimized andThe the plasma plasma chemical chemical processes experiments predominate. and the chromatographic analysis of chitin oligosaccharidesAcknowledgments: were The supported plasma by RFBRchemical grant experiments 15-08-05724_a and and RSFthe grantchromatographic 16-14-00046, respectively.analysis of chitin oligosaccharidesAcknowledgments: were Thesupported plasma by RFBRchemical grant experiments 15-08-05724_a and and RSFthe grantchromatographic 16-14-00046, respectively.analysis of chitin oligosaccharides were supported by RFBR grant 15-08-05724_a and RSF grant 16-14-00046, respectively. Author Contributions: Tatiana Vasilieva, Dmitry Chuhchin, Sergey Lopatin, Valery Varlamov, and Michael VasilievAuthor Contributions:conceived and designedTatiana Vasilieva, the experiments; Dmitry Chuhchin,Tatiana Vasilieva, Sergey Lopatin,Dmitry Chuhchin,Valery Varlamov, Sergey Lopatin,and Michael and MichaelVasiliev Vasilievconceived performed and designed the experiments;the experiments; Tatiana Tatian Vaasilieva, Vasilieva, Dmitry Dmitry Chuhchin, Chuhchin, Sergey Sergey Lopatin, Lopatin, Valery and Varlamov,Michael Vasiliev Andrey performed Sigarev, and the Michael experiments; Vasiliev Tatiana analyzed Va thesilieva, data; Dmitry Andrey Chuhchin, Sigarev contributed Sergey Lopatin, analysis Valery tools; TatianaVarlamov, Vasilieva, Andrey Dmitry Sigarev, Chuhchin, and Michael and VasilievSergey Lopatin analyzed wrote the data;the paper. Andrey Sigarev contributed analysis tools; Tatiana Vasilieva, Dmitry Chuhchin, and Sergey Lopatin wrote the paper.

Molecules 2017, 22, 1908 13 of 15

Author Contributions: Tatiana Vasilieva, Dmitry Chuhchin, Sergey Lopatin, Valery Varlamov, and Michael Vasiliev conceived and designed the experiments; Tatiana Vasilieva, Dmitry Chuhchin, Sergey Lopatin, and Michael Vasiliev performed the experiments; Tatiana Vasilieva, Dmitry Chuhchin, Sergey Lopatin, Valery Varlamov, Andrey Sigarev, and Michael Vasiliev analyzed the data; Andrey Sigarev contributed analysis tools; Tatiana Vasilieva, Dmitry Chuhchin, and Sergey Lopatin wrote the paper. Conﬂicts of Interest: The authors declare no conﬂict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

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Sample Availability: Samples of the compounds are available from the authors.

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