Peiyi Li,Ruiyan Liu,Haozhe Lei,Miaomiao Zhou,Boxing Jian,Xinping Li,2,3

1.College of Bioresources Chemical and Materials Engineering,National Demonstration Center for Experimental Light Chemistry Engineering Education,Shaanxi University of Science and Technology,Xi'an,Shaanxi Province,710021,China

2.Key Laboratory of Paper Based Functional Materials of China National Light Industry,Xi'an,Shaanxi Province,710021,China

3.Shaanxi Provincial Key Laboratory of Papermaking Technology and Specialty Paper Development,Xi'an,Shaanxi Province,710021,China

Abstract:Cellulose nanofiber(CNF)was isolated from Okara using deep eutectic solvent(DES)with high-speed stirring.The composite hydrogels obtained by using different proportions of CNF and sodium alginate(SA)had different properties.The CNF/SA composite hydrogels were analyzed using Fourier transform infrared spectroscopy and scanning electron microscopy and tested for compression properties,rheological properties,water content,and swell ing degree.Physical cr osslinking bet ween SA and Ca2+,and different degrees of hydrogen bond formation between SA and CNF were observed.The CNF/SA composite hydrogel have great potential as reinforcements in eco-friendly composite hydrogels for diverse applications.

Keywords:composite hydrogel;cellulose nanofiber;sodium alginate;Okara

1 lntroduction

Nanocellulose has been widely used as reinforcement material in tissue engineering and some biodegradable films[1-4]because of their excellent mechanical properties.Owing to its non-toxic nature and excellent biocompatibility,nanocellulose has extensive prospects for application in biomedical polymer materials,such as cell culture scaffolds,antimicrobial agents,and materials for drug delivery[5-6]. In addition,nanocellulose is biodegradable and can be made into films to replace plastics.Nanocellulose is often combined with other polymers(such as polylactic acid and polyhydroxy fatty acid ester) to create a biodegradable composite membrane[7-8].Because of its nanoscale size,nanocellulose has a high surface area to volume ratio,low thermal expansion coefficient,high Young's modulus,and high tensile strength.After a series of chemical modifications,it can be used widely in electrochemistry,electronic devices,and other fields[9-10].

Nanocellulose can be prepared by different preparation methods resulting in somewhat different physical and chemical properties,and can be classified into three categories:cellulose nanocrystals(CNCs),cellulose nanofibers(CNFs),and bacterial nanocelluloses(BNCs).BNCs are mainly synthesized by acetic acid bacteria and other bacteria[11].Currently,there are many methods for preparing CNFs.The production of CNFs by mechanical methods uses no chemicals and is environmentally friendly,but consumes a lot of energy,which limits its applications[12-13].Therefore,chemical pretreatment is often adopted to reduce the energy consumption[14].Several existing chemical reagents,such as acidified sodium chlorite, sodium carboxymethyl sulfate, hydrogen peroxide, and TEMPO[15],have been used to pretreat lignocellulosic biomass in order to reduce the energy consumption.Furthermore,the properties of CNFs prepared by the chemical-mechanical method are generally better than those prepared by the pure mechanical method[16].In a previous study,our group successfully separated microfibrillated cellulose(MFC)and CNF from Okara using a chemical-mechanical method and studied their film-forming properties[17-20].Although cellulose has been successfully extracted using these pretreatments,there are still some practical problems,such as a long processing time and production of chemical waste materials.Among new pretreatment schemes,organic solvent pulping and ionic liquid pretreatment are considered to be effective methods for separating lignocellulosic components according to the concept of green chemistry.However,it is still expensive for the industrial production of CNFs.Therefore,costeffective and simple methods are still needed[21].

Compared with ionic liquids,deep eutectic solvents(DESs)are easy to prepare,biodegradable,and environmentally friendly[22].In practical applications,DESs have unusual properties such as excellent solubility,negligible vapor pressure,high thermal stability,and flammability[23],which enable their use for chemical reactions under various environmental conditions and a variety of industrial applications.CNFs prepared by DES were introduced into hydrogels.Compared with traditional hydrogels,the CNF-based hydrogels exhibited better water absorption,better salt resistance,and higher degradation rate.In our previous study,we demonstrated the pretreatment of Okara with a choline chloride-oxalic acid solvent,which was also used in this study[24].

The key to the preparation of Nanocellulose-based hydrogels lies in effective dispersion of nanocellulose in the system and promotion of interaction between the nanocellulose and polymer.The main methods are the blending method and the surface graft copolymerization method of nanocellulose.Ruiz-Palomero et al[25]mixed graphene quantum dots(GQDs)with CNC to prepare GQD/CNC hydrogels.It not only avoids the aggregation of GQDs,but also enhances their luminescent properties,leading to an excellent sensing ability for trichlorophenol(TCP).Yang et al[26]mixed CNFs into polyethylene glycolmodified hydrogels through a blending method and found that CNFs improved the connectivity and the swelling properties of hydrogels.Park et al[27]used a TEMPO/NaBr/NaClO system to obtain TEMPOoxidized BNC(TOBNC)under alkaline conditions at room temperature and then cross-linked them with sodium alginate(SA)in the presence of Ca2+to form nanocellulosic hydrogels.Compared with the SA absorbent material,the compressive strength and chemical stability of the TOBNC/SA composite hydrogels increased,indicating that TOBNC played an important role in enhancing the structure and mechanical and chemical stability of the TOBNC/SA composite hydrogels.

CNF in aqueous solution can form hydrogels with a stable three-dimensional network structure via physical entanglement and hydrogen bonding.In this study,CNFs were prepared from Okara by DESs pretreatment and high-speed shearing.Composite hydrogels were prepared via physical crosslinking with SA,which has excellent biocompatibility and bioadhesion effects.The mechanical and swelling properties of the CNF/SA composite hydrogels were studied,and the structure of the CNF/SAcomposite hydrogels was characterized.

2 Experimental

2.1 Reagents and raw materials

Raw material:Okara was obtained from a soybean milk factory in Heilongjiang Province,with 10% water content.

Reagents:Choline chloride was purchased from Adamasbeta(Shanghai)Chemical Reagent Co.,Ltd.Oxalic acid was purchased from Tianjin Damao Chemical Reagent Factory.SA was purchased from Tianjin Fuchen Chemical Reagent Factory,and calcium chloride was purchased from Tianjin Tianli Chemical Reagent Co.,Ltd.All of the reagents were analytically pure.

2.2 Methods

2.2.1Synthesis of DES

Choline chloride and oxalic acid were added to a roundbottom flask at a molar ratio of 1∶1.The optimum reaction temperature was 80℃,and the stirring speed was 40 r/min.After stirring with a magnetic stirrer at constant temperature for a certain time,it became a clear,transparent solution.

2.2.2Pretreatment of Okara by DES

After synthesis of DES,the temperature in the oil bath was raised to 100℃.According to the mass ratio of Okara and DES of 1∶20,a certain amount of Okara was added.The reaction time was set to 30 min,and the stirring speed was set to 40 r/min to allow it to fully react.At the end of the reaction,cellulose was precipitated using distilled water as a reverse solvent.The samples were washed with distilled water until the filtrate was neutral,and the solids were collected to obtain DES-pretreated Okara cellulose.

2.2.3Preparation of Okara CNF

The DES-pretreated Okara cellulose was adjusted to 3%mass concentration and poured into a high-speed mixer.Okara CNF was obtained at a rotating speed of 6000 r/min for a duration of 3 min.Schematic preparations for DES-pretreated Okara cellulose and CNF are shown in Fig.1.

Fig.1 Schematic preparations for DES-pretreated Okara cellulose and CNF

2.2.4Preparation of CNF/SA composite hydrogel

Suspensions of CNF and SA with mass ratio ranging from 1∶1 to 1∶6 were prepared,and mixed thoroughly using a magnetic stirrer for 20 min.Calcium chloride was dissolved in deionized water and slowly added to the mixture.After standing for 12 h,a CNF/SA composite hydrogel was formed(Fig.2).The properties of the composite hydrogels were tested,and the effects of CNF and SA on the properties of CNF/SA composite hydrogels are discussed below.

Fig.2 Schematic preparation of CNF/SAcomposite hydrogel

3 Results and discussion

3.1 Effect of DES pretreatment and properties of CNF hydrogel

The influence of DES pretreatment on Okara composition is presented in Table 1.The holocellulose content in Okara was 70.64%,and increased to 95.81%after DES pretreatment.Theα-cellulose content in Okara was 62.91%and increased to 92.60%after DES pretreatment.The DES pretreatment removed a significant amount of the protein,fat,and other impurities from Okara.

Table 1 Effects of DES on Okara composition before and after pretreatment

The prepared CNF suspension can form a hydrogel under static conditions,but the gelation time is long and the gelation strength is low.Fig.3 shows the progress in formation of CNF hydrogel.Fig.3(a)shows a newly prepared CNF suspension,Fig.3(b)shows the result after standing for 24 h,and Fig.3(c)shows the result after standing for 72 h.It can be seen that the self-made CNF formed a gel after standing for several hours,but the strength was poor.

Fig.3 Formation process of CNF hydrogel

3.2 Properties of CNF/SAcomposite hydrogel

3.2.1Appearance morphology

By adding SA in different dosages,CNF/SA composite hydrogels were prepared,as shown in Fig.4.It can be

Fig.4 CNF/SAcomposite hydrogels with different SA dosages(CNF∶SA=1∶1(a),1∶2(b),1∶3(c),1∶4(d),1∶5(e),and 1∶6(f),respectively)

seen that the color of the six CNF/SA composite hydrogels was uniformly yellowish brown,which was caused by the DES pretreatment of Okara.According to our experience,the longer the pretreatment time of DES,the darker the color of CNF.When the mass ratio of CNF∶SA was 1∶1,the strength of CNF/SA composite hydrogel was low,and its gel structure was easily to be destroyed.When the mass ratio of CNF∶SA was changed from 1∶2 to 1∶6,the increase of SA dosage significantly improved the strength of CNF/SA composite hydrogel.Therefore,the performance of composite hydrogels with CNF and SA mass ratios ranging from 1∶2 to 1∶6 was tested in the following experiments.

3.2.2Compression performance

The compression strength of CNF/SA composite hydrogel was determined based on the stress-strain curve.The effect of SA dosage on the strength of CNF/SAcomposite hydrogel is shown in Fig.5.It was found that the addition of SA significantly improved the strength of CNF/SA composite hydrogels.When the mass ratios of CNF∶SA were 1∶2 and 1∶3,the strain of CNF/SA composite hydrogels was approximately 40%.When the mass ratio of CNF∶SA was 1∶3,the stress of CNF/SA composite hydrogel increased by 4 kPa compared to that with a mass ratio of 1∶2.With an increase in the dosage of SA,the strain and stress of CNF/SA composite hydrogel increased.This is because SA has a higher degree of chemical crosslinking with Ca2+.When the mass ratio of CNF∶SA was 1∶5,a CNF/SA composite hydrogel with the highest strain and strain was obtained,and the strain reached 57%and the stress reached 0.056 MPa.The improvement in compression strength can be attributed to an increase in internal forces in the hydrogel,and the physical activity crosslinking by the nanofillers prevents the hydrogel from breaking.The formation of hydrogen bonds increases the internal strength of CNF/SA composite hydrogel,and increases its pressure-bearing capacity.An excessive addition of SA led to a decrease in the strength of CNF/SA composite hydrogel.When the mass ratio of CNF∶SA was 1∶6,the strain of CNF/SA composite hydrogel was 50%.

Fig.5 Effect of SAdosage on the strength of the CNF/SA composite hydrogels

3.2.3Rheological properties

The mechanical properties of CNF/SA composite hydrogels can also be expressed in terms of their viscoelasticity.The storage modulus(G')and loss modulus (G") reflect the viscoelasticity of the hydrogel.TheG'andG"values of CNF/SA composite hydrogels at 25℃are shown in Fig.6.All samples showG'values higher thanG"values over the entire angular frequency range.A higherG'value indicates that the hydrogel has more elastic behavior at the test temperature,which results in the formation of a more stable elastic network structure.BothG'andG"values are angular frequency-dependent, which is a characteristic of the viscoelastic behavior of solids.

Fig.6 Effect of SAdosage on the rheological properties of CNF/SAcomposite hydrogels

The rheological properties of CNF/SA composite hydrogels gradually increased with an increase in the dosage of SA.When the mass ratio of CNF∶SA was 1∶2,theG'value of the hydrogel was 4.83 kPa at a angular frequency of 6.3 rad/s.When the mass ratio of CNF∶SA was 1∶6,theG'value of the hydrogel was 14.64 kPa at the same angular frequency,which was 3.03 times higher.The increase in theG'value is assumed to be caused by the formation of hydrogen bonding due to cross-linking between SA and CNF.The formation of hydrogen bonds provides a tighter network structure inside the hydrogel,resulting in higher mechanical strength.According to the study of rheological properties,it was found that the optimal rheological properties of CNF/SA composite hydrogel were obtained when the mass ratio of CNF∶SA was 1∶6.

3.2.4Swelling properties

The swelling degree and water content are very important characteristics for hydrogels,especially in the field of biomedical applications.The water content of CNF/SA composite hydrogel with different dosages of SA is shown in Fig.7(a).Without the addition of SA,the water content of CNF hydrogel was only 85.4%.The addition of SA significantly increased the water content of CNF/SA composite hydrogel,but a change in the dosage of SA had no significant effect on the water content of the CNF/SA composite hydrogel,with values ranging from 96.1% to 97.7%.The physical crosslinking resulting from the addition of CNF can increase the density of the polymer network and increase the solids content of the hydrogel,which reduces the water content of the hydrogel.Therefore,the pure SA hydrogels have the highest water content.However,the mixture of CNF and SA forms hydrogen bonds,thus increasing the water content of CNF/SA composite hydrogel compared with CNF hydrogel.

To study the swelling degree of CNF/SA composite hydrogels, all the freeze-dried hydrogels were immersed in deionized water for 48 h to achieve swelling equilibrium.Freeze-drying changed the threedimensional network structure of CNF/SA composite hydrogel into a porous material,which explains the difference in the swelling degree and water content.Fig.7(b)shows the effect of SA dosage on the swelling degree of CNF/SA composite hydrogel.The swelling degree of CNF/SA composite hydrogel increased with the increase of SAdosage,but it was still far lower than that of the pure CNF hydrogel when the dosage of SA was low.CNF can form a hydrogen bond with the SA matrix,which can improve the swelling degree of CNF/SA composite hydrogel to a certain extent.When the mass ratio of CNF∶SA was 1∶5,the swelling degree of CNF/SA composite hydrogel reached a maximum of 1274.4%.However,a further increase in SA dosage led to a decrease in the swelling degree of CNF/SA composite hydrogel.This is because when the mass ratio of CNF∶SA was 1∶5,the hydrogen bonds formed between SA and CNF reach a maximum,so the swelling degree also reaches a maximum.Therefore,the best swelling properties of CNF/SA composite hydrogel are achieved at a mass ratio of CNF∶SA of 1∶5.

Fig.7 (a)Water content of the CNF/SA composite hydrogel with different dosages of SA;(b)effect of SAdosage on swelling degree of CNF/SAcomposite hydrogels

3.3 Characterization of CNF/SAcomposite hydrogels

When the Okara cellulose was dissolved by DES pretreatment,the degree of polymerization of Okara cellulose reduced and its crystallinity improved;thus,the macroscopic scale of Okara cellulose reduced and its dispersibility improved. Therefore, in the subsequent mechanical treatment,Okara CNF can be obtained by light mechanical treatment.As shown in Fig.8(a),the average diameter of Okara CNF was approximately 27 nm,and its size homogeneity and morphology were excellent.

Fig.8 (a)Morphology and size distribution of Okara CNF;SEMimages of CNF hydrogel(b),SAhydrogel(c),and CNF/SA composite hydrogel(d);(e)FT-IR spectra of CNF,Okara cellulose,SAhydrogel,and CNF/SAcomposite hydrogel

As shown in Fig.8(b),the three-dimensional network structure of CNF hydrogel showed incomplete dispersion and disordered entanglement.The surface was relatively smooth.Fig.8(c)shows the image of SA hydrogel surface obtained by scanning electron microscopy(SEM);particles observed on the surface are speculated to be caused by shrinkage of the sample after freeze-drying.Fig.8(d)shows an SEM image of CNF/SA composite hydrogel(CNF∶SA=1∶5).It can be seen that because of the addition of SA,several CNF particles are entangled and then cross-linked together,and the three-dimensional network structure formed at the same time was compact and regular.This explains the high strength of CNF/SA composite hydrogel.

The CNF/SA composite hydrogel sample(CNF∶SA=1∶5)with the highest compression strength was selected for Fourier transform infrared spectroscopy(FT-IR)analysis.As shown in Fig.8(e),after DES pretreatment,the characteristic peaks for Okara cellulose do not completely disappear after reformation of the CNF/SA composite hydrogel.The absorption peak of the C—O stretching vibration is located at 1045 cm-1.After DES pretreatment,the vibration at this position is significantly weakened,which is assumed to be due to the formation of free hydroxyl groups from fracture of Okara cellulose.The DES pretreatment influences the characteristic groups of Okara cellulose.The peak at 1538 cm-1is the absorption peak of protein amide II,which is mainly the out-of-plane bending vibration of the N—H bond and the stretching vibration of the C—N bond.Compared with the Okara cellulose,the peaks of CNF/SA composite hydrogel after DES pretreatment gradually disappeared in this area,and the protein was effectively degraded in Okara cellulose.The absorption peak of protein amide III bands is mainly in the wavenumber range of 1200-1400 cm-1,which is caused by in-plane bending vibration of the N—H bond at 889 cm-1as aβ-glycosidic bond vibration(characteristic peak of carbohydrate)[28].There is also an absorption peak at 3400-3600 cm-1,indicating the existence of O—H stretching and vibration in the hydrogel.The absorption peak at 2900 cm-1corresponds to the stretching vibration absorption of the C—H bond of methyl and methylene groups.

By comparing the FT-IR spectra of the SA hydrogel and SA/CNF composite hydrogel,it can be seen that the—OH absorption peak of the SA hydrogel at 3400 cm-1is weaker,shorter,and wider.This is because SA cross-linked with Ca2+and consumed a large amount of—OH.However,there is a large number of—OH in the nanocellulose structure of SA/CNF composite hydrogels,so SA/CNF composite hydrogels can be used as carriers for controlled release of pharmaceutical compounds by cross-linking with active substances.

4 Conclusions

Cellulose nanofiber(CNF)was isolated from Okara using deep eutectic solvent(DES)with high-speed stirring.The composite hydrogels obtained by using different proportions of CNF and sodium alginate(SA)had different properties.When strength and swelling dregree of SA/CNF composite hydrogel are required,a CNF∶SA mass ratio of 1∶5 can be used.When high rheological properties of SA/CNF composite hydrogel are required,a CNF∶SA mass ratio of 1∶6 can be used.The SA/CNF composite hydrogels have great potential as eco-friendly, reinforced composite hydrogels for diverse applications.

Acknowledgments

This work was supported by the Foundation of State Key Laboratory of Pulp and Paper Engineering(201819),the project of Shaanxi Provincial Department of Education Key Laboratory Research Open Fund(Grant No.17JS017),and the Project of Shaanxi University of Science and Technology Research Initial Fund(Grant No.BJ15-29).