polymers

Article Effects of Different Denaturants on Properties and Performance of Soy -Based Adhesive

Li Yue 1,2, Zhang Meng 1,2, Zhang Yi 1,2, Qiang Gao 1,2,*, An Mao 3,* and Jianzhang Li 1,2

1 Key Laboratory of Wood Material Science and Utilization, Beijing Forestry University, Beijing 100083, China 2 Ministry of Education, Beijing Key Laboratory of Wood Science and Engineering, College of Materials Science and Technology, Beijing Forestry University, Beijing 100083, China 3 Key Laboratory of State Forestry Administration for Silviculture of the lower Yellow River, College of Forestry, Shandong Agricultural University, Taian 271018, China * Correspondence: [email protected] (Q.G.); [email protected] (A.M.); Tel.: +86-010-62336912 (Q.G.)

 Received: 24 May 2019; Accepted: 24 July 2019; Published: 30 July 2019 

Abstract: Chemical modification of soy protein, via crosslinking, is the preferred method for creating non-toxic, renewable, environmentally friendly wood adhesives. The denaturing process of protein is important for the adhesive performance improvement. In order to investigate the effect of different denaturing agents on the performance of soy protein-based adhesives before and after crosslinking modification. In this study, three different denaturing agents— (U), sodium dodecyl sulfate (SDS), and sodium hydrogen sulfite (SHS) and an epoxide crosslinking agent—Triglycidylamine (CA) were used to prepare soy protein-based adhesives. The results showed: (1) The denaturing agent unfolded protein molecules and exposed more hydrophobic groups to prevent water intrusion, which was mainly a contribution for the water resistance and performance improvement of soy protein-based adhesives. The wet shear strength was improved up to 91.3% (denaturing by urea). (2) After modifying by the crosslinking agent, the properties and performance improvement was due to the fact that the active groups on soybean protein molecules reacted with the crosslinking agent to form a crosslinking structure, and there is no obvious correlation with the hydrophobic groups of the protein. (3) The unfolded soybean protein molecules also expose hydrophilic groups, which facilitates the reaction between the crosslinking agent and protein to form a denser crosslinking structure to improve the performance of the adhesive. Particularly, after denaturing with SHS, the wet shear strength of the plywood bonded by the SPI-SHS-CA adhesive increased by 217.24%.

Keywords: soy protein; protein surface hydrophobic index; hydrophobic groups; wet shear strength; crosslinking structure

1. Introduction The wood-based panel industry in China primarily uses formaldehyde synthetic resin adhesives and their modified products (e.g., urea-formaldehyde, phenolic, and melamine-formaldehyde resins) [1]. As such, throughout the wood-panels’ production and use life-cycle, formaldehyde and other harmful substances are released into the environment, thereby generating a pollution problem and endangering human health. In addition, aldehyde adhesives are derived from unsustainable petroleum resources [2]. Between the depletion of global petrochemical resources and the increasingly serious environmental pollution problems they cause, development of renewable and environmentally friendly wood adhesives has become an important consideration for the wood industry going forward. Protein adhesives are made from raw materials that are available in abundance [3]. Moreover, they have the advantages of being non-toxic, renewable, and environmental- friendly [4,5]; and as such, have become a research hotspot in recent years.

Polymers 2019, 11, 1262; doi:10.3390/polym11081262 www.mdpi.com/journal/polymers Polymers 2019, 11, 1262 2 of 13

Soy protein is a main byproduct of agriculture [6]. Thus, it is an abundantly available, renewable, and low cost option that shows significant potential for development [7]. However, due to its low adhesion strength, poor water resistance, high viscosity, and low solid content, the soy protein adhesive has limited application in industry [8,9]. Earlier studies have attempted numerous and varied methods to chemically modify soy protein in order to improve its bonding performance—such as denaturing agent- [10,11], crosslinking- [12,13], grafting- [14,15], and nano-fiber modification [16], etc. However, the effects of different modification methods on the properties of soybean protein-based adhesives have hardly been reported. Therefore, the effects of different denaturing agents and crosslinking agents on the properties and performance of soybean protein-based adhesives were investigated. Moreover, such knowledge is useful and important for appropriate modification methods in the soy protein-based adhesive preparation and to develop more effective modification methods. In this study, urea, SDS and SHS are common denaturants used to enhance the performance of soybean protein-based adhesives. Previous studies [17] have shown that urea mainly destroys hydrogen bonds between protein molecules, SDS as a surfactant denatures , and SHS enables protein molecules to unfold by breaking disulfide bonds between molecules and hydrolyzes protein molecules [18]. Based on this, three denaturants (urea, SDS and SHS) were individually incorporated into an SPI adhesive solution in this study; which was subsequently mixed with the epoxy crosslinker (CA) to prepare three modified soy protein-based adhesives. Finally, the different denaturants’ effects on the soy protein’s surface hydrophobicity, residual rate, fracture surface micrograph and resultant adhesive performance were quantitatively assessed.

2. Materials and Methods

2.1. Materials SPI with >85% crude protein content was purchased from Xiang Chi Grain and Oil Co., Ltd. (Shandong, China) and milled into 250-mesh flour. Urea and SHS were obtained from Lan Yi Chemical Co., (Beijing, China); and SDS was purchased from Tianjin Chemical Reagent Co., (Tianjin, China). Poplar (Populus tomentosa Carr.) veneers (40 cm 40 cm 1.5 mm, 8% moisture content) were × × purchased from the Hebei Province, China. CA was synthesized in the laboratory. analysis was carried out using ion-exchange chromatography in an automatic amino acid analyzer (Hitachi 835-50, Japan). The SPI amino acid composition is presented in Table1.

Table 1. The amino acid content of SPI.

Amino Acid Name SPI Tyrosine 3.25% Glycine 3.54% Leucine 6.81% Proline 4.54% Alanine 3.73% Phenylalanine 4.43% Valine 5.37% Isoleucine 3.97% Serine 4.53% Threonine 3.33% Aspartic acid 10% 16.65% 2.22% 6.74% Lysine 5.37% Methionine 1.03% Polymers 2019, 5, x FOR PEER REVIEW 3 of 14

Polymers 2019, 11, 1262 3 of 13 Methionine 1.03%

2.2.2.2. Preparation Preparation of of Cross-Linker Cross-Linker Triglycidylamine Triglycidylamine (CA) (CA) CA was was synthesized synthesized using using a similar a similar process process reported reported in Connolly’s in Connolly’s research research [19] and [19] the synthesis and the synthesisprocess was process illustrated was illustrated in Figure 1in. Figure Aqueous 1. Aqueous ammonia ammonia (A, 25 wt (A, %) 25 was wt dropwise%) was dropwise added into added an intoepichlorohydrin an epichlorohydrin solution solution (E, 99.9 wt(E,%) 99.9 with wt a%) molar with ratio a molar of 4:1 ratio (A:E) of in4:1 a (A:E) three-necked in a three flask-necked at a stirrer flask atof a 180 stirrer r/min. of 180 In addition, r/min. In theaddition, mixture the reacts mixture at a reacts temperature at a temperature range from range 50 to from 65 ◦C 50 until to 65 all °C ammonia until all ammoniawas added was and added further and reacted further for reacted 2 h at 60 for◦C. 2 h Since at 60 the °C. reaction Since the was reaction highly was exothermic, highly exothermic, an external anwater-cooling external water circulator-cooling was circulator required to was maintain required the temperature. to maintain The the residual temperature. epichlorohydrin The residual and epichlorohydrinammonium hydroxide and ammonium were removed hydroxide by a vacuumwere removed distillation by a vacuum process atdistillation 45 ◦C, leaving process a colorlessat 45 °C, leavingsyrup consisting a colorless of tris-(3-chloro-2-hydroxypropyl) syrup consisting of tris-(3-chloro amine.-2-hydroxypropyl) Then, a sodium amine. hydroxide Then, solution a sodium of hydroxide50 wt % (weight solution ratio of of 50 syrup:NaOH wt % (weight solution ratio of= syrup:NaOH10:1) with was solution added = to 10:1) facilitate with the was epoxy-ring added to facilitateclosure reaction, the epoxy which-ring tookclosure place reaction, under which vigorous took stirring place under at 50 ◦vigorousC for 2 h stirring and cooled at 50 to°C 25 for◦C 2 toh andobtain cooled CA. to The 25 feather °C to obtain of the CA. resultant The feather CA was of asthe followed: resultant pHCA= was9.0, as viscosity followed: of pH 1200 = mPa9.0, viscositys, epoxy · ofvalue 1200= mPa·s,0.52%. epoxy value = 0.52%

FigureFigure 1. 1. CrossCross-linking-linking Agent (CA)(CA) SynthesisSynthesis Schematic.Schematic. 2.3. Preparation of Adhesive 2.3. Preparation of Adhesive For the SPI adhesive, 15 g of SPI was added to 85 g of deionized water and stirred for 10 min at For the SPI adhesive, 15 g of SPI was added to 85 g of deionized water and stirred for 10 min at 25 C in order to form a homogeneous system. 1 g of denaturation agent (either urea, SDS, or SHS) 25 °C◦ in order to form a homogeneous system. 1 g of denaturation agent (either urea, SDS, or SHS) was mixed with the SPI adhesive and stirred for an additional 20 min at 25 C to develop a series of was mixed with the SPI adhesive and stirred for an additional 20 min at 25 °C◦ to develop a series of adhesives. As a control, the SPI adhesive without denaturant was also stirred at the subsequent 20 min. adhesives. As a control, the SPI adhesive without denaturant was also stirred at the subsequent 20 The resulting SPI adhesives were labeled “SPI”, “SPI-Urea”, “SPI-SDS”, and “SPI-SHS” to reflect the min. The resulting SPI adhesives were labeled “SPI”, “SPI-Urea”, “SPI-SDS”, and “SPI-SHS” to reflect different denaturing agents. 5 g of CA were then mixed with each SPI adhesive, which were stirred for the different denaturing agents. 5 g of CA were then mixed with each SPI adhesive, which were an additional 5 min at 25 C to develop a second series of adhesives. The resulting adhesives were stirred for an additional ◦ 5 min at 25 °C to develop a second series of adhesives. The resulting labeled “SPI-CA”, “SPI-Urea-CA”, “SPI-SDS-CA”, and “SPI-SHS-CA”. The adhesive formulations are adhesives were labeled “SPI-CA”, “SPI-Urea-CA”, “SPI-SDS-CA”, and “SPI-SHS-CA”. The adhesive shown in Table2. formulations are shown in Table 2. Table 2. Formulations used for adhesive samples. Table 2. Formulations used for adhesive samples. Formulation Sample Formulation Sample SPI Deionized Water Denaturing Agents CA SPI Deionized water Denaturing agents CA 1 SPI 15 g 85 g 2 SPI-Urea1 SPI 15 gg 85 85 g g 1 g Urea 23 SPI SPI-SDS-Urea 15 gg 85 85 g g 1 1 g g Urea SDS 4 SPI-SHS 15 g 85 g 1 g SHS 35 SPI SPI-CA-SDS 15 gg 85 85 g g 1 g SDS 5 g CA 6 SPI-Urea-CA4 SPI-SHS 15 gg 85 85 g g 1 g UreaSHS 5 g CA 7 SPI-SDS-CA5 SPI-CA 15 gg 85 85 g g 1 g SDS 5 gg CACA 8 SPI-SHS-CA 15 g 85 g 1 g SHS 5 g CA 6 SPI-Urea-CA 15 g 85 g 1 g Urea 5 g CA 7 SPI-SDS-CA 15 g 85 g 1 g SDS 5 g CA 2.4. Surface Hydrophobicity Measurement 8 SPI-SHS-CA 15 g 85 g 1 g SHS 5 g CA The adhesive samples were placed in an oven at 120 2 C to cure completely then ground into ± ◦ ~200 mesh powder in ceramic mortar to measure the surface hydrophobic index, using an analytical

Polymers 2019, 11, 1262 4 of 13 method reported by Mozafarpour et al. [20]. Sodium 8-anilino-1-naphthalenesulfonate (ANS) was used as the hydrophobic fluorescent probe. At room temperature, the soybean protein samples to be tested were prepared into a 10 mg/mL (Dissolve 3 g sample in 300 mL phosphate buffer solution) solution with a phosphate buffer solution of 0.01 mol/L and a pH of 7.6. The solution was homogenized for 1 min to be fully dissolved, centrifuged at 8000 r/min for 20 min, and the protein concentration in the solution was determined by the coomase bright blue method. The supernatant was diluted into different gradients with the same concentration of the phosphate buffer solution. Four millilitres solution of different concentrations was taken and 20 µL 8 mmol/L ANS solution (0.01 mol/L, pH 7.6 phosphate buffer solution was prepared) was added. The fluorescence intensity (FI) in the sample was detected by a fluorescence spectrophotometer within 8–15 min after the shock, and the phosphate buffer solution was used as a blank control. The fluorescence detection conditions were: Excitation wavelength = 390 nm, emission wavelength = 490 nm, and interstitial width = 5 nm. The protein’s relative fluorescence intensity value was the sample’s fluorescence intensity value minus the reagent’s blank value. The protein’s relative fluorescence intensity was used to make the sample concentration curve, and the curve fitting was performed by the least squares method. The initial slope of the line was the surface hydrophobicity index of the sample.

2.5. Residual Rate Test Adhesive samples were placed in an oven and maintained at 120 2 C until a constant weight ± ◦ (M) was reached. Next, the samples were immersed in water for 6 h in an oven maintained at 60 2 C, ± ◦ then dried at 105 2 C for 3 h until a constant weight (m) was reached. The residual rate was ± ◦ calculated as m/M and reported as a percentage.

2.6. Three-Ply Plywood Preparation and Evaluation Three-ply poplar plywood was fabricated under the following production conditions: Coating 2 density (each surface) = 180 g/m , hot pressing time (1.0 MPa) = 5 min, temperature = 120 ◦C. Plywood samples were stored at room temperature for 12 h after hot pressing. The wet shear strength was measured in accordance with the Chinese National Standard (GB/T 17657-2013) [21]. Sixteen plywood specimens (25 mm 100 mm) were cut from as-prepared plywood × panels, and plywood specimens were submerged in water for 3 h at 63 2 C. After the plywood ± ◦ specimens were cooled for 10 min to room temperature, their wet shear strengths were tested using a tensile machine at an operating speed of 10.0 mm/min. The wet shear strengths were calculated using Equation (1) [21]: Tension Force (N) Wet shear strength (MPa) = (1) Bonding Area (m2)

2.7. Fourier Transform Infrared (FTIR) Spectroscopy The adhesive samples were placed in an oven at 120 2 C to cure completely then ground into ± ◦ ~200 mesh powder in ceramic mortar. The powder was mixed with KBr crystals at a mass ratio of 1:70, and was pressed in a mold to form a sample folium. The FTIR spectra of various powdered adhesives were recorded using a Thermo Nicolet 6700 FT-IR (Nicolet Instrument Corporation, Madison, WI, 1 1 USA) within a range from 4000 to 400 cm− at a 4 cm− resolution, using 32 scans.

2.8. Scanning Electron Microscopy (SEM) For observing the section morphology of the adhesive, the fracture surface of cured adhesive was analyzed using SEM. The adhesive samples were placed in an oven at 120 2 C to cure completely. ± ◦ The cured adhesive samples, about 1.5 mm in thickness, were fractured and their fracture surface were examined. The fracture surfaces were placed on an aluminum stub, and a 10 nm gold film was coated on using an ion sputter (HITACHI MCIOOO, Ibaraki, Japan). The coated fracture surfaces were Polymers 2019, 11, 1262 5 of 13 observed by a Hitachi S-4800 emission scanning electron microscope (Hitachi Scientific Instruments, Tokyo, Japan).

2.9. X-ray Diffraction (XRD) The adhesive samples were placed in an oven at 120 2 C to cure completely then ground into ± ◦ ~ 200 mesh powder in ceramic mortar. X-ray diffraction (XRD) patterns were recorded on an XRD diffractometer (XRD-6000, Shimadzu, Kyoto, Japan) using a cobalt source and a 0.2 theta scan ranging from 5◦ to 60◦ at 45 kV and 30 mA. The sample determination index was carried out using the program Jade 5.0. The degree of crystallinity is calculated as the ratio of the sum of crystalline band areas to the total area under the XRD patterns [22].

2.10. Apparent Viscosity The apparent viscosity of the adhesive was determined using the rheometer (HAAKE RS1, Waltham, UK) with a parallel plate (35 mm diameter). The distance was set to 1 mm for all measurements. The 1 experiments were conducted under a steady shear flow at 25 ◦C. Shear rates ranged from 0.1 to 60 s− 1 in 10 s− increments. The average value was reported.

2.11. Statistical Analysis All experiments were performed in triplicates. The results were expressed as a mean standard ± deviation and processed using the Origin Pro Version 7.5 software (Origin Lab Corporation, Northampton, UK). The one-way analysis of variance was used for determining the significant difference at P < 0.05.

3. Results and Discussion

3.1. The effects of Denaturing Agent on SPI Adhesive The purpose of the protein surface hydrophobicity measurement is to indirectly reflect the change of surface hydrophobicity by denaturing agents. The interaction of hydrophobic groups in soybean protein molecules is extremely significant for maintaining the soybean protein’s quaternary structure [23]. Thus, hydrophobic characterization of the protein surface can reflect higher structure changes in the protein. Protein surface hydrophobicity is characterized using the surface hydrophobic index (S0). The hydrophobic fluorescent probe (ANS) binds to aromatic amino acids at an excitation wavelength of 390 nm and has a maximum absorption at 470 nm. The fluorescence intensity is proportional to the surface hydrophobicity of the protein [24]. Results for the first set of adhesive samples is presented in Figure2. The S 0 of the SPI adhesive is 1577, which is due to being comprised of 28.59% hydrophobic amino acids (tyrosine, phenylalanine, valine, leucine, isoleucine, alanine, methionine) (Table1). The soybean protein S0 increased to reach a maximum of 4572 after urea-denaturation. This increase is explained by the interaction between the urea and protein hydroxyls, which destroys the hydrogen bond, permitting protein molecule unfolding. The addition of SDS increased the protein’s S0 to 2787, because SDS destroyed the protein’s structure and caused the hydrophobic groups to evaginate. The addition of SHS caused the protein’s S0 to increase, reaching 4082. This phenomenon occurs because SHS destroys the disulfide bond inside the protein molecule, resulting in protein molecule unfolding and the exposure of hydrophobic groups inside protein molecules. At the same time, due to the unfolding of the protein molecule, the hydrophilic group inside the protein is exposed. Polymers 2019, 5, x FOR PEER REVIEW 6 of 14

Polymers 2019, 11, 1262 6 of 13

6000 )

0 5000 4572 4082 4000

3000 2787

2000 1577

Surface hydrophobicity index (S index hydrophobicity Surface 1000

0 SPIs SPIs-Urea SPIs-SDS SPIs-SHS Adhesive sample

Figure 2. Surface hydrophobicity index of the cured adhesive samples. Figure 2. Surface hydrophobicity index of the cured adhesive samples. The apparent viscosity reflects the fluidity of the protein adhesive. Too high viscosity makes the poor flow quality of the protein adhesive and too low viscosity will make the protein adhesive in the The apparent viscosity reflects the fluidity of the protein adhesive. Too high viscosity makes the curing process not form an adhesive layer, which will result in a poor bonding performance of the poor flow quality of the protein adhesive and too low viscosity will make the protein adhesive in the prepared plywood [25]. The apparent viscosity results of the sample with different denaturing agents is curing process not form an adhesive layer, which will result in a poor bonding performance of the shown in Table3. After adding urea, the initial viscosity of the sample decreases to 48,720 mPa s, which prepared plywood [25]. The apparent viscosity results of the sample with different denaturing· agents is because the urea reduces the intermolecular force as a dispersant. The addition of SDS increases is shown in Table 3. After adding urea, the initial viscosity of the sample decreases to 48,720 mPa·s, the viscosity of the sample to 61,463 mPa s, because SDS can increase the force between the protein which is because the urea reduces the· intermolecular force as a dispersant. The addition of SDS molecules. The addition of SHS sharply reduced the viscosity of samples to 6381 mPa s, because SHS increases the viscosity of the sample to 61,463 mPa·s, because SDS can increase the ·force between the could hydrolyze protein molecules and reduce the molecular weight of proteins. The pH values of protein molecules. The addition of SHS sharply reduced the viscosity of samples to 6381 mPa·s, the samples measured in the experiment are all between 6.8 and 7.2, so the change of viscosity is not because SHS could hydrolyze protein molecules and reduce the molecular weight of proteins. The related to the pH value. pH values of the samples measured in the experiment are all between 6.8 and 7.2, so the change of viscosity is not related to the pH value. Table 3. The residual rate and initial viscosity of the cured adhesive samples.

SamplesTable 3. The residual SPI rate and initial SPI-Urea viscosity of the cured SPI-SDS adhesive samples. SPI-SHS Initial viscosity (mPa s) 53672 48720 61463 6381 samples · SPI SPI-Urea SPI-SDS SPI-SHS InitialResidual viscosity rate (%) (mPa·s) 84.8 536720.05 83.7 0.0248720 82.5 0.0461463 79.4 0.076381 ± ± ± ± Residual rate (%) 84.8 ± 0.05 83.7 ± 0.02 82.5 ± 0.04 79.4 ± 0.07 TheThe ATR-FTIR ATR-FTIR spectroscopy spectroscopy results results of ofthe the SPI SPI modified modified with with di ff differenterent denaturing denaturing agents agents is is 2 shownshown in Figurein Figure3. Active 3. Active groups groups –NH –NH2, –COOH,, –COOH, and and –OH –OH were were identified identified in in the the SPI; SPI; as wereas were the the 1 −1 threethree characteristic characteristic peptide peptide bond bond peaks peaks –C =–OC=O stretching stretching peak peak at 1627.35at 1627.35 cm −cm(amide(amide I) [I)26 [26],], N–H N–H 1 −1 stretchingstretching peak peak at 1515.69 at 1515.69 cm − cm(amide(amide II) II) [27 [27],], and and the the C–N C–N stretching stretching peak peak and and N–H N– bendingH bending 1 −1 vibrationvibration peak peak at 1234.68at 1234.68 cm −cm(amide(amide III) III) [28 [28].]. The The main main characteristic characteristic peaks’ peaks’ shape shape still still appears appears after after addingadding the the denaturant, denaturant, indicating indicating that that the the soy soy protein’s protein’s molecule molecule primary primary structure structure was was almost almost not not 1 −1 destroyed.destroyed. After After the the addition addition of urea,of urea, amide amide I, II I, and II and III shiftedIII shifted from from 1627.35 1627. to35 1622.02 to 1622.02 cm −cm, 1515.69, 1515.69 1 −1 1 −1 to 1531.19to 1531.19 cm −cm, and, and 1234.68 1234.68 to to 1235.20 1235.20 cm cm− , respectively., respectively. Similarly, Similarly, the the addition addition of denaturantof denaturant agents agents (SDS(SDS and and SHS) SHS) also also caused caused the the amide amide I, II I, andII and III toIII shift.to shift. This This indicates indicates that that adding adding a denaturant a denaturant causescauses the the soy soy protein protein molecules molecules to stretchto stretch and and expose expose more more active active groups; groups; thus, thus, the the protein’s protein’s higher higher structurestructure is destroyed is destroyed to someto some extent. extent.

Polymers 2019, 5, x FOR PEER REVIEW 7 of 14

Polymers 2019, 11, 1262 7 of 13

d 1231.24 1632.99

c 1515.78

1157.23 b

1519.36 1632.93

1235.20 Transmittance a 1531.19 1234.68 1622.02

1627.35 1515.69

4000 3500 3000 2500 2000 1500 1000 Wavenumber (cm-1) Figure 3. Attenuated total reflection (ATR)-FTIR spectroscopic results from the cured adhesive samples: (a) SPI adhesive, (b) SPI-Urea adhesive, (c) SPI- sodium dodecyl sulfate (SDS) adhesive, (d) SPI- sodium Figure 3. Attenuated total reflection (ATR)-FTIR spectroscopic results from the cured adhesive hydrogen sulfite (SHS) adhesive. samples: (a) SPI adhesive, (b) SPI-Urea adhesive, (c) SPI- sodium dodecyl sulfate (SDS) adhesive, (d) SPIX-ray- sodium diffraction hydrogen patterns sulfite and (SHS) the adhesive. calculated crystallinity results of the cured adhesive samples are presented in Figure4. The crystallinity and residual rate can reflect the degree of ordered arrangement of proteinX-raymolecules. diffraction Thepatterns purpose and of the the calculated measurement crystallinity of the residual results rate of the indirectly cured adhesive reflects the samples degree areof modified presented protein in Figure molecule 4. The expansion crystallinity by denaturant and residual agents rate (Urea, can SDS, reflect SHS) the and degree results areof ordered showed arrangement of protein molecules. The purpose of the measurement of the residual rate indirectly in Table3. Figure4 shows that the SPI samples have two distinct peaks at 9 ◦ and 20◦, which are the reflectsprotein molecule’sthe degree αof-helix modified structure protein and moleculeβ-sheet structure, expansion respectively by denaturant [29]. Afteragents the (Urea, addition SDS, of SHS) urea and results SDS, the are crystallinity showed in Table and residual 3. Figure rate 4 shows of the that modified the SPI adhesive samples was have reduced two distinct when peaks compared at 9° andwith 20°, the purewhich protein are the adhesive. protein molecule’s The crystallinity α-helix was structure reduced and by 19.36%β-sheet andstructure, 11.26% respectively respectively, [29]. and Afterthe residual the addition rate was of urea reduced and toSDS, 83.7% the andcryst 82.5%allinity respectively. and residual This rate is of because the modified the urea adhesive destroys was the reducedhydrogen when bond compared within the with protein the moleculespure protein and adhesive. SDS evaginated The crystallinity the proteins’ was hydrophobic reduced by groups,19.36% andresulting 11.26% in respectively, a destroyed and protein the residual molecule rate and was loosely reduced structured to 83.7% protein. and 82.5% The respectively. crystallinity This of the is becausSHS modifiede the urea adhesive destroys was the 11.73%, hydrogen which bond decreased within the by 42.33%protein tomolecules reach the and maximum SDS evaginated and residual the proteins’rate is decreased hydrophobic to 79.4%. groups, On the resulting one hand, in a SHS destroyed destroys protein the disulfide molecule bond and between loosely the structured protein protein.molecules, The and crystallinity on the other of hand,the SHS the modified protein hydrolyzes adhesive was and 11.73%, its molecular which weight decreased decreases. by 42.33% Under to reachsuch conditions, the maximum it is and diffi residualcult to form rate an is decreased ordered structure, to 79.4%. like On thatthe ofone pure hand, proteins, SHS destroys during the disulfidecuring process. bond between the protein molecules, and on the other hand, the protein hydrolyzes and its molecularSEM imagesweight ofdecreases. the fracture Under surface such ofconditions, the cured it adhesive is difficult are to shown form inan Figure ordered5. Manystructure, circular like thatpores of werepure observedproteins, during on the the fractures curing surfaceprocess. of the pure protein adhesive, which are easy to be invaded by water and reduce the water-resistance of the SPI adhesive. After adding the urea and SDS denaturation, the fracture surface pores of the adhesive decreases and the surface becomes smooth, compact and continuous, which prevents the invasion of water and improves the water resistance of the adhesive. After the addition of SHS, the fracture surface of the adhesive became smooth and compact, but some small crystals appeared. This may attribute to the aggregation of small molecules hydrolyzed by SHS through hydrogen bonding during the curing process.

Polymers 2019, 5, x FOR PEER REVIEW 8 of 14

200 3000 SPIs SPIs-Urea SPIs-SDS 2500 SPIs-SHS

Polymers 2019, 5, x FOR2000 PEER REVIEW 8 of 14 90 Polymers 2019, 11, 1262 8 of 13 1500

0 1000 20 3000 SPIs Linear intensity (counts) intensity Linear SPIs-Urea 500 SPIs-SDS 2500 SPI sample SPIs SPIs-Urea SPIs-SDS SPIs-SHS SPIs-SHS Crystallinity(%) 20.34 19.89 18.05 11.73 0 2000 1090 20 30 40 50 60 2θ(o) 1500

Figure 4. X1000-ray diffraction pattern and the crystallinity of the cured adhesive samples. Linear intensity (counts) intensity Linear SEM images of the fracture surface of the cured adhesive are shown in Figure 5. Many circular 500 pores were observed on theSPI fractures sample surfaceSPIs ofSPIs the-Urea pureSPIs-SDS proteinSPIs-SHS adhesive, which are easy to be invaded by water and reduceCrystallinity(%) the water-resistance20.34 19.89 of the SPI18.05 adhesive.11.73 After adding the urea and SDS denaturation, the fracture0 surface pores of the adhesive decreases and the surface becomes smooth, compact and continuous, which10 prevents20 the invasion30 of water40 and improves50 the60 water resistance of o the adhesive. After the addition of SHS, the fract2θure( ) surface of the adhesive became smooth and compact, but some small crystals appeared. This may attribute to the aggregation of small molecules Figure 4. X-ray diffraction pattern and the crystallinity of the cured adhesive samples. hydrolyzed by SHS through hydrogen bonding during the curing process. Figure 4. X-ray diffraction pattern and the crystallinity of the cured adhesive samples.

SEM images of the fracture surface of the cured adhesive are shown in Figure 5. Many circular pores were observed on the fractures surface of the pure protein adhesive, which are easy to be invaded by water and reduce the water-resistance of the SPI adhesive. After adding the urea and SDS denaturation, the fracture surface pores of the adhesive decreases and the surface becomes smooth, compact and continuous, which prevents the invasion of water and improves the water resistance of the adhesive. After the addition of SHS, the fracture surface of the adhesive became smooth and compact, but some small crystals appeared. This may attribute to the aggregation of small molecules Figure 5. Fracture surface micrographs of the cured adhesive samples: (a) SPI adhesive, (b) SPI-Urea hydrolyzed by SHS through hydrogen bonding during the curing process. adhesive,Figure 5. Fracture (c) SPI-SDS surface adhesive, micrographs (d) SPI-SHS of the adhesive. cured adhesive samples: (a) SPI adhesive, (b) SPI-Urea adhesive, (c) SPI-SDS adhesive, (d) SPI-SHS adhesive. The wet shear strengths of the plywood bonded of different denaturing agents (Urea, SDS, SHS)The adhesive wet shear are strengths shown in of Figure the plywood6. The SPI-Urea bonded of adhesive different due denaturing to the fact agents that (Urea, urea breaks SDS, SHS) the hydrogenadhesive are bond shown between in Figure protein 6. The molecules, SPI-Urea which adhesive makes due the to proteinthe fact moleculesthat urea breaks unfold the and hydrogen exposes thebond hydrophobic between protein group, molecules, resulting in which the wet makes shear the strengths protein of moleculesthe plywood unfold increasingto and exposes 0.88 MPa. the Duehydrophobic to the action group, of SDS, resulting the hydrophobic in the wet shear group strengths in the SPI-SDS of the adhesive plywood was increasing evaginated,to 0.88 exposing MPa. Due the hydrophobicto the action of group SDS, within the hydrophobic the protein group molecule, in the resulting SPI-SDS in adhesive the wet shear was strengthsevaginated, of exposing the plywood the increasingtohydrophobic 0.72group MPa. within This the is consistentprotein molecule, with the resulting surface hydrophobicityin the wet shear analysis.strengths Theof the addition plywood of SHSincreas decreasesingto 0.72 the MPa. wet shearThis is strength consistent of plywoodwith the bondedsurface hydrophobicity to 0.29 MPa, because analysis. SHS The can addition hydrolyze of Figure 5. Fracture surface micrographs of the cured adhesive samples: (a) SPI adhesive, (b) SPI-Urea proteinSHS decreases molecules the andwet breakshear proteinstrength chains, of plywood making bonded it diffi cultto 0.29 to formMPa, an because effective SHS adhesive can hydrolyze layer in adhesive, (c) SPI-SDS adhesive, (d) SPI-SHS adhesive. theprotein curing molecules process. and break protein chains, making it difficult to form an effective adhesive layer in the curingTo sum process. up, the improvement of the water resistance of the adhesive and the bonding performance of the preparedThe wet shear plywood strengths are mainly of the plywood due to the bonded protein of molecule different unfoldingdenaturing and agents more (Urea, hydrophobic SDS, SHS) groupsadhesive exposed are shown by the in desaturating Figure 6. The agents. SPI-Urea adhesive due to the fact that urea breaks the hydrogen bond between protein molecules, which makes the protein molecules unfold and exposes the hydrophobic group, resulting in the wet shear strengths of the plywood increasingto 0.88 MPa. Due to the action of SDS, the hydrophobic group in the SPI-SDS adhesive was evaginated, exposing the hydrophobic group within the protein molecule, resulting in the wet shear strengths of the plywood increasingto 0.72 MPa. This is consistent with the surface hydrophobicity analysis. The addition of SHS decreases the wet shear strength of plywood bonded to 0.29 MPa, because SHS can hydrolyze protein molecules and break protein chains, making it difficult to form an effective adhesive layer in the curing process.

Polymers 2019, 5, x FOR PEER REVIEW 9 of 14 Polymers 2019, 11, 1262 9 of 13

1.0 0.88

0.8 0.72

0.6 0.46

0.4

0.29 Wet shear strength (MPa) strength Wet shear 0.2 Polymers 2019, 5, x FOR PEER REVIEW 10 of 14

0.0 SPIs SPIs-Urea SPIs-SDS SPIs-SHS Adhesive samples

Figure 6. The wet shear strengths of plywood bonded by modified SPI adhesive. Figure 6. The wet shear strengths of plywood bonded by modified SPI adhesive. 3.2. The effects of Denaturing Agent on SPI/CA Adhesive The majorTo sum functional up, the groups improvement of soy protein of the molecules water resistance include hydroxylof the adhesive (–OH), andcarboxyls the bonding (–COOH), performance of the prepared plywood are mainly due to the protein molecule unfolding and more amines (–NH ), and sulfhydryls (–SH) [30]. These groups are associated with side chains of amino acids hydrophobic2 groups exposed by the desaturating agents. and terminal groups of proteins. The characteristic peak in CA is the epoxy group, which appeared at 1 940 cm3.2.− . The Figure effects7 shows of Denaturing the ATR-FTIR Agent on spectra SPI/CA ofAdhesive adhesives with CA additive. After the incorporation of CA, its characteristic epoxy group peak disappeared, indicating that CA was cross-linked with The major functional groups of soy protein molecules include hydroxyl (–OH), carboxyls (– active groups on SPI molecules. When compared to the adhesives without CA, it becomes evident COOH), amines (–NH2), and sulfhydryls (–SH) [30]. These groups are associated with side chains of 1 that theamino epoxy acids group and disappearedterminal groups and of proteins a new peak. The characteristic at 1738 cm− peakappeared in CA is [23 the], epoxy which group, wasrelated which to the carbonylappeared group at 940 of cm the−1. Figure ester bond 8 shows and the originated ATR-FTIR fromspectra the of esterificationadhesives with reactionCA additive. between After the epoxy 1 groupsincorporation and carbonyl of groups CA, its ofcharacteristic SPI. The absorption epoxy group band peak at disappeared, 1396 cm− was indicating significantly that CA weakened was cross - [22], whichlinked suggested with active that the groups amino on SPI group molecules. was transformed When compared bythe to the reaction adhesives with without the epoxy CA, it group.becomes The cross-linkingevident structurethat Figurethe epoxy formed7. The group denaturation is conductive disappeared and crosslinking to and improve a new mechanism peak the water at of1738 soy resistance cmprotein−1 appeared molecule. of the [23], adhesive which andwas the wet shearrelated strength to the carbonyl of the prepared group of plywood.the ester bond The and crosslinking originated from mechanism the esterification of the soy reaction protein between molecule −1 is shownepoxy in Figure groups8 . and carbonyl groups of SPI. The absorption band at 1396 cm was significantly weakened [22], which suggested that the amino group was transformed by the reaction with the epoxy group. The cross-linking structure formed is conductive to improve the water resistance of the adhesive and the wet shehar strength of the prepared plywood. The crosslinking mechanism of the soy protein molecule is showng in Figure 7.

f e

d

c Transmittance

b 1738 a

1396

4000 3500 3000 2500 2000 1500 1000 Wavenumber (cm-1)

Figure 7. ATR-FTIR spectroscopic of the cured adhesive samples: (a) SPI adhesive, (b) SPI -CA adhesive, (c) SPI-Urea (U) adhesive, (d) SPI-Urea-CA adhesive, (e) SPI-SDS adhesive, (f) SPI-SDS-CA adhesive, (g) SPI-SHS-CAFigure 8. ATR adhesive,-FTIR spectroscopic and (h) SPI-SHS-CA of the cured adhesive. adhesive samples: (a) SPI adhesive, (b) SPI - CA adhesive, (c) SPI-Urea (U) adhesive, (d) SPI-Urea-CA adhesive, (e) SPI-SDS adhesive, (f) SPI-SDS-CA adhesive, (g) SPI-SHS-CA adhesive, and (h) SPI-SHS-CA adhesive.

SEM images of the fracture surface of the cured adhesive are shown in Figure 9. It can be observed that the fracture surface of the adhesive with CA becomes more compact and continuous than that without CA, which is beneficial to improve the water-resistant bonding performance of the adhesive. Compared with that without CA, a ductile fracture was observed on the fracture surface of the SPI-Urea-CA and SPI-SDS-CA adhesive, which indicated that the generation of the crosslinking structure improved the performance of the adhesive. Compared with that without CA, the fracture surface of the SPI-SHS-CA adhesive becomes more even and uniform, which indicates that the unfolded protein molecules and small molecules generated by the hydrolysis form a uniform cross-

PolymersPolymers2019 2019, 11, 5,, 1262x FOR PEER REVIEW 10 10of of14 13

Figure 8. The denaturation and crosslinking mechanism of soy protein molecule. Figure 7. The denaturation and crosslinking mechanism of soy protein molecule. SEM images of the fracture surface of the cured adhesive are shown in Figure9. It can be observed that the fracture surface of the adhesive with CA becomes more compact and continuous than that without CA, which is beneficial to improve the water-resistant bonding performance of the adhesive. Compared with that without CA, a ductile fracture was observed on the fracture surface of the SPI-Urea-CA and SPI-SDS-CAh adhesive, which indicated that the generation of the crosslinking structure improved the performanceg of the adhesive. Compared with that without CA, the fracture surface of the SPI-SHS-CA adhesive becomes more even and uniform, which indicates that the unfolded Polymers 2019, 5, x FOR PEER REVIEWf 11 of 14 protein molecules and small molecules generated by the hydrolysis form a uniform cross-linking e linkistructureng structure in the hot-pressing in the hot-pressing process, process, which is which conducive is conducive to improving to improving the water-resistant the water- adhesiveresistant bonding performance of the adhesive. adhesive bonding performanced of the adhesive.

c Transmittance

b 1738 a

1396

Figure 9. Fracture surface4000 micrographs3500 3000 of2500 the cured2000 adhesive1500 samples:1000 (a) SPI-CA adhesive, Figure(b) SPI-Urea-CA 9. Fracture adhesive, surface micrographs (c) SPI-SDS-CA ofWavenumber the adhesive, cured adhesive ( d(cm) SPI-SHS-CA-1) samples: adhesive. (a) SPI-CA adhesive, (b) SPI- Urea-CA adhesive, (c) SPI-SDS-CA adhesive, (d) SPI-SHS-CA adhesive.

The residual rate is used to characterize the degree of cross-linking between protein molecules The residual rate is used to characterize the degree of cross-linking between protein molecules and resultsFigure are 8. ATR shown-FTIR in Tablespectroscopic4. After of the the addition cured adhesive of CA, samples: the modified (a) SPI adhesives’ adhesive, residual(b) SPI - rates andincreased resultsCA toadhesive, are 89.3%, shown 91.9%,(c) SPIin Table-Urea 91.5%, (U)4. andAfter adhesive, 90.2%, the addition respectively,(d) SPI-Urea of CA,-CA and theadhesive, the modified wet shear(e) SPI adhesives’ strength-SDS adhesive, of residual the plywood(f) rates increasealso improvedd to 89.3%, to meet 91.9%, the 91.5%, interior and use 90.2%, requirement respectively, ( 0.7 and MPa) the [25 wet]. Thisshear improvement strength of the came plywood about SPI-SDS-CA adhesive, (g) SPI-SHS-CA adhesive, and≥ (h) SPI-SHS-CA adhesive. alsobecause improved the CA to epoxy meet groupthe interior crosslinked use requirement with the amino-, (≥0.7 MPa) carboxyl-, [25]. This and improvement other active groups came about in the becauseproteinSE moleculetheM imagesCA epoxy to form of thegroup a network fracture crosslinked structure,surface with of which the the curedamino is conducive adhesive-, carboxyl to are improving-, and shown other inthe active Figure adhesive groups 9. Ithydrolysis can in bethe proteinstability,observed molecule and that thus the improvingto fracture form asurface the network bonding of the structure, adhesive strength which ofwith the CA prepared is becomes conducive plywood. more to compact improving The wet and shear the continuous adhesivestrengths hydrolysisofthan the plywoodthat withoutstability, bonded CA, and bywhich thus the modifiedisimproving beneficial SPI tothe adhesive improve bonding with the strength water CA is-resistant shown of the inprepared bonding Figure 10 performanceplywood.. The SPI-Urea-CA, The of thewet shearSPI-SDS-CAadhesive. strengths Compared adhesive of the plywood with wet shearthat bondedwithout strength bCA,y wasthe a ductilemodified improved fracture SPI by adhesive 21.59%,was observed andwith 45.83% CA on theis shown comparedfracture in surface Figure with of the10. Theadhesivethe SPI SPI-Urea-Urea without-CA,-CA CAandSPI added.- SDSSPI--SDSCA This- CAadhesive is adhesive, because wet ureawhichshear can indicatedstrength break the wasthat hydrogen improvedthe generation bond by 21.59%, betweenof the crosslinking and the 45.83% protein comparedmoleculesstructure withtoimproved make the theadhesive the protein performan without moleculesce CAof the unfoldadded. adhesive. and This SDS Comparedis because can make ureawith the canthat protein breakwithout molecules the CA, hydrogen the unfold fracture bond as a betweensurfactant,surface the of so theprotein the SPI availability- moleculesSHS-CA adhesive of to moremake hydrophilic becomesthe protein more groupsmolecules even facilitates and unfold uniform, and cross-linking SDS which can indicates make with CAthe that protein to form the moleculesaunfolded denser crosslink unfoldprotein asmolecules structure, a surfactant, and which small so resulted the molecules availability in the generated wet of moreshear by hydthestrength hydrolysisrophilic of the groups form SPI-U-CA a facilitates uniform (from cross cross 0.88- - linking with CA to form a denser crosslink structure, which resulted in the wet shear strength of the SPI-U-CA (from 0.88 to 1.07 MPa) adhesive and the SPI-SDS-CA adhesive (from 0.72 to 1.05 MPa) increased. SHS broke the disulfide bond in the protein molecule and hydrolyzed protein molecules, allowing the protein molecule chains to unfold and break protein chains. Simultaneously, the hydrophilic groups of the protein molecule chains were cross-linked with CA to improve the wet shear strength of the plywood from 0.29 to 0.92 MPa compared with the SPI-SHS adhesive.

Table 4. The residual rate of the adhesive: The SPI-CA, SPI-Urea-CA, SPI-SDS-CA, and SPI-SHS-CA adhesive, respectively.

Adhesive SPI-CA SPI-Urea-CA SPI-SDS-CA SPI-SHS-CA Residual rate (%) 89.3 ± 0.08 91.9 ± 0.05 91.5 ± 0.07 90.2 ± 0.03

Polymers 2019, 11, 1262 11 of 13 to 1.07 MPa) adhesive and the SPI-SDS-CA adhesive (from 0.72 to 1.05 MPa) increased. SHS broke the disulfide bond in the protein molecule and hydrolyzed protein molecules, allowing the protein molecule chains to unfold and break protein chains. Simultaneously, the hydrophilic groups of the protein molecule chains were cross-linked with CA to improve the wet shear strength of the plywood from 0.29 to 0.92 MPa compared with the SPI-SHS adhesive.

Table 4. The residual rate of the adhesive: The SPI-CA, SPI-Urea-CA, SPI-SDS-CA, and SPI-SHS-CA adhesive, respectively. Polymers 2019, 5, x FOR PEER REVIEW 12 of 14 Adhesive SPI-CA SPI-Urea-CA SPI-SDS-CA SPI-SHS-CA Residual rate (%) 89.3 0.08 91.9 0.05 91.5 0.07 90.2 0.03 ± ± ± ±

1.2

1.07 1.05

1.0 0.92

0.8 0.73

0.6

0.4 Wet shear strength (MPa) strength Wet shear

0.2

0.0 SPIs-CA SPIs-Urea-CA SPIs-SDS-CA SPIs-SHS-CA Adhesive samples

Figure 10. The wet shear strengths of plywood bonded by modified SPI adhesive of with CA. Figure 10. The wet shear strengths of plywood bonded by modified SPI adhesive of with CA. To sum up, the addition of the crosslinking agent (CA) improves the water resistance of soy protein-based adhesives and the bonding performance of the prepared plywood, which is mainly To sum up, the addition of the crosslinking agent (CA) improves the water resistance of soy becauseprotein the-based active adhesives groups on and the proteinthe bonding molecules performance are reacted of withthe prepared epoxy groups plywood, to form which a crosslinking is mainly structure,because and the has active no obvious groups correlation on the protein with hydrophobic molecules are groups reacted of soy with protein. epoxy After groups the toaddition form a ofcrosslinking the crosslinking structure, agent, and the activehas no groups obvious of correlation the protein with molecule hydrophobic unfolded groups can cross-link of soy protein. with epoxy After groupsthe addition to form of a the crosslinking crosslinking structure agent, the in theactive curing groups process, of the which protein improves molecule the unfolded water-resistant can cross- bondinglink with performance epoxy groups of the to preparedform a crosslinking plywood. structure in the curing process, which improves the 4.water Conclusions-resistant bonding performance of the prepared plywood.

4. ConclusionsThe following conclusions are drawn from the experimental results of this study: (1) The protein molecule hydrogen bond is destroyed under the action of urea and SDS causes The following conclusions are drawn from the experimental results of this study: the hydrophobic group to evaginate, which increases the surface hydrophobic index of the SPI-Urea (1) The protein molecule hydrogen bond is destroyed under the action of urea and SDS causes adhesive (189.92%) and SPI-SDS adhesive (76.73%) compared with the SPI adhesive. The unfolded the hydrophobic group to evaginate, which increases the surface hydrophobic index of the SPI-Urea protein molecules expose more hydrophobic groups, which improved the bonding performance adhesive (189.92%) and SPI-SDS adhesive (76.73%) compared with the SPI adhesive. The unfolded of the prepared plywood to 0.88 MPa (SPI-Urea adhesive) and 0.72 MPa (SPI-SDS adhesive). The protein molecules expose more hydrophobic groups, which improved the bonding performance of addition of the crosslinking agent CA makes the SPI-Urea adhesive and SPI-SDS adhesive form a the prepared plywood to 0.88 MPa (SPI-Urea adhesive) and 0.72 MPa (SPI-SDS adhesive). The denser crosslinking structure during the hot-pressing process and improves the water resistance of the addition of the crosslinking agent CA makes the SPI-Urea adhesive and SPI-SDS adhesive form a adhesive. Compared with those without CA, the residual rate of the adhesive increased by 9.56% and denser crosslinking structure during the hot-pressing process and improves the water resistance of the adhesive. Compared with those without CA, the residual rate of the adhesive increased by 9.56% and 10.49% and the wet shear strength increased by 21.59% and 45.83%, respectively. Consistent with the fact that the fracture structure occurs on the fracture surface. (2) By breaking the disulfide bond between the protein molecules and causing protein hydrolysis, SHS expands and destroys the protein molecular chains to a large extent, so that the surface hydrophobic index increases to 4082. Due to the sharp decline in the initial viscosity of the SPI-SHS adhesive system, it is difficult to form an effective adhesive layer in the curing process, so the wet shear strength of the adhesive decreases to 0.29 MPa. The unfolding of the protein molecular chain and the hydrolyzed small molecule makes it easier for the hydrophilic group to cross-link with the crosslinking agent (CA) and form a cross-linked structure, which improves the degree of cross- linking of protein molecules (the residual rate increased from 79.4% to 90.2%) and the prepared plywood’s bonding strength (the wet shear strength increased from 0.29 to 0.92 MPa).

Polymers 2019, 11, 1262 12 of 13

10.49% and the wet shear strength increased by 21.59% and 45.83%, respectively. Consistent with the fact that the fracture structure occurs on the fracture surface. (2) By breaking the disulfide bond between the protein molecules and causing protein hydrolysis, SHS expands and destroys the protein molecular chains to a large extent, so that the surface hydrophobic index increases to 4082. Due to the sharp decline in the initial viscosity of the SPI-SHS adhesive system, it is difficult to form an effective adhesive layer in the curing process, so the wet shear strength of the adhesive decreases to 0.29 MPa. The unfolding of the protein molecular chain and the hydrolyzed small molecule makes it easier for the hydrophilic group to cross-link with the crosslinking agent (CA) and form a cross-linked structure, which improves the degree of cross-linking of protein molecules (the residual rate increased from 79.4% to 90.2%) and the prepared plywood’s bonding strength (the wet shear strength increased from 0.29 to 0.92 MPa). (3) The modified SPI adhesive with a denaturation agent (urea, SDS, SHS) can improve the bonding performance of the prepared plywood, which is mainly because of improving the water resistance of the adhesive. The addition of the crosslinking agent improves the bonding performance of the prepared plywood, which is mainly due to the formation of the crosslinking structure in the curing process by the reaction between the active groups on the soybean protein molecules and the crosslinking agent.

Author Contributions: Y.L. and Q.G. conceived the project, designed the experiments, and analyzed the data and wrote the main paper; M.Z. and Y.Z. performed the experiments; Q.G., A.M. and J.L. supervised and directed the project; all authors reviewed the manuscript. Funding: This work was supported by the National Key Research and Development Program of China (2016YFD0600705), Beijing Forestry University Outstanding Young Talent Cultivation Project (2019JQ03004) and the National Natural Science Foundation of China (31722011). Conflicts of Interest: The authors declare no conflict of interest.

References

1. Chen, M.; Luo, J.; Shi, R.; Zhang, J.; Gao, Q.; Li, J. Improved Adhesion Performance of Soy Protein-Based Adhesives with a Larch Tannin-Based Resin. Polymers 2017, 9, 408. [CrossRef][PubMed] 2. Gao, Q.; Liu, C.; Luo, J.; Li, X.; Chen, L.; Wang, W.; Li, J. Effects of resin open time and melamine addition on cold pre-pressing performance of a urea–formaldehyde resin. Eur. J. Wood Wood Prod. 2018, 76, 1253–1261. [CrossRef] 3. Zhang, M.; Zhang, Y.; Chen, M.; Gao, Q.; Li, J. A High-Performance and Low-Cost Soy Flour Adhesive with a Hydroxymethyl Melamine Prepolymer. Polymers 2018, 10, 909. [CrossRef][PubMed] 4. Kaith, B.S.; Jindal, R.; Bhatia, J.K. Morphological and thermal evaluation of soy protein concentrate on graft copolymerization with ethylmethacrylate. J. Appl. Polym. Sci. 2011, 120, 2183–2190. [CrossRef] 5. Pradyawong, S.; Qi, G.; Sun, X.S.; Wang, D. Laccase/TEMPO-modified lignin improved soy-protein-based adhesives: Adhesion performance and properties. Int. J. Adhes. Adhes. 2019, 91, 116–122. [CrossRef] 6. Wang, F.; Wang, J.; Chu, F.; Wang, C.; Jin, C.; Wang, S.; Pang, J. Combinations of soy protein and polyacrylate emulsions as wood adhesives. Int. J. Adhes. Adhes. 2018, 82, 160–165. [CrossRef] 7. Roman, J.K.; Wilker, J.J. Cooking Chemistry Transforms Proteins into High-Strength Adhesives. J. Am. Chem. Soc. 2019, 141, 1359–1365. [CrossRef][PubMed] 8. Li, J.; Luo, J.; Li, X.; Yi, Z.; Gao, Q.; Li, J. Soybean meal-based wood adhesive enhanced by ethylene glycol diglycidyl ether and diethylenetriamine. Ind. Crop. Prod. 2015, 74, 613–618. [CrossRef] 9. Luo, J.; Luo, J.; Yuan, C.; Zhang, W.; Li, J.; Gao, Q.; Chen, H. An eco-friendly wood adhesive from soy protein and lignin: performance properties. RSC Adv. 2015, 5, 100849–100855. [CrossRef] 10. Mo, X.; Sun, X.; Wang, D. Thermal properties and adhesion strength of modified soybean storage proteins. J. Am. Oil Chem. Soc. 2004, 81, 395–400. [CrossRef] 11. Zhang, L.; Zhang, B.; Fan, B.; Gao, Z.; Shi, J. Liquefaction of soybean protein and its effects on the properties of soybean protein adhesive. Pigment Resin Technol. 2017, 46, 399–407. [CrossRef] 12. Xu, Y.; Xu, Y.; Zhu, W.; Zhang, W.; Gao, Q.; Li, J. Improve the Performance of Soy Protein-Based Adhesives by a Polyurethane Elastomer. Polymers 2018, 10, 1016. [CrossRef][PubMed] Polymers 2019, 11, 1262 13 of 13

13. Zheng, P.; Li, Y.; Li, F.; Ou, Y.; Lin, Q.; Chen, N. Development of Defatted Soy Flour-Based Adhesives by Acid Hydrolysis of Carbohydrates. Polymers 2017, 9, 153. [CrossRef][PubMed] 14. Liu, H.; Li, C.; Sun, X.S. Improved water resistance in undecylenic acid (UA)-modified soy protein isolate (SPI)-based adhesives. Ind. Crop. Prod. 2015, 74, 577–584. [CrossRef] 15. Zhang, Y.; Zhang, M.; Chen, M.; Luo, J.; Li, X.; Gao, Q.; Li, J. Preparation and characterization of a soy protein-based high-performance adhesive with a hyperbranched cross-linked structure. Chem. Eng. J. 2018, 354, 11. [CrossRef] 16. Eslah, F.; Jonoobi, M.; Faezipour, M.; Ashori, A. Chemical modification of soybean flour-based adhesives using acetylated cellulose nanocrystals. Polym. Compos. 2018, 39, 3618–3625. [CrossRef] 17. Schmid, M.; Prinz, T.K.; Stabler, A.; Sangerlaub, S. Effect of Sodium Sulfite, Sodium Dodecyl Sulfate, and Urea on the Molecular Interactions and Properties of Whey Protein Isolate-Based Films. Front. Chem. 2016, 4, 49. [CrossRef][PubMed] 18. Liu, T.; Zhao, M. Physical and chemical modification of SPI as a potential means to enhance small peptide contents and antioxidant activity found in hydrolysates. Innov. Food Sci. Emerg. Technol. 2010, 11, 677–683. [CrossRef] 19. Connolly, J.M.; Alferiev, I.; Eidelman, N.; Sacks, M.; Palmatory, E.; Kronsteiner, A.; DeFelice, S.; Xu, J.; Ohri, R.; Narula, N. Triglycidylamine Crosslinking of Porcine Aortic Valve Cusps or Bovine Pericardium Results in Improved Biocompatibility, Biomechanics, and Calcification Resistance. Am. J. Pathol. 2005, 166, 1–13. [CrossRef] 20. Mozafarpour, R.; Koocheki, A.; Milani, E.; Varidi, M. Extruded soy protein as a novel emulsifier: Structure, interfacial activity and emulsifying property. Food Hydrocoll. 2019, 93, 361–373. [CrossRef] 21. Test Methods of Evaluating the Poperties of Wood-Based Panels and Surface Decorated Wood-Based Panels; GB/T 17657; Standardization Administration of The People’s Republic of China: Beijing, China, 2013. 22. Luo, J.; Li, L.; Luo, J.; Li, X.; Li, K.; Gao, Q. A High Solid Content Bioadhesive Derived from Soybean Meal and Egg White: Preparation and Properties. J. Polym. Environ. 2016, 25, 948–959. [CrossRef] 23. Chen, L.; Chen, J.; Ren, J.; Zhao, M. Effects of ultrasound pretreatment on the enzymatic hydrolysis of soy protein isolates and on the emulsifying properties of hydrolysates. J. Agric. Food Chem. 2011, 59, 2600–2609. [CrossRef][PubMed] 24. Li, R.; Wang, X.; Liu, J.; Cui, Q.; Wang, X.; Chen, S.; Jiang, L. Relationship between Molecular Flexibility and Emulsifying Properties of Soy Protein Isolate-Glucose Conjugates. J. Agric. Food Chem. 2019, 67, 4089–4097. [CrossRef][PubMed] 25. Liu, C.; Zhang, Y.; Li, X.; Luo, J.; Gao, Q.; Li, J. “Green” bio-thermoset resins derived from soy protein isolate and condensed tannins. Ind. Crop. Prod. 2017, 108, 363–370. [CrossRef] 26. Xu, Y.; Xu, Y.; Han, Y.; Chen, M.; Zhang, W.; Gao, Q.; Li, J. The Effect of Enzymolysis on Performance of Soy Protein-Based Adhesive. Molecules 2018, 23, 2752. [CrossRef] 27. Liu, C.; Zhang, Y.; Li, X.; Luo, J.; Gao, Q.; Li, J. A high-performance bio-adhesive derived from soy protein isolate and condensed tannins. RSC Adv. 2017, 7, 21226–21233. [CrossRef] 28. Li, X.; Li, J.; Luo, J.; Li, K.; Gao, Q.; Li, J. A Novel Eco-friendly Blood Meal-based Bio-adhesive: Preparation and Performance. J. Polym. Environ. 2017, 26, 607–615. [CrossRef] 29. Xu, L.; Liu, Y.; Yang, M.; Cao, W.; Zhang, H.; Xia, N.; Li, T.; Zhao, X. Properties of soy protein isolate/nano-silica bilayer films during storage. J. Food Process Eng. 2018, 42, e12984. [CrossRef] 30. Luo, J.; Luo, J.; Li, X.; Li, K.; Gao, Q.; Li, J. Toughening improvement to a soybean meal-based bioadhesive using an interpenetrating acrylic emulsion network. J. Mater. Sci. 2016, 51, 9330–9341. [CrossRef]

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).