Protein-based glyoxal–polyethyleneimine-crosslinked adhesives for wood bonding

ABSTRACT Corn gluten protein, potato protein, and pea protein isolate were used as natural raw materials for preparing bio-based environmentally friendly adhesives. Glyoxal and polyethyleneimine (PEI) were used as crosslinking agents. Dry and wet bonding performances of the adhered wood samples were tested for different protein-based adhesives, i.e., protein alkaline-modified adhesives, protein–glyoxal adhesives, and protein–glyoxal–PEI adhesives. The results indicate that crosslinking by glyoxal and polyethyleneimine effectively improves the bonding performance of pea, corn, and potato protein-based adhesives after water storage.


Introduction
Wood adhesives currently used in plywood and other wood composite panels often contain formaldehyde. Formaldehyde-based adhesives typically show high adhesion strength; however, they may be able to emit formaldehyde (especially urea-formaldehyde resins). [1] Another disadvantage of these adhesives is that they are mainly fossil-based. Plant proteins are renewable biopolymers and a sustainable alternative to fossil-based sources. Therefore, protein-based components are currently considered as attractive raw materials for wood adhesives due to increasing environmental issues. To compete in bonding performance with formaldehyde-based adhesives, proteins need to be modified. [2,3] As a first step to increase raw material's bonding properties, protein structures need to be unfolded to be able to interact with crosslinkers. Due to their globular structure, the reactive functional groups are unavailable, which greatly limits their reactivity. There are a number of methods reported in the literature to expose functional groups of protein molecules to their surfaces [4,5] and to unfold protein structures by chemical [6] or thermal and mechanical treatments. [6][7][8] Methods for chemical denaturation and hydrolysis of proteins include treatment by alkali, surfactants, and organic solvents. Alkaline treatment has been extensively used to denature various types of proteins. [9,10] This type of protein denaturation enhances adhesive properties by exposing specific functional groups to the surface as a result of unfolding protein molecules, causing increased intermolecular interaction with the medium. [4] Another challenge of using proteins for adhesives is the presence of a large number of hydrophilic groups in their structure, which increases water uptake by adhesives. [11][12][13] Water resistance can be improved by adding active substances, which can crosslink protein functional groups. [4,14,15] Glyoxal is one of the most reactive crosslinkers. [4,[16][17][18] It can react with functional protein groups such as lysine and arginine. [19] To react with hydroxyl groups of protein, crosslinkers with active amino groups are needed. For this purpose, polyethyleneimine (PEI) can be used. [20] This water-soluble polymer has been used for improving paper wet strength by crosslinking cellulose [14] and for wood adhesives based on hyper-branched polyethyleneimine, urea, and glyoxal. [21] The most extensively studied plant protein is soy protein due to its successful application in wood adhesives in North America. [22,23] While a large part of soy is grown for biodiesel production, other protein sources such as wheat are mainly grown for food and feed. [4,24] In this work, we compared alternative raw materials from plant sources abundant in Europe, namely, corn, potato, and pea proteins, which were denatured and adjusted to acidic conditions (close to isoelectric point) before crosslinking, and investigated their ability to be used in plywood board production. Adjusting proteins to pH close to isoelectric point allows to increase their water resistance by increasing their hydrophobicity. [25] While pea protein is mainly produced for food and feed and is close in its composition to soy protein, [11] potato and corn protein are obtained from the starch production as a by-product. From these three protein sources, corn is the cheapest.
Polyethyleneimine was earlier considered as a crosslinker in a combination with acid anhydrides or glutaraldehyde. [20,26] In this work, combinations of polyethyleneimine and aqueous glyoxal as a more cost-effective modifying agent were used to improve the wet strength of the protein-based adhesives.

Materials and methods
Agrana Research and Innovation Center GmbH (Tulln-an-der-Donau, Austria) kindly provided potato protein (around 80% total protein content) and corn gluten (around 62% total protein content), and Emsland-Stärke GmbH (Emlichheim, Germany) kindly provided pea protein isolate (around 80% total protein content). Glyoxal 40% water solution analytical grade was purchased from Carl Roth (Karlsruhe, Germany) and polyethyleneimine (PEI) was purchased from Sigma Aldrich (St. Louis, the USA). Rotary cut sheets of birch (Betula) veneer with dimensions of 500 mm × 500 mm and a thickness of 1.5 mm were stored in standard climate (20°C, 65% r.h.) prior to the experiments.

Protein characteristics
To determine the protein raw material dispersibility, the protein dispersibility index (PDI) method was used. [27] This method measures the amount of protein dispersed in water after high-speed blending, followed by centrifugation. Each protein was dispersed in amounts of 20 g dry powder in 300 ml of water and blended using an Ultra-TURRAX blender (IKA, Germany) at 8500 rpm for 10 minutes. After this, dispersions were centrifuged for 10 minutes at 1000 rpm. [27] The nitrogen content of the supernatant liquid was determinded by the BCA (bicinchoninic acid assay) method. [28] The PDI was calculated as percentage of the original protein content of the sample.

Preparation of adhesives
In order to expose more active groups of the protein, raw materials were treated under alkaline conditions. Potato and corn proteins were treated 25 mass% in 1 M NaOH solution at 60°C for 45 minutes. Pea protein differs from corn and potato in dispersibility and particle size distribution and requires a shorter alkaline pretreatment of 10 minutes in order to avoid excessive hydrolysis and degradation as was investigated earlier. [28] Then, the pH was shifted to 4 with 60% formic acid to precipitate unfolded proteins and obtain protein adhesives. The mixtures were stirred for 30 minutes to obtain homogeneous adhesives based on pea protein (named pea), potato protein (named pot), and corn protein (named corn). Glyoxal 40% water solution was added to protein adhesives in a protein to glyoxal ratio of 1:0.2 based on dry mass and stirred for 30 minutes until a homogeneous state was achieved to obtain protein-glyoxal adhesives abbreviated pea-g, pot-g and corn-g. Polyethyleneimine was added to protein-glyoxal adhesives prior to application on a wood substrate in two ratios of protein to PEI, which were 1: 0.05 and 1: 0.10 (based on dry mass), respectively. The adhesives composed of the mixture of protein-glyoxal-PEI are abbreviated as pea-g-PEI, pot-g-PEI and corn-g-PEI.

Tensile shear strength testing
To determine dry and wet tensile shear strength of the adhesives, an Automated Bonding Evaluation System (ABES, Adhesive Evaluation Systems, Inc., USA) was used. Beech veneers with dimensions of 117 mm × 20 mm × 0.6 mm, beforehand stored in standardized climate conditions (20 ± 2°C, 65 ± 4% relative humidity), were used as wood substrate. Adhesives were spread on the substrate with an amount of ~150 g/m 2 (dry weight) with an overlapping area of 200 mm x 5 mm and pressed at 110°C for 5 minutes, resulting in a fully cured stated for all protein samples. The time was chosen based on ABES curves showing fully cured stated for all protein samples between 3 and 5 minutes. Consequently, 5 minutes were chosen as comparable and sufficient for curing all samples. Dry strength was measured immediately after hot pressing and 10 s of cooling. Wet tensile shear strength was measured in wet state after 2 hr and 24 hr water storage at room temperature.
For additional wet strength evaluation, a standard test [29] for plywood was performed on five-layer plywood panels of 500 mm × 500 mm size. The panels were produced on a laboratory-scale with a hydraulic hot press (Langzauner GmbH, Lambrechten, Austria) at a pressing temperature of 120°C, a pressure of 1.5 MPa and a pressing time of 10 minutes. The adhesives were applied on the surface of the birch veneers onefold, with a glue application amount of 240 g/m 2 (wet) resulting in 75 g/m 2 based on dry mass. The fiber orientation of the veneers was placed perpendicular to each other to form plywood boards. Two 5-layered panels were produced for every setting.
The quality of the bonding was assessed by evaluating the tensile shear strength of plywood according to EN 314-2. [29] The sample size used was 7 (±1) mm × 25 mm × 150 mm. Bond strengths were tested for the middle layer of the panels. All the samples were stored for 2 weeks in standard climate before testing. For wet strength testing, bonded plywood specimens were stored in water at 20 ± 3°C for 24 hr and tested in wet state (treatment 5.1.1 according to EN 314-1), and for the boiling test, the specimens were stored in boiling water for 6 hr and then cooled to 20 ± 3°C in water for 1 hr (treatment 5.1.2 according to EN 314-1). A Zwick/Roell 100 universal testing machine (Zwick GmbH & Co. KG, Ulm, Germany) equipped with a load cell with a load capacity of 5 kN and a crosshead was used for induction and measurement of the applied force. The tensile shear strength was calculated by dividing the maximum force by the measured overlap area.

Reactivity determination by DSC
The differential scanning calorimetry measurement (DSC) was performed using a calorimeter type DSC 214-Polyma (Netzsch, Selb, Germany). Adhesive samples were heated from 20°C to 200°C at a heating rate of 10 K/ min in highly pressurized steel crucibles. For data analysis, the software Netzsch-Proteus-80 was used.

Chemical structure analysis by FTIR
Fourier-transform infrared spectroscopy (FTIR) measurements were performed using a PerkinElmer Frontier spectrophotometer (Massachusetts, USA). All adhesive samples were cured at 110°C before measurement and then grained and applied as powder. The spectrum was obtained in transmission with a scanning range of 650-4000 cm −1 by combining 32 scans at a resolution of 2 cm −1 .

Adhesive surface homogeneity analysis by SEM
Scanning electron microscopy (SEM) imaging was carried out using an electron microscope TM3030 (Hitachi, Velizy, France) at an accelerating voltage of 15 kV under vacuum. All adhesive samples were preliminarily cured at 110°C and coated before imaging, with a 4-nm-thick platinum layer using EM ACE200 sputter coater (Leica, Wetzlar, Germany).

Adhesive bonding performance
To estimate the influence of crosslinkers on the ultimate bonding performance, the ABES method was used as described in Section 2.3. All adhesives reached their strength plateau at a maximum of five-minute curing time. Therefore, for further performance analysis, a consistent press time of 5 minutes was applied for all adhesives. The dry tensile shear strength values for pea-glyoxal-PEI, pot-glyoxal-PEI, and cornglyoxal-PEI are approaching a level of around 5.5 -5.9 MPa. (Figure 1). While all three pure protein adhesives show a practical absence of bond strength in wet state, glyoxal greatly improves wet bonding performance of the protein adhesive. Pea-glyoxal adhesive shows better wet strength than pot-glyoxal and corn-glyoxal. The wet strength of pea protein crosslinked with glyoxal still remains at a level of 3 MPa after 24 hr in water. The addition of PEI shows further but less pronounced improvement in the case of pea. For potato-and corn protein-based adhesive, the wet strength after 24 hr in water is clearly enhanced by PEI-glyoxal compared to protein-glyoxal only. Corn protein still shows the lowest wet strength among all; however, its level after 24 hr is still around 2MPa and thus clearly improved compared to the protein alone. One of the reasons for higher strength of pea with glyoxal can be better dispersibility (Table 1). Another reason can be the availability of amino groups, participating in the crosslinking. [22,30] The main amino acids of pea protein isolate according to information provided by the supplier are asparagine and glutamine, which have available amino groups. The amino groups are reacting preferably with glyoxal forming C=N linkage. [19] Corn and potato protein have more diverse amino acid content with a lot of hydrophobic groups in the case of corn. [30,31]   As PEI showed positive effects on the performance of the adhesive systems, the effects of its quantity were investigated as well. The increase of the amount of PEI added to protein-glyoxal adhesives did not cause a further improvement of the bonding performance as can be seen in Figure 2, which was also observed in Xi et al. [20] These findings are in line with what was observed for wheat-glutaraldehyde-PEI adhesive in earlier work. [20] One of the reasons can be a possible shortening of pot life of the adhesive with increasing PEI amount, as a more intensive thickening of adhesive was observed after adding an increased amount of PEI.
The standard method according to EN 314-2 was performed on five-layered birch plywood as described in Section 2.3. For the plywood board testing, the best performing adhesive mixture for each raw material was selected. In each case, the component ratio was protein:glyoxal:PEI corresponding to 1:0.2:0.05 based on dry mass. As a tendency, plywood bonded with pea-based adhesive performed best under all conditions (Figure 3), fulfilling the requirements for class 1: dry interior application (EN 314-2) based on the limited amount of samples tested. While plywood samples produced with all protein adhesives show a modest wet strength after 24 hr of water storage, there is a distinct difference for samples after the boiling test. While potato-and corn-based adhesives did not show any remaining strength, pea protein-based adhesive showed tensile shear strength with an average value of around 0.9 MPa. However, no wood failure was observed for wet test results.
A similar tendency of better performance of pea protein-based adhesives and lowest performance of corn protein-based adhesives is observed for both ABES and plywood tests. The results of tensile shear strength for plywood boards are lower than those tested by ABES, which is mainly attributed to the veneer and wood fiber orientation of the samples. While for ABES specimens both adherents are oriented parallel to the load direction, in plywood single layers are oriented perpendicular to the load direction, featuring significantly lower wood adherend strength. Another possible factor might be the lower amount of adhesive applied on plywood compared to the same area for veneer samples used for ABES. The amount was limited by application specifics and to avoid excessive steam formation. However, wet strength results for pea protein-based adhesive after 24 hr in water are closer than other proteins to urea formaldehyde wet strength, which is typically in a range of 2 MPa [32] .

Crosslinking modifications of proteins
The proposed reactions between amino groups of protein and glyoxal and between polyethyleneimine and carboxyl groups of protein are illustrated in Figure 4. Additionally, reactions between PEI and glyoxal are also probably taking place (Figure 4(3)).
Also, interactions between wood surface and adhesive are taking place, such as polar attractions, hydrogen bonds and Van-der-Waals forces. [33] Polar protein groups interact with the polar wood surface. According to manufacturers, polar amino acids such as glutamic acid, lysine and cysteine [23] are mostly present in pea and potato proteins, whereas corn protein contains more nonpolar amino groups such as methionine, which are also present in the content of potato protein. Therefore, less interaction with wood surface can be expected for corn and potato proteins.
The interactions between proteins and crosslinking agents were investigated by DSC and FTIR methods.
The DSC spectra show pronounced exothermic peaks for potato and corn proteins with glyoxal and polyethyleneimine (Figure 5), indicating a noticeable chemical reaction. This is in line with results from wet strength for corn-g-PEI and pot-g-PEI. In the case of pea protein, the peak for protein-glyoxal adhesive is slightly more pronounced than the peak for protein-glyoxal-PEI, and the peak onset starts already before 100°C. Although the weak signals would indicate only limited crosslinking, the bonds formed with these adhesives prove otherwise as they demonstrate pronounced wet strengths.
Glyoxal is a reactive agent, which contains two aldehyde groups. Therefore, it can react with protein amino groups. [34,35] In Figure 6, FTIR spectra of the adhesives are shown.
The wide peak near the 3300 cm −1 region characterizes the O-H and N-H groups, which were then able to react with polyethyleneimine and formed C-O and C-N bonds. A peak at about 2930 cm −1 was attributed to -CH 2 groups. The main absorption peaks at 1655, 1560, and 1242 cm −1 are characteristic of the amide I (C=O stretching), amide II (N-H bending or C-N stretching), and amide III (C-N and N-H stretching). [36] For pea protein-based adhesives, peaks at 1057 cm −1 and 1351 cm −1 can be observed appearing for glyoxal-and PEI-modified adhesives. According to Yue-Hong et al., [19] protein functional (1) (2) (3) groups, crosslinked with glyoxal, can form the Schiff-base structure. This can be observed at 1057 cm −1 and 1351 cm −1 , attributed to C=N stretching and C-N bending modes, caused by this formation.
The addition of glyoxal leads to an increase in the signal at 1057 cm −1 , which can originate from C=N stretch, as well as primary alcohols which are the intermediate structure in the crosslinking reaction. The peaks at around 850 cm −1 and 925 cm −1 , which are attributed to new C-C vibrations according to Ciannamea et al., [36] appear for all proteins, modified with glyoxal and PEI, most intensively for corn protein-glyoxal-PEI adhesive.
Analyzing the consistency of cured adhesives by scanning electron microscopy ( Figure 7) shows that protein adhesives from corn and potato before addition of glyoxal and PEI have a lot of small cracks, while pea protein creates relatively closed surface despite the presence of lumps. The reason can be higher dispersibility of pea protein as raw material compared to potato and corn proteins and, hence, its ability to form a more homogeneous dispersion. The addition of glyoxal leads to "smoother" adhesives although with some minor cracks for corn-g adhesive (Figure 7h). Cracks were not observed for any protein sample crosslinked with glyoxal and PEI. These results may support understanding of wet strength performance, where pea protein shows better wet strength compared to potato and corn already before the addition of crosslinkers ( Figure 1a) and corn protein shows lowest wet strength compared to others as those seem to be prone to cracks.
As shown before, the strength of pea protein modified with glyoxal significantly improved. The addition of polyethyleneimine has a less pronounced influence on wet strength of pea-protein-based adhesive. One reason might be the involvement of the most active groups in crosslinking between amino groups of protein and glyoxal, which leaves a smaller number of crosslinking possibilities for polyethyleneimine. The achieved wet strength results are in line with earlier described improvement for wheat protein, crosslinked by glutaraldehyde and PEI. [20] However, the wet strength for pea and potato protein is higher than earlier described. Reactivity of PEI with pea proteinglyoxal was found to be lower than with corn-glyoxal and with potato-glyoxal. These results showed that crosslinker reactivity can significantly differ depending on protein source and its dispersibility.

Conclusion
With the aim of improving primarily the wet strength of veneer-based adhesive joints, we found that combining glyoxal and polyethyleneimine significantly improved wet bonding performance of the investigated protein adhesives. Corn gluten protein, potato protein and pea protein crosslinked with glyoxal and polyethyleneimine show bond strengths after immersion 24 hr storage in water on a level around 2 MPa for corn- based adhesives and close to 3 MPa for potato-and pea protein-based adhesives at the ratio of components protein:glyoxal:PEI which was 1:0.2:0.05 (dry matter) based on ABES testing method. Glyoxal was found to form pea protein-based adhesive bonds with wet strength around 2.9 MPa after 24 hr storage in water, which is clearly higher than for potato (around 2MPa) and corn (around 1 MPa) proteins based on ABES testing method. Polyethyleneimine was found to be an effective crosslinker for all investigated proteins, including hydrophobic materials such as corn gluten. The results for wet strength after 24 hr storage in water are around 2.5 MPa for pea and potato protein and around 1.9 MPa for corn protein based on ABES testing method. This can make corn gluten from starch production a potential raw material for wood adhesive. Standard bonding evaluation tests were performed on five-layered plywood boards. Pea protein-based adhesives showed wet strength around 1.5 MPa after 24 hr in water and around 0.9 MPa after 6 hr boiling test, which fulfill the requirements for dry interior use (class 1) and close to requirement level for covered exterior (class 2). [29] In contrast, potato and corn proteins bonded boards delaminated after the boiling test and showed the wet strength on a level around 1.1 MPa for potato protein-based adhesive and 0.9 MPa for corn protein-based adhesive and therefore fulfilled the requirements for dry interior use only. [29]