Natural Tannins As New Cross-Linking Materials for Soy-Based Adhesives

polymers

Article Natural Tannins as New Cross-Linking Materials for Soy-Based Adhesives

Saman Ghahri 1,* , Xinyi Chen 2, Antonio Pizzi 2 , Reza Hajihassani 1 and Antonios N. Papadopoulos 3,*

1 Wood and Forest Products Research Division, Research Institute of Forests and Rangelands, Agricultural Research, Education and Extension Organization (AREEO), Tehran 19395-1113, Iran; [email protected] 2 LERMAB, University of Lorraine, 88000 Epinal, France; [email protected] (X.C.); [email protected] (A.P.) 3 Laboratory of Wood Chemistry and Technology, Department of Forestry and Natural Environment, International Hellenic University, GR-661 00 Drama, Greece * Correspondence: [email protected] (S.G.); [email protected] (A.N.P.)

Abstract: Human health problems and formaldehyde emission from wood-based composites are some of the major drawbacks of the traditional synthetic adhesives such as urea formaldehyde resins. There have been many attempts to decrease formaldehyde emission and replace urea formaldehyde resins with bio-based adhesives for wood-based composites. Because of some weakness in soy- based adhesive, chemicals have been used as modifiers. Modified soy-based adhesives without any formaldehyde have been successfully used to prepare wood panels. To achieve this, different synthetic cross-linking chemicals such as phenol formaldehyde resins and polyamidoamine-epichlorohydrin were used. However, in reality, what we need are totally green adhesives that use natural materials. In our previous research work, the use of tannins in combination with soy-based adhesives to make Citation: Ghahri, S.; Chen, X.; Pizzi, wood composites was investigated. Thus, in this research work, the feasibility of using three types A.; Hajihassani, R.; Papadopoulos, of natural tannins (quebracho, mimosa and chestnut tannins) as cross-linking materials for soy A.N. Natural Tannins as New adhesive was studied. The chemical bond formation and adhesion behaviors of tannin-modified soy Cross-Linking Materials for Soy-Based Adhesives. Polymers 2021, adhesives were also investigated by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight 13, 595. https://doi.org/10.3390/ Mass Spectrometry (MALDI-ToF-MS) and thermo-mechanical analysis (TMA). The results showed polym13040595 that at ambient temperature, both ionic and covalent bonds formed between tannin constituents and amino acids; however, at higher temperature, covalent bonds are largely predominate. Based on Academic Editor: Fernão the results obtained from the thermo-mechanical analysis, the modulus of elasticity (MOE) of soy D. Magalhães adhesive is increased by adding tannins to its formulation. In addition, the chemical bond formation was proved by MALDI-ToF-MS. Received: 2 February 2021 Accepted: 15 February 2021 Keywords: tannin; soy protein; cross-linking material; wood adhesive; wood based composites Published: 16 February 2021

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional affil- iations. Soybeansare an ideal raw material for making wood adhesives because they are abundant, renewable, environmentally friendly and readily available [1]. Soy-based wood adhesives have many advantages such as low cost, easy handling and low pressing temperature [1]. Recently, researchers have done a considerable amount of work using different methods and adding different chemicals to soybean flour and soybean protein cross-linking Copyright: © 2021 by the authors. as bio-based wood adhesive for wood-based composites [2–6]. Licensee MDPI, Basel, Switzerland. Despite the great potential of soybeans—in the form of soy flour (SF) and isolated This article is an open access article soy protein (ISP)—as non-formaldehyde adhesives for wood gluing, a major drawback distributed under the terms and conditions of the Creative Commons is their low water resistance [7]. Some researchers have already focused themselves on Attribution (CC BY) license (https:// improving the moisture resistance of these adhesives [8,9]. Several chemical modification creativecommons.org/licenses/by/ strategies involving introduction of phenolic, thiol, maleyl, amine and hydroxyl groups 4.0/). into soy proteins have been attempted to improve the adhesive properties of ISP with

Polymers 2021, 13, 595. https://doi.org/10.3390/polym13040595 https://www.mdpi.com/journal/polymers Polymers 2021, 13, 595 2 of 15

limited success [10–15]. A number of cross-linking agents were also used to increase the water resistance of soy adhesives. To this purpose, several additives like polyamidoamine- epichlorohydrin (PAE), sodium dodecyl sulfate (SDS) and other synthetic materials like phenol formaldehyde (PF) resins have been used [16,17]. The main issue in using synthetic cross-linking materials for soy adhesive is their lower environmental friendliness and their dependence on fossil resources. Therefore, chemicals not depending on oil are needed to improve soybean flour while maintaining its acceptable adhesion properties. Consequently, there is great interest in substituting synthetic chemicals for soy adhesives modification to obtain high-performance bio-based adhesives. Tannins are natural products obtained from plants and are very widespread in nature [18]. Tannins are, after lignins, a major source of polyphenolic components all over the world [19], but they have a considerably greater extraction potential than lignins. Vegetable tannins have been used to tan leather either alone or accompanied by other tanning agents for several thousand years. They are natural products obtained from plants and are very diffuse in the whole plant kingdom [18]. The term natural vegetable tannin is used loosely to define two broad classes of chemical compounds of mainly phenolic nature, namely, condensed or polyflavonoid tannins and hydrolysable tannins [18]. Industrial polyflavonoid tannin extracts are mostly composed of flavon-3-ols repeating units, and two types of phenolic rings having different reactivates with formaldehyde are present on each flavon-3-ol repeating unit, namely A-rings and B-rings, with each repeating unit being linked 4,6 or 4,8 with the units, which precede and follow it [20]. Previous research works showed that the addition of tannin to the materials based on the blends of collagen and chitosan leads to modification of several properties, such as water swelling and mechanical performance [21]. Both condensed polyflavonoid tannins and hydrolysable tannins have already been successfully used as wood composite adhesives [22–31]. Synthesized tannin resin from larch tannin extract with 60.1 wt% has already been successfully used as a modifier of ISP adhesive. The results obtained showed higher crosslinking density and better plywood water uptake resistance and shear strength as well [32]. Moreover, the results of cured ISP-condensed tannin bio-adhesive showed a high cross-linking density, which was attributed to two factors: first, that the imine group (H2C=N–CH2+) generated by hexamine decomposition reacted with the nucleophilic site of the tannin’s A-ring to form an amino methylene bridge [33]. The bridge had the ability to bond with other condensed tannins oligomers and finally increase the crosslinking density of the resultant adhesive. Secondly, the orthoquinone obtained via pyrocatechol oxidation on the tannin’s B-ring led to covalent interaction and cross-linking reaction with ISP molecules, which further increased the cross-linking density of the resultant adhesive [34]. Because of the tannin’s phenolic nature, due to their similarity to phenol in PF resins, tannins are a good choice for soy adhesive modification. In previous research, tannin- modified soy-based adhesives were successfully used for wood-based composites, and satisfactory results were achieved [35–37]. The problem of synthetic adhesives is outlined in the introduction. They are petroleum derived; hence, they are a finite source, and they use and emit formaldehyde, now classified as toxic and carcinogenic. Adhesives based on other aldehydes are slower reacting, thus the work outlined here is aimed at offering a totally bio-sourced environment adhesive for bonding wood panels. The aim of this research work is to apply different net natural tannins (hydrolysable and condensed tannin) as cross-linking agents of soy adhesives (isolated soy protein and soy flour) and to show the great potential of tannins (quebracho, mimosa and chestnut tannins) as cross-linkers for soy adhesives which used for wood composite bonding.

2. Materials and Methods Defatted soy ﬂour (SF) with 47 wt% protein content was purchased from BEHPAK Co. (Iran), and isolated soy protein (ISP) with 90 wt% protein content used in this study Polymers 2021, 13, 595 3 of 15

was donated by ADM Co. (USA). The three tannins used were mimosa bark tannin (Acacia mearnsii, formerly mollissima, de Wildt) extract, chestnut wood tannin (Castanea sativa) and quebracho wood tannin (Schinopsis balansae) extract (produced by SILVA Chimica, S. Michele Mondovi, Italy). The other chemicals were supplied by ACROS Organics.

2.1. Adhesive Formulation Adhesive formulations were described in previous research work [34]. Different amounts of SF and ISP (7.35 g) were added to distilled water (29.4 g) and mixed with a mechanical stirrer (700 rpm speed) at room temperature (21 ± 2 ◦C) to produce soy slurry with 20 wt% solid content. The tannin solution (5, 10 and 15 wt% based on soy dry weight) was then added to the soy slurry and stirred for 30 min. The tannin solutions in water were prepared at 45 wt% concentration. In addition, 6.5 wt% hexamethylenetetramine (hexamine) was used as a hardener; the percentage was based on the dry weight of tannins. Hexamine was added to the adhesives as a 40% aqueous solution. Finally, in the last stage of the adhesive’s preparation, the hardener was added and the pH adjusted to 7 with a 50 wt% NaOH water solution. ISP adhesive was prepared according to the same procedure.

2.2. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-ToF) Mass Spectrometry Mimosa tannin extract was reacted at ambient temperature and at 80 ◦C for 1 h with soy protein isolates (ISP). Test procedure was performed according to the method as previously described by Chen et al. [37]. Samples for MALDI-ToF analysis were prepared by ﬁrst dissolving 7.5 mg of sample powder in 1mL of a 50:50 v/v acetone/water solution. Then 10 mg of this solution was added to 10 µL of a 2,5-dihydroxy benzoic acid (DHB) matrix. The locations dedicated to the samples on the analysis plaque were ﬁrst covered with 2 µL of a NaCl solution 0.1 M in 2:1 v/v methanol/water, and pre-dried. Then 1.5 µL of the sample solution was placed on its dedicated location and the plaque was dried again. Red phosphorous was to standardize the MALDI equipment. MALDI-ToF spectra were obtained using an Axima-Performance mass spectrometer from Shimadzu Biotech (Kratos Analytical Shimadzu Europe Ltd., Manchester, UK) using a linear polarity-positive tuning mode [37].

2.3. FTIR Analysis Fourier transform infrared (FTIR) analysis of the reacted product of tannin + ISP reacted at 80 ◦C for 1 h. The addition of an acid catalyst (pTSA) was carried out using a Shimadzu IR Afﬁnity-1 (Shimadzu Europe Ltd., Manchester, UK) spectrophotometer. A blank sample tablet of potassium bromide, ACS reagent from ACROS Organics (Geel, Belgium), was prepared for the reference spectra. Similar tablets were prepared by mixing potassium bromide with 5% by weight of the sample powders. The spectra were plotted in percentage transmittance by combining 32 scans with a resolution of 2.0 cm−1 in the 400–4000 cm−1 range.

2.4. Thermomechanical Analysis The adhesives were tested dynamically by thermo-mechanical analysis (TMA) on a Mettler 40 apparatus. Different samples of two beech wood plies, each 0.5 mm thick, bonded with each adhesive system, for sample dimensions of 17mm × 5mm × 1 mm, were tested in non-isothermal mode between 25 ◦C and 250 ◦C at a heating rate of 10 ◦C/min in three-point bending. A force varying continuously between 0.1 N, 0.5 N, and back to 0.1 N was applied on the specimens with each force cycle of 12 s (6 s/6 s). The classical mechanics relation between force and deﬂection E = [L3/(4bh3)][∆F/(∆f)] (where L is the sample length, b and h are the sample width and thickness, ∆F is the variation of the force applied, and ∆f is the deﬂection obtained) allows the calculation of the modulus of elasticity (MOE) E for each case tested. Different formulations of adhesives were tested by TMA [35]. Polymers 2021, 13, 595 4 of 15

Polymers 2021, 13, 595 7 of 15 3. Results and Discussion The main components found in the different tannin extracts used coincided with what was obtained by previous work by MALDI-ToF analysis [20,38,39]. Figure1 shows the NH MALDI-ToF spectra for ISP modiﬁed by tannin extracts. The results indicateNH that new com- OH O pounds have formed by reactionsNH of differentO hydrolysable andO condensed polyﬂavonoid HO tanninHN extractsO with soy protein amino acids. TheNH effect is particularlyOH evident when the HN reaction is done at 80 ◦HNC withNH a condensedNH tannin where theNH predominant linkages are O O OH NH2 O O covalent while theOH ionic linkages present for reactionsOOH at ambient temperature have disap- OHpeared.HO The interactionO between tannin constituents and amino acids occurs by reaction of OH the carboxyl group (-COOH) of gallic acid of chestnut tannin with amino groups of amino acids side chains. QuebrachoOH and mimosa tannins react with amino acid functional groups and with theOH hydroxyl groups (-OH) on phenolic rings. (VII).

Data: NO.10001.K21[c] 4 Dec 2020 15:06 Cal: 20201204 4 Dec 2020 15:04 Shimadzu Biotech Axima Performance 2.9.3.20110624: Mode Linear, Power: 75, P.Ext. @ 2300 (bin 78)

%Int. 360 mV[sum= 72070 mV] Profiles 1-200 Smooth Av 50 -Baseline 150

136.7 153.6 100

90 154.7

50 176.7

30 155.7 20 272.7 137.7 175.7 198.7 10 103.8 145.7 158.7 177.6 216.7 278.7 129.7 202.7 0 116.8 100 120 140 160 180 200 220 240 260 280 3001[c] m/z . (a)

Data: NO.10001.K21[c] 4 Dec 2020 15:06 Cal: 20201204 4 Dec 2020 15:04 Shimadzu Biotech Axima Performance 2.9.3.20110624: Mode Linear, Power: 75, P.Ext. @ 2300 (bin 78)

%Int. 11 mV[sum= 2182 mV] Profiles 1-200 Smooth Av 50 -Baseline 150

326.0 100 364.8

352.8 70

60 307.9 330.6 50 380.8 312.7 40 326.9 368.8 30 466.9 300.7 354.8 20 361.6 390.6 412.8 316.7 331.7 482.9 10 410.7 424.8 303.8 318.7 346.7 360.8 376.7 392.8 440.8 0 300 320 340 360 380 400 420 440 460 480 5001[c] m/z (b)

Figure 1. Cont.

Polymers 2021, 13, 595 5 of 15 Polymers 2021, 13, 595 8 of 15

Data: NO.1-4000001.K21[c] 4 Dec 2020 15:11 Cal: 20201204 4 Dec 2020 15:04 Shimadzu Biotech Axima Performance 2.9.3.20110624: Mode Linear, Power: 90, Blanked, P.Ext. @ 2300 (bin 78)

%Int. 8.6 mV[sum= 1719 mV] Profiles 1-200 Smooth Av 50 -Baseline 150

889.1 100

905.1 80

60 907.7 50 873.1 819.6 40 775.5 863.6 951.8 30 731.5 804.3 995. 705.1 20 749.4 837.5 925.5 793.5 881.6 969.7 721.1 857.1 10 809.1 0 700 750 800 850 900 950 10001[c] m/z (c)

Data: NO.1-4000001.K21[c] 4 Dec 2020 15:11 Cal: 20201204 4 Dec 2020 15:04 Shimadzu Biotech Axima Performance 2.9.3.20110624: Mode Linear, Power: 90, Blanked, P.Ext. @ 2300 (bin 78)

%Int. 2.5 mV[sum= 501 mV] Profiles 1-200 Smooth Av 50 -Baseline 150

1177.4 100 1039.9 90

80 1193.5 1083.9 70

1161.5 60

50 1145.6 1013.7 40 1057.5 1101.6 1465.5 30 1172.2 1216.4 1260.3 1015.6 1055.5 1150.0 1449.7 20

0 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 15001[c] m/z (d)

FigureFigure 1. MALDI-ToF 1. MALDI-ToF spectra spectra of tannin-ISPof tannin-ISP adhesive. adhesive.( (a)) 100 100–300–300 Da Da range. range. (b) ( b300) 300–500–500 Da range. Da range. (c) 700 (c–)1000 700–1000 Da range. Da range. (d) 1000–1500(d) 1000–1500 Da range. Da range.

Table 1. Assignment of structures to relevant MALDI-ToF peaks from Figure 1 (a to d). There are a number of compounds formed and detected by the MALDI-ToF analysis

of tannin and ISP, as depicted in Figure1. TheNH MALDI-ToF2 spectra clearly show that amino acids are linked to flavonoid monomers and dimers. In addition, the results reveal soy OH protein polymer chains up to four amino acids linked to flavonoid monomers or dimers, and flavonoid dimers linked to two peptidicO chains as well. Regarding covalent bonds, their formation is favored at high temperature. There are two main reactions occurring: 131 Da= Leucine, no Na+ one is the amination of the tannin, and this can happen with any amino group of an amino acid. The MALDI analysis indicates that many amino acids when alone react with tannin flavonoid monomers through their amino group. However, when the same amino group is linked as it is in the peptide chain of the protein, the only free amino groups that can react in this way are those of the side chain of arginine because they are not involved in the peptidic bond of the skeletal chain of the protein. The reactions of amines with tannins are well documented, it is facile, both using ammonia or primary amines and demonstrated with other techniques other than MALDI [40,41]. Polymers 2021, 13, 595 8 of 15

Data: NO.1-4000001.K21[c] 4 Dec 2020 15:11 Cal: 20201204 4 Dec 2020 15:04 Shimadzu Biotech Axima Performance 2.9.3.20110624: Mode Linear, Power: 90, Blanked, P.Ext. @ 2300 (bin 78)

%Int. 8.6 mV[sum= 1719 mV] Profiles 1-200 Smooth Av 50 -Baseline 150

889.1 100

905.1 80

60 907.7 50 873.1 819.6 40 775.5 863.6 951.8 30 731.5 804.3 995. 705.1 20 749.4 837.5 925.5 793.5 881.6 969.7 721.1 857.1 10 809.1 0 700 750 800 850 900 950 10001[c] m/z Polymers 2021, 13, 595 6 of 15 (c)

Data: NO.1-4000001.K21[c] 4 Dec 2020 15:11 Cal: 20201204 4 Dec 2020 15:04 Shimadzu Biotech Axima Performance 2.9.3.20110624: Mode Linear, Power: 90, Blanked, P.Ext. @ 2300 (bin 78) In the case of arginine, its side chain -NH2 groups can substitute the -OH of the %Int. 2.5 mV[sum= 501 mV] Profiles 1-200 Smooth Av 50 -Baseline 150 ﬂavonoids [40]. The reaction of the carboxylic acid groups of the side chains of aspartic 1177.4 100 and glutamic acid to form an ester is favored with the alcoholic -OH group on the C3 of 1039.9 the heterocyclic ring of the ﬂavonoids. This is logical because, as the phenolic hydroxyl 90 groups have more acidic character, they will be much less likely to react with the carboxylic 80 acid groups1193.5 of the side chains of aspartic and glutamic acids. Reaction with the phenolic 1083.9 70 hydroxyl groups, however, cannot be excluded, but the MALDI technique cannot ascer-

1161.5tain it. It is also evident that this esterification reaction can really occur at the elevated 60 temperatures experienced by the adhesive during adhesive hot pressing. 50 1145.6 The MALDI-ToF analysis shows the peaks corresponding to the molecular weight of 1013.7 the amino acids and tannin extracts and molecular weight of new compounds resulting 40 1057.5 from bonds between them (with and without Na+) in Figure1. In addition, the assignments 1101.6 1465.5 30 1172.2 for the peaks1216.4 of the1260.3 MALDI spectra are shown in Table1. It must be stressed that the 1015.6 1055.5 1150.0 1449.7 20 reaction products between single amino acids and a flavonoid monomer or dimer does not count for the cross-linking of the soy adhesive by the tannin because the reaction in 10 these cases appears not to be able to occur on the –NH- or –COOH that are in the skeletal 0 1000 1050 1100 1150chain of1200 the protein1250 as these1300 are blocked1350 as they1400 participate1450 to the1500 peptidic1[c] bond. Thus, the structures that dom/z not count for cross-linking are of the type such as for the 390 Da peak, and many others (Table1) that correspond to an open fisetinidin structure linked to leucine (d) (without Na+)(I). This clearly shows covalent bond formation between a tannin extracts Figure 1. MALDI-ToF spectra of tannin-ISP monoflavonoid(fisetinidin)adhesive.(a) 100–300 Da range. with (b) 300–500 an amino Da acidrange. -NH (c) 2700–1000group substitution Da range. of the -OH of the (d) 1000–1500 Da range. flavonoids as follows:

TableTable 1. Assignment 1. Assignment of structures of structures to relevant to relevant MALDI-ToF MALDI-ToF peaks peaks from from Figure Figure 1 (a1a to to d). Figure 1d.

NH2 Polymers 2021, 13, 595 9 of 15 OH Polymers 2021, 13, 595 9 of 15

131 Da= Leucine, no Na+ 131 Da= Leucine, no Na+ NH O OH H2N NHNH O NH

NH2 O OH H2N NH NH 146 Da = glutamic acid, no Na+ NH2 O + 154 Da = Leucine with Na + 146146 Da Da = = glutamic glutamic acid,acid, no no Na Na+ 155–156154 Da Da = = aspartic Leucine withacid Nawith+ Na+ 154 Da = Leucine with Na+ 175.7 Da155–156 = no Na+, Da =arginine, aspartic acidcalculated with Na 174.5+ Da + 202.7175.7 Da155–156 Da= withNa+, = no Da Na+, = Tyrosine,calculatedaspartic arginine, acid calculated with 174.5Na 201.8 Da Da, 175.7202.7 Da Da = = no withNa+, Na+, arginine, Tyrosine, calculated calculated 201.8174.5 Da, Da OR no Na+,Tryptophan, calc. 204 Da 202.7 DaOR = withNa+, no Na+,Tryptophan, Tyrosine,calculated calc. 204 Da 201.8 Da, 273 Da Fisetinidin, no NA++ OR no273 Na+,Tryptophan, Da Fisetinidin, no calc. NA 204 Da 326326 Da Da delphinidin delphinidin +Na+Na++ 273 Da Fisetinidin, no NA+ 308308 Da Da = arginine-leucine = arginine-leucine with with Na+ (calculate(calculate 310 310 Da) Da) 326 Da delphinidin +Na+ O 308 Da = arginine-leucine with Na+ (calculate 310 Da) O OH HO NH OH OHOH HO NH OH OH OH + 390390 Da Da= Open = Open fisetinidin-leucine ﬁsetinidin-leucine no NaNa+(calculated(calculated 389.5 389.5 Da), Da), OH 390 Da = Open fisetinidin-leucine no Na+(calculated 389.5 Da), NH O O NH H2N NHNH O NH O OH

NH2 O NH H2N NH NH OOH OH 439 Da = aspartic-leucineNH2 arginineO with Na+ OOH OH 439 Da = aspartic-leucine arginine with Na+ OH OH NH O HO O OH HN NHNH O OH HO O NH2 OH HN NH OH 467 Da = robinetinidin-arginine with Na+,CalculatedNH2 469 Da OH OH 467 Da = robinetinidin-arginine with Na+,Calculated 469 Da OH OH NH O HO O OH HN NHNH O OH HO O NH2 OH HN NH OH OH NH2 OH 483 Da = delphinidin-argininewithNa+(calculated 485 Da) OH OH 483 Da = delphinidin-argininewithNa+(calculated 485OH Da) OH OH NH O CO OH HO O OH NH NHNH O NH CO

HO O NH2 OH NH NH NH OH NH2 OH

Polymers 20212021,, 1313,, 595595 99 ofof 1515 Polymers 2021, 13, 595 9 of 15

NH O NH O OH H N NH NH OH H22N NH NH H22N NH NH NH2 O NH2 O 2 146 Da = glutamic acid, no Na++ 146 Da = glutamic acid, no Na++ 154 Da = Leucine with Na++ 154 Da = Leucine with Na++ 155–156 Da = aspartic acid with Na++ 155–156 Da = aspartic acid with Na++ 175.7 Da = no Na+, arginine, calculated 174.5 Da 175.7 Da = no Na+, arginine, calculated 174.5 Da 202.7 Da = withNa+, Tyrosine,calculated 201.8 Da, 202.7 Da = withNa+, Tyrosine,calculated 201.8 Da, OR no Na+,Tryptophan, calc. 204 Da OR no Na+,Tryptophan, calc. 204 Da 273 Da Fisetinidin, no NA++ 273 Da Fisetinidin, no NA++ 326 Da delphinidin +Na++ 326 Da delphinidin +Na++ 308 Da = arginine-leucine with Na+ (calculate 310 Da) 308 Da = arginine-leucine with Na+ (calculate 310 Da) O O OH OH Polymers 2021, 13, 595 HO NH OH 7 of 15 HO NH OH HO NHOH OH OH OH OH OH 390 Da = Open fisetinidin-leucineTable 1. Cont. no Na++(calculated 389.5 Da), 390 Da = Open fisetinidin-leucine no Na++(calculated(calculated 389.5389.5 Da),Da),

NH O O NH O O NH H N NH NH NH OH H22N NH NH OH H22N NH NH OH NH2 O NH2 O 2 OOH OOH 439 Da = aspartic-leucine arginine with Na+ ++ 439439 Da Da = =aspartic-leucine aspartic-leucine argininearginine with with Na Na++ OH OH OH OH OH NH O NH O HO O HO O HO O HN NH OH HN NH OH NH2 OH NH2 OH 2 OH 467467 Da Da = robinetinidin-arginine = robinetinidin-arginine withwith Na Na+,Calculated++,Calculated 469 469 Da Da 467 Da = robinetinidin-arginine with Na++,Calculated,Calculated 469469 DaDa OH OH OH OH OH NH O NH O HO O HO O HO O HN NH OH HN NH OH NH NH22 OH NH22 OH OH OH + 483483 Da Da = delphinidin-argininewithNa = delphinidin-argininewithNa +(calculated+(calculated 485 485 Da) Da) 483 Da = delphinidin-argininewithNa++(calculated(calculated 485485 Da)Da) OH OH OH OH OH OH NH O CO NH O CO HO O HO O NH NH NH NH NH NH Polymers 20212021,, 1313,, 595595 NH 1010 ofof 1515 NH22 OH NH22 OH OH OH 575575 Da Da = leucine-delphinidin-arginine, = leucine-delphinidin-arginine, no no Na Na+,++, Calculated, CalculatedCalculated 575 575575 Da DaDa OH OH NH

HO O COOH NH NH

COOH NH22 OOC OH NH22 + 601 601Da Da= aspartic = aspartic acid-delphinidin-arginine, acid-delphinidin-arginine, with with Na Na,++ calculated,, calculatedcalculated 601 601601 Da. Da.Da. OH OH O NH O HN HN O HO HN NH OH

NH22 NH NH22 OH OH + 632632 Da Da = arginine-delphinidin-arginine = arginine-delphinidin-arginine no no Na Na,++ calculated,, calculatedcalculated 632 632632 Da DaDa O OH OH OH NH O HN O H NH NH OH

NH22 OH OH 632 Da= Delphinidine-Arginine-Phenylalanine- with Na++,, calculatedcalculated OH OH NH O O HO O NH HN NH NH OH

NH22 O OH OOH 688.8 Da = no Na++,, calculatedcalculated 688.7688.7 Da.,Da., robinetinidin-arginin-leucin-asparticrobinetinidin-arginin-leucin-aspartic acid.acid. Thus,the flavonoid attached to a peptide chain fragment. OH OH NH O O HO O NH HN NH NH OH

NH22 O OH OOH OH 705 Da = no Na++,, calculatedcalculated 704.7704.7 Da,Da, delphinidin-delphinidin-arginine-leucin-aspartic acid. Thus,,thethe flavonoidflavonoid attachedattached toto aa peptidepeptide chainchain fragment,fragment, withwith NaNa++,, calculatedcalculated 728.5.728.5.

NH OH NH O O HO O NH OH HN NH NH NH

NH22 O O OH OOH 857 Da = no Na++,, calculatedcalculated 858858 Da,Da, fisetinidin-arginine-leucin-asparticacid-tryptophan.fisetinidin-arginine-leucin-asparticacid-tryptophan. Thus,the flavonoid attached to a peptide chain fragment.

Polymers 2021, 13, 595 10 of 15 Polymers 2021,, 1313,, 595595 10 of 15 Polymers 2021, 13, 595 10 of 15

+ 575 Da = leucine-delphinidin-arginine, no Na +, Calculated 575 Da 575 Da = leucine-delphinidin-arginine, no Na+, Calculated 575 Da OH OH OH OH OH NH NH HO O COOH HO O COOH HO O NH NH COOH NH NH COOH NH2 OOC COOH NH2 OOC COOH NH2 OOC OH NH2 OH NH2 OH NH2 + 601 Da = aspartic acid-delphinidin-arginine, with Na +, calculated 601 Da. 601 Da = aspartic acid-delphinidin-arginine, with Na+, calculated 601 Da. OH OH OH O OH NH O O NH O HN HN O HO HN HN O NH OH Polymers 2021, 13, 595 HO HN HN O HN NH OH 8 of 15 HO HN NH OH NH2 NH NH2 NH2 NH OH NH2 NH2 NH OH NH2 OH OH OH + 632 Da = arginine-delphinidin-arginineTable 1. Cont. no Na +, calculated 632 Da 632 Da = arginine-delphinidin-arginine no Na+, calculated 632 Da O OH O OH OH OH OH NH O OH NH O HN O H HN O NH NH OH H NH NH OH NH2 OH NH2 OH NH2 OH OH OH + 632 Da= Delphinidine-Arginine-Phenylalanine- with Na+ +, calculated 632632 Da= Da= Delphinidine-Arginine-Phenylalanine- Delphinidine-Arginine-Phenylalanine- with with Na Na, calculated+, calculated OH OH OH OH OH NH O O OH NH O O NH O O HO O NH HO O HN NH NH NH OH HO O HN NH NH NH OH HN NH NH OH NH2 O OH NH2 O OOH OH NH2 O OOH OH OOH + 688.8+ Da = no Na +, calculated 688.7 Da., robinetinidin-arginin-leucin-aspartic acid. 688.8 Da = no Na688.8, calculated Da = no 688.7 Na+ Da.,, calculated robinetinidin-arginin-leucin-aspartic 688.7 Da., robinetinidin-arginin-leucin-aspartic acid. Thus, the flavonoid acid. attached to a peptide Thus,the flavonoid attachedchain fragment. to a peptide chain fragment. Thus,the flavonoid attached to a peptide chain fragment. OH OH OH OH NH O O NH O O HO O NH HO O NH NH NH OH HO O HN NH NH NH OH HN NH NH OH NH2 O OH NH2 O OOH OH NH2 O OOH OH OOH OH OH + + 705 Da = no Na705, calculated Da = no Na 704.7+, calculated Da, delphinidin-arginine-leucin-aspartic 704.7 Da, delphinidin-arginine-leucin-as acid. Thus, thepartic flavonoid acid. attached Thus,the to a peptide chain 705 Da = no Na+, calculated 704.7 Da, delphinidin-arginine-leucin-aspartic acid. Thus,the 705 Da = no Na , calculated 704.7 Da, delphinidin-+ arginine-leucin-as+ partic acid. Thus,the flavonoid attached tofragment, a peptide with chain Na fragment,, calculated with 728.5. Na +, calculated 728.5. flavonoid attached to a peptide chain fragment, with Na+, calculated 728.5. NH NH OH NH OH NH O O OH NH O O NH O O HO O NH OH HO O HN NH NH NH NH OH HO O HN NH NH NH NH OH HN NH NH NH HN NH O O PolymersPolymers 20212021,, 1313,, 595595 NH2 O O 1111 ofof 1515 OH NH2 O OOH O OH 2 OOH + OH OOH 857 Da = no Na , calculated 858 Da,+ fisetinidin-arginine-leucin-asparticacid-tryptophan. Thus, the flavonoid attached to a peptide 857 Da = no Na +, calculated 858 Da, fisetinidin-arginine-leucin-asparticacid-tryptophan. 857 Da = no Na+, calculated 858 Da, fisetinidin-arginine-leucin-asparticacid-tryptophan.chain fragment. Thus,the flavonoid attached to a peptide chain fragment. Thus,the flavonoid attached to a peptide chain fragment. OHOH NHNH OHOH NHNH OO OO HOHO OO NHNH OHOH HNHN NHNH NHNH NHNH NH O O NH22 O O OHOH OOHOOH 873 Da = no Na+, calculated873873 DaDa == no 874no Na+,Na+, Da, robinetinidin-arginine-leucin-asparticacid-tryptophan. calculatedcalculated 874874 Da,Da, robinetinidin-arginine-leucin-asparticacid-robinetinidin-arginine-leucin-asparticacid- Thus, the flavonoid attached to a tryptophan.tryptophan. Thus,Thus, thethe flavonoidflavonoidpeptidechain attachedattached fragment. toto aa peptidepeptide chainchain fragment.fragment.

OHOH NHNH OHOH NHNH OO OO HOHO OO NHNH OHOH HNHN NHNH NHNH NHNH NH O O NH22 O O OHOH OOHOOH OHOH + 889 Da = no Na , calculated 890 Da, delphinidin-arginine-leucin-asparticacid-tryptophan.+ Thus, the flavonoid attached to a peptide 889889 DaDa == nono NaNa+,, calculatedcalculated 890890 Da,Da, delphinididelphinidin-arginine-leucin-asparticacid-n-arginine-leucin-asparticacid- chain fragment. tryptophan.tryptophan. Thus,Thus, thethe flavonoidflavonoid attachedattached toto aa peptidepeptide chainchain fragment.fragment. 1128 Da = no Na+, calculated 1129 Da, fisetinidindimer-arginine-leucin-aspartica cid-tryptophan. Thus, theflavonoid dimer 11281128 DaDa == nono NaNa++,, calculatedcalculated 11291129 Da,Da, fisetinidindimer-arginine-leucin-asparticafisetinidindimer-arginine-leucin-aspartica cid-cid- attached to a peptide chain fragment. tryptophan.tryptophan. Thus,Thus, theflavonoidtheflavonoid dimerdimer attachedattached toto aa peptidepeptide chainchain fragment.fragment.

OHOH NHNH OHOH NHNH OO OO HOHO OO NHNH OHOH HNHN NHNH NHNH NHNH HOHO OH NH O O OH NH22 O O HOHO OHOH OOHOOH HOHO OO OHOH

OHOH OHOH 11941194 DaDa =no=no NaNa++,, calculatedcalculated 11941194 Da,Da, delphinidindimer-arginine-leucin-asparticacid-delphinidindimer-arginine-leucin-asparticacid- tryptophan.Thus,tryptophan.Thus, thethe flavonoidflavonoid dimerdimer attachedattached toto aa peptidepeptide chainchain fragment.fragment.

NHNH OHOH OO NHNH OO OO HNHN OO NHNH OHOH HOHO HNHN NHNH NHNH NHNH O OH NH2 O O O OH NH2 O O OHOH OOHOOH OHOH HOHO OO OHOH

OHOH OHOH 12601260 DaDa == nono NaNa++,, calculatedcalculated 12611261 Da,Da, asparticaspartic acid-cacid-catechin-fisetinidiatechin-fisetinidin-arginine-leucin-n-arginine-leucin- asparticaspartic acid-tryptophan.acid-tryptophan. Thus,Thus, thethe flavonoidflavonoid dimerdimer attachedattached toto aa peptidepeptide chainchain fragmentfragment ++ cross-linkedcross-linked withwith anotheranother aminoacid.aminoacid.

NHNH NHNH OHOH OO NHNH OO OO HOHO HNHN OO NHNH OHOH HNHN HNHN NHNH NHNH NHNH O O OH NH O O O O OH NH22 O O OHOH OOHOOH OHOH HOHO OO OHOH

OHOH OHOH 14491449 DaDa == nono NaNa++,, calculatedcalculated 14491449 Da,Da, Tryptophan-asparticTryptophan-aspartic acid-catechin-fisetinidin-acid-catechin-fisetinidin- arginine-leucin-asparticarginine-leucin-aspartic acacid-tryptophan.id-tryptophan. Thus,Thus, thethe flavonoidflavonoid dimerdimer linkedlinked toto twotwo peptidepeptide chainchain fragmentsfragments == crocross-linkss-link proteinprotein tannintannin proved.proved.

SeveralSeveral bandsbands areare observedobserved inin thethe FTIRFTIR spectrumspectrum inin FigureFigure 2.2. SomeSome areare asas follows:follows: cmcm−−11;; 17351735 cmcm−−11 (shoulder, (shoulder, C=OC=O esters),esters), 17111711 cmcm−−11 (shoulder, (shoulder, C=OC=O acidacid inin C-C-COOH)C-C-COOH) 16801680 cmcm−−11 (C=O(C=O amideamide andand C=OC=O inin -COOH-COOH ofof acid),acid), 16251625 cmcm−−11 (primary(primary amineamine asymmetricasymmetric

Polymers 2021, 13, 595 11 of 15

Polymers 2021,, 1313,, 595595 11 of 15

OH NH OH OH NH O O NH HO O OH NH OH HN NHNH O NH O NH HO O NH OH NH NH2 NH O NH O OH HN NH NH OOH NH NH2 O O 873 Da = no Na+,OH calculated 874 Da, robinetinidin-arginine-leucin-asparticacid-OOH 873 Datryptophan. = no Na+, Thus,calculated the flavonoid 874 Da, robinetinidin-arginine-leucin-asparticacid- attached to a peptide chain fragment. tryptophan. Thus, the flavonoid attached to a peptide chain fragment. OH NH OH OH NH O O NH OH HO O NH O NH O OH HN NHNH O NH O NH HO O NH OH NH2 O O OH HN NH NH OOH NH NH O O OH NH2 O O OH OOH OH + Polymers 2021, 13, 595 889 Da = no Na , calculated 890 Da, delphinidin-arginine-leucin-asparticacid- 9 of 15 889 tryptophan.Da = no Na+ ,Thus, calculated the flavonoid 890 Da, delphinidi attached ton-arginine-leucin-asparticacid- a peptide chain fragment. 1128 Datryptophan. = no Na+, calculated Thus, the 1129flavonoid Da, fisetinidindimer-arginine-leucin-aspartica attached to a peptide chain fragment. cid- 1128tryptophan. Da = no Na +Thus,, calculated theflavonoid 1129 Da, dimer fisetinidindimer-arginine-leucin-aspartica attached to a peptide chain fragment. cid- Table 1. Cont. tryptophan. Thus, theflavonoid dimer attached to a peptide chain fragment. OH NH OH OH NH O O NH HO O OH NH OH HN NHNH O NH O NH HO HO O OH NH2 O NH O OH HO OH HN NH NH OOH NH HO HO O OH NH2 O O HO OH OH OOH HO O OH OH OH OH 1194 Da =no Na+,OH calculated 1194 Da, delphinidindimer-arginine-leucin-asparticacid- 1194 Da =no Na1194+, calculated tryptophan.Thus,Da =no Na 1194+, calculated Da, delphinidindimer-arginine-leucin-asparticacid-tryptophan.Thus, the flavonoid 1194 Da, dimer delphinidindimer-arginine-leucin-asparticacid- attached to a peptide chain fragment. the flavonoid dimer attached to a peptide chain fragment. tryptophan.Thus, the flavonoid dimer attached to a peptide chainNH fragment. OH O NH O O NH HN O OH NH OH HO O HN NH NH O NH O NH O HN O OH NH2 O NH O OH HN O OH NHOOH OH HO HN NH NH NH OH HO O O OH NH22 O O OH OH OOH OH HO O OH OH OH OH 1260 Da = no Na+, calculatedOH 1261 Da, aspartic acid-catechin-fisetinidin-arginine-leucin- 1260 Da = no1260 Na+aspartic, Da calculated = no acid-tryptophan. Na 1261+, calculated Da, aspartic 1261 Thus, acid-catechin-fisetinidin-arginine-leucin-aspartic Da, the aspartic flavonoid acid-c dimeratechin-fisetinidi attached to an-arginine-leucin- peptide acid-tryptophan. chain Thus, the flavonoidaspartic dimer acid-tryptophan. attachedfragment to a peptide +Thus, cross-linked the chain flavonoid fragment with another dimer + cross-linked attached aminoacid. with to a another peptide aminoacid. chain fragment + cross-linked with another aminoacid. NH fragment + cross-linked with another aminoacid. NH OH NH O NH O O NH HO HN O OH NH OH HN O HN NH NH O NH O NH HO O O HN O OH NH2 O NH O OH HN OH HN NH NH OOH NH O O OH HO O OH NH2 O O OH OH 2 OOH Polymers 2021, 13, 595 OH OOH 5 of 15 OH HO O OH OH OH OH + 1449 Da = no Na ,1449 calculated Da = 1449no Na Da,+, Tryptophan-asparticcalculated 1449OH Da, acid-catechin-fisetinidin-arginine-leucin-aspartic Tryptophan-aspartic acid-catechin-fisetinidin- acid-tryptophan. Thus, the flavonoid dimer linked to two peptideextracts chain fragmentsmonoflavonoid(fisetini = cross-link proteindin) tanninwith an proved. amino acid -NH2 group substitution of the - 1449arginine-leucin-aspartic Da = no Na+, calculated acid-tryptophan. 1449 Da, Tryptophan-aspartic Thus, the flavonoid acid-catechin-fisetinidindimer linked to two OH of the flavonoids as follows: arginine-leucin-asparticpeptide chain ac fragmentsid-tryptophan. = cross-link Thus, theprotein flavonoid tannin dimer proved. linked to two peptide chain fragments = cross-link protein tannin proved. Several bands are observed in the FTIR spectrum in FigureO 2. Some are as follows: cm−1;Several 1735 cm bands−1 (shoulder, are observed C=O esters), in the 1711 FTIR cm spectrum−1 (shoulder, in Figure C=O acid 2. Some in C-C-COOH) are as follows: 1680 cmcm−−11; (C=O1735 cmamide−1 (shoulder, and C=O C=O in esters),-COOH 1711 of acid),cm−1 (shoulder, 1625 cm− C=O1 (primaryOH acid in amine C-C-COOH) asymmetric 1680 cm−1 (C=O amide and C=O in -COOHHO of acid), 1625 NHcm−1 (primary amine asymmetricOH OH

(I).

Equally, there are manyEqually, peaks ofthere single are amino many acidspeaks or of peptide single amino fragments acids that or peptide have fragments that have not reacted with the ﬂavonoidsnot reacted of with the tannin. the flavonoids In addition, of the other tannin. peaks In correspondaddition, other to the peaks correspond to the several amino acids andseveral tannin amino extract acids monomers and tannin and extract oligomers monomers of different and molecularoligomers of different molecular weight such asthose atweight 131 Da, such 146 Da,asthose 154 Da,at 131 156 Da, Da, 146 175.7 Da, Da, 154 202.7 Da, 156 Da, Da, 204 175.7 Da, 273 Da, Da, 202.7 Da, 204 Da, 273 Da, 326 Da. In addition, peptide326 Da. two In aminoaddition, acids peptide dimers two such amino as arginine-leucine acids dimers such with as Na+(II) arginine-leucine with Na+(II) were assigned in Table1were from assigned Figure1 a,b.in Table 1 from Figure 1a,b.

NH O OH H2N NH NH

NH2 O (II).

In the same type of peptide fragments the peak at 439 Da (III) assigned to the trimer of aspartic, leucine and arginine (with Na+) from the soy protein polymer chain (Table 1, Figure 1b) and others.

NH O O NH H2N NH NH OH

NH2 O OOH

(III).

The type of linkages that will count for cross-linking the main body of the protein are, for example, the link represented by the peak at 467 Da (IV) assigned to a flavonoid robinetinidin linked to an arginine (with Na+) (Table 1, Figure 1b).

Polymers 2021, 13, 595Polymers 2021, 13, 595 5 of 15 5 of 15

extracts monoflavonoid(fisetiniextracts monoflavonoid(fisetinidin) with an aminodin) acidwith -NH an amino2 group acid substitution -NH2 group of thesubstitution - of the - OH of the flavonoidsOH of as the follows: flavonoids as follows:

O O

OH OH HO HONH NH OH OH OH OH

OH OH

(I). (I).

Equally, there areEqually, many peaksthere are of singlemany aminopeaks ofacids single or peptideamino acids fragments or peptide that havefragments that have not reacted withnot the reacted flavonoids with of the the flavonoids tannin. In of addition, the tannin. other In peaksaddition, correspond other peaks to the correspond to the several amino acidsseveral and amino tannin acids extract and monomers tannin extract and monomersoligomers ofand different oligomers molecular of different molecular Polymers 2021, 13, 595 weight such asthoseweight at 131such Da, asthose 146 Da, at 154131 Da, 156146 Da, 175.7154 Da, Da, 156 202.7 Da, Da, 175.7 204 Da,10 Da, of202.7 15273 Da, 204 Da, 273 Da, 326 Da. In addition,326 Da.peptide In addition, two amino peptide acids two dimers amino such acids as arginine-leucine dimers such as arginine-leucine with Na+(II) with Na+(II) were assigned inwere Table assigned 1 from Figurein Table 1a,b. 1 from Figure 1a,b.

NH NH O O OH OH H N NH NH H2N NH 2 NH NH O NH2 O 2 (II). (II).

In theIn same the typesame of type peptideIn of the peptide fragmentssame typefragments theof peptide peak the atpe fragments 439ak at Da 439 (III) Dathe assigned (III)peak assigned at to439 the Da to trimer (III)the trimerassigned to the trimer of aspartic,of aspartic, leucine leucine andof arginineaspartic, and arginine (withleucine (with Na+) and Na+) from arginine thefrom soy(with the protein soy Na+) protein polymerfrom polymerthe chainsoy protein chain (Table (Table 1polymer, 1, chain (Table 1, FigureFigure1b) and 1b) others. and others.Figure 1b) and others.

NH NH O O O O NH NH NH NH OH H2N NH H2N NH OH

NH2 NHO2 O OOHOOH

Polymers 2021, 13, 595 (III). (III). 6 of 15

The type of linkages that will count for cross-linking the main body of the protein The typeThe oftype linkages of linkages that will that count will count for cross-linking for cross-linking the main the bodymain ofbody the of protein the protein Polymers 2021, 13, 595 are, for example, the link represented by the peak at 467 Da (IV) assigned6 of 15 to a flavonoid are, forare, example, for example, the link the represented link represented by the by peak the atpe 467ak at Da 467 (IV) Da assigned (IV) assigned to a ﬂavonoid to a flavonoid robinetinidinrobinetinidin linked linked torobinetinidin an arginineto an arginine (withlinkedOH Na+)(with to an (TableNa+) arginine (Table1, Figure (with 1, 1Figure b).Na+) (Table1b). 1, Figure 1b). OH OH NH O HO O OH HN NHNH O OH HO O NH2 OH HN NH OH

(IV). NH2 OH

Similarly, the 483 Da (V) peak assigned to a delphinidin-arginine dimer (with Na+). (IV).

Similarly,Similarly, the 483 Dathe (V)483 peak Da (V) assigned peak assigned to a delphinidin-arginine to a delphinidin-argini dimerne (with dimer Na+). (with Na+).

OH OH OH OH NH NH O O HO HO O O HN HNNH NH OH OH NH 2 NH2 OH OH OH OH

(V). (V). There are also several peaks in which a flavonoid is linked to a peptidic fragment containingThere arginineare also severalsuch as peaksthe peak in whichat 575 Da,a flavonoid a leucine-delphinidin-arginine is linked to a peptidic oligomer,fragment containingno Na+ (VI). arginine such as the peak at 575 Da, a leucine-delphinidin-arginine oligomer, no Na+ (VI). OH OH OH OH OH NH OH O CO NH O CO HO O HO O NH NH NH NH NH NH NH2 OH NH2 OH OH

OH (VI). (VI). This shows that tannin flavonoid units are indeed linked and cross-link through the aminoThis acids shows side thatchains tannin of the flavonoid main skeletal units peptidicare indeed chain linked of the and protein. cross-link There through are many the aminopeaks andacids structures side chains confirming of the main this, skeletal such as peptidic the peaks chain at 601 of the Da, protein. 632 Da, There688 Da, are 705 many Da, peaks857 Da, and 873 structures Da, 889 Da, confirming 1146 Da, this,1161 such Da, 1177as the Da, peaks 1194 at Da, 601 1260 Da, 632Da andDa, 6881449 Da, Da 705 (Table Da, 8571). For Da, example, 873 Da, 889 the Da,species 1146 at Da, 1449 1161 Da Da, (VII) 1177 indicates Da, 1194 clearly Da, 1260 that Da covalent and 1449 cross-linking Da (Table 1).of theFor protein example, with the a speciescondense atd 1449 tannin Da is(VII) indeed indicates possible. clearly that covalent cross-linking of the protein with a condensed tannin is indeed possible.

Polymers 2021, 13, 595 6 of 15

OH OH NH O HO O HN NH OH

NH2 OH

(IV).

Similarly, the 483 Da (V) peak assigned to a delphinidin-arginine dimer (with Na+).

OH OH NH O HO O HN NH OH

NH2 OH OH Polymers 2021, 13, 595 11 of 15 (V).

ThereThere are are also also several several peaks peaks in in whichwhich aa ﬂavonoidflavonoid is linked to to a a peptidic peptidic fragment fragment containingcontaining arginine arginine such such as as the the peak peak at at 575 575 Da, Da, a leucine-delphinidin-argininea leucine-delphinidin-arginine oligomer, oligomer, no Na+no (VI).Na+ (VI).

OH OH OH NH O CO HO O NH NH NH

NH2 OH OH

(VI).

ThisThis shows shows that that tannin tannin ﬂavonoid flavonoid unitsunits areare indeedindeed linked and and cross-link cross-link through through the the aminoamino acids acids side side chains chains of of the the main main skeletal skeletal peptidicpeptidic chain of of the the protein. protein. There There are are many many peakspeaks and and structures structures conﬁrming confirming this, this, such such asas thethe peaks at 601 601 Da, Da, 632 632 Da, Da, 688 688 Da, Da, 705 705 Da, Da, 857857 Da, Da, 873 873 Da, Da, 889 889 Da, Da, 1146 1146 Da, Da, 1161 1161 Da, Da, 1177 1177 Da, Da, 1194 1194 Da, Da, 1260 1260 Da Da and and 1449 1449 Da Da (Table (Table1 ). For1). example, For example, the species the species at 1449 at Da1449 (VII) Da indicates(VII) indicates clearly clearly that covalentthat covalent cross-linking cross-linking of the Polymers 2021, 13, 595 7 of 15 proteinof the with protein a condensed with a condense tannind istannin indeed is indeed possible. possible.

NH NH OH O NH O O HO HN O NH OH HN HN NH NH NH

O O OH NH2 O O OH OOH OH HO O OH

OH OH (VII).

Data: NO.10001.K21[c] 4 Dec 2020 15:06 Cal: 20201204 4 Dec 2020 15:04 ShimadzuSeveral Biotech bands Axima are Performance observed 2.9.3.20110624: in the FTIR Mode spectrum Linear, Power: in Figure 75, P.Ext.2. Some@ 2300 are(bin 78) as follows: cm%Int.−1; 1735360 cm mV[sum=−1 (shoulder, 72070 mV] C=O Profiles esters), 1-200 1711Smooth cm Av− 501 (shoulder,-Baseline 150 C=O acid in C-C-COOH) 136.7 −1 153.6 −1 1680100 cm (C=O amide and C=O in -COOH of acid), 1625 cm (primary amine asymmetric −1 −1 −1 −1 90 stretch R-CH2-NH2), 1530 cm154.7(amide), 1480 cm (amide), 1333 cm and 1292 cm −1 −1 −1 (secondary80 amine, R-NH-R), 1165 cm , 1150 cm , 1030 cm (primary amine asymmetric −1 −1 stretch70 R-CH2-NH2), 1000 cm , 850 + 700 cm (primary amine).

50 176.7

30 155.7 20 272.7 137.7 175.7 198.7 10 103.8 145.7 158.7 177.6 216.7 278.7 129.7 202.7 0 116.8 100 120 140 160 180 200 220 240 260 280 3001[c] m/z . (a)

Data: NO.10001.K21[c] 4 Dec 2020 15:06 Cal: 20201204 4 Dec 2020 15:04 Shimadzu Biotech Axima Performance 2.9.3.20110624: Mode Linear, Power: 75, P.Ext. @ 2300 (bin 78)

%Int. 11 mV[sum= 2182 mV] Profiles 1-200 Smooth Av 50 -Baseline 150

326.0 100 364.8

352.8 70

Polymers 2021, 13, 595 12 of 15

stretch R-CH2-NH2), 1530 cm−1(amide), 1480 cm−1(amide), 1333 cm−1and 1292 cm−1 (secondary amine, R-NH-R), 1165 cm−1, 1150 cm−1, 1030 cm−1 (primary amine asymmetric stretch R-CH2-NH2), 1000 cm−1, 850 + 700 cm−1 (primary amine). In the FTIR, there are indications that support what was found by MALDI ToF analysis, namely that reactions occur through the primary amines in ISP by forming secondary amines, this being indicated by the two characteristic bands at 1333 and 1292 cm−1. That esterification of the tannin hydroxyls by the protein side chain acids does also occur is demonstrated by the 1735 cm−1 shoulder. These bands appear to support the Polymers 2021, 13, 595 MALDI results that the reaction of the protein with the flavonoid units of the tannin can 12 of 15 occur both through the amino group of a protein side chain or by esterification with the acid group of a side chain of the protein.

◦ Figure 2. FTIRFigure of reaction 2. FTIR product of reaction mixture product of condensed mixture tanninof conden withsed ISP tannin at 80 withC without ISP at 80°C catalyst without (lower catalyst spectrum) and ◦ with catalyst (higher(lower spectrum)spectrum) bothand with at 80 catalystC. (higher spectrum) both at 80°C.

The TMA resultsIn the for FTIR, different there are tannin-modifi indications thated soy support adhesives what are was shown found byin Figure MALDI 3. ToF analysis, The influencenamely of different that reactionsconstituents occur of ches throughtnut tannin the primary extract amines(Chest), in quebracho ISP by forming (Qub) secondary −1 and mimosaamines, (Mimo) this tannins being on indicated the modulus by the of twoelasticity characteristic (MOE) of bands SF and at 1333ISP adhesives and 1292 cm . That esterification of the tannin hydroxyls by the protein side chain acids does also occur is as a function of temperature is depicted in Figure−1 3a,b. When comparing the MOE content of different demonstratedadhesive formulations, by the 1735 it is cm clearshoulder. that the maximum These bands MOE appear values to supportof resins the MALDI results that the reaction of the protein with the flavonoid units of the tannin can occur both prepared with ISP are higher than those obtained with SF adhesive. The proportion of through the amino group of a protein side chain or by esterification with the acid group of tannins added varied from 5% to 10% to 15% by weight. All the adhesives tested, show an a side chain of the protein. increase in the maximum MOE value with increasing proportions of both hydrolysable The TMA results for different tannin-modified soy adhesives are shown in Figure3. and condensed. The highest MOE for both tannin-modified ISP and SF adhesives were The influence of different constituents of chestnut tannin extract (Chest), quebracho (Qub) obtained by the addition of 10% wt quebracho tannins (5660 MPa for ISP and 3799 MPa and mimosa (Mimo) tannins on the modulus of elasticity (MOE) of SF and ISP adhesives for SF), 5% wt mimosa tannin (5397 MPa for ISP and 3655 MPa for SF) and 10% wt to 15% as a function of temperature is depicted in Figure3a,b. When comparing the MOE content wt chestnut tannin for ISP (5069 MPa) and SF (3341 MPa). of different adhesive formulations, it is clear that the maximum MOE values of resins prepared with ISP are higher than those obtained with SF adhesive. The proportion of tannins added varied from 5% to 10% to 15% by weight. All the adhesives tested, show an increase in the maximum MOE value with increasing proportions of both hydrolysable and condensed. The highest MOE for both tannin-modified ISP and SF adhesives were obtained by the addition of 10% wt quebracho tannins (5660 MPa for ISP and 3799 MPa for SF), 5% wt mimosa tannin (5397 MPa for ISP and 3655 MPa for SF) and 10% wt to 15% wt chestnut tannin for ISP (5069 MPa) and SF (3341 MPa).

Polymers 2021, 13, 595 13 of 15 Polymers 2021, 13, 595 13 of 15

6000 Tannin 0 % 5000 Tannin 5 % Tannin 10 % Tannin 15 % 4000

3000

MOE (MPa) MOE 2000

1000

0 ISP ISP-CHEST ISP-QUB ISP-MIMO Materials

(a)

6000 Tannin 0 % 5000 Tannin 5 % Tannin 10 % 4000 Tannin 15 %

3000

MOE (MPa) MOE 2000

1000

0 SF SF-CHEST SF-QUB SF-MIMO Materials

(b)

Figure 3.EffectEffect of of different different tannins tannins extracts extracts on on MOE MOE of of ISP ISP ( (aa)) and and SF SF ( (bb).).

Based onon thethe results results obtained obtained from from MALDI-TOF MALDI-TOF mass mass spectrometry, spectrometry, covalent covalent and ionic and bondsionic bonds can form can form between between soy amino soy amino acids acids and tanninsand tannins (Figure (Figure3). For 3). these For these reasons, reasons, the MOEthe MOE of tannin-modified of tannin-modified soy floursoy fl adhesiveour adhesive is increased. is increased. In previous In previous studies studies on tannin on tannin and proteinand protein interactions, interactions, different different bonds suchbonds as hydrogen,such as covalent,hydrogen, ionic covalent, and hydrophobic ionic and bondinghydrophobic between bonding tannin between and protein tannin were and reported protein [21were,42, 43reported], and the [21,42,43], reaction betweenand the tanninsreaction andbetween ammines tannins has and been ammines recently has studied been andrecently codified studied in depth and codified [44]. Condensed in depth polyflavonoid[44]. Condensed quebracho polyflavonoid and mimosa quebracho tannins and when mimosa compared tannins to hydrolysable when compared chestnut to tanninhydrolysable showed chestnut better reactiontannin showed with soy better protein. reaction This with happened soy protein. because This of thehappened higher reactivitybecause of of the aromatic higher -OHreactivity groups of inaromatic condensed -OH tannins groups than in condensed the aromatic tannins and aliphaticthan the -OHaromatic groups and in aliphatic hydrolysable -OH tannins. groups Betweenin hydrolysable two industrial tannins. polyflavonoids, Between two quebrachoindustrial appearspolyflavonoids, to react wellquebracho with soy appears protein. to Compared react well towith the soy more protein. branched Compared structure to of the mimosa more tanninbranched oligomers, structure quebracho of mimosa tannin tannin oligomers oligomers, are predominantly quebracho tannin linear oligomers and the inter are flavonoidpredominantly link in linear quebracho and the tannin inter structureflavonoid is link more in easily quebracho hydrolysable. tannin structure These structural is more

Polymers 2021, 13, 595 14 of 15

differences also contribute to the signiﬁcant differences in reaction of two condensed tannins with soy (SF and ISP) in slurry systems.

4. Conclusions Three types of natural tannins—chestnut tannin extracts, quebracho and mimosa condensed polyﬂavonoid tannins—were used as cross-linking agents for SF and ISP adhesives for wood bonding. MALDI-ToF mass spectrometry clearly shows that ionic and covalent bonding between amino acids and tannin constituents are formed at ambient temperature and that covalent bonds do predominate at elevated temperature as present in the hot-pressing of wood panels. They are the bonds exclusively between the –NH2 and –COOH groups of side chains of amino acids in the peptide chain that participate in the tannin/protein cross-linking; thus, the amino acids arginine, aspartic acid and glutamic acid are the only ones able to participate. The TMA results also indicate a higher MOE and better adhesion properties of soy adhesives by adding tannins to their formulation. The quebracho tannin showed higher MOE for both SF and ISP adhesives. Higher MOE was an evidence to conﬁrm bond formation determined by MALDI-ToF. The results obtained from this research clearly show the potential of tannins as cross-linkers for soy-based adhesives to produce totally green adhesives for wood bonding.

Author Contributions: Methodology, S.G. and A.P.; validation, S.G., A.P., A.N.P., X.C., R.H.; investigation, S.G. and A.P., and X.C.; writing—original draft preparation, S.G., A.P., and A.N.P. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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