Covalent and Ionic Bonding between Tannin and Collagen in Leather making and shrinking: A MALDI-ToF study

Collagen powder hydrolysates were reacted with a solution of commercial mimosa bark tannin extract. The mixture was prepared at ambient temperature and prepared at 80°C to determine what reactions, if any, did occur between the collagen protein through its amino acids and the polyphenolic condensed tannin. The reaction products obtained were analyzed by matrix assisted laser desorption ionization time-of- ﬂ ight (MALDI ToF) mass spectrometry. Reactions between the two materials did appear to occur, with the formation of a relatively small proportion of covalent and ionic linkages at ambient temperature but a considerable proportion of covalent linkages tannin-protein amino acids and the disappearance of ionic bonds. The linkages between the two materials appeared to be by amination of the phenolic – OHs of the tannin by the amino groups of the non-skeletal side chains of arginine, and by esteri ﬁ cation by the – COOH groups of glutamic and aspartic acid of the aliphatic alcohol-OH on the C3 site of the ﬂ avonoid units heterocycle of the tannin. The proportion of covalent linkages increases markedly and predominate with increasing temperatures. This tightening of the tannin-protein covalent network formed may be an additional contributing factor both to leather wear resistance and performance as well to leather shrinking when this is subjected to excessive temperatures.


Introduction
Vegetable tannins, both hydrolysable and condensed ones, are polyphenolic materials that have been used for a long time for the manufacture of heavy-duty leather. The structures of hydrolysable and condensed tannins are well studied too [1][2][3][4][5][6][7][8][9]. The interaction between any of the two types of vegetable tannins and the collagen of hides to prepare leather has always been known to depend on the strong complexation of the tannin with the collagen protein by the innumerable secondary forces acting between them. While this is age-old acquired and recognized knowledge, the effect of temperature on both vegetable tanning and on excessive temperatureinduced shrinking of tanned leather has been ascribed to many existing effects, such as moisture loss and leather drying and others [10][11][12][13][14][15]. Such effects exist, are real and have been studied [10][11][12][13][14][15]. However, the possibility of the influence on these effects of the formation of temperature-induced covalent bonds between tannin and collagen has never really been considered.
Recently, research work on the interaction of hydrolysable and condensed tannins with soy protein in the field of wood adhesives [16][17][18] has brought such a hypothesis to the fore also for other proteins interaction with tannins. This work has shown that at ambient temperature tannins do appear to form both ionic and covalent bonds with soy protein [16][17][18]. While the use of the analyzed sample showed bonds formed at room temperature the subsequent use of the resins so formed as thermoset wood adhesives for wood panels would appear to indicate that such bonds might persist or even form in greater proportion even at higher temperatures.
The presence in leather of the combination of tannin with collagen, this being a proteic material, renders probable that also in this case covalent and ionic bonds between the two substances do occur at ambient and higher temperature. These might play a role in leather stability as well as in its shrinking under excessive temperature conditions. The latter might be due to the contribution of either the progressive increase as a function of temperature of covalent bonds protein/tannin or to the tightening due to the tannin structure rearrangement when this is covalently bonded to collagen..
The work presented here it is only aimed at, and limited to determine if covalently bonded structures occurs between collagen and tannin in view to render possible to understand in future their contribution to leather shrinking and stability as a function of the temperature.

Preparation
The pure collagen powder hydrolysates were added to water and mixed with a mechanical stirrer. The tannin solution (15 wt% based on collagen dry weight) was then added to the collagen hydrolysates slurry and stirred for 60 minutes. The tannin solution in water was prepared at 45 wt% concentration. The same procedure was followed for the preparation at 80°C, but the mixture was heated at 80°C for 60 min rather than at ambient temperature. The pH of the solutions were adjusted to 7 before analysis.

MALDI ToF Analysis
Samples for matrix assisted laser desorption ionization time-of-flight (MALDI-ToF) analysis were prepared by first dissolving 7.5 mg of the samples in 1 mL 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 sample holder were first covered with 2 µL of a NaCl solution 0.1M 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 used 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. The measurements were carried out making 1000 profiles. The spectra were precise at +1 Da.

Results and Discussion
The experiment conducted were to examine by matrix assisted laser desorption ionization time of flight (MALDI ToF) mass spectrometry the products obtained by reaction of a condensed tannin with collagen at ambient temperature and at 80°C to determine if covalently co-reacted structures occur, in which cases and to what extent in the two cases. The tannin used is mainly composed of four different flavonoid units linked either C4-C6 or C4-C8, namely robinetinidin, fisetinidin, catechin and gallocatechin, with the first two being strongly predominant [19][20][21][22] their respective percentages in mimosa tannin being approximately 68%, 22%, 5%, 5% by weight [19][20][21]. The M.W. of robinetinidin and catechin is the same. Both flavonoids are present in mimosa tannin but robinetinidin is greatly predominant. Any structure in the Tab. 1 and Tab. B1 in which one of the two is mentioned could also be the other. Due to its predominance robinetinidin should be the favourite one for this tannin.

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The MALDI ToF spectrum in Fig. 1 of the reaction at ambient temperature between collagen and mimosa condensed tannin showed only a limited amount of covalent co-reaction between tannin and single amino acids. These are shown in Tab. 1. There are three considerations to keep in mind from the results in Tab. 1.   It is then evident from Tab. 1 and Fig. 1 that at ambient temperature that valid covalent and ionic linkages between tannin units and the body of the protein do exist but their number appears to be relatively low. These strong linkages probably also contribute to the good performance and durability of vegetable tanned leather.
The situation appears to be rather different at the higher temperature of 80°C used. In Figs. A1a-A1h, and in Tab. B1 are shown the links formed between tannins monomers and oligomers with both amino acids and short peptide sequences of predominant amino acids present in collagen. Again, it is necessary to distinguish between two cases: (i) Amino acid monomers reacted with the tannin by the -COOH and the -NH 2 groups of the amino acid that enter in the formation of the skeletal peptide chain of the protein, and (ii) -COOH and -NH 2 groups of the amino acid side chains that are and will not be involved in the skeletal peptide sequence of the protein. The first of these will not contribute to the cross-linking of the protein, thus while formed they should not be considered. The second ones of these instead, if formed, will actively participate to cross-liking of the protein. Thus, only arginine, glutamic acid and aspartic acid, in relative proportions 6.1%, 12.2% and 7.3% respectively in collagen, belong to the second category, the first due to its -NH 2 and =NH side chain groups capable of reacting with the acid phenolic -OH of the tannin [23-26], and the other two due to the presence in each of an extra -COOH side chain group leading to esterification of the aliphatic alcoholic -OH on the C3 site of tannin flavonoids. It is already evident from this latter list that a lot more reaction products between tannin and collagen amino acids do occur at 80°C (Tab. B1, Figs. A1a-A1h) than at ambient temperature (25°C) (Tab. 1, Fig. 1). It is equally evident that at 80°C, differently than at ambient temperature, no ionic bound reaction products are formed, the totality of the reaction products being covalently bound. This appear to indicate that as vegetal tanned leather is heated further covalent cross-linking by the tannin does occur tightening the network and contributing to the phenomenon of heat induced leather shrinkage.
As regards the reaction products found, a few are noticeable indicating the capacity of covalent crosslinking of the protein by the tannin. Thus the two species 601 Da and 632 Da, respectively being assigned to the sequence aspartic acid-gallocatechin-arginine and arginine-gallocatechin-arginine are the first ones indicating that cross-linking can occur.
As one considers the species formed at progressively larger molecular masses, the interpretation of the actual structure can be variable. An example of a flavonoid monomer linked to a long peptide chain, such as the species at 889 Da can be assigned to a peptide chain fragment of sequence-arginine-leucin-aspartic acid-glycin-glutammic acid having reacted with a gallocatechin yielding structures, among other possibilities, such as: The residual groups in the above amino acids sequence can also react, forming species of higher molecular weight indicating that extensive covalent cross-linking can in reality occur between tannin and protein as for example in the structure assigned to the peak at 1805 Da, this being an example of how the body of the protein is cross-linked by the tannin.

Conclusions
All the above indicates that in the tanning of hides with vegetal tannins a certain number of covalent and ionic bonds do occur between tannin and protein contributing to leather solidity and wear resistance. The proportion of these linkages increases with increasing temperatures of tanning, and in particular the proportion of tannin-protein covalent linkages does increase until these become the only one presents. While such covalent cross-linking may well be a contributing factor to the stability and performance of leather, when this becomes excessive as leather is subjected to higher temperatures, may well be also a strong additional, contributing factor to leather shrinking.
Funding Statement: The authors received no specific funding for this study.

Conflicts of Interest:
The authors declare that they have no conflicts of interest to report regarding the present study.