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-flight (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 esterification by the –COOH groups of glutamic and aspartic acid of the aliphatic alcohol-OH on the C3 site of the flavonoid 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.
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 [
Recently, research work on the interaction of hydrolysable and condensed tannins with soy protein in the field of wood adhesives [
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.
Commercial Mimosa tannin extract (
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 min. 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.
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.
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 [
Peak | Species |
---|---|
370 Da | fisetinidin-proline |
374 Da | fisetinidin-threonine |
376 Da | gallocatechin-alanine OR robinetinidin-serine |
380 Da | fidetinidin- +serine, ionic bond |
388 Da | fisetinidin- +proline, ionic bond |
390 Da | fisetinidin-aspartic acid |
392 Da | fisetinidin- +threonine ionic bond |
396 Da | robinetinidin- +serine ionic bond |
400 Da | robinetinidin-serine + Na+ |
408 Da | fisetinidin+ -aspartic acid, ionic bond |
412 Da | fisetinidin-aspartic acid +Na+ |
418 Da | robinetinidin-glycine-alanine |
426 Da | glutamic acid-fisetinidin +Na+ |
437 Da | robinetinidin- +glycine-alanine, ionic bond OR robinetinidin-phenylalanine |
440 Da | gallocatechin+ -aspartic acid, ionic bond |
442 Da | robinetinidin-glutamic acid +Na+ |
454 Da | gallocatechin+ -glutamic acid, ionic bond |
458 Da | gallocatechin-glutamic acid +Na+ |
469 Da | robinetinidin-arginine +Na+ |
484 Da | glycine-fisetinidin-glutamic acid +Na+ |
The MALDI ToF spectrum in
There are flavonoid monomers linked to amino acids by covalent bonds and linked by ionic bonds. However, in the case of the covalent bonds that would count on linking the protein structure to a flavonoid tannin only the ones between amino acids possessing a side chain with a –NH2 [
Valid covalent linkages between the protein and tannin flavonoid units are then the species at 390 Da, 412 Da 426 Da, 442 Da, 458 Da and 484 Da in
It is then evident from
The situation appears to be rather different at the higher temperature of 80°C used. In
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 (
As regards the reaction products found, a few are noticeable indicating the capacity of covalent cross-linking 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:
These types of structure are recurrent starting from 688 Da. The long peptide chains as in species at 889 Da have several useful reaction sites on which tannin chains can react to contribute to protein cross-linking. Thus, a species such the one at 1260 Da can be interpreted as belonging either to a peptide chain liked to a flavonoid dimer or as linking through different groups two flavonoid monomers, as follows:
A sequence of aspartic acid-gallocatechin dimer-arginine-leucin-aspartic acid-glycin-glutammic acid
and/or
A sequence of aspartic acid-catechin-arginine-leucine-aspartic acid-glycine-glutamic acid-catechin
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.
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.
And any other combinations collagen protein, condensed tannin |
Note: 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