Is reliming necessary for oxidative dehairing?14 July 2008
In our November/December edition we published details of an IULTCS presentation: Alternatives to sulfide dehairing: use of oxidative agents by William N Marmer and Robert L Dudley, Eastern Regional Research Center, USDA Agricultural Research Service. They also provided a related visual display: Oxidative dehairing: to relime or not to relime.
Abstract Reliming serves several purposes and, traditionally, immediately follows the removal of hair (liming/dehairing: 2% sodium sulfide, 2% lime, and 1% sodium carbonate are used at ERRC). Reliming lowers the pH of the hide and opens up the collagen fibres, allowing the uptake of both calcium and water. Opening up the collagen fibres also aids in the removal of non-collagenous proteins and any residual sulfide. The oxidative protocols developed for the tannery usually use 4% sodium hydroxide. This concentration is sufficient to open the collagen fibre and there is an uptake of water by the hide during the dehairing process. Is this opening up of the fibre during dehairing sufficient for the removal of the non-collagenous proteins? The authors extracted proteins from cattle hide using each of these four solutions: 2% lime, 0.1 and 0.01% NaOH and 4 M guanidine. All four mixtures extracted bovalbumin, bovine d2bdu (a bovine allergen) and bovallergin NID. Additional proteins were extracted; they were all bovine serum albumins; no other non-collagenous proteins were isolated. From the limited data they concluded that hides oxidatively dehaired do not require a relime step. Introduction Research focused on the replacement of sulfide in the dehairing process with an oxidative chemical. The authors developed a number of useful formulations for the beamhouse as well as a rapid oxidative dehairing formulation specifically for a packing plant.1,2 One of the many advantages of using an oxidative formulation for the dehairing of cattle hides is the absence of sulfide in the waste stream. Using this process, however, does not reduce the total amount of water used, as immediately after dehairing a ‘reliming' phase was used to swell the hide and remove the non-collagenous materials from the hide. Hydroxide ion (HO-) can cause protein fibrils to take up water and swell.3 In the oxidative dehairing formulations the source of hydroxyl ion is 4% NaOH. This concentration is sufficient to swell the hair on the hide and to hydrolyse the keratin molecule.4 It is speculated that the base would also readily hydrolyse any non-collagenous proteins present in the hide, thus facilitating their removal. It was observed in the studies that collagen, under the prescribed dehairing conditions, is resistant to base hydrolysis. This was shown by the results on the swelling of hides as well as the identification of proteins isolated from the solutions. Experimental Hides from a local abattoir were soaked, washed, and fleshed before further use. The hides were cut into approximately 1.5 x 1.5 in (3 x 3 cm) pieces and placed flesh side down in polystyrene weighing dishes that had been rinsed with 50% acetonitrile/ 50% H2O with 0.1% trifluoroacetic acid. Next, 100% floats of the extraction solution were added to the dishes and the mixtures were agitated. The hide pieces were removed after four hours, blotted dry and weighed. The % swelling was determined as 100X(we-wo)/wo, wherein wo = initial weight of the hide and we = weight of the extracted hide. The isolation of the proteins involved the filtration of the extraction solution to remove any particulate matter, dialysis of the filtrate against water, and finally the lyophilisation of the retentate. The proteins were cleaned using a ReadyPrep 2-D Cleanup Kit (BioRad, Hercules, CA). Separation of the proteins in each isolate was achieved with gel electrophoresis and using either ReadyGELs 4-15% Tris (BioRad) or NuPAGE 12% Bis-Tris Gels (Invitrogen, Carlsbad, CA). The gel data are only qualitative, as the concentration of each of the extracted proteins was not normalised. The purified proteins were isolated from the gels using standard procedures and analyzed using MALDI-TOF/ TOF mass spectrometry. Results and discussion All of the extraction solutions used in this study, even water alone, induced water uptake (swelling) by the hides. In the case of NaOH, the correlation between base concentration and swelling is 1.0M (4%) NaOH>0.1M NaOH>0.01M NaOH. The amount of water uptake from the 1.0M NaOH is probably too low, as after four hours extracting the hide, the hair on the hide piece had started to pulp. Solubilisation of the hair would result in a low hide weight. This potential loss of weight is reflected in the large standard deviation of the average % swelling. The 8M urea extraction solution yielded the smallest amount of hide swelling followed by water and then 4M guanidine HCl. The swelling of the hide by lime was comparable to the swelling observed for 0.01M NaOH. The extracted solutions went through a number of purification steps before the isolation of the proteins. One of the steps was, after centrifugation, the dialysis of the extraction solution. The dialysis either removed the base or removed the salts used in the extracted mixture. Solid protein precipitated out of both the 8M urea and 4M guanidine HCl extraction solutions. This precipitate was either a high molecular weight protein that could not be extracted by the basic extraction solutions or were proteins that had not undergone hydrolysis. A 4-15% Tris-HCl gel of the proteins extracted with 0.01M NaOH, 4M guanidine HCl, 0.1M NaOH and 2% lime was run. The gel contained several bands; four were selected for MALDI-TOF/TOF analysis. Bands A and B were common to all the protein extracts while B and C was absent in the lime-extracted proteins. B and D were very faint, with the highest concentration occurring in the protein extracted by 4M guanidine HCl. The authors were unable to identify the protein responsible for Band D. They did, however, obtain positive protein identification for the other bands. They assigned band A to a bovalbumin (MW 69248.4) and band B to Bovine d2bda (MW 17184.5), a bovine allergen that has a 64.3% amino acid identity with human psoriasin; a calcium-binding protein expressed in keratinocytes.5 Band C was another allergen, bovallergen NID (MW 11536.6), also known as bovine lipocalin allergen Bos D2. The biological function of Bos D2 is not known, but most lipocalin proteins are ligand binding proteins capable of binding and transporting small hydrophobic molecules.6 Next the isolated proteins were studied on a NuPAGE 12% Bis-Tris gel. The gel exhibited several bands that had not been evident on the previous gel. The concentration of the isolated protein were increased when this gel was run. It is interesting that a number of these bands were not present in the proteins isolated from the 2% lime extraction solution. Unfortunately it was not possible to identify all of the bands on the gel as the concentration of the protein was too low. The identified proteins (bands D, F, and G) were all bovine serum albumin precursor (MW 69348.4). This protein is not found in the proteins isolated from the 2% lime extraction solution, though the protein was present in the 4M guanidine solution. Thus this protein is not a base hydrolysis product. Efforts continue to identify the remaining bands that were found in the gel. Conclusions The authors demonstrated that all four extraction formulations can successfully remove non collagenous proteins from cattle hide but so far have not been able to identify any proteins other than bovalbumin and two allergens. In addition, they found that the bovine hides take up water when treated with dilute solutions of bases. The swelling opens up the hide, preparing it for further tanning steps. They concluded that a relime step is not required after an oxidative dehairing. Work is ongoing but from the limited data collected so far they conclude that hides oxidatively dehaired do not require a relime step. References 1. W N Marmer and R L Dudley, The use of oxidative chemicals for the removal of hair from cattle hides in the beamhouse. JALCA 2004. 99(9): p386-392 2. W N Marmer and R L Dudley, Oxidative dehairing by sodium percarbonate. JALCA 2005. 100: p427-431 3. J H Bowes and R H Kenten, The swelling of collagen in alkaline solutions; swelling in solutions of univalent bases. Biochem. J, 950. 46(5): p524-529 4. W N Marmer and R L Dudley, The oxidative degradation of keratin (wool and bovine hair). JALCA 2006. 101(11): p408-415 5. J Rautiainen et al, cDNA cloning and protein analysis of a bovine dermal allergen with homology to psoriasin. J. Investigative Dermatol., 1995. 105(5): p660-663 6. J Rouvinen et al, Probing the molecular basis of allergy. Three-dimensional structure of the bovine lipocalin allergen Bos d 2. J. Biol. Chem., 1999. 274(4): p2337-2343.