Elastin in lamb pelts: its role in leather quality

Introduction Elastin is the major non-collagen fibrous protein present in lambskin after beamhouse processing, comprising 2.7% of the protein material in the grain (Keller, 1990). The role of elastin in lambskin processing has been the subject of particular interest as previous work has mostly been associated with the processing of bovine material (Webster et al, 1987, Alexander et al, 1991). However, the quantity and location of elastin in lambskin is different from that in hide and separate consideration should, therefore, be made in terms of processing to accommodate for these differences. There are unanswered questions about the role of elastin during mottle formation and the persistence of elastin during beamhouse processing through to pickle. Furthermore, some authors have claimed that elastin is not 'tanned' by basic chrome sulfate and that the removal of residual elastin from wet-blue material gave additional area gains (Addy et al, 2002, Rasmussen, 2002). The aim of this work is to outline some of the impacts of removing elastin from lamb pelts during beamhouse processing to enable more informed decisions when processing lambskins. Distribution in skin The amount of elastin throughout a skin was quantitatively measured in lambskin using high performance liquid chromatography (HPLC) (Lowe et al, 2000). Samples of measured area and ~2g wet weight were dried, defatted, and then hydrolysed in 6M HCl. Solid phase extraction was then carried out on the hydrolysate with cellulose to purify the elastin-specific amino acids desmosine and iso-desmosine. The amount of elastin was then determined by detection of desmosine at 268nm on the HPLC. It was determined that lambskin had a lower concentration of elastin than hide and that it was in highest concentration in the butt. Elastin content increased with the age of the animal. Interestingly, this is inversely correlated with the observed propensity to form growth lines during processing since new born lambs are more susceptible to growthiness compared with lambs which are more susceptible than sheep. The lower initial amount of elastin in lambskin could make the skin more susceptible to any possible adverse effects when a fraction is removed during beamhouse processing. Distribution and orientation of elastin in ovine skin Immunohistology has previously been used to investigate connective tissue components in bovine hides and kangaroo skins (Stephens et al, 1991). In the present work, samples of ovine skin were examined using immunohistology in order to investigate the location and orientation of elastin during beamhouse processing. Samples were prepared from lambskins at different stages during beamhouse processing including green skin, painted and pulled skin, and pickled pelt. Samples of skin were cut and marked for orientation to the spine and fixed in Bouin's fluid (saturated picric acid : acetic acid : water, 75:25:1) for 24 hours and then transferred to 70% ethanol to await processing. Samples were processed and embedded in paraffin wax. Sections were cut at 5mm thickness, floated on warm water, attached to glass slides and air-dried overnight before carrying out immunocytochemistry (ICC). The biotin-streptavidin detection system was used as follows: Sections were de-paraffinised and were then equilibrated in phosphate buffered saline (PBS) (0.01M), pH7.2 for 1 minute. The section was encircled with a PAP pen to create a fluid barrier to hold the reagents. Non-specific binding sites were then blocked by the addition of 1% bovine serum albumin (BSA) for 5 minutes followed by incubation in a humidity chamber for 1 hour. The sections were then drained and washed in three changes of PBS for 1 minute each. Anti-elastin antibodies (Sigma anti-elastin E4013 from mouse diluted 1:200 in BSA) were then added to the sections and then were incubated in a humidity chamber for 1 hour after which they were drained and washed in three changes of PBS. Biotinylated IgG (Amersham anti-mouse from sheep RPN1001 diluted 1:200 in BSA) was then added and the sections were incubated in a humidity chamber for 30 minutes. They were then drained and washed in three changes of PBS followed by biotin-streptavidin-peroxidase preformed complex (Amersham biotin-streptavidin- peroxidase RPN1.034 diluted 1:200 in BSA). Sections were then drained and washed in three changes of PBS then reacted in 3,3 diaminobenzidine (DAB) for approximately 3 minutes. The reaction was halted by immersion in PBS. The sections were rinsed in tap water and counter stained with Mayers haemalum for 1 minute, then blued in Scotts tap water for 2 minutes. Sections were then rinsed in tap water, dehydrated, cleared and mounted in DPX. The sections were examined under a light microscope at 20x to 40x magnification. The elastin appeared stained brown/black. The staining showed that most of the elastin appeared in the upper grain, where more appeared below the grain surface in the reticular layer and comparatively less elastin in the papillary layer immediately adjacent to the grain surface. Only small amounts of filamentous elastin associated with collagen bundles appeared to extend further down into the corium. In addition, it appeared that the orientation of elastic fibres depended on their location, with fibres appearing parallel to the spine in the lower grain and perpendicular to the spine in the upper grain. The elastin appeared to be relatively unaffected during the high pH stages of the lime sulfide beamhouse processing but its visibility became more pronounced after painting and pulling. After bating with a pancreatic bate, the elastin was less easily viewed using this technique. However, it was seen in the pickle that more elastin was removed from the layer of the grain immediately adjacent to the grain surface as compared with lower in the grain. Effect of varying elastin removal during beamhouse processing on leather quality Earlier investigations highlighted two important facts (Stone et al, 1982, Lowe, 1997). First, that high ionic strength solutions inhibit elastase activity without impacting on the general protease activity and second, that the presence of anionic surfactants enhances elastase activity. These two observations allowed bating trials to be conducted using the same enzyme but with modified elastase activity. Method Forty-eight lambskins from the same line of stock (Romney cross) were obtained. The skins were then processed through liming using the standard processes detailed in the appendix. The limed skins were washed and then delimed with CO2 without bating. After deliming, the skins were separated into four groups of twelve skins and bated with 0.01% of a high elastase bacterial bate with the following additives: (a) low elastase process - 2.5% NaCl (b) normal process - no additive (c) high elastase process - 0.1% sodium dodecyl sulfate (d) very high elastase process - 0.5% sodium dodecyl sulfate Each group was bated for 75 minutes at 35°C. Samples of bate liquor were taken for HPLC analysis of soluble elastin components as described above and the groups of skins were washed out of bating separately then recombined for pickling. The pickled skins were assessed for mottle, growth lines in the neck area (neckiness) and elastin content as described above and then processed together to dyed crust. Looseness was assessed with a 'break' scale where a score of 1 represented little or no looseness and a score of 5 represented a skin that was very loose (Lowe and Cooper, 1998). The results are illustrated in Figure 1, which shows the levels of elastin released during the bating process. This clearly illustrates the significant increase in groups with respect to the levels of elastin breakdown associated with increasing elastase activity. At the highest elastase level (0.5% SDS) over half of the elastin in the skin had been destroyed. Figure 2 shows the effect of elastin removal on the mottle and neckiness of the pickled pelts. Contrary to expectation, increasing the removal of elastin appeared to have led to increased growthiness of the pickled pelts. The expectation had been that the removal of elastin, which shrinks and becomes brittle upon drying, would improve the flatness of pickled pelts. In fact removing elastin exacerbated the mottling of skin during processing. The results suggest that residual elastin contributes positively to the character of the final ovine crust leather. For the very high elastin removal, there was a significant increase in looseness. However, there was no significant difference between the groups in tear strength, grain strength and softness. Effect of removal of elastin during beamhouse processing on area To investigate the impact of elastin removal on mottle formation and on area of sheepskins, a two level factorial experiment was carried out investigating a number of methods by which the removal of elastin can be altered during bating. The details for each factor investigated are given below. Bate type It is known that different bate types have different relative elastase activities even when normalised to the same general protease activity (Lowe, 1997). The bate types used were a pancreatic bate enzyme (Tanzyme, Tryptec Biochemicals) with low elastase activity and a bacterial bate (Pyrase 100L, Novozymes) which had high elastase activity as measured relative to the pancreatic enzyme. The pancreatic bate was adopted as the standard bate enzyme and was used to define the standard activity at an application rate of 0.1% w/v. Deliming method Altering the deliming from the use of ammonium salts to carbon dioxide increased the elastin removal during bating (Lowe, 1997). Ammonium sulfate (2% ) was used for the ammonium salt deliming and CO2 gas was injected into a recycled float stream until the pH reached 8.0 for CO2 deliming. Bate level It was expected that changing the amount of bate offered would increase both the general bate activity and the elastase activity during bating. The variables of bate treatment were carried out using 75% and 150% of the standard application rate of the pancreatic enzyme. Exposure time Changing the time of bate addition during the deliming/bating procedure may further expose any differences in the effects of general activity relative to elastase activity. The total deliming time given to the batches was 75 minutes. Bate was either added immediately (giving the full 75 minutes exposure time) or after delime had proceeded for 45 minutes (giving 30 minutes exposure time). The values used for the different factors are summarised in Table 1. Deliming was carried out separately on each of the 16 groups depending on the experimental factors. All batches were delimed for 75 minutes at 35°C and then washed 3 times with 100% water. All the groups were then combined for pickling. The amount of elastin remaining in the pickled pelts after processing was measured in stained histological sections using the scale illustrated in Table 2. Each sample was assessed on four different sections. In addition, each high/low pair (four pairs for each factor) were directly compared for remaining elastin. Elastin removal The results for the amount of elastin present in the pickled pelts were aggregated for each of the factors and the differences in the levels of elastin observed between high and low experimental levels was found to be significant at the 95% confidence level except for 'delime type' which was only significant to an 85% confidence level. In each case, the level of elastin removal was related to the level of elastase activity exposure as would be expected. * Higher bate levels gave more elastin removal * Longer exposure time to bate gave more elastin removal * CO2 deliming gave more elastin removal by comparison with ammonium salt deliming * The bacterial bate gave more elastin removal than the pancreatic bate even though the general activity of the two bates had been normalised Area For every individual skin the area was measured at the pulled slat stage and at the pickled pelt stage. All wet pelts were lightly slicked onto the area measuring table, avoiding any undue stretching out and measured electronically. During processing there was an overall increase in area. The 'pickle yield' was defined as the percentage increase in area between pickle and slat or: Pickle yield = pickle area-slat area over slat area x 100% The average pickle yield found in this work was 15.5%. The data for all the skins were analysed together and the results for each of the main effects are plotted in Figure 3. The variable factor is plotted as 'lower' or 'higher' on the x-axis and the resulting pickle yield on the y-axis. The colours represent the different variables. Note, all main effects had a p value of less than 0.0005 giving a 99.95% confidence level. The following points were noted: * When the general level of bate activity was increased by increasing the bate offer, the area yield increased as would be expected * When bate was added part way through deliming, the area yield was reduced. This is probably because less complete bating was allowed to proceed. This again shows that increasing the duration of bating increased the area Neither of the above factors are elastase specific. Altering these factors merely altered the total general proteolytic activity during bating. * A major difference between the two bates used was the relative level of elastase activity Increasing elastase activity during bating by changing bates had a detrimental effect on the pickle area. It is important to note that the two bate types were applied at levels giving the same activity as measured using the hide powder azure test. The point is that at the same level of general activity against skin substance a greater removal of elastin during bating resulted in a reduction in area. * Using CO2 deliming gave less area than ammonium salt deliming. There are two possible reasons for this. The pH difference: at the beginning of deliming the pH of ammonium salt deliming is higher than the pH of CO2 deliming so it is possible that the relatively low pH during the beginning of CO2 deliming has moved conditions outside the optimal range for the two enzymes used and thereby reduced the total general activity of bating resulting in a slight reduction in area. Secondly, CO2 deliming results in a higher level of elastase activity during bating and again it can be seen that the conditions which caused more elastin removal correlated with reduced area. To summarise: * Increasing the general bating activity increased the area * Increasing the elastase activity relative to general activity reduced the area It appears as though two competing effects are occurring simultaneously. One effect involves the general activity of the bate, possibly a collagenase activity, which has the effect of relaxing and spreading out the skin and another effect involving the removal of elastin during bating which appears to be opposed to the relaxation and spreading out of the pickled pelt. It was noted above that excessive removal of elastin from ovine material can increase growthiness and mottle of the skin and it is possible that the elastase related effect, in which a reduction in area can be seen, is related to this effect. Perhaps, as elastin is removed during bating, growthiness increases which draws in the skin and reduces the area. Low elastase activity Overall the results show that removal of elastin from lambskin in the alkaline state, when the structure is under swelling tension, is likely to exacerbate mottle and looseness, demonstrating that application of elastase activity during alkaline process stages is contraindicated. Therefore, enzyme process stages such as bating or enzyme liming should be carried out with care and enzymes used should be of low elastase activity. Traditional ammonium salt deliming has a positive effect on retention of elastin by inhibiting undesirable elastase activity at this stage whereas CO2 deliming provides no inhibitory effect on elastase activity. While no area gains appear possible, through the use of a high elastase treatment prior to pickling some gains are possible on pickled product. Relationship between mottle in final crust leather and initial elastin content of green skin Up until now, the impact of elastin during the formation of mottle has been unclear. In order to examine the relationship between endogenous elastin and mottle observed in the crust leather, 120 lambskins from a variety of breeds were sampled and analysed for elastin content and processed to crust using the following procedure: Standard painting and liming process - standard delime, bate, and pickle process - aqueous degrease - chrome tannage - retannage to crust. The details of which can be found in the appendix. The crust leathers were then assessed for mottle by grading the skins against a fixed set of five representative samples with a range of degrees of mottle, where one represented a skin with low or no mottle and a score of five represented a skin with a high level of mottle. The results show a clear relationship between the quantity of elastin and the degree of mottle observed in the final crust leather with a significant negative correlation (>99.9% confidence). The greater the quantity of elastin that was initially present in the skin, the less was the propensity to form mottle during processing. Conversely skins which had less elastin present in the fresh material showed a greater chance of forming mottle during processing. Certain claims have been made recently about the effect of enzymatic removal of elastin after tanning has been carried out and the collagen has become cross-linked (Addy et al, 2002, Rasmussen, 2002). To investigate the impact of enzymatic elastin removal from ovine material after tanning, the following experiment was carried out: Twenty-four pickled pelt grain splits were obtained from a sheep pelt splitting operation. The grains were placed into three groups designated: controls, pickle and wet-blue, each having eight skins assigned. The three different groups were then converted through to crust using the following processes: Controls Aqueous degrease - chrome tannage - retannage to crust Pickle Aqueous degrease - enzyme treatment - chrome tannage - retannage to crust Wet-blue Aqueous degrease - chrome tannage - enzyme treatment - retannage to crust The processes for aqueous degreasing, chrome tannage, and retannage to crust were the same for each of the three groups and are given in the appendix. The experimental bating treatments were the same whether applied before or after chrome tannage. The enzyme treatment involved 0.5% addition of enzyme product (Novocor AX, Novozymes) to 100% float at pH5.8 at 35°C for 90 minutes. Details of the experimental enzyme treatment are also given in the appendix. The areas were measured in the pickle, in the wet-blue, after neutralisation of the wet-blue, at the wet crust stage, and the dried crust after staking. The yield at each stage was measured as the percentage change in area after the process in question compared with the measurement taken prior to the process. Skin samples were taken in the pickle and in the wet-blue after the enzyme treatment. The samples were sectioned, stained and assessed for elastin removal. Results The data illustrated in Figure 4 show a significant improvement in wet-blue area yield in comparison with the controls when the enzyme treatment was applied to neutralised pickle grains (the pickle group). The increase in yield over the control pickles was 8+-3%. After chrome tanning and neutralisation, the third group (wet-blue) received the same enzyme treatment. The areas were then measured before and after neutralisation and treatment (Figure 5). Again, the yield was calculated as a percentage of the final wet-blue area on the original pickle area. When measured immediately after treatment, the results showed a 4% increase in area for wet-blue skins that received the enzyme treatment in comparison with the controls. All the skins were then processed to crust and the areas were then measured on the wet crust and again after toggle drying. The final overall area yields from pickled grain through to crust are illustrated in Figure 6. The results illustrated in Figure 6 show that the areas gained by application of the enzyme treatment to the neutralised pickle remained through to the finished crust, but when the enzyme treatment was applied to the neutralised wet-blue, there were no significant gains in crust area. In order to investigate the ability of this enzyme treatment to remove elastin from wet-blue material the levels of elastin were assessed microscopically for each pelt immediately prior to retannage. The results of this elastin assessment were not significant. Conclusions Application of high elastase enzyme treatments to wet-blue ovine material provides no advantage to area in the final crust. An enzyme treatment to remove elastin from tanned material has previously been found to give area gains when applied to wet-blue bovine material. However, it was found in this work that while area gains were observed immediately after the enzyme processing of wet-blue lambskin, the area gain was temporary and was lost during the crusting process. However, when the enzyme treatment was applied to the neutralised pickle, this resulted in an increase in area that persisted through to the final dried crust as has been observed previously (Alexander et al, 1991). It is clear that the claimed beneficial effects of elastin removal obtained on cattle hides such as increased area and flatness are not fully reproducible on lambskins. There is also an accumulation of evidence that removal of elastin during early pelt processing is detrimental to the quality of the final leather because it induces looseness and increases mottle in the final product. Appendix Standard painting and liming process * Skins are painted with 300g/m2 250g/l sodium sulfide flake 50g/l hydrated lime 5g/l pregelled starch Hold over night * Pull wool from skins and place in drum * 80% water Run 30 minutes Adjust sodium sulfide concentration to 2% w/v * Run overnight Drain 5 x 200% washes at 25°C for 20 minutes each Drain Standard delime, bate and pickle process * 100% water at 35°C 2% ammonium chloride 0.05% pancreatic bate Run 75 minutes Drain 3 x 100% washes at 25°C for 20 minutes each * 90% water 20% sodium chloride Run 10 minutes * 10% water 2% sulfuric acid Run 3 hours Aqueous degrease * 100% water at 35°C 4% non-ionic detergent Run 90 minutes * Drain 5 x 100% washes at 25°C for 15 minutes each Chrome tannage * 100% water at 35°C 8% common salt 1% di-sodium phthalate 1% formic acid Run 10 minutes * Add 5% chrome sulfate powder (33% basic) Run 30 minutes * Add 0.6% magnesium oxide Heat to 40°C Run overnight * Drain * 300% water at 25°C Run 10 minutes Retan to crust * 150% water at 25°C 1% sodium formate 0.4% sodium bicarbonate Run 45 minutes * 6% syntan Run 30 minutes * Drain * 300% water at 50°C Run 10 minutes * Drain * Add 100% water at 60°C 0.2% ammonia solution (30% w/w) * Add 20% water at 25°C 5% fatliquor 0.5% formic acid (diluted 1:10 in water) Run 90 minutes * Drain 1 x 300% washes at 25°C for 30 minutes Enzyme treatment * Drain float liquor * 100% water at 25°C Liquor adjusted to pH5.8 with 1% sodium formate 1% sodium bicarbonate Run 90 minutes * Drain * Add 100% water at 35°C 0.5% Enzyme (Novocor AX, Novozymes) Run for 90 minutes * Drain * 2 x 150% washes at 25°C for 30 minutes each All percentages based on drained pickle weight. &rtreturn;