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To explore this, an experiment was carried out to monitor the damage to the grain surface in regions of skin that had been depilated for different times. Two lambskins were obtained fresh from the cooling bath at a cooperating meat processing plant. From these skins the flank region and back bone region were excised. The pieces were then treated to the same depilation procedure as described in section 3.2.1

The pieces from the backbone region were held for 16 hours then the wool was pulled. Two depilation regimes were used: the pieces from the flank region were divided into two parts. Depilation after 5 hours (designated as “immediate”) and depilation after 16 hours (designated as “delayed”). Immediately after depilation all the pieces were pickled in the LASRA standard pickle with the addition of 2% formic acid. The skins were then processed through to crust leather using the standard LASRA process (See Appendix sections 8.2.5, 8.2.4, and 8.2.6). The crust samples were then observed under light microscope to characterise the level of damage that occurred.

The experiment was carried out three times and the results from examination of the grain surface of each piece are described in Tables 14 to 16.

Table 14: Grain quality after enzyme wool depilation (Experimental replicate 1)

Skin/Position Procedure Interfollicular pads Follicle mouths 1/Left flank Immediately depilated Inconsistent damage

(some regions damaged some undamaged)

Inconsistent damage (some regions damaged

some undamaged) 1/Left flank Delayed depilation Heavily damaged Heavily damaged 1/Backbone-neck Delayed depilation Some undamaged

regions surrounding non- depilated regions

Some undamaged regions surrounding non-

depilated regions 1/Backbone-butt Delayed depilation Some undamaged

regions surrounding non- depilated regions

Some undamaged regions surrounding non-

depilated regions 1/Right flank Immediately depilated Inconsistent damage

(some regions damaged some undamaged)

Irregular follicle mouths

1/Right flank Delayed depilation Heavily damaged Heavily damaged 2/Left flank Immediately depilated Inconsistent damage

(some regions damaged some undamaged)

Inconsistent damage (some regions damaged

some undamaged) 2/Left flank Delayed depilation Inconsistent damage

(some regions damaged some undamaged)

Inconsistent damage (some regions damaged

some undamaged) 2/Backbone-neck Delayed depilation Inconsistent damage

(some regions damaged some undamaged)

Irregular follicle mouths

2/Backbone-butt Delayed depilation Inconsistent damage (some regions damaged

some undamaged)

Irregular follicle mouths

2/Right flank Immediately depilated Some scuffing Regular / some scuffing 2/Right flank Delayed depilation Some scuffing Slightly irregular/ some

scuffing

The results of the first two replicates were not consistent. In general skin samples pickled soon after wool release (i.e. immediately depilated flank samples or samples from the backbone-butt area) had less damage than those flank samples left 11 hours longer than necessary to remove the wool. These results were not always observed however. In some samples heavy damage was observed irrespective of depilation time. In other flank samples only modest levels of damage was observed irrespective of depilation time. For this reason the experiment was repeated giving the results shown in Table 16.

Table 15: Grain quality after enzymatic depilation (Experimental replicate 2)

Skin/Position Procedure Interfollicular pads Follicle mouths 1/Left flank Immediately depilated Heavy damage Heavy damage 1/Left flank Delayed depilation Mostly undamaged Irregular, Some damage 1/Backbone-neck Delayed depilation Scattered damage, some

undamaged

Some damage, some irregular 1/Backbone-butt Delayed depilation Scattered damage, some

undamaged

Some damage, some irregular 1/Right flank Delayed depilation Very heavy damage Barely discernable 1/Right flank Immediately depilated Very heavy damage Barely discernable

2/Left flank Delayed depilation Slight flattening, scattered heavy damage

Some damage, some irregular 2/Left flank Immediately depilated Damaged Heavy damage 2/Backbone-neck Delayed depilation Mostly undamaged Some damage

2/Backbone-butt Delayed depilation Mostly undamaged Irregular, Some damage 2/Right flank Immediately depilated Mostly heavy damage Heavy damage 2/Right flank Delayed depilation Very heavy damage Barely discernable

Table 16: Grain quality after enzyme wool loosening (Experimental replicate 3)

Skin/Position Procedure Interfollicular pads Follicle mouths 1/Left flank Delayed depilation Most pads remain Some damage to follicle

mouths 1/Left flank Depilated by sulfide after

enzyme exposure

No residual wool severe damage, much of the

surface removed

Severe damage

1/Backbone-neck Delayed depilation Heavy damage all over some very small regions

of intact grain enamel immediately adjacent to

residual wool

Completely eroded

1/Backbone-butt Delayed depilation Medium damage all over some very small regions of intact grain enamel immediately adjacent to

residual wool

Completely eroded

1/Right flank Depilated by sulfide after enzyme exposure

No residual wool severe damage, much of the

surface removed

Severe damage

1/Right flank Immediately depilated Some damage in follicle mouths much of interfollicular pads still viable some regions of heavy damage much

Skin/Position Procedure Interfollicular pads Follicle mouths 2/Left flank Immediately depilated No depilation observed

at all on this piece

No depilation observed at all on this piece 2/Left flank Delayed depilation Heavy damage to whole

piece only some small regions of grain surface

remain

Heavy damage

2/Backbone-neck Delayed depilation Medium damage all over some very small regions of intact grain enamel immediately adjacent to

residual wool

Completely eroded

2/Backbone-butt Delayed depilation Mild damage all over some small regions of

intact grain enamel immediately adjacent to

residual wool

Completely eroded

2/Right flank Delayed depilation Medium damage all over some very small regions of intact grain enamel immediately adjacent to

residual wool

Completely eroded

2/Right flank Immediately depilated Medium damage all over some very small regions of intact grain enamel immediately adjacent to

residual wool

Completely eroded

In addition to the skin pieces described in table 15, an additional sample was excised from the neck of skin one and treated using a conventional lime sulfide depilation process (as described in Appendix 8.2.1). This piece had undamaged grain indicating that the damage observed on the enzyme depilated pieces was solely associated with exposure to enzymes.

In the third experiment (Table 16) two skin pieces were exposed to enzyme solution (for 30 minutes) and then immediately treated with a conventional lime/sulfide depilatory without the holding period that the enzyme depilated pieces received. The intention of this work was to determine the damage to the skin caused by the initial exposure of the skins to the enzymes. These pieces, however, showed levels of damage to the grain that far exceeded those that had been wholly enzyme depilated. Entire portions of grain material were missing, leaving behind a ragged corium at the surface. This is in contrast to the excellent grain surface of the pieces that were not exposed to enzyme. Possible reasons for this observation include:

1. Immediate damage to the grain surface by the enzyme which was exacerbated by the action of the sulfide ions.

2. Modification of the activity of the enzyme by sulfide ions to increase its activity

3. Modification of the grain collagens by sulfide to make them more susceptible to

damage by the enzyme.

It is known that sulfide breaks the disulfide cross links in proteins helping the

denaturation process (O'Flaherty et al., 1956). Due to the denaturing impact of sulfide

ions on the enzyme for example it is unlikely that further enzyme activity produced this damage during the conventional processing step. In recent work (Allsop, 2007) has shown that sulfide depilation followed by enzyme processing in the place of the sulfide based liming process can be carried out with little or no damage to the skin. As such, modification of skin proteins by sulfide is probably not the cause of the damage observed here.

The first reason is therefore the most likely cause of damage; Removal or modification of components by the enzyme process causes the collagens in the skin to become susceptible to damage by sulfide ions. The nature of these modifications however is unknown at this time. In order to carry out subsequent processing using lime and sulfide the nature of these modifications would need to be identified, which is outside the scope of the work. It can be speculated that enzymatic hydrolysis of some proteins in the grain surface, not susceptible to the sulfide ions existent during conventional lime/sulfide depilation, may expose disulfide bonds within the grain surface that are integral to the integrity of the surface structure. In this way damage to the grain by subsequent alkaline sulfide processing may then occur (i.e. Enzymatic removal of specific proteins from the surface may enable the damage to the structures that maintain the surface character).

Over all, the results showed there was consistent damage to the pieces that depilated early, regardless of subsequent treatment. Evidence that wool sheath release does precede grain layer damage was observed however in skin pieces incorporating regions that were not depilated. These samples generally had small areas surrounding the non depilated region that were undamaged. The best pieces were those along the back bone that had barely depilated ie. depilated regions on the backbone adjacent to un-depilated regions. Typical examples of this band of undamaged region, fully damaged regions

located far from the non-depilation portion and conventionally depilated skin are illustrated in Figure 36, Figure 37, and Figure 38 respectively.

Figure 36: Region of undamaged skin surface (red arrows) located between non- depilated skin (green arrow) and damaged skin (blue arrows) (bar is 200 um). Damage becomes progressively worse the further away from the non- depilated region (not shown)

The band of undamaged skin (Figure 36) was measured on the pieces of skin from the final replicate of experiment above. It was found to be 1.50mm ± 0.16mm (95% confidence). This indicates that some (but not all) of the damage caused to the surface during the enzyme depilation process can be reduced by halting the enzyme activity just as depilation is achieved. The thickness of the band of undamaged skin between the non-depilated and damaged areas is related to the time at which depilation can occur before damage occurs. Hence if this time can be made as large as the width of the sigmoid curve (as shown in Figure 34), a successful enzymatic depilation could be carried out with this enzyme. The total area of the skins for that experiment were 0.75 and 1.1m² for each of the duplicate skins respectively.

Figure 37: Typical damage observed on the surface of enzyme depilated skins at a distance at least 5 cm away from the non-depilated regions (bar is 200 um).