A METHOD FOR TESTING THE
PERFORMANCE
OF ENZYMES CELLULASE ON DENIM FABRICS
Preface about this work
1 - Abrasion - basic evaluation
2 - Effect of redeposition - Apparent abrasion
3 - Effective abrasion and "shade loss"
4 - Shade loss
5 - Effective abrasion
6 - Conclusions
Preface
What this document is?
The work published here below refers to the comparison between two competing enzymes used in formulations of chemicals for the "Stone Wash" treatment of the Denim fabrics (jeans).
The realization of this job, which I made in the 1999, required the construction of a laboratory machine used to reproduce the abrasion of clothes against pumice stones typically found in industrial machines.
Some change from the original document.
The reason of the publication of this document in the web is obviously different from the original purpose of achieving data useful for the formulation of enzymatic products. For that reason, specific data as well as the commercial names of the products will not appear here. The two cellulases will be therefore named "ENZYME A" and "ENZYME B".
Why this document in the web?
Mainly for the following 3 reasons:
A - It may help to understand why measurements the enzyme activity often lead to data that are not significant under the practical point of view.
B - It helps to understand why comparisons made in different conditions may lead to contradictory results.
C - Definitely, it helps to understand how complex is the right evaluation of the performance of a cellulase.
ENZYME A - ENZYME B
1 -
ABRASION - BASIC EVALUTATIONS
A set of test using different dosages of enzyme has been conducted on standard pieces of Denim fabric. The temperature and the pH employed for the ENZYME A were 55 °C and 6,5 respectively, while a temperature of 45 °C and a pH of 6,7 have been chosen for the ENZYME B. All the tests had duration of 60 minutes. Following the guidelines of the supplier, 0,56 gr/lit of anhydrous CaCl2 have also been added in the baths where the ENZYME B enzyme was present.
Using a standard procedure, magnified images of each fabric sample have been obtained through a scanner and then loaded into an image processing software. For each fabric image, the program produced a histogram with statistical information. Finally, using a simple mathematical formula, the average brightness value provided with each single histogram was related with the abrasion scale reported in the vertical axis of the graphs below.
Graph. 1
Note : in industrial processes, dosages of enzymes required to reach a given degree of abrasion are usually lower than those reported in the graph. Substantial differences between the laboratory tests and the industrial processes can be also introduced by the effects of the backstaining, which is much more evident in the in the industrial washing machines, due to the higher weight ratio fabric/liquor that leads to a higher color release.
COMMENTS
At low dosages, the cellulase ENZYME B produces a degree of abrasion that is about the double of that shown by the ENZYME A. The difference of performance existing between the two products becomes very little at middle dosages, where the curve of the ENZYME A shows a flat zone. In the other hand, the right side of the graph shows how the abrasion seems to be more dependent on ENZYME A dosages than those of the ENZYME B product.
Anyway, it is necessary to consider that the average brightness value read instrumentally by the optical systems may also be influenced by factors that have nothing to do with the abrasion. A serious comparison between two cellulases that lead to significant differences in the fabric image should take in account both the effects of the backstaining and those derived from possible lost of tone of the cast.
2 - EFFECTS OF THE
BACKSTAINING - APPARENT ABRASION
GENERAL CONSIDERATIONS
At parity of abrasion, the average brightness value measured by any optical system becomes lower when the redeposition of the indigo dye occurs. Under these circumstances, it is not correct to relate directly the instrumental data to the effective abrasion, but rather to the "apparent abrasion".
The apparent abrasion takes therefore in account the loss of brightness and contrast induced by the backstaining, and it is useful to evaluate the quality of the abrasion, rather than the abrasion itself.
Since the tests conducted in the laboratory involve a very little amount of indigo dye released by the fabric samples, further tests were made adding in the baths a given amount of indigo dye.
Graph. 2
COMMENTS
The first test made employing 5 gr/lit of ENZYME B has shown how even a high concentration of indigo in the bath do not affect the look of the fabric in a significant way. Surprisingly, the presence of the dye did not lower the average brightness value, and the fabric still showed an excellent contrast. Due to that observation, no further measurements were made for the ENZYME B in way to build its "apparent abrasion curve", assuming it to be the same of the Graph 1 (displayed as non-continuous curve in the Graph 2).
Differently from the previous case, the look of the fabrics treated with ENZYME A has demonstrated to be sensitive at the presence of indigo dye in the bath. As result, the lost of contrast and brightness leads the ENZYME A apparent abrasion curve to stay at values that are much inferior than those of the ENZYME B for the full range of dosages.
It is worth to mention that the amount of indigo dye added in the baths reproduced the severe conditions of industrial processes, in which a very low weight ratio liquor/fabric is often used. Moreover, no surfactants were added in the baths to prevent the redeposition of the dye. Therefore, the continuous curves of the Graph 2 represent two extreme situations, while between them it should be included the full range of the possibilities met in the industrial "stone wash" treatments.
3 - EFFECTIVE ABRASION AND "SHADE LOSS"
GENERAL CONSIDERATIONS
When one is interested in evaluating the effective abrasion, the backstaining should be limited as much as possible, because it can significantly diminish the average brightness value read instrumentally. Under this point of view, the abrasion tests made in the laboratory constitute already a good starting point, because the very high weight ratio liquor/fabric assures a minimum concentration of indigo dye in the bath. In the other hand, enzymes sometime affect the color of the background, thus introducing another factor that may have influence on the average brightness value. The right interpretation of the histogram profiles can help to evaluate the entity of the shade loss, and it can also allow to process and "adjust" the images in way to obtain new data related to the effective abrasion.
Figure 1 - Evaluation of the Shade Loss through histograms. In the lower part it is represented the flow adopted to "normalize" the images and evaluate the Effective Abrasion.
4 - SHADE LOSS
CONSIDERAZIONI GENERALI
All the images of the fabric samples related to the Graph 1 were loaded into the image processing software. According to the scheme of the previous chapter, for each sample histogram it was selected the blue channel, then it was read the brightness value where the main peak (related to the background) was centered. The values of the background brightness were finally used to build the graph below. The entity of the shift of the main peak toward higher values of brightness is related to the color pull, and therefore low values are preferred.
Graph. 3
COMMENTS
At low to middle degree of abrasion, the color pull caused by the two enzymes is similar. Anyway, at high dosages the ENZYME A leads to a significant shade loss that results in lighter background. It is interesting to notice how the dependence of the shade loss on the dosages of the ENZYME A seems to become stronger after 2 grams/liter. Under these circumstances, the change of slope of the ENZYME A abrasion curve noticed on the Graph 1 at middle dosages may be at least in part due to the increase of the shade loss, and not at all to a real increase of abrasion. On the other hand, after a limited initial shade loss, the ENZYME B seems to preserve the background color on very dark tones, and this remains true even for very high dosages of the product, as demonstrated by the flatness of its curve.
As result, the ENZYME B product gives a better background image, contributing to a higher contrast than it does the ENZYME A. Due to the shade loss noticed at high dosages of the latter, the effective abrasion curve of the cellulase ENZYME A may differ from that displayed on the Graph 1.
5 - EFFECTIVE ABRASION
GENERAL CONSIDERATIONS
The shade loss is responsible of errors when evaluating the abrasion of a sample of fabric through measurement of the average brightness values. As general rule, higher shade loss lead to higher average brightness values, resulting in an overvaluation of the abrasion measured instrumentally. This consideration becomes particularly important when one want to compare the abrasion induced by two enzymes that affect the background in a significantly different way.
In way to attempt making the measurements less dependent from the shade loss, a particular image process system has been applied to those images which background has shown excessive lost of brightness (See image 1).
The Graph 1 has been therefore rebuilt on the basis of the new data acquired.
Graph. 4
COMMENTS
The profile of the curves slightly differs from those drawn in the Graph 1. In particular, the only appreciable difference is related to the maximum dosage of the ENZYME A, where a significant shade lost was noticed.
Note : the method adopted to "normalize" the background color of the Denim images is still temporary, and it will be refined as soon as some tests made specifically for that purpose will be completed.
6 - CONCLUSIONS
At low dosages the product ENZYME B is able to produce higher abrasion than ENZYME A does, while at medium to high dosages the performance of the two products is similar. The ENZYME A seems to promote the redeposition of the indigo dye on the fabric, and therefore the formulations where that enzyme is included require the presence of specific non ionic surfactants. Under these circumstances, the apparent abrasion produced by the ENZYME A depends much on the formulation and on the operative conditions employed in the industrial processes. The ENZYME B has demonstrated to produce an abrasion that is not affected by the redeposition of the indigo dye.
When low "liquor/fabric" weight ratios are used, or when the formulation do not contain a very efficient non-ionic surfactant in way to prevent the backstaining, then at parity of dosages the apparent abrasion produced by ENZYME A is lower than that belonging to the ENZYME B counterpart.
The backstaining induced by the ENZYME A is also responsible of lost of contrast, which is very well noticeable even without the help of any optical instrument. High dosages of ENZYME A have also affected the color of the background, causing further lost of contrast.
On the other hand, the absence of backstaining in the fabrics treated with the ENZYME B cause the contrast to remain on very high values. Simultaneously, high dosages of ENZYME B did not caused a significant shade loss in the background, which remain very dark.
The tests have focused how the biggest difference existing between the two products is mainly related to the different contrast produced on the fabrics, while a substantial difference of the degree of the effective abrasion has been noticed only at low dosages.
Dr. Gianni Maiani - © Copyright 1999
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