**2.4. Soap degumming (conventional method)**

Silk fabrics were soaked overnight in a solution of 5 g. l -1 Marseille soap at pH 9.5 at a liquorto-fabric ratio 40:1. Next day the silk fabrics were degummed in a boiled alkaline solution containing 10 g. l -1 Marseille soap and 1 g. l -1 sodium carbonate for 2 h at a liquor-to-fabric ra‐ tio 40:1 and pH 9.5. Degummed silk fabrics were first rinsed at 50o C with 1 ml. l -1 ammonia and consequently two times at 40o C with 1 ml. l -1 ammonia. Finally the fabrics were rinsed with cool water.

#### **2.5. Enzymatic degumming**

A fabric sample of approximate weight 3.0 g was immersed in Tris-HCl buffer (50 mM) at pH 8.0. A non-ionic wetting agent (Sadopane SF 0.1% w/v) and the appropriate amount of enzyme(s) supplemented the solution. The liquor-to-fabric ratio was adjusted to 40:1, and the mixture was incubated at 50o C and 50 rpm. Blank samples were obtained by treating silk with buffer alone, without enzyme. Enzyme dosage and treatment time were changed. Inac‐ tivation of the enzyme(s) was carried out in hot distilled water for 10 min. At the end of the treatment, silk fabrics were rinsed with distilled water and dried at room temperature. All degumming tests were performed in duplicate.

#### **2.6. Bleaching of degummed silk fabrics**

The enzymatic and conventional degummed silk fabrics were bleached using: 0.4% (o.w.g) bleaching agent (Belphor BH); 0.5% (o.w.g) stabilizator (Sifa FL); 20 ml. l -1 H2O2; 3 g. l -1 Na2CO3; liquor-to-fabric ratio (30:1); wetting agent Clariant Sandoclean PC-FL (1 g. l -1).

The bleaching agent was mixed with the stabilizator, H2O2 and the Na2CO3. The mixture was stirred and heated at 40o C. The silk fabrics were soaked in the mixture and stirred until the temperature reached 90o C. The fabrics were left for 100 min. The fabrics were removed and rinsed initially with hot tap water and finally they were immersed in water with 2-3 drops of formic acid.

#### **2.7. Weight loss and degumming efficiency determination**

Fabric weight loss was recorded as dried sample weight loss. The drying conditions were 105o C in an air-circulated oven for 1 h. The samples were weighed, after cooling in a desicca‐ tor. The following equation (Eq. (1)) was used to calculate the weight loss (wt%):

$$\text{W}\% = (\frac{\text{W1} - \text{W2}}{\text{W1}}) \times 100\tag{1}$$

where, *W1* and *W2* are the weights of the fabric before and after treatment, respectively [59].

The efficiency of the degumming was calculated through a comparison of the enzyme proc‐ ess for silk fabrics with the standard method (degumming using Marseille soap), using the following equation (Eq. (2)):

$$\text{Design}Eff = \frac{\mathcal{W}\_{\text{E}}}{\mathcal{W}\_{\text{MST}}} \tag{2}$$

where, *DegumEff* is the Degumming Efficiency (%), *WE* is the percentage of weight loss by the enzyme treatment and *WMTS* is the percentage of weight loss by the Marseille soap treat‐ ment [51].

The degumming with Marseilles soap was taken as the standard 100% weight loss.

#### **2.8. Wettability: Drop test**

**2.3. Enzyme activities**

242 Eco-Friendly Textile Dyeing and Finishing

containing 10 g.

with cool water.

l

**2.5. Enzymatic degumming**

the mixture was incubated at 50o

was stirred and heated at 40o

the temperature reached 90o

drops of formic acid.

degumming tests were performed in duplicate.

**2.6. Bleaching of degummed silk fabrics**

and consequently two times at 40o

The proteolytic activity was assayed spectrophotometrically with azocasein as a substrate [57]. One unit of activity was defined as the amount of enzyme required to produce a 0.1

Lipase activity was determined against p-nitrophenyl- propionate (pNPP) at pH 7.0 and 25°C [58]. The release of p*-*nitrophenol was monitored spectrophotometrically at 410 nm with the aid of a microplate reader (Molecular Devices Corporation, Sunnuvale, USA). The reaction was initiated by adding 10 μL of properly diluted enzyme to 190 μL of substrate solution (0.4 mM). Control reactions with inactivated lipase were used to correct nonenzy‐ matic pNPP hydrolysis. One unit of activity was defined as the amount of enzyme which

to-fabric ratio 40:1. Next day the silk fabrics were degummed in a boiled alkaline solution

A fabric sample of approximate weight 3.0 g was immersed in Tris-HCl buffer (50 mM) at pH 8.0. A non-ionic wetting agent (Sadopane SF 0.1% w/v) and the appropriate amount of enzyme(s) supplemented the solution. The liquor-to-fabric ratio was adjusted to 40:1, and

with buffer alone, without enzyme. Enzyme dosage and treatment time were changed. Inac‐ tivation of the enzyme(s) was carried out in hot distilled water for 10 min. At the end of the treatment, silk fabrics were rinsed with distilled water and dried at room temperature. All

The enzymatic and conventional degummed silk fabrics were bleached using: 0.4% (o.w.g)

The bleaching agent was mixed with the stabilizator, H2O2 and the Na2CO3. The mixture

and rinsed initially with hot tap water and finally they were immersed in water with 2-3

bleaching agent (Belphor BH); 0.5% (o.w.g) stabilizator (Sifa FL); 20 ml.

Na2CO3; liquor-to-fabric ratio (30:1); wetting agent Clariant Sandoclean PC-FL (1 g.

l

l

C with 1 ml.

l

C and pH 8.0).


C with 1 ml.

l


l -1). l -1


l



C and 50 rpm. Blank samples were obtained by treating silk

C. The silk fabrics were soaked in the mixture and stirred until

C. The fabrics were left for 100 min. The fabrics were removed

increase in absorbance at 440 nm under the assay conditions (50o

released 1 μmol of product per minute under the conditions described.

tio 40:1 and pH 9.5. Degummed silk fabrics were first rinsed at 50o

**2.4. Soap degumming (conventional method)**

Silk fabrics were soaked overnight in a solution of 5 g.


Wettability of the fabric was measured, by means of the ''drop test" before and after the de‐ gumming process. The dried samples at room temperature were tested using AATCC Test Method 39-1980 (evaluation of wettability) [60]. The time period (in sec) between the contact of the water drop with the fabric and the disappearance of the water drop into the fabric was counted as the wetting time. The time of drop disappearance was averaged from meas‐ urements in different points of the fabric sample. Wetting times equal or less than 1 sec were considered as indication of adequate absorbency of the fabrics [61]. All measurements were performed in triplicate.

#### **2.9. Whiteness**

The whiteness index (Berger degree) of the fabrics was determined using a reflectance meas‐ uring Datacolor apparatus at standard illuminant D65 (LAV/Spec. Excl.,d/8, D65/10o) [62].

#### **2.10. Crystallinity index (CrI)**

An X-ray diffractometer (Siemens D5000), was used in order to determine the Crystallini‐ ty Index, using copper Ka radiation. The angles scanned were 10–30o at 0.01o /s. The Crystallinity Index was determined according to the empirical method of Segal et al. [63] applying Eq. (3)

$$rCrI = (\frac{I\_{002-I\_{A00}}}{I\_{002}}) \times 100\tag{3}$$

activity of the enzyme. The pH optimum of papain was found 8.0 while temperature opti‐

remaining activity was measured after incubation for 24 h at the above mentioned tempera‐

**Enzyme Origin Characteristics Activitya pHa T (oC)a**

Papain *Carica papaya* Cysteine protease 1.15 U.

**3.2. Protease treatment of silk fabrics: effect of enzyme dosage**

loadings (expressed as Units of protease per g of silk fabric, U.

ciency nearly 99%) was obtained at enzyme loading of 75 U.

temperature is needed to attain complete degumming (110-120o

fabrics treated with Marseille soap attained a value of 20.4% (w/w).

(genetically modified)

Optimum pH and temperature values in parentheses.

(Fig. 2a) and degumming efficiency (Fig. 2b).

8.0L *Bacillus* sp. Serine-type protease 19.3 U.

C. Papain was stable at pH values of 7.0 and 8.0 and temperatures of 30-50o

Physichochemical and Low Stress Mechanical Properties of Silk Fabrics Degummed by Enzymes

Lipase 6.1 U.

Raw silk fabrics were degummed with Papain and Esperase® 8.0L using different enzyme

cal (weight loss, crystallinity index, whiteness, whiteness after bleaching) and low-stress me‐ chanical properties (Kawabata evaluation system) of the preterated fabrics were assessed. Untreated and conventionally degummed (using Marseille soap) silk fabrics were used as

The weight loss (or degumming loss) represents a quantitative evaluation of the degum‐ ming efficiency after standard or enzymatic degumming. The effect of enzyme dosage on the extent of sericin removal was studied by treating silk fabric samples for 60 min with dif‐ ferent amounts of the two proteases. The results are depicted in Figure 2 both as weight loss

Degumming loss increased linearly as the amount of papain increased, attaining a value of 10.2 % (w/w) (degumming efficiency 50%). Esperase® 8.0L was more efficient in sericin re‐ moval as judged by the weight loss (Fig. 2a). Maximum value (20.1%, w/w, degumming effi‐

Without enzymes, the degumming loss was negligible, owing to the low treatment tempera‐ ture. In fact, it is well known that sericin can be removed by using water alone, but high

mg−1 dry

ml-1 7.0-8.0

7.0-9.0 (8.0)

http://dx.doi.org/10.5772/53730

7.0-9.0 (9.0)

fabric) and the physicochemi‐

fabric. Degumming loss of silk

C, under pressure).

matter

ml-1

g-1

g-1

C (the

245

30-50 (50)

30-50 (50)

(10.0) 30-50 (70)

mum at 50o

Esperase®

controls.

*3.2.1. Weight loss*

a

tures and the pH values).

Lipolase® Ultra 50T Aspergillus oryzae

**Table 6.** Enzymes used for silk degumming

where I002 is the peak intensity from the lattice plane, and Iam is the peak intensity of amor‐ phous phases.

Triplicate sets of data were used to establish the relative error associated with the X-ray dif‐ fraction method [63].

#### **2.11. The Kawabata evaluation system**

Basic mechanical properties namely tensile, bending and shearing were measured by the KES-FB system under high sensitivity conditions. The temperature was 20 ± 0.5o C, and rela‐ tive humidity is 65% ± 5. The properties measured were shear rigidity (*G*, gf. cm. degree-1), bending rigidity per unit length (*B*, gf. cm2.cm-1) and extensibility (*EMT*, %) at 500 gf. cm-1. All measurements were performed in triplicate.
