The use of enzymes in the silk industry is relatively unexplored and it has generated a lot of interest in recent years (Gulrajani et al. 2000). Proteinases, enzymes characterized by their proteolytic activity, have the potential to effect partial solubilization of the proteinaceous gum sericin involved in binding the silk strands together in cocoon, an essential process in the silk cocoon cooking and reeling. As such, pineapple fruit pulp extract (simply pineapple extract) having proteolytic activity due to presence of cysteine proteinases (Rowan and Buttle, 1994) may also find application in silk industry in general and tasar silk cocoon cooking and reeling in particular. The oak tasar (Antheraea proylei J.) silkworm, the larvae of which feed on leaves of oak tree Quercus species (Family-Fagaceae), is an important source of tasar silk (Singh and Singh, 1998). Unlike the mulberry (Bombyx mori L.) silk cocoons, the oak tasar silk cocoons cannot be satisfactorily softened by boiling in plain water (Jolly et al. 1979). The cocoons have to be softened by more drastic boiling off techniques (Pandey and Goel, 1990). Generally the cocoons are cooked in presence of strong alkali agent or other harsh chemicals (Tikoo and Goel, 1987; Das, 1993; Iizuka, 1993; Moon et al. 1996; Chattopadhyay, 1997). Since the chemical methods reduce the quality of the tasar silk thread in many ways (Tikoo and Goel, 1987), an alternative method for the oak tasar cocoon cooking based on the proteolytically active pineapple extract may be developed for better results. In the present investigation, a study was undertaken to enzymologically characterize the pineapple extract with the aim of developing an effective oak tasar (Antheraea proylei J.) silk cocoon cooking method based on pineapple extract.

Matherial and Methods

Pineapple pulp

Fresh and ripe (mature and yellow) fruit of pineapple Ananas comosus (L.) Merr. cv. Queen was purchased from the markets in and around Imphal, Manipur, India and the fruit pulp was prepared from it.

Silk cocoon

The cocoons produced by the oak tasar silkworm Antheraea proylei J. fed on Quercus serrata (Thunb.) leaves were used in the present investigation. The cocoons were obtained from Regional Tasar Research Station, Imphal, India.

Preparation of pineapple extract

The pulp (15 g) of ripe (mature and yellow) pineapple fruit was homogenized with distilled water (100 mL). The resulting homogenate was strained through a coarse cotton cloth and then centrifuged to collect the supernatant as the pineapple extract.

Proteinase assay

The proteinase activity of the pineapple extract, subjected to various experiments of the present investigation, was assayed by a modification of the azocasein method of Rowan and Buttle, 1994.

Testing for applicability of pineapple extract in oak tasar cocoon cooking and reeling

The oak tasar silk cocoons were initially boiled for 30 min and then subjected to soaking in distilled water or pineapple extract in the absence or presence of 9.8 mM sodium carbonate either at room temperature (26-31ºC) or 60ºC for different time periods ranging from 0.5 to 20 hrs. At the completion of each of the soaking step, the cocoon samples were taken out, washed in tap water, semi-dried, deflossed, and then single silk filament reeling was performed on an epprouvette machine in the Reeling Section, Regional Tasar Research Station, Imphal, India.

Results and Discussion

The proteolytic activity exhibited by the pineapple extract was enhanced by addition of sodium carbonate, with its optimum concentration being 9.8 mM (0.125%). The optimum temperature was found to be 60ºC either in the absence or presence of 9.8 mM sodium carbonate. A higher activity was observed in the presence than in the absence of sodium carbonate in the temperature range 30-90ºC. Only up to 13% of the activity was lost when the extract was incubated for 30 min at designated temperatures up to 60ºC in the absence of sodium carbonate. The result was found to be consistent with that reported earlier (Greenberg, 1955). At higher temperatures, the activity was lost faster. However, 20% residual activity remained even after 90ºC incubation for 30 min. The overall thermal stability of the proteinase activity in the presence of 9.8 mM sodium carbonate was more or less the same when compared with that of the pineapple extract alone in the incubation temperature range 30-60ºC. However, the stability was found to be appreciably lower in the presence of sodium carbonate in the higher incubation temperature range 70-90ºC. The time courses of loss of the proteinase activity in pineapple extract maintained at 60ºC in the absence or presence of 9.8 mM sodium carbonate were also studied. The proteinase activity was lost relatively slowly retaining 80-83% activity after incubation for 1 hr at 60ºC either in the absence or presence of 9.8 mM sodium carbonate. The overall stability of the proteinase activity at 60ºC was only slightly lower in presence than in absence of the sodium carbonate concentration.

Boiling of the oak tasar cocoon in water yielded a brown coloured extract. The effects of the cocoon extract on the proteinase activity in the pineapple extract and on its time course of thermal inactivation were studied. The cocoon extract was found to have little effect on the proteinase activity of the pineapple extract either at room temperature (26-31ºC) or at 60ºC in the absence or presence of 9.8 mM sodium carbonate. With respect to the time course of thermal inactivation, it was found that the cocoon extract does not appreciably alter the rate of loss of the proteinase activity at 60ºC in the absence of sodium carbonate. On the other hand, the proteinase activity was lost faster in the presence of sodium carbonate under the same experimental conditions. More than 80% of the activity, however, remained after half-an hour incubation.

By considering the above enzymological characteristics of the pineapple extract, different oak tasar silk cocoon cooking media were constituted by taking the pineapple extract with or without 9.8 mM sodium carbonate at room temperature (26-31ºC) or 60ºC. No single filament reeling could be performed due to lack of minimal softening of the cocoons soaked in distilled water following an initial 30 min boiling. On the other hand, the cocoons soaked in the pineapple extract for 20 hrs at room temperature or for 4 hrs at 60ºC following the initial 30 min boiling were softened at least minimally and they could be subjected to single silk filament reeling. The two cocoon cooking procedures gave more or less the same reeling performances monitored by the reeling parameters - number of ends feeding/cocoon, filament length, recovery %, NBFL and reelability %. The overall reeling performance was further improved by incorporating 9.8 mM sodium carbonate into the pineapple extract cocoon-cooking medium. Besides the improvement in the reeling performances, the overall cocoon cooking time was markedly reduced. These experimental results were suggestive for an applicability of pineapple extract with or without sodium carbonate as an effective agent for oak tasar (Antheraea proylei J.) silk cocoon cooking and reeling.

Acknowledgements

The authors gratefully acknowledge the financial assistance from DBT, New Delhi, India (in the form of a project grant), and that from DST, New Delhi, India (in the form of FIST grant). They also thank Dr. N. Ibohal Singh, Deputy Director, RTRS, Imphal, India for providing some laboratory facilities.

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