FACILITATE REELING

TECHNICAL FIELD

The present invention relates to a method for demineralizing wild silk cocoons before the silk is further processed and to materials with improved properties produced according to the method. This method makes it possible both to reel cocoons from species which cannot be reeled with prior arts and to wet reel cocoons from other which can only be dry reeled or semi-dry reeled with the prior arts. Compared with the prior arts the present invention gives silk fibres with improved quality and reduced or abolished toxicity and allergenicity.

BACKGROUND OF THE INVENTION

The term wild silk refers to silks that are derived from wild silkmoth cocoons rather than silk derived from the domesticated silkmoth, Bombyx mori. Wild silk worm cocoons for the manufacture of silk are usually gathered directly from the wild though partial domestication has been achieved for several wild species. There are three families of wild silkmoths: Saturniidae, Lasiocampidae, and Thaumetopoeidae. In addition the Bombycid silkmoth Bombyx mandarina is found in the wild and has not been domesticated and can therefore be considered to be a wild silkmoth. In India, China and Japan, the cocoons of several species of moths are currently exploited commercially for the production of wild silks and many other species have potential for exploitation in other parts of the world. In addition, there is growing interest particularly in Africa in the potential of wild silkmoth species for the production of commercial wild silks though the industries for these are at an early stage of development (Paterson B. Volume 161, Issue 3696, October 2002, Page 67 Prospects for wild-silk production in South Africa). Many species of wild silkmoth are indigenous and adapted to less developed parts of the World where some are regularly found at high population density and are sometimes present as pests of economic importance. The cocoons of wild silkmoths are generally difficult to soften and this makes reeling from the cocoon difficult or impossible and in consequence most of the wild silk produced commercially is not reeled but carded and spun into staple silk as disclosed by Currie , R. "Silk, chapter 1" in "Silk, mohair, cashmere and other luxury fibres",

Woodhead Textiles Series No. 19, editor, Franck R R, Chemical Rubber Company, Boca Ratan , USA (2001) and by Kumar A and Choudhury R, Textile Preparation and Dyeing Science Publishers, U.S. (2006). Compared with reeling, particularly wet reeling, carding and spinning is disadvantageous as silks prepared in this way have reduced strength , toughness and resistance to wear caused by the shortness of the staple fibre and by damage to the fibres resulting from the carding process and the harsh treatments required to enable the silks to be carded. Several factors contribute to the difficulty of softening wild silk cocoons to enable them to be reeled of which the most important is that the sericin gum which cements the fibroin brins together in the cocoon is relatively insoluble and resistant to swelling compared to that of the domesticated Bombyx mori silk worm. This resistance is thought to result from chemical interaction between silk sericin and inorganic minor components or tannins contained in the wild silk as disclosed by Mondal, M., Trivedy, K., and Kumar, S.N. "The silk proteins, sericin and fibroin in silkworm, Bombyx mori Linn., a review", Capsian J. Env. Sci, 5, (2) 63-96 (2007). This makes the cocoon impossible or difficult to reel with existing methods. Where reeling is possible it is usually achieved by dry or semi-dry reeling and the additional mechanical force required for this compared with conventional wet reeling is likely to damage the silk. In some wild silks it is necessary to resort to prolonged boiling in alkaline solutions and/or treatment with aggressive enzymes or prolonged retting to soften the sericin before the silk can be carded or reeled directly. These treatments are likely to cause significant degradation to the fibroin brins leading to reduced tensile strength, toughness and resistance to wear.

The Lasiocampid silks are particularly difficult to reel as the sericin appears to be particularly hard and resistant to swelling. The sericin of the Lasiocampid silkworm Gonometa postica is difficult to remove (Hess, K. P. 1959. Textile Fibres and Their Use. 6th Edition. USA: J. B Lippincott Company). The sericin of Gonometa rufobrunnea could only be removed by the harsh treatment of heating for 1 hour to 95 °C in a strongly alkaline soap solution as disclosed by Freddi, G., Bianchi Svilokos, A., Ishikawa, H. & Tsukada, M. Chemical composition and physical properties of Gonometa rufobrunnea silk. Journal of Applied Polymer Science 48, 99-106 (1993). Brins of Gonometa rufobrunnea and Gonometa postica degummed by heating to 95°C in a strongly alkaline solution showed low ultimate strength (290 and 178 MPa respectively) and low mean moduli (1.2 and 1.4 GPa) as disclosed by Colomban, P., Hung, M. D., Riand, J., Prinsloo, L. C. & Mauchamp, B. "Nanomechanics of single silkworm and spider fibres: A Raman and micro-mechanical in situ study of the conformation change with stress". Journal of Raman Spectroscopy 39, 1749-1764 (2008). This may be partly attributable to damage during degumming and carding.

The cocoons of Gonometa spp cannot be softened and reeled with existing methods. Even after prolonged boiling, the silk of Gonometa postica cannot be reeled and so must be carded and spun. Alternatively retting for 2-3 weeks buried in moist and warm soil is required to remove the sericin before the silk can be carded and spun.

Saturniid cocoons are also difficult to soften and reel. For example Antheraea mylitta cocoons require prolonged cooking or cooking in soda followed by dry reeling or prolonged boiling in soap and hydrogen peroxide as disclosed by Moor , MA and Mitra, G. 2007 Indian Silk Volume 46, Issue 1, May 2007, Pages 20-22 . Philosamia ricini cocoons are usually carded and spun as a staple because they cannot be wet reeled using existing methods as disclosed by Tuskes, PM, JP Tuttle and MM Collins. 1996. The wild silk moths of North America. Cornell University Press. ISBN 0-8014-3130-1 Satumids of Western Palearctic . As a further example, Antheraea proylei cocoons could not be reeled by soaking in distilled water for 20 hrs at 26-31 °C or for 4 hrs at 60°C after an initial boiling for 30 minutes at 60°C and could only be reeled by prolonged treatment with a pineapple extract containing bromelain as disclosed by Sing, L.R., Devi, Y.R. and Devi, S.K. in their article "Enzymological characterization of pineapple extract for potential application in oak tasar {Antheraea proylei J.) silk cocoon cooking and reeling" in the Electronic Journal of Biotechnology Vol. 6 No. 3, Pages 35-44, 2003. These authors disclose that treatment with bromelain is disadvantageous in that it reduces the tenacity of silk fibers obtained from Antheraea proylei cocoons.

Thaumetopoeid cocoons are also difficult to degum. Those of Anaphe sp. could only be carded and spun after prolonged boiling in concentrated sodium carbonate solution Kato, H. , Hata , T. Yasuta, K. And Kanda C. (1999) Degumming, dyeing and structural properties of wild silkmoth cocoon spinning yarns from Anaphe panda, Cricula trifenestrata and Attacus atlas Linnaeus, Journal of Sericulture Science of Japan. 68, (5), 405-415.

Calcium oxalate has been shown to be present in the cocoon of Antheraea assama, as disclosed by Freddi, G., Gotoh, Y., Mori, T., Tsutsui, I. & Tsukada, M. Chemical structure and physical properties of Antheraea assama silk. Journal of Applied Polymer Science 52, 775-781 (1994) while the hardness of Antheraea mylitta cocoons has been attributed to the presence of these crystals by Shamitha, G and Rao AP (2006) "Studies on filaments of tasar silkworm Antheraea mylitta D (Andhra ecorace). Calcium oxalate has also been identified in the cocoon of the Lasiocampid Malacosoma neustria testacea as disclosed by Ohnishi, E., Takahashi, S. Y., Sonobe, H. & Hayashi, T. Crystals from cocoons of Malacosoma neustria testacea. Science 160, 783-784 (1968) who comment that it is difficult to remove but do not suggest a function for it in the cocoon, considering it only as an excretion from the silk worm's Malpighian tubules. Akai, H. & Nagashima, T. report the presence of calcium containing crystals in the cocoon of the Lasiocampid Gonometa sp. in their paper "Calcium Crystals of Cocoon Shell from African Gonometa silkmoth (Lasiocampidae) Int. J. wild silkmoth & Silk 8, 1-5 (2003). Although the authors suggested the packing of these crystals between the silk fibers might account for the difficulty of reeling the hard outer layer of the cocoon, they did not suggest that agents to remove calcium minerals or tannins might facilitate reeling.

While it is widely understood that sericin, calcium oxalate and tannins and other substances present in wild silk cocoons may have toxic, irritant or allergenic effects the literature does not suggest how these may be removed from cocoons under mild conditions. Furthermore, it is clear that existing methods of reeling silk from wild silkmoth cocoons from the Saturniid family damage the silk resulting in frequent breaking of the silk baves during reeling. This increases the time required for finding fresh ends and results in considerable wastage of unreelable or partially reelable cocoons. Damage to the baves during the dry or semi-dry reeling of wild silks also reduces the strength and toughness of the baves and fibres formed from them with a consequent reduction in resistance to abrasion and wear in wild silk fabrics. The damage also results in longitudinal splitting or partial transverse fracture of the fibre and the production of several forms of imperfection in the yarn including short regional thickenings known as slubs. All these imperfections can reduce the hand, lustre and value of the yarn and the woven fabric. They can also reduce the ease with which twists, yarns and braids can be formed.

Bombyx mori cocoons can be easily reeled by softening the cocoons in warm water. In contrast this simple method of softening cocoons does not generally permit reeling of wild silks necessitating the use of prolonged cooking or boiling in alkaline solutions with or without soaps or the use of aggressive enzymatic treatments or retting to enable sufficient removal or softening of the sericin to enable the fibres to be separated. These treatments are likely to damage the silk fibres. The difficulties of reeling wild silkworm cocoons usually makes it necessary to card the silk and spin it into yarns with very short staples. These yarns have very uneven thickness and greatly reduced strength, toughness resistance to wear and lustre compared to the very long fibres in wet reeled Mulberry silk. Where conventional treatment of silks does permit reeling the softening or removal of the sericin is often inadequate necessitating the use of the greater force involved in dry or semi-dry reeling. This in turn is likely to damage the silk fibres.

Thus obtaining undamaged, long silk fibres by reeling of wild Silk moth cocoons represents an unsolved problem. It is clear that an efficient method for doing this would greatly encourage the development of a sustainable wild silk industry in less-developed parts of the world with significant social and economic benefit enabling it to compete with other silk industries.

BRIEF DESCRIPTION OF THE INVENTION

It is therefore the object of the present invention to provide a method for the efficient reeling of undamaged, long silk fibres from wild Silk moth cocoons by first removing all or substantially all of the mineral matter from them under mild conditions and in so doing, to produce wild silk singles, twists, yarns and cloths with reduced numbers of imperfections, and improved lustre, tensile properties, length, resistance to abrasion and wear, and improved biocompatibility. For some wild Silks including, by way of example only, those of the Lasiocampidae this method makes it possible for the first time to reel the fibres as baves or singles thereby obviating the necessity for damaging degumming and carding treatments needed which yield short staples for spinning into yarns. For other wild silkmoth cocoons including, by way of example only, those of certain Satumiids the method of demineralizing cocoons is highly advantageous in that it enables wild silk cocoons to be wet reeled efficiently with existing equipment used for reeling Mulberry silk thereby reducing damage to fibres resulting from dry or semi-dry reeling or carding. The method of demineralizing cocoons therefore produces silks with improved mechanical properties, resistance to wear and abrasion, and improved lustre and hand. It is also highly advantageous in that the process removes the toxic calcium oxalate, tannins, polyphenols and other toxic agents thus greatly increasing the biocompatibility of wild silks and thereby making them suitable for medical uses and akin the silk fibres less irritant when woven into apparel. The method of demineralizing of cocoons is also advantageous in that it reduces the labour and cost required for producing fibres from wild Silk and reduces wastage by enabling all or most of the silk to be reeled from cocoons. The method is further advantageous in that it enables the dissolution and regeneration of certain wild silks which cannot be regenerated under mild conditions with the prior arts enabling the formation of regenerated silk materials with improved properties and reduced cost.

Thus the method and materials produced by it overcome the disadvantages and limitations inherent in existing processes and in the silks obtained from them.

DETAILED DESCRIPTION OF THE INVENTION In the first aspect of the invention, wild silk cocoons are treated by immersing them in a demineralizing agent preferably an aqueous solution containing 0.2-1 M

ethylenediaminetetraacetic acid (EDTA) or one of its salts or a combination of EDTA with one of its salts or with another chelating agent capable of removing divalent cations or with an aqueous solutions containing citric acid or an aqueous solution containing a fruit juice containing citric acid at a pH between 5 and 10, preferably pH 7. Such solutions containing citric acid could, by way of example only, be lemon juice, orange juice or mausammi juice. Less preferably by way of example other demineralizing agents can be used instead including, by way of example only, deionised water, formic acid or solutions containing aluminium nitrate, phosphate ions or magnesium ions. It is to be understood that a combination of one or more demineralizing agents can be used to remove the calcium oxalate and other minerals from the cocoon and partially remove or soften the matrix between the silk fibres. In one embodiment the invention provides a method to facilitate the reeling or carding of the cocoons of wild silkworms by first treating them with an aqueous solution of a demineralizing agent comprising one or more of the following: ethylenediaminetetraacetic acid

ethylenediaminetetraacetic acid salt

ethyleneglycoltetracetic acid

ethyleneglycoltetracetic acid salt

citric acid

a fruit juice containing citric acid

deionized water

aluminium nitrate

phosphate ions

magnesium ions.

In another embodiment the invention provides a material prepared from wild silk cocoons treated with a demineralizing agent as herein defined.

In each case treatment with the demineralizing agent is carried out at temperatures between 10 and 100°C and preferably between 15 and 60°C. Treatment under mild conditions with the demineralizing agent disclosed here advantageously avoids the degradation to the silk fibres caused by the use of alkaline solutions, soaps, enzymes, retting or prolonged or high temperature treatments.

To facilitate wet reeling it is advantageous for the demineralizing agent to leave at least some of the gum intact while softening it. Failure to leave some of the gum intact allows collapse of the cocoon fibres into a compact and readily tangled mass from which it is difficult to reel or find fresh ends. Failure to soften the gum is also disadvantageous in raising the force required for reeling leading to a greater risk of damaging the fibres. It will be clear from this that demineralisation as described above is distinguished from degumming in that it leaves some or substantially all of the gum intact.

It is further advantageous if part or all of the treatment of the cocoons by immersion in one or more of the demineralizing agents described above is carried out under reduced pressure to facilitate the removal of air from the wall of the cocoon and thereby the penetration of the demineralizing agent.

It is further advantageous if the cocoons are subjected to an initial treatment with a dilute solution of ammonia or an ammonium salt or a combination of both or other dilute mildly alkaline solution or buffer both with a pH not in excess of 8.5 to facilitate removal of tannins or other polyphenols present in the cocoons before demineralizing of the cocoon. This initial treatment can be applied with or without the use of a vacuum to facilitate penetration of the solution. It will be understood that removal of tannins and/ or other substances in this way reduces the toxicity of the silk and helps the subsequent softening and swelling of the matrix between the sericin fibres and subsequent removal of mineral in the demineralizing agent.

Highly advantageously, demineralizing the wild silk cocoons as disclosed in this invention makes it possible to reel most or substantially all of the silk from the cocoon using standard equipment used for the wet reeling of Bombyx mori silk. Demineralizing in this way permits the cocoons of some species of wild silkmoth to be reeled for the first time and for other wild silk species to be reeled without recourse to damaging reagents or retting procedures. It also allows the damaging dry reeling or semi-dry reeling to be replaced by gentler wet reeling. Moreover it avoids the use of other damaging treatments required in some species to enable the silk to be carded. It is to be understood that the method of demineralizing the cocoons facilitates the subsequent degumming of the silk in skeins or as silk fabrics both prepared from either reeled or carded and spun silk. Thus demineralizing the cocoons enables the fibres or yarns or threads or tops or noils or silk fabrics prepared from them to be more completely or more rapidly or more fully degummed or degummed under mild conditions using sodium carbonate with or without soap solutions or enzymes or other reagents used in the mulberry silk industry and known to a person skilled in the art. It will also be understood that the methods for subsequent treatment or handling silk currently used in the silk industry such as finishing and dyeing can also be applied to silk obtained from demineralised cocoons.

Demineralisation of wild silks according to the invention is also highly advantageous for the purpose of preparing regenerated silk solutions in that it enables the silk to be dissolved under milder conditions than in the prior arts. A person skilled in the art will understand that the use of mild conditions in this way greatly improves the properties of the regenerated silk materials obtained.

In a second aspect of the invention the silk fibres obtained by reeling wild silk cocoons after demineralizing as disclosed here with or without subsequent degumming in solutions of sodium carbonate or solutions of sodium carbonate with or without added soap solutions or solutions containing one or more enzymes have advantageous properties compared with fibres obtained using the conventional methods for the preparation of wild silks according to the previous arts. By way of example only the average ultimate tensile strength of Gonometa postica silk fibres prepared by demineralizing with EDTA and subsequently degumming with sodium carbonate according to the current invention was 400 MPa (n= 100) compared to a value of 178 MPa reported by Colomban et al. (op. cit.) obtained for fibres degummed by heating for 1 hour to 95°C in a strongly alkaline soap solution and carding. Similarly the average Young's modulus of fibres prepared according to the present invention was 8.3 GPA (n = 100) compared with 1.4 GPa for fibres prepared with the prior art. Thus the current invention produces fibres of superior tensile strength and Young's modulus compared with the prior art. Load to break and elongation to break of Gonometa postica silk obtained using the protocol described below is significantly higher (both p<0.0001) compared to fibres obtained by carding after treating cocoons with fresh pineapple extract containing bromelain according to the previous art.

A further advantageous property of the silk produced by demineralizing and degumming according to the present invention as described below in Protocol 1 is that all mineral matter including the toxic calcium oxalate is entirely removed from wild silk. To illustrate this, by way of example only, Figure 1 shows that after demineralisation no calcium oxalate could be detected in Gonometa postica silk using a Bruker D8 powder X- ray spectrometer. As calcium oxalate is highly toxic its removal greatly improves the biocompatibility wild silks prepared in this way. In a further aspect of the invention demineralised and degummed fibres prepared according to the present invention can be dissolved in a chaotropic agent under milder conditions than those required in the prior arts. These milder conditions comprise a reduction in the temperature of the chaotropic agent and/or a reduction in the

concentration of the chaotropic agent and in some cases the use of an aqueous solution of lithium bromide in place of the more damaging lithium thiocyanate solution. Thus the milder conditions required for dissolving demineralised silks compared with those of the prior arts enables the formation of regenerated silk materials with improved properties and reduced cost. Silk solutions prepared from demineralised silk prepared according to the invention can be dialysed against deionised water and concentrated as will be understood by a person skilled in the art. Such a person will also know a plurality of ways for using resulting concentrated silk solution to form materials.

In a further aspect of the invention, material prepared according to any aspect of the invention can be used in the manufacture of medical devices or for non-medical uses. The invention is illustrated by way of the following non-limiting examples: PROTOCOL 1. DEMINERALIZING AND DEGUMMING WILD SILK COCOONS

1. Freshly spun cocoons of Gonometa postica were obtained before or after emergence of the moth. To maintain the continuity of the fibre, cocoons were harvested with secouters while still attached to a small piece of branch. For the same reason, cocoons with holes produced by eclosion of parasitic wasps were not used. Cocoons from which the moths had not emerged were stored at -25°C to kill the pupae but other methods of doing this can be used instead as would be understood by a person skilled in the art. For hatched cocoons, pupal shells were removed through the emergence hole using forceps.

2. Optionally cocoons were immersed in aqueous 0.1 to 0.001 M ammonium chloride or ammonium hydroxide solution for 1 hour at 20°C under vacuum. The cocoons were kept under the surface of the solution by a piece of plastic mesh to encourage penetration by the solution. Thereafter the cocoons were thoroughly washed with tap water at room temperature. For some purposes this step can be omitted.

3. A solution of 1M ethyl enediaminetetraacetic acid (EDTA) was adjusted to pH 10 with a concentrated sodium hydroxide solution. The cocoons were immersed in this for 48 hour at 40°C on shaker/stirrer and under vacuum as in step 2 above.

The cocoons were then thoroughly washed with tap water containing calcium ions at room temperature. The use of tap water instead of deionized water improves the ease of subsequent reeling possibly by replacing calcium ions removed by the EDTA. A better characterised solution containing calcium can be used in place of tap water.

4. The cocoons were kept wet and were then ready to be reeled or carded all at room temperature and using standard methods as used by a person skilled in the art. All or substantially all of the silk in un-emerged cocoons could be reeled into a yarn after demineralizing, sometimes without the necessity of finding a new end. 5. Degumming was carried out for one hour with 0.5% sodium carbonate solution at 80°C. For preparation of regenerated silk cocoons demineralised as in step 3 were degummed directly, omitting step 4.

PROTOCOL 2. TENSILE TESTING WILD SILKS 1. Single brins or baves of Gonometa postica silk were obtained respectively with and without degumming using Protocol 1 shown above. For comparison with the industrial method for degumming Gonometa postica silk we used an industrial yarn. The yarn was prepared by degumming cocoons in pineapple extract according to a standard protocol. The silk was then hand carded and hand spun into a yarn. Brins for mechanical testing were gently teased out of the yarn.

2. Hatched cocoons of Antheraea mylitta and A. pernyi were demineralised as in protocol 1. For comparison, industrially reeled A. pernyi and A. mylitta silk skeins were used directly as obtained from commercial suppliers.

3. Samples were mounted without preload onto cardboard frames to give an initial gauge length of 15 mm. The fibres were equilibrated at 40% RH and 26°C for five minutes prior to tensile testing under the same environmental conditions on an Instron Universal Materials Testing Instrument at a strain rate 50% per minute using a 5 N load cell.

4. Cross sectional areas were obtained by clamping orientated silk threads in a holder and cutting them across with a new razor blade before sputtering them with gold and mounting them in a scanning electron microscope whose magnification had been calibrated with a standard TEM grid. As it was impractical to obtain cross sectional areas for each sample adjacent to each test sample, normalized cross-sectional areas were obtained by averaging 150 brins for each sample.

5. Engineering ultimate tensile strengths are reported here. Stress strain curves were plotted using Excel. The J-integral method was used to determine the energy to break. Exelstat 7.5.2 was used for statistical analysis of the data with ANOVA (two tailed). EFFICIENCY OF REELING

Demineralised Gonometa postica cocoons could be completely reeled as shown in Table 1.

Table 1: Reeling data of four different G. postica cocoons is shown. Both the total length of silk reeled from each cocoon and a visual estimate of the completeness of the reeling are

tabulated.

Similar results were obtained for demineralised Antheraea mylitta, A. pernyi, Cricula andrei and Samia canningii cocoons.

RESULTS OF TENSILE TESTING

The effect of combined demineralization and degumming using Protocol 1 on the stress strain curve of Gonometa postica silk filaments is shown in Figure 2. The shape of the stress strain curve is much more consistent in the demineralised and degummed fibres compared to those degummed according to the prior art with pineapple juice. This probably results from highly variable damage during the carding used to separate the filaments. The effect of demineralization on its own, combined demineralization and degumming using sodium carbonates solution, and degumming without demineralization using the pineapple juice treatment of the prior art on the tensile properties of Gonometa postica silk filaments is shown in Table 2.

Table 2: The effect of demineralisation, combined demineralisation and degumming according to the present method and treatment with pineapple juice according to the prior art on the tensile properties. Each box shows the mean, standard deviation and one tailed probability (ANOVA).

Compared with the prior art, Gonometa postica silk filaments prepared by demineralizing the cocoon are highly significantly stronger (p<0.001), 0.056N against 0.080N (Table 2). However, the breaking stress of the demineralised silk filaments is significantly less (p<0.05) due to the greatly reduced cross sectional area of the filaments prepared according to the prior art.

Figure 3(c) shows that pineapple juice produces a highly significant (p<0.001 ) reduction in the cross sectional area of the fibres (125 μπι ² ) in comparison to the two other treatments. This may result from the high efficiency of pineapple juice for removing the sericin gum, most probably because of the active bromelain in this extract. The enzyme might also contribute to the greater variability of the mechanical properties of the silk treated with pineapple juice compared with the demineralised/degummed filaments. Although the breaking energy (Figure 3(f)) is not significantly greater (p>0.05) compared with the pineapple degummed fibres the demineralised/degummed filaments do have a significantly larger (p<0.001) elongation to break (Figure 3(e)).