Biospinning: Change of Water Contents in Drawn Silk

TOSHIHISA TANAKA1, MASATOSHI KOBAYASHI1, SHUN-ICHI INOUE1, HIDETOSHI TSUDA1, JUN MAGOSHI1,2

1Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST), Japan

2National Institute of Agrobiological Sciences (NIAS), 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan

Received 26 August 2002; revised 13 September 2002; accepted 12 November 2002

ABSTRACT: Changes of the water content in drawn silk during drying were investigatedby thermal analysis and 1H pulse NMR. Water in liquid silk by drawing extruded fromthe inside of the silk filament into ambient air. The water contents in the drawn silkdecreased with drying time. Assuming the nonfreezing water has a concentration of 10wt % in the liquid silk, the percentage distribution of water in liquid silk is composedof 10 wt % nonfreezing water, 40 wt % freezing water, and 30 wt % free water. This 40wt % freezing water in the liquid silk may be important for the formation of fine poreson the surface of drawn silk. The apparent pore radius, which was calculated from theresults of thermal analysis, on the surface of drawn silk decreased to 5.0 nm and finallyto 2.0 nm. The calculated apparent fine pore formed on the surface by drawing was 4.0nm. © 2002 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 274–280, 2003Keywords: biopolymer; drawing; differential scanning calorimetry (DSC); 1H pulseNMR

INTRODUCTION

The larvae of domestic and wild silkworms, otherinsects, and spiders produce excellent, fine silkfibers. Silk fiber has many good mechanical andphysical properties for physical chemistry andmaterial science.

The silk glands of a silkworm are a pair oftubes lying on the larvae (Fig. 1). The liquid silkin the silk gland of a silkworm is a highly viscousaqueous solution of two separate proteins, fibroinsurrounded by sericin. The fibroin and sericin aresynthesized in the posterior division (P) and mid-dle division (M) of the silk gland, respectively.These proteins are stored mainly in the middledivision before spinning. In the anterior part ofmiddle division (MA) or anterior division (A), theliquid silk undergoes a gel–sol transition. Liquid

silk is in a liquid state as it flows in the narrowducts to the spinneret. Then silk fiber is producedfrom the spinneret that consists of a silk pressand an orifice. The principal factor of silk-fiberformation involves the action of shearing stressand elongational stress acting on silk fibroin,causing the liquid silk to crystallize.

Silkworms perform very accurate molecularorientation control with several sophisticatedspinning techniques.1 The process of structuralformation from liquid silk to silk fiber is remark-ably complex. The silkworm’s spinning methodincludes the evaporation of water after extrusioninto ambient air. The liquid silk in the silk glandhas 20–25 wt % protein concentration. Water inthe liquid silk evaporates rapidly after extrusionwhen it comes in contact with ambient air (dryspinning and porous spinning). The fine poresformed during evaporation supposedly providethe silk fiber with its excellent moisture uptake,dye-ability, and drape characteristics. The poros-ity of a cocoon filament was analyzed by the ap-parent density measurements2 and small-angle

Correspondence to: T. Tanaka (E-mail: toshihm@ nias.affrc.go.jp)Journal of Polymer Science: Part B: Polymer Physics, Vol. 41, 274–280 (2003)© 2002 Wiley Periodicals, Inc.

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X-ray analysis.3 In this study, we evaluate thechanges of the water content in drawn silk bythermal analysis and 1H pulse NMR to clarify theprocess of silk-fiber formation.

EXPERIMENTAL

Samples were prepared from the liquid silk ofdomestic silkworms, Bombyx mori. The liquid silkwas obtained from the middle part of the middledivision (MM, refer to Fig. 1) in the silk gland ofmature silkworms one day before spinning or co-cooning.

The liquid silk was drawn into fibers after re-moving the tissue portion of the silk gland. Thedrawing conditions were at a rate of about 50cm/min at 25 °C, and the draw ratio was 20 times.The drawn silk was dried at 25 °C under ambientair or at 105 °C under vacuum. Ambient air in theexperimental laboratory was controlled at 25 °Cand about 50–60% (relative humidity) by air-con-ditioning equipment. The drawn silk was ob-served with a light microscope equipped with po-larizer and analyzer. The orientation of thedrawn silk after drying was observed by X-raydiffraction. A Rigaku X-ray generator was oper-ated at 35 kV and 20 mV, with Ni-filtered Cu K�radiation. Thermal analysis of the drawn silk wasperformed by differential scanning calorimetry(DSC) with a Seiko DSC 6100 instrument. Sam-

ples were hermetically sealed in an aluminumcapsule. The standard heating rate of 5 °C/minwas used. The temperature range was from �150to 100 °C. At the end of each DSC measurement,a small hole was made in the aluminum capsule,the sample was dried under vacuum in an oven at105 °C, and the weight of the water in the sam-ples was determined. 1H pulse NMR measure-ments of the liquid silk were carried out with aBruker minispec pc20 spectrometer operating at20 MHz. 1H pulse NMR T2 values were measuredby the Carr–Purcell–Meiboom–Gill (CPMG)method4,5 at 40 °C. The 1H NMR 90° pulse widthwas 3.2 �s, the acquisition number was 4, and therepetition time was 10.0 s. The 1H pulse NMR T2values were measured by the solid-echo pulse se-quence6,7 at 40 °C to obtain information about themolecular motion of water in the drawn silk. The1H NMR 90° pulse width was 3.2 �s, the acquisi-tion number was 8, and the repetition time was4.0 s. Samples of the drawn silk for 1H pulse NMRwere prepared by drying at 25 °C under ambientair for 3 days, at 25 °C under vacuum for 24 h, andat 105 °C under vacuum for 2 days.

RESULTS AND DISCUSSION

After removing the tissue portion of the silkgland, the liquid silk was drawn at 25 °C underambient air. The water in the samples extrudedinto the surface from the inside of the silk fila-ment by drawing. The drawn silk contained wa-ter, was opaque, and had little orientation as ob-served by polarizing light microscopy.

The water content in the drawn silk decreasedwith evaporation into ambient air and drasticallydecreased to a drying time of 30 min (Fig. 2). Thewater content measured to an almost constantvalue in the drying time of 120 min. The evapo-rated water content in the drawn silk dried at 25°C was a total of about 60 wt %. Here, it set thestandard for the weight of the drawn silk dried at25 °C under ambient air to come to equilibrium inweight. Therefore, the water content in the sam-ples was obtained from the critical weight afterdrying at 25 °C for 3 days. However, the watercontent in the drawn silk soon after drawing maybe contained about 70 wt %, whereas the driedsilk at 120 °C under vacuum for 12 h contained 10wt % water.8

The drawn silk dried at 25 °C for 3 days wastransparent and had a uniaxial orientation to thedrawn direction as observed by polarizing light

Figure 1. Photograph of the silk gland of the domes-tic silkworm, Bombyx mori. The silk gland is composedmainly of posterior, middle, and anterior divisions. Themiddle division is composed of posterior, middle, andanterior parts.

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microscopy. From the X-ray diffraction patterns,the molecular orientation of silk fibroin in thedried silk also exhibited a uniaxial orientation aswell as the typical �-form structure of silk fibroin(Fig. 3). The silk fibroin molecules in the liquidsilk before drawing have a random coil and hy-drated �-form structure. Those in the silk fila-ment after drawing have the �-form structure,uniaxially oriented to the drawing direction.

In the DSC curves of the drawn silk with dry-ing times between 0 and 4 min (Fig. 4), a broadendothermic peak exists around 0 °C attributed tomelting of free water, which is adsorbed on thesurface of the drawn silk without interaction withprotein. There are two endothermic peaks in theDSC curves of the drawn silk with drying timesbetween 5 and 9 min (Fig. 5). The endothermicpeak in the higher-temperature region is as-signed to melting of free water, whereas the en-dothermic peak in the lower-temperature regionis assigned to melting of freezing water in thedrawn silk. In the DSC curves of the drawn silkwith a drying time of over 10 min, there is no peakrelated to melting of water in the drawn silk (Fig. 6).

Figure 7 shows the relation between the calcu-lated amounts of respective water in drawn silkas a function of the drying time. The amounts offreezing water and free water were calculatedfrom area aggregates of peaks from the DSC re-sults. The amount of total water was calculated

from the weight of water in samples before andafter drying at 105 °C under vacuum. Theamounts of the freezing and free water decreasedwith increasing drying time. From a drying timeof 5 min, the freezing water and free water con-tents were calculated from the areas of two endo-thermic peaks, respectively. Nonfreezing water,which is the restricted or noncrystallized water byhydrogen bonding with the protein, reveals nopeak in the DSC curves on thermal change.9–11

The amount of nonfreezing water was calculatedby subtracting the amounts of freezing and freewater from the total water. The calculated non-freezing water content in the drawn silk gradu-ally increased with increasing drying time. It hasa maximum value around 7–8 min and then lev-els off to approximately 10 wt %. As another ap-proach for determining the amount of nonfreezingwater, Figure 8 illustrates the relation betweenthe nonfreezing water ratio and the water ratio.The water ratio was obtained from dividing thetotal water (free, freezing, and nonfreezing water)weight by the dried sample weight. The nonfreez-ing water ratio was obtained from dividing the

Figure 3. X-ray diffraction pattern of the drawn silkafter drying at 25 °C for 3 days to come to equilibriumin weight. The meridian is the drawing direction ofliquid silk.

Figure 2. Change of the water content in the drawnsilk after drying by mass measurements. It set thestandard for the weight of the drawn silk dried at 25 °Cfor 3 days to come to equilibrium in weight. The trans-verse axis is the drying timescale.

276 TANAKA ET AL.

nonfreezing water weight by the dried sampleweight. This also shows a maximum at 0.5 g/g forthe water ratio and finally leads to 0.1 g/g for thenonfreezing water ratio. Therefore, the nonfreez-ing water appears to be restricted to some extent(ca. 10 wt %) in the �-form structure of a silkfibroin molecule.

Additional information about water in liquidsilk can be obtained from 1H NMR T2 values by

the CPMG method. The 1H NMR T2 signal decaycurve was composed of three components, that is,a short T2 component corresponding to the immo-bile component, an intermediate T2 componentcorresponding to a quasi-mobile component, and a

Figure 4. DSC curves of the drawn silk after dryingfor 0–4 min. The standard heating rate of 5 °C/min wasused. The temperature range measurement was from�150 to 100 °C. Time is the drying time.

Figure 5. DSC curves of the drawn silk after dryingfor 5–9 min. The standard heating rate of 5 °C/min wasused. The temperature range measurement was from�150 to 100 °C. Time is the drying time.

Figure 6. DSC curves of the drawn silk after dryingfor 10–30 min. The standard heating rate of 5 °C/minwas used. The temperature range measurement wasfrom �150 to 100 °C. Time is the drying time.

Figure 7. Change of amounts of respective water inthe drawn silk after drying calculated from DSC andmass measurement results. It set the standard for theweight of the drawn silk dried at 105 °C under vacuumfor 2 days to come to equilibrium in weight. The trans-verse axis is the drying timescale.

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long T2 component corresponding to the mobilecomponent. T2 values and component ratios were164 ms and 18.2%, 448 ms and 51.3%, and 995 msand 30.5%, respectively. A short T2 componentwas assigned to the 1H NMR signal of silk proteinbecause the liquid silk is a high-polymer concen-tration, about 20 wt %, and has restricted mobil-ity. The intermediate and long T2 componentswere assigned to 1H NMR signals for the bondingand free water in the liquid silk, respectively. Theintermediate T2 component corresponds to thenonfreezing and freezing water obtained by ther-mal analysis.

For the drawn silk dried at 25 °C, the 1H NMRT2 signal decay curve by solid-echo pulse se-quence was composed of two components. Each T2value and component ratio were 17.1 �s and94.3% as well as 65.9 �s and 5.7%. The signaldecay curve for the drawn silk dried at 25 °Cunder vacuum also was composed of two compo-nents. These values were 16.9 �s and 96.3% aswell as 58.2 �s and 3.7%. Eventually, for thedrawn silk dried at 105 °C under vacuum, thesignal decay curve had one component (T2 � 25.6�s). This means that the short T2 component wasattributed to the 1H NMR signal of silk protein.The long T2 component was attributed to non-freezing water in the drawn silk. This conclusion

was also supported from the T2 value attributedto silk protein (T2 � 11 �s), and the dried silk had6–10 wt % water.8

These results from NMR and thermal analysisfor the drawn silk can estimate the componentratio of water in liquid silk. Assuming the non-freezing water has a concentration of 10 wt % inthe liquid silk, these results indicate that theoverall water content in the liquid silk is com-posed of 10 wt % nonfreezing water, 40 wt %freezing water, and 30 wt % free water. The 10 wt% nonfreezing water is restricted to protein and isdifficult to remove without treatment of 105 °Cunder vacuum. The 30 wt % free water in liquidsilk evaporates rapidly into ambient air after ex-trusion. The 40 wt % freezing water in the liquidsilk may be important for the formation of finepores on the surface of drawn silk.

Silk fiber has fine pores formed by evaporation.The porosity in the domestic silk fiber, Bombyxmori, was 5.2% by apparatus density measure-ment with a mixture of water and carbon tetra-chloride.2 The microporous size in the drawn wildsilk, Antheraea pernyi, was also measured bysmall angle X-ray diffraction.3 In scanning elec-tron microphotographs of the wild silk fibers, An-theraea pernyi, round or elliptical shaped pores(diameter: 0.1–0.3 �m) were found in cross sectionsof the cocoon filament. However, the domestic silkfibers, Bombyx mori, have no porous structure inthe cocoon filament by electron microscopy.12,13 Thedifferences of morphology between the domesticand wild silk fiber have occurred because of thesecretory mechanism of silk fibroin in the silkgland.13,14 What information could we obtain fur-ther for the pore size in drawn domestic silk? Weestimated the pore size in the drawn domestic silkof Bombyx mori by applying the thermal resultsto the following equation.

Assuming a pore shape, a pore volume distri-bution (PVD) curve as a function of pore size canbe determined from the DSC curves on freezingand melting.15–17 In this study, the shape of poresin drawn silk was simply assumed to be cylindri-cal, and the pore size was directly calculated fromthe melting data (heating process) of thermalanalysis.

The abscissa of the DSC curve, temperature T,can be transformed into pore radius R to obtainthe PVD with the following equation:

R ���T�

�T � �

Figure 8. Relation between nonfreezing water ratioand water ratio. The nonfreezing water ratio is calcu-lated from dividing the nonfreezing water weight bythe dried sample weight. The water ratio is calculatedfrom dividing the total water weight by the dried sam-ple weight.

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where R is the pore radius, �T is the depression ofthe melting temperature, and �(T) is a coefficientgiven as a function of temperature, which wasdetermined to be 33.30–0.3181 �T during melt-ing (in nm-K) for the case of a cylindrical pore. �Tis determined from the difference between themelting point of free water and that of freezingwater. The first term on the right-hand side of theequation represents the pore radius of freezingwater, whereas the second term, �, is the thick-ness of nonfreezing water adsorbed on the surfaceof the pores. In the literature on the PVD curvesfor silica gels,15,16 the � values were calculated byrepetitive optimization. However, it was difficultto determine the � values in the drawn silk be-cause the nonfreezing water could exist not onlyon the pore surface but also in the drawn silkmatrix. According to the article describing the �values in poly(methyl methacrylate) mem-branes,17 the hypothesis of two or three monolay-ers of adsorbed water on some materials has beenproposed. Therefore, the � values for drawn silkwould also be assumed to be equal to 1.0 nm,which corresponds to nearly three monolayers.The error based on the assumption could be �0.5nm, or it could be negligible. Therefore, we calcu-lated the pore size of drawn silk by direct appli-cation of the preceding equation.

Figure 9 portrays the change of the apparentpore radius in drawn silk with drying time, whichwas calculated from applying the results to an

approximate equation. The apparent pore radiuson the surface of the drawn silk decreased to 5.0nm for 6 min and finally to 2.0 nm after 10 min.This change of the apparent pore radius suggeststhat the calculated pore size may contain the wa-ter layer and/or the distribution of pore sizes onthe surface. Therefore, the calculated pore sizewith a drying time of 5 min contains the waterlayer. The calculated apparent fine pore formedon the surface by drawing is 4.0 nm. These calcu-lated values support the pore size in wild silkfibers by scanning electron microscopy.12,13 Be-cause the order of the calculated pore size is verysmall, it may be difficult to observe such finepores in the drawn domestic silk of Bombyx moriby electron microscopy.

The silk fibroin contains mainly glycine, ala-nine, and serine, which are hydrophobic aminoacids. The crystallized silk fibroin is an insolu-ble protein, whereas the liquid silk is soluble inwater. As identified previously, the water con-tent in liquid silk is approximately 80 wt %, andthe water molecule is released into ambient airby drying. The existence of three components ofwater in liquid silk is important for the silkfiber and unique as a hydrophobic polymer. Allwater molecules in films (10 wt % nonfreezingwater) are bound to silk fibroin molecules withrapid rotation of the methyl groups of the ala-nine residues.8 With this situation, however, itis not known how amino acid composition in silkfibroin related with three components of waterduring spinning.

The authors are grateful to M. A. Becker for her sug-gestions and to Yoshiko Magoshi for her kindness inproviding the silkworms.

REFERENCES AND NOTES

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2. Ishikawa, H.; Sofue, H.; Matsuzaki, K. ResearchReports of the Textile and Sericulture. ShinshuUniversity, 1960; Vol. 10, pp 176–183.

3. Hirabayashi, K.; Ishikawa, H.; Kakudo, M. SEN-IGAKKAISHI 1969, 25, 440–446.

4. Carr, H. Y.; Purcell, E. M. Phys Rev 1954, 94,630.

5. Meiboom, S.; Gill, D. Rev Sci Instrum 1958, 29, 688.6. Powles, J. G.; Strange, J. H. Proc Phys Soc 1963,

82, 6.7. Mansfield, P. Phys Rev 1965, 961, 137.

Figure 9. Apparent pore radius in the drawn silkafter drying calculated from the results of the thermalanalysis. The transverse axis is the drying timescale.

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8. Asakura, T.; Demura, M.; Watanabe, Y.; Sato, K.J Polym Sci Part B: Polym Phys 1992, 30, 693–699.

9. Nakamura, K.; Nishinari, Y.; Hatakeyama, T.;Hatakeyama, H. Thermochim Acta 1965, 267,343.

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13. Akai, H. Int J Wild Silkmoth Silk 2000, 5, 71–84.14. Akai, H.; Nagashima, T.; Aoyagi, S. Int J Insect

Morphol Embryol 1993, 22, 497–506.15. Ishikiriyama, K.; Todoki, M.; Motomura, K. J Col-

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