Low Tensile Strength Due to Fragile Points on Silkworm Cocoon Filaments

Transcription

1 Journal of Insect Biotechnology and Sericology 84, (2015) Low Tensile Strength Due to Fragile Points on Silkworm Cocoon Filaments Keisuke Mase *, Eiji Okada, Tetsuya Iizuka, Takako Miyajima and Toshio Yamamoto National Institute of Agrobiological Sciences (NIAS), 1-2 Ohwashi, Tsukuba, Ibaraki, , Japan (Received October 27, 2014; Accepted April 15, 2015) In order to develop a new silkworm race that produces strong silk fibers, we investigated the physical properties of the cocoon filaments of our breeding strains, which includes a wide variation in filament size. Our strains showed the largest variation in tensile strength ( gf/d), being dependent on filament size, with the weakest cocoon filament being that of PCG, which spins an ordinary size filament (3.8 denier). The tensile strength of the PCG cocoon filament correlated negatively with the sample length, suggesting many fragile points on the filament. A non-uniform shape with many thin constrictions was also observed in PCG cocoon filaments under microscopic analysis. Key words: tensile strength, fragile points, silkworm cocoon filament, interracial variation INTRODUCTION The Japanese silk industry has a strong imperative to breed a new silkworm race that will produce superior silk features which can be differentiated from ordinary silk material. Generally, the tensile strength, elongation, and Young s modulus are the most important physical characteristics of a fiber. Extremely strong raw silk has been in high demand and was once used to make parachutes. In the present, continuing developments are expected for uses such as lightweight ballistic material, stronger suture thread for operations, and biodegradable fishing line. Silk thread, with an average strength of 3-4 gf/d in a state of dryness, is weaker than chemical threads such as polyester ( gf/d), but is a relatively strong natural thread when compared with cotton (3 gf/d) or wool (2 gf/d). Moreover, the elongation of dried silk thread (15-25%) is higher than that of cotton (3~7%), although it is lower than that of wool (25~35%). In short, silk is a natural thread that combines a comparatively high strength with elongation (Shao and Vollrath, 2002; Keten et al., 2010). Silk thread is usually made of five to a dozen cocoons by reeling, and a cocoon consists of a long filament. Filament strength is defined as the tensile intensity (gf/d) that can be endured around a unit of the cross section. The cocoon filament intensity of the inner layer of the cocoon shell is the strongest, because the filament size generally becomes thinner the farther inside the layer is from the middle of the cocoon shell. This means that the strength, *To whom correspondence should be addressed. Dr. Keisuke Mase Nihon University, College of Humanities and Sciences, Sakurajyosui, Setagaya-ku, Tokyo, , Japan Fax & Tel: mase.keisuke@nihon-u.ac.jp elongation, and Young s modulus of a cocoon filament depend on the size of the filament. This is likely due to the higher crystallinity of thinner filaments produced by a higher spinning speed during the development of the filament structure (Iizuka, 1980). However, there are wide variations among silkworm strains regarding the physical properties of their cocoon filaments. In those variations, cocoon filament strength and the dynamic modulus of elasticity have a significant negative correlation with filament size, while elongation has a positive correlation (Iizuka, 1980). In the present study, we have investigated the diversity of the physical properties of cocoon filaments in our silkworm breeding strains to develop a new silkworm race that will produce a stronger filament. Additionally, we have clarified a factor that influences cocoon filament strength. MATERIALS AND METHODS We used 31 Japanese and 33 Chinese silkworm strains, which are maintained at our laboratory, to investigate the interracial variations of the physical properties of cocoon filaments. Silkworm larvae were reared on mulberry leaves and cocooned at 25 C at 70% humidity. Ten dried (105 C, 3 hrs) cocoons were used for each strain. After cooking the cocoons for 13.5 min, each silk sample was prepared from the middle layer of the cocoon shell in a 40 C water bath at a speed of 77 m/min using a multi-end reeling machine twisted three times using the kennel system. To establish the conditions needed for constant measurement, the raw silk samples were kept at 20 C, 55~65% relative humidity, for 2 more hrs until physical testing. Ten centimeters of raw silk was sacrificed to measure the samples physical properties; the filament size (the denier) was estimated on both sides of the sample. The average tensile strength, elongation, and Young s modulus of the 15 samples were measured using the tensile testing ma-

2 44 Mase et al. chine Tensilon STA-1150 (Orientec) at a strain rate of 150 m/min. Regression analysis of each of the physical properties of filament size, with respect to Japanese and Chinese strains, were performed separately because the cocoon filament of Japanese strains are generally thicker than those of Chinese strains. The physical properties of three strains, C515, J510, and PCG were given additional investigation because the cocoon filaments of these strains have unique characteristics: the C515 larva spins a super-thin cocoon filament (1.7 denier); the J510 larva makes a thick filament (5.5 d); and PCG filament breaks easily during the reeling process in spite of its ordinary size (3.8 d). The 30 lines of raw silk prepared from the 10 dried cocoons were bundled into one skein; they were also measured for their tensile strength, elongation, and Young s modulus. Moreover, in order to investigate the influence of the sericin layer on their physical properties, these raw silk skeins were adequately degummed twice in 0.2% Marseilles soap and 0.05% NaCO3 at 95 C for 40 min in a state that fixed both ends of the skein with a clip. Their physical properties were also measured using Tensilon STA It can be generally assumed that the longer a filament is, the easier it is to cut if there are many fragile points on the filament. The cocoon filaments of these three strains were also investigated in samples of different lengths. Five cocoons were randomly chosen from one strain. A 100 m cocoon filament was prepared from the middle layer of a dried cocoon using a single cocoon reeling machine, and the filament size was calculated from its weight. From this 100 m filament, 10 samples of each length, 10, 20, and 30 cm, were prepared and their tensile strength, elongation, and Young s modulus were measured using a tensile testing machine. The results for each cocoon filament were calculated as the average of 10 samples. The lateral morphologies of these filaments were photographed under a microscope. RESULTS We are maintaining several silkworm strains with wide variations in filament size, from a 1.3 denier Chinese strain to a 5.5 denier Japanese strain. Investigating our strains showed that one cocoon filament, which is the average of 10 cocoons, was able to endure a tensile force from 5.3 gf (Chinese strain) to 19.2 gf (Japanese strain) (Fig. 1A). The strength of a single cocoon filament showed an extremely high positive correlation with filament size, in both Japanese and Chinese strains, indicating that thicker cocoon filaments can bear heavier pulling weights. On the other hand, filament strengths showed higher scores with thinner cocoon filaments, since tensile strength is defined as the force that can be resisted by the weight per unit of the cross-sectional area up to the breaking point by pulling tension (Fig. 1B). For a typical example, the superthin filament strain C515, which spins a cocoon filament of 1.7 denier in size, showed the highest strength (4.46 gf/ d), whereas the thicker filament strain J510 (5.5 d) was estimated to have a lower strength (3.52 gf/d). As for this tendency, the Chinese strains (r = ***) had higher correlation coefficients than the Japanese strains (r = **), although both were lower than the strength of a single cocoon filament. Young s modulus values for both strains were also negatively correlated with filament size, as were the filament strengths (Fig. 1D, Chinese strains: r = **, Japanese strains: r = ***). On the other hand, there is no significant correlation (r = ns) between elongation and filament size, although a small positive correlation was seen in the Japanese strains (Fig. 1C, r = *). Among the strains we investigated, the cocoon filament of C515 has the highest strength with a thinner size; J510 has a lower overall strength due to the thickness of the filament. Meanwhile, the ordinary-sized filament (3.8 d) spun by the Chinese strain PCG showed the lowest strength (3.31 gf/d), suggesting that tensile strength is influenced by more than just filament size alone. Then we compared the physical properties between the raw and degummed silk from the three strains in order to investigate the influence of sericin. Tensile strength was significantly increased after degumming (C515: 13%, J510: 13%, and PCG: 18%, as shown in Fig. 2A), but the order of strength did not change among the three strains (C515 > J510 PCG). This is considered to be based mainly on the fact that the filament size was reduced by degumming, while sericin seemed to have little influence on differences in the raw silk strength between these strains. On the other hand, although the elongations decreased and Young s modulus increased after degumming for the three strains, the degree of alteration among the three strains was different. The elongation of C515 decreased remarkably (20% ***), while its Young s modulus increased (17% **) after degumming, whereas PCG displayed no significant difference between raw and degummed silk (Fig. 2B and 2C). Next, to investigate why the PCG filament has the lowest strength, we performed physical testing on different filament lengths prepared from cocoons of the C515, J510, and PCG strains. If a cocoon filament has many fragile points that are equally easy to break physically, then we would predict a longer filament sample to break more easily, indicating a lower tensile strength. We investigated the physical properties of the 10, 20, and 30 cm cocoon filament samples from the middle layer of an arbitrarily picked cocoon of each of the three strains. The tensile strengths of the C515 and J510 cocoon filaments showed practically no distinct relationship with the sam-

3 Fragile points on weak cocoon filaments 45 Fig. 1. The relationship between each physical property and cocoon filament size in our breeding strains. A: endurable weight of a single cocoon filament, B: cocoon filament strength, C: cocoon filament elongation, D: cocoon filament Young s modulus. C515 is a super-thin filament strain (1.7 d). J510 is a fat filament strain (5.5 d). The PCG larva spins cocoon filament of ordinary size (3.8 d) but the reelability is very low (38%). *, **, and *** indicate the significance of regression equation at 5%, 1%, and 0.1% levels, respectively. ple length (Fig. 3A and 3B), whereas the longer-filament PCG sample had a notably lower tensile strength (Fig. 3C). Therefore, the strength of the cocoon filament seems to have a negative correlation with the sample length on average (r = **) in PCG strains, but not in the C515 and J510 strains. Incidentally, something similar to a nonuniform shape with many thin constrictions (11.5 constrictions/5 cm) was observed on a PCG cocoon filament under a microscope, although C515 (1.0 constriction/5 cm) and J510 (3.5 constrictions/5 cm) cocoon filaments were relatively smooth and uniform (Fig. 3J, 3K, and 3L). The elongation of J510 and PCG showed a negative correlation with the sample length (Fig. 3E: J510 r = *** and Fig. 3F: PCG r = ***), although that of C515 was not statistically significant (Fig. 3D: C515 r = ns). On the other hand, this clear association was not recognized for the Young s modulus (Fig. 3G, 3H, and 3I). DISCUSSION A stronger silk thread material can be obtained by bundling several strong and thin filaments. This is useful in practice because it also keeps the filament size deviation to a minimum. Moreover, twisting the bundle of silk yarn, even to 500 T/m, can also raise the strength and elongation a little more (Nakajima et al., 2011). Therefore, a silkworm race that can produce a strong, thin cocoon filament as a material for high-strength silk is desired. In the breeding material strains investigated in this study, we confirmed that thinner filaments have a higher strength, and those of the super-thin strain C515 showed an especially high strength and a uniform shape with few fragile points (1 constriction/5 cm). The practical hybrid race Hakugin, for which C515 is used as a parental race, also produces a super-thin cocoon filament with a very low deviation of single cocoon filament size (Yamamoto et al., 1999). The cocoon filament characteristic of Hakugin is thought to originate from C515. Recently, C515 was also

4 46 Mase et al. used as a host strain for developing a novel transgenic silkworm that produces a high-toughness silkworm silk containing some spider dragline silk (Kuwana et al., 2014). The cocoon filament structures of silkworms and Antheraea become progressively thinner, and their crystallinity, elastic modulus, strength, and density are higher, while their elongation is conversely lower (Iizuka, 1980). This tendency is believed to depend on mechanical denaturation, in which the cocoon filament is formed by the mechanical shear stress applied against the liquid silk during spinning. In a silkworm cocoon filament, progressive crystallization is caused by a higher spinning speed as well as its increase in intensity (Shao and Vollrath, 2002). In addition to a crystalline region, an amorphous region of the silk is also thought to contribute to the mechanical properties of silk fibers (Iizuka, 1980). A recent study of spider silk shows a clear influence from the intermolecular association between beta-sheets in a crystalline region on strength (Hayashi et al., 1999). Moreover, it has been reported that the size and direction of a crystalline structure are also important factors related to strength (Du et al., 2006). In the present study, strength and Young s modulus depended on the filament size related to the crystallinity of the structure, although elongation was not clearly correlated with it (Fig. 1). However, some strains that deviated from the regression line were seen in the strength and Young s modulus, unlike the strength of a single cocoon filament, indicating an interracial variation caused by something other than the filament size. Specifically, the cocoon filament of PCG had a remarkably low strength despite its ordinary filament size. This exceptional cocoon filament was easy to snap when it was long (Fig. 3C), suggesting that it is not uniform, but instead has many fragile points (11.5 constrictions/5 cm). Since silkworms spin cocoon filaments using an S-shaped motion, fibroin fibers are crooked in a small pitch (Iizuka, 1980). The non-uniformity of PCG cocoon filaments that was observed under microscopic analysis suggests that the larva could not constantly perform the spinning motion for some genetic reason (Fig. 3L). Actually, the reelability of PCG cocoons is as low as 38%. This low reelability may be due to low cocoon filament strength. On the other hand, the elongation of the C515 filament was almost constant (Fig. 3D), although J510 and PCG showed a negative correlation with the sample length (Fig. 3E and 3F). Elongation is given as the extension percentage for the sample length until it breaks. Therefore, since it is easily broken at its fragile points by pulling strongly, the elongation a longer PCG filaments is considered to have decreased. The filament strength of J510 also showed a weak negative correlation, and was not statistically significant (Fig. 3B). These results indicate that the physical Fig. 2. Influence of sericin on the physical properties of a silk skein for three strains. A: filament strength, B: filament elongation, C: filament Young s modulus. The effect of degumming was statistically evaluated using a t-test. *, **, and *** indicate significant differences at 5%, 1%, and 0.1% levels, respectively. properties of the cocoon filament are influenced by not only the filament size but also the non-uniform shape of the filament. As sericin, unlike fibroin, is not a crystallized fiber, it was believed that there was no difference in the elastic modulus on each part of the cocoon filament (Iizuka, 1980). However, it is sufficiently likely to have an affect

5 Fragile points on weak cocoon filaments 47 Fig. 3. Changes in physical properties with different lengths of cocoon filament samples for three strains (C515: A, D, G, J; J510: B, E, H, K; PCG: C, F, I, L). A, B, C: cocoon filament strength; D, E, F: cocoon filament elongation; G, H, I: Young s modulus; J, K, L: photograph of a cocoon filament shape under a microscope. Bars indicate 10 μm. Each line on the respective graphs is the result from the average of 10 sample filaments. *, **, and *** indicate significant correlations at 5%, 1%, and 0.1% levels, respectively. on the physical properties by attaching itself to the surface of the fibroin fiber. Sericin s influence was seen on the strength, elongation, and Young s modulus in the present results and an increasing viscoelasticity recovery with degumming has also been reported (Shao and Vollrath, 2002). Unlike its influence on strength, the degree of sericin s influence on elongation and Young s modulus is different between the strains, suggesting that there are other reasons, in addition to the filament s size, for it to become thin with degumming. To obtain silk thread with a high strength, our present study leads us to recommend using a silkworm race that will produce thinner and more uniform cocoon filaments. Obtaining a cocoon filament with such a high intensity seems to not only offer a differentiated special silk material, but to also contribute to an effective reeling process related to productivity. ACKNOWLEDGMENTS We gratefully thank Dr. T. Kameda of the National Institute of Agrobiological Sciences (NIAS), Japan for his beneficial technical advice on this study.

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