The apparent variability of silkworm (Bombyx mori) silk and its relationship with degumming
Graphical abstract
Variation induced in the tensile properties of silkworm (Bombyx mori) silk fibers by degumming includes the appearance of a characteristic two-yield points stress-strain curve.
Introduction
The excellent mechanical properties, biocompatibility and environmentally friendly processing conditions of silk fibers have prompted an active area of research in the field of Biomimetics [1], [2], [3], [4], [5], [6], [7], [8]. Not surprisingly the outstanding properties of these materials are the consequence of their basic roles in the biology of the producing organisms, mainly silkworms and spiders [9]. In addition, silks must be effectively spun under very constrained conditions in terms of both building materials and metabolic energy available for their production [10], [11], [12], [13], [14]. In particular, silkworm silk fibers play a critical role as building elements of the cocoon, a protective case that must protect the worm from predators and other environmental threats during its metamorphosis.
The cocoon is made up of two different families of proteins: fibroins and sericins [15]. Fibroins make up silkworm silk fibers and comprise three different proteins – heavy and light chain fibroins, and hexamerin. Among them, heavy chain fibroin is assumed to play the most critical role in determining the behavior of the solid fibers. Heavy chain fibroin (henceforth referred to simply as fibroin) is a high molecular weight (Mw ∼ 350 kDa) protein with a large amount of repetitions of the motif GAGAGS [16] in its sequence. A combination of pH changes [17], [18], variation in the concentration of ions [19], and mechanical stresses [20] in the gland duct convert the initial protein solution into a fiber in a process which implies the formation of β-nanocrystals. These nanocrystals are the consequence of piling up antiparallel β-sheets formed by regions of the chain that contain repetitions of the GAGAGS motif [21], [22]. It is generally accepted that the nanocrystals are embedded in a different phase conventionally labelled as amorphous. Several microstructural features, including the presence of nanodomains [23], [24], nanofibrils [23], [25], [26], and a skin/core structure [27] were proposed to account for the organization between both phases in silkworm and spider silks. However, and although it is apparent that the tensile behavior of the amorphous phase is controlled by weak interactions such as hydrogen bonds [28], its detailed microstructural organization is still under debate. Sericins are the other proteinaceous component of the cocoon and comprise a group of hydrophilic moieties that are secreted as a coating on the fibroin fibers. Their biological function is to act as cement and maintain the structural integrity of the cocoon as a whole, by gluing the fibroin fibers together.
Essentially all applications of silkworm silk imply the removal of the sericin coating in order to obtain the individual fibroin fibers to which the term silkworm silk conventionally refers. As a matter of fact, the development of a technologically efficient process, known as degumming [29], that allows this removal is one of the key features that explains the historical and economic significance of silkworm silk. In its simplest version degumming consists of immersing the cocoon in boiling water for several minutes. Addition of salts, such as sodium carbonate, or detergents is customary to improve the yield of the process. Usage of this procedure was so extended that the characterization of silkworm silk was systematically performed on fibers previously subjected to a degumming process. Consequently, the properties of these degummed fibers were assumed to represent the intrinsic properties of the material [30]. However, the availability of silkworm silk fibers obtained by forced silking and not subjected to a degumming process [28], [31] showed that the properties exhibited by degummed fibers were different from those of the native material. In particular, it was shown that the as spun material showed very reproducible properties, in contrast with the accepted consideration of silkworm silk as a highly variable material.
Given the complex organization of silkworm silk, it is not surprising that exposure to water and high temperature might exert some significant effects on the microstructure and tensile behavior of silk fibers [32]. The aim of the present work is to study how variability arises in silkworm (Bombyx mori) silk fibers from their originally reproducible behavior as a consequence of degumming. In addition, a proper characterization of the as spun and degummed fibers provides new insights in the relationship between tensile behavior and microstructure in silkworm silk.
Section snippets
Silk retrieval and preparation of samples
B. mori silkworms were kept in captivity as described elsewhere [33]. As soon as one silkworm started spinning, it was placed onto a horizontal, black, vinyl surface in order to prevent cocoon formation. The as-spun fiber was fixed to the mobile crosshead of a mechanical testing machine (Instron 4411), which was used for forcible spinning at a constant speed of 250 mm/min.
Forcibly silked fibers were cut into 30 mm long fragments and then mounted onto plastic frames [30]. As-spun samples were
Mechanical tests
The true stress-true strain curves of the Iw and Dg samples are shown in Fig. 1a and those of RDg samples in Fig. 1b. In both cases, the curves of the Ct samples are shown for comparison. The main mechanical parameters are indicated in Table 1. From the mechanical data, it is apparent that Ct samples, which correspond to the native condition of the material, show the highest values of elastic modulus, yield stress and yield strain. These values, which are notable for a semicrystalline polymer,
Discussion
Contrary to its widespread consideration as a highly variable material, silkworm silk fibers show very reproducible properties when the behavior of as spun (i.e. not subjected to degumming or any other physicochemical treatment) fibers are compared. The stability in the performance of the as spun fibers is probably the consequence of the critical role played by silkworm silk during the development of the worm. In particular, the requirement of creating a stiff case to protect the pupa during
Conclusions
Traditionally silkworm silk fibers are obtained from cocoons through a degumming process. During degumming the exact local conditions to which a given thread is exposed are not controlled and, consequently, can vary from fiber to fiber. As shown above, the variety of effects exerted by degumming and related treatments on silkworm silk fibers justifies the apparent low reproducibility of this material. This low reproducibility is further promoted by naturally occurring variations in the cross
Acknowledgements
The authors are grateful to José Miguel Martínez for his help with the artwork. The work was funded by Ministerio de Economía y Competitividad (Spain) through projects MAT2012-38412-C02-01 and DPI2013-46485, by the Comunidad de Madrid (Spain) (Grant S2011/BMD-2460) and by Fundación Marcelino Botín and by Banco Santander through its Santander Universities Global Division.
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