High-Yield Degumming Methods for Preparing Sericin from Cocoons of Mulberry and Non-Mulberry Silkworms

Journal of Insect Biotechnology and Sericology 88, 1-6 (2019)

High-yield degumming methods for preparing sericin from cocoons of mulberry and non-mulberry silkworms

Mayumi Karimazawa1, Shouji Urita2, Tetsuro Yamashita3 and Koichi Suzuki1＊

1 Biococoon Laboratories Inc., Research and Development Center, in collaboration with Morioka City and Iwate University, Ueda 4-3-5, Morioka 020-8551, Japan 2 Negishi 5-28, Date 960-0466, Japan 3 Department of Applied Biochemistry, Faculty of Agriculture, Iwate University, Ueda 3-18-8, Morioka 020-8550, Japan (Received March 11, 2018; Accepted December 10, 2018)

We developed high-yield degumming methods for preparing sericin from the cocoons of the mulberry silkworm Bombyx mori and the non-mulberry silkworms of Antheraea yamamai, and Cricula trifenestrata. We achieved sericin yields of 54%-55% from B. mori cocoons extracted in 0.24% NaOH, from A. yamamai cocoons extracted

in 0.025% NaOH + 0.475% Na2CO3, and from C. trifenestrata cocoons extracted in 0.5% Na2CO3. In all three ex- tracts, analysis of amino acid compositions indicated a high degree of sericin purity and Tricine–SDS-PAGE analysis indicated degradation to heterogeneous polypeptides. Thus, sericin preparations produced from the cocoons of silkworms by high-yield degumming methods offer promise for biomedical and industrial applications. Key words: Sericin, Degumming, High Yield, Mulberry Silkworm, Non-Mulberry Silkworm

hot water, in dilute sodium carbohydrate, in aqueous INTRODUCTION urea, by autoclaving, and combinations of these methods Silk sericins offer promise not only as components of (Padamwar and Pawar, 2004; Yun et al., 2013). However, cosmetics and biomaterials, but also in medical and phar- it is necessary to prepare more efficient methods of -seri maceutical uses (Zhang, 2002; Padamwar and Pawar, cin recovery to test the biological effects of sericin prepa- 2004), on account of their indigestible, fibrous, and anti- rations. Here, we tested high-yield degumming methods. oxidative characters (Kato and Imai, 2002). Dietary sericin derived from the cocoon of the mulber- MATERIALS AND METHODS ry silkworm Bombyx mori prevented the development of colon tumors in mice (Zhaorigetu et al., 2001) and im- Materials proved lipid and carbohydrate metabolism in rats fed a Cocoons of B. mori were provided by Nichihara R&D high-fat diet (Okazaki et al., 2010). Intragastric perfusion Labs Inc. Those of A. yamamai were obtained from of the same sericin prevented blood glucose elevation in Fukushima Prefecture. Those of C. trifenestrata were har- type 2 diabetes model rats and protected against diabetes- vested in Yogyakarta district, Indonesia. induced injury to the sciatic nerve and nerve cells (Song et al., 2013). Oral administration of it significantly elevat- Degumming methods ed acetylcholine content in rat brains and reversed the im- Each cocoon was cut into quarters and gently washed pairment of learning and memory in Alzheimer’s disease in water (100 vol.) at 65°C for up to 3 min (Fig. 1). The B. model rats (Yellamma, 2014). In comparison, little work mori cocoons were boiled in 0.16%, 0.24%, or 0.32% has been reported on the functional properties of sericins NaOH (50 vol.) at 98°C for 1, 2, 3, 5, 7, or 10 min. The of non-mulberry silkworms, although sericins from three A. yamamai cocoons were boiled in five concentrations of such silkworms—Antheraea yamamai (Cui et al., 2009), NaOH + Na2CO3 (50 vol.; 0.010% + 0.490%, 0.025% + Antheraea mylitta (Dash et al., 2008a; 2008b; Yun et al., 0.475%, 0.050% + 0.450%, 0.100% + 0.400%, 0.200% + 2013; Khyade 2016), and Cricula trifenestrata (Sunarintyas 0.300%) at 98°C for 7 min; the solution was held at 25°C et al., 2012; Liu et al., 2016)—are studied in in vitro cell for 10 min and then at 10°C for 1 h, and then centrifuged lines for biomedical applications, but the conditions for at 800 × g for 5 min. The C. trifenestrata cocoons were their extraction are not yet standardized. boiled in 0.3%, 0.5%, or 0.7% Na2CO3 (50 or 75 vol.) at Before sericins can be extracted, cocoons must be de- 98°C for 30, 40, or 90 min. All solutions were filtered gummed. Many methods are used, including extraction in through No. 1 filter paper (Advantec). Figure 1 shows representative methods for preparing sericin from each kind *To whom correspondence should be addressed. of cocoon. All filtrates were then dialyzed against water Fax: +81-19-613-5570. Tel: +81-19-613-5564. through a 10-kDa cutoff membrane for 3 days. The seri- Email: [email protected] cin preparations were lyophilized and stored at −20°C. 2 Karimazawa et al.

Fig. 1. Procedures for degumming cocoons of (A) B. mori, (B) A. yamamai, and (C) C. trifenestrata. Inset photo- graphs show sericin solution (a, b, c) and powder (a’, b’, c’) from the cocoons of each species. For details see Materi- als and Methods.

Amino acid analysis centage of initial weight by a basic method. About 1 mg of each sericin preparation was hydrolyzed with 6 N HCl (1 mL) at 110°C for 24 h in an evacuated, Ultraviolet absorbance spectra sealed glass tube. Then 50 μL of the hydrolysate was tak- Each sericin extract was dissolved in double-distilled en for the determination of amino acid composition on a water at 0.1 mg/mL for analysis of the UV absorbance JLC-500/V analyzer (JEOL) (Cui et al., 2009). spectra by an ultraviolet spectrophotometer (Jasco V-570 UV-Vis, Japan) at 25°C in the range of 200 to 500 nm at Electrophoretic analysis a scan rate of 1000 nm/min. Each sericin was separated by denaturing Tricine–SDS- PAGE (Schägger and von Jagow, 1987) in 10% acryl- Calculations amide gel, which was then stained with 0.25% Coomassie We calculated the boiling-off loss ratio as {(initial dry brilliant blue R-250. weight of cocoon shells − final weight of degummed shells) ÷ (dry weight of cocoon shells)} × 100 (Gamo and Determination of protein content Hirabayashi, 1984; Rao et al., 2004; Kweon et al., 2012; Protein preparation was precipitated according to the Gowda et al., 2013; Shin et al., 2015), and estimated the modified method of Jonscher et al. (2014) with TCA. Pre- extraction yield of sericin as {(sericin content ÷ boiling- cipitated samples were dried in an oven at 95°C to a off loss content) × 100} (Wu et al., 2007; Silva et al., constant weight. Protein content was calculated as the per- 2012; Gimenes et al., 2014). Degumming methods for sericin preparations 3

Fig. 2. Representative yields and boiling-off losses determined by many combinations of cocoons, degumming chemical liquor ratio, and boiling time. A: Boiling of B. mori cocoons in 0.24% NaOH for 1-2 min (range, 1-10 min)

yielded 54.8%. B: Boiling of A. yamamai cocoons in 0.025% NaOH + 0.275% Na2CO3 for 7 min (range, 1-10 min) yield-

ed 54.1%. C: Boiling of C. trifenestrata cocoons in 0.5% Na2CO3 for 40 min yielded 54.6%. All values are expressed as means ± SEM.

In processing C. trifenestrata cocoons, we overcame RESULTS two critical points: (1) Although the degumming liquor ra- We investigated many combinations of cocoons, de- tio of 50 volumes sufficed for B. mori (NaOH) and A. ya- gumming chemicals, and boiling time. The apparent yield mamai (NaOH + Na2CO3), C. trifenestrata needed 75 of sericin from B. mori was 54.8% in 0.24% NaOH at volumes of Na2CO3 (Figs. 1, 2, 3A). (2) When sericin so-

1-2 min (Fig. 2A; data for 1-2 min shown) and decreased lution degummed in 0.3% Na2CO3 for 40 min was lyophi- to 51.1% at 10 min. All yields were >50% regardless of lized, the degummed silks still had an insoluble mesh the short processing times (1-10 min) in 0.24% NaOH. structure (Fig. 3B; and the silk degummed for 90 min be- The optimum yield of sericin from A. yamamai cocoons came glutinous), regardless of a better yield of sericin in was 54.1% in 0.025% NaOH + 0.475% Na2CO3 at 7 min 0.3% Na2CO3 than in 0.5% Na2CO3 (Fig. 2C). boiling time (Fig. 2B, data for 7 min shown) and de- Although the extractions and yields differed according creased to 35.1% in 0.200% NaOH + 0.300% Na2CO3 at to the degumming methods and cocoon properties, the 7 min. identity of the sericins can be confirmed by the amino 4 Karimazawa et al.

Fig. 3. Effects of Na2CO3 concentration for sericin preparation on quality of degummed silk of C. trifenestrata co-

coon. A: 50 volumes of 0.5% Na2CO3 was insufficient; 75 volumes was needed. B: 0.3% Na2CO3 (left) at 40 min re- tained insoluble mesh structure. acid composition. The main amino acid in all three seri- Table 1. Amino acid composition of each sericin preparation cins was serine (B. mori, 30.8%; A. yamamai, 23.4%; C. Mol% Amino acid trifenestrata, 26.8%; Table 1). The contents of glycine, as- B. mori A. yamamai C. trifenestrata partic acid, and threonine were higher than those of the Asp 17.7 16.3 11.1 other amino acids, accounting for 17.2%/17.7%/7.7%, re- Thr 7.7 15.0 10.8 spectively, in B. mori, 19.8%/16.3%/15.0% in A. yamamai, and 21.5%/11.1%/10.8% in C. trifenestrata. Additionally, Ser 30.8 23.4 26.8 tyrosine and alanine were low (2.8% and 6.0% in B. mori, Glu 5.3 6.4 3.45 4.9% and 3.1% in A. yamamai, and 3.6% and 1.8% in C. Gly 17.2 19.8 21.5 trifenestrata). This characterization of amino acid compo- Ala 6.0 3.1 1.8 sition indicates that all three preparations have a high de- Val 3.6 1.0 0.6 gree of sericin purity, with little or no contamination by Cys Nd 0.1 Nd degummed silk or fibroin (Wu et al., 2007). Ile 0.9 1.1 0.6 We also analyzed the sericin preparations by denaturing Leu 1.4 0.9 0.5 Tricin–SDS-PAGE. Clear protein bands were not detected; instead, broad smeared bands appeared at <25-kDa in B. Tyr 2.8 4.9 3.6 mori, <75-k Da in A. yamamai, and <37-k Da in C. tri- Phe 0.4 0.3 Nd fenestrata (Fig. 4). These broad smeared bands suggest His 1.2 2.6 3.4 the degradation of the sericin proteins (Gupta et al., Lys 1.8 1.4 1.3 2014). Thus, the three sericin preparations would be ex- Arg 3.0 3.7 1.8 pected to show absorption at 280 nm (near-ultraviolet re- Each optimum yield of sericin preparation as shown in Fig. 2 was gion) due to electron transfer by aromatic amino acids, as used for the determination of amino acid composition: extracted reported in B. mori (Gulrajani et al., 2009) and A. yama- from B. mori cocoons in 0.24% NaOH for 1 min, from A. yamamai, mai (Shin et al., 2015). Indeed, the sericin preparations in 0.025% NaOH + 0.475% Na2CO3 for 7 min, and from C. trifenestrata cocoons in 0.5% Na2CO3 for 40 min, respectively. from B. mori and A. yamamai cocoons had an absorption Nd, Not determined. peak at 280 nm, but that from C. trifenestrata had unclear Mol% is a representative value of three experiments. absorption (Fig. 5). This discrepancy remains to be probed. Degumming methods for sericin preparations 5

ences among sericins obtained by different degumming DISCUSSION methods. Protein sizes of 65-400 kDa or 5-100 kDa in The optimum extractions and the resultant yields of ser- B. mori were reported (reviewed by Zhang, 2002, and icin differed according to degumming methods and co- Sehnal, 2008). We previously showed the presence of a coons (Fig. 1), but the amino acid compositions indicated 41-kDa protein deprived from sericin produced from A. a high degree of sericin purity with little or no contamina- yamamai by degumming in 0.5% Na2CO3 (Cui et al., tion by degummed silks (Table 1). 2009). Yamada and Tsubouchi (2001) indicated a complex The denaturing Tricine–SDS-PAGE analysis also shows mixture of >400 kDa in C. trifenestrata by degumming in the degradation of the three sericin preparations (Fig. 4), 8 M aqueous urea. Thus, many degumming methods pro- but comparison of our data with other data reveals differ- vide guidance for the preparation of sericin extraction, and comparative analyses of extraction conditions are critical, because the methods used for extraction and recovery can affect the final properties of the sericin (Gupta et al., 2014). We achieved high yields of sericin: 54.8% in 0.24% NaOH for 1 min from B. mori, 54.1% in 0.025% NaOH +

0.475% Na2CO3 for 7 min from A. yamamai, and 54.6%

in 0.5% Na2CO3 for 40 min from C. trifenestrata (Fig. 2). Reported sericin extraction yields range from 14.0% to 30.0% in B. mori (Silva et al., 2012; Ko et al., 2013; Gupta et al., 2014; Gimenes et al., 2014), but are unknown in A. yamamai (Shin et al., 2015) and C. trifenestrata (Yamada and Tsubouchi, 2001), although the boil-off ratio

in 5% Na2CO3 from the former cocoons and a complex mixture of sericin proteins in 8 M aqueous urea from the latter are confirmed. Consequently, our high yield- sug gests that extraction conditions determine sericin properties and economic value (Gupta et al., 2014). Zhang (2002) and Sehnal (2008) proposed many potential practical uses of silk sericins. We proposed that 41- kDa protein derived from A. yamamai could be used as a culture supplement for Schneider S2 cells and mouse lym- phocytes (Cui et al., 2009). Our group also showed that Fig. 4. Tricine–SDS-PAGE analyses of sericin preparations from cocoons. M, molecular markers (10 μg); 1, B. mori sericin produced by the same degumming methods as used (20 μg); 2, A. yamamai (20 μg); 3, C. trifenestrata (20 μg). here with C. trifenestrata cocoons reduced proliferation of

Fig. 5. UV spectra of sericin preparations from the cocoons of B. mori, A. yamamai, and C. trifenestrata. 6 Karimazawa et al. feline kidney cells (Liu et al., 2016). Our high-yield de- Ko, J.S., Yoon, K., Ki, C.S., Kim, H.J., Bae, D.G., Lee, K.H., gumming methods may provide promising materials for Park, Y.H. and Um, I.C. (2013) Effect of degumming condi- biomedical and industrial applications. tion on the solution properties and electrospinnablity of re- generated silk solution. Int. J. Biol. Macromol., 55, 161-168. Kweon, H.Y., Lee, K.-G., Park K.Y., Kang, S.-W. and Seok, ACKNOWLEDGEMENTS Y.-S. (2012) Degumming characteristics and color stability of goldensilk cocoon. Int. J. Indust. Entomol., 24, 1-5. This study was supported in part by JSPS KAKENHI Liu, W., Karimazawa, M., Ozaki, T., An, Y., Miyazaki, M., Grant Number 23228001. We thank DKS Co, Ltd. (Daiichi Suzuki, K., Tsutsumi, K. and Yamashita, T. 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