Food Sci. Technol. Res., +, (.), ,33ῌ-*,, ,**0

Note

Identification of Soluble Proteins and Interaction with Mannan in Mucilage of opposita Thunb. (Chinese Tuber)

ῌ Takao MYODA , Yosuke MATSUDA, Tomonori SUZUKI, Tomoyuki NAKAGAWA, Takeshi NAGAI and Toshio NAGASHIMA

Department of Food Science and Technology, Faculty of Bioindustry, Tokyo University of Agriculture, +30 Yasaka, Abashiri, Hokkaido *33ῌ,.3-,

Received February 1, ,**0; Accepted September ++, ,**0

In this study, we analyzed the influence of proteins and polysaccharides on the viscous properties of mucilage extracted from Dioscorea opposita Thunb. (Chinese yam). The viscosity of the mucilage was greatly reduced by treatment with protease or mannanase, although not by treatment with cellulase. These results show that the interactions with mannan of certain soluble proteins in the mucilage play an important role in its viscosity, so we identified the major soluble proteins present. Chinese yam mucilage contained at least nine types of major soluble proteins, some of which showed a high percentage of identity with dioscorin, mannan-binding lectin and other functional proteins in the N-terminal amino acid sequence. From these findings, it was suggested that the viscosity of Chinese yam mucilage may be caused by interaction between mannan and soluble proteins such as mannan-binding lectin.

Keywords: Dioscorea opposita, mucilage viscosity, soluble protein, mannan, dioscorin, mannose-binding lectin

Introduction al. (,**-) reported that yam tuber mucilage exhibited an- The genus Dioscorea, which includes 0** to 1** species, giotensin converting enzyme inhibitory activities. More- is widely distributed throughout the world. About /* spe- over, the low starch content of mucilage means that it cies are edible and have common names, and about +, of may be useful in the nutraceutical and cosmeceutical in- these are of economic significance as human food (Coursey, dustries (Fu et al., ,**0). +301). These tubers have been utilized as an important In view of the above, e#orts have recently been made to source of food in many tropical and subtropical countries distribute ground Chinese yam widely through food mar- of Africa and Oceania (especially Melanesia), and also in kets by processing it in various forms, such as freezing or parts of Southeast Asia, since yams are rich in starch and drying. However, processing such as freezing or freeze- provide many vitamins (B+ and C) and minerals such as drying reduces the viscosity of ground Chinese yam, re- potassium, iron, and zinc. Moreover, it has been reported ducing its quality. For this reason, it is necessary to deter- that yams contain diosgenin, which is a steroid saponin. mine the primary cause of viscosity in Chinese yam, in Diosgenin is an important raw material for the industrial order to avoid these processing risks. production of steroid drugs, and is obtained after hydrol- Various studies have been conducted on the mecha- ysis of yam saponins (Hanhn et al., +321; Yang et al., ,**-). nism of viscosity of Chinese yam mucilage. For exam- Therefore, yams are an important crop in the food and ple, it has been reported that yam mucilage consists of chemical industries. glycoproteins and polysaccharides such as mannan and Dioscorea opposita Thunb. (Chinese yam), which is native cellulose (Misaki et al., +31,; Satoh, +301; Hironaka et al., to China and has been cultivated in Asia, is in particularly +33*; Tsukui et al., +333a, +333b). However, there have common use as a food product. Fresh ground Chinese- been no reports on how these factors are related to the yam tuber is used in Japan as a component in meals of viscosity of yams, and the soluble proteins that are con- yam rice boiled with and grated yam soup, as a tained in the mucilage have not been identified. fried food or served in sweetened vinegar, and as a thick- In the present study, we analyzed the relationship of ener for buckwheat flour. Yams contain a large amount proteins and polysaccharides with the viscous properties of water-soluble mucilage that gives a unique texture. of mucilage extracted from Chinese yam, and identified Mucilage is also known to play some functional roles, as the major soluble proteins in the mucilage. well as yam starch and diosgenin. For example, Lee et Materials and Methods ῌ To whom correspondence should be addressed. Material Fresh tubers of Chinese yam were harvested E-mail: [email protected] in Abashiri, Hokkaido, Japan, in November ,**-, and stored 300 T. MYODA et al. at .῍ until use. The whole tubers were peeled, ground, ase (Protease M, Amano Enzyme, Nagoya, Japan) derived and homogenized gently in an equal volume of distilled from Aspergillus oryzae, cellulase (Sigma Chemicals, St. water at .῍ for - h. The homogenate was centrifuged at Louis, MO) derived from Aspergillus niger, and manna- +2,***ῌg, at .῍,for.* min, and the supernatant was nase (Cellulosin, HBI Enzymes, Hyogo, Japan) derived saved as mucilage and used in the experiments. from Aspergillus niger] were used to treat the mucilage. Measurement of viscosity The viscosity of the muci- Enzyme treatments were performed at enzyme-substrate lage was measured by B type viscometers at 0* rpm on ratios of +** U protease: ,* mL mucilage and /* U manna- Rotor No. + at ,/῍ for , min (Toki Sangyo Co., Tokyo, nase or cellulase: ,* mL mucilage at -/῍ for , h, and the Japan). viscosity of the reaction mixture was measured every -* Enzyme treatments Three kinds of enzymes [prote- min. To achieve inactivation, solutions of each enzyme

Fig. +. E#ects of treatment with (A) protease, (B) mannanase and (C) cellulase on viscosity of Chinese yam mucilage. All experiments were performed in 0ῌ1 replications. Bars indicate standard error. Identification of Soluble Proteins and Interaction with Chinese Yam Tuber 301 were heated in a boiling water bath for ,* min. All ex- periments were performed in 0 or 1 replications. Determination of N-terminal amino acid sequences Elec- trophoresis of the soluble proteins in mucilage was per- formed on a +*ῌ (w/v) polyacrylamide gel containing SDS. The soluble proteins separated by SDS-PAGE were trans- ferred to a polyvinyledene defluoride (PVDF) membrane, and their N-terminal amino acid sequences were deter- mined using an automated protein sequence analyzer, Model .11A/+,*A (Applied BioSystems, Foster City, CA, USA).

Results and Discussion E#ect of treatment with several enzymes on viscosity of mucilage We investigated whether the viscosity of Chi- nese yam mucilage was influenced by treatment with vari- ous enzymes, including protease, mannanase and cellu- lase, in order to observe the influence of soluble proteins, mannan or cellulose on its viscosity. Fig. ,. SDS-PAGE of proteins from Chinese yam mucilage. Treatment of the mucilage with protease significantly (A) Marker and (B) proteins of Chinese yam mucilage. reduced its viscosity for -* min, and the viscosity reduc- tion rate was about 1*ῌ after +,* min (Fig. +A), although the treated mucilage still contained the major polysaccha- GKIKIGINGFGRIGRLVARV, which matched perfectly that rides (data not shown). No soluble proteins were detected of glyceraldehyde --phosphate dehydrogenase from sev- in mucilage treated with protease (data not shown). More- eral . Since both proteins are enzymes in the gly- over, treatment of mucilage with mannanase significantly colytic pathway of plants, it seems that they serve to as- reduced its viscosity, similarly to treatment with protease similate a large amount of starch in germination. Bands (Fig. +B), although the treated mucilage still contained the D and E had the same N-terminal amino acid sequence, major soluble proteins (data not shown). In contrast, the VEDEFSYIEGNPNGPENWGN, which agreed with that of addition of cellulase to the mucilage had no e#ect on its the dioscorin from D. batatas (Gaidamashvili et al., ,**.). viscosity (Fig. +C). Previously, Satoh reported that the Dioscorin is the major tuber storage protein in several viscosity of mucilage in Ichoimo, a di#erent variety of D. yam species. Harvey reported that a group of dioscorins opposita, was reduced by cellulase treatment (Satoh, +301). accounted for over 2*ῌ of the total soluble protein in tu- These facts suggest that the interaction of some soluble bers of D. rotundata (Harvey, +32-). In Chinese yam muci- proteins, but not cellulose, with mannan plays an impor- lage, as well as in other yams, dioscorin was the most domi- tant role in the viscosity of Chinese yam mucilage, al- nant protein (Fig. ,). Dioscorin has been reported to have though the mechanism of viscosity is very complex and several important functions: for example, as a storage di#ers among varieties of D. opposita. protein, carbonic anhydrase and trypsin inhibitor (Hou, Analysis of protein components in Chinese yam muci- ,***; Shewry, ,**-), angiotensin converting enzyme inhi- lage In a previous study, expression of yam viscosity was bitor (Hsu et al., ,**,), and antioxidant in tubers (Hou et attributed to mannan (Satoh, +301). The sugar composi- al., ,**+;Houet al., +333). Finally, the N-terminal amino tion of Ichoimo mucilage was mostly mannose, with a acid sequence of band F was DFILYSGESLRSGQSLTYAS, small proportion of glucose and galactose. However, there which shows high homology with that of the mannose- have been few reports on the importance of soluble pro- binding lectin from D. polystachya (3/ῌ sequence identity) teins in yam viscosity, and the major proteins in the muci- (Gaidamashvili et al., ,**.). Mannose-binding lectin is a lage had not been identified. Therefore, we examined the storage protein, and plays an important defensive role as composition of major soluble proteins in the mucilage. it has anti-insect, anti-fungal, and anti-microbial proper- Soluble protein in the mucilage was subjected to SDS- ties, as well as being toxic to birds and mammals (Peu- PAGE (Fig. ,). The mucilage contained at least nine types mans and Van Damme, +33/). of major soluble proteins, and six major protein bands, In this study, we showed that both soluble proteins and corresponding to /* kDa, ./ kDa, ., kDa, -, kDa, ,- kDa mannan were required for expression of viscosity in the and +* kDa, were named A to F, respectively (Fig. ,). mucilage of Chinese yam. This means that interaction Among these proteins, band D, corresponding to -, kDa, between soluble proteins and mannan is likely to be the was dominant. Next, the major soluble proteins were iden- key to viscosity in the mucilage. We think that mannose- tified (Table +). The N-terminal amino acid sequence of binding lectin, which has the ability to cross-link with band B was KGVLKAVENVNIIIGPALVG and it showed polysaccharide, may be an important factor in yam vis- high similarity to enolase from the rubber tree Hevea cosity, although there have been no reports clarifying the brasiliensis (3/ῌ sequence identity) (Wagner et al., ,***). relationship between these proteins and viscosity. In addi- The N-terminal amino acid sequence of band C was tion, because dioscorin is a mixture of subunits that 302 T. MYODA et al.

Table +. Analysis of N-terminal amino acid sequences.

.3 .3/0ῌ.30* appear to associate to form monomers, dimers, tetramers Food Chem., , . Hou, W.C., Liu, J.S., Chen, H.J., Chen, T.E., Chang, C.F. and Lin, Y. and high-Mr polymers (Harvey, +32-), it is also thought H. (+333). Dioscorin, the major tuber storage protein of yam that dioscorin links with mannose-binding lectin, increas- (Dioscorea batatas Decne) with carbonic anhydrase and trypsin ing the viscosity of the yam. We are currently examin- inhibitor activities. J. Agric. Food. Chem., .1, ,+02ῌ,+1,. ing about the relationship between these proteins and Hsu, F.L., Lin, Y.H., Lee, M.H., Lin, C.L. and Hou, W.C. (,**,). Both mucilage viscosity. dioscorin, the tuber storage protein of yam (Dioscorea alata cv. This information may help to reveal the mechanism of Tainong No. +), and its peptic hydrolysates exhibited angio- viscosity of yams and contribute to research on yams not tensin converting enzyme inhibitory activities. J. Agric. Food. Chem., /*, 0+*3ῌ0++-. only in food science but also in agricultural fields such as Lee, M.H., Lin, Y.S., Lin, Y.H., Hsu, F.L. and Hou, W.C. (,**-). The breeding. mucilage of yam (Dioscorea batatas Dence.) tuber exhibited angiotensin converting enzyme inhibitory activities. Bot. Bull. References Acad. Sinica (Taiwan), .., ,01ῌ,1-. Coursey, D.G. (+301). “Yams. An account of the nature, origins, Misaki, A., Ito, T. and Harada, T. (+31,). Constitutional studies on cultivation and utilisation of the useful members of the Dio- the mucilage of ‘Yamanoimo’ Dioscorea batatas Dence, Forma scoreaceae.” Tropical Agriculture Series, Tropical Products Tsukune. Agric. Biol. Chem., -0, 10+ῌ11+. Institute, Longmans, London. Peumans, W.J. and Van Damme, E.J. (+33/). Lectins as Fu, Y.C., Ferng, L.H. and Huang, P.Y. (,**0). Quantitative analysis defense proteins. Plant Physiol., +*3, -.1ῌ/,. of allantoin and allantoic acid in yam tuber, mucilage, skin and Satoh, T. (+301). E#ects of enzymes on mucilage in Ichoimo. bulbil of the Dioscorea species. Food Chem., 3., /.+ῌ/.3. Nippon Kagaku Kaishi, 22, 323ῌ33+ (in Japanese). Gaidamashvili, M., Ohizumi, Y., Iijima, S., Takayama, T., Ogawa, Shewry, P.R. (,**-). Tuber storage proteins. Ann. Bot., 3+, 1//ῌ03. T. and Muramoto, K. (,**.). Characterization of the yam tuber Tsukui, M., Nagashima, T., Sato, H. and Kozima, T. (+333a). Elec- storage proteins from Dioscorea batatas exhibiting unique trophoretic analysis of glycoprotein from yam (Dioscorea op- lectin activities. J. Biol. Chem., ,13, ,0*,2ῌ,0*-/. posita Thunb.) mucilage. J. Food Preservation Sci., ,/, ,2-ῌ,20 Hanhn, S.K., Osiru, D.S., Akoroda, M.O. and Otoo, J.A. (+321). Yam (in Japanese). production and its future prospect. Outlook Agric., +0, +*/ῌ++*. Tsukui, M., Nagashima, T., Sato, H.,Kozima, T. and Tanimura W. Harvey, P.J. and Boulter, D. (+32-). Isolation and characterization (+333b). Characterization of yam (Dioscorea opposita Thunb.) of the storage protein of yam tubers (Dioscorea rotundata). mucilage and polysaccharide with di#erent varieties. J. Jpn. Phytochemistry, ,,, +021ῌ+03-. Soc. Food Sci. Technol., .0, /1/ῌ/2* (in Japanese). Hironaka, K., Takada, K. and Ishibashi, K. (+33*). Chemical compo- Yang, D.J., Lu, T.J. and Hwang, L.S. (,**-). Isolation and identific- sition of mucilage of Chinese yam (Dioscorea opposita Thunb. ation of steroidal saponins in Taiwanese yam cultivar (Dio- cv. Nagaimo). J. Jpn. Soc. Food Sci. Technol., -1, .2ῌ/+ (in scorea pseudojaponica Yamamoto). J. Agric. Food Chem., /+, Japanese). 0.-2ῌ0.... Hou, W.C., Chen, H.J. and Lin, Y.H. (,***). Dioscorins from di#er- Wagner, S., Breiteneder, H., Simon-Nobbe, B., Susani, M., Krebitz, ent Dioscorea species all exhibit both carbonic anhydrase and M., Niggemann, B., Brehler, R., Scheiner, O. and Ho#mann- trypsin inhibitor activities. Bot. Bull. Acad. Sinica (Taiwan), .+, Sommergruber, K. (,***). Hev b 3, an enolase and a new cross- +3+ῌ+30. reactive allergen from hevea latex and molds. Purification, Hou, W.C., Lee, M.H., Chen, H.J., Liang, W.L., Han, C.H., Liu, Y.W. characterization, cloning and expression. Eur. J. Biochem., ,01, and Lin, Y.H. (,**+). Antioxidant activities of dioscorin, the 1**0ῌ1*+.. storage protein of yam (Dioscorea batatas Decne) tuber. J. Agric.