353-12(07-39).fm

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1 Kr. J. Micrbil. Bitechnl. Vl. 35, N. 3, (2007) Bacillus subtilis PUL-Al Biplymer yw p 1 Á 2 Á 1,2 * 1 w ww tƒœw, 2 w m y l Physicchemical Prperties f a Biplymer Flcculant Prduced frm Bacillus subtilis PUL-A. Ryu, Mi-Jin 1, Eun-Kyung Jang 2, and Sam-Pin Lee 1,2 *. 1 Department f Fd Science and Technlgy, Keimyung University, Daegu Krea, 2 Traditinal Micrrganism Resurces Center, Keimyung University, Daegu , Krea Sybean milk cake (SMC) was used fr the slid-state fermentatin by Bacillus subtilis PUL-A islated frm sybean milk cake. In the presence f 5% glutamate the maximum prductin f biplymer (59.9 g/ kg) was perfrmed by fermentatin at 42 C fr 24 hr. The recvered biplymer was cnsisted f 87% γ-plyglutamic acid with mlecular weight f daltn and ther biplymer. The biplymer slutin shwed the great decrease in cnsistency belw ph 6.0, regardless f the mlecular weight f PGA. Biplymer slutin has a typical pseudplastic flw behavir and yield stress. The cnsistency f biplymer slutin was greatly decreased by increasing heating time and temperature in acidic cnditin cmpared t the alkaline cnditin. In kalin clay suspensin, the flcculating activity f biplymer was the highest value with 15 mg/l biplymer and 4.5 mm CaCl 2, but decreased greatly with FeCl 3. The flcculating activity f biplymer was maximum at ph5, but decreased drastically by heating at 60~100 C. In particular, biplymer with native PGA shwed the efficient flcculating activity cmpared t that f mdified biplymer cntaining lw mlecular weight f PGA Key wrds: Sybean milk cake, biflcculant, Bacillus subtilis, cnsistency wš ey tù œw wš. w š w š pullulan, micrbial cellulse, xanthan srk ply-γ-glutamic acid(pga) [22]. w w š ww, w w 2 y w š, ƒ yw z [9-10, 21, 27]. p PGA mt natt, w glutamic acid w biplymers ³ w 100-2,000 kda [8]. š PGA y š, w ƒw š. p PGA ee,,, drug carrier, w, ƒ w *Crrespnding authr Tel: , Fax: splee@kmu.ac.kr ƒw» t, yt, ƒš [19]. w PGA w PGA ƒ x¾ y wš ù ü ƒ w»s w w s PGA w š[1]. PGA w submerged fermentatin(smf) ƒ šƒ w. ù (sybean) w š z(slid state fermentatin, SSF) û» wš š SmF ùkù»s w [2, 23-24]. w x š s» y j š w [14, 20]. w w, p ³ w y ww š[3] ƒ, mgztl w ³ w, š³ z mw» y w š [15-17]. š³ w y

2 204 RYU et al. mw» ww y ƒw, l w ƒ vw.» w w ³ w z w w z w, wš yw p š, ƒ šw. z w ()t(, û) wš, sdium mnglutamate Yakuri (Kyt, Japan), k(defatted sy flur, DSF) Archer Daniels Midland C.(St. Luis, MO, USA)l w. Acid clay HPLC Sigma (St. Luis, MO, USA) w, p w. ³ kl š³ z ³ w Bacillus subtilis PUL-A w API kit 16S rdna sequencing mw w. ³ MRS agar w 42 C 24 w yy k z, ³w 50mL k 5% (w/v) w 42 C 24 (180rpm)w kl w. z 50 g 250 ml f 121 C 15 w þƒw starter 1% ³ glutamate (35%, w/v) 5% ƒw 42 C 24 zw. š z 5g 100 ml ƒw ³y k z (sieve, 0.99 mm) w k z 15,000rpm 20 w ³ debris wš zw. zw 2 v w w isprpanl ƒw ek z z w 2 v isprpanl ƒw zw. z œ» (VO-200, Sunil Eyela C., Ltd. Krea) w» k z w (100mesh) mg y d š w. š w w š HCl ph 3.0 w z 4 C 16 ew 15,000 rpm, 15 w. n w z w š w[4]. PGA d w š gel permeatin chrmatgraphy w dw,» RI (Waters 410, USA) w, GPC clumn OHpak SB 800 HQ series clumn(sb 802.5, SB 805, Shdex, Japan) w 0.1M Na 2 SO 4 /0.05% (w/v) NaN 3 (glacial acetic acid ph 4 ) 1.0 ml/min [6]. tš dextran (American Plymer Crpratin, USA) w w. PGA w w PGA t w GPC wš peak area w w. d z 5 g 45 ml ƒw ³y w z w w. 13 ml w mx (HAAKE RheStress 1, Germany) spindle (Rtr DG43 DIN Titan) w measuring cup DG43 w dw. d 20 C ( γ ) -1 1~100s d mw p w š, Pwer law mdel dw[5]. Pwer law mdel : σ=k r» γ (s -1 ), σ (Pa), K (cnsistency index, Pa s n ), n (flw behavir index). ph y š 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 3.0% w y dw, ph y y 1% š w dw. ph 6N HCl 6N NaOH w, 60 C, 80 C, 100 C ƒƒ 5, 10, 20, 30, 60 w z dw. š y d yd w t kalin clay w. Test tube( mm) kalin clay xk(5g/l) 4.65 ml š 90mM CaCl µl ƒw z w š 100 µl ƒw. yww z 5 ew 1mL

3 w 550nm Ÿ dw, š w Ÿ y ùkü[25]. d Ÿ w y(flcculating Activity: FA) [9, 11]. PROPERTIES OF BIOPOLYMER PRODUCED FROM BACILLUS SUBTILIS 205 Flcculating activity = 1/OD - 1/(OD 550 ) c OD 550 = Sample 550nm O.D. (OD 550 ) c = Cntrl 550nm O.D.» ph y» w y w dw» w kalin clayxk t w, x» w.» CaCl 2 w» w dw.» AlCl 3 6H 2 O, FeCl 3 6H 2 O, FeSO 4 7H 2 O, CaCl 2, MgCl 2, NaCl, KCl» w. š y ph w kalin clayxk, š CaCl 2 ph HCl NaOH ƒƒ 2 10 w» dw, x x w 3 w xw. š ³ w qš ƒ t ³ w MRS agar plate ³ w z clny xk,, j» 30 ³ w. w 30 ³ 5% k w starter w 1% w (42 C, 24 h)w z w ³ 2 w. ³ ƒ w PUL-A ³ API kit 16S rdna sequence w y, w Bacillus subtilis Z % ùkü(data nt shwn). ³ Bacillus subtilis PUL-A w(genbank Accessin Number, EU144043). zl š B. subtilis PUL-A ³ w zl š, glutamate ƒ y w. y 42 C 37 C ƒƒ 3.32 Pa s n, 3.10 Pa s ù n kü. ù ¼ w. z Fig. 1. Changes in the cnsistency index f SMC fermented by B. subtilis PUL-A accrding t fermentatin time and temperature. Fig. 2. Effect f glutamate cntent n the prductin f PGA and cnsistency index in the SMC fermented by B. subtilis PUL-A. 42 C ww, B. subtilis PUL-A w 42 C 24 zk z ƒ ùkû (Fig. 1). š³ w, q z w ³ ƒ š[15]. Glutamate ƒ z y Fig. 2 glutamate ƒ ƒw ƒw ùkû, glutamate 5% ƒw 3.72 Pa s ùkü n, z w š 59.9 g/kg, PGA w 52.2 g/kg 87% ùkü. Glutamate ƒƒ 5% glutamateƒ ƒ w PGA

4 206 RYU et al. ƒƒ ùkü. ù glutamate ƒ 5% ƒ z PGA w. š w š³ glutamate ³ x B. subtilis PUL-A z w ƒ glutamate z w š PGA w. ù gq ƒ glutamate ƒ 5% ƒw š z wj ùkû[7, 17]. z w glutamate dw glutamate 5% ƒ 95% y ùküù, glutamateƒ ƒ glutamate f ùkû, g q w š³ z w w [7]. ƒ glutamate z PGA w, glutamate ƒ 5%ƒ. ph š g e zw w z š glutamic acid w PGA 87% sww. Bacillus subtilis zk z PGA w 65% sww w PGAw ùk üš [17], GPC w PGA ùkû. ph y š dw, Fig. 3 ph 6» w w ù ph 6 w ƒw ùkû. w PGA w y ùkù 6. š ph y š w e w. PGA glutamic acid γ-carbxyl grup y š ph ƒš [28]. glutamic acid carbxyl grup y ü w w, carbxyl grup pk ph e w PGA ü w w w q w. š p ƒ ƒw x l ƒ p ùkü, w ƒ w(yield stress) Fig. 3. Effects f ph n the cnsistency index and mlecular weight f the biplymer prduced by B. subtilis PUL-A.Triangular, mlecular weight; circle, cnsistency index. Fig. 4. Changes in the cnsistency index f biplymer accrding t the heat-treatment at different ph. *Biplymer slutin: 1% (w/v)

5 PROPERTIES OF BIOPOLYMER PRODUCED FROM BACILLUS SUBTILIS 207 Fig. 5. Effect f biplymer cncentratin n flcculating activity. Flcculating activities in kalin clay suspensins cntaining 4.5 mm Ca 2+ and varius cncentratins f the biplymer were measured. Diamnd, 1,300 K; triangular, 64 K; square, 24 K. ƒ w ùkü. ph ƒ š e w» w 1% š ph ƒƒ 2-7 w z, 60, 80, 100 C ww Fig. 4 ùkü. ph 5w 80 C 100 C w w ùk û 20 ƒ ùkû. e ùkü ph C w ùkü. š phƒ w ùkûù, ph w yw ùkû. š y š y 4.5 mm CaCl 2 ww kalin clay suspensin (5g/L) w š ƒw dw. y ƒ š 15 mg/l ¾ ƒwù z w(fig. 5). š ƒ y š ƒ ƒw kalin clay t ƒ» [18] ƒ. y e ùkü š 15 mg/l ùkû, PGA(24K, 64K) w y y j w ùkû, 24K 64K š 5mg/L y ùkü (Fig. 5). Table 1. Effect f varius catins n flcculating activity f biplymer. Fig. 6. Effect f initial ph n flcculating activity f biplymer. Flcculating activities in kalin clay suspensins cntaining 4.5 mm Ca 2+ and 0.1% f the biplymer were measured. y e» ph w š y Ca 2+ Mg ƒw j 2+ ƒwù, 3ƒ 3+ Fe 1ƒ Na ƒ + w w w ùkû(table 1). y 2+ ùkü Ca mM ùk ü. Ca Mg 2+ kalin clay ww PGA carbxyl grup û negative charge y y w wj [12-13]. w 3ƒ 3+ Fe ƒ w y w w kalin clay suspensin FeCl 3 ƒw phƒ 2.66 w w». (reactin mixture) phƒ š y e w mw» w, kalin clay suspensin, 90mM CaCl 2 š (15mg/L) yww HCl NaOH w ph w z y dw. ph 5 y ùkü, w w ùkû(fig. 6). y ùkü ph 5 š 60 C, 80 C, 100 C w y dw, ¼ y w w ùkü (Fig. 7). w PGA y, 100 C ƒw PGA yƒ j w, 80 C w w w. 60 C 60 š PGA yƒ. Flcculating activity (1/OD) Suspensin Al 3+ (AlCl 3 ) Fe 3+ (FeCl 3 ) Fe 2+ (FeSO 4 ) Ca 2+ (CaCl 2 ) Mg 2+ (MgCl 2 ) Na + (NaCl) K + (KCl) Kalin clay

6 208 RYU et al. y z ùkû. w m y l x. REFERENCES Fig. 7. Changes in flcculating activity and mlecular weight f flcculant fllwing heat-treatment. After heating f biplymer slutin (1g/L, ph 5) at 60, 80 and 100 C, flcculating activity in a kalin clay (clse symbl) and mlecular weight(pen symbl) were measured. Square, 100 C; diamnd, 80 C; circle, 60 C. š sw PGAƒ PGA j» w q. w yƒ wš y û w. PGA ƒ y j w e, y w ù yw ƒ y w ùkü[26]. l w Bacillus subtilis PUL-A ³ w zl biplymer flcculant glutamate w 5% ƒw z 42 C 24 z w PGA 52.2g/ kg ùkü. biplymer flcculant PGA w 87%, daltn ùkü. PGA ph y ph 6» w wù, y ùkù. Biflcculant p ƒ ƒw ƒ ƒ x ƒ p ùkü, w ƒw. Biflcculant phƒ wù ƒw ph w w. Kalin clay suspensin w y biflcculant ƒ 15 mg/l y ù kü, y 2+ Ca ƒw ƒ ƒ w 4.5mM ùkû. ù 3ƒ Fe ƒw y w w 3+. y ph 5 ƒ ùkû, 60~100 C w y w w. w PGA native š PGAƒ 1. Ashiuchi, M., T. Kamei, D. H. Baek, S. Y. Shin, M. A. Sung, K. Sda, T. Yagi, and H. Misn Islatin f Bacillus subtilis(chungkkjang), a ply-γ-glutamate prducer with high genetic cmpetence. Appl. Micrbil. Bitechnl. 57: Ashk, P., R. S. Carls, and M. David New develpments in slid state fermentatin:i-biprcesses and prducts. Prcess. Bichem. 35: Baek, J., I. S. Lee, and S. P. Lee Characterizatin and fermentatin characteristics f lactic acid bacteria islated frm sybean curd residue(biji). J. Krean. Sc. Fd. Sci. Nutr. 31: Chi, S. H., J. S. Park, K. S. Whang, M. H. Yn, and W. Y. Chi Prductin f micrbial biplymer, ply(γglutamic acid) by Bacillus subtilis BS 62. Agric. Chem. Bitechnl. 47: Genc, M., M. Zrba, and G. Oza Determinatin f rhelgical prperties f bza by using physical and sensry analysis. J. Fd. Eng. 52: Hng, X., J. Min, L. Hui, L. Dingqiang, and O. Pingkai Efficient prductin f ply(γ-glutamic acid) by newly islated Bacillus subtilis NX-2. Prc. Bichem. 40: Jang, E. K., J. H. Se, S. C. Park, B. S. Y, and S. P. Lee Characterizatin f mucilage prduced frm slid-state fermentatin f sybean grit by Bacillus firmus. Fd. Sci. Bitechnl. In Press. Available 31 Octber. 16(5). 8. Kunika, M. and K. Furusawa Ply (-glutamic acid) hydrgel prepared frm micrbial ply(-glutamic acid) and alkane diamine with water-sluble carbdiimide. J. Appl. Plym. Sci. 65: Kurane, R., K. Takeda, and T. Suzuki Screening fr and characteristics f micrbial flcculants. Agr. Bil. Chem. 50: Kurane, R., K. Teda, K. Takeda, and T. Suzuki Culture cnditins fr prductin f micrbial flcculant by Rhdcccus erythrplis. Agr. Bil. Chem. 50: Kurane, R., K. Hatamchi, T. Kakun, M. Kiyhara, M. Hiran, and Y. Taniguchi Prductin f a biflcculant by Rhdcccus erythrplis S-1 grwn n alchls. Bisci. Bitech. Bichem. 58: Levin, S. and W. T. Friesen Flcculatin f cllid particles by water sluble plymers. pp In Y. A. Attia (ed.), Flcculatin in bitechnlgy and separatin system,

7 PROPERTIES OF BIOPOLYMER PRODUCED FROM BACILLUS SUBTILIS 209 Elsevier, Amsterdam. 13. Levy, N., S. Magdasi, and Y. Bar-Or Physic-chemical aspects in flcculatin f bentnite suspensins by a cyanbacterial. Water Res. 26: Oh, O. and J. S. Han A survey n perceptin and usage f Bizi(sybean curd residue). Int. J. Hum. Ecl. 42: Oh, S. M., J. H. Se, and S. P. Lee Prductin f fibrinlytic enzyme and peptides frm alkaline fermentatin f sybean curd residue by Bacillus firmus NA-1. J. Krean. Sc. Fd. Nutr. 34: Oh, S. M., C. S. Kim, and S. P. Lee Functinal prperties f sybean curd residue fermented by Bacillus sp. J. Krean. Sc. Fd. Nutr. 53: Oh, S. M., E. K. Jang, J. H. Se, M. J. Ryu, and S. P. Lee Characterizatin f the γ-plyglutamic acid prduced frm slid-state fermentatin f sybean milk cake using Bacillus sp. Fd. Sci. Bitechnl. 16: Serita, H Flcculatin f kalin suspensin with graft cplymers f chitsan. Kbunshi Rnbunshu. 52: Shih, I. L. and Y. T. Van The prductin f ply-γglutamic acid frm micrrganisms and its variatin applicatins. Biresur. Technl. 79: Shurtlff, W. and A. Ayagi Tfu and Symilk Prductin, pp New Age Fd Study Center, Lafayette, CA, USA. 21. Takagi, H. and K. Kadwaki Purificatin and chemical prperties f a flcculant prduced by Paecilmyces. Agr. Bil. Chem. 49: Ashiuchi, M. and Misn, H Plysaccharides and plyamides in the fd industry: prperties, prductin, and patents. pp A. Steinbuchel and S.K. Rhee (eds.). Wiley-VCH Verlag GmbH, Weinheim, Germany. 23. Vérnique, B. M., O. Olivier, and C. Pierre Sensrs and measurements in slid state fermentatin: a review. Prcess Bichem. 38: Xu, J., S. Chen, and Z. Yu Optimizatin f prcess parameters fr ply γ-glutamate prductin under slid state fermentatin frm Bacillus subtilis CCTC Prcess Bichem. 40: Yki, H., O. Natsuda, J. Hirse, S. Hayashi, and Y. Takasaki Characteristics f a biplymer flcculant prduced by Bacillus sp. PY-90. J. Ferment. Bieng. 79: Yki, H., T. Arima, J. Hirse, S. Hayashi, and Y. Takasaki Flcculatin prperties f ply(γ-glutamic acid) prduced by Bacillus subtilis. J. Ferment. Bieng. 82: Zajic, J. E. and A. Leduy Flcculant and chemical prperties f a plysaccharide frm Pullularia pullulans. Appl. Micrbil. 25: Zanuy, D., C. Aleman, and S. Munz-Guerra On the helical cnfrmatin f un-inized ply(γ-d-glutamic acid). Int. J. Bilg. Macrml. 23: (Received Aug. 8, 2007/Accepted Sep. 9, 2007)