[J. Ferment. Technol., Vol. 65, No. 6, 711--715. 1987]
Continuous Blue Pigment Formation by Gardenia Fruit Using Immobilized Growing Cells SHIGEAKI FUJIKAWA, SHIGEYUKI NAKAMURA, KUNIMASA KooA, a n d JUN-ICHI KUMADA
Research Lab. of Alcoholic Beveragesand Fermentation Technology, Suntory Ltd., 1-1-1 Wakayamadai Shimamoto-eho, Mishima-gun, Osaka 618, Japan Bacillus subtilis no. 24 was used as a microorganism which hydrolyzes geniposide and forms a blue pigment. This microorganism possessed fl-glucosidase activity during aerobic growth (log phase) and assimilated geniposide as a carbon source. The growth of this cell was depressed by genipin formed by the hydrolysis of geniposide. Blue pigment was formed continuously for 20 d in medium containing geniposide, yeast extract and Polypepton, using growing cells immobilized in calcium-alginate gel.
The blue pigment produced by gardenia fruit (Gardenia jasminoides) is a w i d e l y used n a t u r a l pigment.X) T h e m e c h a n i s m o f b l u e p i g m e n t f o r m a t i o n b y g a r d e n i a fruit is as follows: G e n i p o s i d e c o n t a i n e d in the fruit is h y d r o l y z e d to g e n i p i n a n d glucose b y flglucosidase, a n d t h e b l u e p i g m e n t is t h e n t b r m e d b y the r e a c t i o n of g e n i p i n w i t h a m i n o acids. 2-4) T h e r e a r e m a n y reports conc e r n i n g the hydrolysis o f geniposide with m i c r o o r g a n i s m s (B. subtilis,5) Hansenula sp.,e) Aspergillus sp.,V) Rhyzopus sp.,7) Monascus sp.S)) a n d cellulase.1) A l t h o u g h hydrolysis w i t h these m i c r o o r g a n i s m s or w i t h a n e n z y m e was c a r r i e d out b y the b a t c h m e t h o d , continuous hydrolysis using i m m o b i l i z e d m i c r o o r g a n i s m or e n z y m e has not y e t b e e n studied. T h e r e a r e m a n y reports o f i m m o b i l i z e d fl-glucosidase.g-l~) A c c o r d i n g to these reports, the e n z y m e was i m m o b i l i z e d after e x t r a c t i o n from the cell or b r o t h . W e t h o u g h t t h a t using the i m m o b i l i z e d g r o w i n g cell system as o p p o s e d to e n z y m e i m m o b i l i z a tion after e x t r a c t i o n w o u l d be b e t t e r for t h e p r o d u c t i o n of g e n i p i n from geniposide. I n a d d i t i o n , geniposide is utilized effectively b o t h for t h e f o r m a t i o n o f b l u e p i g m e n t a n d
for cell growth. Therefore, continuous hydrolysis o f geniposide e m p l o y i n g i m m o b i lized g r o w i n g cells was a t t e m p t e d in o r d e r to p r o d u c e b l u e p i g m e n t continuously. Materials and Methods Materials Geniposlde and genipin were prepared from gardenia fruit, x6) Sodium alginate was purchased from Wako Pure Chemical Industries, Ltd., (Osaka). Microorganism B. subtilis no. 24 from the stock culture of Suntory Ltd. was used. Analytical methods Geniposide and genipin concentrations were measured by high performance liquid chromatography (HPLC), TM as was the glucose concentration (column: Biorad HPX-87H, Eluent: 1/100 N H~SO4, detector: IR). The blue pigment was measured by absorbance at 590 nm using a Shimadzu Spectrophotometer UV-240. The living cell number was measured by a Spiral plater (Model D Spiral System Instruments Inc.). Cell number, geniposide and genipin concentrations in the gel beads were measured after the beads were dissolved in 0.8% NaC1 solution with a homogenizer. Determination of ~-glucosidase activity during cultivation B. subtilis was pre-incubated in YPD
medium (yeast extract (Daigo) 1%, Polypepton (Difco) 2%, glucose 2%, CaCls 0.02%) for 1 d at 30°C. After pre-incubation, the cells were suspended
in Y P G e m e d i u m (yeast ext. 1%, Polypepton 2°0, geniposide 2 % , CaC12 0.02%, p H 5.7) to give a concentration o f 7 × 107 cells/ml a n d then shaken at 30°C. fl-Glucosidase activity of the snpernatant The medium was filtered through a Millipore filter (0.45/~m). Geniposide was a d d e d to produce a concentration o f 26 m M in this m e d i u m (1 ml). R e d u c e d geniposide was measured by H P L C after 60 rain at 30°C. O n e unit o f fl-glucosidase activity is defined as the a m o u n t o f e n z y m e degrading 1/~mol o f geniposide per m i n at 30°C. ~-Glucosidase activity of resting cells Cultured m e d i u m (5 ml) was centrifuged at 3000 r p m for 15 m i n . Cells were washed twice with sterilized water a n d then 1/20 M acetete buffer (pH 5.5) c o n t a i n i n g geniposide ( 2 0 m M ) was a d d e d to the cells. T h e decrease in geniposide concentration was m e a s u r e d by H P L C after 60 rain at 30°C.
I m m o b i l i z a t i o n o f B . s u b t i l i s and the preincubation of immobilized cells Sodiumo/ alginate ~2/o) was dissolved in water a n d autoclaved. Pre-incubated B. subtilis was a d d e d to yield a concentration of 3 × 10 ¢ cells/ml o f s o d i u m - a l g i n a t e solution. T h e cells were i m m o b i l i z e d in calcium-alginate beads dropped into a CaCI2 solution. Immobilized cells (30 ml) were p r e - i n c u b a t e d in 120 m l o f Y P D m e d i u m for 2 d at 30°C with shaking.
B. subtilis Pre-incubated i m m o b i l i z e d B. subtilis ( 3 0 m l ) w~.s inoculated in 1 2 0 m l of YPGe-conc medium (yeast ext. 1.25%, Polypepton 2.5%, geniposide 3 2 . 5 m M , CaC12 0.025%) a n d shaken at 30°C.
C o n t i n u o u s h y d r o l y s i s o f g e n i p o s i d e and the formation of blue pigment Pre-incubated i m m o b i l i z e d B. subtilis (30 ml) was added to 120 ml of Y P G e m e d i u m in a Sakaguchi flask a n d shaken for 2 d at 30°C. T w o glass tubes were put into this
[J. Ferment. Technol.,
flask t h r o u g h a cotton wool stopper. T h r o u g h one tube, Y P G e m e d i u m was fed continuously at a rate o f 30 or 80 ml per d. T h r o u g h the other tube, m e d i u m was allowed to flow out continuously at a rate o f 30 or 80 m l per d (Fig. 1).
Results and Discussion fl-Glucosidase activity o£ B. s u b t i l i s Table 1 shows the /3-glucosidase activity of growing B. subtilis, the resting cells and the supernatant of the medium. /~-Glucosidase activity of the growing cells (calculated from the time course of the rate of decrease of geniposide in the medium) was 2.8× 10-11 /~mol/min/cell during the log phase of growth (8h). The /3-glucosidase activity of the resting cells was less than that of the growing ceils. No /3-glucosidase activity in the supernatant of the medium was observed. The /3-glucosidase activity of the growing B. subtilis was the highest.
Time course study of geniposide hydrolysis by immobilized B . s u b t i l l s Calcium-alginate immobilized B. subtilis cells (5 × 109 cells/ml gel) were aerobically cultivated in YPGe medium. Figure 2 shows the time courses of the living cell number, and the glucose, blue pigment, genipin and geniposide concentrations in the medium and gel beads. The geniposide concentration in the medium began to decrease 2 h after inoculation and had almost stopped decreasing by 21 h. The decrease in the geniposide concentration commenced again after 46 h and no geniposide was detected after 53 h. The increase in the genipin concentration continued for 8 h and corresponded to the decrease in the geniposide concentration. However, the genipin concentration began to T a b l e 1.
Fig. l. A: B: C: D: E: F: G:
C o n t i n u o u s cultivation system. M e d i u m vessel Peristaltic p u m p I m m o b i l i z e d cells Sakaguchl flask (Shaking) Water bath Peristaltic p u m p Product receiver
B. subtilis ~3-glucosidase activity. /~-Glucosidase activity
2.8 × 10-11/~mol/min cell
R e s t i n g cells
1.0 × 10-11
Cells incubated for 8 h in YPGe.
Blue Pigment Formation
Vol. 65, 1987]
o~ 5 :[~¢ ~[
o 10 9
~ io7 =-~ ~= Io~ ~ io-' I(3':
20 50 40 5o Incubationtime(h)
Fig. 2. Time course of cell number, geniposide, genipin and blue pigment cone. during incubation of immobilized B. subtilis. O, geniposide cone. in broth; O, geniposide conc. in immobilized gel; Vl, genipin cone. in broth; . , genipin cone. in immobilized gel; @, living cell number in broth; O, living cell number in immobilized gel; ,~, blue pigment cone. in broth; , glucose cone. in broth decrease after 10h. T h e decrease in the genipin concentration was due to the reaction of genipin with an amino acid contained in the medium forming the blue pigment. T h e genipin concentration in the gel was higher than that in the medium. T h e blue pigment formed corresponded to the decrease in the geniposide concentration. Glucose was not detected during cultivation. T h e living cell n u m b e r in the medium decreased below 104cells/mi after 2 1 h , however, it increased after 23 h until 50 h. T h e living cell n u m b e r in the gel also began to decrease after 8 h and stopped decreasing after 30 h. T h e living cell concentration in the gel (I0 e cells/ml gel) was higher than that in the medium. T h e rate of decrease of the living cell n u m b e r in the gel was lower than that in the medium. From these profiles of the living cell n u m b e r and genipin concentration, we believe that this cell hydrolyzed geniposide to
form glucose and genipin. Glucose was assimilated by the cell while the genipin killed the B. subtilis. Therefore, in order to hydrolyze geniposide in a stable state with a continuous reactor using immobilized B. subtilis, it is important to supply geniposide at a flow rate which does not cause the accumulation of genipin in the medium or gel. Continuous blue pigment formation using i m m o b i l i z e d B. subtilis Figures 3 and 4 show the time courses of the blue pigment, genipin and geniposide concentrations and the living cell n u m b e r in the gel by continuous cultivation at a flow rate of 80 and 30 ml/d, respectively. When the flow rate was 80 ml/d (Fig. 3), the geniposide concentration was about 10 m M after 65 h. T h e concentration of genipin, formed by the hydrolysis of geniposide, varied between 0-3 raM. The living cell n u m b e r in the gel varied between 0-5 x 108 cells/ml gel. Blue pigment formation was approximately O D 80. On the other hand, when the flow rate was 30 ml/d (Fig. 4), the geniposide and genipin concentrations were stable and less than 2 m M for 20 d. The living cell n u m b e r in the gel was also stable and was around 5 x 108 cells/ml gel. T h e concentration of blue pigment remained unchanged at O D 120 for 20 d, The results shown in Figs. 3 and 4 can be interpreted as follows; at a high flow rate (80 ml/d), the rate of genipin formation was higher than the rate of reaction of genipin with the amino acids, therefore genipin accumulated in the medium. O n the other hand, at a low flow rate (30 ml/d), genipin did not accumulate in the medium because the rate of genipin formation was lower than the rate of reaction of genipin with the amino acids. T h e accumulation of genipin caused cell death and resulted in a cyclical profile for each analytical value. T h e flow rate that did not cause the accumulation of genipin resulted in the continuous formation of the blue pigment following the steady state hydrolysis of geniposide.
[J. Ferment. Technol.,
~oo "~ o
2-- . . . . . . .
50 60 70 Running lime ( h )
Continuous geniposide hydrolysis and blue pigment formation by immobilized
B. subtilis with high feed rate.
Medium containing yeast ext. 1%, Polypepton 2%, geniposide 26 raM and CaCI2 0.02% was fed continuously. Retention time was 2 d. O, geniposide cone.; ff], genipin cone.; A, absorbance at 5 9 0 n m ; Q, living cell number in the gel.
~,o?~ •~,@l -'i
240 Running time ( h )
Continuous genlposide hydrolysis and blue pigment formation by immobilized
B. subtilis with low feed rate.
Medium containing yeast ext. 1%, Polypepton 2%, geniposide 26 r a m and CaCI~ 0.02% was fed continuously. Retention time was 5 d. O, geniposide cone.; VI, genipin cone.; A, absorbanee at 5 9 0 n m ; O, living cell number in the gel
Vol. 65, 1987]
Blue Pigment Formation
Acknowledgments We would like to thank Prof. S. Nagai of Department of Fermentation Technology, Hiroshima University, for many useful suggestions. References 1) Okuyama, H.: Koukai-Tokkyo-Kouho 52-53934 (1977). 2) Inouye, H., Takeda, Y., Inoue, K., Kawamura, I., Yatuzuka, M., Touyama, R., Ikumoto, T., Shingu, T., Yokoi, T.: 26th Symposium on the Chemistry of Natural Products, Kyoto, p. 577 (1983). 3) I)jerassi, C., Takano, T., James, A.N., Zalkow, L. H., Eisenbraun, E.J., Shoolery, J.N.: J. Org, Chem., 26, 1192 (1961). 4) Djerassi, C., Dray, J.D., Kincl, F.A.: J. Org. Chem., 25, 2174 (1960). 5) Kimura, K.: Koukai-Tokkyo-Kouho 51-6230 (1976). 6) Mikami, Y., Yajima, I.: Koukai-Tokkyo-Kouho 54-96532 (1976). 7) Hasegawa, K. : Koukai-Tokkyo-Kouho 54-
152026 (1979). 8) Ogasawara, I., Shikano, M., Suzuki, S.: Tokkyo-Koukoku 59-16751 (1984). 9) Bissett, F., Sternberg, D." Appl. and Environ. Microbiol., 35, 750 (1978). 10) Woodward, J., Krasniak, R., Smith, R.D., Spielberg, F., Zachry, G.S.: Biotechnol. Bioeng. Syrup., 12, 485 (1982). 11) Lee, J. 1VL,Woodward, J. : Biotechnol.Bioeng., 25, 2441 (1983). 12) Miyairi, S., Sugiura, M.: J. Ferment. Technol., 56, 303 (1978). 13) Vernardos, D., Klei, H.E., Sundstrom, D.W.: Enzyme Microbiol. Technol., 2, 112 (1980). 14) Sundstrom, D. W., Klei, H. E., Coughlin, R. W., Biederman, G.J., Brauwer, C.A.: Bioteehnol. Bioeng., 23, 473 (1981). 15) VanDongen, D.B., Coony, D.O.: Biotechnol. Bioeng., 14, 1253 (1977). 16) Fujikawa, S., Fukui, Y., Koga, K., Kumada, J.: J. Ferment. Technol., 65, 419 (1987). (Received January 29, 1987)