- Email: [email protected]
Production of γ-aminobutyric acid from alcohol distillery lees by Lactobacillus brevis IFO-12005
JOURNALOF BIOSCIENCEAND BIOENGINEERING Vol. 93, No. 1,95-97. 2002
Production of y-Aminobutyric Acid from Alcohol Distillery Lees by Lactobacihs brevis IFO- 12005 SADAJI YOKOYAMA,‘*
JUN-ICHI HIRAMATSU,’
AND
KIYOSHI HAYAKAWA*
Alcoholic Beverages and Food Research Laboratories, Takara Shuzo Co. Ltd., 3-4-l Seta, Otsu-shi, Shiga 520-2193, Japan’ and Kyoto Prefectural Comprehensive Center for SmaN and Medium Enterprises, 17 Chudori, Minami Machi, Shimogyo-ku, Kyoto 600-8813, Japan2 Received 16 August 2001iAccepted 24 October 2001
Lactobacillus brevis IFO-12005 showed good growth in rice shochu distillery lees (kome shochu kasu). Almost all of the free glutamic acid (10.50 mM) in shochu kasu was converted to y-aminobutyric acid (GABA) within 2 d of stationary culture at 30°C. The amount of GABA in the kome shochu kasu medium finally reached 10.18 mM. After centrifugation of the broth culture, the supernatant fraction was treated with a flocculation agent to form a clear solution, then passed through a column containing a synthetic adsorbents, SP-207 to remove the yellow pigment and flavors which are unnecessary from a sensory perspective. An economical and simple production process for GABA was established. [Key words: y-aminobutyric
decarboxylase,
acid, GABA, alcohol distillery lees, shochu kasu, Lactobacillus GAD, glutamic acid]
Shochu is a Japanese distilled alcoholic beverage which is produced from rice, barley, buckwheat, sweet potato, and sugar cane in Kyushu and Okinawa, Southern islands of Japan. There are two types (Ko and Otu) of shochu in Japan. Ko type is distilled using continuous distillation apparatus, and Otu type is distilled using a pot still distillation apparatus. Almost all of fermentation of Ko type and Otu type shochu are done in foreign countries and in Japan, respectively. Production of Otu type shochu reached 358,050 kl in the year 2000 (1). A large amount of shochu distillery lees (hereafter called shochu kasu) is discharged into the environment during Otu type shochu making. The treatment of shochu kasu has become a major problem because the biological oxygen demand (BOD) values, and suspended solid (SS) values, which differ according to the raw materials used, are between 36,000 and 78,000 mgfl, and 20,000 and 48,000 mgll, respectively (2). Therefore, a novel effective process for the treatment of shochu kasu is highly desirable. There have been some reports about the utilization of shochu kasu. Shochu kasu is thought to be a safe and nutritional natural food by-product, but had been dumped into the ocean as fish bait until recently. Shochu kasu is not only free of harmful substances, but may also contain useful bioactive substances such as growth factors for chick muscle cells (3), and growth-stimulating factors for lactic acid bacteria and Bzjidobacteria (4). A saccharifying enzyme (5), a protease (6) and fungal proteins (7) were produced by Aspergillus fungi grown in shochu kasu. Chitosan production from shochu kasu by fungi has also been reported (8). We previously reported the production and some properties of low-salt seasoning from shochu kasu (9). Rice (kome) or
brevis, glutamate
barley (mugi) shochu kasu was hydrolyzed by soy sauce koji (solid culture of Aspergillus sojae) with wheat gluten in 10% NaCl and 5% ethanol. Kome shochu kasu seasoning contained 1.77 mg protein/ml of IC,, value for angiotensin I converting enzyme (ACE), and contained 33.3 mg of yaminobutyric acid (GABA)/lOO ml, being equivalent to 133 mg/lOO g dry wt. (9). GABA has several physiological functions such as neurotransmission, and induction of hypotensive effects, diuretic effects, and tranquilizer effects, particularly with regard to sleeplessness, depression and autonomic disorders observed during the menopausal or presenium periods (1 O-12). It has been reported that the amount of GABA was approximately 20, 150, and 400 mg/lOO g dry wt. in red mold rice, Gabaron tea, and soaked rice germ, respectively (13-16). GABAproducing lactic acid bacteria which rapidly produced a large amount of GABA from glutamic acid (Glu) by glutamate decarboxylase (GAD) [EC 4.1.1.151 were also isolated (17). In the case of Lactobacillus brevis, 4 of the 8 strains tested produced GABA, with some yielding 50 mM GABA from 59 mM Glu in glucose-yeast extract-Polypepton medium (17). However, some problems still remain, such as the high cost of the culture medium. It seems that an economical and simple process of natural GABA production without Glu supplementation could be achieved if GABA could be produced by lactic acid bacteria using shochu kasu as a growth medium. There have been no reports concerning the microbial production of GABA from shochu kasu. In the present study, the production of GABA from kome shochu kasu by lactic acid bacteria without addition of Glu was investigated with a view designing an advanced process for the utilization of shochu kasu. L. brevis IFO-12005 was used in this study. The GAD of this strain has been purified and its enzymatic properties reported (18). The kome sho-
* Corresponding author. e-mail: [email protected] phone: +81-(0)77-543-7225 fax: +81-(0)77-543-7286 95
96
YOKOYAMA ET AL.
chu kusu used has a pH of 4.1, a total nitrogen (TN) content of 0.67%, a formol nitrogen (FN) content of 0.18%, total sugar (TS) content of 3.64%, and a moisture .content of 90.5%. The shochu kasu supernatant had a pH of 4.2, a TN content of 0.32%, a FN content of 0.15%, a TS content of 0.72%, and a moisture content of 95.3%. The preculture of L. brevis IFO-12005 was grown without agitation at 30°C for 24 h in GYP medium (1% glucose, 1% yeast extract, 0.5% Polypepton, 0.2% sodium acetate, 20 ppm MgSO,.7H,O, 1 ppm MnSO,.4H,O, 1 ppm FeSO,.7H,O, 1 ppm NaCl, pH 6.8). An inoculum of 1% was then transferred into the shochu kasu medium. The medium contained only shochu kasu which was adjusted to pH 5.2 using 48% NaOH and autoclaved at 120°C for 15 min. The decrease in the Glu concentration, and increase in that of GABA throughout the cultivation were monitored using thin layer chromatography (TLC) and a Hitachi amino acid analyzer L-8500A. TLC was carried out using cellulose F aluminium plates (Merck Co., Germany), and the solvent system used was n-butanol-acetic acid-water (3 : 2 : 1). Glu was also measured by a calorimetric method using the glutamate dehydrogenase-NAD’-diaphorase system (Boehringer Marmheim, Germany). After stationary cultivation, the broth culture was centrifuged at 10,000 xg for 30 min, and the supernatant was treated with a flocculation agent (colloidal silicon oxide), Koporokku SA (Otsuka Chemical Co., Osaka). After allowing to stand at 5°C for 24 h, the precipitate was discarded by filtration using no. 2 filter paper (Advantec Co., Osaka). The clear filtrate was then passed through a column containing synthetic adsorbents, SP-207 (Mitsubishi Chemical Co., Tokyo) (column dimentions: 1.5 cm+ x 5.7 cm, 10 ml resin) to remove the yellow pigment and flavors which are unnecessary from a sensory perspective. L. brevis IFO-12005 showed almost the same levels of growth (OD,,, of 2.18) in the kome shochu kusu supernatant without any other ingredients added, as in the GYP medium (GD,,, of 2.24) after stationary cultivation for 2 d. Thus, it seems that kome shochu kasu is useful as a growth medium for this lactic acid bacterium. Then, kome shochu kasu was used without separating out the solid particles to omit the centrifugation step. This kome shochu kasu contained 10.50 mM free Glu as determined by the assay system using glutamate dehydrogenase. Almost all of the Glu was converted to GABA after 1 d of cultivation (Fig. 1). The GABA concentration reached 10.05 mM and 10.18 mM after 1 and 2 d cultivation, respectively. Fine-grained particles in the supernatant of broth culture were effectively precipitated by the flocculation agent (Fig. 2). Addition of 0.2% Koporokku SA decreased the A,,, value to 5% of the original value. The cleared solution was yellow and imparted unnecessary flavors from a sensory perspective. In order to use GABA-containing solutions in the production of liquors and beverages, a light color is preferable from the consumer’s point of view. Thus, a decolorization method was investigated. This yellow pigment was effectively removed by application of the supernatant to a column containing the synthetic adsorbents, SP-207 (Fig. 3). When 20 bed volumes of samples were treated with this resin, the A,,, value was reduced by about 75%. Resin-treated solution was suitable for application to the production of liquors and beverages. In addition,
J. BIOSCI.BIOENG., Solvent front --t
Glu+
Origin-+ 0
1
h
d
2
d
FIG. 1. Thin layer chromatogram of shochu kusu broth culture. Immediately after inoculation of precultured Lactobucillus
1.2 r
l
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
(
0.8
Final K of flocculation agent added
FIG. 2. Effect of treatment with flocculation agent. Colloidal silicon oxide, Koporokku SA was added to the centrifuged broth culture to a final concentration of 0 to 0.7%, and the supematants were allowed to stand at 5°C for 1 d. After removal of the precipitate by filtration through a filter paper, the A,, and GABA concentration were assayed. C/C,: The ratio of A,, (closed circle) and GABA concentration (open circle) before (C,) and after (C) treatment with the flocculation agent. The A,, and GABA concentration of the test sample before treatment were 0.547 and 10.18 mM, respectively.
VOL.
NOTES
93,2002 1.2
7. Morimura, S., Kida, K., Nakagawa, M., and Sonoda, Y.: Production of fimgal protein by Aspergillus awamori var.
1.0
kawuchi grown in shochu distillery wastewater. J. Ferment.
0.8
Bioeng., 78, 160-163 (1994). 8. Yokoi. H.. Aratake. T.. Nishio. S.. Hirose. J.. Havashi. S.. and Takasaki, Y.: Chitosan production from shochu ‘distillery wastewater by funguses. J. Ferment. Bioeng., 85, 246 249 (1998). 9. Yokoyama, S. and Tarumi, S.: Production and some properties of low-salt seasoning from shochu distillery waste. Seibutsu Kogaku, 79,211-217 (2001). 10. Stanton, H. C.: Mode of action of gamma aminobutyric acid on the cardiovascular system. Arch. Int. Pharmacodyn., 143, 195-200 (1963). 11. Omori, M., Yano, T., Okamoto, J., Tsushida, T., Murai, T., and Higuchi, M.: Effect of anaerobically treated tea (gabaron tea) on blood pressure of spontaneously hypertensive rats. Nippon Nogeikagaku Kaishi, 61, 1449-1451 (1987). (in Japanese) 12. Okada, T., Sugishita, T., Murakami, T., Murai, H., Saikusa, T., Horino, T., Onoda, A., Kajimoto, O., Takahashi, R., and Takahashi, T.: Effect of the defatted rice germ enriched with GABA for sleeplessness, depression, autonomic disorder by oral administration. Nippon Shokuhin Kagaku Kogaku Kaishi, 47,596-603 (2000). (in Japanese) 13. Kono, I. and Himeno, K.: Changes in y-aminobutyric acid content during beni-koji making. Biosci. Biotechnol. Biothem., 64,617-619 (2000). 14. Tsushida, T., Murai, T., Omori, M., and Okamoto, J.: Production of a new type of high level of y-aminobutyric acid. Nippon Nogeikagaku Kaishi, 61, 817-822 (1987). (in Japanese) 15. Saikusa, T., Horino, T., and Mori, Y.: Distribution of free amino acids in the rice kernel and kernel fractions and the effect of water soaking on the distribution. J. Agric. Food Chem., 42, 1122-1125 (1994). 16. Saikusa, T., Okada, T., Murai, H., Ohmori, M., Mori, Y., Horio, T., Ito, M., and Onoda, A.: The effect of defatting with organic solvent on accumulation of 4-aminobutyric acid (GABA) in the rice germ. Nippon Shokuhin Kagaku Kogaku Kaishi, 48, 196-201 (2001). (in Japanese) 17. Hayakawa, K., Ueno, Y., Kawamura, S., Taniguchi, R, and Oda, K.: Production of y-aminobutylic acid by lactic acid bacteria. Seibutsu Kogaku, 75,239-244 (1997). 18. Ueno, Y., Hayakawa, K., Takahashi, S., and Oda, K: Purification and characterization of glutamate decarboxylase from Lactobacillus brevis IFO-12005. Biosci. Biotech. Biothem., 61, 1168-1171 (1997). I
8
97
0.6
;j 0.4 0.2
0
10
20
30
40
50
60
70
80
Feed (EV)
FIG. 3. Effect of application to the synthetic adsorbents, SP-207. To a 10 ml volume of column, 75 bed volumes (BV) of clear broth culture were fed at a flow rate of 40 ml/h. The A,,, and GABA concentration of the eluted fractions were assayed. C/C+ The ratio of A430 (closed circle) and GABA concentration (open circle) before (C,) and after(C) SP-207 column treatment. The A,,, and GABA concentration of the test sample before treatment were 0.405 and 10.18 mM, respectively.
nary tests, mugi shochu kasu which contained 6.80 mM free Glu, produced 6.30mM GABA by 15. brevis IFO-12005 after 2 d of stationary culture at 30°C. REFERENCES 1. Nikkan Keizai Tushinsha: Shurui Shokuhin Tokei Geppo, No. 6,29-32 (2001). (in Japanese) 2. Nihon Jozokyokai: Jorhu haieki seibun, Jozobutu no Seibun, 139-141 (1999). (in Japanese) 3. Mahfudz, L.D., Nakashima, K., Ohtsuka, A., and Hayashi, K.: Growth factors for a primary chick muscle cell culture from shochu distillery by-product. Biosci. Biotech. Biochem., 61, 1844-1847 (1997). 4. Fur&a, Y., Takashita, H., Omori, T., Sonomoto, K., Ishizaki, A., Shimoda, M., and Wada, H.: Growth-stimulating effect of shochu wastewater on lactic acid bacteria and Bzjidobacteria. Ann. N.Y. Acad. Sci., 864,276279 (1998). 5. Morimura, S., Kida, K., Yakita, Y., Sonoda, Y., and Myoga, H.: Production of saccharifying enzyme using the wastewater of a shochu distillery. J. Ferment. Bioeng., 71, 329-334 (1991). 6. Morimura, S., Kida, K., and Sonoda, Y.: Production of protease using wastewater from the manufacture of shochu. J. Ferment. Bioeng., 77, 183-l 87 (1994).
,
,
I
II
*
Production of y-Aminobutyric Acid from Alcohol Distillery Lees by Lactobacihs brevis IFO- 12005 SADAJI YOKOYAMA,‘*
JUN-ICHI HIRAMATSU,’
AND
KIYOSHI HAYAKAWA*
Alcoholic Beverages and Food Research Laboratories, Takara Shuzo Co. Ltd., 3-4-l Seta, Otsu-shi, Shiga 520-2193, Japan’ and Kyoto Prefectural Comprehensive Center for SmaN and Medium Enterprises, 17 Chudori, Minami Machi, Shimogyo-ku, Kyoto 600-8813, Japan2 Received 16 August 2001iAccepted 24 October 2001
Lactobacillus brevis IFO-12005 showed good growth in rice shochu distillery lees (kome shochu kasu). Almost all of the free glutamic acid (10.50 mM) in shochu kasu was converted to y-aminobutyric acid (GABA) within 2 d of stationary culture at 30°C. The amount of GABA in the kome shochu kasu medium finally reached 10.18 mM. After centrifugation of the broth culture, the supernatant fraction was treated with a flocculation agent to form a clear solution, then passed through a column containing a synthetic adsorbents, SP-207 to remove the yellow pigment and flavors which are unnecessary from a sensory perspective. An economical and simple production process for GABA was established. [Key words: y-aminobutyric
decarboxylase,
acid, GABA, alcohol distillery lees, shochu kasu, Lactobacillus GAD, glutamic acid]
Shochu is a Japanese distilled alcoholic beverage which is produced from rice, barley, buckwheat, sweet potato, and sugar cane in Kyushu and Okinawa, Southern islands of Japan. There are two types (Ko and Otu) of shochu in Japan. Ko type is distilled using continuous distillation apparatus, and Otu type is distilled using a pot still distillation apparatus. Almost all of fermentation of Ko type and Otu type shochu are done in foreign countries and in Japan, respectively. Production of Otu type shochu reached 358,050 kl in the year 2000 (1). A large amount of shochu distillery lees (hereafter called shochu kasu) is discharged into the environment during Otu type shochu making. The treatment of shochu kasu has become a major problem because the biological oxygen demand (BOD) values, and suspended solid (SS) values, which differ according to the raw materials used, are between 36,000 and 78,000 mgfl, and 20,000 and 48,000 mgll, respectively (2). Therefore, a novel effective process for the treatment of shochu kasu is highly desirable. There have been some reports about the utilization of shochu kasu. Shochu kasu is thought to be a safe and nutritional natural food by-product, but had been dumped into the ocean as fish bait until recently. Shochu kasu is not only free of harmful substances, but may also contain useful bioactive substances such as growth factors for chick muscle cells (3), and growth-stimulating factors for lactic acid bacteria and Bzjidobacteria (4). A saccharifying enzyme (5), a protease (6) and fungal proteins (7) were produced by Aspergillus fungi grown in shochu kasu. Chitosan production from shochu kasu by fungi has also been reported (8). We previously reported the production and some properties of low-salt seasoning from shochu kasu (9). Rice (kome) or
brevis, glutamate
barley (mugi) shochu kasu was hydrolyzed by soy sauce koji (solid culture of Aspergillus sojae) with wheat gluten in 10% NaCl and 5% ethanol. Kome shochu kasu seasoning contained 1.77 mg protein/ml of IC,, value for angiotensin I converting enzyme (ACE), and contained 33.3 mg of yaminobutyric acid (GABA)/lOO ml, being equivalent to 133 mg/lOO g dry wt. (9). GABA has several physiological functions such as neurotransmission, and induction of hypotensive effects, diuretic effects, and tranquilizer effects, particularly with regard to sleeplessness, depression and autonomic disorders observed during the menopausal or presenium periods (1 O-12). It has been reported that the amount of GABA was approximately 20, 150, and 400 mg/lOO g dry wt. in red mold rice, Gabaron tea, and soaked rice germ, respectively (13-16). GABAproducing lactic acid bacteria which rapidly produced a large amount of GABA from glutamic acid (Glu) by glutamate decarboxylase (GAD) [EC 4.1.1.151 were also isolated (17). In the case of Lactobacillus brevis, 4 of the 8 strains tested produced GABA, with some yielding 50 mM GABA from 59 mM Glu in glucose-yeast extract-Polypepton medium (17). However, some problems still remain, such as the high cost of the culture medium. It seems that an economical and simple process of natural GABA production without Glu supplementation could be achieved if GABA could be produced by lactic acid bacteria using shochu kasu as a growth medium. There have been no reports concerning the microbial production of GABA from shochu kasu. In the present study, the production of GABA from kome shochu kasu by lactic acid bacteria without addition of Glu was investigated with a view designing an advanced process for the utilization of shochu kasu. L. brevis IFO-12005 was used in this study. The GAD of this strain has been purified and its enzymatic properties reported (18). The kome sho-
* Corresponding author. e-mail: [email protected] phone: +81-(0)77-543-7225 fax: +81-(0)77-543-7286 95
96
YOKOYAMA ET AL.
chu kusu used has a pH of 4.1, a total nitrogen (TN) content of 0.67%, a formol nitrogen (FN) content of 0.18%, total sugar (TS) content of 3.64%, and a moisture .content of 90.5%. The shochu kasu supernatant had a pH of 4.2, a TN content of 0.32%, a FN content of 0.15%, a TS content of 0.72%, and a moisture content of 95.3%. The preculture of L. brevis IFO-12005 was grown without agitation at 30°C for 24 h in GYP medium (1% glucose, 1% yeast extract, 0.5% Polypepton, 0.2% sodium acetate, 20 ppm MgSO,.7H,O, 1 ppm MnSO,.4H,O, 1 ppm FeSO,.7H,O, 1 ppm NaCl, pH 6.8). An inoculum of 1% was then transferred into the shochu kasu medium. The medium contained only shochu kasu which was adjusted to pH 5.2 using 48% NaOH and autoclaved at 120°C for 15 min. The decrease in the Glu concentration, and increase in that of GABA throughout the cultivation were monitored using thin layer chromatography (TLC) and a Hitachi amino acid analyzer L-8500A. TLC was carried out using cellulose F aluminium plates (Merck Co., Germany), and the solvent system used was n-butanol-acetic acid-water (3 : 2 : 1). Glu was also measured by a calorimetric method using the glutamate dehydrogenase-NAD’-diaphorase system (Boehringer Marmheim, Germany). After stationary cultivation, the broth culture was centrifuged at 10,000 xg for 30 min, and the supernatant was treated with a flocculation agent (colloidal silicon oxide), Koporokku SA (Otsuka Chemical Co., Osaka). After allowing to stand at 5°C for 24 h, the precipitate was discarded by filtration using no. 2 filter paper (Advantec Co., Osaka). The clear filtrate was then passed through a column containing synthetic adsorbents, SP-207 (Mitsubishi Chemical Co., Tokyo) (column dimentions: 1.5 cm+ x 5.7 cm, 10 ml resin) to remove the yellow pigment and flavors which are unnecessary from a sensory perspective. L. brevis IFO-12005 showed almost the same levels of growth (OD,,, of 2.18) in the kome shochu kusu supernatant without any other ingredients added, as in the GYP medium (GD,,, of 2.24) after stationary cultivation for 2 d. Thus, it seems that kome shochu kasu is useful as a growth medium for this lactic acid bacterium. Then, kome shochu kasu was used without separating out the solid particles to omit the centrifugation step. This kome shochu kasu contained 10.50 mM free Glu as determined by the assay system using glutamate dehydrogenase. Almost all of the Glu was converted to GABA after 1 d of cultivation (Fig. 1). The GABA concentration reached 10.05 mM and 10.18 mM after 1 and 2 d cultivation, respectively. Fine-grained particles in the supernatant of broth culture were effectively precipitated by the flocculation agent (Fig. 2). Addition of 0.2% Koporokku SA decreased the A,,, value to 5% of the original value. The cleared solution was yellow and imparted unnecessary flavors from a sensory perspective. In order to use GABA-containing solutions in the production of liquors and beverages, a light color is preferable from the consumer’s point of view. Thus, a decolorization method was investigated. This yellow pigment was effectively removed by application of the supernatant to a column containing the synthetic adsorbents, SP-207 (Fig. 3). When 20 bed volumes of samples were treated with this resin, the A,,, value was reduced by about 75%. Resin-treated solution was suitable for application to the production of liquors and beverages. In addition,
J. BIOSCI.BIOENG., Solvent front --t
Glu+
Origin-+ 0
1
h
d
2
d
FIG. 1. Thin layer chromatogram of shochu kusu broth culture. Immediately after inoculation of precultured Lactobucillus
1.2 r
l
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
(
0.8
Final K of flocculation agent added
FIG. 2. Effect of treatment with flocculation agent. Colloidal silicon oxide, Koporokku SA was added to the centrifuged broth culture to a final concentration of 0 to 0.7%, and the supematants were allowed to stand at 5°C for 1 d. After removal of the precipitate by filtration through a filter paper, the A,, and GABA concentration were assayed. C/C,: The ratio of A,, (closed circle) and GABA concentration (open circle) before (C,) and after (C) treatment with the flocculation agent. The A,, and GABA concentration of the test sample before treatment were 0.547 and 10.18 mM, respectively.
VOL.
NOTES
93,2002 1.2
7. Morimura, S., Kida, K., Nakagawa, M., and Sonoda, Y.: Production of fimgal protein by Aspergillus awamori var.
1.0
kawuchi grown in shochu distillery wastewater. J. Ferment.
0.8
Bioeng., 78, 160-163 (1994). 8. Yokoi. H.. Aratake. T.. Nishio. S.. Hirose. J.. Havashi. S.. and Takasaki, Y.: Chitosan production from shochu ‘distillery wastewater by funguses. J. Ferment. Bioeng., 85, 246 249 (1998). 9. Yokoyama, S. and Tarumi, S.: Production and some properties of low-salt seasoning from shochu distillery waste. Seibutsu Kogaku, 79,211-217 (2001). 10. Stanton, H. C.: Mode of action of gamma aminobutyric acid on the cardiovascular system. Arch. Int. Pharmacodyn., 143, 195-200 (1963). 11. Omori, M., Yano, T., Okamoto, J., Tsushida, T., Murai, T., and Higuchi, M.: Effect of anaerobically treated tea (gabaron tea) on blood pressure of spontaneously hypertensive rats. Nippon Nogeikagaku Kaishi, 61, 1449-1451 (1987). (in Japanese) 12. Okada, T., Sugishita, T., Murakami, T., Murai, H., Saikusa, T., Horino, T., Onoda, A., Kajimoto, O., Takahashi, R., and Takahashi, T.: Effect of the defatted rice germ enriched with GABA for sleeplessness, depression, autonomic disorder by oral administration. Nippon Shokuhin Kagaku Kogaku Kaishi, 47,596-603 (2000). (in Japanese) 13. Kono, I. and Himeno, K.: Changes in y-aminobutyric acid content during beni-koji making. Biosci. Biotechnol. Biothem., 64,617-619 (2000). 14. Tsushida, T., Murai, T., Omori, M., and Okamoto, J.: Production of a new type of high level of y-aminobutyric acid. Nippon Nogeikagaku Kaishi, 61, 817-822 (1987). (in Japanese) 15. Saikusa, T., Horino, T., and Mori, Y.: Distribution of free amino acids in the rice kernel and kernel fractions and the effect of water soaking on the distribution. J. Agric. Food Chem., 42, 1122-1125 (1994). 16. Saikusa, T., Okada, T., Murai, H., Ohmori, M., Mori, Y., Horio, T., Ito, M., and Onoda, A.: The effect of defatting with organic solvent on accumulation of 4-aminobutyric acid (GABA) in the rice germ. Nippon Shokuhin Kagaku Kogaku Kaishi, 48, 196-201 (2001). (in Japanese) 17. Hayakawa, K., Ueno, Y., Kawamura, S., Taniguchi, R, and Oda, K.: Production of y-aminobutylic acid by lactic acid bacteria. Seibutsu Kogaku, 75,239-244 (1997). 18. Ueno, Y., Hayakawa, K., Takahashi, S., and Oda, K: Purification and characterization of glutamate decarboxylase from Lactobacillus brevis IFO-12005. Biosci. Biotech. Biothem., 61, 1168-1171 (1997). I
8
97
0.6
;j 0.4 0.2
0
10
20
30
40
50
60
70
80
Feed (EV)
FIG. 3. Effect of application to the synthetic adsorbents, SP-207. To a 10 ml volume of column, 75 bed volumes (BV) of clear broth culture were fed at a flow rate of 40 ml/h. The A,,, and GABA concentration of the eluted fractions were assayed. C/C+ The ratio of A430 (closed circle) and GABA concentration (open circle) before (C,) and after(C) SP-207 column treatment. The A,,, and GABA concentration of the test sample before treatment were 0.405 and 10.18 mM, respectively.
nary tests, mugi shochu kasu which contained 6.80 mM free Glu, produced 6.30mM GABA by 15. brevis IFO-12005 after 2 d of stationary culture at 30°C. REFERENCES 1. Nikkan Keizai Tushinsha: Shurui Shokuhin Tokei Geppo, No. 6,29-32 (2001). (in Japanese) 2. Nihon Jozokyokai: Jorhu haieki seibun, Jozobutu no Seibun, 139-141 (1999). (in Japanese) 3. Mahfudz, L.D., Nakashima, K., Ohtsuka, A., and Hayashi, K.: Growth factors for a primary chick muscle cell culture from shochu distillery by-product. Biosci. Biotech. Biochem., 61, 1844-1847 (1997). 4. Fur&a, Y., Takashita, H., Omori, T., Sonomoto, K., Ishizaki, A., Shimoda, M., and Wada, H.: Growth-stimulating effect of shochu wastewater on lactic acid bacteria and Bzjidobacteria. Ann. N.Y. Acad. Sci., 864,276279 (1998). 5. Morimura, S., Kida, K., Yakita, Y., Sonoda, Y., and Myoga, H.: Production of saccharifying enzyme using the wastewater of a shochu distillery. J. Ferment. Bioeng., 71, 329-334 (1991). 6. Morimura, S., Kida, K., and Sonoda, Y.: Production of protease using wastewater from the manufacture of shochu. J. Ferment. Bioeng., 77, 183-l 87 (1994).
,
,
I
II
*