- Email: [email protected]
Utilisation of soybean residue for the production of β-fructofuranosidase
Bioresource Technology 41 (1992) 231-233
Utilisation of Soybean Residue for the Production of fl-Fructofuranosidase Sachio Hayashi, Katsuhiro Matsuzaki, Toshikazu Kawahara, Yoshiyuki Takasaki & Kiyohisa Imada Department of Industrial Chemistry, Faculty of Engineering, Miyazaki University, 1-1 Gakuen Kibanadai Nishi, Miyazaki, 889-21, Japan
Abstract An industrial waste, soybean residue, was used for the production of a useful microbial enzyme, flfructofuranosidase, by fermentation. The enzyme was produced by Aspergillus japonicus using soybean residue (3-15%, w/v) as the only medium component other than sucrose. Sucrose at 15% (w/v) was the best carbon source for enzyme production. The maximum enzymatic activity reached approximately 9 × 103 U/50 ml medium after 48 h. The cell growth reached at least 0.6 g dry cells~50 ml medium after 120 h. The fermentation by Asp. japonicus to produce fl-fructofuranosidase was suggested as a method to recycle soybean residue. Key words: Soybean residue, fl-fructofuranosidase, A spergillus japonicus.
INTRODUCTION A large amount of soybean residue arises from the food industry, especially the preparation of tofu (an Asian food which is produced from soybean) (Kinoshita et al., 1985; H-Kittikun & Tani, 1986). The residues are treated at great expense to prevent pollution because they have little commercial value. However, the residue is a good nutritional source for microorganisms (Kinoshita et al., 1985; Kato et al., 1986). Some useful materials, such as riboflavin (Kinoshita et al., 1985), lipase (H-Kittikun & Tani, 1986) and okaramin (Hayashi et al., 1988), have been produced using soybean residue and cultures of microalgae (Wong, 1985), and a lactic acid fermentation (Kato et al., 1986) has been reported. There is, however, no report on the production of fl-fructofuranosidase, which has a
high fructosyl-transferring activity, using soybean residue, fl-Fructofuranosidase, which produces fructo-oligosaccharides from sucrose, has become important because the products have favourable properties as foods (increase of intestinal Lactobacillus bifidus, diminution of dental caries) (Hidaka et al., 1987). In previous papers, we reported on the production and properties ef flfructofuranosidase from Aureobasidium sp. (Hayashi et al., 1989, 1990, 1991b, c) and from Aspergillusjaponicus (Hayashi et al., 1991 a ). In the present paper, we describe the utilisation of waste soybean residue as a medium for production of an important enzyme, fl-fructofuranodiase, by Aspergillus japonicus.
METHODS Microorganism and enzyme production Aspergillus japonicus MU-2 (Hayashi et al., 1991a), maintained on 5% (w/v) sucrose, 1% (w/v) yeast extract, 0.1% (w/v) K2HPO4, 0.1% (w/v) MgSO4.7H20, 0.05% (w/v)KCI, 0.001% (w/v) FeSO 4. 7H20, 1.8% (w/v) agar at pH 6"5-7, was grown in 50 ml of medium which consisted of sucrose and soybean residue in the concentration stated in Results and Discussion, in a 200 ml flask shaken at 30°C for 48-120 h. The cells were harvested from the culture broth by centrifugation as a mixture with the soybean residue and were then lyophilysed to estimate their dry weight. The enzyme was solubilised from the dried cells by 0-03% (w/v) cetyl pyridinium chloride monohydrate and ultrasonic disintegration as described previously (Hayashi et al., 1991a) to determine the enzymatic activity. The soybean residue used was obtained from a tofu factory (Miyazaki, Japan) and then lyophilysed before utilisation for fermentation.
231 Bioresource Technology 0960-8524/92/S05.00 © 1992 Elsevier Science Publishers Ltd, England. Printed in Great Britain
S. Hayashi, K. Matsuzaki, T. Kawahara, Y. Takasaki, K. Imada
232
Enzyme activity assay The enzyme was assayed in a reaction mixture consisting of 0.1 ml of enzyme solution, 0.4 ml of 75% (w/v) sucrose, 0.5 ml of 0.15 M Mcllvain buffer (citric acid/Na2HPO4, pH 5) at 60°C for 20 min and terminated by boiling for 10 min. The glucose released in the reaction mixture was assayed by the glucose oxidase method (Wako, glucose test B) and one unit of enzymatic activity was defined as the quantity of enzyme responsible for the transfer of 1/~mol fructose in 1 min. The values given are means from at least two experiments.
2). However, there was no significant effect of the increase in concentration of the residue. Time course of enzyme production From Fig. 3, it can be seen that the maximum enzymatic activity was approximately 9 x 103 U/flask. Cell growth was estimated to reach at least 0.6 g dry cells/flask after 120 h because the weight of the solid from the culture broth, which was a mixture of cells and remains of the soybean residue, increased up to approximately 2.1 g dry weight/flask. The result suggests that soybean
RESULTS AND DISCUSSION -r
121.
Effect of carbon sources on enzyme production Of the various carbon sources used for enzyme production by Asp. japonicus using soybean residue as a medium component (Table 1) sucrose was the best. Lactose and soluble starch gave approximately 32% and 24% of the enzyme production on sucrose, respectively. Glucose, which gave a good enzyme production when yeast extract was used as a nitrogen source (Hayashi et al., 1991a), was not a good carbon source with soybean residue. Fructose, which is a good carbon source for Aureobasidium sp. (Hayashi et al., 1991 c), was not good for Asp. japonicus. The optimum sucrose concentration for enzyme production (Fig. 1) was 15% (w/v). This result was almost identical to the value (15-20%) on the optimal yeast extract medium for Asp. japonicus (Hayashi et al., 199 l a ). Effect of soybean residue concentration on enzyme production Enzyme production was carried out at concentrations of soybean residue from 3 to 15% (w/v) (Fig.
A
3 2 XlO 3 I0
u~
5
o
o
I
I
I
10
20
30
Sucrose conc. ( % ) Fig. 1. Effect of sucrose concentration on rfructofuranosidase production by Asp. japonicus. Basal medium consisted of 3% (w/v) soybean residue. Cultivations were carried out for 48 h at 30°C. Symbols: enzymatic activity, o; final pH, zx.
4 -r I3.
3 X10 3
10
Table 1. Effect of various carbon sources on fl-ffuctofuranosidase production by Asp. japonicus
0--0
"0
5
l0
x'3
13
Carbon source (15%)
Final pH
Enzymatic activity (x 103 U/flask)
Sucrose Lactose Soluble starch Maltose Galactose Glucose Fructose Glycerol
2'5 3.0 2.8 3.0 3.3 3.2 3"3 3"3
9'0 2.9 2.2 1.9 1.5 1.4 1-2 0.2
T h e basal medium consisted of 3% (w/v) soybean residue. Cultivations were carried out for 48 h at 30°C.
5 ._>
46
<
0 0
Soybean
residue
conc.
15 (*/,)
Fig. 2. Effect of soybean residue concentration on flfructofuranosidase production by Asp. japonicus. Basal medium consisted of 15% (w/v) sucrose. Cultivations were carried out for 48 h at 30°C. Symbols: enzymatic activity, o; final pH, zx.
fl-Fructofuranosidasefrom soybean residue
a favourable medium for fl-fructofuranosidase production. For these reasons, fermentation by Asp. japonicus is considered to be a very useful method of utilising soybean residue.
10 Q_
233
6
2 XlO 3 10
-&
REFERENCES
v
2 O~
~
5
[
~
)
>.
0
0 0
40 Time
60
120
( h )
Fig. 3. Time course of fl-fructofuranosidase production by Asp. japonicus. The medium used consisted of 15% (w/v) sucrose and 3% (w/v) soybean residue. Symbols: enzymatic activity, o; dry weight, n; final pH, A.
residue is a good medium component not only for enzyme production but also for cell growth. The present fermentation was successfully carried out with soybean residue as the only medium component other than sucrose. Salts and expensive nitrogen sources, such as yeast extract, used in the optimal medium (Hayashi et al., 1991a) are not required. This reduces the expense of treating soybean residue. We also tried to produce fl-fructofuranosidase from Aureobasidium sp. (Hayashi et al., 1990) (another strain which produces high enzymatic activity on yeast extract) on soybean residue but could not do so. So, it is considered that Asp. japonicus is a special strain which can be used for fermentation to utilise a waste, soybean residue, and that soybean residue, which can be obtained at little expense, is
Hayashi, H., Takiuchi, K., Murao, S. & Arai, M. (1988). Okaramine B, an insecticidal indole alkaloid, produced by Penicillium simplicissimum AK-40. Agric. Biol. Chem.. 52, 2131-3. Hayashi, S., Imada, K., Kushima. Y. & Ueno, H. (1989). Observation of the chemical structure of fructooligosaccharide produced by an enzyme from Aureobasidium sp. ATCC 20524. Curr. Microbiol., 19, 175-7. Hayashi, S., Nonoguchi, M., lmada, K. & Ueno, H. (1990). Production of a fructosyl-transferring enzyme by Aureobasidium sp. ATCC 20524. J. Ind. Microbiol., 5, 395-400. Hayashi, S., Matsuzaki, K., Takasaki, Y. & lmada, K. (1991 a). Production of fl-fructofuranosidase by Aspergillus japonicus. World J. Microbiol. Biotechnol., 8, l 55-9. Hayashi, S., Nonoguchi, M., Takasaki, Y. & lmada, K. (1991b). Purification and production of fl-fructofuranosidase from Aureobasidium sp. ATCC 20524. J. Ind. Microbiol., 7, 251-6. Hayashi, S., Shimokawa, Y., Nonoguchi, M., Takasaki, Y. & lmada, K. (1991c). Nutritional status of Aureobasidium sp. ATCC 20524 for the production of /3fructofuranosidase. World J. Microbiol. Biotechnol., 7, 522-5. Hidaka, H., Eida, T., Adachi, T. & Saitoh, Y. (1987). Industrial production of fructooligosaccharides and its application for humans and animals. Nippon Nogeikagaku Kaishi, 61,915-23. H-Kittikun, A. & Tani, Y. (1986). Preliminary study on microbial production of lipase using soybean residue from tofu making process. Atom. Rep. ICBiotech., 9,275-6. Kato, T., Shiga, I. & Terasawa, K. (1986). Preservation of okara (soybean residue from soy mash) by lactic acid fermentation. Nippon Shokuhin Koq~,o Gakkaishi, 33, 837-41. Kinoshita, S., H-Kittikun, A. & Pinthong, R. (1985). Production of riboflavin from waste of tofu (soybean curd). Atom. Rep. ICBioteeh., 8,322-4. Wong, M. H. (11985). Cultivation of microalgac in refuse compost and soybean waste extract. Agric. Wastes, 12, 225-33.
Utilisation of Soybean Residue for the Production of fl-Fructofuranosidase Sachio Hayashi, Katsuhiro Matsuzaki, Toshikazu Kawahara, Yoshiyuki Takasaki & Kiyohisa Imada Department of Industrial Chemistry, Faculty of Engineering, Miyazaki University, 1-1 Gakuen Kibanadai Nishi, Miyazaki, 889-21, Japan
Abstract An industrial waste, soybean residue, was used for the production of a useful microbial enzyme, flfructofuranosidase, by fermentation. The enzyme was produced by Aspergillus japonicus using soybean residue (3-15%, w/v) as the only medium component other than sucrose. Sucrose at 15% (w/v) was the best carbon source for enzyme production. The maximum enzymatic activity reached approximately 9 × 103 U/50 ml medium after 48 h. The cell growth reached at least 0.6 g dry cells~50 ml medium after 120 h. The fermentation by Asp. japonicus to produce fl-fructofuranosidase was suggested as a method to recycle soybean residue. Key words: Soybean residue, fl-fructofuranosidase, A spergillus japonicus.
INTRODUCTION A large amount of soybean residue arises from the food industry, especially the preparation of tofu (an Asian food which is produced from soybean) (Kinoshita et al., 1985; H-Kittikun & Tani, 1986). The residues are treated at great expense to prevent pollution because they have little commercial value. However, the residue is a good nutritional source for microorganisms (Kinoshita et al., 1985; Kato et al., 1986). Some useful materials, such as riboflavin (Kinoshita et al., 1985), lipase (H-Kittikun & Tani, 1986) and okaramin (Hayashi et al., 1988), have been produced using soybean residue and cultures of microalgae (Wong, 1985), and a lactic acid fermentation (Kato et al., 1986) has been reported. There is, however, no report on the production of fl-fructofuranosidase, which has a
high fructosyl-transferring activity, using soybean residue, fl-Fructofuranosidase, which produces fructo-oligosaccharides from sucrose, has become important because the products have favourable properties as foods (increase of intestinal Lactobacillus bifidus, diminution of dental caries) (Hidaka et al., 1987). In previous papers, we reported on the production and properties ef flfructofuranosidase from Aureobasidium sp. (Hayashi et al., 1989, 1990, 1991b, c) and from Aspergillusjaponicus (Hayashi et al., 1991 a ). In the present paper, we describe the utilisation of waste soybean residue as a medium for production of an important enzyme, fl-fructofuranodiase, by Aspergillus japonicus.
METHODS Microorganism and enzyme production Aspergillus japonicus MU-2 (Hayashi et al., 1991a), maintained on 5% (w/v) sucrose, 1% (w/v) yeast extract, 0.1% (w/v) K2HPO4, 0.1% (w/v) MgSO4.7H20, 0.05% (w/v)KCI, 0.001% (w/v) FeSO 4. 7H20, 1.8% (w/v) agar at pH 6"5-7, was grown in 50 ml of medium which consisted of sucrose and soybean residue in the concentration stated in Results and Discussion, in a 200 ml flask shaken at 30°C for 48-120 h. The cells were harvested from the culture broth by centrifugation as a mixture with the soybean residue and were then lyophilysed to estimate their dry weight. The enzyme was solubilised from the dried cells by 0-03% (w/v) cetyl pyridinium chloride monohydrate and ultrasonic disintegration as described previously (Hayashi et al., 1991a) to determine the enzymatic activity. The soybean residue used was obtained from a tofu factory (Miyazaki, Japan) and then lyophilysed before utilisation for fermentation.
231 Bioresource Technology 0960-8524/92/S05.00 © 1992 Elsevier Science Publishers Ltd, England. Printed in Great Britain
S. Hayashi, K. Matsuzaki, T. Kawahara, Y. Takasaki, K. Imada
232
Enzyme activity assay The enzyme was assayed in a reaction mixture consisting of 0.1 ml of enzyme solution, 0.4 ml of 75% (w/v) sucrose, 0.5 ml of 0.15 M Mcllvain buffer (citric acid/Na2HPO4, pH 5) at 60°C for 20 min and terminated by boiling for 10 min. The glucose released in the reaction mixture was assayed by the glucose oxidase method (Wako, glucose test B) and one unit of enzymatic activity was defined as the quantity of enzyme responsible for the transfer of 1/~mol fructose in 1 min. The values given are means from at least two experiments.
2). However, there was no significant effect of the increase in concentration of the residue. Time course of enzyme production From Fig. 3, it can be seen that the maximum enzymatic activity was approximately 9 x 103 U/flask. Cell growth was estimated to reach at least 0.6 g dry cells/flask after 120 h because the weight of the solid from the culture broth, which was a mixture of cells and remains of the soybean residue, increased up to approximately 2.1 g dry weight/flask. The result suggests that soybean
RESULTS AND DISCUSSION -r
121.
Effect of carbon sources on enzyme production Of the various carbon sources used for enzyme production by Asp. japonicus using soybean residue as a medium component (Table 1) sucrose was the best. Lactose and soluble starch gave approximately 32% and 24% of the enzyme production on sucrose, respectively. Glucose, which gave a good enzyme production when yeast extract was used as a nitrogen source (Hayashi et al., 1991a), was not a good carbon source with soybean residue. Fructose, which is a good carbon source for Aureobasidium sp. (Hayashi et al., 1991 c), was not good for Asp. japonicus. The optimum sucrose concentration for enzyme production (Fig. 1) was 15% (w/v). This result was almost identical to the value (15-20%) on the optimal yeast extract medium for Asp. japonicus (Hayashi et al., 199 l a ). Effect of soybean residue concentration on enzyme production Enzyme production was carried out at concentrations of soybean residue from 3 to 15% (w/v) (Fig.
A
3 2 XlO 3 I0
u~
5
o
o
I
I
I
10
20
30
Sucrose conc. ( % ) Fig. 1. Effect of sucrose concentration on rfructofuranosidase production by Asp. japonicus. Basal medium consisted of 3% (w/v) soybean residue. Cultivations were carried out for 48 h at 30°C. Symbols: enzymatic activity, o; final pH, zx.
4 -r I3.
3 X10 3
10
Table 1. Effect of various carbon sources on fl-ffuctofuranosidase production by Asp. japonicus
0--0
"0
5
l0
x'3
13
Carbon source (15%)
Final pH
Enzymatic activity (x 103 U/flask)
Sucrose Lactose Soluble starch Maltose Galactose Glucose Fructose Glycerol
2'5 3.0 2.8 3.0 3.3 3.2 3"3 3"3
9'0 2.9 2.2 1.9 1.5 1.4 1-2 0.2
T h e basal medium consisted of 3% (w/v) soybean residue. Cultivations were carried out for 48 h at 30°C.
5 ._>
46
<
0 0
Soybean
residue
conc.
15 (*/,)
Fig. 2. Effect of soybean residue concentration on flfructofuranosidase production by Asp. japonicus. Basal medium consisted of 15% (w/v) sucrose. Cultivations were carried out for 48 h at 30°C. Symbols: enzymatic activity, o; final pH, zx.
fl-Fructofuranosidasefrom soybean residue
a favourable medium for fl-fructofuranosidase production. For these reasons, fermentation by Asp. japonicus is considered to be a very useful method of utilising soybean residue.
10 Q_
233
6
2 XlO 3 10
-&
REFERENCES
v
2 O~
~
5
[
~
)
>.
0
0 0
40 Time
60
120
( h )
Fig. 3. Time course of fl-fructofuranosidase production by Asp. japonicus. The medium used consisted of 15% (w/v) sucrose and 3% (w/v) soybean residue. Symbols: enzymatic activity, o; dry weight, n; final pH, A.
residue is a good medium component not only for enzyme production but also for cell growth. The present fermentation was successfully carried out with soybean residue as the only medium component other than sucrose. Salts and expensive nitrogen sources, such as yeast extract, used in the optimal medium (Hayashi et al., 1991a) are not required. This reduces the expense of treating soybean residue. We also tried to produce fl-fructofuranosidase from Aureobasidium sp. (Hayashi et al., 1990) (another strain which produces high enzymatic activity on yeast extract) on soybean residue but could not do so. So, it is considered that Asp. japonicus is a special strain which can be used for fermentation to utilise a waste, soybean residue, and that soybean residue, which can be obtained at little expense, is
Hayashi, H., Takiuchi, K., Murao, S. & Arai, M. (1988). Okaramine B, an insecticidal indole alkaloid, produced by Penicillium simplicissimum AK-40. Agric. Biol. Chem.. 52, 2131-3. Hayashi, S., Imada, K., Kushima. Y. & Ueno, H. (1989). Observation of the chemical structure of fructooligosaccharide produced by an enzyme from Aureobasidium sp. ATCC 20524. Curr. Microbiol., 19, 175-7. Hayashi, S., Nonoguchi, M., lmada, K. & Ueno, H. (1990). Production of a fructosyl-transferring enzyme by Aureobasidium sp. ATCC 20524. J. Ind. Microbiol., 5, 395-400. Hayashi, S., Matsuzaki, K., Takasaki, Y. & lmada, K. (1991 a). Production of fl-fructofuranosidase by Aspergillus japonicus. World J. Microbiol. Biotechnol., 8, l 55-9. Hayashi, S., Nonoguchi, M., Takasaki, Y. & lmada, K. (1991b). Purification and production of fl-fructofuranosidase from Aureobasidium sp. ATCC 20524. J. Ind. Microbiol., 7, 251-6. Hayashi, S., Shimokawa, Y., Nonoguchi, M., Takasaki, Y. & lmada, K. (1991c). Nutritional status of Aureobasidium sp. ATCC 20524 for the production of /3fructofuranosidase. World J. Microbiol. Biotechnol., 7, 522-5. Hidaka, H., Eida, T., Adachi, T. & Saitoh, Y. (1987). Industrial production of fructooligosaccharides and its application for humans and animals. Nippon Nogeikagaku Kaishi, 61,915-23. H-Kittikun, A. & Tani, Y. (1986). Preliminary study on microbial production of lipase using soybean residue from tofu making process. Atom. Rep. ICBiotech., 9,275-6. Kato, T., Shiga, I. & Terasawa, K. (1986). Preservation of okara (soybean residue from soy mash) by lactic acid fermentation. Nippon Shokuhin Koq~,o Gakkaishi, 33, 837-41. Kinoshita, S., H-Kittikun, A. & Pinthong, R. (1985). Production of riboflavin from waste of tofu (soybean curd). Atom. Rep. ICBioteeh., 8,322-4. Wong, M. H. (11985). Cultivation of microalgac in refuse compost and soybean waste extract. Agric. Wastes, 12, 225-33.