Bioresource Technology 93 (2004) 59–62
Production of fructo-oligosaccharides from molasses by Aureobasidium pullulans cells H.T. Shin a, S.Y. Baig a, S.W. Lee a, D.S. Suh b, S.T. Kwon b, Y.B. Lim c, J.H. Lee
Department of Food and Life Science, Sungkyunkwan University, 300 Chunchun-dong, Jangan-gu, Suwon 440-746, South Korea Department of Genetic Engineering, Sungkyunkwan University, 300 Chunchun-dong, Jangan-gu, Suwon 440-746, South Korea c NEL Biotech Co., Ltd., 800-15 Duksan-ri, Samjuk-myon, Ansung 451-882, South Korea Institute of Life Science and Technology, Sungkyunkwan University, 300 Chunchun-dong, Jangan-gu, Suwon 440-746, South Korea b
Received 20 April 2003; received in revised form 30 June 2003; accepted 2 October 2003
Abstract Three diﬀerent strains of Aureobasidium pullulans were grown in batch cultures to compare their abilities for the production of fructo-oligosaccharides. Speciﬁc intracellular enzyme activity was the highest with strain KCCM 12017 and enzyme production was closely coupled to growth. Using A. pullulans cells, 166 g/l fructo-oligosaccharides was produced from 360 g/l molasses sugar as sucrose equivalent at 55 C and pH 5.5 after 24 h incubation. 2003 Elsevier Ltd. All rights reserved. Keywords: Aureobasidium pullulans; Enzyme reaction; Feed additive; Fructo-oligosaccharides; Molasses
1. Introduction Prebiotics are deﬁned as nondigestible food ingredients that beneﬁcially aﬀect the host by selectively stimulating the growth and/or activity of one or a limited number of bacterial species (Gibson and Roberfroid, 1995). Nondigestible oligosaccharides such as fructooligosaccharides (FOS) (Hidaka et al., 1987; Spiegel et al., 1994) and isomalto-oligosaccharides (Kohmoto et al., 1988; Shi et al., 2001) are prebiotics. The FOS, in which 1–3 fructose units are bound at the b-2,1 position of sucrose, are mainly composed of 1-kestose (GF2 ), nystose (GF3 ) and fructofuranosylnystose (GF4 ). The FOS have been shown to stimulate the growth of endogenous biﬁdobacteria. Therefore, the FOS are currently used in many food products. In addition, it has been reported that the addition of FOS to the diets of some animals results in improvements in feed eﬃciency, reduced diarrhea, and reduced smell in feces (Hidaka et al., 1987; Spiegel et al., 1994) although no improvements were achieved for lactating cows (Kobayashi and Eida, 1990) and weanling pigs (Farnworth et al., 1992; Houdijk et al., 1998). *
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0960-8524/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2003.10.008
Food-grade FOS are being produced commercially from pure sucrose using an intracellular enzyme from Aspergillus (Hidaka et al., 1988; Hirayama et al., 1989) or Aureobasidium (Yun et al., 1990). In order to use the FOS as a feed additive extensively, it is necessary to reduce production costs. This goal can be achieved by process improvements together with strain development and genetic manipulation (Rehm et al., 1998; Yanai et al., 2001). In the present study, an evaluation with a most promising strain of Aureobasidium pullulans was carried out on molasses as a source of sucrose because pure sucrose is a rather expensive material for feedgrade FOS production.
2. Methods 2.1. Strains Various strains of A. pullulans used in this study were obtained from Korea Research Institute of Bioscience and Biotechnology (Daejon, Korea) and the Korean Federation of Culture Collections (Seoul, Korea). These strains were maintained on agar plates containing stock medium (sucrose 50 g/l, yeast extract 2 g/l; pH 5.5) at 4 C and subcultured biweekly.
H.T. Shin et al. / Bioresource Technology 93 (2004) 59–62
2.2. Materials 1-Kestose, nystose, and fructofuranosylnystose were purchased from Wako Pure Chemical Industries (Osaka, Japan) and molasses was obtained from CJ Corporation (Seoul, Korea). Other chemicals used were reagent grade. 2.3. Culture conditions Five milliliter of inoculum grown on a seed medium containing sucrose (10 g/l) plus yeast extract (2 g/l) for two days were transferred to a 250 ml ﬂask containing 45 ml of main growth medium and cultured at 28 C for three days at 100 rpm (Shaking Incubator Model SKI/ 1000R, Johnsam, Korea). The main medium consisted of (g/l): sucrose, 100; yeast extract, 10; K2 HPO4 , 5; MgSO4 Æ 7H2 O, 0.5; and NaNO3 , 15. 2.4. Enzyme assay During the cultivation, 5 ml of the culture broth was centrifuged using a table-top centrifuge (5000 rpm) for 10 min at room temperature and the supernatant was collected for the determination of extracellular enzyme activity. The cells centrifuged from the 5 ml culture broth were washed and resuspended up to 5 ml with saline and then used for the determination of intracellular enzyme activity. Enzyme (fructosyltransferase EC 126.96.36.199) activity was determined by a method described previously (Jung et al., 1989). One unit (1 U) deﬁned as the amount of enzyme activity required to produce 1 lmol of glucose/min under the following conditions: (a) pH 5.5, (b) temperature 55 C, (c) reaction time 1 h, (d) reaction mixture consisting of the following composition: 7.5 ml of sucrose 800 g/l, 2.3 ml of 0.1 M citrate buﬀer (pH 5.5), and 0.2 ml enzyme sample. The enzyme reaction was stopped by heating at 100 C for 15 min and the released glucose from the enzyme reaction was measured by glucose oxidase–peroxidase (Sigma catalog number 315-100) at 505 nm using a spectrophotometer (Hewlett Packard 8453, Germany).
containing 10 ml of substrate. Throughout the course of this work 10 units of cells/g sucrose were employed. The enzyme reaction was stopped by heating at 100 C for 15 min and the reaction products were analyzed. 2.6. Analytical methods Dry cell weights (DCW) were determined for 5 ml samples, washed with 5 ml of NaCl (8.5 g/l solution) and once with distilled water, and dried at 105 C for 20 h. Morphological changes during the growth of A. pullulans were examined by means of a light microscope (Olympus BX-50, Japan). Enzyme reaction products were analyzed by HPLC (Hitachi L-6200, equipped with a RI-detector, Japan) using the Kromasil 100-10 NH2 (250 · 4.6 mm) column (Eka Chemicals, Sweden). A mixture of acetonitrile/distilled water (7:3, v/v) was used as the mobile phase at a ﬂow rate of 1 ml/min. The column temperature was controlled at 40 C.
3. Results and discussion 3.1. Comparison of enzyme production by diﬀerent A. pullulans strains Three diﬀerent strains of A. pullulans were grown in batch cultures to compare their abilities to produce enzyme. Data for growth and enzyme production by different strains are summarized in Table 1. It can be seen from Table 1, there was considerable variation between the diﬀerent strains. Compared with A. pullulans KCTC 6789, strains such as KCCM 12017 and KCTC 6353 had higher enzyme production rates. When strain KCCM 12017 and KCTC 6353 were compared, the ﬁnal cell concentration with KCCM 12017 was much lower and, therefore, the speciﬁc intracellular enzyme activity was highest with KCCM 12017. For this reason, strain KCCM 12017 was chosen as the most promising strain for the production of FOS and was used throughout the course of this work.
2.5. Reaction kinetic studies
3.2. Batch culture kinetics of A. pullulans
Unless otherwise speciﬁed, enzyme reactions were carried out at 55 C and pH 5.5 for 24 h in test tubes
A typical time course for the growth of A. pullulans KCCM 12017 on sucrose 100 g/l medium is shown in
Table 1 Growth and enzyme production by various A. pullulans strains grown on sucrose 100 g/l medium for three days at 28 Ca Strains
KCCM 12017 KCTC 6353 KCTC 6789
28.0 59.4 46.1
Data are averages of two experiments.
Enzyme activity (U/ml) Intracellular
67.0 60.0 16.6
54.0 55.0 8.9
121.0 115.0 25.5
Speciﬁc enzyme activity (U/mg DCW) 2.4 1.0 0.4
H.T. Shin et al. / Bioresource Technology 93 (2004) 59–62
Fig. 1. Unlike the results of Hayashi et al. (1990), both intra- and extracellular enzyme productions were closely coupled to growth. Cell concentration and intracellular enzyme activity increased up to 28 g/l and 67 U/ml, respectively. It was evident that not only intracellular enzyme but also extracellular enzyme could be used for the enzymatic production of fructo-oligosaccharides (data not shown). Typical morphological changes during the batch growth of A. pullulans were observed. At the initial stages of growth (0–15 h) mycelia cells were dominant in the culture and they accounted for about 60–70% of the culture cell population. However, yeast-
Enzyme activity (U/ml)
like cells and chlamydospores were dominant in the culture during the latter stages of growth. 3.3. Batch production of fructo-oligosaccharides from molasses Molasses is a mixture of sucrose and reducing sugars such as glucose and fructose. Molasses has also many unidentiﬁed components, with particulate material. The ratio of glucose plus fructose to sucrose with molasses is generally in the range of 0.1–0.2. In order to investigate enzyme inhibition caused by glucose or fructose in molasses, initial reaction rates (reaction time of 1 h) were measured as shown in Table 2. A major product was GF2 and detectable GF3 was produced only after 1 h reaction. Comparing GF2 production with sucrose, reaction rates with sucrose supplemented with glucose or fructose were decreased by 14% and 9%, respectively. Total FOS (GF2 + GF3 + GF4 ) production after 24 h was also decreased due to the inhibition caused by
300 Sugar (g/l)
Dry cell weight (g/l)
Fig. 2. Batch enzyme reaction kinetics with molasses (sugar 360 g/l as sucrose equivalent) at 55 C and pH 5.5. Glucose (m), sucrose (d), 1kestose (s), nystose (n) and fructofuranosylnystose (j).
Table 2 Data for enzyme reaction studies with sucrose 410 g/l with or without sugara Reaction products after 1 h GF2 (g/l) Sucrose Sucrose + glucoseb Sucrose + fructosec a
81 70 74
GF3 (g/l) 4.2 2.5 3.3
Data are averages of two experiments. Glucose was added to 50 g/l to the reaction system. c Fructose was added to 50 g/l to the reaction system. b
Fig. 1. Batch culture kinetics of A. pullulans KCCM 12017 on sucrose 100 g/l medium at 28 C. Intracellular enzyme activity (d), extracellular enzyme activity (s) and dry cell weight (n).
Reaction products after 24 h GF4 (g/l)
– – –
120 110 100
86 82 84
GF4 (g/l) 9.6 8.2 9.8
H.T. Shin et al. / Bioresource Technology 93 (2004) 59–62
glucose or fructose as illustrated in Table 2. It is worthwhile to note that sucrose as a starting substrate undergoes a number of enzyme steps to yield GF2 , GF3 , and GF4 (Jung et al., 1989). Signiﬁcant amounts of GF2 and GF3 with small amounts of GF4 accumulated after 24 h incubation. Similar results with pure sucrose were also obtained with Aspergillus niger (Hidaka et al., 1988), Aspergillus japonicus (Hayashi et al., 1992), and A. pullulans (Yun et al., 1990; Hayashi et al., 1991). In Fig. 2, a typical batch time-course kinetic study with molasses using A. pullulans KCCM 12017 cells is shown. The peak concentration of GF2 occurred at 5 h. Thereafter, the concentration declined, presumably because GF2 was being converted more rapidly to GF3 and GF4 than it was being formed. The total FOS (GF2 + GF3 + GF4 ) formed after 24 h incubation was 166 g/l from molasses sucrose 360 g/l. Therefore, the total FOS formed accounted for 46% of the sucrose as a substrate in molasses. As molasses is used as a feed additive, therefore, there is no need to isolate fructo-oligosaccharides from the reaction mixture. It is concluded that FOS can be produced practically from molasses as a cheap source of sucrose although the enzyme reaction is inhibited to some extent by other sugars such as glucose and fructose in molasses.
Acknowledgements This work was supported by Korea Research Foundation Grant (KRF-2001-005-G00005). References Farnworth, E.R., Modler, H.W., Jones, J.D., Cave, N., Yamazaki, H., Rao, A.V., 1992. Feeding Jerusalem artichoke ﬂour rich in fructooligosaccharides to weanling pigs. Can. J. Anim. Sci. 72, 977–980. Gibson, G.R., Roberfroid, M.B., 1995. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J. Nutr. 125, 1401–1412.
Hayashi, S., Nonokuchi, M., Imada, K., Ueno, H., 1990. Production of fructosyltransfering enzyme by Aureobasidium sp. ATCC 20524. J. Ind. Microbiol. 5, 395–400. Hayashi, S., Nonokuchi, M., Takasaki, Y., Ueno, H., Imada, K., 1991. Puriﬁcation and properties of b-fructofuranosidase from Aureobasidium sp. ATCC 20524. J. Ind. Microbiol. 7, 251–256. Hayashi, S., Matsuzaki, K., Takasaki, Y., Ueno, H., Imada, K., 1992. Puriﬁcation and properties of b-fructofuranosidase from Aspergillus japonicus. World J. Microb. Biot. 8, 276–279. Hidaka, H., Eida, T., Adachi, T., Saitoh, Y., 1987. Industrial production of fructooligosaccharides and its application for human and animals. Nippon Nogeik. Kaishi. 61, 915–923. Hidaka, H., Hirayama, M., Sumi, N., 1988. A fructooligosaccharideproducing enzyme from Aspergillus niger ATCC 20611. Agric. Biol. Chem. 52, 1181–1187. Hirayama, M., Sumi, N., Hidaka, H., 1989. Puriﬁcation and properties of a fructooligosaccharide-producing b-fructofuranosidase from Aspergillus niger ATCC 20611. Agric. Biol. Chem. 53, 667–673. Houdijk, J.G.M., Bosch, M.W., Verstegen, M.W.A., Berenpas, H.J., 1998. Eﬀect of dietary oligosaccharides on the growth performance and faecal characteristics of young growing pigs. Anim. Feed Sci. Technol. 71, 35–48. Jung, K.H., Yun, J.W., Kang, K.R., Lim, J.Y., Lee, J.H., 1989. Mathematical model for enzymatic production of fructo-oligosaccharides from sucrose. Enzyme Microb. Technol. 11, 491– 494. Kobayashi, S., Eida, T., 1990. Eﬀect of fructo-oligosaccharides on milk-yield and milk-component of dairy cows. Asian–Austral. J. Anim. Sci. 3, 21–25. Kohmoto, T., Fukui, F., Takaku, H., Machida, Y., Arai, M., Mitsuoka, T., 1988. Eﬀect of isomalto-oligosaccharides on human fecal ﬂora. Biﬁdo. Microﬂora 7, 61–69. Rehm, J., Wilimitzer, L., Heyer, A.G., 1998. Production of 1-kestose in transgenic yeast expressing a fructosyltransferase from Aspergillus foetidus. J. Bacteriol. 180, 1305–1310. Shi, B., Shan, A.S., Tong, J.M., 2001. Inﬂuence of dietary oligosaccharides on growth performance and intestinal microbial populations of piglets. Asian–Austral. J. Anim. Sci. 14, 1747– 1751. Spiegel, J.E., Rose, R., Karabell, P., Frankos, V.H., Schmitt, D.F., 1994. Safety and beneﬁts of fructooligosaccharides as food ingredients. Food Technol. 48, 85–89. Yanai, K., Nakane, A., Kawate, A., Hirayama, M., 2001. Molecular cloning and characterization of the fructooligosaccharide producing b-fructofuranosidase gene from Aspergillus niger ATCC 20611. Biosci. Biotechnol. Biochem. 65, 766–773. Yun, J.W., Jung, K.H., Oh, J.W., Lee, J.H., 1990. Semibatch production of fructooligosaccharides from sucrose by immobilized cells of Aureobasidium pullulans. Appl. Biochem. Biotechnol. 24– 25, 299–308.