Production of fructosylxylosides by Scopulariopsis brevicaulis sp.

JOURNALOF FERMENTATION AND BIOENGINEERING Vol. 79, No. 3, 242-246. 1995

Production

by Scopulariopsis brevicau Iis sp .

of Fructosylxylosides HIROYUKI

TAKEDAP

AND SHINICHI

Department of Molecular Chemistry, Faculty of Engineering, Hokkaido

KINOSHITA* University, Kita-ku, Sapporo 060, Japan

Received 29 August 1994IAccepted 7 December 1994

A 1-kestose-producing strain, Scopulariopsis brevicaulis, produced two oligosaccbarides, fructosylxyloside (FX) and fructosylfructosylxyloside (FFX) from sucrose and o-xylose. FX is known as an anti-cariogenic sugar, but FFX is found to be a new sugar. FX inhibited the production of insoluble glucan, the presence of which in the mouth contributes to the development of dental caries, from sucrose by Streptococcus mutans but FFX did not. However, neither oligosaccharide was utilized as a substrate for the production of insoluble glucan. We determined the optimal culture conditions for the production of oligosaccharides to be initial sugar concentrations of 80 g/Z sucrose and 80 g/Z o-xylose, initial pH of 7, and incubation temperature of 30%. Under the optimal culture conditions, 50.3 g/l fructosylxyloside and 9.8 g/Z fructosylfructosylxyloside were produced. [Key words: xylose, fructosylxyloside,

Scopulariopsis brevicaulis]

jar fermentor (Tokyo Scientific Instrument Corp., Tokyo) it was incubated at 30°C in 5 I of medium containing appropriate o-xylose and sucrose concentrations with an aeration rate of 2.5 //min. Other conditions were the same as described above and in the previous paper (1). Preparation of crude enzyme After the culture of this fungus, lOOmI of the culture medium was filtered. The mycelia were washed with distilled water twice and 7.5 g of wet mycelia was obtained. These were ground in a mortar with sea sand for 5 min at room temperature and were suspended in McIlvaine buffer (PH 7) to a final volume of 50ml, which was used as a crude enzyme. This preparation showed 0.19 unit/ml of p-fructofuranosidase activity. The enzyme reaction was carried Enzyme reaction out at 40°C for 5 h in a test tube containing 1 ml of the crude enzyme of S. brevicaufis, 0.5 ml of 200 g/l sucrose solution, and 0.5 ml of each 200 g/l saccharide solution. The reaction was terminated by boiling at 100°C for 5 min. After the filtration through a membrane filter of 0.45 pm pore size, the formation of oligosaccharide in the filtrate was analyzed by high-performance liquid chromatography (HPLC). Analysis of oligosaccharides Thin-layer chromatography Sugar samples were spotted on a thin-layer chromatography plate (Kieselgel 60, 100 X 100 mm, Merck Corp., Germany). After development by the ascending technique with an isopropanol, n-butanol and water mixture (12 : 3 : 4) at room temperature for 3 h, the products were detected by spraying with anisaldehyde-HzS04 followed by heating at 1lO’C for 20 min. High-performance liquid chromatography Sugars were determined by a Waters M-600 chromatograph (Nihon Millipore L;d., Tokyo) using a Waters /*-Bondasphere 5 ,U NH,-1OOA (3.9 x 50 mm) column, refractometer (ERC-7515A, Erma Cr. Inc., Saitama), and recorder (C-RSA) with 75% acetonitrile as an eluant at flow rate of 1.0 ml/min at 30°C. Methods for isolation of oligosaccharides After adjusting the mixture to 30% oligosaccharide, 200,~l of each sample was injected into a Waters M-600 chromatography system equipped with a separation column

We reported (1) the production of I-kestose from sucrose by Scopulariopsis brevicaulis, the determination of optimal culture conditions and the crystallization of l-kestose. As this fungus (1) showed transfructosylation activity, we can expect the production of other oligosaccharides when other sugars are added together with sucrose. Among many sugars tested, a large amount of fructosyloligosaccharides were formed from o-xylose in the presence of sucrose. We isolated the oligosaccharides produced from sucrose and o-xylose by chromatography and identified them as fructosylxyloside and fructosylfructosylxyloside by carbon-13 nuclear magnetic resonance (IV-NMR) spectra and other determinations. Fructosylxyloside was shown to have a strong inhibitory effect on the synthesis of o-glucans from sucrose by o-glucosyltransferase from Streptococcus mutans (2-4). Thus this oligosaccharide is known to be useful in protection against the development of dental caries even in the presence of sucrose. Fructosylxyloside is known to be produced by levansucrases from Aerobacter levanicum (5) and Bacillus subtilis var. saccharolyticus (6) and P-fructofuranosidases of Penicillium sp. K-25 (7) and Penicillium frequentans T- 1 (8). However, this oligosaccharide is presently not commercially available. Hence in this study we investigated the feasibility of industrial production of fructosylxylosides by S. brevicaulis, and determined the optimal culture conditions for the production. We also studied the inhibitory effect on insoluble glucan production by S. mutans. MATERIALS

AND METHODS

The seed Microorganism and culture conditions culture of S. brevicaulis was incubated in a 5OOml conical flask containing 100 ml of the medium composed of 100 g/l sucrose, 15 g/l yeast extract, 0.6 g/l urea, 1 g/l K2HP04 and 0.3 g/l MgS04.7H20 (pH 7.0) at 30°C for 24-72 h on a reciprocal shaker at 120rpm oscillation. After inoculating 200ml of the seed culture in to a IO-1 * Corresponding author. § Presnt address: Dept. Gen. Foods, the Hokuren Fed. Agric. Corp., Chuo-ku, Sapporo.

242

VOL.

FRUCTOSYLXYLOSIDE

79, 1995

(PA-43, 20x 250 mm, Yamamura Chem. Lab. Co. Ltd., Kyoto). Acetonitrile (65%) was used at a flow rate of 9.99 ml/min. Oligosaccharide fractions were collected by monitoring the refractometer (Waters R401) after several injections of sample. Fifty mg of each 13C-NMR spectrum measurements saccharide was dissolved in 1 ml of D20 and placed into a quartz capillary tube, and its r3C-NMR spectrum was measured with an NMR spectrophotometer (Bruker AM-500) operated at 125 MHz. In these measurements deuterated TPS {(CH&SiCDr-CD,COONa (Wako Pure Chem. Ind. Ltd., Osaka)} was added as an internal standard. S. Method for measurement of insoluble glucan mutans Ingbritt was cultured in 2ml of heart infusion broth containing appropriate sugars. The culture supernatant contained the soluble glucan and the precipitate contained the insoluble glucan. The insoluble glucan in the precipitate was solubilized by adding 2 ml of 0.5 N sodium hydroxide and this supernatant fraction was used as an insoluble glucan fraction. The amount of insoluble glucan was calculated using dextran (Mw lOO,OOO-200,000) as a standard from the absorbance at 720nm of a 1 : 1 mixture of the glucan solution and methanol (9). RESULTS

AND DISCUSSION

Oligosaccharide production by the enzyme reaction Since S. brevicaulis produced 1-kestose mainly from sucrose (l), we considered that the enzyme involved in this sugar production might have some transglycosidation activity. Therefore, we tested for this activity using the crude cell extract with various saccharides. The enzyme reaction was carried out according to the method described. Oligosaccharides and 1-kestose were detected in the reaction mixtures of n-xylose, raffinose, melibiose, and isomaltose, and in these reactions the production of 1-kestose appeared to decrease some extent. However, no oligosaccharides other than 1-kestose were detected in the reaction mixtures of D-glucose, o-fructose, o-galactose, o-glucosamine, lactose, and maltose. Concentrations of other oligosaccharides except l-kestose were calculated using sucrose as a standard. From these analyses, we found that oligosaccharide production was the highest with o-xylose and the level of production increased in the following order: o-xylose> raffinose > melibiose > isomaltose (Table 1). Although no oligosaccharides other than I-kestose were detected in reactions with sucrose and o-glucose, a small amount of 1-kestose was produced as shown Table 1. This indicated that o-glucose inhibited I-kestose formation from sucrose. Upon further investigation, we focused on the oligosaccharide production from o-xylose and sucrose because o-xylose is available in large amounts for industrial production and less expensive than other saccharides tested here. Production of xylose-containing oligosaccharides by culture method As described in the previous paper (1) the oligosaccharide productions were carried out at pH 7 and 30°C for 72 h in five 500-ml conical flasks containing 1OOml of the medium, containing lOOg/l sucrose, 100 g/l D-xylose and 15 g/l yeast extract, with 12Orpm oscillation. Saccharides in the culture filtrate were analyzed by HPLC, which showed unknown

TABLE 1.

PRODUCTION

Oligosaccharide production by transfructosylation sucrose to various sugars

Sugar addition None D-Xylose Raffinose Melibiose Isomaltose D-GhCOSe

o-Fructose o-Galactose D-Glucosamine Lactose Maltose

Production

243 from

(g/f)

1-Kestose

Other oligosaccharidesa

14.6 5.0 6.0 6.2 8.4 1.1 10.1 13.9 13.8 13.1 13.4


a Concentration of oligosaccharide was calculated using sucrose as a standard except in the case of I-kestose. b Glucose could be converted to sucrose which is not detectable since sucrose is a substrate. The reaction conditions were described in Materials and Methods.

oligosaccharides X0-1 and X0-2, 1-kestose, sucrose, Dxylose, o-fructose, and D-glucose at concentration of 47.2, 4.5, 12.1, 31.0, 65.2, 7.5, and 2Sg/l, respectively. We attempted to purify these unknown sugars from the culture filtrate. Identification Isolation of oligosaccharides To remove o-xylose, D-fructose, o-glucose and proteins from the culture filtrate, it was clarified according to the method described in the previous paper (1) using calcium oxide. After desalting the clarified solution with ion-exchange columns (Diaion PK-220 and WA-30, Mitsubishi Chem. Corp., Tokyo), the solution was concentrated to 60 Brix (weight percentage). The sugar composition of this syrup was 49% X0-1, 5% X0-2, 33% sucrose and 13% l-kestose (dry weight percentage). X0-1 and X0-2 were isolated by HPLC with a YMC packed column using 65% acetonitrile as eluent at a flow rate of 9.99 ml/min. Aliquots (200 ~1) of each sample were subjected to HPLC 40times. Under these conditions, we obtained amorphous X0-1 (1,023 mg) and X0-2 (110 mg), after collection and evaporation. The purity of the oligosaccharides were confirmed by thin-layer chromatography and HPLC. Hydrolysis of oligosaccharides by jl-fructofuranosidase Fifty mg of amorphous X0-1 was dissolved in 1.0 ml buffer (pH 7) containing 250 units of invertase (Boehringer Mannheim Corp., Germany), and the mixture was incubated at 30°C for 20min. The reaction products were analyzed by HPLC and found to contain 152 pmol o-xylose and 151 pmol D-fructose. No other sugars were detected. X0-2 was also treated as above and found to contain 97 pmol o-xylose and 196 pmol Dfructose. From these experiments X0-1 was found to be a disaccharide consisting of o-xylose and o-fructose whose ratio was 1 : 1, and X0-2 was found to be a trisaccharide consisting of o-xylose and n-fructose whose ratio was 1 : 2. Reducing power of the oligosaccharides The reducing power of X0-1 and X0-2 was tested by the Somogyi-Nelson method, and neither sugar was found to show reducing power. Therefore, it seems that D-xylose and n-fructose are linked through reaction of reducing groups. Considering the 13C-N_MR spectrum measurements

FX and I-kestose (g/I)

b

r

L

r

b

c

.A

c

I:

_

_

FRUCTOSYLXYLOSIDE

VOL. 79, 1995 TABLE 3.

Sucrose Sucrose and Sucrose and Sucrose and Sucrose and FX FFX 1-Kestose

20

40

60

80

100

Time(h)

FIG. 4. Production of xylose-containing oligosaccharides using a jar fermentor under optimal conditions. Culture conditions: 30°C pH 7.0.

8Og/l D-xylose, the FX production level was the same as that with 80 g/l sucrose although the 1-kestose production increased slightly with culture time (data not shown). At 8Og/l sucrose, FX production increased with increase in xylose concentration, however, the D-xylose utilization efficiency for FX production was decreased from 36% to 25%. Hence, we selected 80 g/l D-xylose in further experiments. with

Effect of initial pH of medium on FX production S. brevicaulis was cultured in 100 ml of medium at various initial pHs in 500-ml conical flasks for 72 h at 30°C. The final amounts of FX are shown in Fig. 3a. The maximum production was obtained at an initial pH of 7.0.

Effect of culture temperature on FX production The culture was carried out at 26-34°C and pH 7.0 for 72 h. Maximum production was obtained at 30°C (Fig. 3b).

Production of fructosylxyloside under optimal conditions The oligosaccharide production was carried out at pH 7.0 and 30°C in 5 1 of medium in a 10-1 jar fermentor containing 80 g/l sucrose, 80 g/l D-xylose, and 15 g/f yeast extract with an agitation rate of 4OOrpm and an aeration rate of 2.5 //min. The FX concentration reached the maximum at 64 h after inoculating 200 ml of the seed culture (Fig. 4). In further experiments the fermentation period was set at 64 h and the amount of FX produced was 50.3 g/l and that of FFX was 9.8 g/l. The following saccharides besides FX and FFX were detected in the broth: 10.8g/Z 1-kestose, 4.8g/l D-fructose, 2.9 g/l D-glucose, 3.5 g/l sucrose, and 51.4 g/l D-xylose. The culture filtrate was clarified, desalted and concentrated to 70 Brix (weight percentage) as described before (1). The sugar composition of this syrup was 67% FX, 13% FFX, 5% sucrose and 15% 1-kestose (dry weight percentage) and the total amount of oligosaccharide syrup was 473 g. Since D-xylose is a popular sugar but rather expensive, it is a problem that half of the D-xylose remains unused at the end of the culture, which was seen in the case of levansucrase or an other /3-fructofuranosidase (7, 8). In this paper we used calcium oxide and high temperature to dissociate residual D-xylose. It is necessary to consider other methods such as chromatography to recover D-xy-

245

Effect of FX and FFX on the synthesis of insoluble glucan from sucrose by S. mutans Relative synthesi;;;

Carbon source (1%)

0

PRODUCTION

insoluble glucan

\

FX FFX palatinose I-kestose

100 21 107 56 106 0 0 0

lose for its reuse.

Inhibition of insoluble glucan synthesis by glucosyltransferase of S. mutans by FX and FFX Although S. mutans is usually observed in the human cavity (12, 13), S. sobrinus was used in the earlier study to test the inhibitory effects of D-glucosyltransferases (2-4). Hence, a study using S. mutans should be performed to further investigate the inhibitory effect. S. mutans Ingbritt was grown for 18 h to the mid-log phase in the presence of log/l sucrose and log/l other sugars, and the synthesis of insoluble glucan by S. mutans was determined. The results are shown as the relative amount of glucan synthesized (Table 3). FX and palatinose showed a strong inhibitory effect on synthesis of insoluble glucan as expected from results of the earlier studies using S. sobrinus (2-4), but FFX shows no such effect. However, this strain could not utilize both fructooligosaccharides as substrates for the synthesis of insoluble glucan. REFERENCES ’ Takeda, H., Sato, K., Kinosita, S., and Sasaki, H.: Production of 1-kestose by Scowlariomis brevicaulis. J. Ferment. Bioeng., 77, 386-389 (1994). 2. Kitahata, S., Yoshikawa, S., Okada, S., Takeuchi, IL., Imai, S., Nisizawa, T., and Araya, 53.:Dental caries and oligosaccharides. J. Jpn. Sot. Starch Sci.. 28. 142-149 (1981). (in Japanese) 3. Nisizawa, T., Takeuchi, K., and Imai, S.: Difference in mode of inhibition between a-D-xvlosvl B-D-fructoside and cY-isomaltosyl j3-D-fructoside in synihesis of glucan by Streptococcus mutans o-glucosyltransferase. Carbohydr. Res., 147, 135-144 (1986). 4. Kisi, M.: Inhibitory effect of xylosyl fructoside on glucosyltransferases from Streptococcus sobrinus. J. Dental Health, 41, 215 (1991). (in Japanese) 5. Hestrin, S., Feingold, D. S., and Aviged, G.: Synthesis of sucrose and other P-fructofuranosyl aldosides by levansucrase. J. Am. Chem. Sot., 77, 6710 (1955). 6. Fujita, K., Hat-a, K., Hashimoto, H., and Kitahata, S.: Studies on production of xylosylfructoside by levansucrase. Nippon Shokuhin Kogyo Gakkaishi, 36, 910-915 (1989). (in Japanese) 7. Kitahata, S., Suetake, S., and Okada, S.: Purification and transfructosylation reaction of fi-fructofuranosidase from Penicillium sp. K25. Nippon Shokuhin Kogyo Gakkaishi, 33, 826-830 (1986). (in Japanese) 8. Do, Y. and Shimura, S.: The synthesis of xylosucrose by ,%fructofuranosidase. Bioindustry, 15, 269-275 (1988). (in Japanese) 9. Kanno, T.: New functions of isomaltosyl-oligosaccharide. Food Chem., 10, 61-66 (1989). (in Japanese) 10. Periin, A. S., Casu, B., and Koch, H. J.: Configurational and conformational influences on carbon-13 chemical shifts of some carbohydrates. Can. J. Chem., 48, 2596-2606 (1970). 11. Jarrell, H. C., Conway, T. F., Moyna, P., and I. Smith, C. P.: 1.

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TAKEDA AND KINOSHITA

Manifestation of anomeric form, ring structure, and linkage in the W-N.M.R. spectra of oligomers and polymers containing o-fructose; maltose, isomahose, sucrose, leucrose, I-kestose, nystose, inulin, and grass levan. Carbohydr. Res., 76, 45-57 (1979). 12. Hamada, S., Masuda, N., Ooshima, N., Sobe, T., and Kotani,

J. FERMENT.BIOENG., K.: Epidemiological survey of Streptococcus mutans among Japanese children. Jpn. J. Microbial., 20, 33-44 (1976). 13. Qureshi, J. V., Goldner, M., Le Riche, W. H., and Hargreaves, J. A.: Streptococcus mutans serotype in young school children. Caries Res., 11, 334 (1977).