Microbial Fructan - ACS Symposium Series (ACS Publications)

Chapter 18

Microbial Fructan Production and Characterization 1

2

Y. W. Han and M. A. Clarke 1

Southern Regional Research Center, U.S. Department of Agriculture, P.O. Box 19687, New Orleans, LA 70179 Sugar Processing Research, Inc., 1100 Robert E. Lee Boulevard, New Orleans, LA 70179

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2

As part of an ongoing study to develop new products from the agricultural resources provided by sugar -producing crops, a search was initiated for microorganisms to produce polymeric compounds for industrial use. A fructan-producing bacterium was isolated from soils and characterized for polysaccharide synthesis. The composition and properties of the polysaccharide produced were studied. The organism, identified as a strain of Bacillus polymyxa. produced a large quantity of polysaccharide when grown on sucrose. The polysaccharide consisted entirely of fructose: methylation analysis showed that the primary fructose linkages were β(2->6) fructofuranosyl linkages. Carbon 13 nmr showed the product to be a levan type fructan. As part of an ongoing study to develop new products from the a g r i c u l t u r a l resources provided by sugar-producing crops, a search was i n i t i a t e d for microorganisms to produce polymeric compounds for i n d u s t r i a l use. Polysaccharides were the f i r s t group of polymers considered. Dextrans, polymers of glucose synthesized from sucrose, are important i n d u s t r i a l polysaccharides

(I). Fructans are natural polymers of fructose. Depending on the linkage types, fructans are c l a s s i f i e d into two groups: the levans, with mostly β-(2-*6) linkages and the i n u l i n s with β-(2-»1) linkages. Many fructans of both types have branched chains. Levans and i n u l i n s of low molecular weight are abundantly found i n plants, while high molecular weight fructans are produced by many microorganieme (2-4).

0097-6156/90/0433-0210$06.00A) © 1990 American Chemical Society

Glass and Swift; Agricultural and Synthetic Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1990.


HAN AND CLARKE

Microbial Fructan

A v a r i e t y of microorganisms produce e x t r a c e l l u l a r polysaccharides i n the form of capsules attached to the c e l l w a l l , or as slime secreted into the growth medium. These materials are used i n the organism's defense mechanism, or as a food reservoir. Some bacteria produce fructan, among which B a c i l l u s spp. predominate. Oral bacteria such as Rothis dentocariosa. Streptococcus s a l i v a r i u s and Odontomvces viscosus accumulate fructan i n human dental plaque (5-7). Several species of yeast and fungi are also known to produce levan (8.9). Most research on the biosynthesis of fructan has been conducted using B a c i l l u s s u b t i l i s . Aerobacter levanicum. and Streptococcus s a l i v a r i u s (10-21). Microbial fructans or levans, l i k e dextran, were f i r s t found i n sugar factories (16.2). These polysaccharides caused d i f f i c u l t i e s i n the beet sugar manufacturing process by increasing the v i s c o s i t y of the processing l i q u o r . Since t h e i r discovery (22). fructans have received l i t t l e attention and have never been exploited f o r i n d u s t r i a l applications. Recently the sugar industry has faced intense competition from high fructose corn syrup, which i s used as a low cost alternative sweetener. In search of new products from sucrose, the p o s s i b i l i t y of producing microbial fructan from sucrose for i n d u s t r i a l applications has been investigated. In t h i s paper, we report the i s o l a t i o n of a levan-producing bacterium and c h a r a c t e r i s t i c s of the fructan i t produces. I s o l a t i o n of a Fructan-Producing

Bacterium

Figure 1 shows the i s o l a t i o n scheme f o r a levan producing bacterium. About 1 g of rotting sugarcane stalks and the adhering s o i l p a r t i c l e s were added to 100 ml of basal medium and incubated at 30°C with constant shaking. The i s o l a t i o n medium consisted of sucrose 150g; peptone, 2g; yeast extract, 2g; K HP0 , 2g; (NH*) S0*, 0.3g; i n a l i t e r of water. The growth culture was then transferred to fresh media every 7-10 days. After several successive transfers, the culture was plated on agar media and the b a c t e r i a l colonies with gummy appearance were selected. These are organisms f o r which sucrose i s the sole carbon source, and which thrive i n high osmotic pressure. The organisms that produce polysaccharide (alcohol p r e c i p i t a t e ) having negative rotation of polarized l i g h t was t e n t a t i v e l y selected as levan producers. The levan was f i n a l l y confirmed by C nmr, infrared, and methylation analyses. A detailed i s o l a t i o n procedure was reported elsewhere (23) and the organisms has been registered at USDA, Northern Regional Research Center, Peoria, I l l i n o i s , and i d e n t i f i e d as NRRL B-18475. a

A

2

x a


212

AGRICULTURAL AND SYNTHETIC POLYMERS


SoU

Δ-Δ

Successive transfer to new media containing high sucrose (8%)

Streak on agar plate

ι

Δ

Transfer to liquid medium

1

y y — y J

ROH (IS vol) Add L5 vol of ethanol; collect ppt; add 0.5% oxalic acid; boil 10 minutes; select those showing negative polarity

j Oxalic add

Figure 1. Isolation of a fhictan-producing bacterium.


18. HAN AND CLARKE

Microbial Fructan


Production of Fructan The B^ polvmvxa (NRRL B-18475) produces a large quantity of fructan when grown on 4Z-16Z sucrose solution. The production scheme i s shown i n F i g . 1. The organism converted the fructose moiety of sucrose to fructan; of the remaining glucose, most were used as the carbon source for microbial growth and a small amount accumulated i n the growth medium. Some acids were produced as evidenced by decrease i n pH i n the growth medium. The composition of the products was monitored by HPLC (Sugar Analyzer, Waters Associates; HPX-87C column, BioRad Corp. with deionized water, 40 ppm i n calcium acetate as mobile phase). During fermentation, the sucrose l e v e l s dropped and fructan started to appear i n 2 days; thereafter, sucrose l e v e l gradually decreased as fructan increased. Glucose was the major by-product. A small amount of fructose and other unidentified fermentation products smaller i n molecular weight were also observed. The pH of the growth medium was controlled, and f e l l from 7.0 to 4.7 due to acid production. In reports of other fructan production, maintaining pH above 5.5 was important because the optimum pH for fruetansucrase i s between 5.57.0 and fructan may be hydrolyzed at a lower pH (2.). Optimum temperature for growth and fructan production was around 30°C. Aeration has been shown to be important i n the biosynthesis of fruetansucrase (24). Polysaccharide production was especially pronounced when the culture was gently shaken during c u l t i v a t i o n , but vigorous a g i t a t i o n and aeration i n h i b i t e d fructan production (23). A small amount of microbial polysaccharide (detected by alcohol p r e c i p i t a t i o n ) was also produced when the organism was grown on lactose, maltose, and raffinose, but no polysaccharide was produced on glucose or fructose. The organism produced polysaccharide from sugarcane juice, but the y i e l d was much less than that from sucrose. High sucrose concentration has been reported to lower the average molecular weight of the fructan synthesized (25). Fructan was harvested by p r e c i p i t a t i o n from the culture broth by addition of ethanol or isopropanol. Acetone and methanol can also be used. The y i e l d and consistency of the product varied depending on the amount of alcohol added. The fructan started to p r e c i p i t a t e at the medium/alcohol v/v r a t i o of 1:1.2, and the y i e l d peaked at about 1:1.5. Further increase i n the r a t i o hardened the fructan and made the product less f l u i d . S l i g h t l y less isopropanol was needed than ethanol to p r e c i p i t a t e levan (fructan). Although most of the b a c t e r i a l c e l l s , unfermented sugars, and other solubles remained i n the aqueous alcohol phase, pre-removal of microbial c e l l s by centrifuging was needed to obtain a pure form of fructan. The product was further p u r i f i e d by repeated p r e c i p i t a t i o n and d i s s o l u t i o n i n water, followed by d i a l y s i s or u l t r a f i l t r a t i o n . The f i n a l product was an


213

AGRICULTURAL AND SYNTHETIC POLYMERS off-white, flaky or powdery material that could be freezeor vacuum-dried, or alternately dried by t r i t u r a t i o n and p u l v e r i z a t i o n i n a high-speed blender with absolute alcohol. In a t y p i c a l fermentation, B. polvmvxa produced about 3.6g of levan (fructan) i n 100 ml of 15Z sucrose i n 10 days (about 46Z y i e l d on available fructose, where 7.89 g fructose are available from 15 g sucrose).


Composition and Properties of the Fructan 6LC analysis of the oxime derivatives of TFA-hydrolyzed fructan shows over 93Z fructose with a small amount of glucose and traces of degradation products. 6LC analysis of oxime derivatives (26), after hydrolysis of the fructan by t r i f l u o r a c e t i c acid, was performed on a Hewlett Packard chromatograph model 5880, with a fused s i l i c a c a p i l l a r y column. The i n i t i a l molecule i n fructan chain formation i s sucrose, therefore terminal glucose groups w i l l be present i n fructan. Some free glucose may have been adsorbed on the crude sample analyzed, because e s s e n t i a l l y no glucose was observed on methylation analysis. At the molecular weight observed (see below), there i s a very small percentage of glucose units as terminal groups. X-ray c r y s t a l structure analysis showed no c r y s t a l l i n i t y . Fructan i s amorphous. X-ray analysis was performed on a General E l e c t r i c X-ray d i f f r a c t i o n refractometer. A 5Z aqueous solution of crude fructan, a f t e r d i a l y s i s through a membrane with 12,000 daltons cut-off, gave a single, sharp clean peak just below 2 χ 10 daltons on Sephacryl S-500. The compound i s stable i n aqueous solution at pH 4.5 f o r up to 36 hours, when monitored by HPLC a n a l y s i s . Fructan i s readily hydrolyzed by 0.5Z oxalic acid (19). I t i s not decomposed by amylase enzymes. The fructan has an o p t i c a l rotation [ a ] * - 47.2. I t i s non-hygroscopic, unusual i n view of i t s high s o l u b i l i t y . Lyophilized sheets of fructan have been maintained under condition of 25°-30°C and 70Z-85Z r e l a t i v e humidity f o r up to 6 months. The s o l u b i l i t y of fructan i s very high: up to 30Z i n cold water, with no apparent v i s c o s i t y increase. I t i s extremely soluble i n hot water. This high s o l u b i l i t y i s c h a r a c t e r i s t i c of β(2->6) linked fructans. e

3

Structure. The " C nmr spectra, shown i n Figure 2, indicates that e s s e n t i a l l y a l l fructose molecules i n the polymers are i n the same conformation. In Table I, nmr peaks from fructan are compared to peaks from known i n u l i n (β-(1-*2) linked) and b a c t e r i a l levan (β-(2-*6) l i n k e d ) . Data c l e a r l y show the fructan to be of the β-(2->6) type ( 27 ). (See Table II.) Nmr C spectroscopy was performed at 100 MHz with a JEOL GX-400 instrument, at 70°C, with internal standard 1,4-dioxane (567.40). X 3


Τ


uvpruj ptqoMnjq aXHVU ONV NVH *8I ς 7


216

AGRICULTURAL AND Table I.

1


I n u l i n (1-2) Levan (2-6) Fructan

62.2 61.A 61.A

Assignment of nmr

2 10A.5 105.1 105.0

SYNTHETIC POLYMERS

peaks

3

A

5

6

78.5 77.5 77.8

76.6 76.6 76.A

87.A 81.3 81.1

63.A 6A.6 6A.2

Methylation analysis was run by the method of Hakomori (28), followed by hydrolysis with t r i f l u o r a c e t i c acid, 8odium borohydride reduction, and a c e t y l a t i o n . 6LC was performed on a Hewlett-Packard 5970, used as an i n l e t for a mass spectrometer. Molecular weight was determined on a Sephacryl S-500 column (2.6 ζ 70 cm), using deionized water as solvent, upward flow, 2.75 ml/min, and detection by r e f r a c t i v e index monitor, Model R-A01 (Waters Associates). Table I I .

Linkages indicated by methylation analysis

β-(2->6) linked fructose

71Z

Branch points (at 1, 2, 6)

12Z

Terminal groups (1 or 2 position)

13Z

Undissolved material

AZ

Branch points are indicated by the presence of 3,A dimethyl substituted fructose, and the degree of branching of 12Z i s supported by the observation of 13Z terminal groups, indicated by tetramethylated fructose residues, substituted at the 1- or 2-positions. The branches are formed by β- (l-*2) linkages with side-chains of β-(2-»6) linked residues. The degree of branching i n fructans has been shown to range from 5-20Z (28). The free hexose probably results from material that was not dissolved during methylation. (See Figure3.) Summary Fructans (levans) are natural polymers of fructose, found i n many plants and microbial products. Like dextrans, they can be formed as an undesirable microbial byproduct i n the processing of sugar juice and have deleterious e f f e c t s on processing. On the other hand, fructans, which



18. HAN AND CLARKE

217

Microbial Fructan

UJL

a.

10

40

"25"

Time (min.) Figure 3a. GLC of alditol acetate derivatives of the hydrolyzed methylated fructan.

TIC of DATA:P-LK-42.0

f V S

χ φ

»

I

ci

φ ô8

I

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Si β

κχ

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Τ

τ

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~"ϊ

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Time (min.)

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Figure 3b. Identification of alditol acetate peaks (expanded section of 3a).


AGRICULTURAL AND SYNTHETIC POLYMERS

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can be produced only from sucrose, have potential i n d u s t r i a l applications as thickeners and encapsulating agents and could provide products of added value from sugar crops. In this study, a fructan-producing bacterium was i s o l a t e d from s o i l and i t s c h a r a c t e r i s t i c s for polysaccharide synthesis and properties of the product were studied. polvmvxa (NRRL B-18475) produced polysaccharide i n high y i e l d when grown on sucrose solution. Hydrolysis and subsequent analyses showed the product to consist e n t i r e l y of D-fructose. C-nmr and methylation analyses indicated the product to be β-(2-»6) linked polymer of fructose, with 122 branching. The polysaccharide has molecular weight of 2 m i l l i o n daltons and i s r e a d i l y soluble i n water, although not hygroscopic. Downloaded by RUTGERS UNIV on May 30, 2018 | https://pubs.acs.org Publication Date: July 13, 1990 | doi: 10.1021/bk-1990-0433.ch018

ia

Acknowledgment The authors acknowledge L. Ban-Koffi and M. Watson f o r t h e i r technical assistance. The authors thank M. A. Godshall f o r 6LC and 6PC analysis; W.S.C. Tsang f o r HPLC data; A.D. French for x-ray c r y s t a l structure analysis; L. Kenne for nmr analysis, and B. Lindberg for methylation analysis and h e l p f u l advice.

Literature Cited 1. 2. 3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Meade, G.P. and Chen, J.P.C. Cane Sugar Handbook, Wiley Interscience: New York, 1985, 11th Edition. Avigad, G., and Feingold, D.S. Biochem. Biophys. 1965, 70, 178. Pontis, H.G., and Del Campillo, E. In Biochemistry of storage carbohydrates in green plants; Dey, P.M. and Dixon, R.A., Eds.; Academic Press: New York, 1985; Chapter 5, pp. 205-227. Vandamme, E.J. and D.G. Derycke. Adv. in Appl. Microbiol. 1983, 29, 139-176. Higuchi, Μ., Iwami, Y., Yamada, T. and Araya, S. Arch. Oral Biol. 1970, 15(6), 565-567. Manly, R.S. and Richardson, D.J. J . Dent. Res., 1968, 47, 1080-1086. Newbrun, E. J . Dent. Child, 1969, 14, 239-248. Fuche, Α., DeBruijn, J.M. and Niedeveld, C.J. Antonie Van Leeuwenhoek. 1985, 51, 333-351. Loewenburg, J.R. and Reese, E.T. Can. J . Microbiol. 1957, 3, 643. Dedonder, R. Meth. in Enzymol. 1966, 8, 500-505. Tanaka, T., Yamamoto, S., Oi, S. and Yamamoto, T. J . Biochem, 1981, 90, 521-526. Hestrin, S., Avineri-Shapiro, D. and Aschner, M. Biochem. J., 1943, 37, 450-456. Mantsala, P. and Puntala, M. FEMS Microbial Lett., 1982, 13, 395-399.



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Microbial Fructan

14. Perlot, P. and Monson, P. Enz. Eng. 7th Int'l. Conf. Annal. 1984, New York Academic Science, 434, 468-471. 15. Yamamoto, S., Iizuka, M., Tanaka, T. and Yamamoto, T. Agric. Biol. Chem. 1985, 49, 343-350. 16. Fuchs, A. Doctoral thesis, Rijksuniversiteitte Leiden, Waltman Ed., Delft, 1959. 17. Robeiro, J.C.C. Guimarues, Borges, W.V., Silv, A.C., D.O. and Crug, C.D. Rev. Microbiol. 1988, 19(2), 196-201. 18. Lyness, E.W. and Doelle, H.W. Biotechnol. Lett. 1983, 5(5), 345-350. 19. Evans, T.H. and Hibbert, H. Adv. Carbohydr. Chem. 1946, 2, 253-277. 20. Feingold, D.S. and Gehatia, M. J . Polymer Sci. 1957, 23, 783-790. 21. Takeshita, M. J . Bacteriol. 1973, 116, 503-506. 22. Lippman, E.O. Chem. Ber., 1881, 14, 1509. 23. Han, Y.W. J . Indus. Microbiol. 1989, In press. 24. Tkachenco, A.A. and Loitsyankaya, M.S. Appl. Biochem. Microbiol. 1979, 14(4), 502-505. 25. Dedonder, R. and Peaud-Lenoel, C. Bull Soc. Chim. Biol. 1957, 39, 483. 26. Schaffler, K.J. and Morel du Boil, P.G. J . Chromatog. 1981, 207, 221-229. 27. Barrow, K.D., Collins, J.G., Rogers, P.L. and Smith, G.M.C. Eur. Jour. Biochem. 1984, 145, 173-179. 28. Lindberg, B., Loungren, J . and Thompson, J.L. Acta Chem. Scand. 1973, 27, 1819-1821. RECEIVED

December 29, 1989


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Microbial Fructan - ACS Symposium Series (ACS Publications)

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