30. 1995 - the structure of the exopolysaccharide produced by lac to bacillus helve tic us 766

8/3/2019 30. 1995 - The Structure of the Exopolysaccharide Produced by Lac to Bacillus Helve Tic Us 766

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C A R BOH YD R A T ER E S E A R C HELSEVIER Carbohydrate Research 276 (1995) 137-154

The structure of the exopolysaccharide produced byLactobaci l lus he lve t i cus 76 6

G e r a r d W . R o b i j n a , J e r r y R . T h o m a s a , H a n H a a s h,D i c k J . C . v a n d e n B e r g b, J o h a n n i s P . K a m e r l i n g a ,* ,J o h a n n e s F . G . V l i e g e n t h a r t a

a Bijcoet Center, Department of BiD-Organic Chemistry, Utrecht University, P.O. Box 80.075, NL-3508 TBUtrecht, The Netherlands

h UnileverResearch Laboratory Vlaardingen, P.O. Box 114, NL-313OAC Vlaardingen, The NetherlandsReceived 19 January 1995; accepted 29 March 1995

AbstractThe exopolysaccharide produced by Lactobacillus helveticus 766 in skimmed milk was found

to be composed of D-glucose and D-galactose in a molar ratio of 2:1. Linkage analysis and 1D/2DNMR studies ( tH and 13C) performed on the native polysaccharide, and on oligosaccharidesobtained from a partial acid hydrolysate, showed the polysaccharide to consist of hexasacchariderepeating units with the following structure:

fl-D-GalfI

3-~ 3)-fl-D-Glcp-(1 ~ 4)-/3-D-Glcp-(1 -~ 6)-a-D-Glcp-(1 ~ 6)-c~-D-Galp-(1 ~ 6)-c~-o-Glcp-(1 -~

Keywords." Lactic acid bacteria; Lactobacillus helveticus; Exopolysaccharide structure

1 . I n t r o d u c t i o n

In the food industry, bacterial exopolysaccharides (EPSs) are extensively used asthicken ing, gelling, and sta biliz ing agents [1]. Recently, a growin g interest has devel-oped in polysaccharides from lactic acid bacteria. Because they are produced by Food

* Corresponding author.0008-6215/95/$09.50 1995 Elsevier Science Ltd. All rights reservedSSDI 0008-62 I 5(95)00171-9


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138 G. W. Robijn et al. / Carbohydrate Research 276 (1995) 137-154

G r a d e m i c r o o r g a n i s m s , t h e s e p o l y s a c c h a r i d e s m a y f o r m a n e w g e n e r a t i o n o f f o o dt h i ckene r s . I n o r de r t o ga i n a be t t e r i n s igh t i n t o t he r e l a t ions h i p be t w een t he s t r uc tu r e s o fpo l ys accha r i de s f r om l ac t i c a c i d bac t e r i a and t he i r r heo l og i ca l p r ope r t i e s , knowl edge o ft he i r p r i m a r y s t r uc t u r e s i s a p r e r equ i s i te .

I n t h i s con t ex t , s t r uc t u r a l s t ud i e s have s o f a r been ca r r i ed ou t on EPSs p r oduced byStreptococcus thermophilus [2], Lactococcus lactis subsp , cremoris SBT 0495 [ 3 ] ,Lactococcus lactis s ubs p , cremoris H414 [ 4 ] , Lactobacillus delbriickii subsp , bulgari-cus rr [5], Lactobacillus helueticus TY1- 2 [ 6 ] , and Lactobacillus sake 0-1 [7] . Here wer e p o r t t h e s t r u c tu r e d e t e r m i n a t i o n o f t h e e x o p o l y s a c c h a r i d e p r o d u c e d b y Lactobacillushelveticus 7 6 6 i n s k i m m e d m i l k .

2 . E x p e r i m e n t a lProduction, isolation, and purification of the exopolysaccharide.--Lactobacillus

helveticus 766 w as g r o wn i n s k i m m ed m i l k f o r 2 days a t 30C ( non - s haken , non - ae r a t ed ) ;t r ic h l o r o a c e t ic ac i d w a s t h e n a d d e d ( f i n a l c o n c e n t r a ti o n 4 % w / v ) , a n d t h e c u lt u re w a ss t i r r ed f o r 2 h . Ce l l s and p r ec i p i t a t ed p r o t e i n s we r e r em oved by cen t r i f uga t i on ( 27 000g , 30 m i n , 4 C) . The s upe r na t an t was co l l e c t ed and t he EPS was p r ec i p i t a t ed wi t h t wov o l u m e s o f c o l d E t O H . A n a q u e o u s s o l u ti o n o f t h e p re c i p it a te d m a t e ri a l w a s e x t e n s i v e l yd i a l y s ed aga i ns t t wi ce - d i s t i l led wa t e r . A f t e r r em ova l o f i n s o l ub l e m a t e r i a l by cen t r i f uga -t i on , t wo vo l um es o f E t OH wer e aga i n added . The r e s u l t i ng p r ec i p i t a t e was d i s s o l ved i nwa t e r and s ubs equen t l y s ub j ec t ed t o a f r a c t i ona l p r ec i p i t a t i on a t 30% , 40% , 50% , 60%and 70% ace t one , wi t h i n t e r m ed i a t e c en t r i f uga t i on . The p r ec i p i t a t ed m a t e r i a l co l l e c t edf r om t he 40% ace t o ne f r ac t ion was f u r t he r pu r i f i ed on a Se phac r y l S - 500 ge l f i lt r a ti onc o l u m n ( 1 5 0 X 2 . 2 c m , P h a r m a c i a ) , e l u t e d w i th 5 0 m M N H 4 H C O 3 , u s in g r e f r a c t iv ei n d e x m o n i t o r in g ( B i s c h o f f 8 1 0 0 R I d e t e c to r ) . A s m a l l a m o u n t o f t h e p u ri f ie d p o l y s a c -cha r i de ( 250 / xg ) was f r ac t i ona t ed on a Sup e r os e - 6 ge l f i lt r a ti on co l um n ( 3 0 1 cm ,P h a r m a c i a ) , e l u t i n g w i t h 5 0 m M N H a H C O 3 , u s i n g a P h a r m a c i a F P L C s y s t e m w i t hr e f r a c t i v e i n d e x a n d U V m o n i t o r i n g ( P h a r m a c i a , s i n g l e p a t h m o n i t o r U V - 1 / 2 8 0 n m ) .

Protein assay.--Protein c o n t e n t s w e r e d e t e r m i n e d w i t h t h e P i e r c e P r o t e i n A s s a yReagen t , u s i ng bov i ne s e r um a l bum i n a s a s t anda r d .Gas-liquid chromatography and mass spectrometry.--GLC a n a l y se s w e r e p e r fo r m e dw i t h a V a r i a n 3 7 0 0 g a s c h r o m a t o g r a p h ( te m p e r a t u r e p r o g r a m , 1 3 0 - 2 2 0 C a t 4 C / m i n ) ,u s i ng an S E- 30 f u s ed - s i l i c a c ap i ll a r y co l um n ( 25 m X 0 .32 m m , P i e r ce ) o r a CPSi l 43

W C O T f u s e d - s il i c a c ap i l la r y c o l u m n ( 2 5 m X 0 . 3 2 m m , C h r o m p a c k ) . D a t a w e r e c o l -l e c t ed and p r oces s ed wi t h a Sh i m adzu i n t eg r a t o r .P a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s w e r e a n a l y z e d b y G L C - M S w i t h a nM D 8 0 0 / 8 0 6 0 s y s t e m ( F i s o n s i n s t r u m e n t s ; e l e c t r o n e n e r g y , 7 0 e V ) e q u i p p e d w i t h a

D B - 1 f u s e d - s il ic a c a p il l a ry c o l u m n ( 3 0 m X 0 . 3 2 m m , J & W S c i en t if ic ) . S a m p l e s w e r ei n j ec t ed u s i ng a s p li t i n j ec t o r ( s p l it fl ow 1 / 1 0 ) , and a tem pe r a t u r e p r og r a m o f 1 40 - 2 20 Ca t 4 C / m i n w a s u s e d .

P o s i t iv e - i o n m o d e F A B m a s s s p e c t r a w e r e r e c o r d e d us i n g M S 1 o f a J E O L J M S -S X / S X 1 0 2 A t a n d e m m a s s s p e c tr o m e t e r ( a cc e le r at in g v o lt ag e , 6 k V ; X e b e a m ) . T h e


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G.W. Rob ijn et al. / Carbohydrate Research 276 (1995) 137-15 4 139m e t h y l a t e d o l i g o s a c c h a r i d e s a m p l e s w e r e m e a s u r e d o v e r a m a s s ra n g e o f m / z 1 0 - 1 5 0 0i n a m a t r i x o f m - n i t r obenz y l a l coho l .

M o n o s a c c h a ri d e a n a l y s i s . -- O l i g o / p o l y s a c c h a r i d e s wer e s ub j ec t ed t o m e t hano l ys i s( m e t ha no l i c 1 M HC1 , 18 h , 85 C) , and t he re s u l t ing m i x t u r e s o f m e t hy l g l ycos i de s we r et r im e t h y l s i | y l a te d ( 1 :1 : 5 h e x a m e t h y l d i s i l a z a n e - c h l o r o t r i m e t h y l s i l a n e - p y r i d i n e ) , an d t h e nquan t i t a t i ve l y ana l yzed by GLC on SE- 30 [ 8 ,9 ] . I n add i t i on , t he abs o l u t e con f i gu r a t i onso f t h e m o n o s a c c h a r i d e s w e r e d e t e r m i n e d b y G L C a n a l y s i s o f t h e t r i m e t h y l s i l y l a t e d( - ) - 2 - b u t y l g l y c o s i d e s [1 0] o n S E - 3 0 .

M e t h y l a t i o n a n a l y s i s . - - S a m p l e s ( na t i ve EPS , pa r t i a l l y hyd r o l ys ed EPS , o r o l i gos ac -cha r i de - a l d i t o l s ) we r e pe r m e t hy l a t ed a s de s c r i bed p r ev i ous l y [ 11 ] . Af t e r hyd r o l ys i s wi t h2 M CF 3CO 2H ( 2 h , 120 C), t he pa r ti a l ly m e t hy l a t ed m o nos acc ha r i de s we r e r educedw i t h N a B D 4. W or k - up , com pr i s i ng neu t r a l i z a t ion and r em o va l o f bo r i c a c id by co -e v a p o r a t i o n w i th M e O H , f o l l o w e d b y a c e t y l a t io n w i t h A c 2 0 ( 3 h, 1 20 C ) y i e l d edm i x t u r e s o f pa r t i a l l y m e t hy l a t ed a l d i t o l a ce t a t e s , wh i ch we r e ana l ys ed by GLC onC P S i I 4 3 a n d G L C - M S o n D B - 1 [ 8 , 1 2 ] .

P a r t ia l a c i d h y d r o l y s i s . - -P o l y s a c c h a r i d e ( 5 0 m g ) w a s h y d r o l y s e d in 0 . 3 M C F 3 C O 2H( 50 m L) f o r 2 h a t 100 C. Then , t he s o l u t i on was l yoph i l i z ed , and t he r e s i duef r ac t i ona t ed on a B i o - G e l P - 2 ( 20 0 - 40 0 m es h ) ge l f i lt r a ti on co l um n ( 90 X 1 .5 cm ) ,c l u t ed wi t h t wi ce - d i s t i l l ed wa t e r , u s i ng r e f r ac t i ve i ndex m on i t o r i ng . Sub f r ac t i ona t i on o fB i o - G e l P - 2 f r a c t i o n s w a s p e r f o r m e d b y h i g h - p H a n i o n - e x c h a n g e c h r o m a t o g r a p h y w i t hp u l s e d a m p e r o m e t r i c d e t e c t i o n ( H P A E C - P A D ) o n a D i o n e x L C s y s t e m , e q u i p p e d w i t h aC a r b o P a c P A - 1 p e l li c u la r a n i o n - e x c h a n g e c o l u m n ( 2 5 c m X 9 m m ) . T h e c o l u m n w a se l ut e d w i th a g r a di e nt o f N a O A c in 0 .1 M N a O H ( 2 0 - 2 5 0 m M N a O A c a t 5 m M / m i n )a t a f l o w r at e o f 4 m L / m i n . P A D - d e t e c t i o n w a s c a rr i ed o u t w i th a g o l d w o r k i n ge l ec t r ode and t ri p l e - pu l s e am pe r o m e t r y ( pu l s e po t en ti a l s and du r a t ions : E j 0 .05 V, 480m s ; E 2 0 . 6 0 V , 1 20 m s ; E 3 - 0 . 6 0 V , 6 0 m s ) w a s u s e d. D a t a w e r e c o l le c t e d a n dpr oces s ed wi t h a Sh i m adzu C- R3A i n t eg r a t o r . I m m ed i a t e l y a f t e r co l l e c t i on , t he f r ac t i onswer e neu t r a l i z ed wi t h 2 M AcOH, f o l l owed by de s a l t i ng on a c a t i on - exchange r e s i n( D o w e x A G 5 0 W - X I 2 , 1 0 0 - 2 0 0 m e s h , H + - f o r m , B i o - R a d ) , a n d ly o p h i li z a ti o n . A l lH P A E C - f r a c t i o n s w e r e r e d u c e d w i t h N a B D 4 in 1 M N H 4O H p r i o r to ana l ysi s . In t heca s e s o f o l i gos accha r i de - a l d i t o l m i x t u r e s , a f u r t he r f r a c t i ona t i on was ca r r i ed ou t onCar boPac PA- 1 us i ng t he s am e cond i t i ons a s de s c r i bed above .

N M R s p e c t ro s c o p y . -- P r o t o n -d e c o u p l e d 7 5 . 4 6 9 - M H z 13C N M R s p e c tr a w e r e re c o r d e df o r s o l u ti o n s in D 2 0 o n a B r u k e r A C - 3 0 0 s p e c t r o m e t e r ( D e p a r t m e n t o f O r g a n icC h e m i s t r y ) , e q u i p p e d w i t h a 5 - m m b r o a d - b a n d p r o b e , a t a p r o b e t e m p e r a t u r e o f 2 7 C .Chem i ca l s h i f t s a r e exp r e s s ed i n ppm downf i e l d f r om t he s i gna l f o r ex t e r na l t e t r am e t hy l -s il an e , b u t w e r e a c t u a ll y m e a s u r e d b y r e f e r e n c e to e x t e rn a l M e O H ( 6 4 9 . 0 0 ). T h e d a t awer e co l l e c t ed i n 16K co m pl ex da t a s e t s and ze r o - f i l l ed t o 64K. Af t e r Fou r i e r tr ans f o r -m a t i on , u s i ng an exponen t i a l m u l t i p l i c a t i on , t he s pec t r a we r e ba s e l i ne co r r ec t ed wi t h af o u r t h - o rd e r p o l y n o m i a l f u n c t i o n .I D / 2 D N M R s p e ct ra w e r e r e co r d e d o n a B r u k e r A M X - 5 0 0 o r a B r u k e r A M X - 6 0 0s p e c t r o m e t e r ( B i j v o e t C e n t e r, D e p a r t m e n t o f N M R S p e c t r o s c o p y ) at a p r o b e t e m p e r a t u r eo f 2 7 C . P r i o r t o a n a l y s i s , s a m p l e s w e r e e x c h a n g e d t w i c e i n D z O ( 9 9 . 9 a t o m % D ,I s o t ec ) wi t h i n t e r m ed i a t e l yoph i l i z a t i on , and t hen d i s s o l ved i n 99 .96 a t om % D20( I s o t ec ). Ch em i ca l s h i f t s a r e exp r e s s ed i n ppm by r e f e r enc e t o i n te r na l a ce t one ( 6


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140 G. W. Ro bijn et al. / Carbohydrate Research 276 (1995 ) 137-1542 . 2 25 ) . R e s o l u t i o n - e n h a n c e d 1 D ~ H N M R s p e c t r a w e r e r e c o r d e d w i t h a s p e c t ra l w i d th o f5 0 0 0 H z ( a t 5 0 0 M H z ) i n 1 6 K c o m p l e x d a ta s e ts . S u p p r es s io n o f t he H O D s ig n a l w a sa c h i e v e d b y a p p l y i n g t h e W E F T p u l s e s e q u e n c e a s d e s c r i b e d [ 1 3 ] . T h e s p e c t r a w e r eb a s e l i n e c o r r e c t e d w i t h a f o u r t h - o r d e r p o l y n o m i a l f i t w h e n n e c e s s a r y .

2 D H o m o n u c l e a r H a r t m a n n - H a h n ( H O H A H A ) s p e c t r a w e r e r e c o r d e d u s i n g M L E Vm i x i n g s e q u e n c e s o f 1 0 0 m s . T h e s p i n - l o c k f i e ld s t r e n g th c o r r e s p o n d e d t o a 9 0 p u l s ew i d t h o f c a . 2 7 / z s . T h e s p e c t r a l w i d t h s w e r e b e t w e e n 2 0 0 8 a n d 2 8 0 8 H z a t 5 0 0 M H z , o r4 8 0 0 H z a t 60 0 M H z i n e a c h d i m e n s i o n . T h e H O D s ig n a l w a s p r e s a tu r a te d f o r 1 sd u r i n g t h e r e la x a t i o n d e l a y . B e t w e e n 3 2 2 a n d 5 1 2 s p e c t r a o f 2 K d a t a p o i n t s w i t h 2 - 6 4s c a n s p e r t 1 i n c r e m e n t w e r e r e c o r d e d .

2 D N u c l e a r O v e r h a u s e r e n h a n c e m e n t s p e c t r o s c o p y ( N O E S Y ) w a s p e r f o r m e d w i t h am i x i n g t i m e o f 2 0 0 m s . T h e s p e c t r a l w i d t h w a s 2 2 5 0 H z i n e a c h d i m e n s i o n . T h e H O Ds i g n al w a s p r e s a t u r a t e d f o r 1 s d u r in g t h e r e l a x a ti o n d e l a y . A t o ta l o f 5 1 2 s p e c t r a o f 2 Kd a t a p o i n t s w i t h 2 4 s c a n s p e r t ~ i n c r e m e n t w e r e r e c o r d e d .

2 D R o t a t i n g - f r a m e n u c l ea r O v e r h a u s e r e n h a n c e m e n t s p e c tr a ( R O E S Y ) w e r e r e c o r d edw i t h m i x i n g t im e s o f 2 2 5 - 2 5 0 m s . T h e s p e c t ra l w id t h s w e r e 5 50 0 H z a t 6 0 0 M H z o r2 5 1 2 - 3 4 0 1 H z a t 5 0 0 M H z i n e a c h d i m e n s i o n . T h e s p i n - l o c k fi e ld s tr e n g th c o r re -s p o n d e d t o a 9 0 p u l s e w i d t h o f c a . 1 1 0 / xs . T h e H O D s i g n a l w a s p r e s a t u r a te d f o r 1 sd u r i n g t h e re l a x a t i o n d e l a y . B e t w e e n 4 2 4 a n d 5 1 2 s p e c t r a o f 2 K d a t a p o i n t s w i th 8 - 9 6s c a n s p e r t ~ i n c r e m e n t w e r e c o l l e c t e d .

2 D D o u b l e - q u a n t u m f i l t e r e d c o r r e l a t i o n e x p e r i m e n t s ( C O S Y ) w e r e r e c o r d e d i n4 4 8 - 5 1 2 d a t a s e ts o f 2 K d a t a p o i n ts ; 4 - 3 4 s c a n s p e r t~ i n c r e m e n t w e r e c o ll e c te d . T h eH O D s i g n a l w a s s u p p r e s s e d b y p r e s a t u r a t i o n f o r 1 s d u r i n g t h e r e l a x a t i o n d e l a y . T h es p e c tr a l w i d t h s w e r e 2 0 0 8 - 2 8 0 8 H z i n e ac h d i m e n s i o n (a t 50 0 M H z ) .A p h a s e - s e n s i t i v e 1 3 C - 1 H 2 D h e t e r o n u c l e a r m u l t i p l e q u a n t u m c o h e r e n c e ( H M Q C )e x p e r i m e n t w i t h i n v e r s e d e t e c t i o n [ 1 4 ] w a s c a r d e d o u t a t a I H f r e q u e n c y o f 5 0 0 . 1 3 9M H z ( 1 2 5 . 7 6 9 M H z f o r 1 3 C ) w i t h a s p e c tr a l w i d t h o f 3 0 12 H z f o r t 2 a n d 1 4 0 0 0 H z f o rt ~ , u s i n g a 5 - m m b r o a d - b a n d p r o b e . T h e H O D s i g n a l w a s p r e s a t u r a t e d f o r 1 s a n d t h es i g n a l s o f t h e 1 2 C - b o u n d p r o t o n s w e r e s u p p r e s s e d u s i n g a T A N G O p u l s e s e q u e n c e . 13CD e c o u p l i n g w a s n o t a p p l i e d d u r i n g t h e a c q u i s i ti o n o f t h e ~ H F I D . A t o ta l o f 5 12 s p e c t r ao f 4 K d a t a p o i n t s w i t h 6 4 s c a n s p e r t I i n c r e m e n t w e r e r e c o r d e d .

A l l 2 D N M R d a t a w e r e p r o c e s s e d o n S i l i c o n G r a p h i c s I R I S w o r k s t a t i o n s ( I n d i g o ,I n d i g o 2 o r 4 D / 3 5 ) , u s i n g T R I T O N s o f t w a r e ( B i j v o et C e n t er , D e p a r t m e n t o f N M RS p e c t r o s c o p y ) . T h e t i m e d o m a i n d a t a s e t s w e r e m u l t i p l i e d w i t h a p h a s e - s h i f t e d s i n e b e l land , a f t e r Fou r i e r t r ans f o r ma t ion an d ze r o - f i l li ng , da t a s e t s o f 1024 2048 po in t s( 2 0 4 8 2 0 4 8 p o i n t s f o r th e H M Q C e x p e r i m e n t ) w e r e o b ta i n e d , w h i c h w e r e b a s e l in ec o r r e c t e d w i t h a f o u r t h - o r d e r p o l y n o m i a l f u n c t i o n w h e n n e c e s s a r y .

3 . R e s u l t s a n d d i s c u s s i o n

I s ol a ti o n , p u r if i ca t i o n , a n d c o m p o s i t i o n o f t h e p o l y s a c c h a r i d e . - - T h e c r u d e e x o -p o l y s a c c h a r i d e ( E P S ) p r o d u c e d b y L a c t o b a c i l l u s h e l v e t i c u s 7 6 6 , o b t a i n e d a s a n e t h a n o lp r e c i p i t a t e f r o m t h e T C A - t r e a t e d m e d i u m , w a s p u r i f i e d b y f r a c t i o n a t e d a c e t o n e p r e c i p i -t a t i o n a n d s u b s e q u e n t g e l f i l t r a t i o n c h r o m a t o g r a p h y o n S e p h a c r y l S - 5 0 0 . T h e p r o t e i n


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G.W. Robijn et al. / Carbohydrate Research 276 (1995) 137-154Table 1Methylation analysis data of native EPS (1), and native EPS after mild acid hydrolysis (2)

141

Derivative Molar amoun ts a1 2

2,3.5,6-Gal b 0.9 0.52,3,4,6-Gal - 0.32,3,4,6-Gic - -2,4,6-G1c 1.0 1.02,3,6-Glc - 0.72,3,4-Gic 1.8 1.82,3,4-Gal 0.9 0.92,6-G1c 1.1 0.4

2,4,6-G1c is taken as 1.0.h t,4-Di-O-acetyl-2,3,5.6-tetra-O-methyl-D-galactitol-l-d, etc.

c on te n t o f the pu r i f i e d m a te r ia l w a s l e ss tha n 1%. Add i t iona l a na lys is o f the ma te r ia l onSupe rose -6 a f fo rd e d the po lysa c c ha r ide a s a sha rp vo id -vo lume pe a k , sugge s t ing ap o l y m e r o f h i g h m o l e c u l a r m a s s .

Monosa c c ha r ide a na lys i s o f na t ive EPS (1 ) , i nc lud ing de te rmina t ion o f a bso lu tec onf igu ra t ions , r e ve a le d a c ompos i t ion o f o -G lc a nd o -Ga l in a mo la r r a t io o f 2 .0 :1 .0 .Me thy la t ion a na lys i s o f 1 (Ta b le 1 ) showe d the p re se nc e o f t e rmina l ga la c to fu ra nose ,3 - subs t itu t e d g luc o pyra nos e , 6 - subs t i tu t e d g luc opyra nose , 6 - subs t i tu t e d ga la c topyra nose ,a nd 3 ,4 -d i subs t i tu t e d g luc o pyra no se (o r 3 ,5 -d i subs t itu t e d g luc o fu ra nose ) in m o la r r a t io so f 1 :1 :2 :1 :1 . Ac c ord ing to NMR e xpe r ime n t s (v ide in f ra ) the d i subs t i tu t e d g luc osy lre s idue i s in the py ra nose r ing fo rm. To ob ta in in fo rma t ion a bou t the pos i t ion o f thea c id - la b i l e t e rmina l g a la c to fu ra no sy l g roup [15 ], a me thy la t ion a na lys i s wa s a l so c a r r ie dou t on EPS a f t e r m i ld a c id hydro lys i s (0.3 M CF 3CO z H, 7 min , 100C; 2) , re ve a l ing apar t ia l conversion of 3 ,4-d isubst i tu ted g lucose in to 4-subst i tu ted g lucose (Table 1) . Thisf ind ing de mons t ra t e s tha t in the na t ive po lysa c c ha r ide the t e rmina l ga la c to fu ra nosy lg roup i s a t t a c he d to 0 -3 o f the 3 ,4 -d i subs t i tu t e d g luc ose re s idue . The t e rmina l ga la c -topyra nose de r iva t ive obse rve d in the me thy la t ion a na lys i s o f 2 o r ig ina te s f rom hyd ro ly t -ica l ly re leased ga lac tose .The 1D IH NMR spe c t rum o f 1 (F ig . 1 ) c on ta ins s ix s igna l s in the a nome r ic r e g ion(6 5 .6 -4 .4 ) , c o r re spond ing w i th a he xa sa c c ha r ide r e pe a t ing un i t . The s ix monosa c c ha -r ide un it s we re a rb i t r a r ily l a be ll e d A- F . Ba se d on i t s downf ie ld c he m ic a l sh i ft and i t ssmal l coupl ing constant , the anomeric s igna l a t 6 5 .488 ( res idue F, 3J 1,2 < 2 Hz ) wa sa ss igne d to H-1 o f the ga la c to fu ra no sy l g roup . The c ou p l ing c ons ta n ts o f the a nom e r icsigna ls a t 6 4 .528 ( res idue A, 3J1,2 7 .7 Hz) and 6 4 .537 ( res idue B, 3J1.2 7 .8 Hz)sugge s t the p re se nc e o f two /3 -he xopyranosy l r e s idue s , whe re a s the c oup l ing c ons ta n tsof the resona nces a t 8 4 .963 ( res idue C, 3Jl. 2 3 .7 Hz) , 6 4 .991 ( res idue D, 3Jl. 2 3 .7 Hz) ,and 6 5 .295 ( res idue E , 3J1.2 3.8 H z ) indicate three o~-hexopyranosyl residues.The ID 13C NMR spe c t rum o f 1 (no t shown) i s in a g re e me n t w i th the sugge s te dhe xa sa c c ha r ide r e pe a t ing un i t , s inc e s ix s igna l s a re obse rve d in the a nome r ic r e g ion (6110-95) . Based on the i r chemica l sh i f t s , the C-1 s igna ls a t 6 98 .00 , 98 .31 , and 99 .66were a ss igne d to three c~-hexopyranosyl res idues (C, D , and E) , w hereas the C-1 s igna ls


6/12

~-o-Galf F1,1 .3---) )-[5-D-G~cp-(~4)-~j-D-G~cp-(1---~6)-~-D-G~cp~(--)6)-~t-D-Ga~p-( -)6)-cx-D-G~ cp-(---~A B C D E

FH-1

i i5.6

D H-4F H-3

E H - 1

5. 3

F H-4C H - 1D H-1 B H - 1 A H .

HO D' 5 ' . o . . . . . ; . 4P P M

FH -2

C H-6DH -5

E H-5' ' 411

2A H-2

A H-5


7/12

143

A

0 i8,

C a r b o P a c P A - 1

I

rain

, / / ,o v

Bio-Gel P- 2

iml ve

B

51 7

4 8 12min

i ]1

12 rain 1'6

W I0

+ + 2 , , . , i , . I ' +

V

U

1'2 I'6 mirl20

C 8 8a

rain

9a

12 16 rain20, I

8 12 16mln

11l l a

1'6 20 rain 24Fig. 2. (A) HPAEC-PAD (analytical run) and Bio-Gel P-2 (preparative run) elution patterns of a partial acidhydrolysate of native EPS. (B) Preparative HPAEC-PAD fractionation patterns of Bio-Gel P-2 fractions II-V.(C) Preparative HPAEC-PAD fractionation patterns of fractions 8-11, after reduction with NaBD 4.

Fraction & - - T h e m a j o r c o m p o n e n t ( 8 a ) o f r e d u c e d fr a c ti o n 8 h a d a m o n o s a c c h a r i d ec o m p o s i t i o n o f g l u c o s e , g a l a c t o s e , a n d g l u c i t o l in m o l a r r a t io s o f 2 :1 : 1 . M e t h y l a t i o na n a l y s i s o f 8 a d e m o n s t r a t e d t h e p r e s e n c e o f t e rm i n a l a n d 6 - s u b s ti t u te d g l u c o p y r a n o s e ,


8/12

CC-D-Galp-(1 - - ~ 6 ) - D - G l c - o l - l - d 3cc-D-Glcp-(l-->6)-D-Gal-oi-l-d~-D-Glcp-(1 -,6)-D-Glc-ol-l-d

I3-D-Glcp-(1---~4)-D-GIc-oI-I-d

t-D-Glcp-(1--->3)-D-Glc-ol-l-d

l~-D -Glcp -(l -->6)-~-D-G lcp-(l --~6)-~-D-Galp-(1 -~6)-D-G lc-ol-l--dB C D E-ol 8 a

13-D-Glcp-(l -~4 -~-D -Glc p-( --~6)-o~-D-Glep-( --h6)-D-Gal-ol- l-dA B C D-ol 9a

~ D - G ~ c p - ( ~ ----> 4 ) - ~ - D ~ G ~ c p - ( ~ - - ~ 6 )- ~ t ~ D - G ~ c p ~ ( ~ -- ~ '6 )-~ t-D - G a ~ p ~ ( ~ - - > 6 ) ~ D - G ~ c - ~ - ~ - dA B C D E-ol lO a

~c-D-G~p-( ~ --~3 )-~ D -G ~cp -( ~ --.~ -~ -D -G ~p -( ~.--*6)-~t-D..G~ep-( ---~6)-D~Ga~-~-~-dE A B C D- o l l l a

13-D-Gall F1$3.~-~3)-~-D-G~p-(~-.-,`4)-~3-D-G~p-(1.-~6)-(z-D-G~ep-(~--)6).cc-D-Ga~p-(1--r6).cc-D-G~p-(1~ 1A B C D E

Fig. 3. Structures of oligosaccharide-alditols 3-1 1a obtained by partial aci d hydrolysis of the nativepolysaccharide, and the structure of the rep eating unit o f n ative EPS (1).

6 - s u b s t it u t e d g a l a c t o p y r a n o s e , a n d 6 - s u b s t it u t e d g l u c i t o l - l - d i n a p p r o x i m a t e l y e q u i m o l a ram oun t s , i nd i ca t i ng a l i nea r te t r a saccha r ide - a ld i t o l . I n t he h igh - m ass r eg ion o f t hep o s i t iv e - i o n m o d e F A B m a s s s p e c t ru m o f p e r m e t h y l a t ed 8 a , a s t ro n g [ M + N a ] p s e u d o m o l e c u l a r i o n a t m / z 9 0 2 w a s d e t e c t e d , c o r r e s p o n d i n g t o a c o m p o s i t i o n o fH e x 3 H e x - o l - l - d . I n th e 1 D I H N M R s p e c t ru m o f 8 a ( F ig . 4 A ) t hr e e a n o m e r i c s ig n a lsa r e obse r ved a t 6 4 . 492 ( r e s idue B , 3 Jl . 2 7 . 9 Hz ) , 6 4 . 944 ( r e s idue C, 3J l . 2 3 .8 Hz ) , a nd6 4 . 9 7 5 ( r e s i d u e D , 3J1, z 3 . 8 Hz ) , i nd i ca t i ng t he p r e sence o f one / 3 - and twoc ~ - h e x o p y ra n o s y l r e s i d u e s . T h e c o m p l e t e p r o t o n a s s i g n m e n t s f o r re s i d u e s B , C , a n d D ,a s p r e s e n te d i n T a b l e 3 , a re b a s e d o n c r o s s -p e a k s o b s e r v e d i n th e 2 D C O S Y , H O H A H A ,a n d R O E S Y s p e c t ra o f 8 a . O w i n g t o th e r e d u ct io n w i th N a B D 4 , 8 a w a s o b t a i n e d a s am i x t u r e o f t w o i s o t o p i c d i a s t e r e o m e r s ( l a b e l e d a a n d b ) . C o n s e q u e n t l y , d i f f e r e n t


9/12

Te2

IHNMRcmicshsao

gvnpeh

ds

dadosd37oanfomapaadhoyeonvE

reda2CC

ncas(Hzae~ .~ t~

Cmp

Rd

H-ab

H-bbH-2

H3

H4

H5

H-6

H-6

3

Go1~

49(37

-

38

38

39

39

37

6Gco

36(63

37

38

38

37

39

36(24-15

4

Gco1-*

49(37

-

35(98

37

34(~94

37

38c

6Go

36

36

39

36

37

41

38a

5

Glc~~

44(79

-

33(95

35

(92

33(93

34

39(23-13

6Gco

36(64

37

38

38

37

39

41(26-10

6

Gc3~

45(79

-

33(93

35

(91

34(96

34

39(~2-15

4Gco

36(69

37

39

38

38(70

38

39

7

Gco~

51(39

-

35(10

37

(93

34(92

38

38

3Gco

36(68

38

39(55

39

(18

37(77

38

38

37 39 37c

36~

37(60

38 37(51

37(60-17

37e

36(61-17

Ix

aInpmreavohsgonenaoa~22

bTagmesoH1ohwosoocdaeeme[H-aaH-bmah

obnec

whnocmp

ceTagmesoH6aH-6mah

obnec

whno

red


10/12

146 G. W. Robi jn et al . / Carbohydrate Resea rch 276 (1995) 137 -15 4

A

5,1

B

DH-1CH-1

~-D-Glcp-(l--~6)-ct-D-Glcp-(1---~6)-~-D-Galp-(1-~6)-D-Glc-ol-l-d 8aB C D E - o lBH-6b

BH-3

CH-6a D -4 C 2 BH-2o . C iI I ~ . . . . ~ 5 i

CH-4/ / , ~ , . 2 , , . ; 0 ,PPM

~-D-G~cp-( ~---~ 3 )-~-D-G~cp-( ~--~4)-~-D-G~cp-( ~---~6)-~-D-G~cp-( ~--~6)-D-Ga~-~-1-dE A B C D-ol l l a

CH-1HOD AH-1BH.

~D-ol-5 ~ H-= H-22:3 ' / / ' ,9 , , ,5 / / ' , ' 2 PPM ,'0 ' ' ' 3~ ' ' ' 3:~ ' ' ' 3, 32F i g. 4 . 5 0 0 - M H z I H N M R s p e c t r a o f ( A ) t e t r a s a c c h a r i d e - a l d i t o l - l - d 8 a a n d ( B ) p e n t a s a c c h a r i d e - a l d i t o l - l - dl l a . recorded in D20 a t 27C.

c h e m i c a l s h i f ts m i g h t b e e x p e c t e d f o r E - o l H - 1 i n b o t h c o m p o u n d s a a n d b . S t a r t in gf r o m t h e w e l l - r e s o l v e d d o u b l e t a t 8 3 . 6 1 3 ( 3 Jl a. 2 6 .3 H z , i n t e n s i t y ~ 0 . 3 H ) , a s s i g n e d t oE - o l H - l ( a ) , E - o l H - 2 w a s d e t e c t e d in th e C O S Y s p e c t r u m , a n d f r o m E - o l H - 2 t h er e s on a n c e s o f E - o l H - l ( b ) a n d E - o l H - 3 w e r e f o un d . I n t he R O E S Y s p e c t r u m a ni n t e r r e s id u a l c o n n e c t i v i t y D H - 1 , E - o l H - 6 b i s o b s e r v e d a t 8 3 .6 8 . O n t h e E - o l H - 6 bt ra c k in th e H O H A H A s p e c t r u m , c r o s s - p e a k s w i t h E - o l H - 6 a , 5 ,4 , 3 a r e o b s e r v e d , t hu sc o m p l e t i n g t h e a s s i g n m e n t o f re s i d u e E - o l . I n a d d i t i o n t o t h e R O E c r o s s - p e ak DH - 1 , E - o l H - 6 b , w h i c h c o n f i r m s t h e D ( 1 --* 6 ) E - o l s e q u e n c e , i n t e r re s id u a l R O E c o n n e c -


11/12

14 7

T a b l e 3t H N M R c h e m i c a l s h i ft s a o f n a t i v e E P S ( 1 ), a n d o l i g o s a c c h a r i d e - a l d i t o l s - l - d 8 a , 9 a , 1 0 a , a n d l l a , o b t a i n e df r o m a p a rt i al a c i d h y d r o l y s a t e o f 1 , r e c o r d e d a t 2 7 C . C o u p l i n g c o n s t a n t s ( H z ) a r e g i v e n i n p a r e n t h e s e sR e s i d u e 1 8 a 9 a 1 0 a l l aA H - I 4 . 5 2 8 ( 7 . 7 ) - 4 . 5 0 9 ( 7 . 7 ) 4 . 5 1 0 ( 7 . 9 )

H - 2 3 . 4 8 2 - 3 . 3 1 3 ( 9 . 2 ) 3 . 3 1 4 ( 9 . 2 )H - 3 3 . 6 2 5 - 3 . 5 0 7 ( 9 . 3 ) 3 . 5 0 9 ( 9 . 3 )H - 4 3 . 6 2 5 - 3 . 4 1 6 ( 9 . 5 ) 3 . 4 1 6 ( 9 . 4 )H - 5 3 . 4 2 4 3 . 4 8 8 3 . 5 0H - 6 a b 3 . 9 2 3 . 9 1 5 3 . 9 1 5H - 6 b ' 3 . 7 3 3 . 7 3 3 . 7 3

B H - 1 4 . 5 3 7 ( 7 . 8 ) 4 . 4 9 2 ( 7 . 9 ) 4 . 5 2 4 ( 7 . 7 ) 4 . 5 1 8 ( 8 . 0 )H - 2 3 . 5 0 2 3 . 3 2 0 ( 9 . 3 ) 3 . 3 6 4 ( 8 . 8 ) 3 . 3 6 7 ( 8 . 6 )H - 3 3 . 8 6 7 3 . 5 0 0 ( 9 . 2 ) 3 . 6 5 3 . 6 5H - 4 3 . 8 0 9 3 . 3 9 3 ( 9 . 7 ) 3 . 6 5 3 . 6 5H - 5 3 . 61 3 . 4 5 6 3 . 5 9 3 . 5 9H - 6 a 4 . 0 2 3 . 9 2 ( 2 . 3 , - 1 2 . 0 ) 3 . 9 8 5 ( - 1 2 . 3 ) 3 . 9 8 8 ( 2 . 1 , - 1 2 . 4 )H - 6 b 3 . 8 7 3 . 7 3 1 ( 5 . 9 ) 3 . 8 21 ( 5 . 1 ) 3 . 8 2

C H - 1 4 . 9 6 3 ( 3 . 7 ) 4 . 9 4 4 ( 3 . 8 ) 4 . 9 5 5 ( 3 . 7 ) 4 . 9 4 3 ( 3 . 7 )H - 2 3 . 5 6 4 3 . 5 6 3 ( 9 . 8 ) 3 . 5 7 4 ( 9 . 8 ) 3 . 5 6 9 ( 9 . 5 )H - 3 3 . 7 1 7 3 . 7 1 5 ( 9 . 3 ) 3 . 7 4 ( 9 . 4 ) 3 . 7 2H - 4 3 . 5 5 3 . 5 4 4 ( 9 . 3 ) 3 . 5 3 2 ( 9 . 4 ) 3 . 5 4H - 5 3 . 8 8 3 . 8 9 3 . 8 8 3 . 8 9H - 6 a c 4 . 1 7 4 . 1 5 2 4 . 1 7 4 . 1 6H - 6 b c 3 . 9 0 3 . 9 1 3 . 9 1 3 . 91

D H - 1 4 . 9 9 1 ( 3 . 7 ) 4 . 9 7 5 ( 3 . 8 ) - 4 . 9 7 5 ( 3 . 8 )H - 2 3 . 8 2 3 . 8 2 5 - 3 . 8 2 5H - 3 3 . 9 0 3 . 9 1 ( 3 . 7 ) - 3 . 9 1 ( 3 . 4 )H - 4 4 . 0 2 5 4 . 0 2 9 - 4 . 0 2 5H - 5 4 . 1 6 4 . 1 7 2 - 4 . 1 8H - 6 a d 3 . 8 9 3 . 8 8 - - 3 . 8 8H - 6 b o 3 . 7 3 3 . 7 2 3 . 7 2

E H - I 5 . 2 9 5 ( 3 . 8 ) -H - 2 3 . 5 8 - - -H - 3 3 . 7 4 - -H - 4 3 . 5 1 5H - 5 4 . 2 0 - - -H - 6 a e 4 . 0 1 -H - 6 b ~ 3 . 7 1 - - -

F H - I 5 . 4 8 8 ( < 2 ) - - -H - 2 4 . 1 9 2 ( 2 . 6 ) - - -H - 3 4 . 0 3 ( 5 . 3 ) - - -H - 4 4 . 3 2 0 ( 5 . 1 ) - - -H - 5 3 . 8 3 - - -H - 6 a 3 . 7 3 ( ~ 5 , ~ 1 1 ) - - -H - 6 b 3 . 6 7 ( ~ 7 ) - - -

D - o l H - 1 a ) - - 3 . 6 7 5 -H - l ( b ) - - 3 . 6 7 5 -H - 2 - - 3 . 9 7 0 -H - 3 - - 3 . 6 8 8 -H - 4 - - 3 . 7 4 0 -H - 5 - - 4 . 1 5 0 -H - 6 a r _ _ 3 . 8 6 0 -H - 6 b t _ _ 3 . 6 5 0 -

4 . 5 3 4 ( 8 . 0 )3 . 4 1 4 ( 9 . 1 )3 . 6 5 ( ~ 9 . 4 )3 . 5 1 1 ( ~ 9 . 4 )3 , 6 33 . 9 13 , 7 34 . 5 2 5 ( 8 . 0 )3 . 3 6 6 ( 9 . 4 )3 . 6 53 . 6 53 . 5 93 . 9 8 53 . 8 24 . 9 5 6 ( 3 . 7 )3 . 5 7 4 ( ~ 9 . 8 )3 . 7 4 ( 9 . 4 )3 . 5 3 2 ( 9 . 4 )3 . 8 84 . 1 73 . 9 1

5 . 3 2 9 ( 3 . 9 )3 . 5 8 8 ( 9 . 8 )3 . 7 4 ( 9 . 6 )3 . 4 7 0 ( 9 . 7 )4 . 0 03 . 8 03 . 8 0

3 . 6 7 53 . 6 7 53 . 9 7 53 . 6 9 03 . 7 4 04 . 1 5 03 . 8 6 53 . 6 5 0


12/12

148 G.W. Robijn et al. / Carbohydrate Research 276 (1995) 137-154Table 3 (continued)Residue 1 8a 9a lO a I l aE-ol H- l(a ) - 3.613 h (6.3) - 3.61J -H -l( b) - 3.71 h - 3.715 i _

H-2 - 3.84 - 3.84 -H-3 - 3.87 - 3.87 -H-4 - 3.725 - 3.73 -H-5 - 3.93 - 3.92 -H-6 a ~ - 3.91 - 3.91 -H-6b g - 3.68 - 3.68 -a In ppm relative to the signal of internal acetone at 8 2.225.b-g The assignments of H-6a and H-6b m ay ha ve to be interchanged within one residue.h.i The assignments of H -I of the two isotopic diastereomers [H-l(a) and H -l(b)] m ay hav e to be interchangedwithin one compound.

t i v i t ie s B H - 1 , C H - 6 b a n d C H - 1 , D H - 6 b a r e o b s e r v e d , s u p p o r t i n g th e B ( 1 ~ 6 ) C ( 1 --*6 ) D s e q u e n c e , a n d t h u s p r o v i n g t h e s t ru c t u r e o f 8 a a s p r e s e n t e d i n F i g . 3 .

Fraction 9 . - - 1 D I H N M R a n a l y s i s o f r e d u c e d f r a c ti o n 9 s u g g e s t e d t h at i t w a sc o m p o s e d o f a m i x t u r e o f o l i g o s a c c h a r i d e - a l d i to l s . I n t h e h i g h - m a s s r e g i o n o f t h ep o s i t i v e - i o n m o d e F A B m a s s s p e c t r u m o f r e d u c e d a n d p e r m e t h y l a t e d f ra c t io n 9 tw os t ro n g [ M + N a ] + p s e u d o m o l e c u l a r i o n s a t m / z 9 0 2 ( r e l a t i v e a b u n d a n c e 1 0 0) a n dm / z 1 1 06 ( r e l a t iv e a b u n d a n c e 2 0 ) w e r e o b s e r v e d , c o r r e s p o n d i n g t o c o m p o s i t i o n s o fH e x 3 H e x - o l - l - d a n d H e x 4 H e x - o l - l - d , re s p e c ti v e ly . T h e 1 D t H N M R s p e c tr u m o f t hem i n o r c o m p o n e n t , o b t a i n e d a f t e r su b f r a c t io n a t i o n o f r e d u c e d f r a c t io n 9 o n C a r b o P a cP A - 1 ( F i g . 2 C ), w a s i d e n t i c a l to t h e s p e c t r u m o f p e n t a s a c c h a r i d e - a l d i t o l - l - d 1 0 a ( v i d ei n fr a ). T h e m a j o r c o m p o n e n t , 9 a , h a d a m o n o s a c c h a r i d e c o m p o s i t i o n o f g l u c o s e a n dg a l a c t i t o l i n a m o l a r r a t i o o f 3 : I . M e t h y l a t i o n a n a l y s i s r e v e a l e d t h e p r e s e n c e o f t e r m i n a l ,4 - s u b s ti t u te d , a n d 6 - s u b s t i tu t e d g l u c o p y r a n o s e , a n d 6 - s u b s ti t u te d g a l a c t i t o l - l - d i ne q u i m o l a r a m o u n t s , s u g g e s t i n g a l i n e a r t e t r a s a c c h a r i d e - a l d i t o l . I n t h e 1 D I H N M Rs p e c t r u m o f 9 a ( n o t s h o w n ) t h r e e a n o m e r i c s i g n a l s a r e p r e s e n t a t 8 4 . 5 0 9 ( r e s i d u e A ,3 J1 . 2 7 . 7 H z ) , 8 4 . 5 2 4 ( r e s i d u e B , 3 j 1.2 7 . 7 H z ) , an d 8 4 . 9 5 5 ( r e s i d u e C , 3 j 1.2 3.7 Hz),r e f l e c t i n g tw o /3 - a n d o n e c ~ - h e x o p y r a n o s y l r e s i d u e s . A l l n o n - a n o m e r i c r e s o n a n c e s f o rt h e r e s i d u e s A , B , a n d C w e r e f o u n d s t a r t i n g f r o m t h e a n o m e r i c s i g n a l s i n t h e 2 D C O S Ya n d H O H A H A s p e c t r a (T a b l e 3 ). A c h a r a c t e r is t ic m u l t i p l e t a t 6 4 . 1 5 0 ( d d d ) , a s s i g n e d toD - o l H - 5 [ 17 ], w a s u s e d a s s t a r t in g p o i n t f o r th e a s s i g n m e n t o f t h e r e s o n a n c e s o f r e s i d u eD - o l . O n t h e D - o l H - 5 H O H A H A t ra c k , c r o s s - p e a k s w it h D - o l H - 6 a , 6 b , 4 w e r e f o u nd ,a n d c o n n e c t i v i t i e s i n t h e H O H A H A a n d C O S Y s p e c t ra a l l o w e d t h e i d e n t if i c a ti o n o fD - o l H - 3 , 2 ,1 . T h e i n t e r r e s i d u a l c o n n e c t i v i t y C H - 1 , D - o l H - 6 b i n t h e R O E S Y s p e c t r u mc o n f i r m s t h e C (1 ~ 6 ) D - o l s e q u e n c e . T h e i n t e rr e s id u a l R O E c r o s s - p e a k s A H - I , B H - 4a n d B H - 1 , C H - 6 b e s t a b l i s h t h e A ( 1 ~ 4 ) B ( 1 ~ 6 ) C s e q u e n c e , p r o v i n g th e st r u c tu r e o f9 a a s g i v e n i n F i g . 3 .

Fraction 1 0 . - - T h e 1 D ~H N M R s p e c t r u m o f r e d u c e d f r a ct io n 1 0 i n d i c a t e d a m i x t u r eo f o l i g o s a c c h a r i d e - a l d i t o l s . I n t h e h i g h - m a s s r e g i o n o f t h e p o s it i v e - io n m o d e F A B m a s ss p e c t r u m o f re d u c e d a n d p e r m e t h y l a t e d f r a c ti o n 1 0 t w o s t ro n g [ M + N a ] + p s e u d o m o l e c -u l a r i o n s a t m / z 9 0 2 ( r e l a t i v e a b u n d a n c e 8 0 ) an d m / z 1 1 0 6 (r e l a t i v e a b u n d a n c e 1 0 0 )

30. 1995 - the structure of the exopolysaccharide produced by lac to bacillus helve tic us 766

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