Effects of Freezing and Frozen Storage on the Characteristics of Pectin

16

Effects o f Freezing a n d F r o z e n Storage o n the Characteristics of Pectin Extracted f r o m C e l l Walls

Downloaded by UNIV OF ARIZONA on December 16, 2012 | http://pubs.acs.org Publication Date: June 5, 1986 | doi: 10.1021/bk-1986-0310.ch016

D. S. Reid, J. M. Carr, T. Sajjaanantakul, and J. M. Labavitch Department of Food Science and Technology, University of California, Davis, Davis, CA 95616 The effect of freezing and frozen storage on the texture of green beans peaches and strawberries has been assessed. The textural changes result from more than solely turgor pressure loss. The pectic component of the cell wall complex has been extracted and characterized as a function of the freezing method and frozen storage conditions. Changes in this pectic fraction could account for some of the textural alteration observed. Texture is an important attribute of the eating quality of fruits and vegetables. An important aspect of texture is firmness. Texture or firmness result from a variety of contributions. Amongst these contributions are the turgor of an intact cell, and the strength of individual cell walls. It has often been remarked that a major textural consequence of freezing and frozen storage on fruits and vegetables is a loss of tissue firmness. Whilst it is known that freezing causes severe damage to the membranes of cells, and is therefore responsible for a loss of turgor, it is less clear whether there is a contribution to loss of firmness from the cell wall component. It should be remembered here that the softening of tissues during ripening has been shown to be largely a result of changes in the cell wall. In particular, it has been demonstrated that there is a correlation between the pectic component of cell walls, and the tissue firmness. In the study described here we wish to investigate whether some of the softening of plant tissues which accompanies freezing and frozen storage is related to changes in the cell wall, in particular whether there are related changes in the pectic fraction of the cell wall. This requires that we in some way assess texture, and that we also quantify and characterize the pectic materials of the tissue. Experimental Procedures In order to assess texture, we have employed a back extrusion cell accessory on an Instron Universal Testing Machine. The cell, 0097-6156/86/0310-0200$06.00/0 © 1986 American Chemical Society In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.


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i l l u s t r a t e d i n f i g u r e 1, i s m o d i f i e d from a d e s i g n d e s c r i b e d by Bourne and Moyer(1968). The I n s t r o n c r o s s - h e a d i s moved a t 100 mm/min. A sample of m a t e r i a l , c o n t a i n e d i n the cup, i s compressed as the p l u n g e r comes down. At l e n g t h , the m a t e r i a l e x t r u d e s back through the annulus between p l u n g e r and cup w a l l . A t y p i c a l f o r c e d i s t a n c e c u r v e i s shown. The r i s e r e f l e c t s the c o m p r e s s i o n , and the p l a t e a u of the back e x t r u s i o n . We use the p l a t e a u f o r c e as a measure of f i r m n e s s . Ten r e p l i c a t e measurements a r e performed. R e p e a t a b i l i t y i s b e t t e r than 4 J . I n o r d e r t o q u a n t i f y and c h a r a c t e r i z e the p e c t i n component of the c e l l , we must use some p r o c e d u r e t o p r e p a r e a s t a b l e c e l l w a l l f r a c t i o n , and t h e n f o l l o w some p r e d e t e r m i n e d e x t r a c t i o n scheme w h i c h a l l o w s f o r the s e p a r a t i o n o f p e c t i c m a t e r i a l s of d i f f e r e n t c h a r a c t e r i s t i c s . The o v e r a l l amount of p e c t i n i n any s i t u a t i o n i s then determined by a s s a y i n g f o r u r o n i c a c i d s . The e x t r a c t i o n s o f the p e c t i c f r a c t i o n s need not be e x h a u s t i v e , but must be r e p e a t a b l e , s i n c e we w i s h to f o l l o w the changes w h i c h may t a k e p l a c e i n the f r a c t i o n s consequent upon f r e e z i n g and f r o z e n s t o r a g e . The s e p a r a t i o n p r o c e d u r e s we have employed are as f o l l o w s . We have chosen a l c o h o l i c e x t r a c t i o n of macerated thawed t i s s u e as a method f o r the p r e p a r a t i o n of a c e l l w a l l m a t e r i a l w h i c h w i l l be s t a b l e , and s u i t e d t o s t o r a g e p r i o r t o f u r t h e r a s s a y . C e l l w a l l m a t e r i a l was p r e p a r e d a c c o r d i n g t o a method m o d i f i e d from t h a t of Ahmed and L a b a v i t c h (1977) by a d d i n g washing s t e p s u s i n g c h l o r o f o r m / methanol and a c e t o n e . A f l o w c h a r t of the p r o c e d u r e i s shown i n f i g u r e 2. The wash s t e p s were i n c l u d e d as we found t h a t m a t e r i a l e x t r a c t e d u s i n g o n l y a l c o h o l tended to form a g l a s s y , i n t r a c t i b l e m a t e r i a l on d r y i n g . T h i s was p a r t i c u l a r l y t r u e of s t r a w b e r r i e s . The m a t e r i a l f o r e x t r a c t i o n g e n e r a l l y has been d r a i n e d d u r i n g the thawing p r o c e d u r e , and i t i s t h i s d r a i n e d , thawed m a t e r i a l w h i c h i s macerated. The d r a i n e d l i q u i d i s c o l l e c t e d s e p a r a t e l y , and i d e n t i f i e d as d r i p . The e x t r a c t i o n of p e c t i c f r a c t i o n s from the c e l l w a l l m a t e r i a l f o l l o w s the t r a d i t i o n a l l o g i c . F i r s t a w a t e r s o l u b l e f r a c t i o n i s p r e p a r e d . T h i s i s o b t a i n e d by t a k i n g lOOmg of c e l l w a l l m a t e r i a l and s h a k i n g i t v i g o r o u s l y w i t h 20ml of w a t e r . A f t e r s t a n d i n g f o r 5 m i n u t e s , the s u p e r n a t a n t f l u i d i s s e p a r a t e d by c e n t r i f u g e , and the s o l i d m a t e r i a l i s t r e a t e d w i t h more w a t e r . The t r e a t m e n t i s performed f o u r t i m e s , and the s u p e r n a t a n t f l u i d s combined as the water - s o l u b l e p e c t i c f r a c t i o n (WSP). The r e s i d u a l s o l i d i s then used t o p r e p a r e the c h e l a t o r s o l u b l e f r a c t i o n (CSP). A s i m i l a r procedure i s used t o t h a t a l r e a d y d e s c r i b e d , e x c e p t i n g t h a t the s o l v e n t i s 0.1M EDTA i n 0.1M T r i s , pH7. A f i n a l f r a c t i o n , (HSP), i s p r e p a r e d by s u b j e c t i n g the r e s i d u e of s t e p 2 to d i l u t e sodium h y d r o x i d e as s o l v e n t . A g a i n , f o u r s t a g e e x t r a c t i o n i s performed. I t s h o u l d be noted t h a t t h e s e p r o c e d u r e s do not e x t r a c t a l l the m a t e r i a l of the c e l l w a l l p r e p a r a t i o n w h i c h c o n t a i n s u r o n i c a c i d r e s i d u e s . The r e s i d u e of s t e p 3 i s found s t i l l t o c o n t a i n u r o n i c a c i d . T h i s can o n l y be brought i n t o s o l u t i o n by u s i n g more extreme methods f o r s o l u b i l i z a t i o n . R e p l i c a t e e x t r a c t i o n s a r e performed. The m a t e r i a l s o b t a i n e d a r e a n a l y s e d f o r p e c t i n c o n t e n t by u r o n i c a c i d a s s a y . The method of Blumenkrantz and Asboe-Hansen (1973) i s employed. F u r t h e r c h a r a c t e r i z a t i o n of the p e c t i c m a t e r i a l i n c l u d e d assay f o r n e u t r a l sugar c o m p o s i t i o n by the a l d i t o l a c e t a t e method of A l b e r s h e i m e t a l ( 1 9 6 7 ) , w h i c h e n t a i l s d e r i v i t i z a t i o n , and

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

CHEMISTRY A N D FUNCTION OF PECTINS


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Figure 1. The back extrusion c e l l .


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AIS P r e p a r a t i o n / P u r i f i c a t i o n


E t h a n o l : C h l o r o f o r m - M e t h a n o l : Acetone

Procedure

1.

Wash and t r i m raw m a t e r i a l .

2.

Weight 100 g raw m a t e r i a l and p l a c e i n Waring b l e n d e r w i t h 400 mis 70% e t h a n o l .

3.

Homogenize f o r 1 minute.

4.

T r a n s f e r s l u r r y t o 50 ml round-bottom

5.

C e n t r i f u g e a t 19,000 x g (12,500 rpm, ss-34 r o t o r ) f o r 10 minutes.

c e n t r i f u g e tubes and c a p .

I 6.

D i s c a r d s u p e r n a t a n t and t r a n s f e r p e l l e t e d m a t e r i a l t o a c o u r s e sintered glass funnel.

7.

Breakup p e l l e t e d m a t e r i a l u s i n g a m e t a l s p a t u l a .

8.

Wash w i t h 2 x 100 ml volumes 70% e t h a n o l ( a p p l y i n g vacuum a f t e r thorough m i x i n g o f t h e s o l i d s and s o l v e n t ) .

9.

Wash w i t h 3 x 100 ml volumes c h l o r o f o r m - m e t h a n o l

10.

Wash w i t h 3 x 100 ml volumes a c e t o n e .

11.

A i r dry residue.

I

I (1:1 v / v ) .

4 \ F i g u r e 2.

Flow c h a r t of e x t r a c t i o n p r o c e d u r e .


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determination of the acetylated neutral sugars, i n our case by c a p i l l a r y GC. In some cases, the extracts were separated further using column chromatography, the column containing DEAE sephadex, and e l u t i o n being by an i o n i c gradient. Some samples were then subjected to gel f i l t r a t i o n on a 1.1 x 60cm Biogel P100 column, i n a 40mM Acetate, 40 mM EDTA, 50 mM NaCl buffer, pH 6.5, i n order to estimate their approximate molecular weight p r o f i l e s .


Materials In order to study the e f f e c t of freezing on texture, and t e x t u r a l l y related parameters, we have chosen three tissue systems. Green beans, strawberries and peaches. Green beens were of the G a l l a t i n variety. Peaches were of the variety Halford. Two v a r i e t i e s of strawberry were studied, Aiko and Pajaro. A l l were obtained as fresh material, and processed i n our p i l o t plant. Blanching, where required, u t i l i z e d a steam blancher. Fast freezing used a Conrad freezer with an a i r blast temperature of -70 C. Products were frozen unwrapped i n a single layer on open mesh trays. Immediately after freezing they were sealed into bags for storage. Slow freezing was i n s t i l l a i r i n a cold room. In this case the materials were sealed into bags p r i o r to freezing. Results Texture i s an important a t t r i b u t e of the three tissues chosen for study. Since blanching i s an appropriate pretreatment to freezing i n some cases, we might f i r s t ask the question " What i s the e f f e c t of blanching on texture?" As blanching i s a p a r t i a l cooking process, not s u r p r i s i n g l y , tissue softening occurs. We have taken samples of green beans and blanched them for increasing times at 100 C. Not surprisingly, measurement of the back extrusion force shows increasing softening with blanch time. At the same time, the blanch liquor has been assayed for pectin content, determined as uronic acid residues. The pectin released from the tissue as a consequence of blanching has been computed. Figure 3 shows the relationship between texture and released pectin. Clearly, the loss i n texture i s accompanied by a release of pectin. Another factor i n the softening, of course, i s the loss i n turgor which accompanies the thermal destruction of the i n t e g r i t y of the c e l l membranes. What happens i f we freeze the green beans? As can be seen from figures 4 and 5, the result of freezing i s a reduction i n back extrusion force, whether the unfrozen material be blanched or not. The greatest reduction i n back extrusion force as a consequence of freezing i s seen for unblanched tissue, and presumably r e f l e c t s i n large measure the loss i n turgor. However, loss of turgor i s not the sole cause of the reduction i n back extrusion force. Consider...blanching, too, destroys turgor, and yet the reduction i n back extrusion force as compared to fresh tissue i s greater for unblanched, frozen tissue than for blanched, unfrozen t i s s u e . Also, there i s a reduction i n back extrusion force i n blanched tissue due to subsequent freezing and thawing. This reduction i s more marked, r e l a t i v e to the i n i t i a l back extrusion force of the blanched t i s s u e , for the more extensive blanches. Since i t i s u n l i k e l y that the turgor contribution can account for



REID ET AL.


o I 0

' ' 1 ' 200 400 600 800 PECTIC SUBSTANCES RELEASED (/ig/g tissues)

Figure 3. Relationship between texture and pectin release during blanching.

200 -

01 0

i

1 2

i

i

I

I

I

3

4

5

6

BLANCH TIME, MIN Figure refers F/T to blanch

4. E f f e c t of blanching and freezing on texture. UF to tissue which i s not frozen a f t e r the blanch treatment, tissue which has undergone a freeze—thaw cycle a f t e r the treatment.



206


t h i s o b s e r v a t i o n , i t would appear t h a t t h i s l o s s i n f i r m n e s s i s due to the c e l l w a l l c o n t r i b u t i o n . Further, there i s a continued decrease i n t i s s u e f i r m n e s s as a consequence o f extended f r o z e n s t o r a g e . I t i s t h e r e f o r e a p p r o p r i a t e t o f o l l o w t h e changes w h i c h take p l a c e i n c e l l w a l l components as a f u n c t i o n o f f r e e z i n g and f r o z e n s t o r a g e , and t r y t o c o r r e l a t e these w i t h o b s e r v a t i o n s r e l a t i n g t o t e x t u r e , t i s s u e s t r u c t u r e , e t c . As a l r e a d y i n d i c a t e d , we have chosen t o f o c u s on t h e p e c t i c f r a c t i o n , s i n c e i t i s amenable to e x t r a c t i o n and f r a c t i o n a t i o n by r e a s o n a b l y s i m p l e methods. EDTA was chosen as c h e l a t o r i n p a r t because we wished t o p e r f o r m o u r e x t r a c t i o n s under as m i l d c o n d i t i o n s as p o s s i b l e , i e a t room t e m p e r a t u r e , and c l o s e t o n e u t r a l i t y , i n o r d e r t o m i n i m i z e c h e m i c a l change i n t h e p e c t i c f r a c t i o n s d u r i n g t h e i r e x t r a c t i o n . As can be seen from f i g u r e 6, t h e e x t r a c t i o n e f f i c i e n c y o f o t h e r commonly used c h e l a t o r s appears t o be l e s s than t h a t of EDTA. T h i s o b s e r v a t i o n i s d i s c u s s e d f u r t h e r by R e i d and C a r r (paper i n p r e p a r a t i o n ) and i n Carr(1984). Our r e s u l t s a r e b e s t p r e s e n t e d i n s t a g e s . F i r s t we s h o u l d c o n s i d e r t h e p e c t i c c o m p o s i t i o n o f t h e unprocessed raw m a t e r i a l . Then we s h o u l d c o n s i d e r t h e changes which take p l a c e i n t h e g r o s s f r a c t i o n s as a consequence o f p r o c e s s i n g and s t o r a g e . To do t h i s we need o n l y t o determine t h e amounts of p e c t i n i n each f r a c t i o n . A f t e r t h i s we s h o u l d c o n s i d e r whether t h e r e have been any c o m p o s i t i o n a l changes i n t h e p e c t i n . T h i s i s b e s t determined by u s i n g column t e c h n i q u e s t o f r a c t i o n a t e t h e t h r e e c a t e g o r i e s o f p e c t i c m a t e r i a l , and a l s o by f u r t h e r a n a l y s i n g t h e p e c t i n s . I f any changes a r e t a k i n g p l a c e as a consequence o f p r o c e s s i n g , we would expect t o see them r e f l e c t e d i n a t l e a s t some o f t h e s e r e s u l t s . T a b l e 1 summarizes t h e d a t a r e l a t i n g t o t h e u r o n i c a c i d c o n t e n t of i n d i v i d u a l p e c t i c f r a c t i o n s from t h e t h r e e t i s s u e s b o t h b e f o r e and a f t e r p r o c e s s i n g . The d a t a f o r t h e p e c t i c f r a c t i o n s from u n f r o z e n t i s s u e i n c l u d e an a s s a y f o r t h e u r o n i c a c i d c o n t e n t of t h e r e s i d u a l m a t e r i a l a f t e r the e x t r a c t i o n of the three s o l u b l e f r a c t i o n s i n o r d e r t o c o n f i r m t h a t we can account f o r a l l t h e u r o n i c acid residues. The t o t a l u r o n i c a c i d c o n t e n t o f t h e o r i g i n a l c e l l w a l l m a t e r i a l i n a l l cases d e f i n e s 100%. As t a b l e 1 shows, t h e d i s t r i b u t i o n s o f p e c t i c f r a c t i o n s from t h e t h r e e t i s s u e s a r e v e r y d i f f e r e n t . A l s o , the r e s i d u a l uronic a c i d content of the extracted c e l l w a l l m a t e r i a l v a r i e s f o r t h e s e t i s s u e s . However, t h e r e s i d u a l assay i n d i c a t e s t h a t we a r e a c c o u n t i n g f o r most o f t h e u r o n i c a c i d containing material. The f r a c t i o n a t i o n procedure e x t r a c t s about 90% of t h e a v a i l a b l e u r o n i c a c i d i n P a j a r o s t r a w b e r r y c e l l w a l l p r e p a r a t i o n , y e t A i k o has an e x t r a c t i o n o f 70%. Only about 50% o f the u r o n i c a c i d i n green bean c e l l w a l l p r e p a r a t i o n i s e x t r a c t e d . The e x t r a c t i o n e f f i c i e n c y f o r peaches i n c r e a s e s d u r i n g s t o r a g e , s u g g e s t i n g t h a t t h e r e i s some change t a k i n g p l a c e . F i g u r e 7 i l l u s t r a t e s t h e change i n u r o n i d e c o n t e n t of t h e f r a c t i o n s o b t a i n e d from t h e c e l l w a l l m a t e r i a l as a f u n c t i o n o f t h e s t o r a g e time a t -20 C f o r t h e s t r a w b e r r y v a r i e t y , A i k o . I t i s c l e a r t h a t t h e major change i s i n t h e u r o n i c a c i d c o n t e n t o f t h e water s o l u b l e f r a c t i o n . T h i s might be e x p e c t e d , i f u r o n i c a c i d c o n t a i n i n g w a l l components a r e c o n s t i t u e n t s of t h e m a t e r i a l l o s t as d r i p . I n t a b l e l a we compare t h e c e l l w a l l f r a c t i o n s o b t a i n e d by homogenizing thawed t i s s u e , from which t h e d r i p has been l o s t , and f r o z e n t i s s u e , w h i c h has l o s t no m a t e r i a l . There i s a l o s s o f u r o n i d e i n t h e


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r

BLANCH TIME (minutes) F i g u r e 5. T e x t u r e l o s s due t o f r e e z i n g p o s t b l a n c h , c a l c u l a t e d as t h e r a t i o o f the l o s s i n back e x t r u s i o n f o r c e consequent upon f r e e z i n g t o t h e back e x t r u s i o n f o r c e a f t e r b l a n c h i n g but p r i o r to f r e e z i n g .

Sodium EDTA

TOTAL (UG) 1980 ±30

EH Ammonium Oxalate

I500±30

•

I Neutral Aqueous

[%] Sodium Hexametaphosphate.... 1610 ± 30

10 -

I

2

3

WATER SOLUBLE PECTIN F i g u r e 6. i n AIS.

CALCIUM PECTATE

E x t r a c t i o n performance of d i f f e r e n t c h e l a t o r s

a t pH7



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CHEMISTRY A N D FUNCTION O F PECTINS

TABLE 1 (a) S t r a w b e r r y b l a s t f r o z e n -20 C s t o r a g e (variety Aiko) unfrozen 1 week 10 mo WSP 48.6 44.5% 31.2% CSP 14.1 15.4 17.4 HSP 9.5 9.8 12.6 Sum 72.2 69.7 61.2 (*)

10 mo(*) 47.2% 14.8 10.1 72.1

AIS p r e p a r e d d i r e c t l y from f r o z e n t i s s u e , w i t h no d r i p loss occuring, unlike regular preparation.

(b) S t r a w b e r r y slow f r o z e n -12 C s t o r a g e (variety Pajaro)

WSP CSP HSP Sum RESIDUAL

unfrozen 44.63% 24.80 24.05 93.48 15

( c ) Peach unblanched


unfrozen 23.56% 8.86 36.09 68.51 22

1 day 46.98% 19.48 23.83 89.79

4 month 47.17% 18.43 21.86 87.46

-20 C s t o r a g e 1 day 26.28% 9.27 40.52 76.07

4 month 23.74% 11.82 52.68 88.24

(d) Green bean 2m b l a n c h s l o w f r o z e n -12 C s t o r a g e


unfrozen 8.81% 19.33 21.33 49.48 36

1 month 8.32% 22.31 18.85 49.48



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AL.


209

thawed t i s s u e . T a b l e 2 shows an a n a l y s i s of c e l l w a l l u r o n i d e from thawed t i s s u e , t o g e t h e r w i t h the u r o n i d e assayed i n the d r i p c o l l e c t e d d u r i n g the thaw. S o l u b l e u r o n i d e i s indeed l o s t i n the d r i p , l e a v i n g the c e l l w a l l m a t e r i a l d e p l e t e d of m a t e r i a l w h i c h would o t h e r w i s e be i n c l u d e d i n the w a t e r s o l u b l e f r a c t i o n . F i g u r e 8 shows t h a t t h e r e i s a c o n t i n u o u s l o s s of t e x t u r e i n s t r a w b e r r i e s d u r i n g f r o z e n s t o r a g e . T h i s i s p a r a l l e d by the l o s s of s o l u b l e p e c t i n . I t s t i l l remains t o be d e t e r m i n e d whether, and i n what manner, the p e c t i n of the t h r e e f r a c t i o n s i s changing i n c h a r a c t e r . G i v e n t h a t the most d r a m a t i c l o s s i n p e c t i c component o f s t r a w b e r r i e s i s i n the WSP, f i g u r e 7, we have f u r t h e r c h a r a c t e r i z e d t h i s f r a c t i o n by s u b j e c t i n g i t t o f r a c t i o n a t i o n u s i n g a DEAE sephadex column e l u t e d by an i n c r e a s i n g i o n i c g r a d i e n t . An amount of s o l u t i o n c o n t a i n i n g about 7mg u r o n i c a c i d i s p l a c e d on the column, and e l u t e d i n i t i a l l y w i t h 0.1M phosphate b u f f e r , pH6.9. Once no more u r o n i c a c i d m a t e r i a l i s found t o e l u t e , the g r a d i e n t i s s t a r t e d , the f i n a l e l u a n t b e i n g 1.2M phosphate b u f f e r , pH6.9. The r e m a i n i n g u r o n i c a c i d on the column e l u t e s i n c h a r a c t e r i s t i c f a s h i o n . As can be seen from f i g u r e s 9-11 t h e r e i s a change i n the e l u t i o n c h a r a c t e r i s t i c of the WSP m a t e r i a l as a f u n c t i o n of s t o r a g e t i m e . We c o n s i d e r i n p a r t i c u l a r the column bound m a t e r i a l , t h a t which does not e l u t e u n t i l the i o n i c g r a d i e n t i s a p p l i e d . These e l u t i o n p a t t e r n s a r e seen i n f i g u r e s 9b, 10b and l i b . The o r i g i n a l e l u t i o n p a t t e r n s f o r the column bound m a t e r i a l show two peaks, a t about 0.3 t o 0.4 M i o n i c s t r e n g t h and a t about 0.6M i o n i c s t r e n g t h . As a r e s u l t of s t o r a g e , the second peak d e c r e a s e s i n s i z e , w h i l s t the i n i t i a l peak remains unchanged. The f r a c t i o n l o s t i s the more a c i d i c m a t e r i a l , and p r o b a b l y r e p r e s e n t s the p r i m a r y rhamno-galacturonan backbone, as i n d i c a t e d by the s i g n i f i c a n t l y h i g h e r p r o p o r t i o n of rhamnose found i n DEAE column bound m a t e r i a l s ( t a b l e 3 ) . Recent o b s e r v a t i o n s i n d i c a t e t h a t t h i s r h a m n o s e - r i c h u r o n i d e m a t e r i a l i s more s l o w l y e x t r a c t e d from the AIS by water than i s the m a t e r i a l w h i c h e l u t e s i n the v o i d volume of the DEAE column. To throw f u r t h e r l i g h t on the changes i n t h i s f r a c t i o n , we have performed g e l f i l t r a t i o n a n a l y s e s t o o b t a i n p r e l i m i n a r y i n d i c a t i o n s of m o l e c u l a r w e i g h t s . WSP m a t e r i a l s c o l l e c t e d from u n f r o z e n , l d a y f r o z e n and 4.5month f r o z e n s t r a w b e r r y samples a l l v o i d e d a P100 column, i n d i c a t i n g m o l e c u l a r w e i g h t s i n e x c e s s o f 100,000, based on e l u t i o n of g l o b u l a r p r o t e i n s of known s i z e . I n t e r e s t i n g l y , m a t e r i a l p r e c i p i t a t e d from the d r i p l o s s f r a c t i o n w i t h e t h a n o l a l s o d i s p l a y e d a major h i g h m o l e c u l a r w e i g h t peak, w i t h i n a d d i t i o n about 20% of the t o t a l u r o n i d e c o n t a i n i n g m a t e r i a l e l u t i n g w i t h the t o t a l l y i n c l u d e d volume of the column. T h i s would c o r r e s p o n d t o a m o l e c u l a r w e i g h t around 10,000. The p e c t i c m a t e r i a l i n the d r i p i s a s i g n i f i c a n t p a r t of the p e c t i n l o s t from the c e l l w a l l i n l o n g term s t o r a g e ( t a b l e 2 ) . S i n c e , d u r i n g f r o z e n s t o r a g e , the d r i p l o s s i n c r e a s e s , the n a t u r e of the u r o n i d e i n the d r i p , and i t s change w i t h time of s t o r a g e , w a r r a n t s f u r t h e r s t u d y . I t would be u s e f u l t o know how the p r o p o r t i o n s of the h i g h and low m o l e c u l a r w e i g h t f r a c t i o n s v a r y w i t h time of s t o r a g e . Conclusions T e x t u r e i s a f f e c t e d by f r e e z i n g i n ways over and above the e f f e c t of f r e e z i n g on t u r g o r . T h i s s u g g e s t s t h a t t h e r e i s a c l e a r



210


STORAGE TIME, WEEKS

F i g u r e 7. U r o n i d e c o n t e n t of AIS f r a c t i o n s of A i k o s t r a w b e r r i e s a f t e r d i f f e r e n t f r o z e n s t o r a g e t i m e s . WSF r e p r e s e n t s water s o l u b l e f r a c t i o n , CSF c h e l a t o r s o l u b l e f r a c t i o n and HSF a l k a l i soluble fracion.

Table 2 Strawberry d r i p a n a l y s i s Y i e l d o f u r o n i d e from lOOg o f f r e s h b e r r y , f r o z e n s t o r e d a t -20 C f o r 9mo, then thawed f o r 90 minutes a t room t e m p e r a t u r e .

T o t a l f r e s h wt ug u r o n i d e / ug AIS Uronide i n f r a c t i o n (g)

Berry 78.4 0.218 0.417

Drip 21.7 0.106 0.040



R E I D ET

AL.

Figure 8.


Texture loss i n frozen storage of strawberries.



n3.0

300


a

20

40 60 80 ELUENT VOLUME (ml)

100

120

F i g u r e 9. DEAE f r a c t i o n a t i o n o f WSP from u n f r o z e n strawberries. ( a ) complete f r a c t i o n a t i o n p r o f i l e (b) e l u t i o n of column bound m a t e r i a l



REID ET AL.


ELUENT VOLUME (ml)

PHOSPHATE

GRADIENT (ml)

Figure 10. DEAE fractionation of WSP strawberries. (a) complete fractionation p r o f i l e (b) elution of column bound material

from 1 day frozen




F i g u r e 11. DEAE f r a c t i o n a t i o n of WSP from 4.5 month f r o z e n strawberries. (a) complete f r a c t i o n a t i o n p r o f i l e (b) e l u t i o n of column bound m a t e r i a l



Rhamnose

4.5

29.1

5.3

31.2

5.6

30.1

unfrozen 0.24 : 1.0 unbound f r a c t i o n

unfrozen 0.21 : 1.0 bound f r a c t i o n

1 day s t o r a g e 0.18 : 1.0 unbound f r a c t i o n

1 day s t o r a g e 0.32 : 1.0 bound f r a c t i o n

4.5 month 0.14 : 1.0 unbound f r a c t i o n

4.5 month 0.13 : 1.0 bound f r a c t i o n 1.2

20.7

17.0

21.5

3.5

15.3

24.3

18.0

Arabinose

3.0

3.1

2.20

Fucose

3.9

16.9

8.6

15.7

5.6

19.6

Xylose

2.1

3.7

8.5

4.9

5.7

6.8

Mannose

19.5

39.0

17.9

40.1

19.6

35.4

Galactose

21.6

14.3

12.4

15.7

12.6

13.4

Glucose

N e u t r a l sugars r a t i o s from a n i o n exchange chromatography f r a c t i o n s of s t r a w b e r r i e s WSP, s t i l l a i r f r o z e n -12c s t o r a g e .

sample t o t n e u t r a l sugars : t o t uronides

T a b l e 3.



216

chemistry and function of pectins

contribution to texture from the cell wall material, a conclusion supported by abundant material following the changes in cell wall pectins during fruit ripening. Pectin is also an important contributer to the texture of tissues which have been frozen. Changes are seen in the pectic material as a consequence of freezing and frozen storage. In strawberries, the WSP fraction, which we assume to be the fraction most loosely associated with the cell wall, shows the most dramatic change. There is a decrease in amount, paralleling a decrease in firmness. The compositional studies of the fraction suggest that the changes include some associated with the pectin rhamno-galacturonan backbone. The chemical change in the backbone is not yet clearly defined. Changes in the sugar distribution and in the uronic acid to neutral sugar ratios are not in evidence as we analyse the deae bound fractions from fruits at progressively longer storage. The molecular weight remains above the cut-off for a P100 column. Further studies are in progress. These include investigation of changes in the degree of methyl esterification of the fractions. The composition of the other fractions is being determined. The uronide fraction of drip is being analysed. The effect of adding calcium, which should interact with pectin, to strawberries prior to freezing them is also being assessed. Initial data indicate that calcium alters the relative amount of pectin extractable into the different fractions, and also alters the back extrusion force, and the nature of the time dependence of texture on frozen storage. Acknowledgments This research was supported by Grant No US-452-81 from BARD-The United States-Israel Binational Agricultural Research and Development Fund. Literature Cited 1. Ahmed, A.E.R.; Labavitch,J.M. J . Food Biochem. 1977, 1, 361. 2. Albersheim, P.; Nevins, D.J.; English, P.D.; Karr, A. Carbohyd. Res., 1967, 5, 340. 3. Blumenkrantz, N.; Asboe-hansen, G. Anal. Biochem., 1973, 54, 484. 4. Bourne, M.C.; Moyer, J.C. Food Technol., 1968, 22, 1013. 5. Carr, J.M. MS thesis, Dept of Food Science, University of California, Davis, 1984. RECEIVED November 19, 1985


Effects of Freezing and Frozen Storage on the Characteristics of Pectin

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