Function of Metal Cations in Regulating the Texture of Acidified

Chapter 11

Function of Metal Cations in Regulating the Texture of Acidified Vegetables

Downloaded by EAST CAROLINA UNIV on October 2, 2016 | http://pubs.acs.org Publication Date: September 7, 1989 | doi: 10.1021/bk-1989-0405.ch011

R. F. McFeeters Food Fermentation Laboratory, Agricultural Research Service, U.S. Department of Agriculture, and Department of Food Science, North Carolina Agricultural Research Service, North Carolina State University, Raleigh, NC 29695-7624 The use of metal ions to modify the texture of f r u i t and vegetables has been investigated and used f o r nearly 50 years. In general, monovalent ions have been found to cause softening while divalent ions, usually calcium, either i n h i b i t softening or in some cases actually increase firmness. However, there have been few instances i n which the e f f e c t of metal ions on tissue softening rates during processing or storage have been measured. The development o f a r a p i d procedure to measure softening rates in cucumber tissue has made it possible t o investigate q u a n t i t a t i v e effects of metal ions on texture. A s a l t softening e f f e c t i n blanched, a c i d i f i e d cucumber t i s s u e was observed when NaCl or other alkali metal chlorides were added. Low concentrations of calcium, strontium, barium, and lanthanide ions i n h i b i t e d the rate of tissue s o f t e n i n g i n the presence of a high concentration of NaCl. The effects of multivalent ions on i n h i b i t i o n of t i s s u e softening has l e d t o the conclusion that the egg box model, which was developed to explain gel formation by calcium ions i n d i l u t e polypectate solutions, is not a suitable model t o explain metal ion effects on cucumber tissue texture. Hopefully, development of this experimental approach will lead both t o improved understanding of the mechanisms of plant tissue softening and t o better means to retain firmness in cucumber pickle products. The a b i l i t y of metal ions t o modify the texture of f r u i t and vegetable tissue has been studied since 1939, when Kertesz (1) found that calcium ions improved the firmness of tomatoes. A very simplified generalization of the effects of metal ions on fruit and vegetable texture i s that monovalent cations, usually Na and K*", cause tissue softening. Examples of this softening effect include results with peas (2,3), dried peas (4), carrots (5), potatoes (6) and green beans (7). On the other hand calcium, a divalent cation, +

This chapter not subject to U.S. copyright Published 1989 American Chemical Society

Jen; Quality Factors of Fruits and Vegetables ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


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e i t h e r causes t i s s u e firming o r prevents softening o f p l a n t t i s s u e s . Calcium e f f e c t s appear t o be general i n p l a n t t i s s u e s . A l l o f the commodities m e n t i o n e d above r e s p o n d t o c a l c i u m a d d i t i o n . I n addition, the texture o f apples (8) and a p r i c o t s (9) i s a f f e c t e d by calcium. The USDA Food Fermentation Laboratory has been concerned over a l o n g p e r i o d o f time w i t h t h e f a c t o r s which a f f e c t t h e t e x t u r a l q u a l i t y o f a c i d i f i e d vegetables, p a r t i c u l a r l y cucumbers. As i s the case with other processed f r u i t and vegetable products, metal ions have been found t o have important t e x t u r a l e f f e c t s on cucumbers. In c o n t r a s t t o observations w i t h other p l a n t t i s s u e s , NaCl has been found t o improve f i r m n e s s r e t e n t i o n i n fermented cucumbers, i n addition to i t s a b i l i t y to select for l a c t i c acid bacteria i n fermentations and i t s use as a f l a v o r i n g (10-13). Table I shews the e f f e c t t h a t NaCl has i n preventing softening during fermentation and storage o f cucumber s l i c e s . The probable reason t h a t s o f t e n i n g i n h i b i t i o n occurs i s that NaCl can i n h i b i t t o some degree the a c t i o n o f p o l y g a l a c t u r o n a s e s which s o f t e n t i s s u e . T h i s i s suggested i n Table I by the f a c t that NaCl was not required t o maintain firmness i n b l a n c h e d cucumber s l i c e s d u r i n g f e r m e n t a t i o n . I n a d d i t i o n , e a r l i e r r e s u l t s o f B e l l and E t c h e l l s (14) show t h a t NaCl reduces the r a t e o f s o f t e n i n g i n p a s t e u r i z e d cucumber t i s s u e t o which a f i x e d amount o f polygalacturonase a c t i v i t y i s added. Calcium i s c u r r e n t l y added t o most commercial cucumber p i c k l e products due t o i t s a b i l i t y t o p r e v e n t t e x t u r e l o s s i n b o t h fermented (10. 11. 15-17) and non-fermented p i c k l e s (18. 19). Table I. Effect of heat and salt on cucumber s l i c e firmness

Firmness score

Heat Treatment

NaCl,

%

rjH

None

0.0 1.4 3.9 6.5

3.5 3.3 3.2 3.2

3.1C 4.6B 5.2B 8.3A

0.0 1.4 3.9 6.5

3.6 3.4 3.3 3.2

7.9A 8.3A 8.6A 8.9A

77°C, 3.5

min

1

^ e a n s f o l l o w e d by d i f f e r e n t l e t t e r s i n d i c a t e t r e a t m e n t s were d i f f e r e n t a t the 0.05 s i g n i f i c a n c e l e v e l . Source: Data are from r e f . 10. I t i s somewhat s u r p r i s i n g , considering the amount of work done and the v a r i e t y o f d i f f e r e n t p l a n t t i s s u e s studied, t h a t there have not been multiple mechanisms proposed t o explain metal ion e f f e c t s . There has been n e a r l y complete agreement t h a t these e f f e c t s occur due t o t h e i n t e r a c t i o n o f m e t a l i o n s w i t h t h e d e m e t h y l a t e d g a l a c t u r o n i c a c i d r e s i d u e s i n t h e p e c t i c substances o f t h e c e l l w a l l . Monovalent ions, i . e . NaCl, are thought t o a c t by d i s p l a c i n g calcium ions from pectate b i n d i n g s i t e s and thereby d i s r u p t i n g the



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p e c t i n g e l s t r u c t u r e . T h i s i s i l l u s t r a t e d by some data of Van Buren (20) on green beans (Fig. 1). A d i r e c t e f f e c t of NaCl on degradation of c e l l w a l l polymers has not been reported. For d i v a l e n t cations, i . e . calcium, Rees and h i s group i n England proposed an "egg box" model (21) t o e x p l a i n the binding of calcium ions t o polypectate i n aqueous s o l u t i o n ( F i g . 2). The key elements of t h i s model are t h a t c a l c i u m c a t i o n s p r o v i d e i o n i c c r o s s - l i n k s as n e g a t i v e l y charged g a l a c t u r o n i c a c i d p o l y m e r i c c h a i n s l i n e up w i t h each o t h e r and provide pockets i n t o which the metal ions can f i t . A t low calcium l e v e l s there w i l l i n i t i a l l y be dimerization of pectate molecules. As more i o n s are bound, the dimers w i l l aggregate i n t o a l a r g e g e l structure. I emphasize that the egg box model was developed t o explain the binding of d i v a l e n t ions i n polypectate solutions, not the e f f e c t of calcium ions on the p h y s i c a l properties o f p l a n t t i s s u e s . A rather l a r g e volume o f data on the i n t e r a c t i o n s of metal ions with p e c t i n , p a r t i c u l a r l y the work of Rees and h i s collaborators (21-26) and Kohn and coworkers (27, 29), a l l seem t o be explainable i n terms of the egg box model. With p l a n t t i s s u e s there i s much l e s s q u a n t i t a t i v e d a t a on i o n b i n d i n g i n t h e t i s s u e s o r i n i s o l a t e d c e l l w a l l s . However, the data which have been o b t a i n e d seem t o be g e n e r a l l y consistent with the egg box model (30-33). As a r e s u l t , the egg box model has often been used t o attempt t o e x p l a i n calcium i o n e f f e c t s on t h e p h y s i c a l p r o p e r t i e s o f b o t h l i v i n g and p r o c e s s e d p l a n t t i s s u e s (34-40). However, c l e a r d e m o n s t r a t i o n s t h a t p h y s i c a l changes i n p l a n t t i s s u e s , e.g. firming, i n h i b i t i o n o f softening, or i n h i b i t i o n of c e l l elongation during growth, are caused by egg box binding of metal ions are lacking. In a few instances data have been published which are a t l e a s t d i f f i c u l t t o r e c o n c i l e with the egg box model. Stoddard e t a l . (41) were unable t o c o r r e l a t e the degree of calcium c r o s s - l i n k i n g t o the r a t e o f c e l l w a l l elongation. Tepfer and T a y l o r (42) found a l a c k of c o r r e l a t i o n between the a b i l i t y of d i v a l e n t ions t o cause pectate g e l a t i o n and t h e i r a b i l i t y t o i n h i b i t the acid-induced elongation of bean hypocotyls. McFeeters e t a l . (19) found t h a t changing p e c t i n m e t h y l a t i o n i n t h e presence o f c a l c i u m i o n s had l i t t l e o r no e f f e c t on the a b i l i t y o f cucumber t i s s u e t o m a i n t a i n firmness d u r i n g storage. A t low m e t h y l a t i o n there should have been more and stronger calcium cross-linkages with p e c t i n and an i n h i b i t i o n of t i s s u e softening. E f f e c t of Monovalent Ions on Cucumber Softening Rates Now l e t us consider recent r e s u l t s from t h i s laboratory concerning t h e e f f e c t s o f m e t a l i o n s on t h e r a t e s o f t e x t u r e changes i n a c i d i f i e d cucumber t i s s u e t e x t u r e . To i n v e s t i g a t e the e f f e c t o f NaCl on cucumber t e x t u r e i n the absence o f enzymatic degradation, the firmness changes i n blanched t i s s u e under a c i d c o n d i t i o n s , pH 3.2-3.8, were measured (43). The procedure was t o blanch cucumber s l i c e s o r mesocarp t i s s u e p i e c e s i n b o i l i n g water f o r 3 min t o i n a c t i v a t e p e c t i n e s t e r a s e (19). A f t e r c o o l i n g , the t i s s u e samples were covered w i t h a b r i n e which contained a c e t i c a c i d and S0 , t o e q u i l i b r a t e a t 0.6% and 200 ppm, r e s p e c t i v e l y , and the appropriate amount of NaCl. The t i s s u e e q u i l i b r a t e d with the b r i n e i n the c o l d and t h e n samples were i n c u b a t e d a t 44C so t h e y would s o f t e n reasonably r a p i d l y . Firmness o f the t i s s u e was e v a l u a t e d a t t h e 2


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QUALITY FACTORS OF FRUITS AND VEGETABLES

I

I

0.25

0.50

—ι 0.75

1— 1.0

NoCl CONCENTRATION IN SOAK SOLUTION, MOLARITY

Fig. 1. Effect of NaCl on the texture and s o l u b i l i z a t i o n of calcium ions from green bean pods, a) Firmness of pods using a Kramer shear press, b) Solubilization of calcium ions from green bean tissue. Reprinted from reference 20.



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beginning o f the 44C storage and a t s e v e r a l subsequent times by a punch t e s t on 15 t i s s u e pieces from d u p l i c a t e j a r s o f sample u s i n g the Instron Universal Testing Machine (44). The f i r s t order r a t e of t h e s o f t e n i n g r e a c t i o n was c a l c u l a t e d from t h e s l o p e o f t h e softening curves (Fig. 3). The r a t e of softening of cucumber mesocarp t i s s u e increased as t h e e q u i l i b r a t e d s a l t c o n c e n t r a t i o n i n c r e a s e d f o r b o t h whole cucumber s l i c e s and cucumber mesocarp t i s s u e p i e c e s ( F i g . 4 ) . Therefore, i f the enzymatic degradation o f c e l l w a l l s i s prevented, cucumber t i s s u e does s o f t e n i n r e s p o n s e t o N a C l , as has been observed i n other f r u i t s and vegetables. There i s a question about the mechanism of the degradative process t h a t leads t o softening i n a c i d conditions. The s a l t softening described f o r other vegetables has been observed under low a c i d c o n d i t i o n s . I t has been proposed t h a t s o f t e n i n g i n those commodities i s caused by ^ - e l i m i n a t i o n degradation o f p e c t i n , though d i r e c t evidence f o r ^ - e l i m i n a t i o n reactions i n vegetable t i s s u e s has not been obtained (7). However, with the e q u i l i b r a t e d pH o f the t i s s u e i n these experiments i n the range o f 3.2-3.8, the pH w i t h i n the cucumber c e l l w a l l i s almost c e r t a i n l y too low f o r ^ - e l i m i n a t i o n t o occur (45). Therefore, there may be a d i f f e r e n t s a l t s o f t e n i n g mechanism i n a c i d i f i e d p l a n t t i s s u e s as compared t o low a c i d vegetables. F i g 4b a l s o shows the degree o f p e c t i n m e t h y l a t i o n i n t h e c e l l w a l l s o f t h e cucumber mesocarp t i s s u e used i n t h i s experiment. Two p o i n t s s h o u l d be remembered. F i r s t , the degree o f methylation i s r e l a t i v e l y constant i n blanched t i s s u e and, secondly, i t i s q u i t e high. Over 60% of the g a l a c t u r o n i c a c i d residues have methyl e s t e r s and are, t h e r e f o r e , uncharged. T h i s becomes an important consideration when we consider the e f f e c t o f calcium ions on the s o f t e n i n g r a t e i n l i g h t o f the requirement o f i o n i z e d carboxyl groups f o r c r o s s - l i n k i n g o f p e c t i n molecules according t o the egg box model. The s a l t softening e f f e c t i n cucumbers was a l s o s i m i l a r t o r e s u l t s i n green beans (46) i n that sodium and potassium ions had s i m i l a r softening e f f e c t s . In f a c t , a t 0.25 M a l l of the a l k a l i metal ions had a s i m i l a r e f f e c t on the r a t e of cucumber t i s s u e softening (Table I I ) . Thus, the s i z e o f the ions do not appear t o a f f e c t t h e i r a b i l i t y t o increase softening rates. Table I I .

E f f e c t of 0.25 M a l k a l i metal ions on the rate of softening of blanched cucumber mesocarp t i s s u e held a t 44°C

Metal Ion None Lithium Sodium Potassium Rubidium Cesium

Softening rate, day"

1

1

0.055A 0.175B 0.194B 0.159B 0.169B 0.166B

Ί

Means f o l l o w e d by d i f f e r e n t l e t t e r s i n d i c a t e t r e a t m e n t s were d i f f e r e n t at the 0.05 s i g n i f i c a n c e l e v e l . Source: Data are from r e f . 43.




130

-0.5 ι 0

' 10

' 20

' 30

' 40

' 50

Time, days

F i g . 3. F i r s t o r d e r p l o t o f cucumber t i s s u e s o f t e n i n g i n a c i d b r i n e with and without added NaCl. The subscript i i s the i n i t i a l f i r m n e s s , and t h e s u b s c r i p t t i n d i c a t e s f i r m n e s s a t any subsequent time. From reference 43.


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Inhibition of Softening Rates by Calcium Once i t had been demonstrated that NaCl enhanced the rate of cucumber tissue softening, the next question was whether and i n what way calcium ions would inhibit the rate of softening. To answer this question a combination of 1.5 M NaCl and 0 to 80 mM calcium ions were added f i r s t to blanched cucumber slices and then to cucumber mesocarp tissue (47) (Fig. 5). In both experiments there was an excellent f i t of a hyperbolic curve to the softening rates as a function of calcium added, since the hyperbolic model accounted for over 99% of the experimental variation. For the cucumber s l i c e s (Fig. 5a), h a l f of the observed i n h i b i t i o n of softening rate occurred at 6.3 mM calcium. For the mesocarp pieces (Fig. 5b), half maximal inhibition occurred at 1.5 mM calcium ion. These results indicated that even i n the presence of high NaCl concentrations lew calcium ion concentrations could saturate seme binding s i t e that resulted i n inhibition of texture loss. A problem with the results from these experiments was that the absolute rates of softening were highly variable from one l o t of cucumbers to another even though the pattern of responses to metal ions was very consistent. Notice, for example, that the softening rates without added calcium differ by a factor of about 15 (Fig. 5) even though the rates at high calcium concentrations were similar. In view of the inhibitory effect of calcium on softening rates, the natural calcium levels i n lots of cucumbers obtained over a two year period were compared to the softening rates i n 1.5 M NaCl, but i n the absence of added calcium. Fig. 6 shows a reasonable hyperbolic relationship between calcium level and softening rates with the model accounting f o r 97% of the experimental v a r i a t i o n . This suggested that a major reason for l o t to l o t variation i n softening of cucumber tissue was the natural variation i n the calcium content of the fruit. The "Ecfcr Box" Model as an Explanation for Softening Inhibition Now l e t us turn our attention to the question of whether i t i s reasonable to attribute the inhibition of softening by calcium ions to cross-linking of pectin molecules i n the c e l l wall according to the egg box model (21). The conditions of the calcium inhibition experiment shown i n Fig. 5b were such that i t would be expected that calcium cross-links would be relatively infrequent. I f we consider the calcium concentration which gives half maximal inhibition of softening, 1.5 mM, there was only a small proportion of calcium ions present relative to 1500 mM sodium ions. The pK of galacturonic acid residues i n pectate solutions i s about 3.6 (48) while the pH i n the experiment was 3.2 so that a large proportion of the carboxyl groups would be protonated and, therefore, uncharged. The degree of pectin methylation was 62%, which means that only 38% of the galacturonic acid residues i n the c e l l wall would be non-esterified even i f the pH were high enough for the carboxyl groups to be ionized. If i t i s assumed that the methoxyl groups i n the t i s s u e were randomly distributed, as has been reported by Anger and Dongowski (49), the selectivity coefficient between sodium and calcium ions binding to pectin w i l l be relatively small (27). Taking a l l of these factors into account i t was estimated that there would be only one calcium


132


0.05

1.00

VEGETABLES

100


d ο

0.0

0.3

0.6

0.9

1.2 1.5

0.0

0.3

[NaCl], M

0.6

0.9

1.2

1.5

[NaCl], M

Fig. 4. E f f e c t of NaCl concentration on the rate of cucumber tissue softening i n acidic conditions, a) Softening rates of cucumber s l i c e s , b) Softening rates and pectin methylation changes i n cucumber mesocarp tissue pieces. From reference 43.

1.00

0.00 20 40 60 Calcium, mM

Θ0

ç

0.00 0

20 40 60 Calcium, mM

80

Fig. 5. Effect of calcium chloride concentration on the rate of cucumber tissue softening i n 0.6% acetic acid and 1.5 M NaCl. a) Softening rates of cucumber s l i c e s , b) Softening rates of cucumber mesocarp tissue pieces. From reference 47.



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cross-link per 2700 galacturonic acid residues. I t seems d i f f i c u l t to explain such a large calcium e f f e c t on texture upon such infrequent cross-linking events i n the c e l l wall. Nevertheless, such an estimate contains many assumptions so that i t could easily be off by a considerable magnitude. Also, i t i s at least possible that i n plant c e l l walls even rare calcium cross-links could have a large effect on texture, though Deuel et a l . (50) and Doesburg (45) suggested that isolated calcium cross-linkages would not give a strong pectin gel structure. Therefore, the effects of other ions on softening rates were evaluated to assess i n another way whether egg box binding i s a reasonable mechanism to explain the e f f e c t on calcium on (^cumber tissue texture under acid conditions. The results of the addition of 10 mM levels of various ions i n place of calcium are shown i n Fig. 7. The ions are shown i n the order of increasing binding affinity to pectate based upon the data of Kohn (28. 29), except for barium and aluminum for which binding data are not available. Calcium, strontium and barium were good softening inhibitors. However, zinc, cobalt and cadmium ions, which have a f f i n i t i e s equal to or greater than calcium, showed l i t t l e or no inhibition of softening. Copper, which binds much more strongly to pectin than calcium, has considerably less of an inhibitory effect than calcium. The conclusion from these results was that l i t t l e or no correlation existed between the affinity of metal ions for pectin and their inhibition of softening. Like the estimate of calcium c r o s s - l i n k frequency, t h i s r e s u l t also appeared to be inconsistent with the egg box model. In this experiment only the a l k a l i n e earth ions, calcium, strontium and barium were good inhibitors of softening so i t appeared that the binding sites for calcium, whatever their chemical structure, were relatively specific for calcium. To t e s t t h i s idea another group of ions, the lanthanides, were tested for their a b i l i t y to i n h i b i t softening. Several lanthanide ions have been found to specifically bind with high a f f i n i t y to calcium sites i n calcium binding proteins such as calmodulin (51). Thus, in their size and electronic structure they seem to be reasonably good analogs of calcium. The lanthanide ions were added to blanched cucumber mesocarp tissue i n the presence of 1.5 M NaCl and their ability to inhibit softening was calculated i n terms of the equivalent amount of calcium required to give the same degree of i n h i b i t i o n of s o f t e n i n g r a t e s . Since a l l of the lanthanides had a calcium equivalence greater than 0 mM, they a l l inhibited cucumber tissue softening to some degree (Fig. 8). Since 10 mM concentrations of each ion were used, any calcium equivalence value greater than 10 mM meant that the ion was a better inhibitor of softening than calcium. The six lanthanides with ionic r a d i i greater than gadolinium exceeded the 10 mM equivalence level. The conclusion from these experiments was that the binding sites which affected texture were quite specific for calcium ions or calcium ion analogs rather than for ions which bound to pectic substances with high affinity. One f i n a l experiment addressed to the question of the egg box model and textural effects was to determine the a b i l i t y of cadmium ions to reverse the effect of calcium ions on softening. Cadmium ion did not inhibit softening (Fig. 7) even though Kohn (29) has demonstrated that i t binds to pectate with greater a f f i n i t y than calcium. I t also has an ionic radius nearly the same as calcium


134



0.8

0.0

1

0.0

' 2.0

' 4.0

' 6.0

"—-J 8.0 10.0

Calcium, mM Fig. 6. Relationship between the natural concentration of calcium ions i n cnacumbers and softening rates of cucumber tissue i n 0.6% acetic acid and 1.5 M NaCl. From reference 47.

1.0

Na

Mg

Ca

Sr Zn

Co

Cd

Cu

Ba Al

Metal Ion Fig. 7. Effect of 10 mM concentrations of divalent and trivalent cations on the rate of cucumber mesocarp tissue softening i n 0.6% acetic acid and 1.5 M NaCl. From reference 47.



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(52) and, o f course, the same charge as calcium. I t was p o s s i b l e t h a t cadmium d i d not i n h i b i t s o f t e n i n g because i t d i d not form c r o s s - l i n k a g e s w i t h p e c t i n i n a way w h i c h was e f f e c t i v e i n i n h i b i t i n g degradation o f the c e l l w a l l s t r u c t u r e . To t e s t t h i s p o s s i b i l i t y i t was reasoned t h a t i f cadmium and calcium ions were added together t o cucumber t i s s u e along with 1.5 M NaCl, the cadmium i o n should i n h i b i t the calcium e f f e c t on s o f t e n i n g i f the egg box model was o p e r a t i v e . S o f t e n i n g r a t e s w i t h 36 c o m b i n a t i o n s o f calcium and cadmium i o n concentrations were measured. The data were analyzed by doing r e c i p r o c a l p l o t s s i m i l a r t o those which are used t o evaluate enzyme i n h i b i t i o n by competitive, non-competitive and uncompetitive i n h i b i t o r s . In the r e c i p r o c a l p l o t ( F i g . 9), h i g h s o f t e n i n g r a t e s a r e t o w a r d t h e x - a x i s a n d h i g h cadmium c o n c e n t r a t i o n s are toward the y - a x i s . The l i n e without c a l c i u m added t o the cucumber t i s s u e shows t h a t the same s o f t e n i n g r a t e œ c u r r e d with 2 t o 80 mM cadmium added. T h i s confirmed the r e s u l t found f o r 10 mM cadmium i n F i g . 7 over a range o f concentrations. The expectation was that i f calcium was having an e f f e c t due t o egg box binding, cadmium ions should competitively d i s p l a c e calcium ions and r e v e r s e the c a l c i u m e f f e c t . T h i s would r e s u l t i n a group o f l i n e s a t d i f f e r e n t concentrations of added calcium a l l i n t e r s e c t i n g a t the y-axis a t the zero added calcium intercept. The f a c t that the r e c i p r o c a l p l o t s were p a r a l l e l t o t h e x - a x i s a t a l l c a l c i u m concentrations i n d i c a t e d t h a t cadmium was not competing w i t h the s i t e through which calcium had i t s t e x t u r a l e f f e c t . Since both ions presumably would bind t o p e c t i n , the simplest c o n c l u s i o n was t h a t calcium had i t s e f f e c t on s o f t e n i n g r a t e s by b i n d i n g a t some s i t e other than p e c t i n carboxyl groups. Summary and

Conclusions

To b r i e f l y summarize the r e s u l t s as they r e l a t e t o the egg box model, e s t i m a t e s o f a v e r y low frequency o f c a l c i u m c r o s s - l i n k s e x p e c t e d under t h e e x p e r i m e n t a l c o n d i t i o n s used, t h e l a c k o f c o r r e l a t i o n between the a f f i n i t y o f metal ions f o r p e c t i n and the a b i l i t y of those ions t o i n h i b i t softening, the apparent s p e c i f i c i t y of softening i n h i b i t i o n by calcium o r calcium analogs, and the l a c k of competition between calcium and cadmium ions, a l l appeared t o be i n c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t egg box b i n d i n g was r e s p o n s i b l e f o r the i n h i b i t i o n o f s o f t e n i n g o f cucumber t i s s u e by multivalent ions. We need t o begin looking f o r other mechanisms by which cucumber t i s s u e s o f t e n i n g can be i n h i b i t e d by c a l c i u m and c e r t a i n other ions. These r e s u l t s do not i n any way suggest that the egg box mechanism i s not a v a l i d mechanism t o e x p l a i n metal i o n binding i n p e c t i n s o l u t i o n s . I t appears, however, t h a t the egg box model cannot be extended t o explain the p h y s i c a l e f f e c t s of calcium ion addition t o cucumber mesocarp t i s s u e i n t h i s s i t u a t i o n . I t a l s o means t h a t we must be c a r e f u l i n t r y i n g t o e x p l a i n t h e t e x t u r a l e f f e c t s o f metal i o n s i n terms o f the egg box model as we study other processing s i t u a t i o n s . The egg box model may be operative i n some s i t u a t i o n s , but not i n a l l . There does not e x i s t a known a l t e r n a t i v e mechanism t o the egg box model which can e x p l a i n the metal i o n e f f e c t s which have been observed. While the experimental r e s u l t s obtained t o date argue r a t h e r s t r o n g l y a g a i n s t the egg box idea, they do not suggest an


136


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30

0.95

1.00

1.05

1.10

1.15

1.20

Ionic Radius, *k

Fig. 8. Comparison of the inhibitory effect of 10 mM lanthanide ions on cucumber t i s s u e s o f t e n i n g rates r e l a t i v e t o the inhibitory effect of calcium ion. From reference 47.

0 Γ 0.0

u

V 0.1

V 0.2

ι 0.3

i= 0Λ

1/Cd, mM

Fig. 9. Evaluation of possible competition between calcium and cadmium ion for inhibition of cucumber mesocarp softening. From reference 47.



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obvious alternative. Some possible mechanisms which can be imagined include: 1. Non-enzymatic degradation of one or more c e l l wall polysaccharides which can be specifically inhibited by calcium and calcium analogs; 2. A heat stable enzyme which degrades a c e l l wall component and which i s i n h i b i t e d by calcium; or 3. A calcium mediated structural protein, similar to the cadherins found i n animal cells (53), which i s involved i n plant c e l l interactions. The fact that i t i s now possible to experimentally affect the rates of softening processes in cucumbers by a variety of metal ions provides us new tools to investigate the chemistry of plant tissue texture. The a b i l i t y to carry out experiments with gram t o kilogram quantities of a single tissue type should aid efforts to isolate and characterize degraded saccharides from softened mesocarp. Several of the lanthanide ions, which proved to be excellent inhibitors of softening, have useful spectroscopic properties, especially their a b i l i t y to perturb NMR spectra (51). This could be a way to probe the interaction of metal ions with c e l l wall components. Questions about the generality of these texture effects i n other f r u i t s and vegetables and the i n t e r a c t i o n between metal ions and other processing variables, such as pH, are experimentally accessible with the techniques now available. Hopefully, as these experimental possibilities are exploited new ideas concerning the mechanisms of tissue softening and the effects of metal ions on softening w i l l emerge. Beyond the interesting s c i e n t i f i c questions concerning the chemistry of texture, the purpose for doing research i n this area i s to have an impact on the processing practices of the industry and ultimately an impact on the quality of products available to people. This sometimes seems to be a distant goal. However, i t should be possible to identify processing operations i n which changes can be made to improve texture retention as we look at interactions between acidification practices, the addition of sodium and potassium salts, and calcium levels i n the tissue, both the natural calcium and that added during processing. Actocwledqments This investigation was supported i n part by a research grant from Pickle Packers International, Inc., St. Charles, I l l i n o i s . Paper no. 12114 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7643. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U. S. Department of Agriculture or North Carolina Agricultural Research Service, nor does i t imply approval of other products that may be suitable. Literature cited 1. 2. 3. 4. 5.

Kertesz, Ζ. I. Canner 1939, 88(7), 26. Blair, J. S.; Ayres, T. B. Ind. Engr. Chem. 1943, 35, 85. Lenz, R. J . ; Weckel, K. G. Research Report N. 27, College of Agriculture, Univ. of Wisconsin, Sept., 1967. Mattson, S. Acta Agric. Suecana 1946, 2, 185. Sterling, C. J. Food Technol. 1968, 3, 367.


138



6.

Hughes, J . C.; Grant, Α.; Faulks, R. M. J . Sci. Food Agr. 1975, 26, 739. 7. Van Buren, J . P. In Chemistry and Function of Pectins; Fishman, M. L.; Jen, J . J., Ed.; American Chemical Society: Washington, DC, 1986; p. 190. 8. Archer, R. W. Canner-Packer Sept., 1962, 131, 28. 9. Mohammadzadeh-Khayat, Α.; Luh, B. S. J . Food Sci. 1968, 33, 493. 10. Fleming, H. P.; Thompson, R. L.; Bell, Τ. Α.; Hontz, L.H. J . Food Sci. 1978, 43, 888. 11. Thompson, R. L.; Fleming, H. P.; Monroe, R. L. J . Food S c i . 1979, 44, 843. 12. Hudson, J . M.; Buescher, R. W. J . Food Biochem. 1985. 9, 211. 13. Fleming, H. P.; McFeeters, R. F.; Thompson, R. L. J . Food Sci. 1987, 52, 653. 14. Bell, Τ. Α.; Etchells, J. L. J . Food Sci. 1961, 26, 84. 15. Buescher, R. W.; Hudson, J . M.; Adams, J.R. J . Food Sci. 1979, 44, 1786. 16. Buescher, R. W.; Hudson, J . M.; Adams, J . R. Lebensm.-Wiss. u. -Technol. 1981, 14, 65. 17. Tang, H. C. L.; McFeeters, R. F. J. Food Sci. 1983, 48, 66. 18. Etchells; J . L., Bell, Τ. Α.; Fleming, H. P. Advisory Statement: Use of Calcium Chloride to Improve the Texture of Pickles; Pickle Packers International, Inc.: St. Charles, IL. 19. McFeeters, R. F.; Fleming, H. P.; Thompson, R. L. J . Food Sci. 1985, 50, 201. 20. Van Buren, J . P. J . Food Sci. 1984, 49, 910. 21. Grant, G. T.; Morris, E. R.; Rees, D. Α.; Smith, P. J . C.; Thom, D. FEBS Letters 1973, 32, 195. 22. Gidley, M. J . ; Morris, E. R.; Murray, E. J . ; Powell, D. A.; Rees, D. A. J.C.S. Chem. Comm. 1979, 1979, 990. 23. Gidley, M. J . ; Morris, E. R.; Murray, E. J . ; Powell, D. A.; Rees, D. A. Int. J . Biol. Macromol. 1980, 2, 332. 24. Thom, D.; Grant, G. T.; Morris, E. R.; Rees, D. A. Carbohy. Res. 1982, 100, 29. 25. Morris, E. R.; Powell, D. Α.; Gidley, J . J . ; Rees, D. A. J . Mol. Biol. 1982, 155, 507. 26. Powell, D. Α.; Morris, E. R.; Gidley, J . J.; Rees, D. A. J . Mol. Biol. 1982, 155, 517. 27. Kohn, R.; Furda, I. Coll. Czech. Chem. Commun. 1967, 32, 4470. 28. Kohn, R. Pure Appl. Chem. 1975, 42, 371. 29. Kohn, R. Carbohy. Res. 1987 160, 343. 30. Knee, M. Phytochem. 1978, 17, 1257. 31. Irwin, P.; Sevilla, M.; Shieh, J . Biochim. Biophys. Acta 1984, 805, 186. 32. Irwin, P.; Sevilla, M.; Stoudt, C. Biochim. Biophys. Acta 1985, 842, 76. 33. Bush, D. S.; McColl, J. G. Plant Physiol. 1987, 85, 247. 34. Van Buren, J . R. J . Texture Studies 1979, 10, 1. 35. D a r v i l l , Α.; McNeil, M; Albersheim, P.; Delmer, D. P. In The Biochemistry of Plants: A Comprehensive Treatise; Stumpf, P. K.; Conn, Ε. E.; Tolbert, Ν. E., Eds.; Academic Press: New York, NY, 1980; Vol. 1, Chapter 3. 36. Knee, M.; Bartley, I. M. In Recent Advances i n the Biochemistry of Fruits and Vegetables; Friend, J . ; Rhodes, M. J . C., Eds.; Academic Press: New York, NY, 1981; p. 133.



11. McFEETERS


139

37. DeMarty, M.; Morvan, C.; Thellier, M. Plant, Cell Environ. 1984, 7, 441. 38. Ferguson, I. B. Plant, Cell Environ. 1984, 7, 477. 39. Jarvis, M. C. Plant, Cell Environ. 1984, 7, 153. 40. Bacic, Α.; Harris, P. J.; Stone, B. A. In The Biochemistry of Plants: A Comprehensive Treatise; Stumpf, P. K.; Conn, Ε. E.; Preiss, J., Eds.; Academic Press: New York, NY, 1988; Vol. 14, Chapter 8. 41. Stoddard, R. W.; Barrett, A. J . ; Northcote, D. H. Biochem. J . 1967, 102, 194. 42. Tepfer, M.; Taylor, I. E. P. Can. J. Bot. 1981, 59, 1522. 43. McFeeters, R. F.; Senter, M.; Fleming, H. P. J . Food Sci. 1989, In press. 44. Thompson, R. L.; Fleming, H. P.; Hamann, D. D.; Monroe, R. J. J. Texture Studies 1982, 13, 311. 45. Doesburg, J. J . Pectic Substances i n Fresh and Preserved Fruits and Vegetables; Institute for Research on Storage and Processing of Horticultural Produce: Wageningen, The Netherlands, 1965; p. 44. 46. Van Buren, J. P. J. Food Sci. 1986, 51, 131. 47. McFeeters, R. F.; Fleming, H. P. J . Agric. Food Chem. 1989, i n press. 48. Cesaro, Α.; Ciana, Α.; Delben, G.; Manzini, G.; Paoletti, S. Biopolymers 1982, 21, 431. 49. Anger, H.; Dongowski, G. Die Nahrung 1985, 29, 397. 50. Duel, H.; Huber, G.; Anyas-Weisz, L. Helv. Chim. Acta 1950, 33, 563. 51. Martin, R. B. In Calcium i n Biology; Spiro, T. G., Ed.; John Wiley & Sons: New York, NY, 1983, p. 235. 52. Shannon, R. D. Acta Cryst. 1976, A32, 751. 53. Takeichi, M. Development 1988, 102, 639. RECEIVED

May 22, 1989


Function of Metal Cations in Regulating the Texture of Acidified

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