Model studies on behaviour of pectic substances in the potato cell wall during boiling

Potato Res. 19 (1976) ." 289-303

Model studies on behaviour of pectic substances in the potato cell wall during boiling

M. J. H. KEIJBETS l, W. PILNIK-" and J. F. A. VAAL 1

Institute for Storage and Processing of Agricultural Produce (IBVL), P.O. Box 18, Wageningen, the Netherlands

z Department of Food Science, Agricultural University, De Dreyen, Wageningen, the Netherlands

Accepted for publication: 10 June 1976

ZusammenJassung, ROsumO p. 301

Summary

Potato cell walls were boiled at pH 6.1 to study the solubilization of pectic substances. It was shown that pectin was degraded by a [3-elimination reaction. Ions accelerated this breakdown, partly by a specific effect. Calcium, cupric and ferrous ions retarded the solubilization of pectin in comparison with potassium. With calcium ions optimum stability against solubilization of pectin could be obtained when similar equivalents of both calcium and pectin acid groups were present. Calcium-binding anions - citrate, phytate and malate - favoured the solubilization of pectin.

Conversion of esterified into free acid groups in the pectin structure increased the binding activity towards calcium, so that less pectin became soluble. It was concluded that calcium was complexed more strongly by pectin than by potato starch.

Introduction

Cohesion between cells is one of the main textural properties o f the cooked pota to tuber. A survey of factors which influence this and other texture characteristics has been given in recent reviews on po ta to texture (Linehan & Hughes, 1969a: Warren & W o o d m a n , 1974; Keijbets, 1974). Pectic substances in pota to cells and plant cells in general funct ion as cohesive agents in the middle lamella or intercellular layer. The role o f these pectic substances in intercellular cohesion (which we prefer to adhesion based on definitions for cohesiveness and adhesiveness as given by Szczesniak (1963)) dur ing cook ing of potatoes, however, is not clear. The reasons for this may be: the use o f inadequate methods o f (subjective) texture measurement and difficulties in pectin analysis, part icularly in po ta to tissue with its vast excess of starch ; the limited knowledge about pectin structure and function in the native cell wall and middle lamella; lack o f knowledge about the mechanisms of degradat ion and solubilization during cooking.

A few at tempts to relate amoun t s and characteristics o f extracted pectin fractions with po ta to texture have been reported. Warren & W o o d m a n (1974) calculated f rom data o f Bettelheim & Sterling (1955) a statistically significant correlat ion coefficient

289

M. J. H. K E I J B E T S , W. P I L N I K A N D J. F. A. V A A L

between pectin characteristics (degree ofesterification, calcium content and intrinsic viscosity) and intercellular cohesion determined with a method based on retained weight after cooking. Sharma et al. (1959) measured intercellular cohesion as hardness-softness with a penetrometer (puncture) and found that within a variety the greatest hardness was correlated with the highest content of insoluble pectin (plus hemicelluloses). In one of three experiments Linehan & Hughes (1969b) established a significant statistical correlation between intercellular cohesion (puncture) and a pectin fraction, soluble in a calcium sequestering agent which was thought to repre- sent the structure of the intercellular cement.

A loss of cellular cohesion of potato tissue during cooking, measured by objective instrumental methods, is rather well documented (see reviews). Some authors noticed at the same time a shift from insoluble pectic substances into water-soluble pectin (Freeman & Ritchie, 1940; Bettelheim & Sterling, 1955; Doesburg, 1961 ; Hughes & Faulks, 1972; Keijbets, 1975). During enzymic maceration of potato tissue, both phenomena also parallel each other (Keijbets, 1974). A cause and effect relationship between reduction of intercellular cohesion and solubilization of pectic substances thus is suggested.

During heating of potato tissue above 60~ the plasmalemma becomes perme- able (Personius & Sharp, 1938) and soluble substances such as ions can diffuse into the intercellular region (Bartolome & Hoff, 1972). The influence of potato constit- uents on solubilization of pectic substances, especially pectic galacturonan 1 was studied here in a model system with potato cell walls and controlled additions of various ions and starch during boiling.

Materials and methods

Isolation o f cell walls

From 2 kg of tubers (variety Bintje, sp.gr. 1.080-1.090; 1971 ; stored 4.5 months at 6 ~ with sprout inhibitors) cell walls were isolated according to Knee & Friend (1968) with some modifications. After the first washings of the disintegrated mass further unbroken cells were mechanically disintegrated in a high speed homogenizer (Bfihler, Tfibingen, FRG) (50000 rev/min) with water-cooling. The washing and blending procedures were repeated until all cells were broken and no starch grains were present. The cell wall material was freeze-dried, ground and stored at room temperature.

Treatments o f cell walls

H cell walls. Calcium and other ions were removed by treating 10 g cell walls with four volumes of 250 ml 70% ethanolic 0.6 M HC1. The cell walls were washed free of

1 The predominant building block of the main chains of the heteropolysaccharide pectin is galac- turonic acid (Pilnik & Voragen, 1970).

290 Potato Res. 19 (1976)

PECTIC SUBSTANCES IN POTATO CELL WALL DURING BOILING

chloride with 70~/o ethanol, 96~ ethanol and ether. Finally this preparation containing free carboxylic acid groups was dried in vacuo overnight.

PE sapon!/~ed H cell walls. Cell walls were incubated with orange PE (MacDonnel et al., 1945) The cell wall pectic galacturonan was almost completely saponified by this treatment. The walls were then treated as for H cell walls.

Chemicals

Pipes or piperazine-N,N'-bis-2-ethanesulphonic acid (BDH Chemicals, Ltd. Poole, England) is an acid with pK, 6.8 at 20~ Tris-pipes buffer was used for boiling studies at pH 6 - 7 because this buffer does not bind the ions Ca, Mg, Cu which were studied in these experiments for their action on pectin solubilization.

Phytic acid was prepared from sodium phytate (Kaufman & Kleinberg, 1970) by ion exchange on Amberlite IR-120 (H +).

H starch was prepared from potato starch (BDH), by a washing procedure with diluted HC1 (Winkler, 1960). Primary calcium starch (phosphate groups only half neutralized) was prepared by titration of H starch in aqueous suspension with satura- ted calcium hydroxide to pH 7 (Winkler, 1960). All other chemicals used were com- mercially available chemicals.

Boiling procedure

Potato cell walls were boiled in 0.02 M Tris-pipes buffer of pH 6.1 in the majority of the experiments. In one experiment the pH influence was investigated by boiling the cell walls in 0.02 M Tris-pipes buffers from pH 6.1 to 6.5, adjusted with 0.02 M Tris.

In another experiment the buffer concentration was varied between 0.02 and 0.3 M at pH 6.1. For boiling 50 mg cell walls were added to 10 ml buffer and refluxed on a hot plate shaker for 30 min. Ions had been added to the buffer during its preparation from a concentrated stock solution (0.3 M). When organic ions were added, the non- esterified carboxyl groups of pectic galacturonan (COO-) were neutralized generally by additional calcium or potassium hydroxide. However, because of the weak buffering capacity of 0.02 M Tris-pipes, HCI was used to adjust the mixture to pH 6.1 particularly when excessive amounts of organic anions were included in the boiling mixtures. Potato starch was added by weighing.

After boiling the mixtures were cooled, filtered over folded paper and the soluble pectic substances in the filtrate determined.

Analyses

Pectin. Solubilized pectic substances, galacturonan and neutral sugars, were determined by a combination of the carbazole and phenol-sulphuric acid reactions

Potato Res. 19 (1976) 291

M. J. H. K E I J B E T S , W. P I L N I K A N D J. F. A. VAAL

(Keijbets & Pilnik, 1974a). Boiled starch had to be removed in two steps before pectin analysis: by freezing-thawing and by enzymic conversion of the most soluble part into gluconic acid, which does not interfere in the colorimetric analyses. For the latter step amyloglucosidase was added to hydrolyse starch to glucose (2 h, 30~ pH 4.0), which was oxidized to gluconic acid by glucose-oxidase (another 4 h, 30~ pH 7.5) (enzymes available from Boehringer, Mannheim, FRG).

Pectin depolymerization by ]3-elimination (transelimination) was measured with the periodate-thiobarbituric acid (TBA) test, which accounts for unsaturated galact- uronosyl residues. The reaction was mainly carried out according to Weissbach & Hurwitz (1959) by application of a prolonged periodate oxidation step of 40 min (Warawdekar & Saslaw, 1959) The.results are expressed as absorbance measured at 552 nm or as absorbance per unit solubilized pectic galacturonan (= relative A552 number). The latter seemed necessary because the periodate-TBA molar extinction coefficient depends on degree of polymerization of unsaturated pectin degradation products (Voragen, 1972; Keijbets, 1974). The molar extinction coefficient increases with degree of polymerization.

Pectic galacturonan content and degree of esterification with methanol were assayed for cell wall material with Cu 2 + ion exchange (Keijbets & Pilnik, 1974a).

The H cell wall preparation contained 16.0~ or 58~ esterified pectic galacturonan (365 ~tmol free carboxylic acid groups or COO- per gram cell wall material). Saponi- fled H cell wall preparation contained 14.7'!~, of 2'!,', esterified galacturonan (818 Ixmol COO-/g).

Starch, P and Ca. The phosphate content of potato starch was also determined by Cu 2 + ion exchange (Keijbets & Pilnik, 1974a), giving results, identical to poten- tiometric titration (Winkler. 1960). Calcium content was assessed using exchange of these ions with diluted HCI and analysis with a colorimetric calcium method of Milligan & Lindstrom (1972). However, 0.2 M sodium hydroxide (low calcium, 5 p.g/g) was used to produce an alkaline pH since it was found to be more effective than sodium sulphide.

BDH starch contained 50.4 bteq P and 12.0 taeq Ca per gram. H en Ca starch both contained 51.4 p.eq P/g whilst Ca starch also contained 26.8 eq Ca/g.

Standard deviations olanalyses (s)

The experiments generally were not duplicated, but their reproductibility was satis- factory. For seven repeated experiments the mean ~ solubilized pectic galacturonan was 46",, with s = 1.6~176 The mean relative A552 number was 0.47 with s = 0.026.

Results and discussion

Mechanism ol'pectin degradation It is a well established fact that esterified pectic galacturonan during heating at a pH

292 Potato Res. 19 (1976)

P E C T I C S U B S T A N C E S IN P O T A T O C E L L W A L L D U R I N G B O I L I N G

Fig. 1. Periodate-TBA absorbance spectrum of pectic galacturonan solubilized from potato cell walls during 60 min of boiling (non-esterified pectic carboxylic acid groups COO- - neutralized by potassium). abso rbance

0.15 V oc-o

I oo o / \ - \

o.o i \ . o-_.o.

wave length (n m)

Absorbance Absoleltion , 4 / l s o r p l i o n : Wave length l'l,'ellenliinge - L o t t g l t e l l F (f ottde.

Abh. 1. Das Pet jodat-TBS-AbsotT~tionsspektrum yon heim Kochen w~Thrend 60 Mitmten aus den Kartq[fi'lzelhl'dnden herausgel6stem pekt inisehem Galaktm'onan : nieht veresterte Pektinkarhons6ure- gruppen - CO O durch Kalium neutralisiert). Fig. 1. Spectre cf absorption (test pt;riodate- TBA ) des peethles soluhh's ( galaeturonanes ) extraites des membranes eelhdaires des tissus de tubercules par une c'ltissoti de 60 minutes ( /es grottpements carbox.l'liques C O 0 - des peetines ilo/1 estOr([iOes Otant neutralisOs pal" rht lwtassiurn).

beyond 4.5 will be depolymerized by 13-elimination (Albersheim et al., 1960). The natural pH of potato tissue, 5.5-6.5 (Burton, 1966), thus is rather suitable for this type of degradation. Typical periodate-TBA spectra after boiling potato cell walls at pH 6.1 confirm this view. An example of such a spectrum is shown in Fig 1.

The reaction rate of [3-elimination degradation at boiling temperature increases as hydroxyl ion concentration or pH increase because hydroxyl ions initiate the reaction (Neukom & Deuel, 1958). Indeed during boiling of cell walls at pH 6. I to 6.5 absorbance at 552 nm (indicative of unsaturated bonds) and solubilization of pectic galacturonan increased with increasing pH (Fig. 2). The absorbance per unit solubilized pectic galacturonan in the periodate-TBA test increased from 0.38 (pH 6.1) to 0.45 (pH 6.5).

An increase in absorbance at 552 nm and in the absorbance per unit solubilized pectic galacturonan (relative A552 number) might have been caused by an increase of the molar extinction coefficient. This assumption, however, is unlikely, because this would mean that the more pectic galacturonan solubilized the larger the molecules. Therefore, it can be concluded that a rise o fpH favoured [3-elimination and solubilization of pectic galacturonan. The 13-elimination reaction finally only proceeds when galacturonan carboxyl groups at C6 are esterified. Cleavage occurs next to an esterified group, which by its electron-withdrawing character renders the proton at C5 sufticiently acidic (Keijbets & Pilnik, 1974b). The DE (degree of esterification) of cell wall galacturonan (58",,) was fairly suited for [3-eliminative degradation during boiling.

Potato Res. 19 (1976) 293

% s o l u b i l i z e d p e c t i c g a l a c t u r o n a n 7O

o

60 o . ~ ~ . . . j ~ ~ 5 0 A b s o r b a n c e

5 5 2

4 0 0 . 2

3 0 . x x

20 x ~ • 0.1

I 0

0 I I I I I 6.0 6.1 6.2 6.3 6.4 6.5

p H

M. J. H. K E I J B E T S , W. P I L N I K A N D J. F. A. V A A L

Fig. 2. Effect of buffer pH on periodate-TBA absorbance (x) and solubilization of pectic galacturonan (o) during boiling of potato cell walls in buffers of increasing pH (adjusted with 0.02 M Tris).

Abb. 2. Einfluss des Puffer-pH aztf die Peljodat-TBS- Absorption (x) und die LSslichkeit yon pektinisehem Galakturonan (o) wdhrend des Kochens yon Karto/]elzellwdnden in PuJferliSsungen mit steigendem pH (eingestellt mit 0,02 M Tris), Fig. 2. Effet du pH de la solution tampon sur le test pr (x ) et la solubility; des pectines (galacturonanes) (o) durant la cuisson des membranes cellulaires dans des solutions tampons de pH croissant (ajust~; avec 0,02 M Tris).

Cations Calcium and magnesium are the major divalent cations in potato tubers (Burton, 1966; Keijbets et al, 1976), while copper and iron are only present in trace quantities. Calcium, cupric and ferrous ions showed a pronounced ability to keep pectic galacturonan insoluble within the walls at ratios cat ion/COO- increasing from 0 to 1 (Fig. 3). When all non-esterified galacturonan carboxyl groups were neutralized (ratio >~ 1) this ability gradually disappeared for calcium. The level of solubilization for potassium, the most frequent cation in the potato, was clearly higher, but potassium had no insolubilizing ability at all, since at ration K + / C O 0 - = 1 similar results were obtained with or without potassium (cf. Fig. 3 and pH 6.1 in Fig. 2). Fig. 4 shows that the periodate-TBA absorbance at 552 nm for calcium even surpassed that for potassium. The relative A552 number increased from 0.47 to 1.21 for Ca and only from 0.41 to 0.76 for K when the ratio cat ion/COO- rose from 1 to 100. Bearing in mind the arguments used in the previous section (mechanism of pectin degradation) for interpretation of periodate-TBA figures, it is clear that increasing the ratio ofcalcium ions beyond l-2 favours [3-elimination of pectic galacturonan during boiling. The enhanced depolymerization .of galacturonan ultimately destroyed the demonstrated ability (Fig. 3) of calcium to retard solubilization of galacturonan when compared with potassium. Increasing levels of potassium ions also favoured [3-elimination leading to slowly increasing loss of pectic galacturonan from the cell walls (Fig. 3). For magnesium similar results as for potassium were found with res- pect to o;, solubilized galacturonan whilst stimulation of [3-elimination ressembled

oj that of calcium. At the ratio Mg 2 +/COO- = 1 and 10, 54 and 52o, respectively of pectic galacturonan became soluble during boiling, while absorbance at 552 nm was 0.093 and 0.147, respectively and the relative A552 number 0.33 and 0.56, respectively.

294 Potato Res. 19 (1976)


Fig, 3. Effect of presence of cations on solubilization of pectic galacturonan during boiling of potato cell wails Quanti~y of cations expressed as ratio of equivMents cation to equivalents pectin COO- . Anions indicated between brackets. Equiv. anions = equiv, cations.

~ solubilized pectic galacturonan

80 [ K*(citrote 3-) 70 I - ~ - " �9 -

30 1 ~" "g~'~6~o /

~ 01.2 015 4 2 5 10 210 50 100 ratio cation/CO0-

Abh. 3. s der Amresenheit yon Kationen at{/die Laslichkeit yon pektinischem Galakmronan w6hrend des Kochens yon Kartq/./elzellw&ulen. Menge tier Kationen, ausge~hOckt ab Verhgihnis yon Kc~tionendquivalenten :u Pektin CO0--,4equivalenten. ,4nionen angegehen m Klammer,l. Aequiv. Anionen = ~Yquiv. Kationen. Fig. 3. E[l~'t de la prOsence de cations 3ur l(l soluhilitO des pectines (galacturonanes) ~htrant la cuisson des membranes eelhdaires. Taux de cations exprimd dana le rapport cations Oquivalents-peetines C O 0 - Oquivalents. Anions huliquOs entre parenthOses. Anions Oquivalents = cations ~;quivalents.

Fig. 4. Periodate-TBA absorbances during boiling of potato cell walls as in Fig. 3.

Absorbance 552 0.3

0.2

/ o Ca2*(C i-)

K, (citrate3-) o ~ / "

-e2+~so >) / ' / o ~ # : - - -~_7_ . . . . . . . . . ~ "

o o

.I 0.2 0.5 I 5 I 20 50 100 ratio cat ion/CO0-

,466. 4. Peljodat- TBS-Absorption w6hrend des Kochens yon l<artq[lelzelhrgmden wie in A bb, 3. Fig. 4, Spectre dabsorption du lest p&iodote-TBA z&rant la ctlissott de.~ membranes uellulairea. comme pour la fig. 3,

Potato Res. 19 (1976) 295

M. J. H. KE1JBETS, W. P I L N I K A N D J. F. A. VAAL

These results confirm earlier findings that several ions stimulated to different extents [3-eliminative breakdown of boiling pectin solutions (Keijbets & Pilnik, 1974b).

Nevertheless, calcium ions have a large capacity to insolubilize pectic galacturonan, even when this is progressively depolymerized. This may be elucidated by showing that at a level of 50~ solubilized pectic galacturonan (Fig. 3), galacturonan was depolymerized extensively in the presence of Ca ions but only to a small extent in the presence of potassium ions (cfratio Ca 100 and K I in Fig. 4). Pectic galacturonan showed optimum stability during boiling at ratio Ca 2 + / C O 0 - = 1-2 (Fig. 3), or when all charges on the pectin molecule were neutralized. This means that the presence of only a few "calcium bridges" will not give maximum stability and that the presence of 'microcrystalline junction zones" (Rees, 1969, 1972) in cell wall and middle lamella describes more appropriately the function of calcium in insolubility of pectin gel structures. An often supposed role of magnesium in insoluble pectin structures ( 'protopectin') (Joslyn, 1962; Hoff & Castro, 1969), similar to that of calcium, could not be established in these experiments.

A n i o n s

Three anions found in potato - citrate, malate and phytate - were investigated in a system in which all pectic carboxyl groups were neutralized by calcium ions. These anions were added as K salts in onefold to tenfold excess.

Citrate and malate chelate calcium (Martell & Calvin, 1962), whereas phytate pre-

Fig. 5. Influence of a,lions on solubilization of pectic galacturonan during boiling of potato cell walls. All pectin COO- groups neutralized by calcium (equiv. COO- = equiv. Ca-" +). Ratio aoion/ COO- expressed as in Fig. 3. Cation is potassium (equiv. K + = equiv, anion). ~ solubil ized pectic go lac turonan 60 " ci trote3"

~ - "~ ' "~x phy tate12"

30 ~ .o chlor ide

2O

I0

I ~ i

o 5 6 ra t io o n i o n / c o o -

Abb. 5. Eh!lluss yon Anionen auJ die L6slichkeit von pekt inischem Galak turomm wdhrend des Kochens yon Zellw&lden. Alle P e k t i n - C O O - - G r u p p e n dutch Calcium (dquiv. C O 0 - = dquiv. Ca 2 +) neutralisiert. Verhdlmis An ion~CO0 - dargestellt wie m Abh. 3. Kation ist Kalium ( dquiv. K + = dquiv. Anion). Fig. 5. h{/luence des anions sur let solubilitt; des pectines (galacturonanes) &o'ant la cuisson des membranes celhdaires. Tous les groupements C 0 0 - sont neutral is& par du calcium ( C O 0 - c;quivalent = Ca z + #quivalent). Rapport a n i o n s ~ C O 0 - exprime; comme pour f ig . 3. Le cation est le potassium ( K + #quivalent = anion #quivalent).

296 Potato Res. 19 (1976)


cipitates calcium at a pH > 5.4 (Kaufman & Kleinberg, 1971). Citrate, phytate and malate - in this order - favoured the solubilization of pectic galacturonan (Fig. 5) compared to chloride which had no effect. The removal of calcium from its binding sites on the galacturonan probably aided in this process. However, a second process is suggested because of the linearly increasing curves for citrate and malate and because citrate surpassed the 50-55~ level of solubilized galacturonan expected on removal of all the calcium ions from their bound sites on the pectic galacturonan (Fig. 3). Indeed, in an experiment without calcium ions, citrate ions proved to stimu- late 13-eliminative breakdown resulting in enhanced solubilization of pectic galacturonan (Fig. 3 and 4). This finding confirms earlier findings of Keijbets & Pilnik (1974b) that citrate and other anions stimulated [3-elimination degradation of boiling pectin.

The calcium binding capacity of organic acids compared to pectic galacturonan has been studied by some workers and good calcium binding activities have been established for citrate (Deuel et al., 1957; Molloy & Richards, 1971). However, the selectivity for calcium will depend on pH and DE (Kohn & Furda, 1967). At 80'~,, DE the latter authors found no longer selective binding of calcium. These attthors also established a decreasing stability of calcium pectinate in solutions of increasing potassium chloride concentration, possibly caused by ion exchange. The results shown here in Fig. 5 do not demonstrate a similar influence of increasing concentration of potassium chloride on solubilization of pectic galacturonan from tSotato cell walls.

Decrease o/'degree o/'ester~/ication When compared with the original H cell wall preparation de-esterification of the wall with pectinesterase to 2"i, DE increased the binding activity of pectic galacturonan towards calcium ions (cf. Fig. 3 and Table 1). At ratio Ca/COO- 1 in the H cell wall preparation 30~ of the pectic galacturonan had been solubilized after boiling (Fig. 3). After de-esterification this decreased to 15",, (Table 1 ; ratio Ca now 0.44 because of liberation of carboxyl groups) and even to 4". with additional Ca ions (Table I ; ratio Ca 1). It should be mentioned, moreover, that as an effect of repeated washings (for replacing all ions by H +) the starting level of solubilization in the de- esterified wall had been raised to 72". compared to 50-55~ in the original H cell wall preparation.

Upon addition of organic anions, at two levels of neutralization of carboxyls by calcium, only moderate increases of solubilization of pectic galacturonan were encountered (cf. Table 1 and Fig. 5). This finding again indicates the increased selectivity of pectic galacturonan towards calcium at low DE (Table 1). Phytate seems to be more (or equally) effective in calcium binding than citrate in these experiments particularly at the lower calcium neutralization level.

Ionic strength That the effect of ions on 13-elimination of pectin (Keijbets & Pilnik, 1974b) must be

Potato Res. 19 (1976) 297

M. J. H. KEIJBETS, W. PILNIK AND J. F. A. VAAL

Table I. Influence of ions during boiling on the solubilization of pectic galacturonan from PE saponified cell walls.

Ratio L Cation Ratio Anion* Additional-" Solubilized 3 cation/COO- anion/Ca-' + CI -,/CO0- galacturonan

0 0.44 0.44 0.44

.44 '.44 .44

73 K Cl - 72 Ca 1 Cl - 15 Ca 5 citrate a- 0.8 12 Ca 10 citrate a - 1.3 14 Ca 5 phytate i ,, - 1.4 18 Ca 10 phytate l : - 2.3 23 K CI - 74 Ca 1 C1 - 4 Ca 5 cit rate a - 1.9 4 Ca 10 citrate a - 2.4 15 Ca 5 phytate ~-'- 2.9 4 Ca 10 phytate ~ -" - 4.7 12

* The organic anions were accompanied by equivalent amounts of K + - Die organischen Anionen waren yon gleichwertigen Mengen K + hegleitet - Les anions organiques ~;taient accompagn~;s p a t des quantitds Oquivalents de K +

Verhiiltnis - Rappor t , 2 Zusdtz l iches - Addi t ionnel . 3 Geh'istes - Soluhle

Tabelle 1. Einfluss von lonen aufdie L6slichkeit yon pektinischem Galakturonan aus PE-verseiften Zellw/inden w~hrend des Kochens. Tableau 1. Influence des ions durant la cuisson sur la solubilit~ des pectines (galacturonanes) pro- venant de membranes cellulaires saponifiees par PE.

part ly ascribed to an unspecific influence of ionic strength, was shown by results of increasing molar i ty of buffer dur ing cell wall boi l ing (Fig. 6). Between 0.02 and 0.1 mol/ l i t re a sharp increase of solubi l izat ion of pectic ga lac tu ronan was observed. At higher concen t ra t ion the solubi l izat ion became stat ionary. The use of a di luted buffer th roughout the experiments, therefore, minimized the inf luence of buffer concent ration.

S t a r c h

Pota to starch was added to cell wall material in boi l ing exper iments in such quant i t ies as starch and pectic ga lac tu ronan roughly appear in pota to tissue. Pota to starch is characterized by or tho-phospha te groups, main ly b o u n d to C6 in some glucose residues of the amylopect in fract ion (Richter et al., 1968). Of the two remain ing acid groups one is fully neutra l ized by cat ions at pH 5 - 6 (Winkler , 1960). Ca lc ium and magnes ium probab ly are the p r e d o m i n a n t cat ions (Richter et al., 1968).

H starch (no cat ions bound) exerted no influence on the control level of 50~ solubi l izat ion of pectic ga l ac tu ronan (Fig. 7; cf. Fig. 3). However, the addi t ion to this

298 Potato Res. 19 (1976)


% solubilized pectic galacturonan 8O

- 0

0 / o

70

60

50

40

30

20

10

x ~ /

x

I 0.1

u

Absorbance 552 0.3

x ~ X 0.2

- - 0.1

I I 0 0.2 0.3

molar i ty Ir is-pipes pH 6.1

Fig. 6. Effect of buffer concentration on solubilization of pectic galacturonan (o) and periodate-TBA absorbance (x) during boiling of potato cell walls.

Abh. 6. Ein/luss der Pl(fferkonzentration ~tl~/ die Lgslichkeit yon pektinischem Galakturonan (o) und Petjodat-TBS-Absorption (x ) wdhrend des Kochens yon Kartof[elzellwdnden. Fig. 6. Effet de la concentration de la solution tampon sur la solubilit6 des pectines (galacturonanes) (o) et I'ahsorption du test p&iodate- TBA (x) chwant la ('ltissotl ds membranes celhdaires.

mixture of calcium ions at ratio Ca 2 + / C O O - = 1 did limit the solubilization to about 30%.When boiling cell walls with Ca starch and BDH starch (52 and 24"i; of phosphate neutralized by calcium, resp.) less than 50'!,, of pectic galacturonan was solubilizied (Fig. 7). Calcium ions must have been transferred from starch phosphate to carboxylic acid groups of galacturonan, by which they obviously are more strongly complexed. It was expected that Ca starch would give a lower level of galacturonan solubilization than BDH starch (less Ca), but this was probably obscured by a different gelling and boiling behaviour of these two starches.

In these experiments no indications were found for other interactions between starch and pectic galacturonan than transfer of ions from starch to galacturonan. Linehan & Hughes (1969b) suggested that amylose chains might act as a cement between potato tuber cells by formation of hydrogen bonds with polysaccharides of the cell wall. However, the results presented in Fig. 7 for H starch do not indicate hydrogen bonding between starch and cell wall polysaccharides leading to obviously less solubilization of pectic galacturonan.

Conclusions

The b e h a v i o u r o f the pect ic subs tances in the p o t a t o cell wall dur ing boi l ing at pH 6.1 - within the no rma l p o t a t o p H range - is inf luenced to a m a r k e d extent by the

presence o f ions.

Potato Res. 19 (1976) 299


% solubil ized pectic galacturonan 6 0 -

5 0

4 0

30

20

10

0200

o o o o o H - s t a r c h

x x - x x x C o - s t a r c h ~ - - . - - _ . A .._.___ - - - - - - .

" - - - - - - - o ~ "'------ zx B D H - s t a r c h

�9 ~ * ' ' " - ~ H - s t a r c h . C a 2 .

I , I ~ ] 300 4 0 0 5 0 0

s ta rch (mg)

Fig. 7. Influence of potato starch on the solubilization of pectic galacturonan during boiling of potato cell walls. H starch + Ca-'+: equiv. Ca -'+ = equiv. CO0-.

Abb. 7. Eil~/luss yon Karto[.]elstdrke at~l die L6sl ichkei t yon pek th l i schem Galak- turonan wdhrend des Kochens yon Kar- tq{lelzellwdnden. H S tdrke + Ca 2 + dquiv. Ca'- + = 6quiv. C 0 0 - . Fig. 7. b!/luence de I'amidon sur la solubility; des pect#Tes (galacturonanes ) durant l(1 cuisson des membranes cellulaires. H amidon + Ca'- + : Ca'- + 6qui- valent = C O 0 - Oquivalent.

Two processes seem involved in the solubilization of the pectic material, i.e. galacturonan. The polymer molecules are depolymerized by chain splitting via 13- elimination. Once the chain length of the degraded parts is small enough and the molecules are no longer bound in the framework of the cell wall gel (Rees, 1969, 1972) by cations, pectic galacturonan is (partly) solubilized.

Increasing pH and increasing concentrations of ions, including ion-specific effects, favour the 13-eliminative degradation of pectic galacturonan in the potato cell wall.

The ions differ in their binding activity towards the pectic galacturonan as well. Of the major potato cations calcium in particular stabilizes the pectin structure in the cell walls. When all charges on the galacturonan are neutralized by Ca, the solubilization during boiling is at a minimum. The stabilization of pectin, due to binding of calcium ions, surpasses the counteracting influence of depolymerization of galacturonan. Neither potassium nor magnesium ions exert a similar influence as calcium.

The solubilization of pectic galacturonan is increased by the calcium binding anions, citrate, phytate and malate. The insolubility, on the other hand, is reinforced by producing more free acid groups on the pectin molecule by saponification. The calcium binding activity of pectic galacturonan then becomes higher.

Potato starch only influenced the level of solubilization of pectic galacturonan by interference of the ions bound by the starch phosphate. Hydrogen bonds between the two polysaccharides do not play a role.

300 Potato Res. 19 (1976)


Zusammenfassung

Modelluntersuchungen iiber das Verhalten yon Pektinsubstanzen in der Karto[.felzell- wand wiihrend des Kochens

Der Verlust der interzellularen Koh~ision, ein Hauptmerkmal der Textur gekochter Kartof- feln, ergibt sich aus dem Verlust der verbinden- den Eigenschaften des interzellular koh~isiven Materials, d.h. der Pektinsubstanzen. In einem Modellversuch wurde der Einfluss von Kartof- felbestandteilen auf die L6slichkeit von Pektin durch Kochen von isolierten Zellw~inden mit verschiedenen Ionen und St~rke untersucht.

Der Mechanismus des Pektinabbaus bei pH 6,1 wurde als eine 13-Elimination angesehen, was dutch ein typisches Perjodat-TBS-Spektrum (Abb. 1) und durch eine erh6hte Reaktionsge- schwindigkeit bei erh6htem pl--I (Abb. 2), die sich in 16slicherem Pektin nach dem Kochen auswirkte, bestfitigt wurde.

Calcium-, Kupfer- und Eisenionen verzOger- ten, im Vergleich zu Kalium, die L6slichkeit von Pektin (Abb. 3). Magnesium verz6gerte die LOs- lichkeit nicht. Calcium jedoch erh6hte, ver- glichen mit Kalium, auch die Reaktionsge- schwindigkeit der [3-Elimination (Abb. 4). Diese gegens~itzlichen Wirkungen yon Calcium zeigen die F~ihigkeit dieses Ions, das Pektin in der Kar- toffelzellwand in einem unl6slichen Zustand zu halten.

Optimale Stabilit~it gegen die Aufl6sung yon Pektin wurde bei einem Verh~iltnis von Calcium zu Pektins~iure~iquivalenten von 1 2 erzielt. Zugabe von Calciumbindenden Anionen, zum Beispiel Zitrat, Phytat und Malat, erh6hte die L6slichkeit des Pektins aus der Zellwand (Abb. 5). Sowohl Kationen wie Anionen beschleunig- ten den 13-eliminierenden Abbau, zum Teil dutch eine unspezifische Wirkung der Ionen- st~irke. Bei Erh6hung der Konzentrat ion des Puffers erh6hte sich denn auch die L6slichkeit des Pektins wfihrend des Kochens (Abb. 6).

Fast vollst~indige Verseifung des zu 58u,, veresterten Zellwandpektins dutch eine Pek- tinesterase (PE) erh6hte die Affinit~it ffir Cal- ciumionen. In Anwesenheit von Calcium wurde weniger Pektin 16slich, w~ihrend die Calcium- bindende F~ihigkeit der organischen Anionen ebenfalls abnahm (Tabelle 1).

Kartoffelstf.rke ohne gebundenes Calcium fibte keinen Einfluss auf die Pektinl6slichkeit aus (Abb. 7). Wenn Calciumionen in der St~irke vorhanden waren, wurden sie als teilweise in die Zellw~inde transferriert angesehen, was durch eine Abnahme der L6slichkeit von Pektin belegt wurde.

R6sum6

Etude sup" le comportement des substances pectiques dans la membrane celhdaire des tissus de pomme de terre pendant la cuisson

La perte de la coh6sion cellulaire, caract6ristique principale de la texture de pommes de terre cuites, r6sulte de la perte des propri6t6s du ci- ment responsable de l 'adherence intercellulaire (substances pectiques). Dans cette 6tude. l'influence des consti tuants du tubercule sur la solubilit6 des pectines a 6t6 observ6e sur des membranes cellulaires isolees, pendant la cuisson, en prdsence de divers ions et d 'amidon.

Le m6canisme de la d6gradation des pectines �9 h pH 6.1 a 6t6 assimil6 /l une r6action de [3- 61imination, ce qui est confirm6 par le spectre

typique du test p6riodate-TBA (Fig. l) et par l 'augmentation du taux de d~gradation en fonction de 1"61+vation du pH (Fig. 2), condui- sant ",i une teneur plus importante en pectines solubles apr~s la cuisson.

Les ions calcium, cuivre et fer, diminuent la solubilit6 des pectines par rapport au potassium (Fig. 3). Le magn6sium n'a pas cette action.

Le calcium, cependant, a aussi augment6 le taux de [3-61imination en comparaison avec le potassium (Fig. 4). Ces effets oppos6s mettent en 6vidence l 'aptitude de cet ion "5. maintenir

Potato Res. 19 (1976) 301

les pectines dans un 6tat insoluble au niveau de la membrane cellulaire.

L 'opt imum de stabilit6 des pectines a dr6 ob- tenu avec un rapport calcium-pectines acides de 1-2. L'addit ion d 'anions organiques s6que- strant le calcium, tels le citrate, le phytate et Ic malate, augmente la solubilit+ des pectines de la membrane cellulaire (Fig. 5). Cations et anions ont acc~l&6 ta r6action de [3-elimination, en pattie par un effet non sp6cifique de force ioni- que. L'~ldvation de la molarit6 des solutions- tampons augmente la solubilite des pectines durant la cuisson (Fig. 6).


La saponification presque totale des 58',/, de pectines est~rifiees, contenues dans la membrane cellulaire, par une pectinest6rase, augmente leur affinit6 pour le calcium. En pr6sence de cet ion les pectines deviennent moins solubles tandis qt, e l'effet s6questrant des anions organiques, vis-il-vis du calcium, diminue 6galement (Ta- bleau 1 ).

L 'amidon libre de calcium n'a pas d'effet sur la solubilit6 des pectines {Fig. 7). Quand l 'ion calcium est li6/~ l 'amidon il peut ~?tre partielle- ment transf6r6 b, la membrane cellulaire et provoquer une diminution de la solubilit6 des pectines.

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Documents

Model studies on behaviour of pectic substances in the potato cell wall during boiling