Potato Res. 17 (1974) : 169-177

Some problems in the in potato tuber tissue

analysis of pectin

M. J. H. KEIJBETS I and W. P I L N I K 2

Institute for Storage and Processing of Agricultural Produce (IBVL), Wageningen, the Netherlands 2 Department of Food Science, Agricultural University, Wageningen, the Netherlands

Accepted for publication: 3 August 1973

Zusammenfasstmg, R~sum~ p. 175

Summary

Simultaneous determination of the degree of esterification and pectin content, without extraction, by Cu 2+ ion exchange is described. The method, based on the high affinity of Cu ~+ for pectic car- boxylic acid groups, is applied to potato tuber tissue and potato, tuber cell wall material, and the results are compared with a combination of pectolytic extraction for pectin content and methanol determination after saponification and distillation. Cu 2+ ion exchange appeared a valuable method for potato cell wall analysis. During analysis of potato tissue phosphate covalently bound to starch interferes in ion-exchange and leads to erroneous results. A means of correction is given for inter- ference of neutral sugars in the carbazole test after pectolytic extraction of pectic substances.

Introduction

Pectic substances play an impor tant role in several plant tissues by their presence in different parts of the cell wall. Here they affect the structural properties and contribute largely to the firmness and coherence of the tissues. Because they can be degraded under food processing conditions, pectic substances also bear an important relation- ship to textural problems in food technology.

Dur ing our work on the influence o f pectin on pota to cooking quality, we needed a reliable method for the determination of pectin content and the degree of esterifica- t ion ( = DE) o f pectin o f pota to tissue.

Determinat ion o f the pectin content o f plant material has been achieved by an already classic method of McCready & M c C o m b (1952), based on extraction and degradat ion of pectic substances by E D T A and pectinase after saponification. The pectic substances are measured colorimetrically with a modification of the Dische (1947) carbazole test ( M c C o m b & McCready, 1952). This procedure was again modified for pota to pectin anaIysis (Potter & McComb, 1957).

The EDTA-pect inase extraction o f pectic substances does not permit the use of the extracted but degraded pectin for determination of the DE. On the other hand it is extremely difficult to extract the pectin complex out of plant tissue without degrada- tion and change in DE.

169

M. J. H. K E I J B E T S A N D W. P I L N 1 K

Hence we looked for a method to measure both pectin content and DE in a simul- taneous procedure without extraction. Taking into account the fact that application of decarboxylation of uronic acids and methoxyl determination by a Zeisel procedure are likely to be reliable only for extracted pectin (Doesburg, 1965), titrimetry and ion exchange seemed the methods of choice.

After preliminary experiments a direct titrimetrical method (Gee et al., 1958) was discarded, because the high starch content of potato tissue leads to erroneous results by alkali absorption during saponification? Ion exchange with sodium acetate (Raunhardt & Neukom, 1964) was also rejected, because of experimental difficulties.

Wunsch (1952) showed that the affinity of Cu 2+ ions for pectin is very high. From pectin solutions Cu-pectinate is precipitated stoichiometrically even at 95 ~ DE and at low pH. On this basis Tibensky et al. (1963) devised a method for analysis of DE of pectin solutions, but surprisingly they did not describe the usefulness of their method for the determination of the pectin content of solutions. We adapted the method of Tibensky et al. for ion exchange and compared the results with extraction by pecto- lytic enzymes for pection content, combined with methanol determination for DE. After liberation of the esterified methanol and destillation, methanol can be deter- mined colorimetrically according to Wood & Siddiqui (1971), although gaschromato- graphical analysis after conversion to the nitrite ester is also a convenient method (Bartolome & Hoff, 1972; Litchman & Upton, 1972).

Experimental

Preparation of samples For the Cu z + ion-exchange analysis alcohol-hTsoluble solids (---= AIS) of tissue are always used. These are prepared by slicing 2-3 kg of peeled potato tubers (variety Bintje was used in the experiments reported here) and taking a sample of 100 g, which is divided into sub-samples of 10 g. These sub-samples are blended with five volumes of 96 % ethanol in a Biihler homogenizer at moderate speed. The solids are collected by filtration through a glass filter (porosity 15-40 ~xm) and washed five times with 50 ml 70 % ethanol.

Potato cell wall material is prepared from freeze-dried potato powder. 20 g of powder are washed on cheese-cloth thoroughly with water to remove starch granules. The residue is disintegrated in the Btihler homogenizer at maximum speed (50000 rev/min) with water-cooling.

After washing out the starch repeatedly on cheese-cloth, disintegration and washing are continued until all cells are broken and starch can no longer be detected under the microscope. The cell walls are freeze-dried.

i After completing this manuscript, a new titrimetric method of pectin analysis came to our attention (D. S. Warren and J. S. Woodward, J. Sci. Fd Agric. 24 (1973) 769-777). Applied to potato tuber tissue, this method will probably suffer from the same disadvantages as mentioned here, notably alkali absorption by starch, and as discussed in the section Results and discussion, interference by phosphate groups attached to starch.

170 Potato Res. 17 (1974)

A N A L Y S I S OF P E C T I N IN P O T A T O T U B E R T I S S U E

Cu z + ion exchange The procedure is carried out on the same glass filters used for AIS preparation, with 10 g fresh weight. The AIS are mixed with 75 ml 1 ~ copper sulphate, which is sucked off after 15-30 min, and another 75 ml added for the same time. During this treat- ment the pH must be under 4, being adjusted if necessary with dilute hydrochloric acid.

The physically adsorbed copper ions are washed out repeatedly with water until the filtrate shows a negative reaction for copper, using test sticks, Quantofix Cu+/ Cu 2+ (Macherey-Nagel). The chemically bound Cu 2+ ions are exchanged for I-[ + ions with 0.6 N HC1 in 70~o ethanol (3 • 50 ml, 5 rain). Before this exchange of Cu 1+ ions, the residue on the glass filter is washed with 35 and 7 0 ~ ethanol (75 ml) to avoid lumping.

The residue is then saponified with 0.1 N NaOH in 60~o ethanol (75 ml, 1 h), washed once with 30 ml 1 N acetic acid in 70 ~o ethanol and subsequently with 70 ~o ethanol several times. The ion exchange procedure with. Cu 2 + has then to be repeated.

The filtrates with the exchanged Cu 2+ ions are made up to 500 ml after addition of 100 ml 2 M NH4OH. The Cu 2 + content is analysed according to a slight modification of the method described by Fries. To I0 ml solution (pH between 8 and 9), 5 ml 0.5 ~o cuprizon (oxalic acid-bis (cyclohexylidene hydrazide)) in 50 ~ ethanol is added and the volume made up to 50 ml. The cuprizon solution is prepared by heating 2.5 g cuprizon in 250 ml ethanol, and then making up to 500 ml with water.

The absorbance is measured after 30 rain at 590 nm against water (Beckman DU). Cu z+ ion exchange of potato cell wall is analysed with 50-100 mg cell wall. The

quantities of solutions are adapted accordingly (25 ml copper sulphate; 3 • 10 ml 0.6 N I-[CI in 7 0 ~ ethanol; 25 ml 0.1 N N a O H in 60~o ethanol etc.).

Pectolytic extraction procedure and methanol determhzation Pectin is extracted according to a simplified modification of the method of Potter & McComb (1957). The use of a special, concentrated pectolytic enzyme preparation, Ultrazym-100 (Dr. Schubert AG, Basel, Switzerland), which contains pectin lyase, polygalacturonases and pectin methyl esterase (Rombouts, 1972), allowed the origi- nal saponification and EDTA steps to be omitted. To 2 g of freeze-dried potato powder (about 10 g fresh weight), 50 ml of water are added; the pH is adjusted to 4 with 1 M citric acid, and 5 ml 0 . 2 ~ Ultrazym-100 are added finally. The sample is incubated overnight at room temperature, filtered through folded paper and the re- sidue washed out until a finalvolume of 100 ml is reached. After appropriate dilution pectin is determined by a modification (Rouse & Atkins, 1955) of the carbazole test, in combination with. the phenol-sulphuric acid test (Dubois et al., 1956), to correct the galacturonic acid content for the interference of neutral sugars in the carbazole test.

For methanol determination another sample of 2 g potato powder is saponified with 50 ml 0.1 N NaOt-[ for one hour, acidified to a pH of about 4 by adding 2.5 ml 2 N sulphuric acid, followed by the addition of 50 ml water (and some anti-foam if necessary) and distillation.

Potato Res. 17 (1974) 171

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

The methanol is collected in 70-75 ml of distillate, which is made up to 200 ml and a final concentration of 1 N sulphuric acid. Methanol is measured colorimetrically according to Wood & Siddiqui (1971).

Results and discussion

Cu 2 + ion e x c h a n g e

The ion exchange procedure, performed before and after saponification, enables the calculation of both the degree of esterification and the pectin content (as polyuronides) f rom the colorimetrical determination of Cu 2+ ions.

A standard curve for the cuprizon-copper reaction has to be prepared, for instance with copper sulphate. An example of calculation of pectin content and DE is given here. The calculation is based on the stoichiometrical reaction of two pectic carboxylic acid groups with one Cu 2+ ion.

DE = (y--x) /y • 100K

176 x + 190 (y--x) % pectin (as polyuronides) = 1000 W ~ " 100K (on fresh weight).

where: x ----- a/635 mmoles polyuronides y = b/635 mmoles polyuronides a = [zg Cu before saponification (standard curve) b = ~g Cu after saponification (standard curve) W = weight of sample AIS as fresh weight in g.

P e c t o l y t i c e x t r a c t i o n p r o c e d u r e a n d m e t h a n o l d e t e r m i n a t i o n

It is well known now that pectic substances are heterogeneous polysaccharides. Neutral sugars occur in side chains, covalently linked to the galacturonan main chain, but they may even be found within this main chain (Pilnik & Voragen, 1970). For this reason neutral sugars are solubilized together with galacturonic acid during the pectolytic extraction. The carbazole test is fairly specific for galacturonic acid (Me- Comb & McCready, 1952) but nevertheless neutral sugars react under the test condi- tions (Table 1). As shown in this table, glucose and galactose can react to an extent of

Table 1. Reaction of 50 ~tg sugar with carbazole-sulphuric acid.

Sugar z As2s

D-galacturonic acid 2 0.616 D-glucose 0.062 D-galactose 0.036

a Z u c k e r - Sucre 2 D-Galakturonsdure - D-acide galacturonique

Tabelle 1. Reaktion yon 50 ~tg Zucker mit Carbazol-Schwefelsfiure. Tableau 1. Rdaction de 50 ~g de sucre avec l'acide carbazole-sulfurique.

172 Potato Res. 17 (1974)

A N A L Y S I S OF P E C T I N IN P O T A T O T U B E R T I S S U E

5-10 ~o of galacturonic acid. Obviously, therefore, neutral sugars can give rise to con- siderable interference when present in excess. For this reason the Rouse & Atkins carbazole test was followed by a neutral sugar test with phenol-sulphuric acid.

Both in the carbazole and the phenol-sulphuric acid test, galacturonic acid and neutral sugars (glucose, galactose) when mixed in different ratios give additive absorbances. Bearing this in mind it is possible to calculate formulae for determining galacturonic acid and neutral sugars from the standard curves.

Neutral sugars as glucose: anhyd, galacturonic acid (~xg/ml) --~ 85 A 5 2 s - - 4.4 A49o

glucose (izg/ml) = 52 A49o -- 38 A 5 2 s

Neutral sugars as galactose: anhyd, galacturonic acid (~.g/ml) -- 84 As_~5 -- 3.3 A49o

galactose (~xg/ml) = 6 0 A 4 9 o - - 46 Asz5

where: A 5 2 s = absorbance, carbazole reaction Aa.9o =- absorbance, phenol-sulphuric acid reaction.

Galactose constitutes the major part of neutral sugars in potato pectin (Hoff & Castro, 1969), hence neutral sugars may preferably be calculated as galactose in potato pectin analysis.

From the peetolytic extraction procedure, combined with methanol determination after saponification and distillation, the degree of esterification of pectic substances can be calculated. In the past methanol has been determined by different colori- metrical methods, usually after oxidation to formaldehyde and reaction with chromo- tropic acid - sulphuric acid (Eegrive, 1937). Wood & Siddiqui (1971) described a method for methanol analysis without the distillation which is necessary when one works with biological fluids. In our experience, however, distillation gives better results because otherwise blanks must be done (unsaponified potato tissue), and also the recovery of methanol, added to potato samples, is nearly 10070 with distillation but only 9 0 ~ without. The pectin content and the DE can be calculated as follows:

f m . M DE - 55070

pectin (anhyd. galacturonic acid) = (fg. G) / (10 4 . W) ~ (on fresh weight). After correction for DE:

pectin (corr.) = 70 pectin (uncorr.) -t- (DE . 8/10 4) x 70 pectin (uncorr.)

where: M = methanol ([zg/ml) (standard curve) G • anh. galacturonic acid ([xg/ml) (formula) fm ---= dilution factor for methanol f, ----- dilution factor for galacturonic acid W ---- fresh weight of sample in g.

Potato Res. 17 (1974) 173

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

Table 2. Pectin analysis of potato tissue (variety Bintje, 1971) (~. pectin on fresh weight basis).

Sp. gr. class I Cu 2+ ion exchange 2 Pectolytic extraction/ methanol

pectin DE pectin (corr.) DE

1.060-1.070 0.38 34 0.37 52 1.070-1.080 0.40 36 0.37 54 1.080-I .090 0.42 25 0.38 52 1.090-1.100 0.42 26 0.41 48 I. 100-1.110 0.45 27 0.40 49

1 Spezifisches Gewicht (Klasse) - Classement pal" poids spOcifique z CuZ+.lonen.Austausch _ Exchange d'ion Cu 2+

Tabelle 2. Pektin-Analyse von Kartoffelgewebe (Sorte Bintje, 1971) ( ~ Pektin auf Basis des Frisch- gewichtes). Tableau 2. Analyse de la pectine dans le tissu de la pomme de terre (vari6t6 Bintje, 1971) ( ~ pectine du poids frais).

A n a l y s i s o f p o t a t o t i ssue a n d p o t a t o ce l l wal l m a t e r i a l b j , the two m e t h o d s

Samples of potato tissue, prepared from five specific gravity classes out of one lot of potatoes, were analysed by Cu 2 § ion exchange and the pectolytic extraction/methanol procedure. From Table 2 it can be seen that the two methods of pectin analysis gave different results. Cu 2 + ion exchange results in a higher pectin content and a lower DE than the pectolytic extraction/methanol procedure. Cu-" § ion exchange also leads to rather low DE's although other authors give higher values (40-60~) (Hoff & Castro, 1969; Jaswal, 1969).

To clarify these contradictory results, cell walls were isolated from potatoes of the specific gravity classes mentioned in Table 2 with a minimum of degradative treat- ments. The pectin of these cell walls can be assumed to be identical to the pectin of the original potatoes. The results of both methods of analysis, applied to cell walls, now show better agreement (Table 3). In particular the DE's are fairly similar, but the pectolytic extraction procedure seems to result in somewhat higher values for pectin content than the Cu z + ion exchange.

Consideration of the results of the pectolytic extraction/methanol procedure in the analysis of potato tissue (Table 2) and potato cell wall (Table 3) demonstrates a satisfactory agreement between DE, determined on whole tissue and on isolated cell walls, although the DE's of the latter are somewhat lower, in particular for the three highest sp.gr, fractions.

The deviating figures given by Cu z § ion exchange on potato tissue (Table 2) must be explained by interference of starch, which is removed with other non-cell-wall sub- stances during cell wall isolation. Starch constitutes up to 80 ~ of potato dry solids and has the additional characteristic of phosphoric acid groups covalently bound to C6 of the glucose of the amylopectin fraction (Greenwood, 1966). Indeed, application of Cu 2+ ion exchange to potato starch (British Drug Houses) demonstrated an ex-

174 Potato Res. 17 (1974)

ANALYSIS OF PECTIN IN POTATO TUBER TISSUE

Table 3. Pectin analysis of potato cell wall material (variety Bintje, 1971) (% pectin on dry weight basis).

Sp. gr. class I Cu 2+ ion exchange z Pectolytic extraction/ methanol

% pectin DE pectin (corr.) DE

1.060-1.070 16.1 53 17.9 55 1.070-1.080 16.6 57 18.6 57 1.080-1.090 16.3 56 16.9 61 1.090-1.100 15.7 59 17.7 55 1.100-1.110 16.1 58 17.2 57

Spezifisches Gewicht (Klasse) - Classement pal" poids spOcifique 2 Cu 2 +-Ionen-Austausch - Exchange d'ion Cu 2 +

Tabelle 3. Pektin-Analyse von Zellwandmaterial der Basis des Trockengewichtes). Tableau 3. Analyse de la pectine dans le matdriel du Bintje, 1971) (pectine en % du poids sec).

Kartoffel (Sorte Bintje, 1971) (% Pektin auf

paroi cellulaire de la pomme de terre (vari6t6

change capac i ty for Cu 2 + ions of 25.3 ~.mol/g. A t i t r imetr ica l es t imat ion of phospha te groups of this po t a to s ta rch accord ing to Wink le r (1960) yielded 23.6 ~mol acid groups /g , in quite close agreement with ion exchange analysis. This means tha t phospha te , cova len t ly b o u n d to po ta to starch, interferes dur ing Cu 2 + ion exchange analysis of po t a to tissue, resul t ing in higher pect in contents and lower DE ' s than found with pectoly t ic ex t r ac t ion /me thano l analysis, because these phospha te groups behave as free or non-ester if ied pectic carboxyl ic acid groups.

I t was out of the scope of our work on po t a to cook ing qual i ty to investigate the appl icab i l i ty of the Cu 2 + ion exchange me thod for o ther p lant tissues. I t seems a rel iable me thod to compa re the results of whole tissue analysis with cell wall analysis after i so la t ion o f cell walls f rom the tissue under invest igat ion, as demons t r a t ed here for p o t a t o tissue.

Especial ly for es tab l i shment of the D E of pectin this precaut ion has to be taken, otherwise great devia t ions from the real value may be encountered.

Acknowledgment

We thank Miss H a n n y Vaal for skilful technical assistance.

Zusammenfassung

Uber einige Prob leme bei der Analyse l,on Pek t i n im Kar to f f e l kno l l engewebe

Eine Ionen-Austauschmethode, basierend auf gleichzeitige Bestimmung des Pektingehaltes und dem Austausch von CuZ+-Ionen mit den S~ure- des Veresterungsgrades (DE = VG) des Pektins gruppen des Pektins, wurde im Hinblick auf die yon Kartoffelknollengewebe ohne Extraktion

Potato Res. 17 (1974) 175

des Pektins untersucht. Diese neue Methode wurde mit der pektolytische Extraktion yon pek- tinischen Substanzen ffir den Pektingehalt, ver- bunden mit der Methanol-Bestimmung nach Verseifung und Destillation ffir den VG von Pektin, verglichen. Beide Methoden wurden bei Kartoffelknollengewebe angewendet. Cu 2 +- lonen-Austausch ergab h6here Pektingehalte und niedrige VG als die pektolytische Extraktion/ Methanol-Analyse (Tabelle 2). lsolierung des Pektins aus Kartoffelgewege in relativ gerei- nigter Form, z.B. aus den Zellw~inden, und Anwendung beider Analysenmethoden, ffihrten zu einer besseren Obereinstimmung des Pektin- gehaltes und des VG (Tabelle 3). Es wurden eini- ge Anhaltspunkte daffir gefunden, dass Phos- phate, die kovalent an die Kartoffelst~rke, die bei der Zellwandprfiparierung entfernt wird, ge- bunden sind, beim CuZ+-Ionen-Austausch st6- rend wirken. FOr die Analyse von Pektin in den

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

Zellwfinden der Kartoffel scheint sich der Cu 2 +- Ionen-Austausch als eine brauchbare Methode zu erweisen.

Die pektolytische Extraktion/MethanoI-Ana- lyse brachte befriedigende Ergebnisse fiir den Pektingehalt und VG yon Kartoffelknollen- gewebe (Tabellen 2 und 3).

Nach der pektolytische Extraktion yon Pek- tinsubstanzen arts Kartoffelknollengewebe wurde die Modifikation der Carbazolreaktion nach Rouse und Atkins zur Bestimmung von Pektin als wasserfreie Galakturonsfiure angewendet. Die Korrektur der erhaltenen Werte mittels eines neutralen Zuckertestes (Phenol-Schwefelsfiure- test) trug zu einer besseren Bestimmung des Pek- tingehaltes im Kartoffelknollengewebe (und in den Zellw~inden der Kartoffel) bei. Ohne diese Korrektur w0rde der Pektingehalt um zirka 2 0 ~ 0berschfitzt.

R6sum6

De quelques problbmes clans/'analyse de la pectine clans le tissu de tubercule de pomme de terre

Les auteurs ont fait des recherches sur une m6- thode d'6change ionique, bas~e sur l'6change de I'ion Cu 2+ avec les groupes d'acide pectinocar- boxylique, dans la ddtermination dans le tissu de tubercule de pomme de terre, ~t la lois de la teneur en pectine, et de son degr6 d'est6rification ( = DE) sans extraction du complexe pectine.

Les auteurs ont compar6 la nouvelle m~thode avec l'extraction des substances pectiques pour ce qui concerne la teneur en pectine, en combi- naison avec leurs d6terminations au m6thanol apr+s saponification et distillation pour ce qui concerne l'ester de pectine (DE). Les deux m6- thodes furent appliqu6es au tissu de tubercule de pomme de terre.

La m6thode de l'6change d'ion Cu 2+ donne des teneurs plus 61ev6es en pectine ,et plus basses en ester de pectine, que l'analyse 'extraction pec- tolytique/m6thanol' (tableau 2). L'isolement, dans les 6chantillons de tissu de pomme de terre, de la pectine dans une forme d6j~. relative- ment purifi6e, comme dans les parois cellulaires, et l'application des deux m6thodes d'analyse ont conduit h une meilleure estimation aussi bien de la teneur en pectine que de DE (Tableau 3). Les

auteurs ont recueilli des preuves que le phosphate est combin~ par ses valences 5_ la f6cule de pore- me de terre qui est enlev6e au cours du traite- ment de la paroi cellulaire, et qu'il interf6re dans l'6change d'ion Cu 2+. L'6change d'ion Cu 2+ se r6v61e une m6thode pleinement utile pour I'ana- lyse de la pectine de la paroi cellulaire de la pomme de terre.

La m6thode'extraction pectolytique/m6thanol' donne des r6sultats satisfaisants pour l'analyse de la pectine et de DE du tissu de tubercule de pomme de terre (tableaux 2 et 3).

Apr6s extraction pectolytique des substances pectiques des tissus de tubercules de pomme de terre, on utilise la r6action du carbazole, modifi6e par Rouse et Atkins pour d6terminer la pectine sous forme d'acide anhydrogalacturonique. La correction des valeurs obtenues par un test de sucre neutre (test b. l'acide ph~nol-sulfurique) contribue h une meilleure d6termination de la teneur en pectine du tissu de tubercule de pomme de terre (et de la paroi cellulaire de pomme de terre). Sans cette correction la teneur en pectine serait surestim6e de 20 ~ environ.

176 Potato Res. 17 (1974)

A N A L Y S I S OF P E C T I N IN P O T A T O T U B E R T I S S U E

References

Bartolome, L. G. & J. E. Hoff, 1972. Firming of potatoes: Biochemical effects of preheating. J. agric. Fd Chem. 20: 266-270.

Dische, Z., 1947. A new specific color reaction of hexuronic acids. J. biol. Chem. 167, 189-198. Doesburg, J. J., 1965. Pectic substances in fresh and preserved fruits and vegetables. I BVT Communi-

cation No 25, Wageningen, the Netherlands. Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers & F. Smith, 1956. Colorimetric method for

determination of sugars and related substances. Analyt. Chem. 28 : 350-356. Eegrive, E., 1937. Reaktionen und Reagenzien zum Nachweis organischer Verbindungen. IV.

Formaldehyd. Z. analyt. Chem. 110: 22-25. Fries, J., n.d. Spurenanalyse. Erprobte photometrische Methoden, E. Merck A. G., Darmstadt,

p. 88. Gee, M., E. A. McComb & R. M. McCready, 1958. A method for the characterization of pectic

substances in some fruit and sugar-beet mares. Fd Res. 23: 72-75. Greenwood, C. T., 1966. Physical and chemical characteristics of potato starch. Proc. PI. Sci. Syrup.,

Campbell Inst. agric. Res., Can-tden, New Yersey. Hoff, J. E. & M. D. Castro, 1969. Chemical composition of potato cell wall. J. agric. Fd Chem. 17:

1328-1331. Jaswal, A. S., 1969. Pectic substances and the texture of french fried potatoes. Am. Potato J. 46:

168-173. Litchman, M. A., & R. P. Upton, 1972. Gaschromatographic determination of residual methanol in

food additives. Analyt. Chem. 44: 1495-1497. McComb, E. A. & R. M. McCready, 1952. Colorimetric determination of pectic substances. Analyt.

Chem. 24: 1630-1632, McCready, R. M. & E. A. McComb, 1952. Extraction and determination of total pectic materials in

fruits. Analyt. Chem. 24: 1986-1988. Pilnik, W. & A. G. J. Voragen, 1970. Pectic substances and other uronides. In: A. C. Hulme (Ed.),

The biochemistry of fruits and their products, Part I. Academic Press, London, Potter, A. L. & E. A. McComb, 1957. Carbohydrate composition of potatoes. Pectin content. Am.

Potato J. 34: 342-346. Raunhardt, O, & H. Neukom, 1964. (3bet Ver~inderungen der Pektinstoffe von ,~pfeln tmd Birnen

whhrend des Reifeprozesses. Mitt. Geb. LebensmUnters. II. n y g . 55: 446--455. Rombouts, F. M., 1972. Occurrence and properties of bacterial pectate lyases. Doctoral Thesis,

Agricultural University, Wageningen, the Netherlands. Rouse, A. H. & C. D. Atkins, 1955. Pectinesterase and pectin in commercial citrus juices as determin-

ed by methods used at the Citrus Experiment Station. Bull. Fta Univ. Agric. Exp. Sin. 570. Tibensky, V., J. Rosik & V. Zitko, 1963. Zur Bestimmung des Veresterungsgrades yon Pektin.

Nahrung 7: 321-325. Winkler, S., 1960. Die Bestimmung des Phosphors~iuregehaltes der Kartoffelstfirke auf komplexo-

metrischem und alkalimetrischem Wege. Stdrke 12 : 35-42. Wood, P, J. & I. R. Siddiqui, 1971. Determination of methanol and its application to measurement of

pectin ester content and pectin methyl esterase activity. Analyt. Biochem. 39: 418--428. Wunsch, G., 1952. (]ber Schwermetallpektinate. Makromolek. Chem. 8: 124-133.

Potato Res. 17 (1974.) 177