J Sci Food Agric 1989,49,15-86

In-Situ Pectin De-esterification of Alkali-Treated Fruit Waste Materials

Karen King,* Grenville Norton, John R Mitchell and John Caygill$

Department of Applied Biochemistry and Food Science, University of Nottingham, School of Agriculture, Sutton Bonington, Loughborough, Leks LE12 5RD, UK

(Received 5 September 1988; revised version received 24 October 1988; accepted 15 November 1988)

ABSTRACT

Waste peel and pulp materials from orange, lime and mango fiuits were treated with sodium carbonate which increased the pH and resulted in the de-esterijication of pectin in situ. Chemical and enzymic (pectinesterase) de-esterification occurred in each of the treated fruit waste materials. A specijic carbonate enhancement of pectinesterase activity which was clearly demonstrated in orange peel was found to a lesser extent in lime pulp but not at all in mango peel. During treatment of the dispersions with Na2C0,, each of the three fiuit waste materials gelled. The pectin degree of esterijication of the orange and lime dispersions was below 50% (low methoxyl pectin) on gelling but the mango peel dispersions gelled at pectin degree of esterification levels above 50% although the gelation mechanism was that of a low D E pectin. The degree of esterijication of the soluble pectin in the mango dispersion was, however, found to be very much lower than the overall pectin degree of esterification determined. I t is suggested that in-situ pectinesterase activity i s restricted to the soluble pectin in mango peel whereas in the citrus residues both soluble and bound pectin are de-esterijied.

Key words: Pectinesterase, pectin, oranges, limes, mangoes.

* Present address: Agricultural and Food Chemistry Research Division, Department of Agriculture for Northern Ireland and the Queen’s University of Belfast, Newforge Lane, Belfast BT9 5PX, UK. $ Present address: Overseas Development and Natural Resources Institute, 56/62 Gray’s Inn Road, London WClX 8LU, UK.

75

J Sci Food Agric 0022-5142/89/$03.50 0 1989 Society of Chemical Industry. Printed in Great Britain

76 K King, G Norton, J R Mitchell, J Caygill

INTRODUCTION

Pectins are used in many food products as stabilisers, thickeners and gelling agents. High methoxyl pectins, with a degree of esterification (DE) above 50%, can be used as gelling agents in a limited range of products such as jams, preserves and jellies, as they require high acidity (below pH 3) and low water activity (high soluble solids such as sugar) to form a gel. Commercially low methoxyl pectins, with a DE below SO%, are produced from high methoxyl pectins extracted from citrus peels and apple pomace. These low methoxyl pectins gel in the presence of multivalent cations (usually calcium) at acid and neutral pH and can therefore be used in a wide range of products (Glicksman 1979).

High methoxyl pectin can be de-esterified using acid, alkali or enzymes. Acid is commonly used commercially as alkali requires careful control to prevent hydrolysis by 1-elimination, reducing the size of the pectin polymer and hence affecting its functional properties. Enzyme de-esterification is effected by pectinesterase (PE: EC 3.1.1.1 1) which has been found in numerous higher plants and some microorganisms.

The treatment of plant materials, usually fruit or vegetable wastes, with sodium carbonate has been used to effect in-situ de-esterification of pectin (Baier and Wilson 1941; Buckley et a1 1978). Raising the pH stimulates de-esterification both by enzymic (pectinesterase) and by chemical means. This type of alkali treatment produces a low methoxyl pectin gelling agent from orange peel suitable for autoclaving in the presence of a phosphate sequesterant (Mitchell et al1978). Speirs (1979) reported that for heat stability the pectin requires a DE below 20% as above this DE hydrolysis of the polymer occurs by p-elimination.

This process of alkali treatment was originally patented by the California Fruit Growers’ Exchange (Baier and Wilson 1941) and used on orange peel but other materials such as pea pods have also been examined (Taylor and Pritchard 1982) using similar treatments. In previous work (King et a1 1986, 1988) the characteristics of pectin de-esterification in situ for lime pulp and mango peel, wastes from fruit processing, were found to be different. Application of exogenous PE to increase the rate of de-esterification in mango peel which contains relatively low levels of endogenous enzyme was found to be unsuccessful (King et a1 1988). This paper examines the treatment of orange peel, lime pulp and mango peel with sodium carbonate to effect pectin de-esterification in situ. Comparisons of the different materials are made in relation to the apparent substrate preference of the endogenous PE and hence its effect on the use of alkali treatment to produce a low methoxyl pectin gelling agent.

EXPERIMENTAL

Materials

Limes Citrus aurantiifolia (Christm) (Persian type) and mangoes Mangifera indica (L) (cv Julie) were obtained through Geest Associates, Spalding, Lincs, UK, and Valencia oranges Citrus sinensis (L) from a local wholesaler. The lime fruits,

Pectin de-ester$cation of alkali-treated fruit waste 77

delivered as a single consignment, were all unripe as defined by a completely green skin. Approximately half of the fruits were ripened (to a minimum of 50% yellow skin) at 20°C over a period of 21 days. Fruits which did not ripen were discarded. The ripe and unripe fruits were processed to obtain pulp as described previously (King et al1986) and the freeze-dried material was stored at - 15°C. The mangoes were classified according to their stage of ripeness as unripe, semi-ripe or ripe, and the peel was removed and dried as described previously (King et al 1988).

The oranges were washed, dried and hand peeled. The peel was minced and frozen with some subsequently being freeze dried and ground to pass through a 1-mm sieve. Both the frozen and dried samples were stored at - 15°C.

Fruit materials containing inactive PE were prepared by adding each of the materials to boiling water and maintaining the temperature at 8CL90"C for 3 4 min. These dispersions were then cooled, freeze dried, and stored at - 15°C.

Determination of in-situ pectinsterase activity by titration

PE activity was determined by titration with sodium hydroxide using an autotitrator as previously described for lime pulp (King et al 1986). Dried fruit material (1.4 g or 0.7 g) was added to 50 mlO.1 M NaCl maintained at 25°C by water circulating through a thermostated vessel. The pH was adjusted to the required value (to give PE activities between pH 6 and 11.0) with 0 1 M NaOH and the sample was titrated with 002 or 001 M NaOH for up to 10min. Heat-treated samples were titrated in a similar manner and the unheated sample activities were corrected. PE activity was calculated as units g - ' dry matter (DM) where one unit is defined as the amount of enzyme which catalyses the release of one mole of methanol or free carboxyl groups per minute.

Galacturonic acid content and degree of esterification of pectin

Pectin was determined as galacturonic acid using a method adapted from Blumenkrantz and Asboe-Hansen (1973) as described previously for lime pulp (King et a1 1986). The pectin DE was calculated from the methanol released following alkaline de-esterification (Mitchell et a1 1978). Galacturonic acid content and pectin DE of the fruit materials were calculated from analyses carried out on hot water soluble and ethylenediaminetetra-acetic acid fractions extracted from the alcohol insoluble solids of the material (King et al 1986).

Determination of methanol release and pectin degree of esterification by gas chromatography

Aliquots (10 ml) of suspensions or gels were rapidly expelled into 1 ml50% v/v HC1. After centrifuging at 1710 x g for 10 min, the supernate was decanted and analysed for methanol by gas chromatography as described previously (King et a1 1986). Residual methanol content was determined by calculation from either the methanol released or following alkali de-esterification as appropriate.

Alkali treatment of fruit materials

Dispersions of the fruit materials were treated with alkali (usually Na,CO,) to raise the pH and hence effect pectin de-esterification.

78 K King, G Norton, J R Mitchell, J Caygill

Orange peel Twenty grams of frozen (homogenised 15 s using Polytron Speed 5) or 4.0 g dried orange peel was suspended in water and 0.5 g Na,CO, dissolved in water was added to give a final volume of 50 ml. The samples were maintained at 25°C in a water bath, and pH and methanol release were followed with time up to 60 min. At 60 min the samples were freeze dried and the pectin DE was calculated from the residual methanol content as described in the section, ‘Galacturonic acid content and degree of esterification of pectin’.

Lime pulp The method used was similar to that for orange peel but using 1.4g dried pulp and either 0-25 g or 0-5 g of Na,CO,. The lower pulp concentration was required as the acidity of the lime pulp resulted in too low a pH (6.5) when using similar pulp and carbonate concentrations to the orange peel. Increasing the level of Na,CO, was impractical as excessive effervescence occurred.

Mango peel Methanol release from mango peel was examined using 1.4 g dried peel suspended in 50 ml distilled water with 0.25 g Na,CO,.

Gelation properties of the three mango peels were examined using titration with sodium hydroxide. Dried peel (1.4 g) was suspended in 50 ml distilled water and titrated at either pH 8.5 or 9.5 for 5 min using 0.1 M NaOH. Aliquots (6 ml) were then removed and centrifuged for 5 min at 1710 x g during which the samples gelled. The remaining dispersions, which had also gelled, were aged at room temperature for 18 h. Methanol content and hence pectin DE were determined by gas chromatography for all samples at gelation and following ageing.

RESULTS AND DISCUSSION

The galacturonic acid content and degree of esterification (DE) of the pectin for each of the fruit materials are presented in Table 1. The heat treatment used to inactivate pectinesterase had no significant effect on either parameter in any of the fruit materials.

Orange peel

During Na,CO, treatment of orange peel the pH decrease (Fig 1) was similar for both the dried and frozen heat-treated peels but that of the unheated frozen peel was greater than that of the dried peel. The methanol content and pectin DE of these samples at 60 min was lower in both the frozen peels compared with the dried (Table 2). The difference between the heat-treated and unheated peels was, however, similar-0.36 and 0.35 % methanol (12 and 13 % DE) respectively for the dried and frozen peels. This suggests that, during freeze drying, the pectin becomes less amenable to de-esterification by both chemical and enzymic means.

Both the unheated peel samples gelled and had to be disrupted to allow sampling to continue. The heat-treated samples, however, did not gel even when the

Pecrin de-esterifcation of alkali-treated fruit waste 79

TABLE 1 Galacturonic acid content and pectin degree of

esterification in fruit materials

Fruit material

Orange peel

Lime pulp: Unripe Ripe

Mango peel Unripe Semi-ripe Ripe

Galacturonic acid Pectin D E content (% D M ) ( % I

17.0 78

25.5 25.3

60 67

11.7 70 16.1 77 13.9 85

incubation period was increased to over 120 min and the pectin DE had dropped to the same level as that in the unheated peel on gelling. This difference is attributed to the mode of pectin de-esterification occurring in each of the peel samples. In the heat-treated peel only chemical de-esterification occurs, whereas in the unheated peel chemical and enzymic (PE) de-esterification will occur. Chemical de- esterification alone results in a random distribution of carboxyl groups along the pectin molecule whereas PE acts sequentially resulting in blocks of de-esterified galacturonic acid residues (Henri et a1 1961; Kohn et a1 1968). Gelation of the peel samples containing enzyme de-esterified pectin supports the hypothesis of Kohn (1975) and Powell et a1 (1982), that gelation of low methoxyl pectin in the presence of divalent cations requires either a minimum sequential array or a block of free carboxyl groups.

0 10 2 0 30 40 50 60 Time (min)

Fig 1. Change in pH of orange peel dispersions treated with sodium carbonate: frozen peel; 0 heat- treated frozen peel; 0 dried peel; 0 heat-treated dried peel; ? gelation. Measurements made at 25°C

with 0.5 g Na,CO,.

80 K King, G Norton, J R Mitchell, J Caygill

TABLE 2 Methanol content and pectin DE of orange peel treated with

Na,CO, for 60 min

Sample Methanol content Pectin DE (% DM) f %)

Dried: Heat-treated 1.33 Unheated 0.97

47 35

Frozen: Heat-treated 0.98 35 Unheated 0.63 22

Samples dispersed in 50ml distilled water with 0.50g Na,CO, and maintained at 25°C for 60min. Residual methanol content and pectin DE determined on freeze-dried material.

In-situ PE activity in orange peel was found to be optimum over a wide range of pH when measured by titration with NaOH (Fig 2). It is apparent from the pH change determined during Na,CO, treatment (Fig 1) that, although total de- esterification would remain relatively unchanged, the proportion due to PE would increase as the pH decreased with time. However, de-esterification rates up to 5 min calculated on the basis of methanol release during Na,CO, treatment of the dried peel were equivalent to 9 and 70 PE units g- D M for the heat-treated and unheated peels, respectively. The rate of de-esterification for the heat-treated peel is similar to that determined by titration (Fig 2) but that of the unheated peel is much higher (70 units g-' compared with 18 units g-' DM). This supports the hypothesis of Speirs

40 L

._ I

?\ P"

Fig 2. Dependence of in-situ activity of orange peel PE on pH. W Freeze-dried peel; 0 heat-treated freeze-dried peel; 0 corrected for chemical de-esterification.

Pectin de-esterijkation of alkali-treated fruit waste 81

(1979) that specific ion activation of PE by the CO2,-/HCO, occurs within orange peel and hence the proportion of enzymic to chemical de-esterification on treatment with Na,C03 will be much greater than that determined by titration using NaOH. This carbonate effect is important when considering the manipulation of the alkali treatment conditions to effect changes in the functional properties of the resulting pectinaceous material. At gelation the pectin DE was 38 % in the dried and 32% in the frozen peel. In the frozen peel the DE had dropped to 22% after 60 min and it seems probable that a DE below 20%, which is reported to be necessary for heat stability (Speirs 1979), could be obtained on further incubation.

Lime pulp

The pH of lime pulp treated with Na,CO, changed little during the time of assay (Table 3). This was attributed to the buffering capacity of the COZ-/HCO; system. At the lower concentration of Na,CO, little change in methanol content was found for the heat-treated pulp whereas at the higher concentration methanol release during the first 5 min was equivalent to 11.3 and 8.9 PE units g- ' DM in unripe and ripe pulp respectively (Fig 3). These rates of de-esterification were similar to those obtained by titration with NaOH of 8.3 and 8.0 units g- ' DM respectively. De- esterification rates equivalent to 47,39,41 and 62 PE units g- ' DM (calculated up to 5 min) were obtained for unripe pulp using 0-25 and 0.50 g Na,CO, and ripe pulp using 0.25 and 0.50 g Na,CO, respectively. These rates of de-esterification were generally higher for the samples at pH 10.0 (0.5 g Na,CO,) than those found by titration with NaOH but the increase was proportionately much less than that found using orange peel. The methanol content and hence pectin DE were similar for the ripe and unripe peels at 60min and lower at the higher Na,CO, concentration. For all unheated samples the pectin DE was at or below the maximum level of 20 % reported by Speirs (1979) for heat stability.

Mango peel

The decrease in methanol content was similar for each of the three types of peel

TABLE 3 Effect of sodium carbonate on pH of lime pulp dispersions

Sample" PH ~

0.25 g Na2C0, 0.5 g Na2C03

Unripe: Unheated Heat treated

7.5 8.5

9.8 10.1

Ripe: Unheated 7.4 9.7 Heat treated 8.2 10.0

Samples contained 1.4 g dried pulp suspended in 50 ml distilled water at 25°C.

82 Mitchell, J Caygili

60

40

20

. l o - YI 0

-60;

I - -40

z 0

- 20

I I I I I

0 10 20 30 40 50 60 Time (min)

Fig 3. Changes in methanol content and pectin degree of esterification for lime pulp treated with sodium carbonate: W 0.25 g Na,CO,; 0 0.25 g Na,CO, heat-treated pulp; 0 030 g Na,CO,; 0 0.50 g

Na,CO,, heat-treated pulp.

when treated with Na,CO, but the decrease in pectin DE was reduced in the ripe peel (Fig 4) apparently due to the lower level of GA determined in the peel. Initial de-esterification rates equivalent to 18,23 and 18 PE units g-' D M were calculated for the unripe, semi-ripe and ripe peels whereas the corresponding heat-treated peels gave levels equivalent to 6 , 11 and 11 PE units g - D M respectively. These levels were similar to those found by titration with NaOH (King et al1988). No activation of PE by carbonate or bicarbonate ions occurred.

As no activation of PE was apparent with the Na,CO,, a comparison between NaOH and Na,CO, was carried out. The pH of the NaOH-treated samples decreased more rapidly (Fig 5 ) due to lack of buffering capacity from the CO:-/HCO; system. The decrease in pectin D E was similar for both alkalis during the first 10 min after which the pectin D E of the NaOH-treated samples changed little whereas that of the samples treated with Na,CO, continued to decrease very slowly resulting in a lower DE at 360 min (Table 4).

This difference in the rate of de-esterification is attributed to. the pH of the respective dispersions. The buffering capacity of the COZ-/HCO; system maintains the pH at a level (8.5) where both enzymic and chemical de-esterification can occur, whereas the NaOH sample drops to a much lower pH (approximately 7) at which de-esterification is negligible by either mechanism. Both unheated samples gelled within 10min when the pectin DE was determined at 52 and 56% for the

Pectin de-esterijkation of alkali-treated j k i t waste 83

0 I I I I I I

-0-

v YI

I I I I 1 I

o 10 20 30 40 50 60 0 0

Time (min)

Fig 4. Changes in methanol content and pectin degree of esterification for mango peels treated with sodium carbonate: (I unripe peel; V heat-treated unripe peel; 0 semi-ripe peel; 0 heat-treated semi-

ripe peel; a ripe peel; 0 heat-treated ripe peel.

I- 10.0

0 20 40 60 80 100 120 Time (min)

Fig 5. pH changes in unripe mango peel dispersions treated with sodium carbonate and sodium hydroxide: mango peel and Na,CO,; 0 heat-treated mango peel and Na,CO,; 0 mango peel and

NaOH; 0 heat-treated mango peel and NaOH; t gelation.

84 K King, G Norton, J R Mitchell, J Caygill

TABLE 4 Changes in methanol content and pectin DE of unripe mango peel treated with NaOH and

Na,CO,

Sample incubation Na,CO, treated NaOH treated and time (min)

Methanol Pectin Methanol Pectin content D E content DE

( % D M ) ( %) (% D M ) (%I Unheated :

0 5

10 120 360

Heated: 0

360

2.05 70 2.05 70 1.66 57 1.64" 56 1.52" 52 1.45 49 1.29 44 1.34 46 1.10 40 1.38 47

2.05 70 2.05 70 1.72 64 1.70 63

'Sample gelled.

Na,CO,- and NaOH-treated samples respectively. These levels are much higher than either the orange peel or lime pulp systems and are also above the DE level of 50% for low methoxyl pectins. This gelation of the mango peel was further examined by titrating samples at known pH (8.5 and 9.5) and isolation of 'soluble' and 'bound' pectin by centrifugation. Gels were formed with the unripe peel after only 8 min titration (Table 5) but were much less stable than the gels formed later by

TABLE 5 Gelation of mango peel dispersion treated with NaOH

Unripe peel Semi-ripe peel Ripe peel PH PH PH

8.5 9.5 8.5 9.5 8.5 9.5

Approximate time to gelation (min)

Gel pH Methanol content at gelation

Pectin DE at gelation

Methanol content after 18 h

Pectin DE after 18 h

(% DM)

(%I

(% DM)

( %)

8

8.31 1.72

(0.33)" 59

1.59 (0.46)

54 (16)

(11)

8

9.18 1.67

(0.38) 57

(13) 1.54

(0.51) 53 (17)

9

8.33 1.79

(0.25) 66

1.53 (0.51)

56

(11)

(21)

9 10

9.12 7.97 1.54 1.45

(0.50) (0.50) 58 63

(19) (22) 1.32 1.52

(0.72) (0.43) 49 66

(28) (19)

5

8.03 1-38

(0.57) 60

1.38 (0.57)

60

(25)

(25)

a Figures in parentheses indicate the change from initial values.

Pectin de-esterification of alkali-treated fiuit waste 85

the semi-ripe and ripe peels. After ageing, the gels formed by the unripe peel had collapsed and considerable syneresis had occurred. The gel formed from the ripe peel, however, was more stable and syneresis was reduced. Centrifuged samples also gelled in the clearer phase at the top of the tube indicating that gelation of the dispersion is due mainly to soluble pectin and that sufficient is solubilised to support gelation.

In this type of system it is difficult to define clearly the ‘soluble’ and ‘bound’ pectin. A situation similar to that proposed by Krop (1974) may apply, namely strands or loops of pectin extending into the serum and being adsorbed on to the pulp particles. This may play an important role in gelation, in addition to that of the soluble pectin, by linking the particulate material.

After 5 min titration at pH 8.5, precipitation with 8076 ethanol and drying, the level of soluble pectin in the unripe and ripe peels was determined at 1.6 and 2.8 % D M with a DE of 5 and 35% respectively. These values further support the hypothesis that the soluble pectin is the major fraction of pectin involved in gelation of the mango peel dispersions and that the overall DE may not be a suitable parameter of reference for assessing the potential functional properties of this type of system. Preferential de-esterification of the soluble pectin is also implied, as suggested earlier (King et a1 1988). This was further substantiated by calculation of the levels of PE activity for the unripe and ripe peels assuming that only the soluble pectin was de-esterified and to the DE determined in the initial 5 min which were 11 and 13 units g-’ DM respectively. These levels of activity were similar to those determined by titration with NaOH (King et al 1988).

Although the systems used here are complex, two mechanisms can be identified to explain the in situ de-esterification of pectin: in the first the methanol content/DE of the total pectin (soluble and bound) decreases, whereas in the second only the methanol content/DE of the soluble pectin (as defined by the centrifugation method described earlier) decreases.

In a system where the first mechanism operates and PE de-esterifies both soluble and bound pectin, the total methanol content will decrease proportionately with the decrease in the D E of the soluble pectin. If gelation occurs when the soluble pectin has attained a certain methanol content/DE, this will be the same as or very similar to that of the bound pectin. Subsequent homogenisation of this system, for example, when used as a food hydrocolloid, would result in the solubilisation of pectin with a similar DE and therefore increase functional properties.

If, however, the second mechanism operates and the soluble pectin is preferentially de-esterified whilst the bound pectin remains relatively unchanged, ie esterified, then two different levels of esterified pectin will be present. Subsequent homogenisation of this system will result in increased heterogeneity of the DE of the soluble pectin as the higher DE pectin is solubilised, which would tend to decrease the functional properties of the material.

These two mechanisms approximate respectively to the citrus and mango systems used in this work. Other factors including differences in cell wall structure such as the association of pectin and other polymers and the location and solubility of the PE in situ will contribute to the complexity of the mechanisms.

/In addition the relative proportions of chemical and enzymic de-esterification

86 K King, G Norton, J R Mitchell, J Caygill

during Na,CO, treatment are important in determining the functional properties, particularly gelation, of the resulting material. Further work is required t o examine the effect of the two different types of de-esterification and their interaction in this type of in-situ system.

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