Extraction and Characterisation of Pectin from Various Tropical Agrowastes

Suhaila Mohamed and Zahariah Hasan

ABSTRACT The extraction of pectin from kalamanzi, pineapple waste and cocoa pods using ethanol, metallic salt and acetone precipitalion were compared and evaluated in terms of yield, colour and gelling characteristics. Acetone precipitation was the best method so was used to extract pectin from other agrowastes. The yields, jelly grades and chemical characteristics of pectins (moisture, ash, total uronic acid, acidic and methylated uranic acids, jelly grade, setting time, gel unit and colour) from the different sources were compared. The overall pectin producing potential in order af merit were kalamanzi (musk lime), jackfruit waste, cocoa pod husk, durian skin, banana skin, reject immature starfruit, melon skin, rambutan pod, pineapple skin and duku skin. Cashew apple gave a very high yield of pectin but the pectin mass was gummy and could not be ground to a powder. canned fruit processing industry has increased to become one of the 10 most 'factory processed' fruits of Malaysia (FAMA, 1988) and the disposal of waste (skin and seed) has become a problem. Utili sing the skin as a source of pectin is a possibility. Although duku is a seasonal fruit it has a very thick skin which can be used as a pectin source, complimentary to the non seasonal sources available. Rambutan and duku have the added advantage of being very prolific fruit producing crops and the skins have a low water content which could reduce the cost of transportation and drying. Recent interest in processing musk melon into juice and other products will result in localised waste (skin) which may be a viable pectin source. Cashew apple is often discarded due to its rather unacceptable astringent taste. Malaysia's sandy ground covers a vast area of 365,000 acres, giving the cashew nut fruit industry good potential. Jackfruit 'wastes' (rind, core and undeveloped perianth), form 59.1 % of the fruit (Bhattia, Sidappa and Lal, 1955) while in the canning of jackfruit, the rejected part is 80-85% of the total fruit (Muhammad, 1972), which may be utilised as a source of pectin. Durian is one of the ten most 'factory processed' local fruits (FAMA, 1988) and has very thick, flexible but tough skin. Since durian consumption results in a lot of waste, it 'would be an advantage if the waste could be processed into useful products. Francis and Bell (1975) reviewed the commercialisation of pectin from breadfruit, jackfruit, papaya, coconut, taro, yam, water hyacinth, kangkong, sweet potato, cassava, banana, guava, sugarcane, tamarind and cocoa pod. A more recent review on pectin from some tropical fruits were made by Hodgson and Kerr (1991). The objectives of this study were: (1) to investigate indigenous plant materials and agrowaste as potential sources for pectin; (2) to characterise the pectin extracted from the various sources; and (3) to assess the potential of the sources as a raw material for pectin manufacture. The plant materials and agrowastes investigated were: Kalamanzi or Musk lime (Citrus microcarpa), pineapple (Ananas comosus) skin, cocoa pod (Theobroma cacao) huskI. immature starfruit (Averrhoa carambola) I rarnbutan . . . ( Nep h e/lllln lappaceum) skm, duku (Lansilim domestlCllm) . . skin, jackfruit (Artacarpus heterophylllls) waste, n:usk:nelon .(Cuclimis mela LJ ski~11durian (Durio zlbethmus) skm, banana (Musa acuml1lata)and cashew (Anacardillm occidelltale) apple.

INTRODUCTION The Statistical Department of Malaysia reports that in 1989 Malaysia imported about 60 tonnes of pectin worth almost RM one million (US5250,000). There is a vast resource of agricultural products and agro\vastes which can be used to produce pectin. A banana processing factory alone produced 1,381 metric tonnes of banana peel waste in 1984. Kalamanzi lime (Limau kasturi), is non seasonal, cheap and a prolific producing crop which is underutilised and would be a good source for pectin production. Cocoa pod husk has long been a disposal and pollution problem to the cocoa industry and could be a cheap source of pectin if it can be processed immediately and the processing modified to suit the nature of raw material (eg: high pigmentation). During pineapple canning, 45% of the total weight of the fruit is wasted and can be used to extract pectin, while pectin can also be obtained from the leaves and stems of the pineapple plant. In order to get carambola of good size, farmers have to limit the numbers of fruits per tree, plucking away large quantit'ies of immature fruits and discarding them. The use of rambutan in the

Based Oll a paper presented at the 'Symposium Sumber Alam Tropika' Kuchim:, C Sarawak, Malaysia, organised by Universiti . (6 - 9 0 ec 1993) . M aIaYSla5arawa k UNIMAS

The authorsarefrom the Facultyof FoodScienceand Bioteknologi, Universiti Pertanian Malaysia, 43400 Serdang, Selal1gorDaml Ehsan, Malaysia.

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MATERIALS

AND METHODS

about 15 seconds whereupon a pectin aluminiumcopper complex separated throughout the liquor as an extremely finely divided continous gel which immediately coagulated into curds and rose to occupy about a quarter of the liquor volume, the final optimum pH of the mother liquor being 3.9 to 4.2 at 25C. The solution was centrifuged at 3500 G for 10 mins to separate the coagulated gel from the mother liquor of sulphuric acid and methanol (10.8% and 70.4% respectively). The gel was then rinsed in ethanol, drained, and fresh 75% alcohol added to make a fluid mixture and enough alkali buffer (ammonium hydroxide, sodium carbonate or a mixture of the hvo), added to adjust the pH. After about 30 minutes, the pH of the suspension was about 5.5 :t 0.3 and the pectin upon drying and preparation into a 1% sol yielded a pH of 3.9 :t 0.3. They were then dried at 60'C and ground. Extraction of pectin using acetone

Samples of citrus peel, pineapple skin, cocoa pod husk, immature starfruit (reject), duku skin, durian skin, cashew apple, jackfruit 'waste' and rambutan skin were obtained locally. The agrowastes were washed then immersed in 95% boiling methanol for 15 mins followed by washings. The peel was then dried at 80'C for 2 days. Three methods
to find

of extraction were initially compared for obtaining good yield and quality pectins. Duplicate extractions were carried out from each source.
the best method

Extraction

using

ethanol-HCl

One hundred grams of ground and dried peel were weighed into a tared 2000 mL beaker containing 500 mL distilled water. Twelve grams of freshly ground sodium hexametaphosphate (BDH) were then added and the initial pH adjusted with 3 :\ HCl to 2.2:t 0.1, heated with constant stirring at 80 :t 5C for 1 hour and the pH checked at intervals of 15 mins. \-Vater loss was replaced at intervals except in the last 20 minute of extraction. The extract was filtered through muslin cloth and the residue washed with 200 mL of warm water. The 'washings were added to the filtrate, which was concentrated by evaporation on a hot plate to approximately one fifth of its initial volume. The concentrated pectin solution was cooled to 50'C and poured into a volume of ethanol in a ratio of 1:3, the ethanol contained 0.5 M HCl. The mixture was stirred for 0.5 hour and allowed to stand for one hour. The precipitate was centrifuged at 3500 G for 15 mins, washed at the same pH with more ethanol-HCl solution and centrifuged at the same speed for 15 mins. Finally the precipitate \vas washed with acetone to remove all traces of HCl and ethanol which could interfere with the methoxvl determination. The precipitate was dried at 60C to constant weight. Extraction of pectin using metallic Smit, 1971) salts (Wiles and

1 kg of the macerated sample was heated at 75C in a vessel containing 4 L water and 12 g of freshly ground sodium hexametaphosphate. The pH was adjusted to 4.0 :t 0.5 when necessary using citric acid or sodium hydroxide. Water lost by evaporation \\'3S replaced at intervals except in the last 20 min of extraction. The extract \vas filtered using a centrifugal separator / filter, lined with cheesecloth. The collected filtrate was concentrated to 1/3 of the original volume. The cooled filtrate \vas poured into 2 volumes of acetone containing 0.5 M HCl (pH of the slurry at about 1) and stirred for 30 min. The precipitate was separated on coarse mesh nylon or muslin cloth and washed at the same pH to remove all but traces of ash. The precipitate was washed repeatedly in 500 mL portions of 70% acetone until it was essentially chloride ion-free or the pH was above 4.0, dehydrated further in 500 mL of acetone and dried overnight at 45C. The precipitate \vas weighed to determine the yield and this pectin was used for further analysis. The dried pectin ,vas tested for ammonia which would interfere with many of the proposed analytical methods. This was done by adding 1 mL of 0.1 N NaOH to a small amount of dried sample. On heating, the presence of ammonia could be detected by moistened litmus paper. Ammonium ion, if present was washed out with acidified 60% alcohol, followed by neutral alcohol to remove the acid and dried. This pectin was used for further analysis.

Samples were treated as above except that they were

alternatively treated with 1% sodium bisulphite instead of sodium hexametaphosphate, and heated at 70-75'C for 1 hour with constant stirring. The extract ,vas filtered through muslin cloth and the residue washed \vith 100 mL of ,varm \vater. The washings were added to the filtrate which was then cooled to 50'C and 1.75'70 (v/v) of the metal salt precipitating solution was added (540 g aluminium sulphate and 45.9 g copper sulphate/LJ over about 1 minute. Immediately thereafter 1.05% v /v of the soda ash solution (169.6 g sodium carbonate/ L) was added and stirred for

Characterisation of pectin
Moisture was determined according to AOAC (1980), with 1 g of ground sample weighed and dried at 105'C for 4 hours to constant weight. For Ash, 1 g of pectin (ground to pass 80 mesh) was weighed in a tared crucible and ignited slowJy, then heated for
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44

about 4 hours at 600'C to constant weight (Owens, 1952). Equivalent Weights were done by weighing 0.5 g pectin (ammonia and moisture free) in a 250 mL conical flask, moistened with 5 mL ethanol. 1 g of sodium chloride were added to sharpen the end point. 100 mL of carbon dioxide free distilled water and 6 drops of phenol red indicator were added. The pectic substances were stirred rapidly to dissolve, then titrated slowly with 0.1 N NaOH until the colour of the indicator changed (pH 7.5) and persisted for at least 30 seconds. The neutralised solution was used for methoxyl determination. Methoxyl contents were determined by adding 25 mL of 0.25 N NaOH to the neutral solution, mixing thoroughly, and allowing to stand for 30 minutes at room temperature in a stoppered flask. 25 mL of 0.25 N HCl was then added and titrated with 0.1 N NaOH to the same end point as before. Methoxyl = mL of alkali X N of alkali X 3.1 X 100 content (%) . welg ht a f samp 1e 0/. 0 R -COOH (A'd' Cl IC uronlC .
aCl'd)

Jelly grade (the quality of pectin) was estimated by measuring the jelly firmness of a standard 65% sugar jelly, pH 3.0 :!: 0.5 on a finger pressure test. "Grade" of pectin means the weight of sugar with which one part by weight of pectin will, under suitable conditions at ambient temperatures, form a satisfactory jelly. Test jellies were made and compared with a jelly made under similar conditions with a standard 100grade pectin sample after an 18-hour time lapse. A piece of jelly was cut and squeezed between the thumb and forefinger until the jelly breaks and compared with the standard jelly. Differences of up to 5% could be detected by an experienced operator. If the jelly did not have the strength of a standard one, a jelly was prepared by increasing the amount of pectin and compared with the standard jelly. Pectin Equivalents based on Jelly grade . Wt x Grade of pectm . b emg use d Grade of pectin to
be used Rate . of Settmg
.

. . W t a f pectm req Ulre d

% R-COOH used for calculating the anhydrouronic acid content was determined by titration with sodium hydroxide. to pH 7.5 using phenol red indicator. . . Molecular weIght of AUA (1 umt) - 176 g.

(Kertesz, .

1951) . . . hme reqUIre d f or

. . . T 0 ta I an h y d fourontc aCId S (ADA) (ara b 1l10se, ga Iactose

an d 0 th er sugars. ) % AUA = % R-COOH + % R-COOCH,
, x 100 176 x O.lv

If t h e Je11 y set In 10 t O:J2 " mIn, . ., . conSI d ere d ' rap! d settmg an d 1 "f t h e the setting of jelly was more than , ., . ., I
me d !Um settmg to s 1ow settmg .

th e pectin "

\vas

25 min, it was

176 x O.lz x 100 + w x 1000

. Je IIy Umt

w x 1000

were h z = mL (titre)
y w

of NaOH

from
from

% R-COOH
% R-OCH

. . . . IS t1e 1 quantIty a f JeIIy w h IC1 1 can be produced from a gIven weIght of raw matenal (FrancIs and Bell, 1975). This unit indicated the economic potential of the pectin sources under study. (Jelly Unit . ld x JeIIY gra d)e. " Yle = pectm
"

=
=

determination. mL (titre) of NaOH determination. weight of sample.

Determination contaminants)

of acid

insoluble

ash

(silicous

Evaluating gel properties: the pectins used were of two types: (1) with pH adjustment in the final extraction procedure. (2) without pH adjustment in the final extraction procedure. The experiment was carried out with the assumption that they are high-ester pectins. The standard formulation for the preparation of jelly without pH adjustment (Table 1 and 2) was 105.0 mL water, 2.5 g pectin and 54.0 g sugar. The amount of pectin that sedimented after forming the gel was measured for solubility of the pectin. The methoxyl content or degree of esterification is an important factor controlling the setting time of pectins, the sensitivity to polyvalent cations and their usefulness in low solids, gels films and fibres and was detennined by saponification of the pectin and titration of the liberated carboxyl group (Gee, McComb and McCready, 1958).

.. The ash was weIghed and treated wIth 0.5 M HCl. The colloidal solution formed was filtered to obtain the insoluble material, ignited and weighed (Gee, et al., 1958).

RESULTS

AND DISCUSSION

Three methods of extraction were initially compared to determine the yield and characteristics of pectin produced from three different sources i.e kalamanzi, pineapple and cocoa pod. Table 1 shows that the pectin from Kalamanzi peel (Limau kasturi) was higher than from pineapple skin and cocoa pods. This yield value of Kalamanzi was comparable to the yield of lime peel Citrus auranUfolia S. (32.25%) obtained by Rouse and Crandall (1978) using nitric acid. The extraction of pectin from cocoa using acetone with

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45

sodium hexametaphosphate gave the best yield (9.6%) together with the most desirable colour. For pineapple skin, the pectin extracted using alcohol gave the highest yield (6.7%) although the colour of pectin was very undesirable i.e. black. Acetone

extraction gave a slightly lower yield value of 5.3% but produced the best colour (pale yellow). Acetone extraction using sodium hexametaphosphate was the best extraction method in terms of yield, colour and cost.

Table 2 indicates that there was only a slight variation .in the solubility of the pectin between Kalamanzi peel, pineapple skin and cocoa pods and that pH affects the strength of the gel. Owens (1952) showed tha t for a lo\v-rnethoxyl pectin, used \"it11 a constant calcium:pectin ratio of 40:1, a maximum gel strength were obtained, with values decreasing on either side of the optimum pH. However, since the formulation for gel preparation was done on the assumption of high-ester pectins, only extraction methods were varied. The extraction method, which did not employ final adjustment of pH, resulted in firmer gels and the pectin had a pH range of 1.9 :t 0.5. Gels from Kalamanzi pectin and pineapple pectin resulted in favourable pale colour and that from cocoa was very dark in colour. Decolourising techniques need be incorporated for the extraction of pectin from cocoa. Moisture values for the pectin from Kalamanzi peel were 3.9 :t 1.3% on drying for two days at 105'C. For pineapple skin pectin it was 11.3 :t 2.7% and for cocoa pods 12.7 :t 7% when dried overnight at 105'C. If the pectin from Kalamanzi peel was not dried for 2 days the moisture content remained too high for it to be ground or reduced in size. Overall, metallic salt extraction produced a higher ash content than acetone extraction. The optimum aqueous extract:acetone ratio was 1:1.5 (Table 3) which was used in all following investigations.

10%, coupled with good jelly grade, considered commercially viable.

is usually

Although cashew apple gave the highest yield, the pectin precipitate was gumlike and ungroundable. This unexpected texture could indicate either high moisture content or the presence of acetone insoluble substances other than pectin occluded in the precipitate. Moisture All the sources except cashew apple, produced pectin having moisture contents from 9% to 14% (Table 4). Commercially, this would be considered a normal moisture content. The moisture content of cashew apple pectin was not measured. Ash The inorganic impurities in commercial pectin are indicated by the ash content. Table 2 shows that the pectins extracted using sodium hexametaphosphate had high ash content and could be as high as 45.9% (pineapple). All the pectins extracted (Table 4) used sodium hexametaphosphate and had quite high ash content indicating high inorganic impurities. However repeated washing with acid only reduced the ash content very slightly and was suspected to be silicous material as the ash behaved and looked like a typical silicous ash. To confirm this, "acid insoluble determination" was carried out on the extracted pectins and sodium hexametaphosphate (the possible source of the contaminant). Table ,5 shows sodium hexametaphosphate contained a considerable amount of acid insoluble ash (9.8%). Rambutan pod pectin extracted using sodium hexametaphosphate also contained a considerable amount of acid insoluble ash (4.9%). The % ash increases as the pectin yield decreases, indicating that the contaminant did not originate from the raw material, but from something that has been added to the raw material in consistent proportion, i.e sodium hexametaphosphate. Upon treatment with hydrochloric acid, material like CaCO, or NazCO, would have dissolved out leaving behind acid insoluble ash, e.g: silica, alumina, various silicates or alumina silicate. However it is beyond the scope of this work to determine the exact silicous material. This "Nork can only caution against using sodium hexametaphosphate having no detailed specification on the labelling. 1% sodium bisulphite can be used instead. %AUA The %AVA indicates the purity of pectin against other organic material i.e neutral polysaccharides such as arabinose, galactose and other sugars which can be calculated by the expression 100% - AVA %. Table 4 shows that the sources investigated produced pectin containing quite good %AUA in the range of 70% to

Yield Table 4 shows that the highest yield was from cashew apple (38.7% dwb), followed by jackfruit pericarp (22.5% dwb) and musk lime (19.8% dwb). Melon skin pericarp-mesocarp (12.5% dwb) and banana skin (11.9% dwb) gave intermediate yields, while cocoa pods (9.7% dwb), young immature starlruit (8.3% dwb), rambutan skin (7.1% dwb) and durian skin (6.2% dwb), gave fairly low yields. Pineapple skin and duku skin both had very low yields (dwb) of 3.7% and 2.7% respectively. A source with a yield of above

ASEAN

Food Journal Vol. 10, No.2,

1995

47

90%, except for jackfruit skin (pericarp) which contained about 52.8% AVA. Despite the low %AUA, jackfruit skin pectin still showed very good pectin potential. Durian skin had the highest %AUA of 90.1%. Methoxyl content is an important factor in controlling the setting time of pectins, their combining power with metallic ions and the ability of the pectin to form gels. All the sources produce pectins with quite high methoxyl content (9-13%) (Table 4) suggesting that they arc all high methoxyl pectin (HMP). They would be expected to exhibit the typical HMP characteristics such as having a rapid /medium gel-setting period, requiring

sugar and acid for gel formation and the ability to disperse more easily than do pectins of less than 7% methoxyl content (Low methoxyl pectin/LMP).

48

Jelly grade (JG) is defined as the parts by weight of sugar with which one part by weight of pectin, under standard conditions, will form a jelly of satisfactory properties. The higher the grade, the less pectin 1s required, but this is not the only criterion for selecting a specific grade. It is apparent (Table 4) that the pectins from musk lime (JG > 100), durian skin ()G 90-100) and jackfruit pericarp (JG 90) had the highest JG, followed by pectins from immature starfruit (JG 80), cocoa pod husk ()G 70-80) and rambutan pod ()G 70-80), and then banana skin (JG 70), melon peri carp and mesocarp (JG 60) and duku skin ()G 60), whilst pineapple skin pectin had the lowest JG (40-60). Comparisons of JG are only valid when the extraction method employed is the same, as JG is much affected by the method of extraction. Setting time (Approximate)

colours of pectins from durian skin (almost colourless), jackfruit waste (nearly colourless), musk lime (off white), melon waste (off white), pineapple (off white) and immature starfruit (off white) were the lightest followed by pectins from cocoa pod husk (clear light brown gel), duku skin (clear light brown gel), banana skin (clear light brown gel) and rambutan pod (rather cloudy light brown gel). Adomoko (1972) obtained a 25-29% dwb yield of crude pectin from the immature husk (ash content 7.9-8.8%) and 8-11 % yield from the ripe cocoa pod husk (ash content 8.9-9.8%) using mild acid extraction. The pectin on precipitation was described as a white gelatinous precipitate, unlike that obtained by Blakemore, Dewar and Hodge, (1966) on a sun-dried material which has undergone a lot of enzymic browning. Sun-drying altered the proto-pectin nature resulting in a reduction in yield and potential gelling power of the isolated pectin (Francis 1965, unpublished). Because pectic substances play an important role during the development of the cell-wall in immature cocoa fruit, the pectic substances form a higher proportion of the husk than in the ripe pod husk. The ripe pods have about 9 times more dry husk material per pod than do the cherelles (immature pod) of the size used, thus the total yield of pectin from the husk of one ripe pod is estimated to be 3 times more than can be obtained from the husk of one immature fruit (Adomoko, 1972). Adomoko also found that extraction with 0.05 N or 0.1 N HCI at 70-80C was too drastic as judged by loss of solubility and the ability to imbibe water. The pectins from some of the sources investigated were quite highly coloured, probably due to the presence of polyphenols or other V'later soluble pigments trapped inside the pectin during precipitation. Better filtration techniques using a filter aid, activated carbon, diatomaceous earth, etc may remove these pigments to improve the colour of pectin extracted.

Commercially marketable pectin product should be able to set satisfactorily \vithin a given time according to the requirement of the product manufacturing procedure. In the manufacture of jam, quick-setting times of a few minutes at 90-95C are desirable so that the fruit particles become embedded throughout the pectin gel and do not float to the top of the container. Slow-set pectins take approximately one hour at 50-60C and are used for jellies which must be filled and cased before setting starts. Table 4 shows that, as expected of high methoxyl pectin, all the sources gave pectins having rather rapid gel-setting periods ranging from 5 to 35 minutes, with banana skin and jackfruit pericarp having a remarkably rapid gel-setting period of about 5 minutes. Jelly unit (JU) Table 4 indicates that the pectin from jackfruit pericarp (JU 2007-2034) and musk lime (> 1980) had the highest jelly unit followed by pectin from banana skin ()U 819-847), melon pericarp and mesocarp (JU 732-768), cocoa pod husk (JU 560-770), immature starfruit (JU 656-664), durian skin ()U 562-620), rambutan skin (JU 364-455), pineapple skin (JU 174-180) and duku skin (JU 162). This unit is useful in assessing the production potential of pectin raw material. It represents the quantity of jelly which can be produced from a given weight of raw material. This unit would be useful in ranking the economic potential of agrowaste sources. Colour Colour of pectin appearance of the having little effect product would be colours of pectins is important as it affects the gel produced. The lighter colours on the final appearance of a food preferred. Table 4 indicates that the vary according to the source. The

CONCLUSIONS The pectin producing potential in order of merit was musk lime, jackfruit \vaste, cocoa pod husk, durian skin, banana skin, reject immature starfruit, melon skin, rambutan pod, pineapple skin and duku skin.

REFERENCES AOAC Association of Official Analytical Chemists. Official methods of analysis, 13th Edition. Washington D.C; 1980. Adomoko, D. Cocoa Pod Husk Pectin. Phytochemistry. 11: 1145-1148; 1972.

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Bhattia, BS., Sidappa, GS., and Lal, G. Composition and Nutritive Values of )ackfruit. Indian). Agric. Sci. 25(4): 6303-6306; 1955. Blakemore, W.R., Dewar E.T. and Hodge R.A. Polysaccharides of the cocoa pod husk. ). Sci. Food Agric. 17:558-560; 1966. FAMA, 1988. Malaysia Federal Agricultural Marketing Authority Kuala Lumpur: Marketing Research Department; 1988. Francis, B.)., and Bell) .K. Commercial Review. Trop. Sci. 17 (1). p. 25;1975. Gee, M., McComb, A and McCready 23, 72; 1958. Pectin: a

Kertesz, 2.1. The Pectic Substances. lnterscience Publishers Inc., New York. p. 485; 1951. Muhammad, Y. Some Major Factors Worth Consideration in Manning for a Diversified Fruit Processing Plant, Ministry of Agriculture and Fisheries, K.L., Malaysia; 1972. Owens, H.S. Methods used at Western Regional Research Laboratory for Extraction of Pectic Materials, AIC-340, Western Regional Research Laboratory, Albany, California; 1952. Wiles KK and Smit C).B. US. Patent 3,622559. Nov 23. Sunkist Growers Inc; 1971. Rouse AH. and Crandal P.G. Pectin content of lime and lemon peel as extracted by nitric acid. ). Food Sci. 43: 72-73; 1978.

KM. Food Res.,

Hodgson AS. and Kerr L.H. Tropical fruit Products. In The Chemistry and Technology of Pectin. Walter KH. ed. Academic Press, San Diego 6786; 1991.

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1995