CHEMICAL COMPOSITION, ENZYME ACTIVITY AND EFFECT

OF ENZYME INACTIVATION ON FLAVOR QUALITY OF

GREEN COCONUT WATER'
CLEBER F. CAMPOS, PAUL0 EDUARDO A. SOUZA, J. VIRGiLIO COELHO and
M. BEATRIZ A. G L ~ R I A Z
Departamento de Alimentos, Faculdade de F a d c i a
Universidade Federal de Minas Gemis
Belo Horizonte, MG, Brazil 30180-112

Accepted for Publication August 22, 1996

ABSTRACT

The chemical composition of green coconut water was determined. Both

polphenobxidase andperoxidase were observed to be present and active in green
coconut water. lhese enzymes showed optimum activity at pH 6.0 and 5.5 and
at temperatures of 25 and 35C, respectively. Among chemical and physical
treatments investigated, heating at 90Cfor 550 s and addition of ascorbic acid
were, individually, the most eflcient for enzyme inactivation. Addition of ascor-
bic acid did not affect sensory properties, however, heat treatment at 90C for
longer than 100 s decreasedhvor quality. Combinations of heat treatment with
potassium metabisupte, ascorbic acid or both additives did not affect jlavor
quality.

INTRODUCTION

Coconut (Cocos nucifera) is a perennial plant, bearing fruit continuously for

up to 60-70 years, 12 to 13 times a year (Banzon 1990).
Brazilian coconut production was estimated to be 603 billion fruits (IBGE 1987),
7 5 1 of which has been used by industries (Maciel et al. 1992). Mature coconuts

'Paper presented at 1994 Institute of Food Technology Annual Meeting, Atlanta, GA, USA.
*Towhom correspondence should be addressed: Departamentode Alimentos, Faculdade de F d i a ,
UniversidadeFederal de Minas Gerais, Av. Oleghio Maciel2360,30180-112 Belo Horizonte, MG,
Brazil, e-mail: gloriam@oraculo.Icc.ufmg.br, fax: 55.031.337 9076.
Journal of Food Processing and Preservation u) (1996) 487-500. AU Rights Resewed.
0 Copyright 1996 by Food & Nutrition Press, Inc.. TrumbuU. Connecticut. 487
488 C.F.CAMPOS, P.E.A. SOUZA, J.V. COELHO and M.B.A. G a F U A

have been widely used in the manufacture of coconut flakes, coconut milk, copra
coconut oil and copra meal cake (Sison 1977; Montenegro 1985; Jayalekshmy
et d. 1986).
The green coconut, at approximately6 to 7 months of development, is receiv-
ing more attentionnowadays, It contains on average400 mL of a sterile, nutritious,
refreshing and delicious drink (Gonzalez 1990). It is relatively high in potassium
and low in sodium (Anzaldo 1985). Green coconut water has been used directly
from the fruit at the production area, along the Brazilian coast, and also in cen-
tral urban areas (Val 1992).
Green coconut water has also earned popularity in the world market (Gonzalez
1990) especially for its healing qualities, such as oral or intravenous rehydra-
tion, drink in cholera cases, combating intestinal warms, and relieving stomach
troubles (Jasper 1979; Montenegro 1985; Jayalekshmy et ul. 1986). It has also
been used in the cultivation of plant and microbial cells because of its growth
promoting factors (Sierro and Velasco 1976; Al-Khayri er al. 1992).
To reduce transportation volume and cost associated with the whole fruit and
to improve the shelf life of green coconut water, its processing is necessary.
However, enzymatic browning may occur affecting its nutritive value, flavor and
appearance (Galeazzi 1984).
The purpose of this study was to determine the chemical and physico-chemical
composition of green coconut water and to investigate the presence and means
of inhibitingenzyme activity without affecting sensory properties of green coconut
water.

MATERIALS AND METHODS

Materials
Green coconut (Cocos nuciferu) was purchased in the retail market of Belo
Horizonte, MG, Brazil. All chemicals were of analytical grade.

Determination of the Chemical and Physico-chemical Composition

Water, total and soluble solids, reducing, non-reducing and total sugars, ash,
lipids, proteins, pH, total acidity, and turbidity were determined according to
AOAC methods (Horwitz 1980). Water content was determined by weight loss
during heating at 70C under vacuum. Total solids was determined by weight dif-
ference. Soluble solids, expressed as 'Brix, was determined with an Abbe refrac-
tometer (aus Jena) at 2OC. Total, reducing and non-reducing sugars were deter-
mined by the gravimetric copper reduction method of M u o n and Walker. Ash
was determined by calcination at 55OC in a muffle furnace of predried coconut
 GREEN COCONUT WATER QUALlTY 489

water. Lipids were extracted using a Soxhlet apparatus and the crude protein con-
tent was calculated from total nitrogen determined by the Kjeldahl methd. The
pH of the samples was measured using an analogic pH meter (Analion). Total
acidity was determined by titrating 10 mL of sample with 0.1 N NaOH solution,
using phenolphthalein as indicator. Turbidity was determined spectrophoto-
metrically at 610 nm (relative to distilled water). Total phenolic compounds were
determined according to Swain and Hillis (1959)and quantified using catechin
as standard.

Enzyme Activity
Polyphenoloxidase activity was determined according to Ponting and Joslyn
(1948)using catechol as the phenolic substrate. A 10 mL test tube containing
5.5 mL of 0.2M phosphate buffer @H6.0) and 1.5mL of 0.2 M phenolic substrate
was placed in a water bath at 25C.After stabilization of the temperature, 1 mL
of coconut water was added to the test tube, the mixture was homogenized and
changes in absorbance were followed at 425 nm in a Shimadzu UV-VIS 160A
spectrophotometer at one minute intervals.
Peroxidase activity was determined according to the technique described by
Ferhrmann and Diamond (1967).A 20 mL test tube containing 7 mL of 0.2 M
phosphate buffer @H 5.5) and 1 mL of coconut water was immersed in a 35C
water bath. After stabilization of the temperature, 1.5 mL of 0.05% guaiacol
(phenolic substrate) and 0.5 mL of hydrogen peroxide (0.1%)were added, the
mixture was homogenized and the changes in absorbance were followed at 470
nm in a Shimadzu UV-VIS 160A spectrophotometer at one minute intervals.
An unit of enzyme activity was defined as the amount of enzymatic extract
capable of increasing absorbance at 425 and 470 nm for polyphenoloxidaseand
peroxidase, respectively, at rates of 0.001 unit per minute.
The optimum pH for activity of the enzymes was investigated by using 0.2
M phosphate-citrate buffer for pH ranging from 3.5 to 8.5 (Campos1993).For
polyphenoloxidase, 5.5 mL of buffer at specific pH values and 1.5 mL of the
phenolic extract were heated to 25C.After stabilization of the temperature, 3.0
mL of coconut water was added and enzyme activity was determined as described
previously. For peroxidase, 7.0 mL of phosphate buffer at specific pH values
and 1.0 mL of coconut water were heated to 35C. After stabilization of the
temperature, 1.5 mL of 0.5% phenolic substrate and 0.5 mL of hydrogen perox-
ide (0.19%) were added, the mixture was homogenized and the activity of peroxi-
dase was determined as described previously.
The optimum temperaturefor activity of the enzymes was investigated from
5 to 6OC (Campos1993).For polyphenoloxidase,5.5 mL of buffer @H 6.0) and
1.5 mL of the phenolic extract were heated to the specific temperatures. After
stabilizationof the temperature, 3 .O mL of coconut water was added and enzyme
490 C.F. CAMPOS, P.E.A. SOUZA, J.V.COELHO and M.B.A. GL6RIA

activity was determined as described previously. For peroxidase, 7.0 mL of

phosphate buffer @H5.5) and 1.O mL of coconut water were heated to the specific
temperatures. After stabilization of the temperature, 1.5 mL of 0.5% phenolic
substrate and 0.5 mL of hydrogen peroxide (0.196) were added, the mixture was
homogenized and the activity of peroxidase was determined as described
previously.

Inhibition of Enzyme Activity

The effect of heat treatment on the activity of polyphenoloxidaseand perox-
idase was determined. Tests tubes containing 10 mL of coconut water were placed
in water baths at temperaturesof 70 to 9OC.As the desired temperaturewas reach-
ed, tubes were collected at specific time intervals, refrigerated immediately and
the extract analyzed for polyphenoloxidase and peroxidase activity (Campos 1993).
The effect of additives on the activity of polyphenoloxidase and peroxidase
was investigated by adding different concentrations of ascorbic, sorbic and ben-
zoic acids, potassium metabisulfite, EDTA, citric acid, carbon dioxide and cys-
teine. The effect of heat treatment at 9OC combinedwith one or two addltives
was also investigated.
After submitting coconut water to the different treatments, polyphenoloxidase
and peroxidase activities were determined under standard assay conditions as de-
fined previously.

Sensory Evaluation
A trained sensory panel of 15 members was used for the evaluation of the
coconut water submitted to the different treatments. Acceptance of samples was
tested by asking panelists to evaluate samples on a 7 points hedonic scale (Moraes
1988).

Statistid Analysis
In order to verify the existenceof significantdifferenceamong samples, analysis
of variance was used and means were compared by Tukey’s test (Moraes 1988).

RESULTS AND DISCUSSION

Chemical and Physko-chemical Composition of Green Coconut Water

Chemical and physiccwhemical parameters, such as soluble solids, total ac-
tivity, pH, transmittance and volume were determined in individual samples of
 GREEN COCONUT WATER QUALITY 49 1

green coconut water in a total of 30 samples (Table 1). It was observed that values
varied significantly among individual samples. However, correlation was observed
among coconut water volume, levels of soluble solids and percent transmittance
(Campos 1993). This is in agreement with results reported by Jayalekshmy et
al. (1986).
A pool of these 30 samples was used for further physicu-chemical analysis (Table
2). The concentration of total sugars was higher than the values described by
Endef (1977) and Sison (1977), however, those of lipids and proteins were lower.
According to Anzaldo (1985) and Jayalekshmy er al. (1986), the composition
of coconut water changes with the stage of development.The levels of total sugars,
total solids and total titratable acidity increase up to the six month of develop
ment, decreasing afterwards. The concentrations of lipid and protein increase at
the final stage of development. Differences in coconut water composition could
also be due to coconut variety and cultivation practices, as well as transportation
and storage conditions (Jayalekshmy et al. 1986).

Enzyme Activity
Polyphenoloxidaseand peroxidase were present in green coconut water show-
ing activities of 32.1 and 114.3 U/mL, respectively. The predominance of perox-
TABLE 1.

CHEMICAL AND PHYSICO-CHEMICAL COMPOSITION OF GREEN COCONUT

WATER

Values
Parameter
Range Mean'

Volume per coconut (mL) 100-600 297 151

Soluble solids (OBrix,2PC) -

4.46 7.02 5.34 2 0.71

Total titratable acidity

(mg citric acid/100 mL) -
13.9 76.8 48.1 i17.8

PH -
4.70 6.40 5.20 _+ 0.45

Transmittance* (%) 26.6 - 95.0 775 i16.6

'Mean standard deviation) of indhidlul vllucs of 30 coconut sample.

'Tmnunittpnce at 610 IUII ~a m c w n of t u r b i .
492 C.F. CAMPOS, P.E.A. SOUZA, J.V. COELHO and M.B.A. GL6IUA

TABLE X

CHEMICAL AND PEYSIC(IcBEMIcAL COMPOSlTION OF A POOL OF WATER

FROM 30 GREEN COCONUTS

Paramer Vnlues'

Water (gD00m.L) 94.20 5 1.90

Total solids (gD00mL) 5.80 2 0.12

Soluble solida (OBrix,UPC) 537 5 0.11

Total sugars (g/l00mL) 530+021

Reducing sugars (g/l00mL) 4.90 5 0.20

Non-reducing sugars (g/l00 mL) a40+0.04

Ash w00mL) +
M 0.01

Protein (mo/100mL) 19.so+aso

Lipids (mg/l00mL) ii.oo+aa

Total phenolics (mg atecbinll00 mL) 6.E6 2 0.55

Total litratable acidity (mg citric .eiW00 mL) 131.202 2.80

PH +
530 0.10

TranrmittancC* (%) 81.00 2 1.70

'M a n of biparcrrdc ,+rElad.rddeviation.

'Tnmmithnce at 610 mu n a mcwuc of aubldiry.

idase over polyphenoloxidase activity was also observed by Silva (1981) in car-
rots and hearts of palm, however, in fruits such as bananas and apples,
polyphemloxidase had hisher activity than peroxik.
The optimum pH for polyphenoloxidase and peroxidase were observed to be
6.0 and 5.5, mpectively (Table 3). According to these data,green coconut water
was observed to have favorable pH for the activity of both enzymes. The o p
timum t e m p e m for enzyme activity was observed to be 25C for polyphewlox-
idase and 35C for peroxidase (Table 4).
 GREEN COCONUT WATER QUALlTY 493

TABLE 3.

EFFECT OF pH ON TEE RELATIVE ACIWlTY OF POLYPHENOL.€)XIDME AND

PEROXIDME IN GREEN COCONUT WATER

Enzyme activity (%)

PH
Polypbenoloridase Peroridme
~

3.5 74.5 2 1.7 74.1 0.2

4.0 80.0 2 0.5 783 2 0.8

4.5 82.5 2 0.7 82.5 2 0.6

5.0 91.2 2 1.2 913 2 1.2

5.5 94.1 2 1.0 100.0 t 1.0

6.0 100.0 2 03 77.0 2 0.2

as 72.2 t 1.1 55.0 2 1.6

7.0 57.6 t 0.2 44.1 t 0.8

7.5 40.7 2 03 31.2 2 0.9

ao 22.5 2 1.5 17.0 2 0.5

85 15.0 2 0.8 3.2 2 0.4

Enzyme Inactivation
The effect of pH on enzyme activity can be seen in Table 3. Lowering the pH
does not seem to be by itself an efficient method of enzyme inactivation.
It was observed that refrigeration is not sufficientto completely inhibit enzyme
activity in green coconut water. According to Table 4, at 5C percentual enzymes
activities of 46 and 39%relative to those at the optimum temperature, were ob-
served for polyphenoloxidase and peroxidase, respectively.
494 C.F. CAMPOS, P.E.A. SOUZA, J.V. COELHO and M.B.A. GL6RIA

TABLE 4.

EFFECT OF TEMPERATURE ON THE RELATIVE ACTIVITY OF

POLYPHENOLOXlDASE AND PEROXIDASE IN GREEN COCONUT WATER

Enzyme activity ("/.)

Temperature (OC)
Polyphenoloxidase Peroxidase

5 46.0 2 1.5 39.0 5 0.9

15 95.0 +- 0.4 63.0 2 0.7

25 100.0 5 1.0 84.0 5 1.1

30 95.7 5 0.7 92.0 2 2.0

35 91.3 5 1.2 100.0 2 1.2

40 87.0 2 0.9 91.7 5 1.5

50 69.0 2 0.7 75.0 2 1.8

60 39.0 5 1.3 24.0 5 0.9

Mean of thm determinations_f standard deviation.

Activity is relative to maximum activity at the optimum temperature: 25OC for polyphenoloxidax and
35OC for peroxidrue.

The effect of heat treatment on enzyme activity is illustrated in Fig. 1 and 2.

It can be observed that heat treatment at 9OC was the most efficient, however,
holding times of 550 and 310 s were necessary for complete inactivation of
polyphenoloxidase and peroxidase, respectively, Polyphenoloxidasewas observed
to be more heat resistant than peroxidase. According to Silva (1981) polyphenol-
oxidase was also observed to be more resistant to heat treatment than peroxidase
in figs. However, other studies indicated peroxidase to be the most heat resistant
of the enzymes. For this reason, peroxidase has been used as a heat treatment
index (Luh and Whitaker 1974; Naveh el al. 1981; Lee and Pennesi 1984; Muf-
tugil 1985).
Among additives investigated, ascorbic acid (Table 5 ) was observed to be the
most efficient in the inhibition of both enzymes. Potassium metabisulfite at con-
 GREEN COCONUT WATER QUALlTY 495

0 100 200 300 400 500 600

Time (seconds)
FIG. 1 . EFFECT OF HEAT TREATMENTS ON THE RELATIVE ACTMTY OF POLY-
PHENOLOXIDASE IN GREEN COCONUT WATER

centration of 15 mg/100 mL inhibited 10096 of polyphenoloxidase, but only 11%

of peroxidase activity (Table 6). Polyphenoloxidasewas observed to be more sen-
sitive to the effects of these additives than peroxidase. According to studies by
Eskin et al. (1971) and Silva (1981) potassium metabisulfite and ascorbic acid
were observed to be the best additives in the inhibition of polyphenoloxidaseand
peroxidase, respectively, in several fruits and vegetables.
Sorbic and benzoic acids, EDTA and cysteine at concentrationsup to 20 mg/100
mL, as well as carbon dioxide and citric acid at concentrations as high as 150
and 240 mg/100 mL, respectively, did not reduce significantly the activity of
polyphenoloxidase and peroxidase in green coconut water (Campos 1993).

Sensory Evaluation
During heat treatment, the longest holding time that could be used at each
temperature without affecting sensory properties were observed to be 100 s at
4% C.F. CAMPOS, P.E.A. SOUZA, J.V. COELHO and M.B.A. GL6RIA

0 100 200 300 400 500 600

Time (seconds)
FIG. 2. EFFECT OF HEAT TREATMENTS ON THB RELATIVE ACl’MTY OF PEROXIDASE
IN GREEN COCONUT WATER

W,110 s at 85C, 130 s at 8OC, 180 s at 75C, and 175 s at 7OC. Therefore,
the higher the temperature and the longer the holding time, the greater the flavor
loss. According to these results, the use of heat for enzyme inactivation can
significantly affect the flavor of cocollllt water.
The study of the effect of pH on the sensory quality of CocOIlllt water Wcated
that by lowering the pH to 4.5, there was a significant change in the flavor qual-
ity of green coconut water.
The study of the effect of additives on the sensory characteristics of coconut
water indicatedthat amcmg mvatigataladditives, only ascorbic acid and potassium
metabisulfitecould be used at concentrationsup to 20 and 10 mgll00 mL, respec-
tively, without affecting cocollllt flavor (p < 0.05, Tukey test). Therefore, ac-
cording to the amount of these additives necessary to inhibit enzyme activity,
only ascorbic acid could be usedwithout afftctiag flavor quality of green coconut
water.
 GREEN COCONUT WATER QUALITY 491

TABLE 5.
EFFECT OF DIFFERENT CONCENTRATIONS OF ASCORBIC ACID ON THE
RELATIVE ACIlMTY OF POLYPHENOLOXIDASE AND PEROXIDME IN GREEN
COCONUT WATER

Enzyme activity(%)

Polyphenoloridme Peroxidare

0.0 100.0 2 1.8 100.0 2 2.0

05 97.1 2 1.0 88.9 2 1.7

1.0 94.22 15 881 2 1.2

2.0 88.4+0.3 86.4 i0.9

3.0 71.8 2 1.8 84.7 2 1.0

5.0 48.4 2 05 813 2 1.4

7.0 223 2 0.9 66.7 2 0.9

10.0 0.9 2 0.0 263 2 2.2

15.0 0.4 2 0.0 0.0 i0.0

20.0 0.0 2 0.0 0.0 2 0.0

Mean of three dtftmhatbas 2 r b a d u d dcvhtbn.

A~tyhrrLdivcdD~tyofcnrymanWoutLdditivcTempmdurr~250Clorpdypbenobnidw
.ad35°C fbr ptroddnsc

The combined effect of heat treatment and addition of one additive on enzyme
activity and flavor quality of green coconut water was investigated. Among possi-
ble combinations, heat treatment associated with addition of 5 mgllOO mL of
potassium metabisulfite or 10 mg/lOO mL of ascorbic acid were the only ones
which did not affect flavor quality of coconut water. Furthermore, no significant
difference on sensory evaluation was observed when heat treatment was com-
bined with both additives at concentrations of 5 and 5 mg/lOO mL or 2.5 and
10 mg/lOO mL of potassium metabisulfite and ascorbic acid, respectively (p <
0.05, Tukey test).
498 C.F. CAMPOS, P.E.A. SOUZA, J.V. COELHO and M.B.A. aL6RIA

TABLE 6.

EFFEm OF DIFFERENT CONCENTRATIONS OF POTASSIUM METABISULFITE

ON THERELATIVE ACIWITY OF POLYPEENOulXIDASE AND PEROXIDME
IN GREEN C0CO"T WATER

Enzyme activity(%)

PolyphenoloxidLIc Peroxidase

0.0 1mot 1.0 100.0 2 1.6

0.5 88.240.6 98750.3

1.0 82.4 4 0.7 97.3 21.6

2.0 76.7 20.8 97.1 2 1.3

3.0 7334a7 %.9 5 0.8

5.0 60.6+_1.6 95.6 1.6

7.0 41.4 5 0.5 955 5 2.0

10.0 26.0 5 0.4 95.4 5 1.4

15.0 0.0 2 0.0 88.6 2 1.7

20.0 0.0 2a0 880Ok 1.0

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