Table 2. Changes in standard curve correlation coefficient (r) through
time.
Time(min) r
Table 3. Activity estimates from assaying known amounts of OPPME;
r = 0.99986 for 0.009, 0.012 and 0.015 units.
0
5
10
15
20
25
30
-0.997149
-0.996521
-0.994151
-0.990756
-0.988862
-0.984617
-0.980215
rimetric estimates were in close agreement, giving a value
of 288.6 ± 11.21 units • ml-1 (n = 16). Colorimetric assay
of 0.006, 0.009, 0.012 and 0.015 units of OPPME indicated
that the microplate assay was accurate between 0.009 and
0.015 units of enzyme (Table 3). Higher concentrations
could be assayed but were limited by the rapidity of the
change in optical density. The activity estimate obtained by
assaying 0.006 units was only one-half of the predicted
value (0.0029 units).
The commercial non-pasteurized orange juice con
tained 0.0133 ± 0.0005 [x equivalents acid released -min-1
• jxl"1 (n= 18) while the hand-expressed juice contained only
0.001 ± 0.00003 fx equivalents acid released -mirr1 ixh1
(n=60). These values are equal to 13.3 units -ml-' and 1.0
unit -ml"1 respectively.
Conclusions
The colorimetric assay for PME activity introduced by
Hagerman and Austin (1986) has been adapted successfully
to a kinetic microplate reader. Results presented here dem
onstrate that the method is accurate and applicable to a
commercial PME as well as a commercial, non-pasteurized
and a hand-expressed orange juice. The sensitivity of the
assay is slightly greater than originally reported by Hager
man and Austin (1986). As little as 0.009 units of PME
could be measured accurately. The assay requires very small
volumes of substrate and enzyme. The microplate format
allows for extensive replication of individual samples, mak-
Added Activity
(units)
Estimated Activity
(units)
0.006
0.009
0.012
0.015
0.00294 ± 0.00022 (n = 16)
0.00898 ± 0.00037 (n = 32)
0.01205 ± 0.00056 (n = 32)
0.01495 ± 0.00042 (n = 16)
ing statistical treatment possible. Finally, adaptation of the
colorimetric PME assay to a kinetic microplate reader saves
a tremendous amount of time. After becoming familiar
with the procedure, 24 samples could be assayed, with each
sample replicated three times, in less than one hour. Addi
tionally, computer software is available to operate the
microplate reader, collect and manipulate data and present
final estimates of enzyme activity.
Literature Cited
1. Baldwin, E. A. and R. Pressey. 1989. Pectic enzymes in pectolyase.
Separation, characterization, and induction of ethylene in fruits. Plant
Physiol. 90:191-196.
2. Fox, J. D. and J. F. Robyt. 1991. Miniaturization of three carbohydrate
analyses using a microsample plate reader. Anal. Biochem. 195:93-96.
3. Hagerman, A. E. and P. J. Austin. 1986. Continuous spectrophotomet-
ric assay for plant pectin methyl esterase. J. Agric. Food Chem. 34:440-
444.
4. Koch, J. L. and D. J. Nevins. 1990. The tomato fruit cell wall. II.
Polyuronide metabolism in a nonsoftening tomato mutant. Plant
Physiol. 92:642.
5. Plantner, J. J. 1991. A microassay for proteolytic activity. Anal.
Biochem. 195:129-131.
6. Rombouts, F. M. and W. Pilnik. 1978. Enzymes in fruit and vegetable
juice technology. Process Biochemistry 13:9-14.
7. Tucker, G. A., N. G. Robertson and D. Grierson. 1982. Purification
and changes in activities of tomato pectinesterase isoenzymes. J. Sci.
Food Agric. 33:396-400.
8. Versteeg, C, F. M. Rombouts, C. H. Spaansen, and W. Pilnik. 1980.
Thermostability and orange juice cloud destabilizing properties of
multiple pectinesterases from orange. J. Food Sci. 45:969-971, 998.
9. Wenzel, F. W., E. L. Moore, A. H. Rouse, and C. D. Atkins. 1951.
Gelation and clarification in concentrated citrus juices. I. Introduction
and present status. Food Technol. 5:454-457.
Proc. Fla. State HorL Soc. 104:104-108. 1991.
CHARACTERIZATION OF THE RIPENING OF
CARAMBOLA (AVERRHOA CARAMBOLA L) FRUIT
Elizabeth J. Mitcham and Roy E. McDonald
Agricultural Research Service
U.S. Department of Agriculture
2120 Camden Road
Orlando, FL 32803
Additional index words: cell wall, firmness, color, ethylene,
respiration, pectin.
Acknowledgements: The authors would like to thank Craig Campbell
of J.R. Brooks & Son for hand-selecting and donating the fruit used in
this study. We also acknowledge the excellent technical assistance of Ms.
Heather Tucker.
Abstract. Carambola fruit were harvested at four stages of
ripeness: dark green (DG), light green, color break (CB) and
ripe. Additional ripe fruit were stored at 21C until overripe
(OR). The ratio of CIE color a/b increased during ripening in
versely to the decrease in fruit firmness. Respiration and
ethylene production of carambola fruit of different ripeness
stages suggested a possible climacteric pattern. However,
daily monitoring of individual fruit respiration and ethylene
production provided inconclusive evidence as to the climac-
teric/nonclimacteric nature of carambola fruit. The cell walls
of DG carambola fruit were comprised mainly of cellulose
(60%) and hemicellulose (27%), with pectin polymers account
ing for only 13%. There was an increase in the proportion of
less tightly bound chelator-soluble pectin and a decrease in
104 Proc. Fla. State HorL Soc. 104: 1991.
covalently-bound pectin during ripening. The amount of total
cell wall uronic acid decreased at the OR stage. The amount
of hemicellulose decreased beginning at the CB stage and the
proportion of cellulose increased throughout the ripening pro
cess.
Although carambolas have been commercially grown in
Asia for many years, the industry in South Florida is rela
tively new. While production has increased rapidly in recent
years, little is known about the physiology of carambola
ripening. Although changes in color, soluble sugars and
acids have been documented during fruit ripening
(Campbell et al., 1989; Vines and Grierson, 1966), there is
little or no information available on the textural changes
that occur during ripening of carambola fruit. The soften
ing process is of great commercial importance because the
postharvest shelf life of the fruit is limited by increased
softness which leads to an increase in susceptibility to
mechanical damage and disease. Softening of fleshy fruits
is mainly due to a modification of cell wall structure. Cell
wall structure and modification thereof have not been inves
tigated in the carambola fruit.
Also, uncertainty exists about the classification of caram
bola fruit as climacteric or nonclimacteric. Vines and Grier
son (1966) observed a respiratory climacteric in 'Golden
Star' carambola and classified carambola as climacteric.
However, Oslund and Davenport (1983) found no evidence
that 'Golden Star' carambola was climacteric. Similarly, Lam
and Wan (1983) found no evidence for a climacteric pattern
in carambola (cv. BIO), and exposure to Ethrel, a treatment
which releases ethylene gas, failed to induce ripening (Lam
and Wan, 1983). It was recently reported that treatment
with 100 ppm ethylene can promote carambola ripening
(Sargent and Brecht, 1990). This type of response to
ethylene treatment is normally associated with climacteric
fruit. It has been suggested that carambola is a climacteric
fruit and that the climacteric occurs very late in the ripening
process.
The objective of this investigation was to increase our
knowledge of the physiology of carambola ripening by
exploring changes in fruit texture and cell wall composition
during ripening, and additionally, to search for evidence
of a climacteric pattern in carambola.
Materials and Methods
Fruit material. Carambola (Averrhoa carambola L., cv.
Arkin) fruit were harvested in Homestead, Fla. at four
different stages of development as determined subjectively
by color development: 1) dark green (DG), no yellow color;
2) light green (LG), green with pale yellow between ribs;
3) color break (CB), mostly yellow with some green remain
ing, orange color between ribs; and 4) ripe (R), yellow to
light orange. Fruit were transported to Orlando, gently
dipped in 0.015% sodium hypochlorite (30C, pH 7.7),
rinsed with deionized water (dH2O) and dried. Fruit were
stored at 21C, 90% RH overnight. Eight to 12 fruit of good
condition representing DG, LG, CB, and R stages were
selected for respiration, ethylene production, color, firm
ness, and cell wall analysis. Additional R fruit were stored
(1 to 3 weeks) until overripe (OR, dark orange color), at
which time they were also sampled.
Respiration and ethylene production. Eight single fruit or
pairs of small fruit at each stage of ripeness were sealed in
Proc. Fla. State Hort. Soc. 104: 1991.
930 ml jars at 21C. After 15 min, 1 ml gas samples were
analyzed for CO2 using a Hewlett Packard 5880 gas
chromatograph equipped with a Porapak Q column and a
thermal conductivity detector (TCD). Nitrogen served as
the carrier gas at a flow of 30 ml-mirr1. Only five measure
ments of CO2 production were obtained from OR fruit as
fruit showing any sign of decay were not used. The same
fruit were then sealed in 930 ml jars at 21C with 30 g of
soda lime (sodium hydroxide with calcium oxide or hydro
xide) to absorb CO2. After 22 to 24 hr, 1 ml gas samples
were analyzed for ethylene using a Hewlett Packard 5880
gas chromatograph equipped with an alumina column and
a flame ionization detector (FID). Nitrogen served as the
carrier gas at a flow rate of 30 ml-min-1.
An additional 20 fruit of various stages of ripeness from
LG to R were monitored daily for CO2 production as de
scribed above. A separate group of 15 fruit were monitored
every second day over a 20-day period for ethylene produc
tion as described above.
Color. After gas measurements, color measurements
(CIE L*a*b) were taken on the same 8 to 12 fruit at each
ripeness stage. A Minolta CR200 Colorimeter fitted with
an aperture plate (11 mm diameter) was used to measure
external color. The mean of three measurements per fruit,
on the side of three wings, were averaged for fruit of each
ripeness stage.
Firmness. Carambola fruit were supported by a triangu
lar platform with the flat side of one wing perpendicular
to a round, convex probe (8 mm diameter) attached to an
Instron Universal Testing Instrument. The Instron was
fitted with a 100 kg load cell and operated at a crosshead
speed of 25 mm-min-'. Resistance to compression (3 mm)
was measured on three wings per fruit.
Cell wall extraction. Fruit were peeled, and seeds and
endocarp removed. The mesocarp from two to three fruit
per stage were combined, frozen in liquid nitrogen, and
stored at —80C for subsequent cell wall extraction. Later,
tissue was thawed, homogenized 3 min with a Polytron in
3 volumes of 80% ethanol, treated in a cell disruption bomb
to break remaining whole cells and rinsed through Mirac-
loth with 2 volumes 20 mM Hepes-NaOH (pH 6.9). The
residue was stirred in 2 volumes phenol:acetic acid:dH2O
(2:1:1, w/v/v) for 20 min, and successively rinsed in 3 vol
umes chloroform:methanol (1:1, v/v) and 3 volumes
acetone. Cell walls were dried in a vacuum oven at 40C
over phosphorous pentoxide.
Cell wall uronide determination. Cell walls (10 mg) were
incubated on ice in 2 ml concentrated sulfuric acid on a
gyratory shaker. Two 500 |xl aliquots of dH2O (4C) were
added slowly and the solution incubated on a shaker until
dissolved (6 hr). Uronic acid concentration was estimated
by the carbazole method (Dische, 1947).
Cell wall fractionation. Cell walls (150 mg) were fraction
ated into chelator-soluble pectin (CSP), covalently-bound
pectin (CBP), a hemicellulosic fraction (HF), and a cellulosic
fraction (CF) as described previously (Mitcham et al., 1989),
except that the HF was extracted using a single incubation
in 8 N KOH for 3 hr. Carbohydrate concentration of cell
wall fractions was determined by the phenol-sulfuric acid
method (Dubois et al., 1956). Uronide concentration was
determined using the carbazole method (Dische, 1947).
Cellulose determination. Cellulose content of crude cell
walls was estimated according to the procedure of Updeg-
raff (1969). Cell walls (10 mg) were incubated in teflon-cap-
105
ped tubes with 1 ml 2 N tritluoroacetic acid (TFA) for 1
hr at 121C to hydrolyze noncellulosic neutral sugars. The
TFA solution was removed and the residue rinsed twice
with dH2O and incubated at 30C for 1 hr with 1 ml 78%
sulfuric acid. The volume was brought to 25 ml with dH2O
and incubated at 121C for 1 hr. Cellulose content was esti
mated using the Anthrone method for total hexose (Spiro,
1966) with glucose as the standard.
Results and Discussion
Color and firmness. CIE color "a" and "b" values both
increased during ripening of carambola fruit (data not
shown); however, the ratio of a/b provided the most linear
relationship to ripeness stages which had been selected
based on subjective color analysis (Fig. 1). Resistance to
compression (firmness) decreased linearly as ripening prog
ressed (Fig. 1). The decrease in firmness was inverse to the
increase in a/b color indicating that determination of ripe
ness stage based on fruit color is very accurate.
Respiration and ethylene production. The rate of CO2 pro
duction of fruit picked at different ripeness stages (Fig. 2)
was 30 ml-kg-'-h"1 at the DG and LG stages, decreased to
20 ml-kg-'-hr1 at the CB stage, then increased to the original
rate by the OR stage. The pattern of CO2 production indi
cated a possible preclimacteric minimum at the CB stage
with a peak in respiration at the OR stage. However, there
is no evidence for a decrease in CO2 production after the
peak. Ethylene production from the same fruit began to
increase at the CB stage and increased sharply at the OR
stage (Fig. 2). The results for ethylene production were
similar to those of Oslund and Davenport (1983).
To search for additional evidence of a climacteric pat
tern in carambola fruit, CO2 and ethylene production was
monitored over time (Fig. 3) in fruit harvested at different
stages of ripeness. No consistent pattern in respiration was
observed in fruit harvested at the CB stage (Fig. 3A) or any
other ripeness stage (data not shown), and we did not ob
serve a peak in CO2 production like that reported by Vines
and Grierson (1966). As fruit became OR, decay often in
terfered with respiration measurements. The presence of
decay was associated with an increase in respiration, which
may have been due to a wound response or to respiration
of the decay organisms.
Ethylene production, however, followed a climacteric
pattern with a sharp peak in production at approximately
-2
-4
-6
DG LG CB R
Stage of Ripeness
OR
Fig. 2. CO2 and ethylene production of carambola fruit at different
stages of ripeness. Each value represents the mean production rate of 5
to 8 individual fruit or pairs of fruit at each stage of ripeness. Vertical
bars represent SE.
the R stage (Fig. 3B). All fifteen fruit monitored underwent
a peak in ethylene production, but the timing and mag
nitude varied with the ripeness stage at harvest. Two CB
fruit underwent an immediate peak in ethylene production
while the ethylene peak of R fruit occurred several days
later and was lower in magnitude (Fig. 3B). It is possible
that R fruit underwent peak ethylene production prior to
harvest. Two LG fruit underwent a peak in ethylene pro-
60
50 g-
40 il zi O -i'
30 § «
20 2
10
20
DG LG CB R
Stage of Ripeness
Days
Fig. 1. Color and firmness of carambola fruit at different stages of
ripeness. Each value is the mean of 3 measurements per fruit and 8 to
12 fruit per stage. Vertical bars represent SE.
106
Fig. 3. A) Daily CO2 production of 3 individual CB carambola fruit.
B) Ethylene production of individual LG, CB, and R carambola fruit measured every second day. Arrows designate the point when decay was
visible.
Proc. Fla. State Hort. Soc. 104: 1991.
duction at the CB to R stage. Most fruit exhibited signs of
decay 2 to 4 days after the peak in ethylene production.
The decay organisms did not appear to contribute to ethylene production since the production rate continued
to decrease after decay began. However, ethylene produc
tion by the carambola fruit may have been a pathogen-in
duced, wound response that occurred before decay became visibly noticeable. Alternatively, normal production of
ethylene by the fruit may have stimulated decay develop ment.
Our data regarding the climacteric nature of carambola
are inconclusive. However, as explained by Biale (1960),
placing a fruit in the nonclimacteric category calls for a
more tentative decision than listing one as climacteric be
cause positive evidence can be experimentally obtained
much more readily than convincing negative proof.
Cell wall composition. The carbohydrate portion of DG
carambola cell walls was comprised largely of cellulose
(60%) and hemicellulose (27%) polymers with pectin polym
ers comprising only 13%. In unripe fruit, a greater percent
age of the pectin was covalently-bound pectin (CBP) which
is more tightly bound within the cell wall (Fig. 4A). The
proportion of loosely associated chelator-soluble pectin
(CSP), which can be solubilized from the cell wall with cal
cium chelators, was lower than CBP in the unripe fruit (Fig.
4 A). However, in OR fruit, the increase in CSP and decrease
in CBP resulted in equal amounts of the two types of pectin
polymers. In addition to a shift in the type of pectin polym
ers present during ripening, there was also a slight decrease
in cell wall uronides, the major component of pectin, after
the R stage (Fig. 4B). The modification of pectin polymers
from CBP to CSP began after the LG stage along with the
decrease in fruit firmness; however, there was no net loss of uronic acid from the cell wall until after the R stage.
The changes in carambola pectin polymers during ri
pening are similar to those found in strawberry fruit. The
amount of cell wall uronide decreases late in ripening (Knee
et al., 1977), and a shift to CSP also occurs during ripening
of strawberry fruit (Woodward, 1972). Woodward pro posed that a change in the cell wall po/ysaccharides takes
place in such a way that the uronide polymers become
rearranged to allow more plasticity in the walls of the fruit.
Neal (1965) suggested that chelation of calcium ions, which
are thought to form crosslinks between carboxyl groups of
polyuronide chains, may lead to softening of the tissue.
In addition to the pectin changes, there was also a de
crease in the amount of hemicellulosic material which began
at the CB stage (Fig. 5A). The amount of cellulosic material
increased linearly throughout ripening (Fig. 5B). The in
crease in the proportion of cellulose may be due to a loss
of other cell wall polymers.
It is interesting that the amount of all four types of cell
wall polymers (mg/150 mg cell wall) increased from the DG
stage to the CB stage (Figs. 4 & 5). These data indicate a
decrease in another cell wall component, perhaps protein.
Conclusions
The development of carambola fruit CIE a*/b* color
was inverse to the decrease in fruit firmness indicating color
is an accurate means by which to determine fruit ripeness.
Carambola cell walls were comprised mainly of cellulose
DG LG CB R
Stage of Ripeness
Fig. 4. A) Galacturonic acid equivalents of chelator-soluble pectin (CSP)
and covalently-bound pectin (CBP) extracted from 150 mg cell wall at
different ripeness stages. B) Total galacturonic acid equivalents extracted
from 10 mg cell wall.
Proc. Fla. State Hort. Soc. 104: 1991.
LG CB R
Stage of Ripeness
Fig. 5. Glucose equivalents of hemicellulosic (A) and cellulosic (B) polymers extracted from 150 mg cell wall at different stages of ripeness.
107
and hemicellulose with only 13% pectin; however, the pro
portion of less tightly bound chelator-soluble pectin in
creased with ripening.
Literature Cited
1. Biale, J. B. 1960. Respiration of fruits. In: Encyclopedia of Plant
Physiology, Vol. XII/2, J. Friend and M.J.C. Rhodes (eds.), p. 536-
592, Springer-Verlag, Berlin.
2. Campbell, C. A., D. J. Huber, and K. E. Koch. 1989. Postharvest
changes in sugars, acids, and color of carambola fruit at various
temperatures. HortScience 24:472-475.
3. Dische, Z. 1947. A new specific color reaction of hexuronic acids. J.
Biol. Chem. 167:189-198.
4. Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers and F. Smith.
1956. Colorimetric method for determination of sugars and related
substances. Anal. Chem. 28:350-356.
5. Knee, M., J. A. Sargent, and D. J. Osborne. 1977. Cell wall metabolism
in developing strawberry fruits. J. Exp. Bot. 28:377-396.
6. Lam, P. F. and C. K. Wan. 1983. Climacteric nature of the carambola
(Averrhoa carambola L.) fruit. Pertanika 6(3):44-47.
7. Mitcham, E. J., K. C. Gross, and T. J. Ng. 1989. Tomato fruit cell
wall synthesis during development and senescence. In vivo radiolabel-
ing of wall fractions using [14C] sucrose. Plant Physiol. 89:477-481.
8. Neal, G. E. 1965. Changes occurring in the cell walls of strawberries
during ripening. J. Sci. Food Agric. 16:604-611.
9. Oslund, C. R. and T. L. Davenport. 1983. Ethylene and carbon
dioxide in ripening fruit of Averrhoa carambola. HortScience 18:229-
230.
10. Sargent, S. A. and J. K. Brecht. 1990. Ethylene pretreatment allows
early harvest of carambola. HortScience 25:1174.
11. Spiro, R. G. 1966. Analysis of sugars found in glycoproteins. In:
Methods of Enzymology, Vol. 8, E. F. Neufeld and V. Ginsburg,
(eds.), p. 4-6, Academic Press, NY.
12. Updegraff, D. M. 1969. Semi-micro determination of cellulose in
biological materials. Anal. Biochem. 32:420-424.
13. Vines, H. M. and W. Grierson. 1966. Handling and physiological
studies with the carambola. Proc. Fla. State Hort. Soc. 79:350-355.
14. Woodward, J. R. 1972. Physical and chemical changes in developing
strawberry fruit. J. Sci. Food Agric. 23:465-473.
Proc. Fla. State Hort. Soc. 104:108-111. 1991.
AN EXPERT SYSTEM FOR HYDROCOOLERS
Jean-Pierre Emond and Khe V. Chau
Agricultural Engineering Dept.
IF AS, University of Florida
Gainesville, FL 32611
Jeffrey K. Brecht
Vegetable Crops Dept.
IF AS, University of Florida
Gainesville, FL 32611
Additional index words, cantaloupe, celery, cucumber,
peaches, sweet corn, cooling.
Abstract. This paper describes an expert system that has been
developed to give the specifications required to cool produce
with a hydrocooler. Provided with a user friendly interface,
this expert system can perform a wide range of tasks such as:
hydrocooler design, precooling process optimization, quality
control, energy conservation and investigation of new applica
tions. In order to execute these tasks, the expert system needs
information provided by the user such as: kind of product,
loading capacity, entering and exit temperature of the product,
water temperature and residence time. Depending on the in
formation given, the expert system will use default values for
the missing information to compute the residence time, product
temperature, refrigeration capacity, water flow rate and the
maximum shelf life of the product if stored at the exit temper
ature. Assumptions made in this expert system are: cooling
efficiency of 50%, 3°F rise in temperature of the water going
through the hydrocooler, exit product temperature determined
by the 7/8 cooling time unless the chilling injury threshold
temperature of the product is higher. Further improvement
will make the software more versatile and applicable to a
wider variety of commodities.
The temperature of fresh fruits and vegetables is the
greatest determinant of the rate of deterioration by decay
Florida Agricultural Experiment Station Journal Series No. N-00536.
108
and senescence, and consequently, of the potential market
life (Kader et al., 1985). Produce temperature control is
critical from the moment of harvest when the process of
deterioration begins; delay in cooling the product can cause
loss of quality.
Several methods for rapid removal of heat from pro
duce are in commercial use (Ryall and Lipton, 1979). The
choice of a particular method depends mainly on the rate
of cooling desired, product surface to volume ratio, product
susceptibility to water damage, availability of equipment,
the value and the perishable nature of the product. It is
very important to select the optimal precooling system to
satisfy the commodity and the market structure needs. A
knowledge-based system for selecting precooling methods
was developed by Morey et al. (1988) to help producers
faced with a decision of selecting a precooling method. This
computer program (Morey et al., 1988) gives the users the
opportunity to specify the crop and information about their
operation, and uses this information to select possible cool
ing methods, which can be room cooling, forced-air cooling,
hydrocooling, vacuum cooling or package icing. However,
the above system does not provide-any pertinent informa
tion on the possible cooling methods selected.
Once a precooling method is selected, the service of an
expert to characterize the system is required. With their
knowledge, experts are able to determine the specification
needed to design and operate a precooling unit. This kind
of expertise can be costly and may not be readily available.
The same problem can be observed for owners of precool
ing units who want to change operating conditions of their
units or to use them for other crops. Only an expert can
provide this kind of information. In many cases, a joint
effort of a team of experts in postharvest physiology and
engineering would be useful. These teams cannot always
be available at the time the information is needed. For these
reasons, computer programs that simulate human experts
in this area are needed. By storing the knowledge of many
experts in its memory bank a computer program ("expert
system") can provide answers to questions from users. Com-
Proc. Fla. State Hort. Soc. 104: 1991.