Lake Alfred AREC Research Report-CS74-1
Presented at the Subtropical Fruits Institute, Homestead, Fla.
February 20, 1974
A PROPOSED METHOD FOR ESTIMATING AVOCADO FRUIT MATURITY
- '- r~r
CHARLES R. BARMORE
A. H. ROUSE
INSTITUTE OF FOOD AND AGRICULTURAL SCIENCES, UNIVERSITY OF FLORIDA,
AGRICULTURAL RESEARCH AND EDUCATION CENTER, LAKE ALFRED
Methods for estimating avocado fruit maturity have been extensively
investigated. At present, the most reliable method for estimating maturity
of a particular cultivar (variety) grown in Florida is based on field
observations during fruit growth and its typical growth characteristics
as observed over several years.
Recently, the authors noted that an enzymatic test could possibly be
used to aid in the estimation of avocado fruit maturity. This test is being
proposed as a supplement to the present method, thereby, strengthening its
The following discussion examines the basis for the test, its validity,
method of analysis, and how it can supplement the present method of estimating
avocado fruit maturity. A cooperative study is proposed between the Avocado
Administration Committee (AAC) and the authors to determine the feasibility
of using this method by the AAC.
Basis for Test
Softening is the most obvious change associated with ripening of
avocados. This is brought about by the action of pectolytic enzymes on
the pectic constituents of the cell walls of fruits. Isolation and
characterization of these enzymes in avocado and other fruits are well
Two enzymes of particular interest associated with the softening process
are PECTINESTERASE (PE) and POLYGALACTURONASE (PG). PG activity does not
become measurable until after harvest, increasing to a maximum when the
fruit becomes soft. In contrast, PE activity is highest in immature fruit,
decreasing as the fruit matures. After harvest, PE activity continues to
decrease, reaching a low plateau when softening is complete. The activity
of PE and PG as the fruit matures and ripens is shown in Fig. 1. Because
A S 0 N D 0 2 4 6 8 10
Fig. 1. A stylized graph showing
the changes in PE and PC during
fruit maturation and ripening of
a typical avocado fruit. PE x---x,
of this particular pattern of PE activity during fruit maturation of a given
variety, this enzyme could possibly be used as an indicator for estimating
avocado fruit maturity. Information from our laboratory and others indicates
that at maturity a specific PE value is reached. A graphic representation
of the relationship between fruit maturation and the PE activity during this
period is shown in Fig. 2. This work was reported by Drs. Zaubermann and
Schiffmann-Nadel of the Volcani Institute of Agricultural Research, Israel.
Their work also supports our concept of using PE as a measure of maturity.
J J A S
0 N D
Fig. 2. The activity of PE in 'Fuerte'
avocado fruit at various stages of
development. (From: Zaubermann and
Schiffmann-Nadel, 1972. Plant Physiol.
Validity of the test is based on the fact that there is a relationship
between PE activity and days required to soften. The latter method is
presently used to check maturity when in question. We have conducted
numerous tests during the past 2 seasons for the purpose of determining
the accuracy of using PE to estimate softening time. With standardized
conditions, we are able to predict softening time within + 1 day. Deviation
from the actual time required is due to the fact that definition of "soft"
Additional support for the test is given in Fig. 3. Three sizes of
'Haile' fruit having an average weight to length ratio of 366 g/13 cm,
422 g/13.3 cm and 530 g/14.3 cm were analyzed for PE activity. The latter
group of fruit was mature and being shipped commercially. Theoretically,
fruit of this cultivar having a PE value of 30 could be considered mature.
At present, size restriction would be necessary to ensure that only such
fruit were harvested.
U J 6 0 \
2 0 -
24 28 32 36 40
Fig. 3. Relationship between dimension
(expressed as weight in grams divided by
length in centimeter) and PE activity of
'Haile' avocado fruit.
Description. The method for PE determination is designed to measure
the rate of demethylation of pectin, thus freeing the carboxyl (acidic)
groups which can be titrated with an alkali. In essence, the higher the
PE activity the greater will be the rate of demethylation, thus requiring
an increasing amount of alkali to maintain the pH of the reaction mixture
at pH 7.5.
Equipment and reagent needs for PE analysis are listed below. All
materials can be purchased through most scientific supply companies.
1. Pectin NF (Sunkist Growers pure citrus pectin).
2. Sodium chloride, reagent grade.
3. Sodium chloride, 2% solution.
4. Sodium hydroxide, 0.05 N.
5. Sodium hydroxide, 0.25 N.
6. Buffer solution, pH 7.0.
1. pH meter with Automatic titrator.
2. Sorvall Omni-mixer.
3. Precision stirrer with rheostat and s.s. stirring rod
or variable speed magnetic stirrer with teflon covered
4. Mettler balance, P 2000.
5. Food chopper.
a) Buret, 25 ml.
b) Beakers, 150 ml, 1000 ml.
c) Graduated cylinder, 50 ml.
d) Mohr pipette, 5 ml.
e) Volumetric flask, 1000 ml.
f) Glass stoppered bottles (3), 1000 ml.
g) Plastic wash bottle, 500 ml.
h) Plastic reagent bottle, 500 ml.
k) Drop bottle, 30 ml.
Representative sample. The sampling procedure is a very important part
of the overall analysis. This procedure must be standardized. Fruit to be
analyzed must be representative in both size and weight of those fruit to
be evaluated for maturity. In addition, the number of fruit sampled must
be sufficient that the PE value obtained is statistically valid. We have
arbitrarily selected a 10 fruit sample, but the actual number should be
determined from a testing program.
Sample preparation. Tissue preparation consists of grinding each fruit
individually in a food chopper and taking a specific aliquot from each
sample. The aliquots are combined and thoroughly mixed. This composite
sample can be stored frozen at-100F or below for at least 1 month without
.significant loss in PE activity.
PE analysis. Directions for the preparation of the solution and
analysis of PE are as follows:
Preparation of Solutions
1. 17 pectin-salt solution--Mix 10 g of citrus pectin with 11.7 g
sodium chloride and slowly add to 800 ml distilled water with
agitation. After completely dispersed make to 1 liter. Add 6
drops of toluenu.
2. 27. sodium chloride solution--Dissolve 20 g sodium chloride in
distilled water and make up to 1 liter.
3. 0.05 N and 0.25 N sodium hydroxide--Dissolve standard strength
ampule to volume for proper normality.
1. From the composite sample, weigh 100 g of chopped avocado and
add 100 g 2% sodium chloride(salt) solution.
2. Homogenize the mixture for 5 minutes.
3. Weigh 10 g sample into a 150 ml beaker, add 50 ml 1% pectin-salt
solution and place under stirrer on pH meter.
4. Adjust to pH 7.8 with 0.25 N sodium hydroxide.
5. When pH returns to 7.5, begin timer (set for 15 minutes).
6. As pH drops below 7.5, add 0.05 N sodium hydroxide solution
dropwise from buret at a rate that will maintain pH 7.5.
7. The pectinesterase activity is read from the chart depending
on the quantity of 0.05 N sodium hydroxide solution used, or the
quantity of sodium hydroxide used is multiplied by the factor
6.7 to give units of PE activity for the sample.
The total time required for the analysis is approximately 20 minutes.
The analysis is automatic once the reaction has been started. Several samples
can be run simultaneously.
Calculation of PE value. The PE value is calculated from the number
of mlof alkali required to maintain the pH of the reaction mixture at
7.5. Activity of the enzyme is expressed as the number of PE units X 104.
This value can be obtained directly from a table or formula as shown in
Establishing Maturity Requiremonts
The proposed method is not suggested as a replacement for the present
method, but as a supplement. The PE value, once determined through a
Tabl.e .' Chart for the determination of pectinesterase activity.
(1 ml 0.05 N NaOH = 0.00067 X 104 = 6.7 PE units)
Reaction Time: 15 min. *10 gram Sample
PE X 104 ml 0.05 N NaOH PE X 104 ml 0.05 N NaOH PE X 104 ml 0.05 N NaOH
6.7 1.0 23.3 3.5 40.0 6.0
7.3 1.1 24.0 3.6 40.7 6.1
8.0 1.2 24.7 3.7 41.3 6.2
8.7 1.3 25.3 3.8 42.0 6.3
9.3 1.4 26.0 3.9 42.7 6.4
10.0 1.5 26.7 4.0 43.3 6.5
10.7 1.6 27.3 4.1 44.0 6.6
11.3 1.7 28.0 4.2 44.7 6.7
12.0 1.8 28.7 4.3 45.3 6.8
12.7 1.9 29.3 4.4 46.0 6.9
13.3 2.0 30.0 4.5 46.7 7.0
14.0 2.1 30.7 4.6 47.3 7.1
14.7 2.2 31.3 4.7 .48.0 7.2
15.3 2.3 32.0 4.8 48.7 7.3
16.0 2.4 32.7 4.9 49.3 7.4
16.7 2.5 33.3 5.0 50.0 7.5
17.3 2.6 34.0 5.1 50.7 7.6
18.0 2.7 34.7 5.2 51.3 7.7
18.7 2.8 35.3 5.3 52.0 7.8
19.3 2.9 36.0 5.4 52.7 7.9
20.0 3.0 36.7 5.5 53.3 8.0
20.7 3.1 37.3 5.6 54.0 8.1
21.3 3.2 38.0 5.7 54.7 8.2
22.0 3.3 38.7 5.8 55.3 8.3
22.7 3.4 39.3 5.9 56.0 8.4
of a 50:50 mixture of 2% sodium chloride plus avocado tissue by weigh
testing program for each cultivar, would be used to determine the actual
date a fruit of a specific size would be mature.
The date of harvest proposed early in the season would be determined
by the present criteria. However, as this date is approached, PE activity
would be determined for the larger fruit in order to judge when they are
actually mature. The date that the fruit reaches maturity could be on,
before, or even after the proposed picking date. This would be valid
since PE is a measure of the number of days required to soften which now
can be determined only after the first suitable picking date is past. Use
of such a maturity standard is shown in Table 2. In this example, the proposed
picking date for 'Lula' for 1974 would be October 1. Fruit at this picking
must be 14 oz in weight with a width of 3-3/8 in. or greater. If on September
29 the PE was down to 45, fruit of the above size would be considered as
mature and allowed to be harvested. Adjustment in size restriction could
be made at any time. If PE was still above 45, picking would be delayed.
Similarly, picking might be advanced if PE was significantly below 45
prior to October 1.
Table 2. Example of the maturity standards
for showing the relationship of PE activity
for several avocado cultivars.
Proposed Fruit size Maximum
picking Weight Width PE
Variety date (oz) (in.) Units X 104
Lula Oct. 1 14 3-3/8 45
Booth 8 Sept. 15 13 3-1/4 75
Haile July 31 17 3-9/16 30
Waldin Aug. 7 15 3-9/16 90
The use of PE as an indicator of maturity has several merits in that
it is relatively a simple and quick test, it is a measure of softening
time, the test is easily standardized, and it would supplement the present
method. We emphasize that to date the method has been tested only in
Our purpose in presenting the method to the Avocado Administration
Committee and growers is to develop a joint effort among those concerned
in examining the feasibility for commercial use. A testing program of
at least 2 years would be required.