Results and Discussion

Standard curves for ethyl butyrate, octanal, linalool, aterpineol, carvone and valencene are given in Fig. 1.
Curves were corrected for purity of standard, internal
standard and fitted by linear regressison. Ethyl butyrate
had the highest response and valencene the lowest.
The percent recovery and the coefficient of variation
for the five volatile compounds added to chilled juice is
given in Table 1. Highest recoveries were for ethyl buty
rate and octanal and lowest recoveries were for a-terpineol
and carvone. The recoveries tend to decrease as the boiling
point of the compounds increases.
Data are presented for an immediate separation of
methylene chloride extract of the distillate and for analysis
of the methylene chloride extract after overnight separa
tion at 2 C. The immediate separation is most applicable
for quality control. The coefficient of variation for ethyl
butyrate was 2.53 to 2.84; for octanal, 2.16 to 4.11; for
linalool, 2.94 to 6.23; for a-terpineol, 2.81 to 8.35 and for
carvone 4.14 to 9.78. These values are reasonable for a
procedure which includes distillation, extraction and gas
chromatographic analysis.
The increase in gas chromatographic peak area for volatiles determined for the chilled juice sample and for the
juice samples with 0.25, 0.50 and 0.75 ppm added volatile
compounds is presented in Fig. 2. There was a linear re
sponse with increase in concentration of the added stand
ards. The concentration of the recovered volatiles for the
five added compounds was determined from the standard
curve (Fig. 3). For the chilled juice, ethyl butyrate concen
tration was 0.98 ppm; octanal, 1.5 ppm; linalool, 1.7 ppm;
a-terpineol, 0.42 ppm and for carvone 0.05 ppm. For the
chilled juice with 0.75 ppm standard added the values de
termined were; ethyl butyrate, 1.58 ppm; octanal, 2.15

ppm, linalool, 2.14 ppm; a-terpineol, 0.74 ppm and for

carvone, 0.37 ppm. Although the concentration for dlimonene is not presented it was determined to be approx
imately 200 ppm, which is in the expected range.
Major differences were found between samples of
evaporator pump-out, chilled juice and orange juice from
concentrate which contained an oxidized oil when analyzed
by this procedure (Fig. 4). The evaporator pump-out
chromatogram indicated only a small amount of dlimonene and the juice sample containing the oxidized oil
indicated significant increases in the carvone peak and the
a-terpineol peak. Other unidentified peaks also increased
in concentration.

The procedure indicates quantitative differences for

important volatile constituents of orange juice samples and
should provide a useful rapid analytical method for quality
control.
Literature Cited
1. Marsili, R. T. 1986. Measuring volatiles and limonene-oxidation prod
ucts in orange juice by capillary gc. LC-GC Mag. Chromatogr. Sci.
4:358-362.
2. Moshonas, M. G. and P. E. Shaw. 1983. Analytical procedures for
evaluating aqueous citrus essences. Instr. Ansalysis of Foods Vol. II:
137-148. Acad. Press.
3. Moshonas, M. G. and P. E. Shaw. 1984. Direct gas chromatographic
analysis of aqueous citrus and other fruit essences. J. Agr. Food Chem.
32:526-530.
4. Moshonas, M. G. and P. E. Shaw. 1987. Quantitative analysis of orange
juice flavor volatiles by direct-injection gas chromatography. J. Agr.
Food Chem. 35:161-165.
5. Schreier, P. 1981. Changes of flavor compounds during the processing
of fruit juices. Proc. Long Ashton Symp. 7:355-371.
6. Scott, C. W. 1968. Fruits and fruit products; collaborative study of
recoverable oil in citrus juices by bromate titration. J. Assoc. Offic.
Anal. Chemists 51:928-931.

Proc. Fla. State Hort. Soc. 101:147-150. 1988.

MEASUREMENT OF ORANGE JUICE COLOR WITH THE

HUNTERLAB LABSCAN REFLECTANCE SPECTROCOLORIMETER
Bela S. Buslig
Florida Department of Citrus
P. 0. Box 1909
Winter Haven, Florida
Charles J. Wagner, Jr.1
U.S. Citrus and Subtropical Products Laboratory2
P. O. Box 1909
Winter Haven, Florida

Additional index words. Model LS-5100.

Abstract. The HunterLab LabScan, Model LS-5100, scanning

laboratory reflectance spectrocolorimeter was adapted to en

able it to accept 1-in. diameter test tubes and was tested in
comparison with the industry standard HunterLab Citrus ColFlorida Agricultural Experiment Station Journal Series No. 9661.
Present address: 1144 Lake Martha Dr. W., Winter Haven, FL 33881.
2U.S. Department of Agriculture, Agricultural Research Service,
South Atlantic Area.

Mention of a trademark or proprietary product is for identification

only and does not imply a warranty or guarantee of the product by the
Florida Department of Citrus or the U. S. Department of Agriculture
over other products which may also be suitable.

Proc. Fla. State Hort. Soc. 101: 1988.

orimeter. Following statistical analysis of the results, regres

sion equations were developed for the expression of color
numbers. This instrument employing the prescribed adapter
and equations was accepted by U.S. and Florida regulatory
agencies as one alternative to the Citrus Colorimeter.
The development of the Citrus Colorimeter (CC) was
the direct result of application of the theoretical and prac
tical considerations involved in the measurement of color
in orange juices (6-9, 11, 12, 17). The announced intention
to discontinue the production of the CC by Hunter As

sociates Laboratory, Inc. (HunterLab) and the continued

necessity for objective measurements of color for grading

purposes encouraged research to evaluate available in
strumentation for the measurement of color values of
orange juices. Various colorimeters and spectrophotometers were tested in comparison with the CC to find suitable
replacements (2-5, 16).

Major advances in computer technology and elec

tronics, since the original design of the CC, yielded signif
icant improvements in color instrumentation. The newer
instruments are much more versatile, with a variety of illu147

minants and color scales available in a single instrument.

However, the CR and CY and the derived CN scales, used
in the citrus industry, are not in their repertoire. Most
color instruments are designed to take measurements from
flat surfaces in contrast to the familiar 1-in. diameter
round tubes employed with the CC. In the interest of un
iformity we tried to maintain sample presentation similar
to the CC with the newer instruments. All of the colorimet
ers or spectrophotometers tested were modified to make
the measurements in the presently used 1-in. diameter
glass test tubes. After a preliminary screening with limited
numbers of juice samples, all promising instruments were
re-evaluated in comparison with the CC. This evaluation
was conducted with sufficient number and color range of
orange juice samples to achieve statistical significance. Mul
tiple linear regression equations were generated to express
color numbers (CN) from each instrument's measurements
in terms of the values obtained from the CC. Earlier pub
lications by us described various additional facets of this
work (2-4, 16).
The present paper shows the results obtained with the
HunterLab LabScan Model 5100, reflectance spectrocolorimeter.
Materials and Methods

The HunterLab LabScan Model-5100 is a sophisticated

multipurpose spectrocolorimeter equipped with 0 illumi
nation and 45 circumferential viewing observer. The sen
sor's 16 station fiber optic viewing ring collects light from
the sample and averages the input from all stations. This
reduces errors caused by surface variation, directional or
textured samples. The instrument continuously scans the
visible spectrum, providing tristimulus values based on
data collected in 10 nm intervals (10). We employed illuminant C and collected data in terms of CIE X, Y and Z
values for direct comparison of the analogous values of the
standard CC. The instrument was modified with a tube
adapter, made from a 1-in. I.D steel tube fastened verti
cally on an 18 mm port plate, permitting the sample tube
to touch the front of the plate. The instrument was tilted
forward to permit vertical orientation of the sample tube
during measurement.
The HunterLab Model D45D2 CC was used as the ex
perimental reference device. This instrument also employs
illuminant C and is equipped with four broad-band filters
corresponding to tristimulus response functions, with 45
source and 0 observer position to examine orange juice
presented in 1-in. diameter selected glass test tubes. Read
ings are normally obtained in terms of CR, CY and CN.
However, for these experiments the X, Y and Z scales were
used to permit direct comparison of the values generated.
The relationships between these values for the CC are
shown in Table 1.
Both the LabScan and the CC were calibrated with the
same OJ4 plastic standard using identical X, Y and Z val
ues. Calibration with the OJ4 tube was performed after
Table 1. Citrus Colorimeter scales.

3U

27

Q
Q

24
21

18
+

15
12
9

148

15

18

21

24

27

X-LABSCAN
Fig 1. X(LabScan) vs X(CC).

each 24 readings. A total of 429 juice samples were meas

ured with the CC and two LabScans, utilizing the 1-in.
diameter glass test tubes, resulting in 858 paired data
points.
All color values used as dependent variables for the
statistical comparisons with the LabScan measurements
were obtained with the CC.
The data analysis and calculations were performed with
the MINITAB Statistical Analysis System (14) on a DEC
VAX minicomputer or the MSTAT: Microcomputer
Statistical Program (13) on a DEC Rainbow 100A micro
computer. All graphics were created with an IBM PC/XT
clone, using the SlideWrite program (1).
Results and Discussion

To maintain comparability with the the CC, we have

used the 1931 CIE illuminant C setting. An adapter was
constructed to enable use of the 1-in. test tube normally
employed with the CC to maintain uniform sample presen
tation throughout the experiments. Since both the
LabScan and the CC can read color values in terms of X,
Y and Z, direct comparison of these values could be made
easily.

rs

26.50

22.50

18.50

14.50

-H-

9.50
CR = 200[(1.277X - .213Z)Y - 1]
CY = 100(1 - .847Z/Y)
CN = 22.51 + .165CR + .111CY
CN = 0.61 + 42.14X/Y-16.43Z/Y

12

12.50

15.50

18.50

21.50

24.50

27.50

Y-LABSCAN
Fig 2. Luminance (Y) values. LabScan vs CC.

Proc. Fla. State HorL Soc. 101: 1988.

0.67

0.58

Q
f

0.49

0.40

0.31

N
0.22
0.1 3
10

11

Z-LABSCAN

more deviation from linearity than the primary values,

with an offset still present. Such results, in part, may be
explained by the convex surface of the sample tubes,
further affecting the amount of light reaching the ob
server. Since both the CC and the LabScan were calibrated
with the same standard OJ4 tube and the calibration values
entered for both were identical, only slope differences due
to the distance ratios should have remained in the plots of

1.06

1.03
1.00

0.97

0.94
0.91

0.97

0.99

1.01

X/Y-LABSCAN
Fig. 4. X/Y(LabScan) vs X/Y(CC).

Proc. Fla. State Hort. Soc. 101: 1988.

0.36

0.45

0.54

0.63

Fig. 5. Z/Y(LabScan) vs Z/Y(CC).

Figs. 1, 2 and 3 graphically show comparison of X, Y

and Z values measured. The correlation coefficients for
the individual color values, X, Y and Z, were 0.984, 0.972
and 0.977, respectively.
The figures show quite acceptable linearity of the plot
ted values for each of these parameters. However, this ob
servation is accompanied by an offset in each of the values
measured. This is probably due to differences in distance
from the samples to the observer between the two types of
instruments. The differences in reflected light intensity,
with other factors behaving in a linear manner, should be
proportional to the ratio of the squares of apparent dis
tance. This would imply that the comparison of the ratios
of X/Y and Z/Y, which are factors in the CR and CY and
consequently of the CN value expressions of the CC (Table
1), should be directly comparable between the CC and the
LabScan, with little or no offset observed. Plotting these
values, shown in Figs. 4 and 5, indicate that these show

0.95

0.27

Z/Y-LABSCAN

Fig. 3 Z(LabScan) vs Z(CC).

0.93

0.18

1.03

1.05

the ratios. The offset remaining indicated perception of

qualitative differences between the plastic tubes and the
orange juices in addition to the surface effects by the two
types of instruments. Regardless, the correlation coeffi
cient for each parameter was sufficiently high to permit
close estimation of the values expected from the CC, based
on measurements with the LabScan.
Multiple linear regression equations were calculated by
using the CC color values (CN) as the dependent variable
with LabScan X, Y and Z values or the calculated X/Y and
Z/Y values as independent variables. The result of the cal
culations, using the X, Y and Z values, are shown in Table
2. The equation generated with the X/Y and Z/Y ratios
yielded a lower correlation coefficient and therefore was
not considered further at this time.
The equation in Table 2 was used to calculate CN val
ues for each of the juices measured and these were plotted
against CN values read directly from the CC. Fig. 6 shows
these results. The diagram shows most significant devia
tion in CN values at the extremes of the practical color
range, with good linearity in the most often measured area.
The correlation coefficient is as shown in Table 2, explain
ing 96.8% of the variation observed.
Following the approval received from the U. S. Depart
ment of Agriculture, Inspection Service to use some new
colorimeters with the applicable adapters and equations
we developed, in late 1985 the Florida Citrus Commission
revised Chapter 20-65 of the Florida Citrus Fruit Laws
(15), concerning the use of colorimeters for official color
score determination. The HunterLab LabScan Model
5100, along with two others were permitted in addition to
the CC.
The equation developed for the LabScan spectrocolorimeter was based on the results from juices col
lected in a single season, therefore the equation may have
to be modified if and when additional data is collected,
although such is not expected. Further examination of the
data to enable us to parallel the CC equation parameters
more closely may also yield improved performance, possi
bly reducing variation at the color range extremes.
Table 2. LabScan regression equation.
CN = 40.10 + 1.90X- 1.55Y- 1.45Z
R = 0.984 s.e. = 0.263

149

4. Buslig, B. S., C. J. Wagner, Jr., and R. E. Berry. 1987. A general

purpose tristimulus colorimeter for the measurement of orange juice
color. Proc. Fla. State Hort. Soc. 100:47-49.
5. Eagerman, B. A. 1978. Orange juice color measurement using gen
eral purpose tristimulus colorimeters. J. Food Sci. 43:428-430.
6. Huggart, R. L. and F. W. Wenzel. 1954. Measurement and control
of color of orange concentrate. Proc. Fla. State Hort. Soc. 67:210-216.

7. Huggart, R. L. and F. W. Wenzel. 1955. Color differences of citrus

juices and concentrates using the Hunter color difference meter.
Food Technol. 9:27-29.

8. Huggart, R. L., R. W. Barron, and F. W. Wenzel. 1966. Evaluation

of the Hunter Citrus Colorimeter for measuring the color of orange

juices. Food Technol. 20:677-679.

9. Huggart, R. L., F. W. Wenzel, and F. G. Martin. 1969. Equivalent
color scores for Florida frozen concentrated orange juice. Proc. Fla.
10

CN-LABSCAN(CALC)
Fig. 6. CN(LabScan-calculated) vs CN(CC).

Literature Cited
1. Advanced Graphics Software. Inc. 1988. Version 2.11. SlideWrite
Plus AGS, Inc. Sunnyvale, CA.
2. Buslig, B. S. and C. J. Wagner, Jr. 1985. General purpose tristimulus
colorimeters for color grading orange juice. Proc. Fla. State Hort.
Soc. 97:74-76.
3. Buslig, B. S. and C. J. Wagner, Jr. 1985. Instrumental measurement
of orange juice color. Food Technol. 39(9):95-97.

State Hort. Soc. 82:200-205.

Hunter Associates Laboratory, Inc. 1987. LabScan Spectrocolorimeters. Bulletin G.C.3.3.10. 8 pp.

11. Hunter, R. S. 1967. Development of the citrus colorimeter. Food

Technol. 21:906-911.

12. Hunter, R. S. 1975. The measurement of appearance. 348 pp. WileyInterscience, New York.

13. Michigan State University. 1985. User's guide to MSTAT: microcom

puter statistical program (Version 4.0). MSU, East Lansing, MI.
14. Minitab Incorporated. 1985. Minitab reference manual. 232 pp.
Minitab Inc., State College, PA.

15. State of Florida, Department of Citrus. 1975. Official rules affecting

the Florida citrus industry. Chapter 20-65.
16. Wagner, C. J., Jr. and B. S. Buslig. 1983. Instruments for color grad
ing orange juices. Proc. 1983 Citrus Technol. Conf. pp. 4-5.

17. Wenzel, F. W. and R. L. Huggart. 1962. Relation between Hunter

color difference meter values and visual color of commercial frozen
concentrated orange juice. Proc. Fla. State Hort. Soc. 75:331-336.

Proc. Fla. State Hort. Soc. 101:150-154. 1988.

EFFECT OF CELL WALL HYDROLYSIS ON BRIX IN CITRUS FRUIT

Ed Echeverria, Jacqueline K. Burns, and
Louise Wicker

University of Florida, IFAS

Citrus Research and Education Center
700 Experiment Station Road
Lake Alfred, FL 33850

Additional index words. Citrus paradisi x Citrus reticulata, Or

lando tangelo, pectinase, pectinesterase, hemicellulase, cellulase.
Abstract. Citrus finisher pulp was incubated in the presence of
various cell wall digesting enzymes and changes in Brix and
solubilized sugars were determined. Cellulysin, hemicel
lulase, or pectinesterase treatments alone did not increase
Brix. Pectinase alone or the four cell wall digesting enzymes
in combination resulted in 0.6 and 1.0 unit increase in Brix,
respectively. Reducing sugars, glucose, and galacturonic acids
were solubilized only following pulp digestion with pectinase
or all four cell wall digesting enzymes. Brix and solubilized
sugars increased during the first 3 hr of digestion, but no

further increases were observed during the remaining 2 hr of

incubation. The direct correlation between the increase in Brix

and sugars solubilized from citrus pulp suggests that products

of cell wall hydrolysis may contribute to the increase in Brix
observed during post harvest storage. Our study also indicates
that pectinase is essential for citrus pulp digestion, but other

Florida Agricultural Experiment Station Journal Series No. 8928.

150

cell wall digesting enzymes are required for total cell wall
solubilization.

Several varieties of citrus fruit continue to accumulate

soluble solids (Brix) during storage (4, 6, 7, 11). In 'Hamlin' orange, for example, the increase in total soluble solids
is accompanied by a parallel increase in sucrose and a con
comitant decline in acid content (4). The presence of all
the gluconeogenic enzymes in mature sweet oranges (5)
suggests the possibility of de novo sugar synthesis from acid
after the fruit is detached from the tree. In some instances,
however, the postharvest increase in Brix gradually
plateaus and declines, only to increase again during the
latter stages of storage (4, 6, 7). At this time, the activities
of the regulatory enzymes of the gluconeogenic pathway
have declined and enzymes of sugar catabolism dominate.
Thus, gluconeogenesis and de novo sugar synthesis do not
seem to be involved in the second and final rise in Brix.
Other varieties, such as 'Robinson' tangerine and Talestine' sweet lime do not show any direct correlation between
the uninterrupted increase in Brix and changes in sugars
after harvest (4). The above information suggests the in
volvement of a separate mechanism responsible for the
increase in Brix during storage of some varieties and dur
ing the final stages of storage in others.
Maturation and senescence in fruit involves a series of
physiological and biochemical events which include
changes in membrane permeability, carbohydrate compo
sition, and cell wall structure (2). In Citrus, degradation of
cellulose, hemicellulose and pectin from cell walls of juice
Proc. Fla. State Hort. Soc. 101: 1988.