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HENDRICKSON AND KESTERSON: GRAPEFRUIT SEED OIL 219

GRAPEFRUIT SEED OIL

Rudolph Hendrickson and J. W. Kesterson sample were washed thoroughly, counted,


weighed, dried and reweighed. After drying the
Florida Citrus Experiment Station
seed samples for 24 to 48 hours in an oven at
Lake Alfred 90-95° C, the seeds were coarsely ground with
a mortar and pestle to facilitate the separation
One of the earliest references to Florida of the kernels from the hulls.
grapefruit seed oil as a commercial product was
that of Jamieson, et al. (4) who in 1930 Similarly, seeds were recovered for compari
analyzed both solvent extracted and Andersen son from shaddock (Siamese pumelo 10044),
expeller oil samples for fatty acid content, which has been listed as the progenitor of grape
iodine number and other chemical and physical fruit although there is some doubt in regard
characteristics. The bitterness of the crude to this relationship.
grapefruit seed oil appeared to preclude its use
The seed oil was expelled from the separated
in anything but soap since a feasible method
kernels in a small-scale press designed for labora
for the removal of this taste was not available.
tory samples. The press, made from a cylindrical
By 1940, Nolte and von Loesecke (6) were able
bar of stainless steel 1 and 7/8 inches in diam
to report on the procedures for the commercial
eter, has a well that is approximately one inch
production, expression, bleaching and wintering
deep in which the seed kernels are pressed by
of grapefruit seed oil in Florida and pointed out
a cylindrical plunger of almost the same diam
further that the extremely bitter crude oil could
eter. Figure 1 more clearly shows the design
be easily refined to a bland tasting product. Ap
as well as the recessed reservoir around the
proximately 90,000 pounds of crude oil were
perimeter to retain the expressed oil. Designed
produced annually in this period when a po
to accommodate the kernels of 30 to 100 seeds,
tential of almost 4,000,000 pounds was theo
it was possible to express two to three ml. of
retically available.
seed oil in one pressing with a hydraulic pres
sure of five tons per square inch.
Interest and production of grapefruit seed
oil in the subsequent 20 years almost disap Analytical Procedures.—The values for refrac
peared until recently public attention was called tive index were obtained using a Bausch and
to the therapeutic usefulness of having a higher Lomb Precision Refractometer having a sodium
proportion of unsaturated fats in the diet as is lamp. All readings were corrected to 25° C.
furnished by some vegetable oils. With the re
newed interest and increasing production of
grapefruit seed oil, there appeared to be a latent
advantage in studying the seasonal and varietal
differences in order to obtain an accurate pic
ture of Florida's seed oil potential. Further
more, the advent of improved gas chroma to-
graphic procedures for the qualitative and
quantitative determinations of fatty acids has
greatly facilitated this study.

Experimental Procedure

Preparation of Samples.—During the 1960-61


season four varieties of grapefruit—Excelsior,
Walters, ^Duncan and Marsh—were picked at
different stages of maturity and juiced to re
cover the seeds. From one to ten fruit were
used per sample depending on how seedy the
variety happened to be. The seeds of each

Florida Agricultural Experiment Station Journal Series No. Figure 1. Small-scale seed oil press designed for labora
1316t tory extractions.
220 FLORIDA STATE HORTICULTURAL SOCIETY, 1961

The iodine absorption numbers were deter tract were injected directly into a Model A-110-
mined by the official Hanus method of AOAC C Aerograph equipped with a one mv. Brown
recorder and disc integrator. Analyses were
(2)-
The fatty acid content was measured by a made using both five- and ten-foot columns
gas chromatographic technique wherein the of diethylene glycol succinate (DEGS) as a 15
samples were first transesterified by a modifica per cent stationary phase on 60/80 fire brick.
tion of the procedure applied by Michaels et al. Column operating conditions were somewhat
(1). Two drops of seed oil were refluxed one variable; temperature being between 194° and
hour in 10 ml. of one per cent sulf uric methanol 224° C, filament current 150 or 200 milliam-
mixture. The mixture was then evaporated to peres and helium flow rate limited to 60 ml.
about one ml. under nitrogen and diluted per minute.
with four ml. of water. The water suspension
Other information such as acid value, saponi-
was extracted twice with 30-60 petroleum ether
fication, unsaponifiable matter, Acetyl, Rei-
(10 ml. each time). The petroleum extract
chert-Meissl and Polenski values for seed oils
was evaporated to dryness under nitrogen and
dissolved in four or five drops of benzene. were not expected to show critical differences
Aliquots of the transesterified benzene ex and as a consequence were not determined.

Table 1. Seed characteristics of grapefruit and shaddock at


different degrees of maturity.

Per Cent
Variety of Date Wt./Fruit No. of Seeds Per Cent Seeds Moisture
Fruit Processed (g) per Fruit (Wet Basis) in Seeds

Excelsior 8/29/60 322 45 5.8 70.1


Excelsior 10/26/60 422 60 4.7 58.0
Excelsior 1/ 3/61 562 64 4.2 54.5
Excelsior 2/10/61 480 60 3.7 55.0
Excelsior 3/23/61 494 49 2.9 57.0

Duncan 8/29/60 436 66 7.3 82.3


Duncan 10/26/60 582 62 4.2 62.0
Duncan 1/ 3/61 643 74 4.5 57.9
Duncan 2/10/61 655 58 3.6 57.0
Duncan 3/23/61 660 41 1.9 55.0

Walters 8/29/60 324 58 7.7 72.6


Walters 10/26/60 460 65 5.2 58.1
Walters 1/ 3/61 694 60 3.1 55.0
Walters 2/10/61 662 55 2.7 53.0
Walters 3/23/61 712 47 2.4 56.0

Marsh 10/ 3/60 380 5.5 .44 62.0


Marsh. 1/ 3/61 508 3 .20 51.6
Marsh 2/10/61 473 4 .30 56.0
Marsh 3/23/61 521 4 .24 54.0

Shaddock 8/29/60 960 105 4.9 52.0


Shaddock 3/23/61 1,524 52 2.0 50.0
HENDRICKSON AND KESTERSON: GRAPEFRUIT SEED OIL 221

Results and Discussion from 53 to 58 per cent moisture; however, they


Physical Characteristics.—The seed character have a much higher moisture content in the
istics of the seedy and seedless varieties of more immature stage and were more difficult to
grapefruit are shown in different degrees of dry without charring.
maturity in Table 1. The weight of a fruit
on each processing date is also recorded for Similar information is presented in Table
comparison. Each of the three seedy varieties 1 for shaddock at two different degrees of
maturity.
was noted to have approximately the same num
ber of seeds per fruit with a trend toward a Refractive Index.—The temperature corrected
fewer number of seeds as full or over-maturity refractive index measurements for these grape
was reached. The percentage of seeds in the fruit seed oils are tabulated in Table 2. In each
seedy varieties was noted to decrease with matur of the four varieties studied, refractive index
ity, which is in accordance with a recent study of the oil increased as the fruit became more
(5) indicating that seeds reach full size sooner mature. There was further, a great similarity
than the fruit. The time of picking would between the indices of the four varieties on any
therefore greatly influence the percentage of processing date. It was tempting, therefore, to
seeds in these varieties and would explain con say that the index of refraction of a grapefruit
flicting reported percentages (3,6). Percentage seed oil would be a measure of maturity. Sam
of moisture in undried fresh seeds was related ples other than those reported have shown that
to maturity as is shown in Table 1. At matur with greater maturity the index decreases or
ity, grapefruit seeds can be expected to have remains unchanged.

Table 2. Physical and chemical properties of grapefruit and


shaddock seed oils produced in Florida.

Variety of Date Iodine Fattv


Fruit Processed No. Palmitic Stearic Oleic

Excelsior 8/29/60 1.4689 98.3 34.6 2.2 22.2 38.0 3.0


Excelsior 10/26/60 1.4689 99.6 34.5 3.6 20.5 36.2 5.2
Excelsior 1/ 3/61 1.4692 99.7 35.8 3.6 22.8 34.2 3.6
Excelsior 2/10/61 1.4695 101.0 31.1 5.0 22.5 37.2 4.2
Excelsior 3/23/61 1.4695 97.8 34.4 4.8 21.2 34.8 4.8

Duncan 8/29/60 1.4690 96.8 35.6 1.6 20.6 39.2


Duncan 3.3
10/26/60 1.4691 IQO.O 36.7 3.6 20.0 34.8 4.9
Duncan 1/ 3/61 1.4692 100.0 35.4 2.7 22.4 36.5 3.0
Duncan 2/10/61 1.4693 100.0 31.1 4.4 20.7 38.7
Duncan 5.1
3/23/61 1.4696 99.5 35.9 3.4 20.6 35.4 4.7

Walters 8/29/60 1.4690 96.0 35.0 2.9 21.1 37.6


Walters 3.4
10/26/60 1.4691 98.1 37.5 3.7 18.6 37.4 2.8
Walters 1/ 3/61 1.4692 98.9 35.2 3.7 22.5 34.6
Walters 4.0
2/10/61 1.4692 100.0 33.0 3.8 20.8 37.7 4.7
.Walters 3/23/61 1.4695 99.6 34.8 3.8 20.6 36.3 4.5

Marsh 10/ 3/60 1.4691 98.5 34.5 3.6 22.9 35.4 3.6
Marsh 1/ 3/61 1.4692 98.8 34.7 2.8 22.2 35.4 4.9
Marsh 2/10/61 1.4693 100.0 35.0 3.1 20.0 37.3 4.6
Marsh 3/23/61 1.4696 101.0 36.4 3.1 19.5 36.4 4.6

Shaddock 8/29/60 1.4690 97.5 30,5 3.2 24.7 38.0 3.6


Shaddock 3/23/61 1.4692 99.4 26.0 4.9 27.2 37.0 4.9
222 FLORIDA STATE HORTICULTURAL SOCIETY, 1961

of manufacture was noted to be important, but


1.4710 O
'o not necessarily controllable.

/o The iodine numbers of the grapefruit and


/

/
shaddock seed oils were noticeably similar, as
.4700 were the refractive indices.
<

( Fatty Acid Composition.-It was necessary to


op/

/
9 establish proper gas chromatographic param
Citrus Seed Oils eters, type of column and retention time of
.4690 OOC
known fatty acids in order to determine fatty
y oo

acid composition which is shown in Table 2.


Each fatty acid was identified by the highly
/o specific retention time of its methyl ester against
1.4680
90 100 110 120 that of a known acid. The quantity of each
Hanus Iodine No. acid was determined by the disc integrator at
Figure 2. A scatter diagram of refractive index measure tachment. The extent to which the methyl
ments versus the Hanus iodine absorption number for num esters of stearic and oleic acid could be separ
erous citrus seed oil samples.
ated determined the optimum column and
Iodine Absorption Number.—The iodine ab operating conditions. A typical chromatogram
sorption numbers for these same seed oils are of a grapefruit seed oil, wherein good separa
shown in Table 2. A similar, but less distinct, tion of the methyl esters of the fatty acids
relationship was noted between iodine numbers, has occurred, is shown in Figure 3. Complete
maturity and variety as was seen for index of separation of the closest peaks was possible with
refraction. The close relationship between re a new DEGS five-foot column operating at ap
fractive index and iodine number is more proximately 190° C, but a longer 10-foot
clearly seen in Figure 2, which is a scatter dia column operated at a higher temperature in
gram of these values for grapefruit as well as sured a quicker analysis with equal resolution
orange, lemon and tangerine seed oils. By even after much use.
virtue of this correlation between iodine num Grapefruit seed oils contain five fatty acids
bers, which measures degree of unsaturation, and the per cent of distribution of these com
and index of refraction it would seem possible ponents are shown in Table 2. There was a
to use the values of refractive index as a rough remarkable similarity between all samples such
measure of unsaturation. There was a tendency that neither variety nor maturity could be
for seed oils made early in the season to have distinguished on this basis. There was a slight
a lower absorption number. Since seed oil trend in samples prepared from the more
manufacturers would be interested in obtaining mature fruit to have a higher linolenic and
an oil having the highest iodine number, time stearic acid content and a correspondingly lower
linoleic acid analysis.
~l 1 :■■■-
The shaddock seed oils contained the same
■ *
MCTHYLATEO ORAfa five fatty acids as grapefruit seed oil in ap
proximately the same percentage, but with

Z__Z__J — - —:— r

|_.. .
palmitic acid being present in somewhat lower
.....
- - "i _|-
1
percentage and oleic acid in higher percentage.
A
.. ..-.- .. ...
1
A typical analysis of a Florida grapefruit
m -v-A seed oil could be expected to have the five
fatty acids, palmitic, stearic, oleic, linoleic and
J • «. _|
linolenic in following proportions: 35, 3.5, 21,
T HE IWWTM

if 1| A* 36 and 4.5 respectively. This is considerably


different from an earlier published analysis (4)
Figure 3. A typical gas chromatographic trace of methyl
ated grapefruit seed oil. Operating parameters were: 5-ft. wherein the older, more difficult ester-fraction
column DEGS at 189° C, helium flow-55 ml. per mm.,
filament current-150 ma., and a chart speed of 20 in. per and lead salt procedure had to be usqd.
hr.
ROUSE, ATKINS, MOORE: PECTINESTERASE STUDIES 223

Summary
The fatty acid chromatographic analyses of
Information as to the number of seeds per grapefruit seed oils from different varieties and
fruit, the per cent of seeds in the fruit and of different maturity showed only small varia
the amount of moisture in the seeds was deter tions. A typical analysis for the fatty acid com
mined for four common varieties of grapefruit ponents was established since it was different
at different stages of development. Seed oils than that reported in earlier work (4).
from these samples were found to have physical
and chemical values so close as to make dif Fatty acid content, refractive indices and
ferentiation between varieties impossible. How iodine numbers were noticeably similar for
ever, an increase in fruit size resulted in a shaddock and grapefruit seed oils such as to ad
noticeable increase in refractive index and ditionally support a definite relationship be
Hanus iodine absorption number with some in tween the two kinds of citrus fruits.
dication that these values may decrease beyond
LITERATURE CITED
full maturity. The close relationship between
refractive index and iodine number would in issue ^9589raPh Research Notes- Mlcro fat analysis. Fall
fer the possibility of using this index as a 2*l Association of Official Agricultural Chemists. Official
methods of analysis 7th Ed., 910 pp. 1950. Washington, D. C.
rough measure of the degree of unsaturation in 3. Fudge, R. B. Relation, of magnesium deficiency in
grapefruit seed oils. grapefruit leaves to yield and chemical composition of fruit.
Fla. Agr. Expt. Sta. Bui. 331: 1-36. 1939
4. Jamieson, G S., W. F. Baughman and S. I. Gertler.
Design data for the construction of a small- Grapefruit Seed Oil. Oil and Fat Industries 7: 181-82. 1930
5. Kesterson, J. W. and R. Hendrickson. Naringin, a
scale laboratory seed oil press were described as bitter principle of grapefruit. Fla. Agr. Expt. Sta Bui.
511: 5-35. 1953.
well as procedures and parameters for the gas 6. Nolte, Arthur J. and Harry W. von Loesecke. Grape
chromatographic analysis of citrus seed oils. fruit Seed Oil, Manufacture and Physical Properties. Ind.
and Eng. Chem. 32: 144-46. 1940.

RECENT STUDIES OF THE EFFECT OF PECTINESTERASE ON THE


STABILITY OF FROZEN CONCENTRATED ORANGE JUICES
A. H. Rouse, C. D. Atkins and E. L. Moore sures but was not effective at higher finisher
Florida Citrus Experiment Station pressures of 7 and 8 psig. The PE activity
was approximately the same in the finished
Lake Alfred products when the juices were heated to 190° F.
prior to concentration.
In 1960, the authors (8) reported that frozen
orange concentrates prepared from Valencia
Wenzel, Moore, Rouse and Atkins (9)
oranges maintained cloud stability for 52 days showed that concentrate produced from un-
or more when placed at 40° F. storage. This heated juices of seedy varieties of citrus are
was accomplished, regardless of finisher pres more susceptible to gelation and clarification
sures used during processing, by inactivating than those made from juices of the less seedy
the enzyme, pectinesterase (PE), in the juice varieties. Because of this and since the pre
viously mentioned studies were made using
prior to concentration and by controlling the
Valencia orange concentrates, Pineapple oranges
PE activity in the pulpy cutback juice. Bissett
were selected for this study.
and Veldhuis (4) extracted Valencia orange
juice at varying finisher pressures and reported One of the purposes of this paper is to show
the ^effect of different PE levels in 42° and
that heating juices to 190° F. was effective
50.7° Brix Pineapple orange concentrates on
in stabilizing the cloud in concentrates pre
the stability of these products during storage
pared from juices at 5 and 6 psig finisher pres-
at 40° F. Concentrates were prepared from
* Cooperative research by the Florida Citrus Experiment juices obtained by using different finisher head
Station and Florida Citrus Commission.
pressures. Data are also presented on the pectic
1333 da Agrlcultural Experiment Station Journal Series, No.
contents and water—insoluble solids found