FLORIDA STATE HORTICULTURAL SOCIETY, 1975
486
6. Dougherty, R. H. and Koburger, J. A. 1972. Preparation
and storage of pasteurized-refrigerated mango fruit. Proc.
Fla. State Hort. Soc. 85:190.
7. Goldweber,
S.
1967.
Thoughts
on
the
industry. Proc. Fla. State Hort. Soc. 80:384.
Florida
mango
8. Harris, H. 1963. Pasteurized refrigerated peach prod
ucts. Alabama Agric. Exp. Sta. Highlights of Agri. Res.
10(2) :11.
9. Krishnamurthy, S., Patwardham, M. V.
manyam, H. 1971. Biochemical changes during
the mango fruit. Phytochemistry 10(11) :2577.
10. Mustard, M. J. 1948. Preservation of
freezing. Fla. Mango Forum Proc. 1948:27.
11. Thomas, P. 1975. Effect of post-harvest
on quality, carotenoids and ascorbic acid content
mangos on ripening. J. Food Set. 40:704.
and Subraripening of
mangos
by
temperature
of Alphonso
LEAVES FOR FOOD: PROTEIN AND AMINO ACID CONTENTS
OF LEAVES FROM 23 TROPICAL AND SUBTROPICAL PLANTS
could
Nancy T. Hall, Steven Nagy
U.S. Citrus and Subtropical Products Laboratory1
Winter Haven
Abstract Leaves are a potential source of low
cost protein. By dry weight, leaves of 23 plant
types contained protein from 6 to 41%, of which
14 contained 20% or more. Notable were castor
bean
(Ricinus
(Momordica
communis)
charantia)
41%,
33%,
balsam
cowpea
pear
(Vigna
sinensis) 32%, and cassava (Manihot esculenta)
32%. The leaves had large quantities of the es
sential amino acids lysine, leucine and isoleucine,
moderate amounts of valine, threonine and phenylalanine, and minor amounts of methionine and
tryptophan. Many nonessential amino acids were
found in moderate quantities. Tyrosine and his-
tidine were low, and cysteine and cystine were
detected at levels that were less than 1% of the
total
amino
acids
recovered.
Leaves
were
es
sentially similar in their amino acid compositions,
although several cultivars showed notable varia
tions in methionine.
Because
food
of
scarcity
the advantages
of exploiting the
light and high yield of leaves due to multiple
cropping.
Kohler and Bickoff (18), contributors to the
Third International Congress of Food Science and
Technology (1970), recognized the research of
Osborne and Wakeman (25) as one of the earliest
in this field. Other early leaf protein investigations
have been covered in a review by Tilley and Ray
mond (30) and more recent work by Pirie (26,27),
Akeson and Stahmann (1), and Oelshlegel et al.
(24) have contributed considerably to knowledge
in this emerging field. The nutritional values of
leaf proteins have been investigated by Waterlow (32), Duckworth and Woodham (11), Gerloff
et al. (15) and Subba Rau et al. (29) while leaf
amino acid compositions have been researched by
Chibnall et al. (10), Gerloff et al. (15) and Byers
(9).
Through extensive research leaf protein con
centrates (LPC) have become a reality. They are
currently used as an animal feed supplement and
being tested for human consumption. Amino acid
compositions of many LPC's have been found to
increasing world
the
provide
year-round availability of high amounts of sun
and Robert E. Berry
development
population and
of
new
be as beneficial as soybean meal, and as digestible
and nutritious as milk. Waterlow (32) found that
protein
when leaf proteins were combined with milk pro
sources has been a high priority research goal
teins in the diets of children suffering from pro
for the past decade. Coordinated by N. W. Pirie
tein malnutrition, weight gains were
of Rothamsted, England, under the International
to those of similarly afflicted children on milk at
Biological Program (28), the development and use
equal
of leaf protein have been actively pursued. Protein
firmed that leaf proteins could provide essential
from leaves that can be grown in tropical areas
levels.
Other
investigations
con
amino acids to supplement those already in the
normal
lOne of the laboratories of the Southern Region, U. S.
Department of Agriculture, Agricultural Research Service.
- The authors thank Dr. Franklin W. Martin and staff, of
the Mayaguez Institute of Tropical Agriculture, Mayaguez,
Puerto Rico, for the samples of dried leaves used in this
study, o
Mention of brand names is for identification only and does
not imply endorsement by the U. S. Department of Agricul
ture.
protein
equivalent
diet.
LPC's
have
been
prepared
from
(19,29), soybean, cowpea and peanut
hyacinth
alfalfa
(5), water
(23), chenopodium, marrow, corn, nas
turtium, red clover, rye grass, sanfoin, turnip and
wheat
(15), ramie, swamp cabbage and brassica
(8,14). Martin et al. (21) initiated a study screen-
HALL ET AL: LEAVES FOR FOOD
487
ing leaf species with characteristics ideal for use
as protein sources in the tropics.
The current study was made to determine the
protein and amino acid contents of 23 types of
tropical
and subtropical
leaves
which
were
r
in
cluded in the study by Martin et al. (21). Leaves
having greater than an arbitrarily selected level
'
h
J J
3 -
.165
<"
32 5 -
2
107
p vine
31.8 i
1.8
.182
of 20% protein were subjected to acidic and basic
p
31.6 1
.5
.107
hydrolyses and their amino acid compositions were
A herb
27-5 -
.3
.2U3
determined by semiquantitative thin-layer chroma-
P vine
2«i.l» -
.0
.162
tree
23-9 -
.3
.128
P vine
23.3 -
.2
.152
tography
(TLC).
This
method
of
amino
acid
analysis was chosen because it is fast, inexpensive
and requires minimal equipment. This information
on protein content
and
amino
acid
composition
Protein Determinations
Leaves, picked at maturity stages known to be
and human
consumption,
were
dried at 58°C. Within 24 hr of sampling for micro
Kjeldahl nitrogen (2) and total free amino acids
determinations
23.3 -
22.U i
.7
.091
21.6 i
.3
.109
20.9 -
.6
.133
20.li -
.1
.12U
P tree
Materials and Methods
animal
shrub
shrub
P herbaceous
these leaf cultivars as future sources of food.
for
.081
P
P
A herb
should be helpful in the nutritional assessment of
safe
chrub
(4), the leaves were redried in
vacuo at 95-100°C for 5 hr. The catalyst in the
micro Kjeldahl total nitrogen method was modified
P shrub
19.5 -
.2
.076
P shrub
17.5 i
.2
N.D.'
N.D.
P herb
16.3 i
.It
P tree
15.5 i
.2
N.D.
P tree
1U.5 -
.3
N.D.
P herb
13.0 i
.2
N.D.
P tree
11. b -
.2
N.D.
P tree
10.8 -
.2
N.D.
P tree
7.5 -
.!»
N.D.
P
5.9 i
.2
N.D.
tree
by using copper sulfate instead of mercuric sulfate (6). The mean crude protein value was ob
tained from 2 to 5 Kjeldahl nitrogen determina
tions on 100 mg samples:
leaves times 6.0 (21).
Amino
For free amino acid determination by formal
titration, three 10-ml aliquots were taken from a
200 ml aqueous leaf homogenate prepared by dis
integrating 5 g of dried leaves in a Waring
Total
free
amino
acids
were
reported
(Table 1) as milliequivalents per gram dry leaves.
Three to 6 samples of the 14 species having
20% or more protein were subjected to both acid
(6N HC1, 22 hr, 110°)
and basic
[250 mg
(OH)2-8H2O)
in
H2O,
dissolved
128°] " hydrolysis
(7,20).
5
ml
24
(Ba
hr,
Following ~ hydrolysis
amino acids were extracted with H2O, CHC13 and
CH3OH
(7:8.7:17.3
v/v/v).
layer was passed through an
cation exchange column
acid
mixtures
(standards
and
leaf
hydrolysates) were separated by two-dimensional
Amino Acid Determinations
blendor.
> acid per gin of dry lei
mg nitrogen/100 mg
The
CH3OH-H2O
Amberlite CG-120
(Rohm and Haas, Phila
delphia, Pa.) which retained the amino acids (16).
These were eluted with IN NH40H, and the efflu
ent concentrated to dryness on a rotoevaporator.
The dried amino acid sample was dissolved in 0.1
N HC1 and refrigerated until analyzed by TLC.
TLC on precoated 250 fi silica gel GF plates
(Analtech, Inc., Wilmington, Del.). Separation
was effected in the first dimension with solvent I
(CHCl3-Me0H-17% NH40H, 2:2:1, v/v/v); the
plates were dried for ca. 1 hr in a forced air-draft
hood, turned 90°, and separated in the second di
mension with solvent II (phenol -H2O, 75:25,
w/w). The plates were dried for an hour and
sprayed with ninhydrin cupric nitrate indicator.
Amino acids were identified by their relative Rf
values and by their specific reaction to the poly
chromatic ninhydrin indicator (22). The analyses
of the amino acids was semiquantitative. A stand
ard curve was prepared for each amino acid re
lating area of chromatographic spot with weight
of the amino acid. Aliquots (0.5, 1, 2 and 4 fil) of
standard amino acid mixtures were spotted on 4
plates and the area of each amino acid spot was
determined from photocopies of the plates by
planimeter tracing. Weights were determined by
the formula (31): VA = m log wt + C. Aliquots
FLORIDA STATE HORTICULTURAL SOCIETY, 1975
488
(0.5, 1, 2, 4 ittl)
of the acid hydrolyzates of each
of the 14 samples were spotted and the correspond
ing weights of
from the
each
amino
standard curves.
acid were
obtained
Weights for
all
the
v Solvent
I
amino acids were totaled and the relative weight
12
percent calculated, e.g., wt specific amino acid/
total amino acid wt x 100. Values were tabulated
(Table 2) and represent the mean of 3 to 6 de
terminations. The coefficient of variation (CV) was
determined for several mean ranges
(MR)
with
the following results: MR 1-3, CV 25-65%; MR
3-8, CV 10-40%; MR 8-17, CV 5-20%.
3o
6
x°y
Results and Discussion
The percentages of protein calculated for the
23 leaf samples ranged from 6 to 41% (Table 1).
Protein content, based on Kjeldahl values, include
protein nitrogen, free amino acid nitrogen, and
nonproteinaceous nitrogen. The free amino acid
contents were noticeably low and ranged from .076
to .243 meq/g dry leaves. The highest protein
content was found in Ricinus communis, the com
mon castor bean. Of the remaining 13 species whose
leaves contained 20% or more protein, 6 of these,
viz., cowpea, cassava, chayote, pigeon pea, banana
,O2O
Solvent U
Ori gin
Fig. 1. Thin-layer chromatographic separation of amino
acids of coffee leaves after acid and basic (dotted areas)
hydrolysis: (1) lysine, (2) arginine, (3) aspartic acid, (4)
serine, (5) glycine, (6) glutamic acid, (7) proline, (8) histidine, (9) threonine, (10) alanine, (11) valine, (12) methionine, (13) tyrosine, (14) leucine and isoleucine, (15) phen
ylalanine, (16) tryptophan, (17) oxidative breakdown prod
ucts of cysteine, (x) ?-aminobutryic acid and (y) tentatively
identified as hydroxyproline.
and coffee, are currently grown for other food uses.
Thus, processing their leaves
for
protein
could
provide an additional product or by-product.
Fig 1 shows a typical TLC amino acid profile
resulting from hydrolysis of coffee leaves. Acid
hydrolysis resulted in nearly complete decomposi
tion
of
tryptophan.
Basic
hydrolysis,
however,
liberated tryptophan intact but caused partial de
composition
cystine.
of
Both
serine,
acid
and
threonine,
basic
arginine,
hydrolyses
and
caused
some oxidative degradation of cystine and cysteine.
For semiquantitation of leaf amino acids,
acid-hydrolyzed mixtures were used.
only
To confirm
the presence of tryptophan and to approximate
its percentage, basic hydrolysis was employed.
Table 2 lists the proximate weight percentage
distribution
of essential
and
nonessential
amino
leaves examined. Methionine values ranged from
1% in castor bean, Ceylon spinach and pigeon
pea to 4% in cassava. This amino acid has often
been noted as a limiting factor in many proteins
of different origin.
In comparing the essential leaf amino acids to
other amino acid sources, the percentages of lysine,
threonine,
valine,
phenylalanine
and
leucine-
isoleucine equaled or exceeded those in the FAO
reference protein
(13).
Methionine content was
lower than the FAO percentage in only 3 of the
leaves;
Ceylon
spinach,
pigeon
pea
and castor
bean. Tryptophan was not definitively quantitated
but probably is at least equal to the 1.4% in the
FAO standard. With the exception of banana and
coffee leaves, the nonessential amino acids, histi-
acids in the 14 types of leaves with highest pro
dine, glycine, alanine, serine, proline and arginine
tein content. Although tryptophan could not be
were similar to the average reported for these
quantitated in the acid hydrolyzed mixture, rela
tive area comparisons of valine and phenylalanine
to tryptophan from a basic hydrolyzed mixture
resulted in an estimate of 1-3% for tryptophan.
Valine and phenylalanine
TLC
standard
curves
were essentially similar to the standard curve of
tryptophan. Leucine and isoleucine were unresolved
by TLC and were reported as an entity. Lysine and
threonine values were similar for all types of
amino acids in leaf protein concentrates (15). In
approximately one-third of the leaves, percentages
of aspartic and glutamic acids were ca. 1 to 2 per
centage points lower than values reported by Gerloff et al. (15) for LPC. These lower values may
be due to greater variation in the TLC technique
than in the ion-exchange method of Gerloff et al.
Banana leaves showed the lowest combined total
of aspartic and glutamic acids. For cowpea, cassa-
HALL ET AL: LEAVES FOR FOOD
489
?varf
1
c
I.yr.iiie
7
Valino
M
7
('
Thvoonine
Thenyl-a-nine
Methionine
8
£
",,
Olutamic acid
C
7
7
C
6
€
n
r
6
15
7
7
ft
7
7
o
C
6
2
■5.
d'-
t/"
15
8
7
j,
6
6
6
11
10
"
6
Aspartic acid
8
7
7
?
5
6
10
10
9
10
f,
9
1*
Alrarlnv:
r.
5
oeri »■;•.'.•
('•
6
6
f.
7
b
r.
fi
5
PvnliiiO
r>
6
(•"
•Vrrlr.irie
"
7
7
c'
iyvosinc
<■■
2
2
2
<__
<1
<1
<1
?
5
D1"
E*
DW
5
3
3
12
10
8
7
7
9
7
9
11
7
7
6
c
5
5
)i
5
9
7
6
6
3
3
<1
<1
9
6
10
)i
,
2
•
2
<"-
<1
<Z
<1
7
7
4
3
k
<1
<1
Cy3tei:ie?ra:id
"A.'\ i:."> aoias ex? r-.-cscd as percent ty veipht of tot-:!
3
7
G
8
7
6
7
11
'
3
8
5
10
^
c
7
5
3
10
7
p
D-
JJyeine
cyst i no
15
0
7
7
<1
7
It
6
5
n::.ir.o aMir: >••?<?■:
*'•:>_• eh G-'r.ir.C! acid pcrccr.t.i^o vni ue represer.4:: the i:.'.:-ar. f-r* '—' '":•:-•:•:•••!:.!!".
"Lourtoe and iroleucino uin-.^lY-a ry TI.C;
expressed as cor.l.?r.-:-d 4 o' •
WTryrihni!}i:.«i detected by ha^ic hydrolysis mvI ert U-ini-.cd as 1-3^.
va, mulberry and pigeon pea, the percentage of
tyrosine
(2%) was ca. 1 percentage point lower
than the average reported by Gerloff et al.
to foods have been extraction, palatability, unde
sirable color due to chlorophyll, biological un
availability of
Any leaf crop with high protein and significant
amounts of most essential amino acids should be
strongly considered as a source of LPC or as a
supplementary food, or both, especially if the crop
can be widely grown in tropical and subtropical
regions. The leguminous plants, cowpea and pigeon
pea, already widely grown for their high protein
seeds, should be considered as a source of supple
amino
acids,
and
the occasional
presence of toxic substances. Many of these prob
lems have been partially solved by new processes
such as the Pro-Xan method (17), separation of
LPC into chloroplastic and cytoplasmic (white or
colorless)
fractions, and heat destruction of
poisonous cyanides (12). Through improved pro
cessing techniques, increased research efforts, and
continued studies on plant hybridization, many of
ly limiting in diets based on cereals such as rice,
the problems that currently prevent the wide
spread use of leaf proteins may be overcome.
Solution of these problems may be the next decade's
maize and wheat
greatest contribution to the world's starving and
mentary amino acids because of their high leaf
protein content and high lysine, which is common
cassava
leaves,
methionine
(5). The nutritional quality of
which
are
high
in
(6 and 4%, respectively)
lysine
and
would en
hance a diet of cassava root (3), a staple food in
the tropics
of America,
Africa,
and
Asia.
Purslane and Ceylon spinach leaves are currently
used as greens in some areas. These crops should
be encouraged to expand because of their high
leaf-protein levels.
The castor bean, grown in India for castor oil
production, has a high leaf-protein
content but
has not been processed because, although the im
mature leaves are safe to consume, a poisonous
alkaloid combined with the protein, ricin, forms in
the mature leaf (21). Research to solve this prob
lem through improved processing techniques
or
plant hybridization could provide access to a new
and potentially valuable protein source.
Other problems encountered in processing leaves
malnourished peoples. The study of leaf amino
acid balance and protein content is a beginning.
Literature Cited
1. Akeson, W. R. and M. A. Stahmann. 1966. Leaf pro
tein concentrates: a comparison of protein production per
acre of forage with that from seed and animal crops. Econ.
Bot. 20:244-250.
2. American Public Health Association. 1965. Standard
Methods for the Examination of Water and Waste Water.
New York. 402-404 p.
3. Anon. 1972. Amino Acid Content of Foods. Food and
Agriculture Organization of the United Nations. Rome, Italy.
46-47 p.
4. Association of Official Agricultural Chemists. 1965. Of
ficial Methods of Analysis. W. Horowitz (ed.), Washington,
D. C. 324 p.
5. Betscart, A. A. and J. E. Kinsella. 1974. Influence of
storage on composition, amino acid content and solubility of
soybean leaf protein concentrate. J. Agric. Food Chem 22*
116-122.
6. Blanded, W. J. and V. W. McLoche (eds.). 1963. Ele
mentary Quantitative Analysis—Theory and Practice. Harper
and Row, New York. 379 p.
7. Brenner, M., A. Niederwieser and G. Pataki. 1969.
Amino acids and derivatives. Thin-Layer Chromatography
E. Stahl (ed.). Springer-Verlag, New York. 730-785 p.
FLORIDA STATE HORTICULTURAL SOCIETY, 1975
490
8. Brown, H. E., E. R. Stein and G. Saldana. 1975. Evalu
ation of Brassica carinata as a source of plant protein. J.
and nonprotein amino acids in citrus leaves as affected by
sample preparation and species differences. Am. Soc. Hortic.
Agric. Food Chem. 23:545-547.
Sci. 96:514-518.
digestibility of some protein fractions from three species of
tropical leaves as feasible sources of protein. In review.
22. Moffat, E. D. and R. I. Lytle. 1959. Polychromatic
techniques for the identification of amino acids on paper
9. Byers, M. 1971. Amino acid composition and in vitro
leaves of various ages. J. Sci. Food Agric. 22:242-251.
10. Chibnall, A. C, M. W. Rees and J. W. H. Lugg. 1963
The amino acid composition of leaf proteins. J. Sci. Food
^U.* Duckworth, J. and A. A. Woodham. 1961. Leaf protein
concentrates I. Effect of source of raw material and methods
of drying on protein value for chicks and rats. J. Sci. Food
Agric. 12:5-15.
12. Eggum, B.
O. 1970. The protein quality of cassava
leaves. Br. J. Nutr. 24:761.
13. Food and Agriculture
Organization.
1965.
Nutrition
Meetings Report, Series No. 37. Food and Agriculture Organi
zation of the United Nations. Rome, Italy.
14. Garcha, J. S., B. L. Kawatra and D. S. Wagle. 1970.
Evaluation of different leaf protein concentrates
essential amino acids Curr. Sci. 39(12) :269-270.
for
some
15. Gerloff, E. D., I. H. Lima and M. A. Stahmann. 1965.
Amino acid composition of leaf protein concentrates. Agric.
16 Kaiser, F. E., C. W. Gehrke, R. W. Zumwalt and K. C.
Kuo (eds.). 1974. Amino Acid Analysis. Analytical Bio
chemistry Laboratories, Inc. Columbia, Missouri. 48 p.
17. Kohler, G. O. 1974. Wet processing of alfalfa. 12th
Tech. Alfalfa Conference, Proc, Overland Park, Kansas.
65-66 p.
18.
,
and
E.
M.
Bickoff.
1971.
Leaf Protein.
Proc SOS/70 Third International Congress Food Science and
Technology. Institute of Food Technology, Washington, D. C.
290-295 p.
19.
.,
and D. deFremery. 1974. Green
leaves—a potential new source of protein for human nutri
tion. Protein Symposium at University of California. Davis,
21. Martin, F. W., L. Telek and R. Ruberte'. 1975. Some
chromatograms. Anal. Chem. 31:926-928.
23. Niyogy, S- C. and J. J. Ghosh. 1975. Biochemical and
nutritional studies on leaf proteins. Final Tech. Report.,
Dept. of Applied Science, Calcutta, University, Calcutta, In
dia.
24. Oelshlegel, F. J., Jr., J. R. Schroeder and M. A. Stah
mann. 1969. Potential for protein concentrates from alfalfa
and waste green plant material. J. Agric. Food Chem. 17:
791-795.
25. Osborne, T. B. and A. J. Wakeman. 1920. The pro
teins of green leaves. J. Biol. Chem. 42:1-26.
26. Pirie, N. W. 1969. The production and use of leaf
protein. Proc. Nutr. Soc. 28:85-91.
27.
. 1969. The present position of research on
the use of leaf protein as a human food. PL Fds. Hum. Nutr.
1:237-246.
28.
. 1971. Leaf Protein: Its Agronomy, Prep
aration and Use. International Biological Program Handbook
20, Blackwell Scientific Publications, Oxford, England. 192 p.
29. Subba Rau, B. H., S. Mahadeviah and N. Singh. 1969.
Nutritional studies on whole extract coagulated leaf protein
and fractionated chloroplastic and cytoplasmic proteins from
lucerne (Medicago sativa). J. Sci. Food Agric. 20:355.
30. Tilley, J. M. A. and W. F. Raymond. 1957. The ex
traction and utilization of leaf protein. Herb. Abst. 27:235245.
31. Truter, E. V. 1963. Thin Film Chromatography. John
Wiley and Sons, Inc., New York. 112-118 p.
32. Waterlow, J. C. 1962. The absorption and retention
of nitrogen from leaf protein by infants recovering from
malnutrition. Br. J. Nutr. 16:531.
20. Labanauskas, C. K. and M. F. Handy. 1971. Protein
SUSCEPTIBILITY OF WEST INDIAN AVOCADOS TO
CHILLING INJURY AS RELATED TO RAPID COOLING
WITH LOW TEMPERATURE AIR OR WATER1
J. J. GAFFNEY
USD A, Agricultural Research Service
Gainesville
C. D. Baird
IF AS, Agricultural Engineering Department
Gainesville
Abstract Removal of field heat from avocados
before shipment reduces decay and retards the
ripening process, thus extending market life. Some
avocados, expecially West Indian varieties, are
subject to chilling injury when stored at tempera
tures lower than 55 °F. The use of low tempera
tures for the cooling medium can greatly reduce
the time required for cooling. Such practice, how
ever, raises the question of whether the fruit
might suffer from chilling injury when cooled in
this way. Studies were conducted, over a period
of three seasons, on the rapid cooling of 12 West
Indian varieties of avocados to determine whether
this manner of cooling would cause chilling in
jury
to the
fruit.
Air
temps
were
as
low
as
17 °F, and water temp used were as low as 33°F.
Fruit were cooled to mass-average temps that gen
erally ranged from 40° to 50 °F but was as low as
lFlorida Agricultural Experiment Stations Journal Series
No 7079. Appreciation is expressed to Dr. Charles Barmore,
AREC, Lake Alfred, Dr. James Soule, Department of Fruit
Crops, University of Florida, and to Dr. Donald Spalding,
ARS USDA, Miami, Florida, for assisting in the chilling
injury evaluations involved in this investigation. Grateful
acknowledgement is given to J. R. Brooks and Son, Inc.,
Homestead, Florida for providing fruit samples and to the
Florida Avocado Administrative Committee, Homestead, for
providing
financial
assistance.
33 °F in one test, and were then allowed to ripen
at 70°F. No definite evidence of chilling injury
was found in any of the samples tested.
Removal of field heat from avocades before
shipment is desirable in order to lower the respira-