NUTRITIVE VALUE OF LEAF PROTEIN CONCENTRATES

PREPARED FROM Anaranthus SPECIES

P. R. CHEEKE1, R. CARLSSON2.3 and G. O. KOHLERI

Department of Animal Science, Oregon State (Jniversity, Corvallis, Ore. 97331;
2Department of Plant Physiology,
University of Lund, Sweden; anct aUSDA-SEA,
Western Regional Research Laboratory, Berkeley, Calif. Oregon Agricubural
Experiment Station Technical Paper No. 5491, received 2l Apr. 1980.
urrrptri 8 Dec. t980.
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CHsrxE,, P. R., CenrssoN, R. eNo KoHr_En, G. O. 198 1. Nutritive value of leaf

protein concentrates prepared fromAmarantftas species. can. J. Animal Sci. 61:
199-204.

Leaf protein concentrate (LPC) samples were prepared from several species of
Amaranthus . Their proximate composition and phenolic content were determined. In
vitro nitrogen digestibility was low (28-48Ea) and was negatively correlated with
phenolic content. The amino acid content ofone sample ofA. hypochondriacus LpC
was measured, and compared favorably with reported values for essential amino acids
for alfalfa LPC and soybean meal. The nutritibnal values of the Lpc samples were
assessed in a rat feeding trial. compared to a soybean meal control diet, the gains with
Amaranthus LPC were poor, ranging from l8 to72vo of the control value. The cause
of the poor growth remains to be determined. The high ash content of the samples, or
the presence of organic factors such as saponins, phenolics and oxalaies are
For personal use only.

possibilities to account for the poor growth rate of rats.

Des 6chantillons dp concentrat de prot6ine foliaire ont 6t6 pr6par6s ir partir de plusieurs
espbces d'Amaranthus et on a 6tabli leur composition imm6diate-et leur Gneur en
ph6nols. Leur digestibilit6 de N in vitro 6tait bass e \28-48vo) et n6gativemenr con6l6e
avec les concentrations ph6noliques. La teneur en acides amin6s d'un 6chantillon de
concentrat obten]u de Amaranthus hypochondriacus se comparait favorablement avec
les valeurs publi6es pour les acides aminds essentiels des Concentrats de prot6ine de
luzerne et du tourteau de soja. La valeur nutritive de divers isolits extrarts
d'Amaranthus a 6t6 mesur6e sur des rats. compar6 d un r6gime t6moin h base de
tourteau de soja, le gain de poids produit par les isolats de prot6ine d',4 maranthus 6tait
m6diocre, se situant entre 18 et78To des valeurs t6moins. La cause de cette oibtre
performance reste i 6lucider. Elle pourrait tenir d la concentration 6lev6e en cendres
ou encore d la pr6sence de facteurs organiques comme les saponines, les ph6nols et les
oxalates.

Green plants synthesize and contain largei
amounts of protein. Forages are not utilized
efficiently as protein sources for humans and
most nonruminant animals, because the
protein is poorly digested, the fibrous feeds
are unpalatable, and they have a low
digestible energy content for animals that do
3Present address (R.C.): CETEC-SDA, 30000 Belo
Horizonte. Brazil.
Can. J. Anim. Sci. 6l: 199-204 (March 19tt)
t99
not digest cellulose. One procedure for
utilizing the protein in green plant tissue for
nonruminants is the production of leaf
protein concentrate (LPC), which is pre-
pared by expressing the plant juice with a
press, and coagulating the soluble protein in
the juice. LPC prepared from alfalfa has
given growth performance similar to that
obtained with soybean meal with swine
(Myer et al. 1975; Cheeke et al. 1977b) and
with poultry (Kuzmicky and Kohler 1977).
Amaranthus species contain large
 200 CANADIAN JOURNAL OF ANIMAL SCIENCE
amounts of extractable protein (Lexander et
al. 1970). They grow in many parts of the
world, including temperate and tropical
regions. Amaranthus species were important
grain crops in South America prior to the
Spanish conquest. Efforts are being made to
develop several Amaranthus species as crop
plants (Cole 1979). Forage types are grown
in Africa and Asia as vegetable crops.
Can. J. Anim. Sci. Downloaded from cdnsciencepub.com by 187.16.184.228 on 04/01/22
Amaranthus has been identified by the U.S.
National Science Foundation as one of six
plants with promising potential as new
crops. The others are kenaf, crambe, pigeon
pea, jojoba, and guayule. Amaranthus
contains saponins, alkaloids, phenolics, and
oxalates (Feine et al. 1919) which might
have adverse effects on LPC quality. The
objective of this study was to evaluate the
quality of Amaranthr.rs LPS as a protein
source, using growing rats as the assay
animal.
For personal use only.
MATERIALS AND METHODS
The plants were grown in piots at Davis, Calif.
Two replicated plots (15 m2) were used for each
species or cultivar. Nitrogen fertilizer was
applied at 200 kg N per ha. The whole plants were
harvested ll wk after planting, chopped, and
ground together with a 2% sodium bisulfite
solution of pH 8.5 (one part fresh weight plus
0. 1 5 part solution) . The material was pressed in a
screw press; the expressed juice was readjusted to
pH 8.5 and quickly heated to 85'C. The
coagulated leaf proteins were collected with a
delaval separator, and the wet LPC was
freeze-dried. Unless otherwise indicated, the
LPC was washed twice with weak acid at pH 4, to
remove phenolic substances, and again freeze-
dried. Samples of LPC from low saponin andhigh
saponin alfalfa were included for comparative
purposes.
The proximate composition of Amaranthus
LPC samples was determined, using Association
of Official Agricultural Chemists ( 1970)
methods. Amino acids were estimated for one
sample of A. hypochondriacus LPC, using a
Beckman Spinco column chromatography au-
toanalyzer after hydrolysis in 6 N HCI for 24 h.
Cysteine and cystine were determined as cysteic
acid and methionine as methionine sulfone, after
oerformic acid oxidation.
In vitro pepsin-trypsin digestibility of Amaran-
thus LPC was determined by the Lexander et al.
( 1970) modification of the Akeson and Stahmann
(1965) method. Protein nitrogen soluble inO.5Vo
trichloroacetic acid was expressed as a percen-
tage of total protein in the LPC.
Phenolics in rhe Amaranthus LPC were
estimated as ethanol-extractable phenolics and as
pepsin-trypsin-released phenolics ln the first
case, the LPC was refluxed three times with 807o
ethanol (1 g LPC, 50 mL ethanol, for 2 h, l5 min
and 15 min). Folin reagent was used to detect
phenolics (Folin and Denis 1915). In the second
case, LPC was digested with pepsin-trypsin
(Lexander et al. 1970). The digest was analyzed
for chlorogenic acid using the method of
Hoepfner (1932) . In both cases, chlorogenic acid
was used as the standard. Results are expressed as
milligrams of chlorogenic acid equivalents per
sram N in LPC.
The LPC was included in rat diets to provide
l0 Vo dietary protein. Corn and soybean meal were
added to provide 67a protein, to give a total
dietary crude protein level of 16Vo. ln addition'
the diets contained 47o mineral mix (Jones and
Foster 1942). l7o vitamin mix (Cheeke et al.
1977a) and4Vo corn oil. The proportions of LPC'
com and soybean meal were variable, and are
listed in Table 3. Five male Long-Evans rats of
about 75 g initial weight were assigned to each
treatment. They were fed ad libitum until all feed
was used; the number of days for each treatment is
indicated in Table 3. This procedure was used
because the quantities of the LPC samples
available were quite limited, so the maximum
amount of feed that could be made in each case
was prepared. The gains were compared to those
of the control animals, over the appropriate time
period. Means were compared by Least Signific-
ant Difference (Steel and Torrie 1960).
RESULTS AND DISCUSSION
The LPC samples were much lower in
protein and higher in ash (Table l) than is
usually the case for LPC, which typically has
values of aboil 50Vo crude protein and
5-107o ash (Pirie 1978). The high ash
content of these samples, which con-
sequently reduces the percentage protein,
may be attributable to a fine dust blowing in
from surrounding fields. However, a high
ash content may be a characteristic of
Amaranthus, since in previous studies
 CHEEKE ET AL. PROTEIN FROM AMARANTHUS SPECIES 201
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 202 CANADIAN JOURNAL OF ANIMAL SCIENCE

(Cheeke and Carlsson 1978), Amaranthus
whole-plant grown elsewhere was found to
have a high ash content. The amino acid
analysis (Table 2) of a sample of Amaran-
thus hypochondriacus compared favorably
in terms of essential amino acid composition
to that of soybean meal, particularly with
respect to lysine and sulfur-containing
amino acids, and was very similar in amino
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However, there was no apparent relationship
between phenolic content and growth rate;
the correlation coefficient for the regression
between gain and phenolic content was low
(r +0.43). This might suggest that
phenolics did not influence the gains, or that
the level in all cases was sufficient to
markedly impair protein utilization' The
effect of phenolics on rat pedormance is not
totally clear; plants or plant meals with high

acid composition to alfalfa LPC (Table 2).

The growth rates of the rats
Amaranthus LPC were quite poor (Table 3)
in most cases. The amino acid analysis
indicates that the A. hypochondriacus LPC
should not be deficient in total essential
amino acids, although their availability
might be low, as suggested by the low in
vitro nitrogen digestibility (Table I ).
Amaranthus species contain phenolics
(Heagauer 1964) which bind proteins and
reduce their digestibility (Pirie 1978). The
content of ethanol-extractable phenolics was
For personal use only.
fed phenolic content usually give poor animal
performance, but chlorogenic acid when fed
to rats at l7o of the diet had no effect on
Protein Efficiency Ratio or digestibility
(Eklund 1915). On the other hand,
Horigome and Kandatsu ( 1968) showed that
phenolic substances allowed to react with
casein in the presence of o-diphenoloxidase
reduced the biological value of the protein.
This did not happen in the absenca of the
phenoloxidase.
The Amaranthus LPC had considerably

negatively correlated with pepsin-trypsin less phenolics than the original plant matter'
nitrogen digestibility (r : -0.83, n : ll). Per gram of dry matter, the ethanol-
extractable phenolic content of whole plant,
press residue and LPC ranged from l1-17,
Table 2. Amino acid composition of LPC prepared 1-9, and 2-8 mg, respectively' Per gram of
ftom Amaranlhus hypochondriacts (g amino acid/16 g
N) compared to that of soybean meal and alfalfa LP
N, the corresponding values were 900-
1100, 700-l100, and 60-140 mg. Thus the
g amino acid/16 g n phenolic level ofthe LPC was about 1O-fold
A. less than for the original plant material.
hypochondriacus Alfalfa SoYbean Either phenolics in the plant were not
Amino acid LPC LPCt meali completely bound to the LPC, or were
Lysine 6.6 5.9 6.5 removed by the acid washing.
Threonine 5.0 ).1 l.J Rats on the diet containing unwashed
Proline 4.6 4.9 6.3 mixed Amaranthus LPC had a very poor
Cysteine t.2 1.2 i.5
growth rate (Table 3), and exhibited a
Methionine 2.4 2.3 l3
Valine 6.9 6.3 5.3 bloated, swollen appearance. Gross exami-
Isoleucine 5.8 5.6 5.3 nation revealed no tissue lessions, but the
Leucine 9.3 9.3 8.2 intestines were distended with fine particu-
Phenylalanine 6.0 5.9 5.3
late matter, which may have been ash, or a
Tyrosine 4.2 4.8 3.3
Histidine 2.4 z.-t z. I poorly digested polysaccharide fraction. Its
Arginine 5.8 6.5 1.3 identity was not pursued.
Aspartic acid 9.9 r0.0 14.0 Saponins are another possibility to
Serine 5.3 4.3 5.6
account for the poor animal performance.
Glutamic acid 10.6 I1.4 20.0
Saponins, assessed by the Trichoderma
Glycine 5.4 \\ \ )
Alanine 6.0 6.3 5.3 bioassay of Pedersen and Wang (197 l), have
been detected inAmaranthus foliage at high
tKuzmicky and Kohler 1977.
tlntemational feed reference number 5 O4 604 levels (Cheeke, unpubl. observ.). Saponin
 CHEEKE ET AL.
-
PROTEIN FROM AMARANTHUS SPECIES 203
Table 3. Diet composition, and rat growth and feed intakes

Diet composition Rat performance

Vo Days Avg daily Gain as Avg daily
Vo Vo soybean on gain Vo of feed intake
Source of LPC LPC corn meal test (9, control (g)
Corn-soy control 69 22 6t 8.3+0.3 .] 100
8.2+0.4a 100
l6t 8.5 +0.3 d 100 22.0+0.6
A . anclancalius 4t.4
44.6 5 l3 5.9+O.3 bc 69 21 .6+ 1 .5
A. ascendens 44.4 60
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41.6 5 lo 5.1+0.9 bc 18.0+2.1

A. flavus 54.3 58
29.'7 7 o 4.8+ 1.0 b 18.1 + 1.1
A. gangeticus 46.2 39.8 5 l5 4.8+0.3 b 56 19.5+ 1.8
A. mantegazzianus 41 .:t 5 12 4.1+0.9 b 55 18.0+2.8
A. Taiwan l2l 43.7 5 IJ 6.1+0.9 c 72 2l.l+1.5
A. cruentus HHI 60.2 21.8 9 l6 1.5+0.8 d 18 15.2+ | .2
A. cruentus HH2 55.2 27.8 8 8 5.4+0.9 bc 66 18.5+1.1
A. cruentus HH3 60.2 21.8 9 l6 )6+11nf 3t t6.l+1.'7
A. hypochondriacus 48.5 36.5 6 l3 4.9+0.9 bf 58 18.9+ 1.3
l. Taiwan 3t +/. I 44.1 ^ l3 6.0+O.6 bc 11 20.5+ | .4
A. cruentus HH3
(unwashed) 5 6.8 26.2 l3 3.8+0.5/ 45 17.6+2.2
Mixed Amaranthus
(unwashed) 68.5 l 1.5 ll t6 2.2+0.4d 26 15.9+ 1.0
High saponin alfalfa 20.6 70.4 16 5.9+O.7 bcg 69 11 .'7+1.2
Low saponin alfalfa 19.3 11.7 6.9+0.4I + I .0
For personal use only.

14 81 19.3
fAverage daily gain for the control group was computed after 6, 9 and l6 days on test.
tSeed obtained from Taiwan; speciei noi identified.
a-g Means followed by different letrers are different (p < 0.01).

content of the LPC samples was not
measured.
The high ash content of the samples may
have been another factor involved in the poor
rat gains. If the ash acted as a non-nutritive
diluent, increased feed intake mieht have
occurred. as rats adjust their feed Intake to
meet caloric needs. Because intake was not
increased (Table 3), factors other than the
ash content may also be contributing to the
poor growth. It is also possible that the high
mineral content may have caused low diet
palatability, or had some other detrimental
effect. While the ash content
samples is higher than generally reported for
LPC (Pirie 1978), these samples may be
reflective of what might be observed under
commercial LPC production.
In a previous study (Cheeke and Carlsson
1978), whole plant Amaranthus meal gave
very poor results when fed to rats. Growth
was poor, and feed intake low. In studies
with Amaranthus seeds (Cheeke and
Carlsson, unpubl. observ.), rat performance
has been poor. The Amaranthus fractions,
whether whole plant, seed, or LPC, seem to
be unpalatable to rats, and have an
unpleasant, earthy taste to the human palate.
Phenolics, saponins, and oxalates are
components of Amaranthas (Hegauer 1964;
Feine et al. 1979), which mighr be
responsible for the poor animal responses.
While several Amaranthus sDecies are
vigorously growing. high yielding plants.
the factor(s) responsible for the poor animal
of these acceptance need to be overcome before
Amaranthus , or LPC prepared from it, can
be successfully utilized as a feedstuff.
AKESON, W. R. and STAHMANN, M. A.
1965. Nutritive value of leaf protein concentrate,
an in vitro digestion study. Agric. Food Chem.
13: 145-148.
ASSOCIATION OF OFFICIAL AGRICUL-
TURAL CHEMISTS. 1970. Official methods of
 CANADIAN JOURNAL OF ANIMAL SCIENCE

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