J Sci Food Agric 1991, 54, 211-219

Afrosimetric Estimation of Threshold Saponin

Concentration for Bitterness in Quinoa (Chenopodium
quinoa Willd)
Michael J Koziol
Latinreco SA, Centre Nestle de Investigacion y Desarrollo Technologico de Alimentos
para America Latina, Casilla 6053-CCI, Quito, Ecuador
(Received 14 March 1990; revised version received 5 June 1990;
accepted 2 July 1990)
ABSTRACT
Taste testings and saponin estimation by a simple afiosimetric method showed
that quinoa containing 0.11 % saponins or less (corresponding to foam heights
of 1.0 cm or less) can be considered sweet. A variation of the standard
afiosimetric method is described which reduces total analysis time fiom 73 to
7 min. Used with the proper precautions, this rapid method, in which quinoa
producing foam heights of 1.3 cm or less is considered sweet, is suitable for
selecting promising low-saponin varieties in field trials and for monitoring the
eficacy of abrasive dehulling (polishing) as a debittering process.
Key words: Chenopodium quinoa, quinoa, afrosimetric assay, bitterness,
saponins.

INTRODUCTION
Quinoa (Chenopodiumquinoa Willd) is an Andean pseudocereal which is currently
attracting considerable international attention as a food supplement not only for its
protein content and quality but also for its fat, vitamins and minerals. For example,
quinoa shows an average protein content of 14.6% (fresh weight) with some
Ecuadorian varieties showing protein contents as high as 21.9 %. This protein is of
good quality, supplying 200% of the histidine, 377 % of the isoleucine, 347 % of the
lysine, 312% of the methionine +cysthe, 363 % of the phenylalanine + tyrosine,
411% of the threonine, 180% of the tryptophan and 346% of the valine
recommended in protein sources for adult nutrition by the FAO/WHO/UNU joint
21 1
J Sci Food Agric 0022-5142/91/$03.50 0 1991 SCI.Printed in Great Britain

M J Koziol

212

expert consultation (1985) (Koziol 1990). Quinoa shows an average fat content of
5.6 % (fresh weight) with linoleic and linolenic acids accounting for about 55 % of
the fatty acids (Kozioll990).It is a good source of vitamins E, B, and B,, folic acid
and biotin, and of potassium, iron, copper, magnesium and manganese (Koziol M
unpublished data).
Quinoa is classified as sweet or bitter depending upon its saponin content.
Although certain sweet varieties have been developed, such as Sajama, Cheweca,
Piartal, Nariiio and the Latinreco varieties V12-1 and V12-2, most of the quinoa
grown in South America is of the bitter variety which poses a problem for its
incorporation into food products.
Fortunately, the saponins are concentrated in the outer layers of the grain
(perianth, pericarp, seed coat and a cuticle-like layer) which facilitates their removal
by abrasive dehulling (Reichert et al 1986) or by the more traditional method of
washing the grains with water. Verification of the efficiency of debittering quinoa
and the continuing research to develop new low-saponin varieties necessitate a
simple and rapid assay which can be used in the factory or field.
Although nine triterpenes have been identified as the sapogenin moieties of
quinoa saponins, with oleanolic acid and hederagenin dominating (BurnoufRadosevich and Delfel 1984; Burnouf-Radosevich et al 1985), the respective
glycosidic moieties have yet to be determined. Without such complete structural
information for the calculation of the molecular weights of the saponins the
analytical methods quantitating only sapogenins are of little practical use (see Price
et all987 for a review of analytical methods). This consideration eliminates many of
the gas and liquid chromatographic and spectrophotometric methods for reasons
other than the paucity of such equipment in local (ie South American) factories and
field stations. Thin layer chromatography without ancillary techniques and pure
standards is qualitative; haemolytic techniques require a standardised supply of
blood and suffer the complication that not all saponins show similar haemolytic
activity; gravimetric methods are fraught with difficulties not least of which is a nonspecific extraction of soluble compounds; and the simplest bioassay of all, tasting
the grain, is complicated by the variation in the human ability to detect bitterness,
fatigue in tasting and by the fact that not all saponins taste bitter.
The property common to all saponins, indeed the very basis for their generic
name, is their ability to produce a soapy lather in water. Although an afrosimetric
analysis also has its complications, it is the method most easily adapted to local
factory and field use.

MATERIALS AND METHODS

Saponin standards
Saponins used to calibrate the method were extracted from commercially available
bitter quinoa representing a mixture of varieties. Whole seeds (30 g) were first
defatted by Soxhlet extraction with diethyl ether for 6 h. Saponins were extracted
with 250 ml of 80% methanol under reflux for 4 h, and the extract was filtered and
then evaporated to dryness. The dried extract was dissolved in n-butanollethanoll

Saponins and bitterness in quinoa

213

water (1:1:1 v) using 5 ml of solvent for each 1 g of extract, and applied to a column
of aluminium oxide of 125 ml bed volume to remove the pigments extracted along
with the saponins. Saponins were eluted from the column with 250 ml of the same
solvent and the eluate was evaporated to dryness. This crude saponin extract was
stored desiccated over silica gel at room temperature (25-27C). Analysis of the
extract showed 15.0% protein (Kjeldahl N x 6.25), 3.8 % ash (incineration for 4 h at
55OOC)and 2.4 % water content (oven drying for 4 h at 102C): the purity of this
crude saponin extract (ie 78.8%) was taken into consideration in preparing all
solutions.
Standard afrosimetric method

Quinoa seeds (0.50+ 0.02 g) are weighed directly into a screwcap test tube (160 mm
long, 16 mm dia). Upon the addition of 5 ml of distilled water the tube is capped and
shaken vigorously (4 shakes s- l, up and down movement) for 30 s. After a 30-min
rest the tube is again shaken vigorously for 30 s. After a second 30-min rest the tube
is shaken vigorously for 3Os, given a last shakedown as one would to an oral
thermometer and allowed to rest for 5 min before reading the foam height to the
nearest 0.1 cm.
Rapid afrosimetric method

As above, except that foam height is read 5-10 s after the initial shaking.
Saponin-sensitive taste panel

To estabiish a panel of tasters sensitive to bitterness a preliminary screening of

volunteers was conducted by offering them once a day (mornings) two glasses of
liquid, identifying the first glass as containing distilled water and asking them if they
detected any bitterness in the liquid in the second glass. The liquid in the second
glass was either distilled water or 10,50,100or 500 mg saponin litre-', presented in
random order on five successive days. The response varied from three sensitive
individuals who detected bitterness at 10 mg saponin litre-' to one insensitive
individual who could not detect bitterness even at 500mg saponin litre-l, a
concentration which all the other volunteers found to be distinctly bitter. This
insensitive individual was exempted from further tastings. The tasting of distilled
water against the distilled water reference disqualified another three volunteers on
the suspicion that they had psychologically conditioned themselves to detect
bitterness in the second glass irrespective of what liquid it contained.
Survivors of the preliminary screening were then subjected to tasting, in triplicate,
four solutions presented in randomly numbered glasses which contained either
distilled water, or 10,50 or 100 mg saponin litre-', and were asked to arrange the
samples in increasing degree of bitterness. The results of these tastings classified the
volunteers into three categories:

(1) those who could not distinguish between distilled water and 10mg saponin
litre-' (both liquids identified as non-bitter) nor between 50 and l00mg
saponin litre-' (both identified as slightly bitter);
(2) those who could not distinguish between distilled water and 10 mg saponin

M J Koziol

214

litre- but who, in all three tastings, successfully distinguished getween 50 and
100mg saponin litre-; and
(3) the three most sensitive individuals who consistently arranged the four liquids
in order of increasing saponin concentrations.
The 10 volunteers who were classified as either category 2 or 3 were elected to the
taste panel.
To train the tasters to identify the characteristic bitterness of quinoa saponins,
pastes were prepared with uncooked quinoa flours by mixing 15 g of flour with
24ml of distilled water. Two varieties of quinoa, Sajama (sweet) and LR-012
(bitter), both purified through years of careful selection by the Agronomy
Department of Latinreco, were used in this part of the study. On three consecutive
days, tasters were presented with a reference paste of Sajama flour and a paste of
98 % Sajama and 2 % LR-012 flours for comparison: all tasters agreed that the latter
was distinctly bitter.
Once trained to identify the bitterness of saponins in the pastes, the tasters were
presented with a reference paste of Sajama flour and four mixtures of Sajama flour
containing either 0.4,0.6,1*0or 1.6% LR-012 flour in randomly numbered cups and
asked to indicate which of the four samples were more bitter than the reference flour
paste. The tastings were conducted in triplicate. Whole quinoa seeds of both the
sweet and bitter varieties were mixed in the same proportions as in the flours and the
saponin content was estimated afrosimetrically.
As expected, detection of bitterness in pastes of uncooked flours was more
difficult than in distilled water, especially as some tasters found the reference flour
paste to be slightly bitter, though of a different nature to the bitterness of saponins.
Subject response also varied; for example, one taster classified the flour mixture
containing 0 6 % LR-012 flour as more bitter than the reference paste only two out
of three times. A subjective decision was taken to classify as bitter a flour paste
which received more than 50% of positive marks (indicating the tasters found it
more bitter than the reference flour) as summed over the triplicate tastings.

RESULTS AND DISCUSSION

The apparent simplicity of an afrosimetric method belies inherent problems. The
production and stability of a foam are dependent upon chemical structure and
surfactant activity, pH, presence of salts and method of agitation (German et a1
1985; Rossi et at 1985).The conditions stipulated for this afrosimetric method were
found optimal for producing a stable, compact foam for most of the varieties of
quinoa assayed. Varieties such as Porotok (Table 1) produced an unstable foam
composed of large air bubbles which rapidly collapsed, making it difficult to obtain
a reliable measurement of foam height. The 6% of the total protein content of
quinoa that is extracted in the course of the standard afrosimetric method most
likely represents albumins +globulins; albumin is known to produce unstable
foams (German et al1985; the albumin +globulin fraction represents about 43 /, of
the total protein content of quinoa: Scarpati de Briceiio and Briceiio 1980).If foam

Saponins and bitterness in quinoa

215

TABLE 1
Estimation of saponin concentrations in quinoa with the standard
afrosimetric method
Quinoa variety

Foam height (cm)

mg Saponin
(g-' j-esh wt)

Mean

SD

'Sweet'
Sajama 01
Sajama 02
Sajama 03
Perulac, polished

0.1"
0.4
0.6
0.2

0.1
0
0.1
0.1

BLD~
0 31
0.57
0.05

'Bitter'
Perulac, whole grains
Porotok
LR-013

1.4
5.6'
7.5'

0-4
0.6
0.5

1.60
ALDd
ALD

Three entries of Sajama were tested. Values represent the means of 4

determinations with standard deviations (SD), except for: "mean of 6
determinations and emean of 21 determinations.
Below the limits of detection of the method.
Unstable foam characterised by large air bubbles.
Above the limits of detection of the method.

instability is due to the extracted albumins, it is curious that this effect is not
observed with other varieties of quinoa, leading to the supposition that some
varieties of quinoa must have radically different protein and/or saponin profiles or
contain some other foam-destabilising factors.
As the foaming behaviour of proteins depends upon their structure (German et a1
1985), it would seem reasonable to assume the same for saponins. There is currently
no information available that compares saponin profiles among varieties of quinoa,
so it was considered more prudent to calibrate this method with saponins extracted
from a mixture of bitter varieties thus averaging at the outset any possible
differences in saponin profiles and foaming behaviour. Such a calibration should be
viewed not as potentially sacrificing specificity but rather as a necessary
generalisation of the applicability of the method given the current small-scale
production of quinoa which presents a mixture of varieties to the commercial
market.
Preliminary investigations showed that although significant differences in foam
height were found within a range of sample weights from 0.45 to 0.55 g, no such
effect was seen within the range 0.48-0.52 g and hence the stipulation of sample
weight of 0.50+ 0.02 g.

Calibration curve
Figure 1 shows the calibration curve obtained using solutions of the crude saponin
extract and following the standard afrosimetric procedure. The levelling off of foam
heights at saponin concentrations greater than 0.4 mg ml-' is probably due to

216

M J Koziol

'1
6-

5-

i5
I

E,0

u.

Saponins

-1

mg mi

Fig 1. Foam production by saponins extracted from quinoa. Solid circles, foam produced by solutions of
the crude saponin extract. The equation for the linear portion of the curve (0-0.4 mg ml- ') used to
estimate saponin concentrations by the standard afrosimetric method is: y = 7.638xf0.173, r =0.992
( P < 0,001, F-test) and standard error of the coefficient (slope)= 0.462. Open squares, foam produced by
the addition of 0.50g of Sajama (sweet quinoa) grains to solutions of the crude saponin extract in an
attempt to improve agitation. The possibility that the saponins were being absorbed by and/or adsorbed
on to the sweet quinoa grains was confirmed by the obtention of similar results using grains of polished
bitter quinoa (open triangles).

problems in obtaining a sufficiently vigorous agitation to produce greater foam

heights. It was thought that, if this were indeed the case, agitation could be
improved by adding 0.50 g of the sweet quinoa, Sajama, to the solutions of the crude
saponin extract. The addition of Sajama grains to solutions containing 1.0 mg crude
saponin ml-' indeed resulted in greater foam heights, but foam heights were
reduced at the lower saponin concentrations (open squares in Fig 1)most likely due
to the saponins being absorbed by and/or adsorbed on to the sweet quinoa grains.
The possibility of such an absorption/adsorption of saponins was confirmed by
repeating the experiment using polished grains of a bitter quinoa in place of the
sweet Sajama grains (open triangles in Fig 1).
The concentrations of saponins in solution can be linearly correlated with foam
heights ranging from 0.2 to 3.0 cm, and tentatively estimated in quinoa grains using

Saponins and bitterness in quinoa

217

the following equation:

mg saponin g-' fresh wt =

(0.646)(foam height, cm) - 0.104

(sample wt, g)

(1)

Saponin concentrations and bitterness

Table 1 shows the results of applying the afrosimetric method to some of the quinoa
varieties from the experimental plots of Latinreco: the varieties were classified as
'sweet' or 'bitter' by taste testing. As quinoa saponins have been so little studied
there are no suitably accurate methods with which to compare this afrosimetric
method. On a dry weight basis, Blanca de Junin, a low-saponin variety of quinoa,
was estimated to contain 0.09 % saponins by TLC (Burnouf-Radosevich and
Paupardin 1983) and 0 1 6 % by haemolysis (Reichert et al 1986). With this
afrosimetric method, Sajama 03 showed 0.07% saponins on a dry weight basis
which, when compared with the estimates of saponin concentrations in Blanca de
Junin, seems a reasonable value for a sweet quinoa.
Whole grains of Perulac, classified as bitter and giving a foam height of 1.4 cm,
were successfully debittered by abrasive dehulling (polishing). The data in Table 1
imply that an inflexion point between sweet and bitter quinoa occurs between
0.06 % and 0.16 % saponin content (foam heights of 0.6 and 1.4 cm, respectively).
Pastes made with sweet quinoa flour (Sajama)doped with flour from a bitter quinoa
(LR-012) were presented to a panel of tasters along with a reference paste of Sajama
flour to determine when bitterness was definitely detected. This point was
arbitrarily set as a flour mixture receiving more than 50% positive marks summed
over three tastings: a positive mark indicated that the flour was judged more bitter
than the reference flour. Saponin concentrations in the flour mixtures were
estimated afrosimetrically using whole grains of both the sweet and bitter quinoas
mixed in the same proportions as in the flours. The results showed that the addition
of 1.6 % bitter quinoa flour to the Sajama flour met the criterion set for bitterness
(Table 2). Quinoa can thus be considered sweet if it contains 0.11% saponins or less
TABLE 2
Determination of the threshold for the detection of bitterness in mixtures
of flours of sweet (Sajama) and bitter (LR-012) quinoa

7; LR-012
in mixture

Saponins in
flour mixture

Corresponding
foam height (cm)

% Positive
responses"

0.4

0.08
0.10

0.8
09
1.o
1.2

26
48

0.6
1.0
1.6

0.11
0.13

42
71

In the taste testing, a positive response indicated that the flour mixture
was judged to be more bitter than the reference flour (Sajama). The
bitterness threshold was arbitrarily set at that point at which a flour
mixture received more than 50% positive responses, summed over three
taste testings. Thus, quinoa is judged bitter if it contains more than 0-11 %
saponins and gives a foam height greater than 1.0 cm.

218

M J Kozioi

on a fresh weight basis. Thus the afrosimetric method can be used as pass/fail test,
classifying as sweet those varieties of quinoa that produce 1.0 cm of foam or less.
Interestingly, these data also underscore the potency of the bitterness of quinoa
saponins, given that only 1.6% of bitter quinoa flour was sufficient to impart an
unacceptable level of bitterness to the flour mixture.

Rapid saponin estimations

The standard afrosimetric method was developed to allow sufficient time for the
extraction of the saponins from the quinoa grains. The total procedure takes about
73min, which was found to be too long to be practical for use in the field for
identifying low-saponin varieties or for assaying the efficacy of abrasive dehulling. A
correlation of foam heights obtained 10 s after the initial 30 s agitation with those
obtained following the standard procedure showed:
(final foam height, cm)= (0.668)(foam height after 30 s, cm) 0.174
(r=0*935) (2)
The number of observations in this correlation was 60, the correlation significant at
P<O.OOl (F-test) and the standard error of the coefficient was 0.03. This equation
can be substituted into eqn (1) to give:
(0.432)(foam height after 30 s, cm) + 0.008
mg saponin g - fresh wt =
(3)
(sample wt, g)

With this rapid afrosimetric method, quinoa is classified as sweet if it gives a foam
height of 1.3 cm or less, and the total time to perform the analysis is reduced to
about 7 min.
However, such rapidity of saponin estimation is not to be had without sacrifices
and there are several caveats to be borne in mind when applying the rapid
afrosimetric method. First, foam height is changing rapidly from both directions,
rapidly decaying at the upper surface while the foam-liquid interface is in the
process of defining itself. These factors exert a combined effect on the uncertainty of
the measurement of foam height. Secondly, the correlation assumes variation in
only one parameter, in this case foam height as influenced by the time of extraction
of the saponins. Yet the efficiency of the extraction is itself a function not only of time
but also of the surface area presented by the quinoa grains in the sample being
analysed, the saponin concentration, how readily the saponins can be leached from
the grains and the foaming properties of the leached saponins.
Nevertheless, this rapid afrosimetric method has been successfully applied in the
field by the agronomists of Latinreco in screening over 5000 individual quinoa
plants grown on our various experimental plots (taking the precaution of retesting
promising individuals with the standard method, of course). It has also been
successfully applied, again with the backup of the standard method, in trials on the
efficacy of abrasive dehulling conducted in our pilot plant.
ACKNOWLEDGEMENTS
We gratefully acknowledge the technical assistance of Sras M E Montalvo, S

219

Saponins and bitterness in quinoa

Fonseca, V Burbano and L Mendoza in preparing and analysing the crude saponin
extract, and thank Ing M Alvarez for testing the method under field conditions.
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