S: Sensory & Food
Determination of Absolute Threshold and
Just Noticeable Difference in the Sensory
Perception of Pungency
L. Orellana-Escobedo, J.J. Ornelas-Paz, G.I. Olivas, J.A. Guerrero-Beltran, J. Jimenez-Castro, and D.R. Sepulveda
Abstract: Absolute threshold and just noticeable difference (JND) were determined for the perception of pungency
using chili pepper in aqueous solutions. Absolute threshold and JND were determined using 2 alternative forced-
choice sensory tests tests. High-performance liquid chromatography technique was used to determine capsaicinoids
concentration in samples used for sensory analysis. Sensory absolute threshold was 0.050 mg capsaicinoids/kg sample. Five
JND values were determined using 5 reference solutions with different capsaicinoids concentration. JND values changed
proportionally as capsaicinoids concentration of the reference sample solutions changed. Weber fraction remained stable
for the ﬁrst 4 reference capsaicinoid solutions (0.05, 0.11, 0.13, and 0.17 mg/kg) but changed when the most concentrated
reference capsaicinoids solution was used (0.23 mg/kg). Quantiﬁcation limit for instrumental analysis was 1.512 mg/kg
capsaicinoids. Sensory methods employed in this study proved to be more sensitive than instrumental methods.
Keywords: chili pepper, just noticeable difference, pungency, sensory threshold
Practical Application: A better understanding of the process involved in the sensory perception of pungency is currently
required because “hot” foods are becoming more popular in western cuisine. Absolute thresholds and differential thresholds
are useful tools in the formulation and development of new food products. These parameters may help in deﬁning how
much chili pepper is required in a formulated product to ensure a perceptible level of pungency, as well as in deciding
how much more chili pepper is required in a product to produce a perceptible increase in its pungency.
Chili pepper fruits are one of the most consumed spices
throughout the world. Chili peppers provide pungency, color,
and aroma to different types of foods and are appreciated not
only as food ingredients, but also because of their pharmaceu-
tical and physiological actions (Garces-Claver and others 2006;
Cisneros-Pineda and others 2007). In the United States there has
been a great increase in the demand of this fruit in recent years
because of the increased multiculturalism, popularity of ethnic
restaurants, and increased use of “ﬂavored” vegetables in the diet
to avoid fats and cholesterol (Lawless 1989). The food industry is
the largest consumer of chili peppers, where the spice is used as
a ﬂavoring agent in sauces, soups, processed meats, snacks, can-
dies, soft drinks, and alcoholic beverages (Mathew and others
The sensory quantiﬁcation of chili pepper’s pungency is used
by the food industry for a variety of purposes including the study
of ingredient variation limits in products containing chili pepper
MS 20110882 Submitted 7/21/2011, Accepted 12/1/2011. Authors Escobedo,
Ornelas-Paz, Olivas, and Sep ´
ulveda are with Centro de Investigacion en Alimentacion
y Desarrollo, A.C. Unidad Cuauhtemoc, Av. Rio Conchos S/N, Parque Industrial,
C.P. 31570. Cd. Cuauhtemoc, Chihuahua, Mexico. Author Guerrero-Beltran is with
Universidad de las Americas Puebla. Santa Catarina Martir, Cholula Puebla. C.P.
82720, Mexico. Author Jim´
enez-Castro is with Univ. Autonoma de Chihuahua.
Av. Escorza No. 900, Zona Centro. C.P. 31000 Chihuahua, Chihuahua, Mexico.
Direct inquires to author Sepulveda (E-mail: firstname.lastname@example.org).
such as salsas and cheese sauces (Meilgaard and others 1991). One
metric of interest that is essential for the determination of these
variation limits is the absolute threshold (Shepherd and others
2008). The ﬁrst method developed for the quantiﬁcation of pun-
gency, known as the Scoville method, is a sensory test based on
the determination of the perception threshold in serially diluted
samples. Pungency thresholds are useful in the determination of
“heat”, which contributes to the sensory properties of a product.
Such values have immediate practical implications for acceptable
compared with unacceptable levels of ﬂavor components (Lawless
and Heyman 1999). There is also another metric that is rele-
vant for food product developers: the just noticeable difference
(JND). JND is the minimal difference that can be detected between
2 stimuli (Lawless and Heymann 1999). JNDs are important, for
example, when a negative change, such as the reduction in an
expensive ingredient in a formula is not readily discernible to
consumers (below JND), or when a positive change, such as an
increment in the amount of a consumers’ favorable ingredient, is
very apparent to consumers (above JND; Chang and Chiou 2006).
Research on pungent food products have been conducted to study
the interaction of chili peppers with other food ingredients such as
cheese sauces, chicken patties, pork patties, sucrose, sodium chlo-
ride, and citric acid, among others (Sizer and Har ris 1985; Carden
and others 1999; Emrick and others 2005; Reinbach and others
There is little investigation on pungency thresholds. JND for
pungency has never been reported and a few studies have re-
ported absolute threshold values. The reported values for absolute
2012 Institute of Food Technologists R
doi: 10.1111/j.1750-3841.2011.02589.x Vol. 77, Nr. 3, 2012 rJournal of Food Science S135
Further reproduction without permission is prohibited
S: Sensory & Food
Pungency sensory perception . . .
threshold have been obtained using different sensory methods such
as magnitude estimation and the method of limits (Krajewska and
Powers 1988; Lawless and others 2000). These are good methods
but may present some limitations. Magnitude estimation meth-
ods produce results having ratio problems such as judges having
a strong favored-number effect (O’Mahony 1982). It is also said
that magnitude methods are incapable of providing stable and re-
producible values for ﬂavor intensity (Meilgaard and others 1991).
Similarly, the method of limits is slightly biased and can be very
biased if subjects falling on the upper or lower limits of the range
under test are not reexamined (Meilgaard and others 1991). On
the other hand, 2 alternative forced-choice sensory tests (2AFC)
may be used to estimate both absolute thresholds and difference
thresholds (JND) in an unbiased manner (Ulrich and Miller 2004).
Prior research shows that this test appears to be an especially sen-
sitive psychophysical tool (Macmillan and Creelman 1991), dis-
courages response biases, produces an especially high level of per-
formance and produces results that are stable and valid (Fausti and
others 1979; James and others 1997; Mojet and others 2001; Braun
and others 2004; Shepherd and others 2008; Ulrich and Vorberg
The objective of this study was to determine the absolute thresh-
old and JND in the sensory perception of chili pepper pungency
using 2AFC tests.
Materials and Methods
HPLC analysis of capsaicinoids
Dry Piquin chili pepper (Capsicum annuum L. var. aviculare)
found at local market was used in this study. High-performance
liquid chromatography (HPLC) was employed to determine its
capsaicin and dihydrocapsaicin content. These 2 capsaicinoids
were quantiﬁed as they are good predictors of the pungency
of chili peppers, constituting about 90% of the total capsaici-
noid content (Reilly and others 2001). Total capsaicinoid content
in this study was deﬁned as the sum of capsaicin and dihydro-
capsaicin found in the studied sample. The capsaicinoids extrac-
tion method used in this study was an adaptation of the method
reported by Cisneros-Pineda and others (2007). Methanol was
used instead of acetone (Kurian and Starks 2002; Ornelas-Paz and
others 2010). Solid chili pepper samples were blended to a ﬁne
powder and 0.6 g of powdered chili were mixed with 10 mL
of methanol HPLC grade and kept for 2 h at 4 ◦C. Samples
were then centrifuged for 5 min at 11000 ×gand the super-
natant was ﬁltered through a polyethylene membrane of 0.45 μg
of pore size previous to analysis by HPLC. Three replicates were
The following HPLC operating conditions, used by
Ornelas-Paz and others (2010), were employed. The HPLC set-
up consisted of a Varian (Model 9012) solvent delivery system and
a Varian (Model 9050) UV-VIS detector (set at a λ=236 nm).
A Supelcosil LC-C18 column (25 cm ×4.6 mm, 5 μm) was
used. The mobile phase consisted of acetonitrile/water/acetic acid
50:50:1 at a ﬂow rate of 1 mL/min. Injection volume was 20 μL,
run time 30 min, and temperature 25 ◦C.
Standards of capsaicin and dihydrocapsaicin (Sigma-Aldrich, St.
Louis, Mo., U.S.A.) were used to identify and quantify these cap-
saicinoids. HPLC grade methanol was used to prepare standard
curve solutions for capsaicin and dihydrocapsaicin. Standard curves
were prepared using concentrations of 5 to 1000 μg/mL. Two
replicates were conducted. A correlation coefﬁcient of 0.9960 was
achieved on the construction of the curves.
Determination of instrumental quantiﬁcation limit (LOQ)
Forty-ﬁve solutions containing different capsaicinoid concen-
trations were analyzed to obtain an instrumental quantiﬁcation
limit for this study. Determination of the signal-to-noise ratio
was performed by comparing measured signals from samples with
known low concentrations of the analyte and by establishing the
minimum concentration at which the analyte can be reliably quan-
tiﬁed, considering a limit signal-to-noise ratio of 20 to 1.
Sensory determination of pungency
Absolute threshold. Sensory tests were performed at a local
market in Cuauhtemoc City, Chihuahua, Mexico. Two hundred
and ﬁfty subjects (18–60 y old; female) volunteered to participate
in the study (50 tests per comparison level). There was no training
session and none of the subjects knew about the objective of the
study. Each subject participated only once in the study.
2AFC were used to determine the absolute threshold of pun-
gency’s sensory perception, which was deﬁned as the concentra-
tion corresponding to 76% correct responses. Paired samples con-
sisted of one pungent sample and a puriﬁed water sample. Pungent
samples were prepared as described in the ASTM method E1083-
00 (Standard Test Method for Sensory Evaluation of Red Pepper
Heat 2000) employing polysorbate-80 for capsaicinoids emulsiﬁ-
cation. Test samples were produced by serial dilutions, obtaining
concentrations of 0.039, 0.043, 0.050, 0.053, and 0.061 mg/kg
Two 10 mL samples (one containing plain water and one con-
taining the stimulus) were presented to each subject randomly in
plastic cups. Subjects swallowed the entire sample, waited 30 sec
and rinsed the palate using spring water. Subjects were asked to
choose the one item they thought was more pungent.
Just noticeable difference
JND tests were also performed at a local market in Cuauhtemoc
City, Chihuahua, Mexico. One thousand two hundred and ﬁfty
subjects (18–60 y old; female) volunteered to participate (50 tests
per comparison level, 5 reference solutions, 5 comparison solutions
per reference solution). There was no training session and none of
the subjects knew about the objective of the study. Each subject
participated only once in the study.
Reference solutions (0.05, 0.11, 0.13, 0.17, and 0.23 mg/kg
total capsaicinoids) were used to obtain different values of JND.
2AFC tests were used to determine JND. In every case, the JND
was deﬁned as the concentration difference where 76% correct
responses were found. Two samples (one containing the reference
solution and another containing a solution of a higher capsai-
cinoids concentration) of 10 mL each were presented to each
judge (5 comparison solutions of higher pungency were used for
comparison against each reference solution). Subjects waited 30
sec between samples and rinsed palate using spring water. Sub-
jects were asked to choose the one item they thought was more
Weber fraction determination
Weber’s law states that the just noticeable difference between
2 stimuli is a ﬁxed proportion of the value of the stimuli being
judged (reference; Goldstein 1980). JND values obtained in this
study were employed to calculate the Weber fraction for pungency
perception. Weber fraction (k) was calculated using
S136 Journal of Food Science rVol. 77, Nr. 3, 2012
S: Sensory & Food
Pungency sensory perception . . .
where Iis the increase in the physical stimulus needed to be
judged different (JND) and Iis the starting level of the stimulus
(reference solution concentration) (Lawless and Heymann 1999).
Data were analyzed by a χ2goodness-of-ﬁt test. Sensory thresh-
old data were analyzed by calculating the correlation between the
proportion of correct responses and total capsaicinoids content
(mg/kg) using Microsoft Excel 2007.
Results and Discussion
The total capsaicinoids content in Piquin chili pepper was
3.6 mg/g (dry matter). Results for the sensory determination of
the absolute threshold are shown in Figure 1. A linear correlation
between the proportion of correct responses and the concentra-
tion of the pungent sample was found. The R2value indicated that
more than 99% of the variation in the proportion of correct re-
sponses can be explained by the variation in capsaicinoids concen-
tration. The proportion of correct responses (n=50) was obtained
for each 1 of 5 studied concentrations. No evidence of carryover
effects was observed as no statistically signiﬁcant preference for
the ﬁrst or second tasted sample was found. Correct responses
increased as capsaicinoid concentration content increased.
The absolute threshold and JND are usually deﬁned as the
stimulus intensity at which the proportion of correct responses
is 76% (Klein 2001). Concentration of stimuli for absolute thresh-
old at 76% correct responses was 0.05 mg/kg total capsaicinoids,
which indicates that pungency is perceptible by human subjects
at that concentration and above. Krajewska and Powers (1988)
found similar results using the method of magnitude estimation.
They reported absolute threshold within a range of 0.039 to
0.078 mg/kg total capsaicinoids using ethanol for capsaicinoids
extraction/emulsiﬁcation. It is interesting to ﬁnd coincidence be-
tween the absolute threshold value found by Krajewska and Powers
Figure 1–Linear response observed between the proportion of correct
responses and the concentration of capsaicinoids in aqueous solutions
compared against pure water in 2AFC paired tests.
(1988) and the one found in this study, because it would be reason-
able to believe that Mexican judges participating in this study, who
consume hot peppers on a regular basis (around 8 kg per year per
capita on average), would present a higher threshold for pungency
perception than American judges due to desensitization caused by
continuous periodic exposure to the stimulus (Lawless and others
1985). Furthermore, more recent studies conducted with Ameri-
can judges (Lawless and others 2000) have found higher threshold
values (0.31 ±0.03 mg/L) when using the method of limits,
both when using ethanol and polysorbate-80 for capsaicinoids ex-
traction/emulsiﬁcation. Lawless and others (2000) compared the
efﬁciency of using ethanol and polysorbate-80 for capsaicinoids
extraction/emulsiﬁcation, but documented that the comparison
did not show clear effects, and no differences were observed.
In comparison with other types of stimuli commonly present
in foodstuffs, it may be said that the human senses are especially
sensitive to capsaicinoids, as absolute threshold values reported in
literature for aqueous solutions of other products are much higher
(that is, sweet: 2% sucrose, sour: 0.07% citric acid, salty: 0.2%
sodium chloride, and bitter: 0.07% caffeine; ASTM 1981).
Deﬁning absolute threshold as the stimulus concentration where
the outcome of 76% of the conducted 2AFC tests is correct is
not the only way to deﬁne absolute threshold values. Exper-
imental data derived from 2AFC tests may also be evaluated
by a χ2goodness-of-ﬁt analysis. Threshold values may be es-
timated by testing against the null hypothesis stating that the
observed proportion of correct responses in the sample corre-
sponds to a sample taken from a population with a 50/50 pro-
portion of correct responses (chance performance in the case
of 2AFC tests). Following this strategy, threshold values may be
estimated with various degrees of conﬁdence, as illustrated in
Table 1. Absolute threshold values derived from this study
(Table 1) may be located in the range 0.043 to 0.50 mg/kg to-
tal capsaicinoids with varying degrees of conﬁdence from 93% to
99.96%, the latter being the conﬁdence level corresponding to the
76% criterion, which may be considered by statistical standards as
Results for JND and Weber fraction are shown in Table 2.
JND values indicate the amount of additional stimulus required to
Table 2– Reference solution values, Weber fraction, and just
noticeable difference concentration values.
concentration for Just noticeable
reference solutions Weber difference (I)
(I) (mg/kg) fraction(k)(mg/Kg)
0.05 0.60 0.030
0.11 0.62 0.068
0.13 0.59 0.077
0.17 0.64 0.108
0.23 0.76 0.174
Table 1–χ2goodness-of-ﬁt test for absolute threshold values corresponding to different signiﬁcance levels.
Number of trials Correct responses Proportion C/I χ2Signiﬁcance level (α) Threshold (mg/kg)
50 32 64/36 3.38 0.0700 0.043
50 33 66/34 4.50.0300 0.044
50 34 68/32 5.78 0.0200 0.045
50 35 70/30 7.22 0.0070 0.046
50 36 72/28 8.82 0.0029 0.048
50 37 74/26 10.58 0.0011 0.049
50 38 76/24 12.50 0.0004a0.050a
C/I, proportion of correct/incorrect responses.
aStatistical signiﬁcance and absolute threshold value corresponding to the absolute threshold value criterion of 76% correct responses.
Vol. 77, Nr. 3, 2012 rJournal of Food Science S137
S: Sensory & Food
Pungency sensory perception . . .
Figure 2–Linear response observed between the
proportion of correct responses and the
differential concentration of capsaicinoids in
aqueous solutions compared against reference
aqueous solutions of 5 different concentrations.
Just noticeable difference values (JND).
perceive a difference between 2 samples, both containing the
stimulus. JND results were congruent with Weber’s law. Calcu-
lated Weber fraction values for suprathreshold scaling functions
(0.59–0.64) were similar among tests conducted with reference
solution ranging from 0.05 to 0.17 mg/kg total capsaicinoids.
However, the reference solution with the highest capsaicinoids
concentration (0.23 mg/kg) showed a different Weber fraction
(0.76). This abnormality may be attributed to the nature of the
stimulus produced by capsaicinoids, which easily implies fatigue
when the aftertaste of the ﬁrst tasted sample interferes as “noise”
with the tasting of a subsequent sample, together with the de-
sensitization produced after capsaicin intake (Stevens and Lawless
1987). Another possible documented explanation for the exis-
tence of deviations from Weber’s law is derived from experimental
evidence that has proven that Weber’s law holds true for most
senses as long as the stimulus intensity is not too far to the thresh-
old (Goldstein 1980). This information suggests that an aqueous
capsaicinoids solution with a concentration of 0.23 mg/kg capsai-
cinoids is probably far enough from the threshold concentration as
to cause deviations from Weber’s law. Finally, in Figure 2 absolute
threshold and JND graphics are shown. Linear responses for every
test conducted with different reference solutions, show a similar
The quantiﬁcation limit of the conducted instrumental analysis
was 0.352 mg/kg for capsaicin and 1.160 mg/kg for dihydrocap-
saicin. Total capsaicinoids quantiﬁcation limit was 1.512 mg/kg.
Quantiﬁcation limits previously reported in literature establish val-
ues of 13 mg/kg with UV detection and 4 mg/kg (Sein and
others 1998) and 5 mg/kg (Chiang 1986) employing a ﬂuores-
cence method. These results are similar in order of magnitude,
to the instrumental values obtained in this study. On the other
hand, Chiang (1986) developed an HPLC-ECD and UV method,
which results in a detection limit as low as 0.06 mg/kg which is
closer to the sensory threshold values found in this study. The re-
sults of this study demonstrate that sensory quantiﬁcation methods
are more sensitive than instrumental methods in the quantiﬁcation
of capsaicinoids. Prior studies conducted on the comparison of
sensory methods compared with instrumental methods, including
taste, odor and texture, have already reported that sensory meth-
ods are more sensitive than instrumental methods (Min 1983; Lu-
cisano and others 1989; Rousset and others 1995; McIlveen and
Armstrong 1996; Bae and others 2002; Prakash and others 2005).
In view of this, an increasing amount of attention is being paid
to correlating instrumental and sensory methods. In fact there is
a desire to interpret the instrumental measurements in terms of
sensory principles (Todd and others 1977; Hoffman and others
1983; Parrish 1996). Although improvements on instrumentation
contribute to the production of better measurements and to low-
ering quantiﬁcation limits, the most important point in the use
of sensory analysis is that only human beings can perceive, ana-
lyze, integrate, and interpret the entire spectrum of characteristics
in one evaluation (Kramer and Szczesniak 1973). Flavor research
has shown that instrumental analysis does not totally reﬂect the
sensory proﬁle of a food product and sensory evaluation gives a
realistic opinion about the likes and dislikes of a particular ﬂavor,
so it is recommended to obtain a blend of both instrumental and
sensory techniques (Hariom and others 2006). Sensory methods
determine the perception of foods through the human senses and
this can only be achieved with one instrument: the human being.
The absolute threshold for the sensory perception of pungency
was 0.050 mg/kg capsaicinoids and instrumental quantiﬁcation
limit for instrumental analysis was 1.512 mg/kg capsaicionids,
which makes sensory methods more sensitive. JND changed pro-
portionally while intensity of the reference sample increased. We-
ber fraction results were similar (0.59–0.64) for the ﬁrst 4 reference
concentrations studied, and slightly higher for the highest studied
reference concentration (0.76). The use of an aqueous vehicle in
this study may represent a limitation when extrapolating its ﬁnd-
ings to a different food system, as it is well known that presence of
lipidic substances modiﬁes the perception of pungency affecting
perception threshold values.
The authors thank Liliana Nunez, Nabil Carmona, Veronica
Gonzalez, and Angel Esparza for their technical assistance and
Consejo Nacional de Ciencia y Tecnologia (CONACYT) for
providing the funding for the graduate studies of author Orellana-
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