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Determination of Absolute Threshold and Just Noticeable Difference in the

Sensory Perception of Pungency

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DOI: 10.1111/j.1750-3841.2011.02589.x · Source: PubMed

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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.

S: Sensory & Food

Introduction such as salsas and cheese sauces (Meilgaard and others 1991). One

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
1971).
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 Sepúlveda 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 Jiménez-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: dsepulveda@ciad.mx).
Quality
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 Harris 1985; Carden
and others 1999; Emrick and others 2005; Reinbach and others
2009).
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


C 2012 Institute of Food Technologists
R

doi: 10.1111/j.1750-3841.2011.02589.x Vol. 77, Nr. 3, 2012 r Journal of Food Science S135
Further reproduction without permission is prohibited
 Pungency sensory perception . . .

threshold have been obtained using different sensory methods such Determination of instrumental quantification limit (LOQ)
as magnitude estimation and the method of limits (Krajewska and Forty-five solutions containing different capsaicinoid concen-
Powers 1988; Lawless and others 2000). These are good methods trations were analyzed to obtain an instrumental quantification
but may present some limitations. Magnitude estimation meth- limit for this study. Determination of the signal-to-noise ratio
ods produce results having ratio problems such as judges having was performed by comparing measured signals from samples with
a strong favored-number effect (O’Mahony 1982). It is also said known low concentrations of the analyte and by establishing the
that magnitude methods are incapable of providing stable and re- minimum concentration at which the analyte can be reliably quan-
producible values for flavor intensity (Meilgaard and others 1991). tified, considering a limit signal-to-noise ratio of 20 to 1.
Similarly, the method of limits is slightly biased and can be very
biased if subjects falling on the upper or lower limits of the rangeSensory determination of pungency
under test are not reexamined (Meilgaard and others 1991). On Absolute threshold. Sensory tests were performed at a local
the other hand, 2 alternative forced-choice sensory tests (2AFC) market in Cuauhtemoc City, Chihuahua, Mexico. Two hundred
may be used to estimate both absolute thresholds and difference and fifty subjects (18–60 y old; female) volunteered to participate
thresholds (JND) in an unbiased manner (Ulrich and Miller 2004). in the study (50 tests per comparison level). There was no training
Prior research shows that this test appears to be an especially sen-session and none of the subjects knew about the objective of the
sitive psychophysical tool (Macmillan and Creelman 1991), dis- study. Each subject participated only once in the study.
courages response biases, produces an especially high level of per- 2AFC were used to determine the absolute threshold of pun-
formance and produces results that are stable and valid (Fausti and gency’s sensory perception, which was defined as the concentra-
others 1979; James and others 1997; Mojet and others 2001; Braun tion corresponding to 76% correct responses. Paired samples con-
and others 2004; Shepherd and others 2008; Ulrich and Vorberg sisted of one pungent sample and a purified water sample. Pungent
2009). samples were prepared as described in the ASTM method E1083-
The objective of this study was to determine the absolute thresh-00 (Standard Test Method for Sensory Evaluation of Red Pepper
old and JND in the sensory perception of chili pepper pungency Heat 2000) employing polysorbate-80 for capsaicinoids emulsifi-
using 2AFC tests. cation. Test samples were produced by serial dilutions, obtaining
concentrations of 0.039, 0.043, 0.050, 0.053, and 0.061 mg/kg
Materials and Methods total capsaicinoids.
Two 10 mL samples (one containing plain water and one con-
HPLC analysis of capsaicinoids taining the stimulus) were presented to each subject randomly in
Dry Piquin chili pepper (Capsicum annuum L. var. aviculare) plastic cups. Subjects swallowed the entire sample, waited 30 sec
found at local market was used in this study. High-performance and rinsed the palate using spring water. Subjects were asked to
liquid chromatography (HPLC) was employed to determine its choose the one item they thought was more pungent.
capsaicin and dihydrocapsaicin content. These 2 capsaicinoids
were quantified as they are good predictors of the pungency Just noticeable difference
of chili peppers, constituting about 90% of the total capsaici- JND tests were also performed at a local market in Cuauhtemoc
S: Sensory & Food

noid content (Reilly and others 2001). Total capsaicinoid content City, Chihuahua, Mexico. One thousand two hundred and fifty
in this study was defined as the sum of capsaicin and dihydro- subjects (18–60 y old; female) volunteered to participate (50 tests
Quality

capsaicin found in the studied sample. The capsaicinoids extrac- per comparison level, 5 reference solutions, 5 comparison solutions
tion method used in this study was an adaptation of the method per reference solution). There was no training session and none of
reported by Cisneros-Pineda and others (2007). Methanol was the subjects knew about the objective of the study. Each subject
used instead of acetone (Kurian and Starks 2002; Ornelas-Paz and participated only once in the study.
others 2010). Solid chili pepper samples were blended to a fine Reference solutions (0.05, 0.11, 0.13, 0.17, and 0.23 mg/kg
powder and 0.6 g of powdered chili were mixed with 10 mL total capsaicinoids) were used to obtain different values of JND.
of methanol HPLC grade and kept for 2 h at 4 ◦ C. Samples 2AFC tests were used to determine JND. In every case, the JND
were then centrifuged for 5 min at 11000 × g and the super- was defined as the concentration difference where 76% correct
natant was filtered through a polyethylene membrane of 0.45 μg responses were found. Two samples (one containing the reference
of pore size previous to analysis by HPLC. Three replicates were solution and another containing a solution of a higher capsai-
conducted. cinoids concentration) of 10 mL each were presented to each
The following HPLC operating conditions, used by judge (5 comparison solutions of higher pungency were used for
Ornelas-Paz and others (2010), were employed. The HPLC set- comparison against each reference solution). Subjects waited 30
up consisted of a Varian (Model 9012) solvent delivery system and sec between samples and rinsed palate using spring water. Sub-
a Varian (Model 9050) UV-VIS detector (set at a λ = 236 nm). jects were asked to choose the one item they thought was more
A Supelcosil LC-C18 column (25 cm × 4.6 mm, 5 μm) was pungent.
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, Weber fraction determination
run time 30 min, and temperature 25 ◦ C. Weber’s law states that the just noticeable difference between
Standards of capsaicin and dihydrocapsaicin (Sigma-Aldrich, St. 2 stimuli is a fixed proportion of the value of the stimuli being
Louis, Mo., U.S.A.) were used to identify and quantify these cap- judged (reference; Goldstein 1980). JND values obtained in this
saicinoids. HPLC grade methanol was used to prepare standard study were employed to calculate the Weber fraction for pungency
curve solutions for capsaicin and dihydrocapsaicin. Standard curves perception. Weber fraction (k) was calculated using
were prepared using concentrations of 5 to 1000 μg/mL. Two
replicates were conducted. A correlation coefficient of 0.9960 was I
k=
achieved on the construction of the curves. I

S136 Journal of Food Science r Vol. 77, Nr. 3, 2012

Pungency sensory perception . . .

where I is the increase in the physical stimulus needed to be (1988) and the one found in this study, because it would be reason-
judged different (JND) and I is the starting level of the stimulus able to believe that Mexican judges participating in this study, who
(reference solution concentration) (Lawless and Heymann 1999). consume hot peppers on a regular basis (around 8 kg per year per
capita on average), would present a higher threshold for pungency
Statistical analysis perception than American judges due to desensitization caused by
Data were analyzed by a χ 2 goodness-of-fit test. Sensory thresh- continuous periodic exposure to the stimulus (Lawless and others
old data were analyzed by calculating the correlation between the 1985). Furthermore, more recent studies conducted with Ameri-
proportion of correct responses and total capsaicinoids content can judges (Lawless and others 2000) have found higher threshold
(mg/kg) using Microsoft Excel 2007. values (0.31 ± 0.03 mg/L) when using the method of limits,
both when using ethanol and polysorbate-80 for capsaicinoids ex-
Results and Discussion traction/emulsification. Lawless and others (2000) compared the
The total capsaicinoids content in Piquin chili pepper was efficiency of using ethanol and polysorbate-80 for capsaicinoids
3.6 mg/g (dry matter). Results for the sensory determination of extraction/emulsification, but documented that the comparison
the absolute threshold are shown in Figure 1. A linear correlation did not show clear effects, and no differences were observed.
between the proportion of correct responses and the concentra- In comparison with other types of stimuli commonly present
tion of the pungent sample was found. The R2 value indicated that in foodstuffs, it may be said that the human senses are especially
more than 99% of the variation in the proportion of correct re- sensitive to capsaicinoids, as absolute threshold values reported in
sponses can be explained by the variation in capsaicinoids concen- literature for aqueous solutions of other products are much higher
tration. The proportion of correct responses (n = 50) was obtained (that is, sweet: 2% sucrose, sour: 0.07% citric acid, salty: 0.2%
for each 1 of 5 studied concentrations. No evidence of carryover sodium chloride, and bitter: 0.07% caffeine; ASTM 1981).
effects was observed as no statistically significant preference for Defining absolute threshold as the stimulus concentration where
the first or second tasted sample was found. Correct responses the outcome of 76% of the conducted 2AFC tests is correct is
increased as capsaicinoid concentration content increased. not the only way to define absolute threshold values. Exper-
The absolute threshold and JND are usually defined as the imental data derived from 2AFC tests may also be evaluated
stimulus intensity at which the proportion of correct responses by a χ 2 goodness-of-fit analysis. Threshold values may be es-
is 76% (Klein 2001). Concentration of stimuli for absolute thresh- timated by testing against the null hypothesis stating that the
old at 76% correct responses was 0.05 mg/kg total capsaicinoids, observed proportion of correct responses in the sample corre-
which indicates that pungency is perceptible by human subjects sponds to a sample taken from a population with a 50/50 pro-
at that concentration and above. Krajewska and Powers (1988) portion of correct responses (chance performance in the case
found similar results using the method of magnitude estimation. of 2AFC tests). Following this strategy, threshold values may be
They reported absolute threshold within a range of 0.039 to estimated with various degrees of confidence, as illustrated in
0.078 mg/kg total capsaicinoids using ethanol for capsaicinoids Table 1. Absolute threshold values derived from this study
extraction/emulsification. It is interesting to find coincidence be- (Table 1) may be located in the range 0.043 to 0.50 mg/kg to-
tween the absolute threshold value found by Krajewska and Powers tal capsaicinoids with varying degrees of confidence from 93% to

S: Sensory & Food

99.96%, the latter being the confidence level corresponding to the
76% criterion, which may be considered by statistical standards as

Quality
overconservative.
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.
Total capsaicinoid
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
Figure 1–Linear response observed between the proportion of correct 0.17 0.64 0.108
responses and the concentration of capsaicinoids in aqueous solutions 0.23 0.76 0.174
compared against pure water in 2AFC paired tests.

Table 1–χ 2 goodness-of-fit test for absolute threshold values corresponding to different significance levels.
Number of trials Correct responses Proportion C/I χ2 Significance level (α) Threshold (mg/kg)
50 32 64/36 3.38 0.0700 0.043
50 33 66/34 4.5 0.0300 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 0.0004a 0.050a
C/I, proportion of correct/incorrect responses.
a
Statistical significance and absolute threshold value corresponding to the absolute threshold value criterion of 76% correct responses.

Vol. 77, Nr. 3, 2012 r Journal of Food Science S137

 Pungency sensory perception . . .
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
S: Sensory & Food
capsaicinoids solution with a concentration of 0.23 mg/kg capsai-
cinoids is probably far enough from the threshold concentration as
Quality
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
tendency.
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
S138 Journal of Food Science r Vol. 77, Nr. 3, 2012
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).
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.
Conclusions
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.
Acknowledgments
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-
Escobedo.
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