Instrumental and Sensory Characterization of Heat-Induced
Odorants in Aseptically Packaged Soy Milk
PATRICIO R. LOZANO,†MARYANNE DRAKE,§DANIEL BENITEZ,#AND
KEITH R. CADWALLADER*,†
Department of Food Science and Human Nutrition, University of Illinois, 1302 West Pennsylvania
Avenue, Urbana, Illinois 61801; Department of Food Science, North Carolina State University,
Raleigh, North Carolina 27695; and Cargill, Inc., 2525 Ponce de Leon Boulevard, Suite 800,
Coral Gables, Florida 33134
Predominant heat-induced odorants generated in soy milk by ultrahigh-temperature (UHT) processing
were evaluated by sensory and instrumental techniques. Soy milks processed by UHT (143 °C/14 s,
143 °C/59 s, 154 °C/29 s) were compared to a control soy milk (90 °C/10 min) after 0, 1, and 7 days
of storage (4.4 ( 1 °C). Dynamic headspace dilution analysis (DHDA) and solvent-assisted flavor
evaporation (SAFE) in conjunction with GC-olfactometry (GCO)/aroma extract dilution techniques
and GC-MS were used to identify and quantify major aroma-active compounds. Sensory results
revealed that intensities of overall aroma and sulfur and sweet aromatic flavors were affected by the
processing conditions. Odorants mainly responsible for the changes in sulfur perception were
methional, methanethiol, and dimethyl sulfide. Increases in 2-acetyl-1-pyrroline, 2-acetyl-thiazole, and
2-acetyl-2-thiazoline intensities were associated with roasted aromas. A marginal increase in intensity
of sweet aromatic flavor could be explained by increases in 2,3-butanedione, 3-hydroxy-2-butanone,
?-damascenone, and 2- and 3-methylbutanal. Predominant lipid-derived odorants, including (E,E)-
2,4-nonadienal, (E,E)-2,4-decadienal, (E,Z)-2,4-decadienal, (E)-2-nonenal, (E)-2-octenal, 1-octen-3-
one, 1-octen-3-ol, and (E,Z)-2,6-nonadienal, were affected by processing conditions. Intensities of
overall aroma and sulfur notes in soy milk decreased during storage, whereas other sensory attributes
did not change. Color changes, evaluated by using a Chroma-meter, indicated all UHT heating
conditions used in this study generated a more yellow and saturated color in soy milk in comparison
to the control soy milk.
KEYWORDS: Thermal processing; cooked off-flavors; soy milk; UHT; storage effect; color
Soy milk, a water extract of soybeans, is an excellent source
of protein and essential fatty acids. It is cholesterol-free and
relatively cheap in comparison with other sources of protein.
Despite the beneficial attributes of soy milk, its consumption
in the Western world has been limited due to its unacceptable
beany flavor (1). A considerable number of studies have been
conducted to determine the volatile compounds responsible for
the beany off-flavor in soy milk. As a result, in the past decade
novel processing technologies have been developed to reduce
beany off-flavors, obtain better yields, eliminate antinutritional
factors, and extend the shelf life of soy milk. High temperature-
short time (HTST) and ultrahigh-temperature (UHT) processing
methods have been a crucial part of this development (1, 2).
The use of high-temperature processing combined with aseptic
packaging has opened new markets and avenues for the
distribution of soy milk (2). Unfortunately, color changes, loss
of nutrients, and creation of “cooked off-flavors” have been
encountered in soy milks produced under these conditions. A
viable solution to this problem has been difficult to address
because little information exists about the chemical reactions
that occur at these temperatures and also due to the uncertainty
of the effect of storage on these heat-induced aroma compounds.
The aim of this project was to characterize the major aroma-
active components generated under UHT conditions, to assess
their impact on the overall soy milk aroma and flavor, and to
determine the effect of short-term storage on key heat-induced
MATERIALS AND METHODS
Chemicals. Analytical grade authentic compounds were obtained
from Aldrich Chemical Co. (St. Louis, MO) except for 2-acetyl-1-
pyrroline, which was a gift from Dr. R. Buttery (USDA, ARS, WRRC,
Albany, CA); ?-damascenone, which was provided by Firmenich Co.
(Princeton, NJ); and δ-octalactone and γ-nonalactone, which were
* Corresponding author [telephone (217) 333-5803; fax (217) 333-1875;
†University of Illinois.
§North Carolina State University.
purchased from Bedoukian Research (Danbury, CT). (E,E)-3,5-Octa-
dien-2-one was synthesized on the basis of a previously published
procedure (3). (Z)-2-Nonenal was synthesized from (Z)-2-nonen-1-ol
(Bedoukian Research Inc.) by oxidation with Dess-Martin periodinane
(0.3 M in dichloromethane; Aldrich Chemical Co.) following the
procedure described by Meyer and Schreiber (4). Odorless distilled
water was prepared by boiling glass-distilled water in an open flask
until its volume was reduced by one-third of the original volume.
Soy Milk Production. The soybean variety selected for this
experiment was VINTON 81 because of its high protein yield (5). Fresh
whole beans from the 2004 harvest were obtained from the Illinois
Center for Soy Foods at the University of Illinois (Urbana, IL).
A standardized soy milk procedure was adopted to consistently
provide a homogeneous product for volatile analysis by eliminating
possible sources of variation during its manufacturing. This procedure
was a combination of two previously published methods (6, 7). It
consisted of a predrying step at 93.3 °C for 10 min applied to the beans
solely to weaken the soybean hulls and minimize the damage of the
rotating drum when the hulls were separated from the cotyledons. After
dehulling, the beans were soaked with odorless distilled water in a
proportion of 1:5 w/w (water to wet soybeans), and the mixture was
held at 4 ( 1 °C for 14 h. The soaked beans were washed with cold
distilled water for 1 min, and the water was discarded. A double “hot-
grinding” step using a 1:7 w/w (odorless distilled water to dry beans)
ratio followed, using a bean disintegrator BMI 300 (Beam Machines
Inc., San Francisco, CA) equipped with a stainless steel mesh screen
no. 40. This step was to create an emulsion and limit lipoxygenase
activity by keeping the temperature at about 80 °C (8). The obtained
slurry was later filtered using a vibration screen separator KM-1-SS
(Kason, Montreal, Canada) with a no. 200 stainless steel mesh screen
to separate the soy milk from the okara.
Heat Treatment. Three heat combinations in the UHT range (143
°C/14 s, 143 °C/59 s, and 154 °C/29 s) and one control in the HTST
range (90 °C/10 min) were selected for this experiment. All treatments
were replicated twice using two different batches of fresh (same day)
soy milk each time. The heating conditions chosen for control (90 °C/
10 min) followed the procedure described by Feng and Acree (9) to
reduce solely the microbial contamination of aerobically processed soy
milk and make it safe for the sensory panelists. The mildest UHT
treatment was chosen to mimic the extreme conditions applied for
sterilizing cow’s milk (143 °C/14 s) (10), whereas the other two UHT
treatments were selected on the basis of experimental (11) and
mathematical modeling data (12) that provide the appropriate holding
time and processing temperature combinations at the UHT range (121-
154 °C) to destroy 90% of the soy milk trypsin inhibitor (TI). This
extent of heat inactivation has been reported to provide adequate TI
destruction where the maximum nutritive value or protein efficiency
in soy milk is acquired (13).
Equipment. A miniature (0.8-1.4 L/min) HTST/UHT processing
system (Microthermics, Raleigh, NC) using a direct steam injection
system, a laminar flow high-efficiency particulate air (HEPA) hood,
and an automatic fill control to simulate aseptic filling was used to
process the soy milk. The system has two stages of heating; an initial
preheating stage to increase the temperature of the soy milk from 4 to
121 °C and a second stage to bring the soy milk to the UHT
temperatures described above. The system was cleaned and sterilized
with an odorless detergent and steam at 125 °C for 20 min, respectively.
One liter glass Pyrex bottles, equipped with Teflon-lined lids and
previously autoclaved (125 °C/20 min) and tested for residual odors,
were used to store the soy milk under aseptic conditions. Samples were
stored at refrigeration temperature (4.4 ( 1 °C) after being processed.
Soy milk samples were analyzed at 0, 1, and 7 days after processing
by sensory and analytical techniques.
Sensory Evaluation of Soy Milk. Eight panelists (females, 45-55
years of age) trained in the Spectrum method of descriptive analysis
for the generation of qualitative and quantitative data evaluated the
products (14). Each panelist had greater than 500 h of previous
experience in the sensory evaluation of food products including previous
experience with fluid milk, soy milk, and soy protein concentrates and
isolates. Panelists received an additional 6 h of training (three 2-h
sessions) focused on soy milk. During the focused training, panelists
evaluated and discussed an array of commercial and experimentally
produced soy milks. Specific attributes, attribute definitions, and
references were developed by the panelists (Table 1). Panelists used
the 15-point universal Spectrum intensity scale (14). Analysis of
variance of data collected from the last part of training indicated that
the panel and panelists could consistently use the attributes to
differentiate the products.
Samples were removed from refrigeration approximately 1 h prior
to evaluation and shaken well to distribute any sediment. Approximately
40 mL was dispensed into 2-oz portion cups labeled with three-digit
random codes and covered. Panelists independently evaluated the
orthonasal aroma and then the flavor (retronasal aromatics and basic
tastes) and viscosity of the samples in a randomized balanced order.
References were available for calibration during evaluation. Distilled/
deionized water and soda crackers were provided for palate cleansing.
Panelists rested 3 min between individual samples followed by a 15
min rest period between each replication. Samples were analyzed in
triplicate by each panelist.
Color Changes. Soy milk changes in coloration were registered by
using a Chroma-meter (Cr 400, Konica Minolta). This instrument
measured the color changes by exposing the soy milk to various
wavelengths and expressing the differences in factors of L (lightness),
a (redness-greenness), and b (yellowness and blueness). For better
understanding of the color change relationship, lightness (L), chroma
(?a2+b2), and hue [tan-1(b/a)] values were calculated.
Instrumental Evaluation of Soy Milk Aroma. Heat-induced
odorants created in soy milk required more than one technique to be
properly isolated. This is because highly volatile components of soy
Table 1. Soy Milk Flavor Lexicon Employed in Descriptive Analysis
Aroma (Evaluated Orthonasally)
overall aroma intensityoverall aroma intensity of the product
Flavors (Evaluated When Product Is Placed in the Mouth)
sweet aromatic associated with cooked oatmeal (retronasally)
sharp aromatic associated with cooked eggs and cooked
aromatics associated with grain
aromatics associated with flour paste
aromatics associated with metal or rare beef juices
mouthfeel sensation of drying, drawing, or puckering of
the tongue or oral cavity
basic taste associated with sucrose
basic taste associated with acid
basic taste associated with bitter compounds
force required to move a spoon back and forth in the sample
degree to which the sample has very fine particles
Quaker oatmeal, 50 g in 500 mL of water
hard-boiled mashed egg, steamed or boiled
broccoli or cabbage
Cheerios, 50 g in 500 mL of water
flour, 60 g in 500 mL of water
1-octen-3-one, 20 ppm in ethanol
1% alum in water or black tea (soak six bags in
500 mL of water for 10 min)
5% sucrose in water
0.08% citric acid in water
0.08% caffeine in water
water ) 1, cream ) 3
chew raw potato cube
aReference taken from Meilgaard et al. (14).
(10) Clare, D. A.; Bang, W. S.; Cartwright, G.; Drake, M. A.; Coronel,
P.; Simunovic, J. Comparison of sensory, microbiological, and
biochemical parameters of microwave versus indirect UHT fluid
skim milk during storage. J. Dairy Sci. 2005, 88, 4172-4182.
(11) Kwok, K. C.; Qin, W. H.; Tsang, J. C. Heat inactivation of trypsin
inhibitors in soymilk at ultra high-temperatures. J. Food Sci.
1993, 58, 859-862.
(12) Kwok, K. C.; Liang, H. H.; Niranjan, K. Mathematical modeling
of the heat inactivation of trypsin inhibitors in soymilk at 121-
154 °C. J. Sci. Food Agric. 2002, 82, 243-247.
(13) Hackler, L. R.; Van Buren, J. P.; Streinkraus, K. H.; El Rawi,
I.; Hand, D. B. Effect of heat treatment on nutritive value of
soymilk protein fed to weanlings rats. J. Food Sci. 1965, 30,
(14) Meilgaard, M.; Civille, G. V.; Carr, B. T. Descriptive analysis
techniques. In Sensory EValuation Techniques, 3rd ed.; CRC
Press: Boca Raton, FL, 1999; pp 187-200.
(15) Cadwallader, K. R.; Baek, H. H. Aroma-impact components in
cooked tail meat freshwater crayfish (Promacambarus clarkii).
In Food FlaVors; Formation, Analysis and Packaging Influences;
Mussinan, C., Contis, E., Ho, C. T., Parliment, T., Spanier, A.,
Shahidi, F., Eds.; Elsevier Science: Amsterdam, The Nether-
lands, 1998; pp 271-278.
(16) Van den Dool, H.; Kratz, P. D. A generalization of the retention
index system including linear temperature programmed gas-
liquid partition chromatography. J. Chromatogr. 1963, 11, 463-
(17) Engel, W. B.; Schieberle, P. Solvent assisted flavour evaporations
a new and versatile technique for the careful and direct isolation
of aroma compounds from complex food matrices. Eur. Food
Res. Technol. 1999, 209, 237-241.
(18) Grosch, W. Detection of potent odorants in foods by aroma
extract dilution analysis. Trends Food Sci. Technol. 1993, 4, 68-
(19) Zhou, Q.; Wintersteen, C. L.; Cadwallader, K. R. Identification
and quantification of aroma-active components that contribute
to the distinct malty flavor of buckwheat honey. J. Agric. Food
Chem. 2002, 50, 2016-2021.
(20) Colahan-Sederstrom, P.; Peterson, G. D. Inhibition of key aroma
compound generated during ultrahigh-temperature processing of
bovine milk via epicatechin addition. J. Agric. Food Chem. 2005,
(21) Benitez, D. Effect of presoak treatment (blanching vs. cold
soaking) and sodium bicarbonate addition during processing on
volatile composition of soymilk. M.Sc. Thesis, University of
Illinois, Urbana, IL, 2003.
(22) Kobayashi, A.; Yohko, T.; Hirata, N.; Kubota, K.; Kitamura,
K. Aroma constituents of soybean milk lacking lipoxygenase
isozymes. J. Agric. Food Chem. 1995, 43, 2449-2452.
(23) Wang, A. H.; Dou, J.; Macura, D.; Durance, T. D.; Nakai, S.
Solid phase extraction for GC analysis of beany flavours in
soymilk. Food Res. Int. 1998, 30, 503-511.
(24) Min, S.; Yu, Y.; Yoo, S.; Martin, S.S. Effect of soybean varieties
and growing locations on the flavor of soymilk. J. Food Sci.
2005, 70, C1-C7.
(25) Wilkens, W.; Lin, F. W. Gas chromatography and mass spectral
analyses of soybean milk volatiles. J. Agric. Food Chem. 1970,
(26) Fujimaki, M.; Arai, S.; Kiriyaga, N. Studies of flavor compounds
in soybean. Part I. Aliphatic carbonyl compounds. Agric. Biol.
Chem. 1965, 29, 855-858.
(27) Macleod, G.; Ames, J. Soy flavor and its improvement. Crit.
ReV. Food Sci. Hum. Nutr. 1988, 27, 219-400.
(28) Guadagni, D. G.; Buttery, R. G.; Turnbaugh, J. G. Odour
thresholds and similarity ratings of some potato chips com-
pounds. J. Sci. Food Agric. 1972, 23, 1435-1444.
(29) Matoba, T.; Hidaka, H.; Narita, H.; Kitamura, K.; Kaizuma, N.;
Kito, M. Lipoxygenase-2 isozyme is responsible for generation
of n-hexanal in soybean homogenate. J. Agric. Food Chem. 1985,
(30) Frankel, E. N.; Neff, W. E. Analysis of autoxidized fats by gas
chromatography-mass spectrometry. Lipids 1981, 16, 279-283.
(31) Badenhop, A. F.; Wilkens, W. F. The formation of 1-octen-3-ol
in soybeans during soaking. J. Am. Oil Chem. Soc. 1969, 46,
(32) Kato, H.; Doi, Y.; Tsugita, T.; Kosai, K.; Kamiya, T.; Kurata,
T. Changes in the volatile flavor components of soybeans during
roasting. Food Chem. 1981, 7, 87-94.
(33) Coghe, S.; Beenot, K.; Delvaux, F.; Vanderhaegen, B.; Delvaux,
F. Ferulic acid and 4-vinylguaiacol formation during brewing
and fermentation: Indications for feruloyl esterase activity in
Saccharomyces cereVisiae. J. Agric. Food Chem. 2004, 52, 602-
(34) Dorfner, R.; Ferge, T.; Kettrup, A.; Zimmermann, R.; Yeretzian,
C. Real-time monitoring of 4-vinylguaiacol, guaiacol, and phenol
during coffee roasting by resonant laser ionization time-of-flight
mass spectrometry. J. Agric. Food Chem. 2003, 51, 5768-5773.
(35) Luttrell, W. R.; Wei, L. S.; Nelson, A. I.; Steinberg, M. P. Cooked
flavor in sterile Illinois soybean beverage. J. Food Sci. 1981,
(36) Boatright, W. L.; Lei, Q. Headspace evaluation of methanethiol
and dimethyl trisulfide in aqueous solutions of soy-protein
isolates. J. Food Sci. 2000, 65, 819-822.
(37) Prentice, R. D. M.; Bryce, J. H. A source of dimethyl disulfide
and dimethyl trisulfide in grain spirit produced with a coffey
still. J. Am. Soc. Brew. Chem. 1998, 56, 99-103.
(38) Buttery, R. G.; Seifert, R. M. Characterization of some volatile
potato components. J. Agric. Food Chem. 1970, 18, 538-539.
(39) Buttery, R. G.; Guadagni, D. G.; Ling, L. C.; Seifert, R. M.;
Lipton, W. Additional volatile components of cabbage, broccoli,
and cauliflower. J. Agric. Food Chem. 1976, 24, 829-832.
(40) Rhim, J. W.; Jones, V. A.; Swartzel, K. R. Kinetics studies in
the colour of skim milk on heating. Lebensm. Wiss. Technol.
1988, 21, 334-338.
(41) Van Buren, J. P.; Streinkraus, K. H.; Hackler, L. R.; El Rawi,
I.; Hand, D. B. Indices of protein quality in dried soymilks. J.
Agric. Food Chem. 1964, 12, 524-528.
(42) Kessler, H. G. Effect of thermal processing on milk. In
DeVelopments in Food PreserVation 5; Thorne, S., Ed.; Elsevier
Applied Science: London, U.K., 1989; pp 91-130.
(43) Kwok, K. C.; MacDougall, D. B.; Niranjan, K. Reaction kinetics
of heat-induced colour changes in soymilk. J. Food Eng. 1999,
Received for review November 1, 2006. Revised manuscript received
February 15, 2007. Accepted February 17, 2007. Funding for this study
was provided by the Illinois Center for Soy Foods, the Illinois Council
on Food and Agriculture Research (C-FAR), and the U.S. Department
of Agriculture (Special Grants Program, Project 2005-345-15767).