1864 J. Agric. Food Chem., Vol. 47, No. 5, 1999
Ko¨hler and Grosch
Ch em ica ls. Acetic acid anhydride, 2,2′-dimethyl-4-(hy-
droxymethyl)-1,3-dioxolane (solketal), 4-(dimethylamino)py-
ridine (DMAP), [2H]chloroform, and fatty acids 6:0, 8:0, 10:0,
12:0, 14:0, 16:0, 18:0, 20:0, 22:0, oleic acid (18:1), and linoleic
acid (18:2) were obtained from Aldrich (Steinheim, Germany).
Dicaprin (di-10:0) and distearin (di-18:0) were obtained from
Sigma (Deisenhofen, Germany). All other chemicals were from
Merck (Darmstadt, Germany).
bath. Boiling distilled water (40 mL) was then added, and the
mixture was immediately vortexed until a homogeneous
emulsion had formed. The emulsion was cooled to room
temperature by rinsing the flask with tap water and then
made up to 50 mL with distilled water (1% w/v, stock solution).
Aliquots of this stock solution (3.2, 6.4, 9.6, and 12.8 mL) were
made up to 16 mL with distilled water. For baking (10 g of
flour) 5 mL of the stock solution or the diluted solutions was
used. In this way concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5%
DATEM based on flour were applied.
Com m er cia l DATEM Sa m p les. Eight DATEM samples
were obtained from two producers of DATEM.
Syn t h esis of 1-Mon oa cylglycer ols. Acetoneketals of
1-Monoacylglycerols. To a solution of fatty acid (15 mmol; 6:0-
20:0, 18:1, and 18:2), solketal (15 mmol), and DMAP (1.875
mmol) in diethyl ether (130 mL) at 20 °C was added dropwise
a solution of N,N′-dicyclohexylcarbodiimide (DCC; 18.75 mmol)
in diethyl ether (15 mL) to keep the temperature below 25 °C
(Eibl et al., 1983). For docosanoic acid (22:0), carbon tetra-
chloride (300 mL) instead of diethyl ether was used as solvent.
After 270 min of stirring at 20 °C, the suspension was filtered
into a separation funnel and the filter was washed with solvent
(25 mL). The solution was then washed twice with hydrochloric
acid (HCl; 75 mL; 0.5 mol/L) and sodium carbonate solution
(75 mL; 0.5 mol/L), respectively. Precipitated fatty acid was
removed by filtration. The organic phase was then washed
twice with sodium chloride (NaCl) solution (10% w/w; 150 mL)
and dried over anhydrous sodium sulfate, and the solvent was
removed by evaporation.
Dough Mixing. DATEM emulsion or water (5 mL, respec-
tively) was cooled in the mixer to 15 °C for 1 min. Flour (10 g;
8.6 g of dry mass), a solution of NaCl and saccharose (1 mL;
NaCl, 200 mg/mL; saccharose, 100 mg/mL), a solution of
L-ascorbic acid (0.05 mL; 4 mg/mL), and yeast (0.7 g) were then
added and mixed for 1 min at 1250 rpm. The final temperature
of the dough was 26 °C.
Dough Handling and Baking. After removal of the dough
from the mixer, it was allowed to rest for 20 min at 30 °C in
a water-saturated atmosphere. The dough was then reshaped
on a dough rounder for eight cycles. The resulting dough ball
was passed through the rolls of a pasta machine to form an
oval dough piece. This was rolled up and after a proofing time
of 35 min at 30 °C in a water-saturated atmosphere, it was
baked for 10 min at 230 °C. The oven (39 × 39 × 33 cm) was
saturated with the vapor of 50 mL of water prior to baking.
The volume of the bread was determined by measuring the
amount of water displaced by the bread at room temperature.
A vessel containing 500 mL of water was put on a balance,
which was then adjusted to 0.0 g. The bread was impregnated
with paraffin at 80 °C for 3 s, allowed to cool for 10 s, and
then immersed in the water. The mass of the displaced water
corresponded to the volume of the bread.
Micr osca le Rh eology. Dough and gluten extensigrams
were measured with the methods reported by Kieffer et al.
(1981a,b) with some modifications. For dough rheology flour
(10 g, 8.6 g of dry mass) and NaCl (0.2 g) were mixed in a
microfarinograph for 1 min at 60 rpm and 22 °C. DATEM
emulsion (5 mL) and distilled water were added, and the dough
was mixed until a maximum consistency of 550 Brabender
units (BU) was reached. The dough was removed, shaped to
an ellipsoid form, and pressed into Teflon forms to give strands
of 53 × 4 × 4 mm. After 40 min of resting in a desiccator at
22 °C in a water-saturated atmosphere, the strands were
measured with an Instron 1122 Micro-Extensograph (Instron,
Bucks, U.K). The extensibility of gluten was determined by
mixing flour (10 g, 8.6 g dry mass) with DATEM emulsion (5
mL) and distilled water for 2 min so that a final dough
consistency of 520-580 BU was reached. The dough was then
washed with distilled water (540 mL) in a glutomatic (Perten
Instruments, Huddinge, Sweden). The residue (gluten) was
centrifuged in a test tube (100 × 13 mm i.d.) for 5 min at 4000g
and 22 °C, pressed into Teflon forms, and treated as described
above.
Ga s Ch r om a togr a p h y/Ma ss Sp ectr om etr y (GC/MS).
GC analyses were performed by means of a gas chromatograph
Type 5300 (Fisons, Mainz, Germany) equipped with a CP-SIL
5CB capillary (10 m × 0.25 mm i.d., 0.12 µm film thickness,
Chrompack, Frankfurt, Germany) equipped with an on-column
injector. An aliquot (0.5 µL) of the sample in chloroform
[0.001% 1-monoacylglycerol (w/v)] was injected on-column at
60 °C. After 1 min, the temperature was raised at 25 °C/min
to 300 °C, which was held for 15 min. Helium at a flow rate of
1.5-2.5 mL/min was used as a carrier gas. An MAT95S mass
spectrometer (Finnigan MAT, Bremen, Germany) was con-
nected with the gas chromatograph. The transfer line was
heated to 300 °C. Negatively charged ions were generated by
chemical ionization with ammonia at 115 eV using a source
temperature of 250 °C, and the scan ran from m/z 100 to m/z
500.
1-Monoacylglycerols. Acetoneketals of the 1-monoacylglyc-
erols 10:0-22:0, 18:1, and 18:2 (4 mmol) were refluxed in
methanol (25 mL)/HCl (2 mL; 1 mol/L) for 10 min. The solution
was transferred into a separation funnel with diethyl ether/
tetrahydrofuran (50 mL; 40:10, v/v), washed once with NaCl
solution (50 mL; 10%, w/w), twice with sodium hydrogencar-
bonate solution (saturated at 22 °C; 25 mL), and twice with
NaCl solution (50 mL; 10%, w/w). The organic phase was dried
over anhydrous sodium sulfate, and the solvent was removed
by evaporation. Acetoneketals of the 1-monoacylglycerols 6:0
and 8:0 (4 mmol) were hydrolyzed according to the method of
Baer and Fischer (1945) in 10% (v/v) acetic acid (6:0, 300 mL;
8:0, 1000 mL) at 60 °C under stirring (6:0, 2 h; 8:0, 4 h). The
resulting solution was extracted five times with diethyl ether
(30 mL) and then treated as described above. The crude
1-monoacylglycerols were dissolved in diisopropyl ether (100
mL) and purified by column chromatography (20 × 3 cm i.d.)
on silica gel G60 (50 g; 1.5% water). The column was flushed
with diisopropyl ether and stepwise eluted with the following
diisopropyl ether/acetonitrile mixtures (v/v): 100 mL 9:1, 50
mL each 8:2, 7:3, 6:4, 5:5, and 4:6. 1-Monoacylglycerols were
eluted in fractions 4-6.
Syn th esis of DATEM. A modified method according to
J acobsberg et al. (1976) was used.
Diacetyltartaric Acid Anhydride. Powdered tartaric acid (27
mmol), orthophosphoric acid (0.46 mmol), and acetic acid
anhydride (90 mmol) were stirred for 25 min until the tartaric
acid had dissolved. The mixture was then heated to 80 °C, a
vacuum of 16 kPa (160 mbar) was applied, and the tempera-
ture was allowed to rise to a maximum of 110 °C while acetic
acid (54.2 mmol, 3.1 mL) was distilled off.
DATEM. 1-Monoacylglycerols or diacylglycerols (1-2 mmol)
were weighed into a pointed flask, and an aliquot of freshly
prepared diacetyltartaric acid anhydride was added. The molar
ratio 1-monoacylglycerol/diacetyltartaric acid was 1:0.95. The
mixture was heated to 110 °C for 5 min or until it had been
melted. The temperature was then increased to a maximum
of 140 °C. At 139 °C a vacuum of 5 kPa was applied and acetic
acid formed was distilled off for 3 min. The vacuum was
removed, and the synthesized DATEM was allowed to cool.
Micr osca le Ba k in g Test. This was performed as a micro
rapid mix test (MRMT) with 10 g of flour according to the
method of Kieffer et al. (1993) with some modifications. The
ingredients based on the flour were as follows: NaCl, 2%;
saccharose, 1%; yeast, 7%; ascorbic acid, 20 mg/kg. The final
water content of the dough was 46.4%.
Ma ss Sp ectr om etr y. Solutions of 1-monoacylglycerols in
chloroform (0.05% w/v) were directly applied to the mass
spectrometer (MAT95S) running in the chemical ionization
mode with isobutane as the reagent gas, generating positively
charged ions.
Preparation of DATEM Emulsions. In a 50 mL volumetric
flask DATEM (500 mg) was heated for 3 min in a boiling water