at 55°C. The suspension was centrifuged at 3,000 rpm (Beckman model J-6B)
for 25 min at 22°C, and the supernatant was filtered. The filtrate was
acidified with acetic acid to pH 4.5, and 95% ethanol (3 volumes) was added in
a thin stream to the rapidly stirred solution. The precipitate was collected
by centrifugation (3,000 rpm, 25 min, 22°C) and dissolved in 0.5 liters of
water. The resulting solution was filtered through a layer of diatomite
filter-aid (Celite, Manville Products Corp.) in a Buchner funnel
The aqueous solution was passed through a column of Amberlite
IR-120(H+) cation-exchange resin to remove proteinaceous material.
All effluent with an acidic pH (pH 3) was collected and dialyzed against
distilled water. The retentate was concentrated and lyophilized
Sulfation with methyl sulfoxide-sulfur trioxide complex. MFP was
dissolved in DMSO (1 g/10 ml). After solution was complete, the reaction
mixture was cooled to 15° to 17°C. The complex (Whistler and Spencer
1961) was added, and the mixture was stirred at 15d° to 17°C for 15 min.
Ice (10 g) and water (25 ml) were added, and the solution was neutralized to pH
7 with 10% ammonium hydroxide solution. A 5% excess of alkali was added, and
the mixture was stirred for 15 min. The Prodomex MFP derivative was
precipitated with ethanol (3 volumes), redissolved, and dialyzed 24 h against a
pH 8 solution of ammonium hydroxide and 48 h against distilled water. The
final product was obtained by freeze-drying.
Sulfation with triethylamine-sulfur trioxide complex. A mixture of
triethylamine-sulfur trioxide complex (Aldrich Chemical Co.) and 250 ml of
dimethylformamide was cooled to 0°C. Prodomex MFP (1 g) was added, and the
reaction was allowed to proceed 24 h at 0°C with stirring. The material was
then dialyzed 24 h against a solution of 10% ammonium hydrogen carbonate, and
48 h against distilled water. The sulfated product was precipitated with
ethanol (3 volumes), collected by centrifugation (3,000 rpm, 25 min, 22°C),
Samples (0.05 g) were analyzed for sulfur content using the method described by
Ma and Crattner (1970). Removal of cationic interferences prior to analysis
was necessary. This was done by adding 0.15 g of cation-exchange resin
(Amberlite IR-120 [H+]) to solutions of samples, stirring for 20
min, and removal of an aliquot after settling (Hoffer et al. 1979).
Emulsions were prepared with a Vibra Cell Sonifier (Sonics & Materials,
Inc.). A mixture of either limonene or olive oil (0.25 ml), and gum solution
(2.25 ml of a 1% w/v solution) was sonicated for two 90-s periods, first
placing the tip of the instrument's tapered probe into the surface of the
mixture, and then lowering the probe into the middle of the sample. The
instrument was used in a continuous mode at a power setting of 3 (75 watts)
(Dea and Madden 1986). In the case of diluted emulsions, 1 ml of emulsion was
diluted with 9 ml of distilled water (1:10 dilution). After 2 min, another
1:10 dilution was made (1:100 dilution) (Prakash et al. 1990).
Emulsion stability was determined by diluting the emulsion and monitoring
turbidity as a function of time at a wavelength of 500 nm with a Varian Model
DMS80 double-beam spectrophotometer (Pearce and Kinsella 1978). Gum arabic was
used as a standard. All three emulsions were diluted appropriately to give a
final 1:1000 dilution.
Marigold flower petal meal contains a water-soluble gum (MFP) that has been
characterized as a protein-polysaccharide. MFP has emulsifying and
emulsion-stabilizing properties equivalent to those of gum arabic towards
limonene and slightly less than those of gum arabic towards olive and castor
oils. It is, however, not possible to prepare concentrated emulsions with MFP
because of the high viscosity of its solutions at concentrations above about
5%. A test of the hypothesis that the reason for the difference in rheological
behavior between MFP and gum arabic, also a protein-polysaccharide, was the
much lower charge density of MFP did not support it for increasing the anionic
charge on MFP to a value close to that of gum arabic reduce