Table of Contents
Medina, A.L. and J.N. BeMiller. 1993. Marigold flower meal as a source of an
emulsifying gum. p. 389-393. In: J. Janick and J.E. Simon (eds.), New crops.
Wiley, New York.
Marigold Flower Meal as a Source of an Emulsifying Gum
Ana L. Medina and James N. BeMiller
- Marigold Flower Polysaccharide (MFP)
- Preparation of MFP
- Sulfation (Medina Fuentes 1991)
- Emulsion Stability
- Fig. 1
Marigold (Tagetes erecta L., Asteraceae) is not only grown as an
ornamental, cut flower, and landscape plant, but also as a source of pigment
for poultry feed. The pigment is added to intensify the yellow color of egg
yolks and broiler skin. It is composed of esters of xanthophyll (lutein).
Finely ground blossom meal, often enriched with an extract, or the extract
itself, usually saponified for better absorption, is added to the feed.
Marigolds are grown for this purpose in various locations in the western
hemisphere, primarily in Mexico and Peru, by and for various companies who
produce feed additives.
One interest of the Whistler Center for Carbohydrate Research is to search for
ways to meet the need for an alternative to gum arabic, the supply of which has
been variable and uncertain. Unique properties of gum arabic are its ability
to emulsify and its ability to form high-solids, low-viscosity solutions. The
marigold flower meal that remains after removal of the xanthophyll esters by
extraction was chosen as a potential source of such a gum because it was
believed to contain a polysaccharide component that had the ability to protect
hydrophobic substances from oxidation. To remove pigment, blossoms, either
fresh or after having been stored in silos, are pressed to remove water. The
resulting cake is dried, pelletized, and extracted with hexane. The remaining
meal was used as a source of MFP in this work (Fig. 1).
Field production of marigolds is well established, especially for the Americas,
and has been studied elsewhere, particularly in South Asia (C.G. Fohner pers.
commun.). Marigolds are sown directly into a finely prepared seed bed. The
crop requires supplemental irrigation. Both overhead and furrow irrigation
should be available. The soil surface must be kept moist for uniform
germination and emergence; irrigation by sprinkler is advised to enhance
seedling establishment and minimize soil crusting. After the stand is
established, water is best applied by furrow irrigation.
Marigolds are usually grown in double rows on 75- or 100-cm beds. The crop is
sown as early as possible, for an early start promotes flowering. Final stands
in the row should be 15 to 25 cm. Under optimal germination rates, the seeding
level should be 0.37 kg/ha.
Phosphorus is required to promote flowering. Nitrogen should be applied two or
three times during the growing season. The effects of nutrients, growth
regulators, planting time, and plant density on plant growth, flower yield and
quality, and seed yield have been reported elsewhere (Parmar and Singh 1983;
Arora and Khanna 1986; Gowda and Jayanthi 1986; Ravindran et al. 1986; Shedeed
et al. 1986; Anuradha et al. 1988a, 1988b, 1990; Yadav and Bose 1988;
Arulmozhiyan and Pappaiah 1989; Girwani et al. 1990; Tolman et al. 1990).
Marigold is often intercropped with other plants. Rotation with marigolds
reduces diseases of other crops (Medhane et al. 1985; Ijani and Mmbaga 1988;
Perwez et al. 1988; Abid and Maqbool 1990), and reduces nematode populations
(Prasad and Haque 1982; Baghel and Gupta 1986; Reddy et al. 1986; Alam et al.
1988). No pesticides are registered in the United States for use on marigolds
grown for xanthophyll production, so proper site selection to minimize pest
problems is important. Marigolds are susceptible to root diseases, but risk
can be minimized by not planting marigolds in fields previously planted to
peppers and by avoiding fields that are prone to standing water. Mites can be
a severe problem on marigolds, but miticides are not registered for use on
marigolds grown for xanthophyll production or feed use.
Cultivation to control weeds is advised until the crop canopy has closed
because as no herbicides are reg-istered for use on marigolds grown for direct
use in poultry feed or for xanthophyll production. Flowers are harvested by
hand when plants have, on the average, two or three fully developed flowers
(about 90 days after planting). Subsequent harvests (up to two) can be made at
intervals of 3 to 5 weeks, depending on plant vigor. Mechanical harvesters are
also used; they generally limit the number of harvests to one because of plant
MFP can be extracted from the meal with warm (50° to 55°C) water
(BeMiller et al. 1989). MFP was determined to be a protein-polysaccharide.
One purified fraction of the polysaccharide portion was found to contain 3.75%
galacturonic acid and neutral sugars in the molar ratio: galactose (15):
glucose (7): arabinose (3). Methylation analysis indicates a highly branched,
acidic arabinoglucogalactan. Partial characterization of the protein part
indicated the presence of at least two very hydrophobic polypeptide
constituents (Wickramasingha 1990).
The crude extract was dark, and a procedure involving an oxidative pretreatment
of the meal prior to extraction of the gum was worked out to give preparations
of minimal color (BeMiller et al. 1989). Both the crude-extract and the
bleached MFP had emulsifying and emulsion stabilization powers for limonene
equivalent to those of gum arabic at equal concentrations (1 and 2.5%) and
slightly less than those of gum arabic for olive and castor oils (BeMiller et
al. 1989). It was not, however, possible to prepare high-solids, low-viscosity
solutions of MFP and, hence, not possible to prepare concentrated emulsions.
It was hypothesized that the difference in negative charge on the two molecules
(ca. 16% uronic acid in gum arabic vs. 3.75% uronic acid in MFP) might be the
reason for their different rheological behaviors. A project was undertaken to
increase the negative charge on MFP by sulfation. The net negative charge was
increased to 14.3 mole% without decreasing its emulsion-stabilizing properties.
The reduction in viscosity was, however, only slight and remained much higher
than that of gum arabic (Medina Fuentes 1991).
MFP wascharacterized from material subjected to two different treatments before
pigment extraction, as well as from fresh petals after removal of pigments.
Storage of blossoms in silos before pigment extraction resulted in a MFP
preparation of slightly less viscosity and protein content as compared to MFP
obtained from nonensiled blossoms. The former MFP also required a slightly
higher concentration to achieve equivalent emulsion stability. MFP obtained
from fresh petals had poorer performance as an emulsifier and higher solution
viscosity (Medina Fuentes 1991). Covalently attached phenolic compounds were
present in all three sources of MFP.
The source of the meal obtained from Prodomex S.A. de C.V. (Prodomex meal) was
marigolds grown near Los Mochis, Sinaloa, México, that had been picked
both by hand and by machine. Fresh blossoms were heated, then pressed. The
resulting cake was dried, extruded into pellets, and extracted with hexane.
The source of the meal obtained from Kemin Industries, Inc. (Kemin meal) was
marigolds grown in Peru. Blossoms were stored in a silo before being pressed,
dried, and hexane extracted. The source of the fresh marigold flowers was
Alternative Agriculture Cooperative, Sedalia, Missouri; they were kept frozen
Removal of pigments from fresh flowers. Petals were separated from the
rest of the flower and extracted in a Soxhlet apparatus with methanol. The
methanol was removed, and the petals were soaked in a mixture (2:1 v/v) of
benzene and ethanol at room temperature for 1 h and then extracted again in a
Soxhlet apparatus with the same solvent. The petals were extracted twice with
hexane in a Soxhlet apparatus. The remaining colorless petals were dried at
Extraction of MFP. Dry meal (300 g) or extracted petals (55 g) were
soaked in water (1,500 ml) for 3 h at room temperature. The suspension of
swollen material was then heated for 3 h with constant stirring in a water bath
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 reduced its viscosity only
- Abid, M. and M.A. Maqbool. 1990. Effects of inter-cropping of Tagetes
erecta on root-knot disease and growth of tomato. Int. Nematol. Network
- Alam, M.M., A.M. Khan, and S.K. Saxena. 1988. Management of plant parasitic
nematodes by different cropping sequences. Indian J. Plant Pathol.
- Anuradha, K., K. Pampapathy, and N. Narayana. 1988a. Effect of N and P2O5 on
the nutrient composition and uptake by marigold (Tagetes erecta L.). S.
Indian Hort. 36:209-211.
- Anuradha, K., K. Pampapathy, and R. Sreenivasalu. 1988b. Effect of N and P2O5
on flowering and yield of marigold (Tagetes erecta L.). S. Indian Hort.
- Anuradha, K., K. Pampapathy, and N. Narayana. 1990. Effect of nitrogen and
phosphorus on flowering, yield and quality of marigold. Indian J. Hort.
- Arora, J.S. and K. Khanna. 1986. Effect of nitrogen and pinching on growth
and flower production of marigold (Tagetes erecta). Indian J. Hort.
- Arulmozhiyan, R. and C.M. Pappaiah. 1989. Studies on the effect of nitrogen,
phosphorus and ascorbic acid on the growth and yield of marigold (Tagetes
erecta L.) cv. MDU 1. S. Indian Hort. 37:169-172.
- Baghel, P.P.S. and D.C. Gupta. 1986. Effect of intercropping on root-knot
nematode (Meloidogyne javanica) infesting grapevine (var. Perlette).
Indian J. Nematol. 16:283-284.
- BeMiller, J.N., A.K. Gupta, and D.S. Wickramasingha. 1989. Structure and
properties of the water-extractable polysaccharide of marigold (Tagetes
erecta) petals, p. 1-14. In: R.P. Millane, J.N. BeMiller, and R.
- Chandrasekaran (eds.). Frontiers in carbohydrate research-1. Elsevier Applied
- Dea, I.C.M. and J.K. Madden. 1986. Acetylated pectic polysaccharides of sugar
beet. Food Hydrocolloids 1:71-88.
- Girwani, A., R.S. Babu, and R. Chandrasekhar. 1990. Response of marigold
Tagetes-erecta to growth-regulators and zinc. Indian J. Agr. Sci.
- Gowda, J.V.N. and R. Jayanthi. 1986. Studies on the effect of spacing and
season of planting on growth and yield of marigold Tagetes erecta Linn.
S. Indian Hort. 34:198-203.
- Hoffer, E.M., E.L. Kothny, and B.R. Appel. 1979. Simple method for microgram
amounts of sulfate in atmospheric particulates. Atmos. Environ. 13:303-306.
- Ijani, A.S.M. and M.T. Mmbaga. 1988. Studies on the control of root knot
nematodes (Meloidogyne species) on tomato in Tanzania using marigold
plants (Tagetes species), ethylene dibromide and aldicarb. Trop. Pest
- Ma, T.S. and R.C. Crattner. 1979. Modern elemental analysis. Marcel Dekker,
- Medhane, N.S., G.B. Jagdale, A.B. Pawar, and K.S. Darekar. 1985. Effect of
Tagetes erecta on root-knot nematodes infecting betelvine. Intl.
Nematol. Network Newslett. 2:11-12.
- Medina Fuentes, A.L. 1991. Marigold (Tagetes erecta) flower
polysaccharide: (1) comparison of preparations from different sources; (2)
viscosity and emulsion stabilizing properties of sulfated gum. MS Thesis,
Purdue University, West Lafayette, IN.
- Parmar, A.S. and S.N. Singh. 1983. Effect of plant growth regulators on
growth and flowering of marigold (Tagetes erecta). S. Indian Hort.
- Pearce, K.N. and J.E. Kinsella. 1978. Emulsifying properties of proteins:
evaluation of a turbidimetric technique. J. Agr. Food Chem. 26:716-723.
- Perwez, M.S., M.F. Rahman, and S.R. Haider. 1988. Effect of Tagetes
erecta on Meloidogyne javanica infecting lettuce. Int. Nematol.
Network Newslett. 5:18-19.
- Prakash, A., M. Joseph, and M.E. Mangino. 1990. The effects of added proteins
on the functionality of gum arabic in soft drink emulsion systems. Food
- Prasad, D. and M.M. Haque. 1982. Reaction on varieties of marigold against
root-knot nematode, Meloidogyne incognita. Indian J. Nematol.
- Ravindran, D.V.L., R.R. Rao, and E.N. Reddy. 1986. Effect of spacing and
nitrogen levels on growth, flowering and yield of African marigold (Tagetes
erecta L.). S. Indian Hort. 34:320-323.
- Reddy, K.C., A.R. Soffes, G.M. Prine, and R.A. Dunn. 1986. Tropical legumes
for green manure II. Nematode populations and their effects on succeeding crop
yields. Agron. J. 78:5-10.
- Shedeed, M.R., K.M. El-Gamassy, M.E. Hashim, and A.M.N. Almulla. 1986. Effect
of some growth regulators on the growth, flowering and seed production of some
summer annuals. Ann. Agr. Sci., Ain Shams Univ. 31:677-689.
- Tolman, D.A., A.X. Niemiera, and R.D. Wright. 1990. Influence of plant age on
nutrient absorption for marigold seedlings. HortScience 25:1612-1613.
- Whistler, R.L. and W.W. Spencer. 1961. Preparation and properties of several
polysaccharide sulfates. Arch. Biochem. Biophys. 95:36-41.
- Wickramasingha, D.S. 1990. Partial characterization of the structure and
properties of the water-extracted polysaccharide of marigold (Tagetes
erecta) flowers. MS Thesis, Purdue University, West Lafayette, IN.
- Yadav, L.P. and T.K. Bose. 1988. Influence of planting time and plant density
on growth, flowering and seed yield in marigold. Bangladesh Hort. 16:17-21.
||Fig. 1. Preparation of MFP. aHand or machine picked.
bProdomex MFP (not ensiled). cKemin Industries MFP
(ensiled). dPigment was removed from fresh petal MFP by extraction
with methanol and benzene-ethanol, in that order, then hexane.
Last update September 12, 1997