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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

  1. CULTIVATION
  2. METHODOLOGY
    1. Marigold Flower Polysaccharide (MFP)
    2. Sources
    3. Preparation of MFP
    4. Sulfation (Medina Fuentes 1991)
    5. Emulsion Stability
  3. CONCLUSION
  4. REFERENCES
  5. 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).

CULTIVATION

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 damage.

METHODOLOGY

Marigold Flower Polysaccharide (MFP)

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.

Sources

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 until used.

Preparation of MFP

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 room temperature.

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 (Wickramasingha 1990).

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 (Wickramasingha 1990).

Sulfation (Medina Fuentes 1991)

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), and freeze-dried.

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).

Emulsion Stability

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.

CONCLUSION

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 slightly.

REFERENCES

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 aw