Annatto Colour for Food – Potentialities and Prospects

Saurabh Sharma

ANNATTO colour has been used as a colour additive in butter, cheese, margarine and other food products (Anon., 1948). It can also be used as tracer in milk, cream (England and Yongue, 1940) and vegetable fat (Anon., 1942). It is colouring matter of vegetable origin derived from pericarp of the seeds of Bixa orellana L. (Anon., 1948). The pericarp contains orange coloured coating of carotenoids. The main pigment in the seed coat is bixin, a mono methyl ester of cis-polyene dicarboxylic acid (Mc Keown and Mark, 1962). Upon hydrolysis, the terminal methyl group splits off resulting into a dicarboxylic acid, the norbixin (Karrer et al., 1929).

Annatto colour is prepared by leaching the pericarp of the seeds with an extractant prepared from food grade solvents (Aparnathi and Sharma, 1991).

In the present article general information on annatto, methods of extraction / quantification and use as a food colour has been briefly discussed.


The total pigment content of annatto seeds varies from 0.73 (Bhalkar and Dubash, 1983) to 1.30 percent (Angelucci et al., 1980) comprising of various carotenoids (Tirimana, 1981) of which the cis-bixin amounts to 82 percent (McKeown and Mark, 1962).

Since bixin is the principal colouring matter, the chemistry and performance of annatto colour is essentially that of the bixin (Marmion, 1979). Bixin is a highly unsaturated compound and its conjugated double bonds act as chromophores (Barnicoat, 1937). Chemically bixin is monomethyl ester of cis-polyene dicarboxylic acid with an empirical formula C25H30O4 (McKeown and Mark, 1962).

The structural formula of cis-bixin, given by McKeown and Mark (1962) is as below:

Upon hydrolysis, the terminal methyl group of bixin splits off to give a dicarboxylic acid, norbixin. In presence of alkali norbixin exists as sodium or potassium salt (Karrer et al., 1929) which is water soluble and believed to be the tinctorial agent present in water soluble annatto colour (Barnicoat, 1937). The norbixin content in commercial annatto cheese colour varies form 0.32 to 1.27 percent (Reith and Gielen, 1971).

The bixin, dissolved in vegetable oil, undergoes complex series of isomerization and degradation reactions when heated to  extraction temperatures (Iversen and Lam, 1953). A yellow pigment (thermal degradation product), trans-bixin and cis-bixin are the major carotenoids in oil-soluble annatto colour (McKeown and Mark, 1962). The total pigment content of the commercial annatto butter colour varies from 0.2 to 2.6 percent, atleast 30 percent of which is bixin (FAO/WHO, 1970).

Manufacturing of Annatto colours 

Annatto colour is prepared by leaching the pericarp of the annatto seeds with an extractant prepared from one or more food grade materials like various organic solvents, edible vegetable oils and fats, and alkaline aqueous or alcoholic solution (Aparnathi and Sharma, 1991). The selection of an extractant is done depending upon the intended use (Marmion, 1979).

Water-soluble annatto colour

The water-soluble annatto colour preparation can be obtained either by direct aqueous-alkali extraction or from organic solvent extract of annatto seeds.

Direct aqueous-alkali extraction

The pericarp of the annatto seeds is extracted by agitation in aqueous-alkali at a temperature not exceeding 70°C. The solution is filtered to remove the insoluble material (FAO/WHO, 1970). The use of sodium bicarbonate (Lakshmana Rao, 1926), sodium carbonate (Anon., 1942) and sodium hydroxide (Bhalkar and Dubash, 1983) has been reported.

For refined product, the pigment in alkaline aqueous extract is precipitated by addition of hydrochloric acid. The precipitates are filtered, dried and washed with petroleum solvent to remove unwanted impurities and odours. The pigment is then mixed with alkali and dissolved in water (Marcus, 1963).

From solvent extracted bixin

Indirect method for preparation of water soluble annatto colour involves extraction of the pigment from the seeds by a suitable organic solvents, removal of the solvent by distillation and then hydrolysis of the pigment bixin with aqueous-alkali solution (Preston and Rickard, 1980; Rama Murthy and Krishna Rao, 1982).

According to Rama Murthy and Krishna- Rao (1982) 1, 2-dichloroethane is the most acceptable as it gives maximum yield of colour and is also a permitted solvent for use in food industry. Use of mixture of ethanol and chloroform (75:25, v/v) has also been found satisfactory (Bhalkar and Dubash, 1983).

Oil-soluble annatto colour

Annatto seeds, immersed in vegetable oil at a temperature not exceeding 70°C, are mechanically abraded to remove the pericarp from the seeds. The slurry of the pigment in oil is heated under vacuum of about 20 mm Hg at a temperature not exceeding 130°C. The solution is filtered to remove insoluble material (FAO/WHO, 1970). A mixture of 2 parts of castor oil, 2 parts of mustard oil and 1 part of sesame oil can be used as a satisfactory solvent for annatto pigment (Titov, 1940). Use of mono and diglycerides, propylene glycol or free fatty acids is also practiced (Preston and Rickard, 1980).

Recovery of Annatto pigment during extraction

The recovery of annatto pigment during extraction of annatto seeds depends upon the pretreatment given to the seeds and conditions followed during extraction. About 61.0, 74.0 and 76.5 percent pigment was recovered when the seeds were extracted with water, sodium hydroxide solution and a mixture of ethanol and chlorofrorm (75:25, v/v) respectively (Bhalkar and Dubash, 1983). Aparnathi (1985) observed a recovery of 93.9 and 74.07 per cent when steeped seeds were extracted with acetone and sodium hydroxide solution (0.1N) respectively.

Factors affecting the storage stability of Annatto colours 

All commercial samples of annatto colour have been found to deteriorate with age. In such solutions considerable amount of precipitates deposit with corresponding decrease in tinctorial power (Barnicoat, 1937). The various factors affecting the storage stability of annatto colour are described-below.

Type of carrier medium

Stability of annatto butter colour is higher than that of the annatto cheese colour presumably due to tocopherols present in oil (Reith and Gielen, 1971).

pH of carrier medium

The tinctorial power of annatto cheese colour has been found to decrease at a faster rate at alkaline pH value than at pH 5.5 (Banicoat, 1937).

Temperature of storage

Temperature has definite detrimental effect on storage stability of annatto colour (Banicoat, 1937; Aparnathi, 1985) and hence these colour preparations should be stored under cold conditions (Reith and Gielen, 1971).

Concentrations of pigment

Annatto pigment decomposes at a faster rate with higher initial concentration of pigment (Reith and Gielen, 1971; Aparnathi, 1985)

Trace metals

The trace metals namely copper and iron have been reported to exert destructive effect on annatto colour (Barnicoat, 1937).

Type of container

Stainless steel containers are superior to glass for storage of annatto colour (Carrie, 1938; Barnicoat, 1945)

Head space oxygen

Atmospheric oxygen bleaches the annatto colour (Barnicoat, 1937). Storage of annatto colour under vacuum is recommended (Barnicoat, 1945).

Exposure to light

Light exerts destructive effect on stability of annatto colour (Barnicoat, 1937). Reith and Geilen (1971) recommended storage of annatto colour in dark.

Extraction of annatto colour for analysis

For qualitative or quantitative analysis of annatto colour, the pigment is extracted from the seeds. For extraction of colour solvent is selected depending upon the nature of the material. To facilitate the extraction in some cases, a preliminary separation is achieved by suitable technique. The methods followed for extraction of colour from various products are described in Table1.

Resolution of annatto colour

The ‘resolution’ of annatto colour is helpful in qualitative and quantitative analysis. For detailed compositional analysis and also for distinguishing annatto from other natural or synthetic colours, generally different chromatographic techniques are used. A summary of these techniques is given in Table 2.

Detection of annatto colour

For detection of annatto colour, use of adsorbing materials, chemical reactions, ultra violet (UV) light and co-chromatographic methods have been reported. Amongst different tests, Carr-Price reaction, in which annatto gives deep – blue colour with concentrated H2SO4, is frequently used. The qualitative tests for detection for annatto colour are out lined in Table3.

Determination of annatto colour

For determination of annatto colour gravimetric, colorimetric and spectral assay methods are followed. The spectral assay method  is relatively simple, rapid and widely accepted (Aparnathi and Sharma, 1991). These methods are given in Table 4.

The spectral assay involves preparation of solution of colour in suitable solvent (Chloroform for total pigment and bixin and O. 1N sodium hydroxide for norbixin), dilution of the solution to obtain absorbance at the specific wavelength against the solvent blank. The following formula is used for calculation.


A    =     Blank corrected sample absorbance

a    =     Absorptivity value in 1/g/cm (282 for total pigment and bixin and 287 for norbixin)

b    =    Absorbance cell path length in cm

c    =    Amount of sample taken in g/L


Annatto is mainly used as a food colour. Its chief application is in colouring butter and cheese. It can be used as a tracer and condiment in food products. It can also be safely utilized as a colournat in drugs and pharmaceutical industries.

Quality standards for annatto

The information on standards and legislations on quality, use and manufacturing aspects of annatto colour is given below:

Prevention of Food adulteration Act/Rules (PFA)

Annatto Colour is included in the list of the natural colouring matter of PFA Act / Rules which state that annatto colour may be used in any article of food (except as otherwise provided in the rules). It is further stated that annatto shall be sold only under Indian Standards Institution certification mark (Rule 48C). Also, according to A.26.09 (Appendix A, PFA Act/Rules) the maximum limits for metallic impurities shall be Arsenic, 3ppm; Lead, 10 ppm; copper, 30ppm; heavy metals 40 ppm.

According to Rule 42 (z): A package containing annatto colour in vegetable oils shall bear the following label. Namely:


Bureau of Indian standards (BIS)

According to BIS (IS: 2557-1963), annatto colour shall be prepared only from the seeds of Bixa orellana L. and shall not contain any extraneous colouring matter. The colour intensity of annatto colour preparations shall be as indicated in Table 5.


Annatto is derived from pericarp of seeds of Bixa orellana L. by leaching the pericarp in  edible vegetable oil (oil - soluble annatto) or in alkaline aqueous solution (water- soluble annatto). Complete absence of any unfavorable physiological action by regular and prolonged consumption may be regarded as the main factor in the choice of dye for food materials. With the present consumer trends where focus in towards the use of natural colouring matter in foods annatto has tremendous potential in domestic and international fronts .

Annatto is used as a colour additive in butter, cheese, margarine and other food products. Apart from food it also find use in some non-food application like cosmetics and drugs. Hence, the diversity of use and easy detectability adds to its bright trading prospects and good scope for marketing in India and abroad.





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