There are several articles describing this method but I need a more detailed protocol.
β-carotene (0.6mg) in 1 mL of chloroform was taken in a amber bottle and mixed with 60 mg of linolenic acid and 600mg of Tween 80 (poly oxy ethelene sorbiton monopalnitate). The chloroform was removed under nitrogen and the resulting solution was immediately diluted with 30ml of triple distilled water and the emulsion was mixed well for 1 min. The emulsion was further diluted with 120ml of oxygenated water and used for assay.
To the samples (200µl), 4ml of freshly prepared emulsion mixture was added and mixed well. A control consisting 200µl of ethanol and 4ml of emulsion was also analyzed. The absorbance of reaction mixture was measured immediately (t=0) at 470nm against blank consisting of emulsion mixture except β-carotene and at 60 min interval for 2 h (t=120). The tubes were placed in a water bath at 50oc between measurements. Measurement of colour was recorded until the colour of β-carotene disappears. The antioxidant activity (AA) of the isolated compounds was evaluated in terms of bleaching the β-carotene using the formula of AA = 100 [1-(Ao-At) / (A00-Aot)], where Ao and Aoo is the absorbance measured at the beginning of the incubation for samples and control respectively. At and Aot are absorbance measured for samples and control after incubation for 120min respectively.
Kumars method above is fine. this is the protocol I used. It is based on the method by Duan et al. (2006) with some modifications. See my paper- "Antioxidant activities assessed using in-vitro and cellular model systems of five brown seaweeds harvested in spring from the west coast of Ireland."
Beta-Carotene Bleaching Assay (BCBA)
The BCB method is based on the loss of the yellow colour of β-carotene due to its reaction with radicals which are formed by linoleic acid oxidation in an emulsion. The rate of β-carotene bleaching is slowed down in the presence of antioxidants. A b-Carotene emulsion was prepared by adding 50 µl b-Carotene, 10 mg linoleic acid, 100 mg Tween-40 and 1.9ml chloroform. A control emulsion was prepared as above but without b-Carotene. The chloroform was evaporated and 100 ml oxygenated distilled water was added to each emulsion. 50 µl of seaweed extracts and standard were added to a 96-well polystyrene plate. 200 µl b-Carotene emulsion was added to each of the sample and standard wells while 200 µl control emulsion was added to the control wells and was measured immediately at 450 nm. The plate was then incubated in the dark at 50°C and read at 30 minute intervals for 420 minutes. The resulted were expressed as % b-Carotene Bleaching inhibition (Jayaprakasha et al, 2001).
Dear all friends,
How I can prepare the linoleic acid for using in beta carotene bleaching assay. I bought it from SIGMA its no. is "L-1376-10MG". Its product info sheet only tells us that it is miscible with ethanol, ether, chloroform.
Plus, in assay its quantity sometime write in "gm" not in liquids (uL). So its confusing me now.
Dear Ashan Hameed,
I think it will help
The density of linoleic acid is given in the product for SIGMA ( "L-1376-10MG) it is 0.902 g/mL at 25 °C(lit.), So based on this you can prepare/ convert the linoleic acid into the grams.
β- Carotene is fat soluble hydrocarbon with yellowish orange color. Linoleic acid hydroperoxides on reaction with β- Carotene bleaches its color. This is due to the fact that β- Carotene forms a complex with linoleic acid and oxidizes it. This consumption of β- Carotene causes the reduction of bright yellow color to light milky color. Now the presence of an antioxidant in the reaction mixture checks the consumption of β- Carotene by acting on linoleic acid free radicals. As the β- Carotene is released from the complex between it and linoleic acid, the yellow color of the reaction mixture is regained. Hence stronger the antioxidant present in the reaction mixture brighter will be the color and higher will be its optical density.
i m not sure about the above given formula. How it works?
Dear All,
I am looking for a process flow for belaching yellow carrot to obtain nearly white carrot fiber for food application. I applied alkaline bleaching with hydrogen peroxide. Hydrogen peroxide has been degarading rapidly, while adding chelating agents, which are compliance with food.
Thanks,
For blank : chloroform + linoleic acid + tween 20?
I'm using tween 20 as i have no tween 40
Yes that is the blank I used.
Tween 20 and 40 are both emulsifiers so hopefully they can used interchangebly .
Dear all,
Can I use Lucarotin 1 CWD / Y for beta-carotene bleaching test ????
I haven’t done this assay in over 8 years so please forgive me if I’m wrong but I think Lucarotin 1 CWD / Y should work once no additional preservatives have been added to it.
Kulisic, T., Radonic, A., Katalinic, V., & Milos, M. (2004). Use of different method for testing antioxidative
activity of oregano essential oil. Food Chemistry, 85, 633–640.
Kulisic, T., Radonic, A., Katalinic, V., & Milos, M. (2004). Use of different method for testing antioxidative
activity of oregano essential oil. Food Chemistry, 85, 633–640.
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