A degradation study of beta carotene in infant food and raw vegetables by ozone treatment


β – Carotene the precursor of Vitamin A is a red-orange pigment present in plants and fruits like carrot, broccoli, lettuce, sweet potato, tomato, and winter squash and gives them their color. More intense the color the more beta carotene it has. This Carotenoid is added to many foods such as margarine, infant formula etc. Carotenoids belong to the most important group of natural pigments as they contribute too many diverse functions due to their high structural diversity. They also possess an immense amount of radical scavenging properties.

Beta carotene is a member of Carotenoids which are terpenoids and are synthesized biochemically from eight isoprene units and thus have 40 carbons.




This structure with 3 to 15 conjugated bonds allows them to have various biological functions. Additionally, due to their high unsaturation level they are susceptible to degradation reaction such as oxidation and hydrolysis which finally modifies their biological functions.

Ozone, widely used for sterilization in food industry as it has high oxidation power and high penetrability which increases microbial security for these foods. In addition, it does not leave behind any toxic residues unlike other types of sanitization agents. However, ozone has the capacity to react with the organic matter present in food, especially the unsaturated compounds, such as Carotenoid pigments, through well-known cycloaddition reaction resulting in carbonyl compounds.

The adverse effect has been revealed on two intervention trials with β – Carotene on two groups i.e. cigarette smokers and asbestos-exposed workers. The detrimental effects were observed related to oxidation products of beta carotene. Their generation of oxidation products confined in the polyene structure of beta carotene that is beneficial for radical scavenging, but is also prone to oxidation. The diversity of cleavage products depends on the dominant degradation mechanism and the bond cleavage would tend to occur either randomly or at defined position of the conjugated electron system.








The aim of this work was therefore to study the effect of ozone treatment in the degradation process of beta carotene.

Materials and methods

Test material: Raw vegetables (carrot, broccoli), infant formulae (Aptamil)

Reagents and standardsBeta carotene standard obtained from sigma Aldrich. All solvents used were of HPLC grade viz.  Tetrahydrofuran (THF), Acetonitrile, methanol, 2-propanol. BHT and ammonium acetate used were of analytical grade.


  1. Balance
  2. Spectrophotometer
  3. Ultrasonic water bath
  4. Rotary evaporator
  5. HPLC system
  6. Micro pipette
  7. Ozone generator

Preparation of standard

10.0 mg of beta-carotene reference material was weighed with an accuracy of 0.01mg in a 10ml volumetric flask and made up to the volume with Tetrahydrofuran. The solution was sonicated for 30 seconds. Resulting stock solution is used for preparation of calibration curve, recovery study and ozone treatment.

Sample preparation

All extractions steps were performed in brown or aluminum-foil wrapped glassware and under dimmed-light conditions to avoid photo-oxidation of the Carotenoids.

  • Infant formulae: 5g of infant formulae was accurately weighed in a round bottom flask and saponified with 5% ethanolic KOH at 80℃ for 45 minutes under reflux. The saponified mixture was transferred to a separating funnel and extracted with petroleum benzene. The extract was water washed and evaporated at 40℃ in rotary evaporator. The dried extract was reconstituted with Tetrahydrofuran. Similarly, the recovery samples and ozone treated samples were extracted.
  • Raw vegetables: 5g of the raw vegetable (washed and grinned finely) was accurately weighed in a 50ml Tarson tube (for each vegetable under study) and extracted 50ml with THF. The sample was extracted at 50℃ overnight on a shaking water bath till the sample became colorless. The extract was them filtered and evaporated to concentrate and reconstituted with THF and used for further study.

Ozone treatment

Two ozone generators were used for ozone treatment viz. Witherpro-7 and Zeon 100.

Each of 50g of Infant formulae was treated with Zeon 100 at a fixed ozone concentration but with variation of time. Vegetables were treated with Witherpro-7. The concentration of oxygen was controlled to treat the samples at different concentration of ozone at different interval of time.






The standard solutions of fixed concentration (10ppm and 5ppm Beta carotene) were also ozone treated at a fixed ozone concentration with variation in time interval to study the degradation pattern in standard solution.

Results: Spectrophotometric analysis at 448nm

Beta carotene (mg/kg) Absorbance at 448nm
0.1 0.0245
0.5 0.0763
1 0.1629
5 0.7232
 10 1.5936

time (sec) conc. (mg/kg)
3 0.5615
5 0.5198
10 0.5019
15 0.0785
20 0.0370

time (sec) conc. (mg/kg)
5 1.2339
10 0.5393
15 0.4925
20 0.4363
25 0.2809

HPLC system conditions

Column: Zorbax C-18 (250mm X 4.6mm X 5µ)

Flow rate: 0.6ml/min

Run time: 20 minutes

Wavelength maxima: 448nm

LC: Isocratic (A)

Mobile phase A: In 1L volumetric flask, 50mg butylated hydroxytoluene (BHT) was added in 20 ml 2-propanol and 0.2ml of triethylamine was added. To this was added 455ml acetonitrile and 450ml of methanol. Mixture was sonicated and volume was made up with methanol. Finally, the 1L mobile phase was filtered and sonicated for further use.













Results: HPLC

Infant formulae:

Time of ozone exposure (minutes) Beta carotene post exposure (mg/kg) Dilution factor Beta carotene post exposure (mg/kg)
0 min 15.999 5 3.2
3 min 13.143 5 2.629
4 min 11.542 5 2.308
5min 10.673 5 2.135








Percentage degradation: (Initial conc. – Final conc.) X 100

Initial conc.

For infant formulae there is a limitation for ozone exposure. Hence Zeon 100 with the capacity of 500mg/hr. of ozone emission was used for a limited span of time. Percentage degradation for infant formulae was found to be 33%.

Degradation in Carrot

Carrot Conc. of ozone (mg/kg) Beta carotene post exposure (mg/kg) Dilution Factor Beta carotene post exposure (mg/kg)
0 0 318.73 10 31.87
1 0.12 282.88 10 28.29
2 0.17 206.06 10 20.61
3 0.2 156.31 10 15.63
4 0.6 79.62 10 7.96







Degradation in Broccoli

Broccoli Conc. of ozone (mg/kg) Beta carotene post exposure (mg/kg) Dilution Factor Beta carotene post exposure (mg/kg)
0 0 810.98 10 81.098
1 0.12 706.37 10 70.637
2 0.17 667.60 10 66.760
3 0.2 213.77 10 21.377
4 0.6 154.62 10 15.462








For vegetables ozonization was done in 1L water by using Wither pro-7 at 4 different concentrations of ozone for a fixed amount of time. The degradation varied from 75 to 80 percent.


The oxidation of beta carotene, under different concentration of ozone was found be varied between 33% in infant formulae to 80% in raw vegetables. The limitations of the food industry remain in the cost effectiveness of the process as ozone exposure has high cost. However, with the developing technology and awareness we can overcome the limitations.

Further potential of the work: Identification of degradation products.

Acknowledgments: I would be thanking the R&D team MSk and Team water house for their continuous support to complete this project.

Contributed by Shraboni Palley.