1. NATURAL COLOURANTS EXTRACTION & CHARACTERIZATION FROM OILSEEDS
Presented by
SRUTHI ROSE THOMAS
REG NO: 11MBT0022
II M.TECH BIOTECHNOLOGY
VIT UNIVERSITY

2. PLACE OF WORK UNDER THE GUIDANCE OF
CSIR-Central Food Technology Research Institute
A Constituent laboratory of Council of Scientific & Industrial Research (CSIR)
Ministry of Science & Technology
Mysore, Karnataka
UNDER THE GUIDANCE OF
EXTERNAL GUIDE
INTERNAL GUIDE
Dr Sridevi Annapurna Singh
Head/ Sr. Principal Scientist
Protein Chemistry & Technology Department
CSIR-CFTRI, Mysore
Prof. Vino S.
Assistant Professor (Senior)
School of Bio Sciences and Technology
VIT University, Vellore

3. INTRODUCTION Colour Most important visual cues of a food
Determines its acceptability & consumability
Modify the way taste, odor & flavor are perceived
Added in food & beverages to fulfill the expectations of consumers

4. Purposes of adding colour additives
Offset colour loss during storage
Correct natural variations
Enhance colours that naturally occur
Provide colour to colourless foods

5. General classification of food colours
Perceived to be safe
Used from time immemorial
More expensive and less stable
Stability and overall cost factor
Potent carcinogens/mutagens
Threats to the environment
Use of unpermitted colours raise safety issues

6. AIM & OBJECTIVES
Isolation of natural colour from hulls of oilseeds – sesame and mustard – by-products of industry
Use of seed hulls for the preparation of natural colours – recovery of value added products from waste
Characterization of isolated extract – physico-chemical, spectral and bioactive properties
Stability of the colour with respect to temperature, pH and light

7. MATERIALS & METHODS Antioxidant Activity Determination
Seed Material
Sesame seed hull
Mustard hull
Preparation of Extracts
Solvent extraction
Alkali extraction
Analysis of Colour
Measurement of visual colour
Absorption spectra of the extracts
Antioxidant Activity Determination
DPPH radical method (Brand-Williams et al., 1995)
FRAP assay (Benzie & Strain, 1996)

8. Validation of antioxidant activity
Lipoxygenase inhibition assay (Axelrod et al., 1981)
Preparation of colour in the solid form & Quantitation
Precipitation
Drying
Identification of the extracted pigments
Chemical tests (Vallimayil et al., 2012)
UV-Visible absorption spectra
FT-IR Spectroscopy
Stability studies
Temperature
Exposure to light

9. RESULTS Pigment extraction
SSHE-A
SSHE-B
SSHE-C
SSHE-D
MHE
The sesame and mustard seed hull extracts with 1% NaOH are visibly darker than the others

10. Colour Attributes
Table: Colour readings (L* values) of extracts of hulls of sesame and mustard with different solvents
Solvent used
SSHE-A
SSHE-B
SSHE-C
SSHE-D
MHE
Ethanol
73.03
72.07
77.92
76.66
74.08
Methanol
76.60
73.16
74.97
74.00
72.36
n-Propanol
77.16
76.61
73.57
74.37
Glacial Acetic acid
73.12
63.47
68.34
61.61
60.60
Acetone
77.80
76.06
78.18
72.71
75.04
Acetonitrile
75.66
74.23
74.60
77.70
74.43
1% NaOH
62.90
22.43
61.70
21.70
47.53
The NaOH extraction process is found to give the best results with the lowest L* values

11. Visible absorption spectra of the sesame seed hull extracts
No distinct peaks were identified for these extracts C D

12. Visible absorption spectra of the mustard hull extracts
Multiple peaks within the visible region
Extracts obatined using methanol, glacial acetic acid and 1% NaOH were similar to the sesame seed hull extracts

13. DPPH radical scavenging activity
Solvent used for
Extraction
DPPH scavenging capacity (%)
SSHE-A
SSHE-B
SSHE-C
SSHE-D
MHE
Ethanol
79.28
13.36
84.48
31.48 -
Methanol
88.95
27.62
83.87
40.44
75.88
n-Propanol
62.93
12.23
60.62
21.49
Glacial Acetic acid
89.85
54.57
89.97
88.45
77.73
Acetone
25.15
1.84
50.92
3.07
Acetonitrile
34.40
2.85
36.79
7.74
1% NaOH*
95.09
95.46
95.15
95.58
95.43
Synthetic antioxidant used for comparison of results
5 µg/ml
15 µg/ml
25 µg/ml
50 µg/ml
75 µg/ml
100 µg/ml
BHT
18.63
49.20
63.94
82.98
88.07
89.68
White varieties had a greater radical scavenging activity than the black varieties
The 1% NaOH extracts of all the varieties showed highest antioxidant activity

14. Ferric reducing/ antioxidant power
Solvent used for
Extraction
Fe2+ equivalent released (µM)
SSHE-A
SSHE-B
SSHE-C
SSHE-D
MHE
Ethanol
615.5
364.5
625
357
675
Methanol
618.5
375.5
609.5
386.5
850
n-Propanol
762.5
204.5
714
61.5
814.5
Glacial Acetic acid
602
385
594
565
836
Acetone 69
65.5
68.5 64
774
Acetonitrile
633.5
160.5
615
355.5
213.5
1% NaOH
3956.5
4876.5
4566.5
5626.5
2217.5
Synthetic antioxidant used for comparison of results
5 µg/ml
15 µg/ml
25 µg/ml
50 µg/ml
75 µg/ml
100 µg/ml
BHT
230
699
737
809
874.5
918.5
Fig: Standard curve obtained using various concentrations of FeSO4.7H2O
Significant Fe3+ reducing capacity was observed for extracts obtained with 1% NaOH

15. Inhibition of lipoxygenase by the NaOH extracts of sesame and mustard seed hulls
The inhibitory activity of the black varieties of sesame seed hull extract was found to be highest followed by mustard hull extract

16. Yield of the colourants in solid form Dry weight (mg colour/g hull)
Sample
Colour
Dry weight (mg colour/g hull)
SSHE-A
Brown
114.1
SSHE-B
Black
89.0
SSHE-C
35.5
SSHE-D
142.6
MHE
53.4
The colour pigment was precipitated, dried and refrigerated in the form of powder

17. Diagnostic tests for the pigments
S No
Test
SSHE (A)
Results
SSHE (D)
MHE 1
Water
Insoluble 2
Organic solvents 3
Alkaline reagents
Soluble 4
Colour
Brown
Black 5
Precipitation in 3N HCl
Negative
Positive 6
Reaction with oxidizing agent (H2O2)
Decolorized 7
Reaction for polyphenols (FeCl3 test)
Brown Precipitate 8
Reaction with ammoniacal silver nitrate solution 9
Reaction with KMnO4
Solution turned brown
The extracts were identified as Melanin-Like Pigments

18. UV-Visible absorption spectra
The absorption of light by melanin is maximum in the UV region and decreased progressively as the wavelength increases
A peak at ~280 nm indicates the presence of proteins associated with the extracts
Log of the optical density of melanin when plotted against wavelength gave linear curve with negative slope

19. FT-IR spectra Sample Match % SSHE-A 58.99 SSHE-B 46.06 SSHE-C 77.96
SSHE-D
81.81
MHE
77.93
(a) Synthetic melanin (b) SSHE-A (c) SSHE-B (d) SSHE-C (e) SSHE-D (f) MHE
FT-IR spectra of SSHE-D, SSHE-C & MHE showed significant match % when compared with synthetic melanin

20. Stability studies – Effect of temperature on colour
Refrigerated
Room temperature
37 °C
Fig: Absorption spectra showing pigment degradation with continuous exposure to different temperature conditions over a period of 36 days

21. Stability studies – Effect of light on colour
Refrigerated
RT - dark
RT - light
Fig: Absorption spectra showing pigment degradation with continuous exposure to different light conditions over a period of 36 days

22. Fig: Effect of temperature and light on the colour pigments, expressed as percentage absorbance over time
Absorption towards the higher wavelengths reduced significantly over time

23. SUMMARY
1% NaOH was found to be the best solvent for extraction of colours from sesame and mustard seed hull
The extracts were found to possess significant DPPH radical scavenging activity
The extracts also possessed Ferric reducing capacity comparable to BHT (synthetic antioxidant)
The antioxidant activity of the NaOH extracts was further validated using an enzyme model in-vitro (LOX inhibition)
The NaOH extracts were partially purified and pigment (Black & Brown coloured) was obtained in powder form
The pigment extracted was identified as Melanin-Like Pigments using chemical tests and spectroscopic properties (UV-Visible & FT-IR)
The samples were tested for stability – Refrigerated samples were found to be most stable while higher temperatures lead to loss of colour over time

24. WORK TO BE DONE Potential applications of the pigments isolated
Writing up of the report

25. REFERENCES
Alan Mortensen (2006). Carotenoids and other pigments as natural Colorants. Pure Appl. Chem. 78 (8):1477–1491.
Axelrod B, Cheesbrough TM & Laakso S (1981). Lipoxygenase from soybeans. Methods in Enzymology 71:
Benzie IFF & Strain JJ (1996). The Ferric Reducing Ability of Plasma (FRAP) as a measure of ‘‘Antioxidant Power’’: The FRAP Assay. Analytical Biochemistry 239: 70–76.
Brand-Williams W, Cuveliar ME & Berset C (1995). Use of free radical method to evaluate antioxidant activities. Lebensm.-Wiss. Technol/Food Science and Technology 28:
Delwiche JF (2012). You eat with your eyes first. Physiology & Behaviour 107 (4):
Ranganna S (2008). Handbook of Analysis and Quality Control for Fruit and Vegetable Products. New Delhi, Tata McGraw-Hill Publishing Company Limited. Edition 2:
Vallimayil J & Eyini M (2012). Physiochemical characterization of melanin pigment by Pleurotus djamor (Fr.) Boedijn. World Journal of Science and Technology 2(7):
Zengyu Yao, Jianhua Qi & Lihua Wang (2012). Isolation, fractionation and characterization of melanin-like pigments from chestnut (Castanea mollissima) shells. Journal of Food Science 77(6):

26. Thank You