NATURAL COLOURANTS EXTRACTION &

CHARACTERIZATION FROM OILSEEDS

Presented by

SRUTHI ROSE THOMAS

REG NO: 11MBT0022

II M.TECH BIOTECHNOLOGY

VIT UNIVERSITY

UNDER THE GUIDANCE OF

2

Dr Sridevi Annapurna SinghHead/ Sr. Principal Scientist

Protein Chemistry & Technology Department

CSIR-CFTRI, Mysore

Email: sridevi@cftri.res.in

Prof. Vino S.

Assistant Professor (Senior)

School of Bio Sciences and Technology

VIT University, Vellore

Email: vinokhanna22@gmail.com

EXTERNAL GUIDE INTERNAL GUIDE

PLACE OF WORK CSIR-Central Food Technology Research Institute

A Constituent laboratory of Council of Scientific & Industrial Research (CSIR)

Ministry of Science & Technology

Mysore, Karnataka

3

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 expectation

s of consumers

INTRODUCTION

4

Colour additive

Offset colour loss during

storage

Correct natural

variations

Enhance colours that

naturally occur

Provide colour to

colourless foods

Purposes of adding colour additives

5

General classification of food colours

Perceived to be safeUsed from time immemorial

More expensive and less stable

Stability and overall cost factorPotent carcinogens/mutagens

Threats to the environmentUse of unpermitted colours raise safety issues

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 6

MATERIALS & METHODS

7

8

9

RESULTSPigment extraction

The sesame and mustard seed hull extracts with 1% NaOH are visibly darker than the others

SSHE-A

SSHE-B

SSHE-C

SSHE-D

MHE

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Colour Attributes

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 74.97 76.61 73.57 74.37

Glacial Acetic acid

73.12 63.47 68.34 61.6160.60

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

Table: Colour readings (L* values) of extracts of hulls of sesame and mustard with different solvents

The NaOH extraction process is found to give the best results with the lowest L* values

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Visible absorption spectra of the sesame seed hull extracts

A

B

C

D

No distinct peaks were identified for

these extracts

12

Multiple peaks within the visible region

Extracts obatined using methanol, glacial acetic acid and 1% NaOH were similar to the sesame seed hull extracts

Visible absorption spectra of the mustard hull extracts

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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

DPPH scavenging capacity (%)  

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

DPPH radical scavenging activity

White varieties had a greater radical scavenging activity than the black varieties

The 1% NaOH extracts of all the varieties showed highest antioxidant activity

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Solvent used for

Extraction

Fe2+ equivalent released (µM)  

SSHE-

A

SSHE-

B

SSHE-

C

SSHE-

DMHE  

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

Fe2+ equivalent released (µM)  

5 µg/ml15

µg/ml

25

µg/ml

50

µg/ml

75

µg/ml

100

µg/ml

BHT 230 699 737 809 874.5 918.5

Ferric reducing/ antioxidant power

0 200 400 600 800 10000.0

0.5

1.0

1.5

2.0 R2=0.998y=0.002x+0.027

Abso

rban

ce a

t 593

nm

Concentration of Fe2+

Fig: Standard curve obtained using various concentrations of FeSO4.7H2O

Significant Fe3+ reducing capacity was observed for extracts obtained with 1% NaOH

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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

0 5 10 15 20 25 30 350

10

20

30

40

50

60

70

R² = 0.995470836449747

R² = 0.97882791322974

R² = 0.985369316815291

R² = 0.959562828930303

SSHE-A

Linear (SSHE-A)

SSHE-B

Volume of inhibitor (µL)

Lip

oxy

ge

na

se in

hib

itio

n (

%)

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Yield of the colourants in solid form

Sample Colour Dry weight (mg colour/g hull)

SSHE-A Brown 114.1

SSHE-B Black 89.0

SSHE-C Brown 35.5

SSHE-D Black 142.6

MHE Brown 53.4

The colour pigment was precipitated, dried and refrigerated in the form of powder

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Diagnostic tests for the pigments

S No Test 

SSHE (A)

Results

SSHE (D)

 

MHE

1 Water Insoluble Insoluble Insoluble

2 Organic solvents Insoluble Insoluble Insoluble

3 Alkaline reagents Soluble Soluble Soluble

4 Colour Brown Black Brown

5 Precipitation in 3N HCl Negative Positive Negative

6 Reaction with oxidizing

agent (H2O2)

Decolorized Decolorized Decolorized

7 Reaction for polyphenols

(FeCl3 test)

Brown

Precipitate

Brown

Precipitate

Brown

Precipitate

8 Reaction with ammoniacal

silver nitrate solution

Negative Positive Negative

9 Reaction with KMnO4Solution turned

brown

Solution turned

brown

Solution turned

brown

The extracts were identified as Melanin-Like Pigments

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UV-Visible absorption spectra

390 440 490 540 590

-3

-2.5

-2

-1.5

-1

-0.5

0

f(x) = − 0.00325044935064935 x + 0.540246103896103R² = 0.999545587654161f(x) = − 0.00332508831168831 x + 0.283009393939394R² = 0.952144442913692

f(x) = − 0.00239203766233766 x + 0.418083116883117R² = 0.926438681743635

f(x) = − 0.00403507402597403 x + 0.520034155844158R² = 0.967695104373952

f(x) = − 0.00257254545454546 x + 0.737247489177491R² = 0.938843669649951

f(x) = − 0.0054498103896104 x + 0.804002337662343R² = 0.985035630044019

SSHE-A Linear (SSHE-A)SSHE-B Linear (SSHE-B)SSHE-C Linear (SSHE-C)

Wavelength (nm)

Lo

g a

bs

orb

an

ce

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

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FT-IR spectra

Sample Match %

SSHE-A 58.99

SSHE-B 46.06

SSHE-C 77.96

SSHE-D 81.81

MHE 77.93

FT-IR spectra of SSHE-D, SSHE-C & MHE showed significant match % when compared with synthetic melanin

(a) Synthetic melanin (b) SSHE-A (c) SSHE-B (d) SSHE-C (e) SSHE-D (f) MHE

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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

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Refrigerated

RT - lightRT - dark

Fig: Absorption spectra showing pigment degradation with continuous exposure to different light conditions over a period of 36 days

Stability studies – Effect of light on colour

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Absorption towards the higher wavelengths reduced significantly over time

Fig: Effect of temperature and light on the colour

pigments, expressed as percentage absorbance over

time

1 2 8 15 22 29 360

20

40

60

80

100

120

Days

Pe

rce

nta

ge

co

lou

r (A

bs

at

45

0 n

m)

1 2 8 15 22 29 360

20

40

60

80

100

120

Days

Pe

rce

nta

ge

co

lou

r (A

bs

at

65

0 n

m)

1 2 8 15 22 29 360

20

40

60

80

100

120

room temperature

refrigerated

37 degrees

dark

light

Days

Pe

rce

nta

ge

co

lou

r (A

bs

at

55

0 n

m)

SUMMARY

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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

WORK TO BE DONE

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Potential applications of the pigments isolated

Writing up of the report

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: 441-451.

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: 25-30.

Delwiche JF (2012). You eat with your eyes first. Physiology & Behaviour 107 (4): 502-504.

Ranganna S (2008). Handbook of Analysis and Quality Control for Fruit and Vegetable Products. New Delhi, Tata McGraw-Hill Publishing Company Limited. Edition 2: 497-525.

Vallimayil J & Eyini M (2012). Physiochemical characterization of melanin pigment by Pleurotus djamor (Fr.) Boedijn. World Journal of Science and Technology 2(7): 76-80.

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): 671-676.

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Thank You