Stabilization and Solubilization of Astaxanthin byStabilization and Solubilization of Astaxanthin byStabilization and Solubilization of Astaxanthin byStabilization and Solubilization of Astaxanthin byStabilization and Solubilization of Astaxanthin by Stabilization and Solubilization of Astaxanthin by

Inclusion Complex Formation with CyclodextrinInclusion Complex Formation with Cyclodextrin

Stabilization and Solubilization of Astaxanthin by Stabilization and Solubilization of Astaxanthin by

Inclusion Complex Formation with CyclodextrinInclusion Complex Formation with Cyclodextrin

S Ki 1 E h Ch 1 Ji i Y 1 M Ji I 2 d H J Ch 1 *Soyoung Kim1, Eunah Cho1, Jimin Yoo1, Man-Jin In2 and Hee Jeong Chae1,*

1Department of Food and Biotechnology and Center for Food Function and Safety1Department of Food and Biotechnology, and Center for Food Function and Safety,

Hoseo University, Asan 336-795, Korea 2Department of Human Nutrition and Food Science, Chungwoon University, Hongseong

1

350-701, Korea

Functional Food & Bioprocess Lab.b i o l a b. h o s e o . a c . k r

OH

OIntroductionIntroduction

C40H52O4 FW 596.9AstaxanthinAstaxanthin HO O

31

85

14

15

7'

8'1'

3'

▣ A carotenoid pigment found in certain marine animals and plants such as shrimp and algae

▣ similar structure to beta-carotene

Astaxanthin (550 times) >> Vit E

▣ protecting DNA, enzyme and cells against oxidative damage

▣ powerful antioxidant properties

▣ Chemical synthetic forms : free

Types of currently available astaxanthinTypes of currently available astaxanthin

Chemical synthetic forms : free

▣ Natural forms

- Krill and crayfish oils (~ 0.15 %), esterified

Ph ffi t ( 0 40 %) f

Dosage of astaxanthin : 2 mg/day

(FDA)

Dosage of astaxanthin : 2 mg/day

(FDA)

2

- Phaffia yeast (~ 0.40 %), free

- Hamatococcus species (1.5-3.0 %), esterified

(FDA)(FDA)

Functional Food & Bioprocess Lab.b i o l a b. h o s e o . a c . k r

Research purposeResearch purpose

The problem of astaxanthinThe problem of astaxanthin

Poor aqueous solubilitylimits its application

in aqueous phaselimits its application

in aqueous phase

Highly unsaturated molecule damaged by heat or lightdamaged by heat or light

WaterWater--solubilizationsolubilizationWaterWater--solubilization solubilization

and stabilization of astaxanthinand stabilization of astaxanthin

f f ti l f d & tif f ti l f d & ti

3

for functional food & cosmeticsfor functional food & cosmetics

Functional Food & Bioprocess Lab.b i o l a b. h o s e o . a c . k r

Material and method

Astaxanthin in dichlo:Ace. ■ Material - Astaxanthin

Cyclodextrin in DW, 50

Astaxanthin : CD = 1: 1 - 1: 200

- α-, β-, γ- cyclodextrin(CD) : Wako, Japan

- HPβ-, HPγ-CD : Sigma, USA

- Cyclo B(β-CD) : Corn products, USA

Stirring (50 , 7hr)

■ Analysis

- particle size : Scanning electron microscope(SEM)

- Change of structure : FT-IR

cooling

g

■ Water-solubility & stablility

evaporating× 1000

4

Inclusion complexInclusion complexAstaxanthin

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FTIR spectra and SEM : β-CD × 200Results

× 500

Results

β-CD HP-β-CDIC

β-CD

β-CDIC

Astaxanthin× 500

1000200030004000

ββ-CDAstaxanthin

5

Wave number(cm-1)

1000200030004000

β-CD I

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FTIR spectra and SEM : HP-β-CD × 2000FTIR spectra and SEM : HP β CD

Astaxanthin : CD = 1: 1

HP-β-CD HP- β -CDIC

HP-β-CD

× 2000

HP-β−CDIC(1:1)HPβ CDIC(1 200)

AstaxanthinHP-β-CDIC

× 2000

1000200030004000

HP-β−CDIC(1:200)AstaxanthinHP-β−CD

6

Wave number(cm-1)

1000200030004000

HP-β-CDIC

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FTIR t d SEM C l B(β CD)FTIR spectra and SEM : Cyclo B(β-CD)

× 1000

CycloB

CycloB IC

CycloB (β-CD)

CycloB(β-CD) IC (1:200)

Astaxanthin× 2000

1000200030004000

CycloB(β-CD)Astaxanthin

7

Wave number(cm-1)

1000200030004000

CycloB IC (β-CD)

Functional Food & Bioprocess Lab.b i o l a b. h o s e o . a c . k r

Water-solubility of inclusion complex in various pH and temperaturey p p p

β-CDIC is 13.6 times of free astaxanthin at 25

β-CDICAstaxanthin CycloB IC

β

β-CDIC is 110 times of free astaxanthin at pH 6.5

120

140 Astaxanthinβ−CDICCycloBIC

140 Astaxanthinβ-CDICC l B IC

110

(§¶/

§¢ in

DW

)

80

100

120 CycloBIC

13.58 (§¶/

§¢ in

DW

) 80

100

120 CycloB IC

2.35 2 2

78.8

Solu

bilit

y

20

40

60

5.17 2.911

3.13

1.61

Solu

bilit

y (

20

40

60

1

2.35 2.2

8.77.9

8Temperature(¡É)4 25 45

01 1.16 1

1

2 6.5 80

pH

1 1

Functional Food & Bioprocess Lab.b i o l a b. h o s e o . a c . k r

Effects of UV irradiation on the long-term stability of synthetic astaxanthin and inclusion complex

Effect of lightEffect of light FTIR spectraFTIR spectra

Astaxanthin

80

100

Astaxanthinβ-CDIC

1 0 0 02 0 0 03 0 0 04 0 0 0

U V i r r a d ia t io nD a r k

stab

ility

(%)

60

W a v e n u m b e r ( c m - 1 )

β-CDICRela

tive

20

40

W b ( 1 )

1 0 0 02 0 0 03 0 0 04 0 0 0

D a r kU V i r r a d i a t i o n

Storage time (days)

0 5 10 15 20 25 300

9

W a v e n u m b e r ( c m - 1 )

=> β-CDIC is 7 times compared with free astaxanthin(10%) after 14 days

Functional Food & Bioprocess Lab.b i o l a b. h o s e o . a c . k r

Effects of temperature on the long-term stability of synthetic astaxanthin and inclusion complex

45℃45℃25℃25℃4℃4℃

100

℃℃

100100

ve s

tabi

lity

(%)

60

80ve

sta

bilit

y (%

)

60

80

ve s

tabi

lity

(%)

60

80

Rel

ativ

20

40

Astaxanthinβ-CDIC

Rel

ativ

20

40

Astaxanthinβ-CDIC

Rel

ativ

20

40

Astaxanthinβ-CDIC

Storage time (days)

0 5 10 15 20 25 300

Storage time (days)

0 5 10 15 20 25 300

Storage time (days)

0 5 10 15 20 25 300

10

=> β-CDIC is stable than free astaxanthin at 45

Functional Food & Bioprocess Lab.b i o l a b. h o s e o . a c . k r

Effect of heating on the stability of synthetic astaxanthin and inclusion complexes

Effect of heatingEffect of heating FTIR spectraFTIR spectra

A hi

100

120Astaxanthinβ-CDICβ(cyclo)IC

Astaxanthin

stab

ility

(%)

60

80

W a v e n u m b e r ( c m -1 )

1 0 0 02 0 0 03 0 0 04 0 0 0

H e a t in gC o n t r o l

Rel

ativ

e s

20

40 β-CDIC

Heating condition

Control 65¡É, 30min 85¡É, 15sec 100¡É, 5sec0

20

W a v e n u m b e r ( c m - 1 )

1 0 0 02 0 0 03 0 0 04 0 0 0

C o n t r o lH e a t i n g

11=> β-CDIC is 9 times compared with free astaxanthin after heating(100 , 5sec)

Functional Food & Bioprocess Lab.b i o l a b. h o s e o . a c . k r

Effects of oxygen on the long-term stability of synthetic astaxanthin and inclusion complex

100

abili

ty (%

)

60

80

⇒ β-CDIC is very stable than free astaxanthin

Rel

ativ

e st

40

Astaxanthinβ-CDIC

after 28 days

0 5 10 15 20 25 300

20β CDIC

12

Storage time (days)

0 5 10 15 20 25 30

Functional Food & Bioprocess Lab.b i o l a b. h o s e o . a c . k r

Effects of pH on the long-term stability of synthetic astaxanthin and inclusion complex

pH 3pH 3 pH 5pH 5 pH 8pH 8

100100100Astaxanthinβ-CDIC

ve s

tabi

lity

(%)

60

80ve

sta

bilit

y (%

)

60

80

ve s

tabi

lity

(%)

60

80

Rel

ativ

20

40

Astaxanthinβ-CDIC

Rel

ativ

20

40

Astaxanthinβ-CDIC

Rel

ativ

20

40

Storage time (days)

0 5 10 15 20 25 300

Storage time (days)

0 5 10 15 20 25 300

Storage time (days)

0 5 10 15 20 25 300

13

=> β-CDIC is stable than free astaxanthin in acidic condition

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Summaryy

▣ FTIR spectrum, SEM

▣ W t l bilit f i l i l (β CDIC & C l B IC)

- Best host material for inclusion of astaxanthin => β-CD- Reaction ratio between astaxanthin and host molecule 1 : 200

▣ Water-solubility of inclusion complex (β-CDIC & CycloB IC)

- at 25 : 63.08 & 34.9 ug/ml 13.6 & 2.9 times of free astaxanthin

- at pH 6.5 : 127.4 & 91.2 ug/ml 110 & 78.7 times of free astaxanthin

- light : 7 times compared with free astaxanthin(10%) after 14 days

▣ Stability of inclusion complex (β-CDIC)

light : 7 times compared with free astaxanthin(10%) after 14 days

- Oxidation : very stable within 21 days

- heating : 9 times compared with free astaxanthin after heating(100 , 5sec)

14

- Storage temperature : stable than free astaxanthin at 45℃