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BUENOS AIRES 2010.9.21

The Novel Hair Dyeing Technology by Using Melanin Precursor Prepared by an

Aspergillus Tyrosinase

Traditional Japanese Sake Brewers

Koike, Kenzo 1 & Hata, Yoji 21 Kao Corporation, Tokyo, Japan.

2 Gekkeikan Sake Company, Kyoto, Japan.

1. Background of Our Hair Research

Hair Research encountered Biotechnology in 2001.

Relationship between melanin and color of the hair

- Melanin type and amounts affect color and brightness of the hair -

Brown

Blond

Black

Red

Less Melanin

Bright

Dark

MoreMelanin

Eumelanin RichPheomelanin Rich

Asian hair has more Eumelanin

Relationship between melanin and color of the hair

- Melanin type and amounts affect color and brightness of the hair -

Brown

Blond

Black

Red

Less Melanin

Bright

Dark

MoreMelanin

Eumelanin RichPheomelanin Rich

Asian hair has more Eumelanin

Relationship between melanin and color of the hair

- Melanin type and amounts affect color and brightness of the hair -

Eumelanin Rich

Brown

Blond

Black

Red

Pheomelanin Rich

Asian hair has more Eumelanin

Less Melanin

Bright

Dark

MoreMelanin

The difference between Gray and Black Hair

- The difference is only melanin -Male hair sample from a Japanese (50’s)

No melanin Melanin Granules

Gray hair Black hair

Melanogenesis Eumelanin biosynthesis

• Eumelanin is a polymer made from Tyrosine or DOPA.• This pathway is initiated by Tyrosinase oxidation reaction.• Melanin precursors are converted to Melanin by Oxygen.

NH2HO2C

OHOH

NH2HO2C

OH

NHHO2C

OHOH

OO

NH2HO2C

NH

OO

HO2C

NH

OHOH

NH

OO

NH

OO

HO2C

NH

OHOH

HO2C

+

-

tyrosinaseO2

Eumelanintyrosine

DOPA

dopaquinone

cyclodopa dopachrome

5,6-Dihydroxyindole

5,6-Dihydroxyindole-2-carboxylic acid

dopaquinonedopa

（DHICA)

tyrosinase

tyrosinase

O2

O2

O2

CO2

G. Prota., “Progress in the chemistry of melanins and related metabolites.” Med. Res. Rev. 8,525-556, 1988

Melanogenesis Eumelanin biosynthesis

• Eumelanin is a polymer made from Tyrosine or DOPA.• This pathway is initiated by Tyrosinase oxidation reaction.• Melanin precursors are converted to Melanin by Oxygen.

NH2HO2C

OHOH

NH2HO2C

OH

NHHO2C

OHOH

OO

NH2HO2C

NH

OO

HO2C

NH

OHOH

NH

OO

NH

OO

HO2C

NH

OHOH

HO2C

+

-

tyrosinaseO2

Eumelanintyrosine

DOPA

dopaquinone

cyclodopa dopachrome

5,6-Dihydroxyindole

5,6-Dihydroxyindole-2-carboxylic acid

dopaquinonedopa

（DHICA)

tyrosinase

tyrosinase

O2

O2

O2

CO2

Melanin precursors

G. Prota., “Progress in the chemistry of melanins and related metabolites.” Med. Res. Rev. 8,525-556, 1988

A new idea for hair dyeing system with melanin (precursor)

Melanin precursor Melanin

NH

OH

OH

NH

NH

NH

O

O

O

O

O

O

Into Hair(Cuticle)

O2 (Air)

Benefits・ Melanin and its precursors are biological substances.・ The Melanin precursor is small enough to penetrate hair.・ It converts to melanin by exposure to air.・ Melanin has a natural dark color.

Issues： The Melanin precursor is very unstable.None of the natural compounds were industrially available.

By the way, we have traditional biotechnology in Japan.

Fungus and yeast have been usedin food manufacturing and various fermentation processes

in Japan for over 1000 years.

Hair Research encountered Biotechnology in 2001.

02. Preparation of Melanin Precursor using fungus

TyrosinaseManufacturing new ingredient from natural resources using same process as in nature.

Collaboration between Kao & Gekkeikan

Japanese Sake manufacturingJapanese Sake manufacturing 13

Japanese Traditional Biotechnology Rice

Saccharomyces cerevisiae (yeast)

Aspergillus oryzaefungas)

酒造り (SAKE Dukuri) Alcohol Fermentation

酵母（KOBO) 麹（KOJI)

Clean Water

日本酒Japanese SAKE

Traditional Brandfrom 17th century(400 yrs)

Black Koji(Irregular)

Koji (Normal)

There was a color problem,occasionally, during 1950s and1960s.

Fungal melanin formation in the process of fermentation.

Black Koji(Irregular)

Koji (Normal)

strained sake

sludge

＜＜SAKE FermentationSAKE Fermentation＞＞Rice

Steamed rice

KOJIYeast

seed

MOROMI

SAKE

seed

Water

Melanin formation

As a result, the Black Koji was identified, and the Melanin formation mechanism was found.

Tyrosinase from Aspergillus oryzae makes melanin.

1.Obata et al, “Cloning of a Novel Tyrosinase-Encoding Gene (melB) from Aspergillus oryzae and Its Over expression in Solid-State Culture (Rice Koji)” J. Biosci. Bioeng. 97400-405 2004.

Tyrosinase was found to be expressed in Black Koji.

Tyrosinase (enzyme)

The Japanese Sake company found atyrosinase strongly expressed in

Black Koji.

Black Koji

Koji : Fermented rice with Aspergillus oryzae

Solid SurfaceCulture

LiquidCulture

New Collaboration research was started

1.Obata et al, “Cloning of a Novel Tyrosinase-Encoding Gene (melB) from Aspergillus oryzae and Its Over expression in Solid-State Culture (Rice Koji)” J. Biosci. Bioeng. 97400-405 2004.

Melanogenesis Eumelanin biosynthesis

• Eumelanin is a polymer made from Tyrosine or DOPA.• This pathway is initiated by Tyrosinase oxidation reaction.• Melanin precursors are converted to Melanin by Oxygen.

NH2HO2C

OHOH

NH2HO2C

OH

NHHO2C

OHOH

OO

NH2HO2C

NH

OO

HO2C

NH

OHOH

NH

OO

NH

OO

HO2C

NH

OHOH

HO2C

+

-

tyrosinaseO2

Eumelanintyrosine

DOPA

dopaquinone

cyclodopa dopachrome

5,6-Dihydroxyindole

5,6-Dihydroxyindole-2-carboxylic acid

dopaquinonedopa

（DHICA)

tyrosinase

tyrosinase

O2

O2

O2

CO2

Melanin precursors

Plant-originated DOPA

Which should be produced?

The dyeing ability of melanin precursors

0

5

10

15

20

25

30

DHICA DHIoleCA DHIole

染色

性（Δ

E）

Dye

ing

abili

ty

DHI

DHICA：5,6-Dihydroxyindline 2-carboxylic acidDHIoleCA ：5,6-Dihydroxyindole 2-carboxylic acidDHI ：5,6-Dihydroxyindole (DHI)Dyeing condition： 3%Ammonia, 0.1% each compound, 3 times

The total system for melanin precursor production

• The product is adjusted forcosmetics materials.

• Tyrosinase is produced byfermentation.

• Dopa is converted toDihydroxyindole in tyrosinaseprocess.

culturewash

catalyst solution

solution of melanin precursor

solid-liquid separation

enzyme reaction

concentration and adjustment

5,6-Dihydroxyindole solution

Production of catalyst(tyrosinase)

DOPAProduction of

melanin precursor

Adjustment of solution

Melanin precursor production by fungus Tyrosinase

DOPA (Plant-origin)

Melanin precursor (DHI)

NH

OH

OH

Melanin Precursor production

Melanin

DOPA

precursor

Melanin precursor was produced from Plant-originated Dopa.

• DOPA (the starting material) is of Plant-origin.• Melanin precursor was produced by using the same process as in nature.• The Melanin precursor is a sustainable compound.

03. Application of Melanin Precursor to Hair Dye

Development of a natural coloring product for gray hair.

New Concept of Natural Hair Dye

• The new dyestuff, 5,6-dihydroxyindole,has been manufactured from natural resources using a similar process to nature.

• It ensures “True Natural Hair Color” by recovering color by melanin.

• Air oxidation system without H2O2– Gentle to hair: less damage– Easy to use: no mixing, no long waiting.

Formulation of the Hair Dye

Foam type: Ease of UseAerosol: maintain stability by Oxygen shutoff

Foamer(surfactant)

Dihydroxyindole(New Dyestuff)

Alkali Solvents

ThickenersAntioxidants

How to Use: 20g of the product was applied to hair. After standing for 5 min. hair is washed with shampoo.

The relationship between DHI conc. and Dyeing ability

0

10

20

30

40

50

60

0 1 2 3 4 5 6

Cycle number

Dye

ing

abili

ty (

△E)

DHI 0.3%

DHI 0.1%

Dyeing image

DHI: 0.1% or 0.3%＋3％ ammoniaProcedure: Chinese grey hair, RT, 5min, shampooed and dried,This treatment was repeated as indicated.

DHI at 0.1 - 0.3% dyed gray hair gradually.

The effect of alkaline and pH in Dyeing ability

pH 10 with ethanolamine was most effective

アルカリとpHの影響　３回染色

0

5

10

15

20

25

30

35

40

45

50

9 9.5 10 10.5 11

ｐH

染色

性⊿

E

モノエタノールアミン

48%苛性ソーダ

炭酸ナトリウム

炭酸グアニジン

28%アンモニア

Dye

ing

abili

ty

Ethanolamine 48% sodium hydroxidesodium carbonateguanidine carbonate

28%ammonia

Melanin precursor in the formulation was stable for 6 months

0

20

40

60

80

100

120

0 20 40 60 80 100

保存日数(日)

染料

（％

）

5℃

RT

40℃

storage period (days)

DH

I

Brown and Ash shades were achieved.

Brown shade Ash shade

DHI: 0.3%＋1％ Ethanolamine + AntioxidantsProcedure: Chinese grey hair, RT, 5min, shampooed and dried,This treatment was repeated up to 5 times.

Melanin precursor is converted to Melanin by air

Dyeing ability: ±

5 min

Just after discharging

Melanin formation by air

Dyeing ability:＋＋

Easy to use: no mixing, no long waiting period

The mechanism for dyeing with melanin precursor

Cross section1. Melanin precursor can penetrate into the cuticle of hair.2. Melanin is formed by exposure to air.3. Gray hair is gradually dyed by step-wise use (3-5 times).4. The final color of hair then looks more natural.

Cortex

Cuticle

Gray hair 3 times 5 times

A partDyeing image

How to Use・ Treatment・ 5 min.・ Shampoo

Dyeing ability

Panelist results

Before 1st 3rd 5th

・ Each repeated treatment brought natural change.・ The color of hair became very natural.・ The color only faded after application was stopped.

Advantages of the new technology

Hair damage levelSkin stainingCysteic acid [mol％]

0.00

0.50

1.00

1.50

2.00

未処理 ﾒﾗﾆﾝ前駆体5回

ﾍｱｶﾗｰ　5回 ﾍｱﾏﾆｷｭｱ5回Non

TreatedDirect Dye

Hair Dye

New Technology

NewTechnology

Direct dye system

Skin → 30 min. → Washing with water

Hair → Hydrolysis → A.A. analysis (HPLC)

Conclusions

A New Natural Hair Dye

• The new dyestuff from natural resources– DHI has been manufactured using a similar process

to nature.

• Restoring Melanin into gray hair.– Recovery of natural color by melanin

• Gentle to hair and Easy to use – Air oxidation system without H2O2

Gracias mucho!!

Thank you for your attention!

Gracias mucho!!Argentina!