○ K. Harada1, T. Fukuda1, R. Wada1, R. Hiraoka1, S. Yokoyama1,

K. Shimada2, N. Kinashi3 and H. Takamura3, ○原田和樹1、福田 翼1、

和田律子1、平岡亮人1、横山 駿1、島田和子2、木梨直子3、高村仁知3

（1National Fisheries Univ., 2Yamaguchi Prefectural Univ., 3Nara Women’s

Univ.,1 水大校・食品科学、2山口県立大学, 3奈良女子大学）

Annual meeting of Japan Society for Bioscience, Biotechnology, and Agrochemistry on 27th March,2013 in Tohoku University at Sendai city.

日本農芸化学会2013年度大会, 2013年3月27日、於：東北大学（仙台市）

Antioxidant activities on the processing product

and fish sauce made from sea urchin gonad

measured by using H-ORAC and L-ORAC methods

（ウニ加工品並びにウニ魚醤のイン・ビトロ系における抗酸化性）

Background

Edible raw sea

urchin gonad

Ethanol-soaked

sea urchin gonad

processing

product in

Shimonoseki city

Fermented fish

sauce of sea

urchin gonad

made from our

university

Ｏｂｊｅｃｔｉｖｅ

We already reported in this annual meeting that the ethanol-soaked sea urchin gonad processing product indicated antioxidant activity, i.e., H-ORAC (Hydrophilic Oxygen Absorbance Capacity) high value. In this annual meeting, we researched the total ORAC value, i.e., H-ORAC value + L (Lipophilic)-ORAC value among raw sea urchin gonad, ethanol-soaked sea urchin gonad product and fermented fish sauce from sea urchin gonad.

Exactly measured sample of about 2 g

Ultrapure water

Homogenization using Polytron

Centrifugation (4℃, 1,970×g, 30 min)

⑥

Preparation of sample for H-ORAC

20 ml

Supernatant (Sample for ORAC measurement)

Freeze-dried sample

Exactly measured sample of about 0.1 g

Hexane

Agitation using vortex instrument for 5 min

Centrifugation (4℃, 1,450×g, 15 min)

⑥

Preparation of sample for L-ORAC

1 ml

Vacuum drying of Supernatant using vacuum centrifuge

Freeze-dried sample

Twice

Addition to 250 μl acetone

+ 7% methylated ß-cyclodextrin (MCD)

HO O

O

CO２H

Fluorescein

Peroxyl Radical (ROO・)

AAPH（Generator of Radical）

37℃

Degradation

Vanishment of fluorescence

Measurement Time

Flu

ore

sce

nt

Inte

nsi

ty

Sample or

trolox

Blank

AUCsample – AUCBlank ⇒ ORAC value (µmol TE/100g）, AUC = area under kinetic curve

TE ⇒ Trolox (水溶性ビタミンE誘導体)Equivalent（当量） （Modified quotation from Watanabe et al.: 化学と生物, 47, 237-243, 2009 ）

Principle of ORAC Method

Net AUCsample

Measurement of ORAC

Mithras LB940 multimode microplate

reader (Berthold Technologies GmbH &

Co. KG, Germany)

Diluted sample solutions, positive control (trolox

solution) or negative control (assay buffer) 20 μl on a well of microplate

Fluorescein solution 200 μl

AAPH solution

75μl, automatic

injection

Measurement of fluorescent intensity

Measurement interval: 2 min Number of times of Measurement: 46 times （90 min） Excitation wavelength: 485 nm Measurement wavelength: 520 nm Detection angle: Top

Heated at 37℃ in the plate reader

Results of H-ORAC

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

Raw Raw added to

salit and EtOH

EtOH-soaked

product (A

company)

EtOH-soaked

product (B

company)

EtOH-soaked

product (C

company)

EtOH-soaked

product

(Average)

EtOH-soaked

product with

'Kazunoko'

(C company)

Fermented

fish sauce (D

company)

H-O

RA

C V

alu

e (

μm

ol TE

/100

g)

n = 3

Results of L-ORAC

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

Raw Raw added

to salit and

EtOH

EtOH-soaked

product (A

company)

EtOH-soaked

product (B

company)

EtOH-soaked

product (C

company)

EtOH-soaked

product

(Average)

EtOH-soaked

product with

'Kazunoko'

(C company)

Fermented

fish sauce (D

company)

L-O

RA

C V

alu

e (

μm

ol TE

/100 g

)

n = 3

Results of Total-ORAC

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

Raw Raw added

to salit and

EtOH

EtOH-soaked

product (A

company)

EtOH-soaked

product (B

company)

EtOH-soaked

product (C

company)

EtOH-soaked

product

(Average)

EtOH-soaked

product with

'Kazunoko'

(C company)

Fermented

fish sauce (D

company)

L-ORAC

H-ORAC

Tota

l-O

RA

C V

alu

e (

μm

ol TE

/100 g

)

n = 3

⑭

1. Total–ORAC value of raw sea urchin gonad and sea urchin gonad

processing product before maturation were near 4,000 μmol TE/100 g

as T-ORAC, the breakdown between H-ORAC and L-ORAC was near

equal.

2. It was assumed that in raw sea urchin gonad α-tocopherol and β-

carotene contributed to the high L-ORAC value, ascorbic acid

contributed to high H-ORAC value.

3. Total-ORAC value of the ethanol-soaked sea urchin gonad processing

product decreased to average 60% as compared with raw sea urchin

gonad, the decreased value was almost L-ORAC value.

4. Fermented fish sauce made from sea urchin gonad indicated high T-

ORAC value of about 4,500 μmol TE/100 g, the almost value indicated

H-ORAC value. It assumed that the ‘Koji’ in fish sauce contributed to T-

ORAC value in part.

This work was supported by ‘Yamaguchi Prefectural Uni Cooperative Society

in Japan’. Also, this work was supported in part by a Grant-in-Aid for

JSPS KAKENHI Grant Number 23500949.

Conclusion