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○ 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