FISHERIES SCIENCE

2002; 68: 430435

Original Article

1,1-Diphenyl-2-picrylhydrazyl (DPPH)
radical scavenging by protein hydrolyzates
from tuna cooking juice
Chia-Ling JAO1 AND Wen-Ching KO2*
1

Department of Food Science and Technology, Tung-Fang Junior College of Technology and
Commerce, Kaohsiung 829 and 2Department of Food Science, National Chung-Hsing University,
Taichung 402, Taiwan 202
ABSTRACT: Protease XXIII, from Aspergillus oryzae, was used to hydrolyze tuna cooking juice at
37C for up to 6 h. The hydrolyzate obtained at the degree of hydrolysis of 25.68% (after hydrolysis
for 2.5 h) displayed the highest 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging effect, reaching
82.19%. Six major fractions (A, B, C, D, E, and F) of this hydrolyzate were obtained by Sephadex
G-25 column chromatography using a 0.05 M phosphate buffer (pH 6.5) as the mobile phase. All six
fractions displayed a scavenging effect for the DPPH radical, but the scavenging effect was only
obvious in two fractions (B and C). After the solid content of hydrolyzates was concentrated from one
to five times, the scavenging effect of the DPPH radical increased from 17% to 75% for fraction B,
and from 13% to 66% for fraction C. Seven anti-oxidative peptides were isolated from the hydrolyzates
(mixture of B and C fractions) by reversed-phase HPLC. The peptide sequences comprised four to
eight amino acid residues, including Val, Ser, Pro, His, Ala, Asp, Lys, Glu, Gly, or Tyr.
KEY WORDS: 1,1-diphenyl-2-picrylhydrazyl radical, protein hydrolyzate, scavenging effect,
tuna cooking juice.

INTRODUCTION
As well as its primary function of supplying
nutrients and energy, protein also has numerous
tertiary functions relating to physiological regulation.1 Various physiological activities have been
found in the hydrolyzates derived from the proteolytic hydrolysis of various food proteins.2 Among
them, a number of functional peptides derived
from milk and soybean have already been identified, and may have anti-oxidant properties,3
reduce blood pressure,4 and regulate cholesterol
in serum.5 Natural proteins can thus be considered
as physiologically functional food.6 Numerous
protein sources, including aquatic species, eggwhite albumin, soy protein, and fish muscle, have
been confirmed to act as anti-oxidants preventing
the peroxidation of lipids or fatty acids.711 The
anti-oxidative activities of amino acids or peptides
*Corresponding author: Tel: 886-4-2287-1690. Fax: 886-42287-3208. Email: wcko@dragon.nchu.edu.tw
Received 18 July 2001. Accepted 22 October 2001.

have thus been investigated to reveal the antioxidative mechanism of protein hydrolyzates.
Successful hydrolysis of proteins also depends
on proteases. Microbial proteases are usually used
for the hydrolysis of proteins. Aspergillus oryzae,
which has been used widely in fermented food, is
a good source of proteases because it produces
more than 10 endo- and exoproteases with a wide
range of optimum pH.12
Cooking is crucial to canned tuna processing,
resulting in approximately 4% water-soluble
protein in cooking juice, including sarcoplasmic
proteins and collagen,13 which are then discarded
in the waste water.14 Recovering these proteins
and utilizing them as foodstuffs is essential to
enhancing their value and reducing waste watertreatment costs. Although several methods of
recovering fish water-soluble proteins from the
waste water of seafood processing plants have
been investigated,13,1517 the recovered proteins
are generally used in animal feeds and fertilizers.
In addition to preparing tuna cooking juice
hydrolyzate using A. oryzae protease, this work

DPPH scavenging

FISHERIES SCIENCE

investigated the 1,1-diphenyl-2-picrylhydrazyl

(DPPH) radical-scavenging capacity of the hydrolyzate. The biologically active substances created
by the proteolytic digestion were also identified.
MATERIALS AND METHODS
Materials

Tuna cooking juice was obtained from a canned

tuna processing plant in Chiayi, Taiwan. Tuna meat
was steamed (100105C) for 34 h, after which the
hot cooking juice was collected and sealed in 400 g
polyethylene bags and then transferred to the
laboratory and stored at 4C overnight. The cooled
cooking juice was then filtered through two layers
of gauze to remove any floating fats and solids, and
the filtrate was collected and stored at 30C until
required for use. The DPPH and protease XXIII
derived from A. oryzae were purchased from Sigma
Chemical Co. (St Louis, MO, USA), the Sephadex G25 column was produced by Pharmacia (Uppsala,
Sweden), and all other chemicals used were
analytical grade products.

Enzymatic hydrolysis

A volume of 2 mL of 0.5% protease solution was

added to 50 mL of treated cooking juice,18 and
aliquots were removed at intervals of 06 h at
37C. Digested hydrolyzates were heated in boiling
water for 5 min to inactivate the protease.
The hydrolyzates were then centrifuged (5000 g,
10 min), and the resultant supernatants were
freeze-dried and stored at 18C until required for
use.3 The approximate degree of hydrolysis of the
tuna cooking juice was determined by a modification of the method described by Boudrant and
Cheftel.19

431

buffer (pH 2.2). Simultaneously, the protein

hydrolyzate was also hydrolyzed in 6 N HCl at
110C for 24 h in a vacuumed sealed tube. Amino
acids obtained without HCl hydrolysis were
termed free amino acids, and the differences
between the values of amino acids with HCl
hydrolysis and free amino acids were termed combined amino acids. Amino acids were measured
using a ninhydrin reagent with an amino acid standard, which was applied using a high-performance
amino acid analyzer (Beckman 6300, Fullerton, CA,
USA) with the following settings: column, cation
exchange resin; analysis temperature, 5070C;
cuvette path length, 12 mm; citrate buffer rate, 20
mL/h; and ninhydrin rate, 10 mL/h.
Scavenging effect on 1,1-diphenyl-2picrylhydrazyl radical
The DPPH radical, which is organic, stable, and has
been proven to be absorptive at 517 nm, is a convenient reagent for the radical scavenging assay,
which is independent of any enzyme.21 When this
compound is stabilized by accepting an electron
or hydrogen radical, its absorptive characteristics
vanish.22 Unlike laboratory-generated free radicals
such as superoxide radical and hydroxyl radical,
DPPH has the advantage of being free of additiveinduced complications such as metal chelation
and enzyme inhibition.
The scavenging effect of the hydrolyzate on the
DPPH radical was estimated using a modification
of Yen and Wus method.23 A volume of 1 mL of
hydrolyzates were first added to a methanolic
solution (0.05 mL) of DPPH radical (with a final
concentration of 10 mM). The mixture was then
shaken vigorously and allowed to stand for 30 min,
after which the absorbance of the resulting solution was measured at 517 nm using a spectrophotomemter (Hitachi U-2000; Hitachi, Tokyo, Japan).
The DPPH scavenging effect (%) was calculated as:
[(OD517 control OD517 sample)/(OD517 control)] 100%. (1)

Analysis of free and combined amino acids

Free and combined amino acids were determined

following the procedure of Moore and Stein.20
Protein hydrolyzate (15 mL) was precipitated with
10 mL of 15% trichloroacetic acid (TCA). After
setting for 30 min, the hydrolyzate was centrifuged
(5000 g) at 4C for 10 min, and this process was
repeated three times to extract the precipitate.
Next, an equal volume of ether was added to the
extracted supernatant to remove the TCA. The
supernatant was then freeze-dried after being
frozen overnight at 30C, and added to a 25-mL
volumetric flask containing 10 mL of 0.2 M citrate

The control used was 0.1% TFA in deionized water,

and the scavenging effect was measured as
described earlier. All tests and analyses were
repeated three times and the results averaged.
Purification of anti-oxidant peptide with
scavenging effect
The anti-oxidant peptide was purified using
column chromatography, as described by Astawan
et al.,1 but with slight modifications. Approximately
0.27 g of freeze-dried powder (obtained from the
protease hydrolyzate) was diluted in 2 mL of dis-

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432

tilled water, fractionated by gel filtration on a

Sephadex G-25 column (2.5 cm 50 cm; Pharmacia, Uppsala, Sweden), and eluted with 0.05 M
phosphate buffer (pH 6.5). Each 5 mL fraction was
collected at a flow rate of 40 mL/h, and absorbance
at 280 nm and the DPPH radical scavenging activity were measured for all fractions.
Samples exhibiting anti-oxidative activity were
further purified using high-performance liquid
chromatography (HPLC; Hitachi), and 4 mL of the
active sample was collected and dried. The residue
was dissolved in 0.5 mL of 0.1% TFA, and the substance was then injected into an RP-18(e) column
(4 mm i.d 250 mm length; Merck, Gibbstown, NJ,
USA), equilibrated with 0.1% TFA, and eluted using
a linear gradient of acetonitrile (020%/50 min) in
0.1% TFA at a flow rate of 0.7 mL/min. The eluted
active fractions were concentrated using a centrifugal concentrator (VaCO I; Zirbus, Hilfe Gottes,
Germany), and then applied to a MICRA NPS RP-18
column (4.6 mm 33 mm; MICRA, Northbrook, IL,
USA). The gradient was then eluted with 035%
acetonitrile at a flow rate of 0.8 mL/min to obtain
purified peptides with a scavenging effect. Each
chromatograph was monitored for its absorbance
of ultraviolet light at 210 nm. Finally, the amino
acid of the peptide was sequenced using a 477-A
protein sequencer chromatogram (Biosystems,
Foster, CA, USA).
RESULTS AND DISCUSSION

C-L Jao and W-C Ko

the hydrolyzates scavenging activities on the

DPPH radical were measured over a 6-h period
(Fig. 1). The initial DPPH scavenging activity of
tuna cooking juice was approximately 18%, and
scavenging effect did not increase with increasing
degree of hydrolysis, whereby hydrolysis proceeded when the degree of hydrolysis reached 32%
at 6 h, and no increase in activity being observed
after 2.5 h (degree of hydrolysis was 25.68%).
Several investigations on protein hydrolyzates
have frequently reported their anti-oxidative properties.7,8,24 The results of these investigations
suggest that the anti-oxidative activity of protein
hydrolyzates is influenced by the levels of amino
acids and peptides, which are primarily influenced
by enzyme hydrolysis.
Isolation of 1,1-diphenyl-2-picrylhydrazyl
radical scavenging activity peptides
To characterize the DPPH radical scavenging activity of peptides derived from tuna cooking juice, the
protein was hydrolyzed with protease XXIII for
2.5 h, and the hydrolyzate (AOH) was then separated by gel filtration chromatography. Figure 2
displays the fraction profile for AOH. Although
continuous, the profile clearly contained six major
sections, which are labeled A (fraction number
1317), B(1837), C(3845), D(4651) E(5260), and
F(6173). In the present study, we used the concentrated portion instead of the concentrated

1,1-Diphenyl-2-picrylhydrazyl scavenging
effect at different degrees of hydrolysis
AOHA

Protease XXIII was screened for the hydrolysis of

tuna cooking juice. The degree of hydrolysis and

40

AOH

100

80

30
25

60

20
40

15
10

20

Scavenging effect (%)

Degree of hydrolysis (%)

35

Absorbance (OD 280 nm)

A
1.5

F
D

1400

0.5
390
4500
0

0
0

0
0

30

60

90 120 150 180 210 240 270 300 330 360 390

Time (min)

Fig. 1 Time-courses of hydrolysis and the scavenging

effect of tuna cooking juice treated with protease XXIII
at 1 : 25(v/v) of enzyme to substrate. The concentration
of protein was 4% and that of enzyme was 0.5%. ()
Degree of hydrolysis; () scavenging effect.

10

20

30

40

50

60

70

80

Fraction NO
Fig. 2 Elution profile of protease XXIII hydrolyzate
separated by using gel filtration on a Sephadex G-25
column. The column (2.5 cm 50 cm) was equilibrated
and eluted using 0.05 M phosphate buffer (pH 6.5) at a
flow rate of 40 mL/h. AOHA, mixture of most active
peptides, which were obtained from fractions B and C.

FISHERIES SCIENCE

DPPH scavenging

90

90

Concentrated once

80

Concentrated twice

70

Concentrated three times

60

Concentrated four times

50

Concentrated five times

40
30
20
10
0
A

AOV

Absorbance (OD 210 nm )

AOIV

20

10

Acetonitrile (%)

AOIII

0
0

10

20

30

60
50
40
30
20
0

Fig. 3 Scavenging effect of various fractions obtained

from the hydrolyzate (AOH) shown in Fig. 2 on
absorbance (at 517 nm) of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical.

AOII

70

10

EF

Section

AOI

Fraction AOI
Fraction AOII
Fraction AOIII
Fraction AOIV
Fraction AOV

80

Scavenging effect (%)

Scavenging effect (%)

100

433

40

50

Time (min)

Fig. 4 Purification of anti-oxidative activity peptides by

high-performance liquid chromatography. The fraction
AOHA shown in Fig. 2 was placed on an RP-18 column
(4 mm 250 mm) and eluted with a linear gradient of
020% (50 min) of acetonitrile.

peptides for each fraction to compare the scavenging effect. If the scavenging effect did not change
with increased number of times concentrated, this
indicated that the peptide fractions had a weak
scavenging effect. When the solid content of
each fraction (48 mg/mL of fraction A, 39 mg/mL
of B, 55 mg/mL of C, 69 mg/mL of D, 61 mg/mL
of E, and 52 mg/mL of F) was concentrated three
times, the scavenging activities of the peptide
fractions reached 2.54% (A), 70.43% (B), 47.54%
(C), 8.72% (D), 19.87% (E), and 8.48% (F). Fractions
B and C (molecular weights ranged from 390 to
1400) had the strongest scavenging effect on the
DPPH radicals (Fig. 3). The mixture of active
peptides; that is, fractions B and C (marked
AOHA), was then collected, concentrated, and
purified by reversed-phase HPLC using a 0.1%
TFAacetonitrile system.

No. times fraction concentrated

Fig. 5 Effect of various peptide fractions obtained from
the AOHA shown in Fig. 4 on absorbance (at 517 nm)
of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical.
Absorbance of the sample was higher than that of the
control, and no scavenging effect was evident.

Figure 4 displays the RP-18 column chromatographic pattern and the yields of five fractions
(indicated as AOI, AOII, AOIII, AOIV, and AOV) for
AOHA. All five fractions were appropriately fractionated, concentrated, and assayed for their
DPPH radical scavenging activity. The scavenging
effect was measured using 2 mg/mL of fraction AOI,
5 mg/mL of AOII, 23 mg/mL of AOIII, 57 mg/mL of
AOIV, and 124 mg/mL of AOV. Fraction AOI displayed an excellent scavenging effect (>80%) at
concentrations of approximately 32 mg/mL (Fig. 5).
Fraction AOII displayed a scavenging effect of
more than 70% at 80 mg/mL, and the scavenging
effects of butyl hydroxyanisol (BHA) and lascorbic acid were 92% and 80%, respectively, at
100 mg/mL. Furthermore, fractions AOI and AOII
clearly contained significantly more anti-oxidant
components than AOIII, AOIV, and AOV, and these
components could react rapidly with DPPH radicals, thus reducing almost all of the DPPH radical
molecules corresponding to available hydroxyl
groups.25 These analytical results confirm that peptides are free-radical inhibitors or scavengers, and
may be primary anti-oxidants. It is has been suggested that the peptides anti-oxidant mechanism
is that of free radical scavenging.10
Amino acid sequences
Peptides in fractions AOI and AOII, totaling
approximately 13 peaks, were collected and
analysed (Fig. 6), and it was found that only five
peaks (P1, P2, P3, P4, and P5) displayed good scavenging effects for the DPPH radical. The active

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434

C-L Jao and W-C Ko

Table 1 Amino acid sequences of isolated anti-oxidative peptides derived from tuna cooking juice with protease XXIII
Peptides
P1a
P1b
P2a
P2b
P3a
P4a
P5a

Structure

Scavenging effect* (%)

Molecular weight

Pro-Ser-His-Asp-Ala-His-Pro-Glu
Ser-His-Asp-Ala-His-Pro-Glu
Val-Asp-His-Asp-His-Pro-Glu
Pro-Lys-Ala-Val-His-Glu
Pro-Ala-Gly-Tyr
Pro-His-His-Ala-Asp-Ser
Val-Asp-Tyr-Pro

66
71
77
74
75
81
74

1010
896
953
766
457
751
544

*The solid content of P1a was 57 mg/mL, P1b was 54 mg/mL, P2a was 61 mg/mL, P2b was 68 mg/mL, P3a was 87 mg/mL, P4a was
95 mg/mL, and P5a was 89 mg/mL.
The anti-oxidative activity fractions of P1, P2, P3, P4, and P5 were concentrated using a centrifugal concentrator, and then applied to
a MICRA NPS RP-18 column (4.6 mm 33 mm), and the gradient eluted with 035% acetonitrile at a flow rate of 0.8 mL/min.

100

60
P1

P5
P3

P2

40
P4

20

Scavenging effect (%)

Absorbance (OD 210 nm)

80

0
0

10

15

20

Time (min)

Fig. 6 Anti-oxidative activity [as measured by 1,1diphenyl-2-picrylhydrazyl (DPPH) radical scavenging

activity] of various peptide fractions obtained from
AOH hydrolyzates by high-performance liquid chromatography. () DPPH scavenging effect (%) = [(OD517
control OD517 sample)/(OD517 control)] 100%. Control, 0.1%
trifluoroacetic acid in deionized water.

peptides were purified further using a MICRO NPS

RP-18 column. P1a, P1b, P2a, and P2b were found to
be the active peptides of the P1 and P2 fractions,
and P3a, P4a, and P5a were the active peptides
derived from the P3, P4, and P5 fractions. The
scavenging effects are shown in Table 1.
Table 1 also lists the amino acid sequences of
the peptides mentioned earlier. The sequences of
P1a and P1b were identical, except that the former
lacked an N-terminal Pro. P1a, P1b, P2a, and P2b contained one His residue, one Pro residue, and one
Glu residue at the C-terminal sequence (-His-ProGlu). Finally, the sequences of P3a and P5a were free
of His residue but did contain Tyr residue. The peptides comprised amino acid residues, including
hydrophobic amino acids, Val or Leu, at the Nterminal positions, and Pro, His, or Tyr in the
sequences.
Several amino acids, such as Tyr, Met, His, Lys,
and Trp, are generally accepted as being anti-

oxidants despite their occasionally pro-oxidative

effects.26 Dipeptides consisting of Ala, Tyr, His,
and Met at the N-terminus on linoleic acid have
been investigated previously by Kawashima et al.,27
revealing that the dipeptides displayed greater
anti-oxidative activities than the constituent
amino acid mixtures in an aqueous solution.
The anti-oxidative activity of histidine-containing peptides has been described elsewhere,28 and
can be attributed to the chelating and lipid radicaltrapping abilities of the imidazole ring. The
anti-oxidative activities of histidine-containing
peptides exceeded that of histidine itself, a
phenomenon that was partly the result of the
increased hydrophobicity of the peptides, which
increased the interaction between the peptides
and fatty acids.3 As proven by the present study, the
sequence of five DPPH radical scavenging-activity
peptides contained histidine residues. Meanwhile,
the remainder of the peptides contained tyrosine
residue, which is a significant source of hydrogen.
Furthermore, all the anti-oxidative peptides
derived from the tuna cooking juice contained
proline residue. Interestingly, prolyl polypeptides
are sensitive to oxygen.29 Uchida and colleagues
have demonstrated that prolyl polypeptides are
sensitive to oxidation by Cu(II)/H2O2 and generate
mainly g-aminobutyric acid, hydro-oxyproline,
aspartic acid, and glutamic acid after hydrolysis of
the oxidized substrates.
CONCLUSIONS
The present study has demonstrated clearly that
protein hydrolyzates of tuna cooking juice, and the
fractions derived from them after separation via
a column chromatographic procedure, possess
strong DPPH radical-scavenging properties that
are comparable to those of BHA and l-ascorbic
acid at the same concentration. These effects
are attributed to the variable hydrogen-donating

DPPH scavenging

FISHERIES SCIENCE

poperties of the active peptides present in the

hydrolyzates/fractions. Hydrolyzates from tuna
cooking juice, and the fractions derived from them,
can be incorporated into lipid-containing foods
as chain-breaking anti-oxidants to minimize
free radical-mediated lipid peroxidation. These
hydrolyzates can also be used as alternatives to
conventional drugs for treating human diseases
associated with free radical-mediated tissue
damage. However, such usage must be adequately
justified by animal and clinical studies, creating a
need for further research.

11.

12.

13.

14.

15.

ACKNOWLEDGMENT
16.

The authors thank the National Science Council of

the Republic of China for financially supporting
this research under Contract No. NSC89-2313-B005-001.

17.

18.

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