ペプチド装置の開発で進む有用ペプチドの機能解明
京都府立大学大学院 生命環境科学研究科 佐藤健司

食品タンパク質の酵素分解物の経口摂取により、血圧低下、食後の髙脂血の改善等の有益な作用が報告されている。これらは医薬品と同様に活性分子と生体の相互作用によると考えられる。しかし、食品タンパク質の酵素分解物中には非常に多くの種類のペプチドが存在する。またほとんどのペプチドは消化・吸収の過程でアミノ酸に分解される。そのため、従来から行われてきた試験管内でのアッセイに基づき活性成分を同定することは非常に困難である。一方、経口摂取によるアッセイで活性成分を同定するためには、かなりの量のペプチドを分画する必要がある。しかし、ペプチドを有害な試薬を用いずに大量に分画することはかなり困難であった。
我々はこの問題を解決するため、ここのペプチドの等電点の差に注目し、ペプチド自身を両性担体として用いる等電点電気泳動法が有効であることを示し、これを Autofocusing と呼んでいる。動物実験のため最大 50 L までのサンプルが分画可能なバッチ型 Autofocusng 装置の開発に成功し、この装置を用いて invivo のアッセイのみで鮫軟骨由来の尿酸低下ペプチド、グルテン由来の肝炎抑制ペプチドの同定に成功している。これらの結果に基づき、in vivo の評価に基づく、in vivo activity-guided fractionation を機能性ペプチドの同定法として提案している。
さらの連続分画が可能な Autofocusing 装置の開発にも成功し、食品加工への応用の可能性をしめしている。

食品成分の摂取により従来の栄養的な価値を超えた健康増進作用が示唆されている。

特に食品タンパク質の酵素分解物であるペプチドは、高血圧の緩和、メタボリックシンドロームの改善、高尿酸血症の緩和、皮膚・関節の状態改善、メンタル面の改善…

•非常に複雑な多数の成分が存在•ヒトへの応用は粗分画物なら容易•ペプチド等は生体中でさらに分解する可能性が高く、vitroとvivoの評価が一致しない可能性が高い。
•Vivoでは混合物が評価に使われ、生体への移行がほとんど不明であり、活性成分は、
ほとんど不明。

食品は基本的に食経験があるものを対象
ヒトでの評価のハードルが比較的低い
活性成分の同定が非常に困難
複雑な構造の成分でも利用可能
化学反応による活性の改変は困難
消化・吸収過程での変化が大きく、それを把握しにくいそのため In vitro high throughput screening Animal experiment Human trialといった従来の発想ではvitroで活性があった成分がHuman trialで活性がなければ、すべての努力が無駄になる可能性がある。
そこで、機能性食品の開発、特にペプチド性の機能性食品のために、Vivoでの評価に基づく 活性成分の同定In vitroの実験でのメカニズム解明のアプローチを提案す
る。

1

提案
•大量分画によるvivoで活性成分の同定

陽極

サンプルコンパートメント(ペプチド水溶液)

陰極•標的組織に移行した活性成分の同定
上記の手法により決定した活性成分を用いてメカニズムの解明

リン酸溶液

水酸化ナトリ
多孔質膜またはアガロース ウム溶液
膜

Autofocusingの原理
化学合成アンフォラ
インを加えることなくサンプルペプチドが両性担体として自らの等電点に移動

1. 大量分画によるvivoで活性成分の同定
微量のペプチドの分画は可能、しかし、vivoでの評価が可能な量のペプチドを毒性の無い試薬をもちいて、低価格で分画することは困難

市販調製用等電点電気泳動装置 Rotofor (50mL sample cell; Bio-Rad)を用いてペプチドのAutofocusing現象を確認

一つの解決として水を溶媒とする大容量
Ampholyte-Free 等電点電気泳動法(Autofocusing) を紹介する。

2

Non-digested
lysozymepH
2.2 2.4 2.8

3.6 4.6

9.4

11.9 12.1 12.1 12.1

Amino acid composition of peptide (%)

100

Lysozyme hydrolysate
Fr. 3 pH 2.3
5 pH 3.1
9 pH 4.0
13 pH 5.3
15 pH 6.3
17 pH 8.9

80
70
60
50
40
30
20
10
0
1

19 pH 9.9

1%リゾチームのトリプシン分解物の Rotofor によるAutofocusing現象 ペプチドおよび未分解リゾ
チームが分画されている。

Window

Plate
20cm

Insert the separator into the tank

5

6

7

8

9

10

hyperuricemic rat

3

*

2

*
1
0
vehicleCathode side4

酸性画分は酸性ペプチドに富む 等電点による分画が生じている

serum uric acid levels(mg/dL)

Separetor

10cm

Anode

3

normal rat

1%agarosesolution

10cm

2

カゼイン分解物のAutofocusing装置による分画

Screen (100mesh)

Schotch tape

25cm

Basic
amino
acid
Nutrial
amino
acid
Acdic
amino
acid

90

vehiclenondigested

digested

the water extract of the shark cartilage (1 g/kg)

No.1

2

Sample compartment
.
.
9 10

オキソン酸誘発高尿酸血症ラットにサメ軟骨の水抽出物の酵素分解物(1
水抽出物の酵素分解物(1 g/kg BW)を摂取させBW)を摂取させると高尿酸血症が改善するが水抽出物では改善
しないペプチドが活性成分か？

3

可能性のあるメカニズム

Dose
mg/dL

•尿中への尿酸排出促進（−）

3.5
3
2.5
2
1.5
1
0.5
0

•食事中のプリン体の吸収阻害（−）•尿酸合成酵素（キサンチンオキシダーゼ）の阻害 (in vitroでは−）メカニズムが不明のアッセイができない

In vitro

pH

9
7
5
3
1
3

Bascic fraction

各画分を酵素分解し、動物に投与

4

5

6

7

8

9

10

Autofocusing (5 L)によるサメ軟骨の水抽出物の分画

Absorbance at 280 nm

3.5
3
2.5
2
1.5
1
0.5
0

11

2

Alcalase digest

塩基性画分

13

1

0.332g/kg

塩基性画分は少量で尿酸低下作用を持つ固形物 (g)

酸性画分

Vihecle

1g/kg

Fr. 1

2.5

Fr. 2

2

Fr. 3

1.5

Fr. 4

1

0.5

26~29
0

36~40
0

100

200

300

400

500

600

塩基性画分の分取用逆相クロマトグラフィーによる分画
矢印からアセトニトリルの濃度勾配開始

45Dose (mg/kg)

427

4.5

2997

180

109

50

*

*

4
3.5

Serum uric acid (mg/100 mL)

YLDNY* P< 0.01

3
2.5
2VehicleBasicfraction

Fr. 1

Fr. 2

Fr. 3

Fr. 4

4.5
4

*P<0.01

3.5
3
2.5
2
1.5
0

1

2

3

4

5

6

Dose (mg/kg)

最も吸着した画分4は 50 mg/kgで尿酸低下作用を持った。

その中の一つYLDNYが5 mg/kg i.p. で有意な尿酸低下作用を持った。50 mg/kg 経口投与でも有意な低下活性を持つ。

Identification of peptides in protease digest of Fr.4
Peak
No..

MW(Da)
720

29-30

a

c

b

30-31

d

SEC (min)

e

31-32

i

l

f
g

k
h

32-33

n

o

m

j
p
q

r

33-34

290

34-35
5.00

7.50

10.00

12.50

Sequence

a

LYP

b

YLDNY

c

DFWRY

d

SPPYWPYe

SLPYWPY

f

INY

g

VYQ

h

YNL

i

LY

j

SIYD

k

FY

l

YL

m

RYL

n

GYL

o

YF

p

YY

q

SNWQ

r

FY

従来の in vitroのスクリーニングに基づくアプローチではこのペプチドの同定は不可能Vivoでの評価に基づく分画により活性ペプチドの同定とそのメカニズムが解明できた
現在、vivoでの評価を基に活性性ペプチドの同定を行っている

15.00

RP-HPLC5

鮫軟骨中に血管新生を抑制し、MMPを抑制する物質が含まれる これは事実。
ただし、これは経口摂取での効果とは関係がない。
しかし、一方で鮫軟骨の経口摂取によるがんの進行抑制が期待されている。しかし、アカデミアではほとんど信じられていない。

L-methionine
BOP

L-methionine

DL-ethionine

12

16 18

Choline 欠乏食

30 32

Atypical
hyperplasia
Cacinoma

P<5%

ｚ

0.20%
サメ軟骨抽出物

0.40%

サメ軟骨水抽出物 (SCPG)の経口投与によるがん進行抑制
発がん処理後のがんの進行が、抑制された

SCPG group

Basal group(0, 0.2, 0.4% the extract)

DL-ethionine

26

Hyperplasia

4
3.5
3
2.5
2
1.5
1
0.5
0
0%

NC PC

実験食

BOP

BOP

Incidence

鮫軟骨の摂取によるがんの進行抑制

50

100

Choline 欠乏食

ハムスターを用いた化学誘発膵管がんモデル
BOP; N-nitrosobis(2-oxopropyl)amine

担がんハムスターの血清のMMP-9阻害活性
SCPG投与でMMP-9 阻害活性が見かけ上上昇

6

13

8

11

6
4

9
7
5

2

3
1

0
1

2

3

4

5

6

7

8

9

10

膵がん発生個数（個）

10

pH 

Chondroichin sulfate  (mg/mL)
Peptide  (mg/mL)

Basic fraction

1.6

膵がん発生頻度（%）

Acidic fraction

100
80

1.2

60

P < 0.05

0.8

40
0.4

20

0

0

基礎食群

酸性画分
塩基性画分 コンドロイチン硫酸
エタノール上清 エタノール沈殿

Basal

AS

AP

B

CS

Fraction number

Autofocusingによる鮫軟骨水抽出物の分画

膵がんの進行抑制はAP:プロテオグリカン画分に認められた。

酸性画分はコンドロイチン硫酸を含む
酸性画分は75%エタノールで沈殿

NC PC

AS:低分子コンドロイチン硫酸と酸性
コラーゲンペプチド
B: 塩基性コラーゲンペプチドAPAS

1(V)*

NC PC

B

CS

1(V)
2(V)
1(V)*

Inhibition (%)

AP: アグリカン様タンパク質を持つプ
ロテオグリカンと少量のコラーゲンペ
プチド

Basal

1(V)
2(V)

90.0
80.0
70.0
60.0
50.0
40.0
30.0
20.0
10.0
0.0

*

Basal

AP

AS

B

CS

血清中のMMP-9 阻害活性もAPの摂取で増加している。

7

担がん状態本プロジェクトは以下のエピソー
ドから始まる

持続的炎症？ ハイポキシア？
アグリカン由来
ペプチド

サイトカイン？の誘導

αマクログロブリンのMMP-9の誘導

ダウンレギュレーション

グルテン酵素分解物中の肝炎抑制ペプチドの同定

8D-ガラクトサミン誘発肝障害モデルラットでのAutofocusing分画物投与による肝障害抑制効果

ガラクトサミン誘発肝障害に対する保護効果
WGH中のペプチドを分画し、いずれの画分がガラクトサミン誘発肝障害を抑制するかを検討

血清AST活性AST activity in serum(Karmen unit/L)

実験

3500
3000
2500
2000
1500

＊

1000

＊

500
0
C

1

2

3

4

5

6

7

8

9

10

Fractin number（千葉大学園芸学部 真田先生より提供）

ピログルタミン酸ペプチド

各FractionのpH、アミノ酸構成、総ペプチド量
O

N

pH

CONHCHR1CONHCHR2CO……..

ピログルタメートアミノペプチ
ダーゼ処理
O

N

COOH

+

アミノ酸構成Coupling Reaction

+
PITC

総ペプチド
量

PeptidePITC ; Phenyl
isothiocyanate

pH 8-9PTC-Peptide

PTC-peptides ;Phenylthiocarbamyl peptides9ピログルタミルアミノペプチダーゼ消化＋: 酵素あり －: 酵素なし

Gln

Gln-Gln

Ile Leu

SEC Fraction

33 min －＋34 min －＋35 min －＋36 min －＋Autofocusing 装置により分画した画分の経口摂取により活性画分を同定してゆき、その中のペプチドを分離同定する。
構造情報からペプチドを化学合成し、活性ペプチドを同定する Activity-guided
fractionationにより消化・吸収を考えた
活性ペプチドの同定が可能である。
またAutofocusing法は機能性成分の濃縮にも利用可能であると考える。

37 min －＋SEC 33-37 min ピログルタミルアミノペプチダーゼ消化

pyroGlu-Leuに有意な肝障害改善活性が認められた。
また、遊離のpyroGluには肝障害改善傾向が見られた。

No

Sample compartment
2… 5%

water

連続式 Autofocusing 装置の開発
ペリスタポンプ
8 ml / min＊1

caseinpeptide

…10

water

First unit
Anode
side

Cathode side

0.1N
H3PO4

0.1N
NaOH

Second
unit
0.1 N
H3PO4

0.1N
NaOH

Third unit
0.1N
H3PO4

0.1N
NaO
H
Fractioncollector

10Amino acid composition (%)

100

80

塩基性アミ
ノ酸

60

40

20

中性アミ
ノ酸

0

酸性アミ
ノ酸
1

2

3

4

5

6

7

8

9

10

Fraction number
連続式 Autofocusing 装置によるカゼインペプチドの分画

Autofocusingは連続分画も可能であり、食品加工技術としても利用可能であると考えている。

11756

November 2007, Vol. 18 (11)Bioactive peptides—
large-scale preparation
Kenji Sato and Kaori Hashimoto

Several scientific studies have revealed that ingestion of some enzymatic hydrolysates of food proteins, namely, mixtures of peptides, produces various beneficial activities beyond basic nutritional values. Moderation of hypertension and hyperlipidemia
is considered as one of these beneficial effects.
The emerging market for nutraceuticals and functional foods
is stimulating the production of enzymatic hydrolysates of milk,
animal, fish, egg, and plant proteins on an industrial scale. Growing worldwide interest in biodiesel and bioethanol production
have encouraged consideration of possible added value to be obtained from the by-products arising from their manufacture. The
production of bioactive peptide fractions from protein by-products is one attractive approach.
The identification of an active peptide from a mixture of
peptides is the initial step required in determining its potential
health beneficial properties. In most cases, the active peptides aretentatively identified by high-performance liquid chromatography (HPLC) separation and in vitro assays using enzyme and cell
culture systems. Unlike many other functional substances, however, peptides are susceptible to degradation during the process ofdigestion and lose their desired activity. Therefore, the beneficial
effects determined in vitro cannot be directly linked to those present following digestion. The potential activity of the peptides
must be evaluated through feeding experiments.
Although liquid chromatography (LC) is the most powerfultool for isolation of peptides, it is a relatively expensive system
for large-scale preparations, especially for the first purification
step. In addition, some solvents frequently used in LC, such as
methanol, acetonitrile, and trifluoroacetic acid are harmful, but
peptide fractions obtained with low selective techniques such as
filtration have been used for feeding experiments. Nonetheless, alarge-scale, low-cost, and biocompatible procedure for peptide
fractionation is needed.

BATCH TYPE AUTOFOCUSING OF
BIOACTIVE PEPTIDES
We have demonstrated that peptides can be fractionated on thebasis of the amphoteric nature (possessing both acidic and basic
properties) of sample peptides without adding chemically synthesized ampholines (having the capacity to act either as an acid or abase) by using a laboratory-scale preparative isoelectric focusing
apparatus. This approach has been referred to as autofocusing,and has advantages in cost and biocompatibility over LC. Some

FIG. 1. Schematic drawing of a batch-type autofocusing apparatus. This apparatus can process up to 50 L. Smaller apparatuseswith a sample compartment of 5 × 10 × 10 cm or 5 × 7 × 8 cm
are also prepared. Total volumes of sample compartments of thethree types of apparatus are approximately 50, 5, and 1 L, respectively (Hashimoto et al., 2005).
preparative matrix-free isoelectric electrophoresis apparatuseshave been developed. The main factor that decreases the resolution of matrix-free isoelectric electrophoresis is diffusion of sample by convection current. To minimize the effect of the convection current, a gravity gradient with sucrose and thin-layer focusing cell have been used. These apparatuses can process a sampleof less than 1 L in volume. However, further scale-up is considered to be difficult due to the structural nature of the apparatuses.
Furthermore, addition of even nontoxic chemicals such as sucrose
to form a gravity gradient is not desirable for subsequent feeding
experiments. Therefore, we have simply used a thin agarose gellayer (matrix) to avoid diffusion. By using this technique, an autofocusing apparatus which can process up to 5 L has been developed.
Figure 1 shows a schematic drawing of the apparatus. A tankis separated by thin agarose gel layers supported on nylon screens
into 12 compartments. The anode compartment is filled with 0.1
N phosphoric acid. The cathode compartment is filled with 0.1 N
sodium hydroxide. Other compartments are used as sample compartments and numbered consecutively from the anode side. The
cell sample compartments or sample compartments Nos. 5 and 6
are filled with 1–10% peptide solution.
In the latter case, other sample compartments are filled withwater. Direct electric current at constant voltage at 300–500 V is

inform

757

FIG. 2. Development of pH gradient (upper) and amino acid composition (lower) of each fraction after autofocusing of a commercial
enzymatic hydrolysate of wheat gluten by the batch-type apparatus
as shown in Figure 1. Amino acid composition is expressed as percentage of acidic, neutral, and basic amino acids.
applied to the electrodes depending on sample condition. Normally, fractionation is completed after 12–24 h.
Figure 2 shows an example of separation of peptides by the
autofocusing apparatus as described in Figure 1. A pH gradient wasformed by the autofocusing of an enzymatic hydrolysate of wheat
gluten at 500 V for 24 h. Peptides in the acidic and basic fractions
are rich in acidic and basic amino acids, respectively, which indicates that peptide separation occurs on the basis of the amphotericnature of sample peptides.
The pH profile obtained by autofocusing of the same proteinhydrolysate may be modified by the concentration of the sample
and by the presence of a salt. Fractions with similar pH collectedfrom different batches have essentially the same peptide composition. Thus, autofocusing allows reproducible large-scale separationof peptides. This approach can be considered to be matrix-free electrophoresis. However, separation occurs only in the thin agarose
gel layer, which allows separation in a shorter time than conventional large-scale free-zone electrophoresis.
Application of autofocusing at the first purification step has
provided a basic peptide fraction, prepared from an enzymatic digest of shark cartilage, that has anti-hyperuricemic activity. Withfurther preparative reversed-phase LC fractionation, we have succeeded in preparing a fraction having significant anti-hyperuricemic activity at 50 mg/kg body weight in an oxonate-induced
rat model. (Oxonate inhibits the activity of uricase, the enzyme responsible for the breakdown of uric acid that is produced normallyin the body and in excessive amounts in the condition of gout.)
Major peptides in the active fraction could be isolated by a seriesof HPLC and identified without difficulty.
On the basis of the amino acid sequence data, the major peptides in the active fraction were chemically synthesized and used
for the feeding experiment. Consequently, a pentapeptide, Tyrosine-Leucine-Aspartic acid-Asparagine-Tyrosine (YLDNY), wasidentified as the active peptide. The in vivo activity-guided largescale fractionation of peptides by combination of autofocusing and
LC followed by identification of the peptides in the active fraction

Bioactive peptides

758

FIG. 3. Schematic drawing of the continuous type of autofocusing
apparatus. In this case, three units of the batch type apparatus withsample compartment (5 × 7 × 8 cm) are used (Hashimoto et al.,2006).
would be an effective approach for identification of bioactive peptides following ingestion.

POTENTIAL OF AUTOFOCUSING FOR
INDUSTRIAL APPLICATION
The autofocusing system can process a relatively large amount of
peptides. The chemicals used, such as sodium hydroxide, phosphoric acid, and agarose, are all food grade. To demonstrate thepotential for industrial application, a prototype of a continuous
type of autofocusing apparatus has also been developed. As
shown in Figure 3, three autofocusing units are connected in tandem. The electrode and sample solutions are continuously delivered to the electrode compartments and sample compartments
Nos. 5 and 6, respectively. Water is delivered to other sample
compartments. The solution in each compartment in the first unit
is continuously delivered to the corresponding compartments ofthe second and third units. The effluents from the third unit arecollected. By using this apparatus, peptides can be continuously
separated for 8 hours. This type of continuous apparatus can fractionate larger amounts of peptides using less electric power compared with the batch-type apparatus having the same sample compartment volume. Thus, the continuous-type apparatus is suitablefor industrial fractionation of peptides. Crude enzymatic hydrolysates of food proteins frequently show a bitter or odd taste,
and most of the constituting peptides are inactive peptides. Separation of the active peptide by autofocusing would improve thetaste of peptide-based products by decreasing the amounts of undesired peptides.

CONCLUSION
A mixture of peptides dissolved in water can be fractionated in a
preparative isoelectric focusing apparatus on the basis of their amphoteric nature. This approach is referred to as autofocusing andhas an advantage in processing cost and biocompatibility over andLC system and can be scaled up. By using thin agarose gel layers
to prevent diffusion of the sample, both batch and continuous
types of autofocusing apparatus have been developed. The batch
type autofocusing apparatus enables in vivo activity-guided fractionation for identification of bioactive peptide surviving ingestion. The continuous one would enable preparation of bioactive
peptide fractions in concentrated form for use as a functional foodingredient. The present approach would be a breakthroughs not
only in basic research but also in industrial processing for development of peptide-based functional foods.

Kenji Sato and Kaori Hashimoto are affiliated with the Department
of Food Sciences and Nutritional Health, Kyoto Prefectural University, Shimogamo, Kyoto 606-8522, Japan. Contact Kenji Sato via email at k_sato@kpu.ac.jp.

Isoelectric Focusing
The technique of Isoelectric Focusing isused routinely to separate molecules basedon differences in their electric charge, andhas found particular application in the separation of proteins and peptides. It is a typeof zone electrophoresis, usually performed
in a gel such as polyacrylamide, starch, andagarose that takes advantage of the fact thata molecule’s electric charge changes withthe pH of its environment.
The separation takes place over a medium that has a pH gradient (usually createdby aliphatic ampholytes possessing bothacidic and basic properties). Passage of anelectric current through the medium creates a “positive” anode and a “negative”
cathode at each end of the gel and allows
negatively charged molecules to migrate
through the pH gradient toward the anodeand the positively charged particles toward
the cathode.As the molecule moves toward
the pole opposite of its charge the pH gra-

November 2007, Vol. 18 (11)information
dient will cause reduction in the degree ofcharge until a net charge of zero is reached,at which point migration of the molecule
will cease.
The particular pH at which a molecule
possess a net zero electric charge is its Isoelectric Point, or pI. Many molecules show
minimal solubility at their pI. An environment with pH value <pI will result in a molecule carrying a net positive charge; with pH
value >pI there will be a net negative charge.
Isoelecric focusing can resolve proteins andpeptides that differ in pI value by as little as
0.01.
Proteins and peptides owe their ability
to carry an electric charge due to varying
ionizable groups on the constituent aminoacids that reflect their particular composition. Further background information isavailable online at netlink: http://instruct1.
cit.cornell.edu/Courses/biobm330/protlab/
IEF.html.  Xiao, Z.,T.P. Conrads, D.A. Lucas.
G.M. Janini, C.F. Schaefer, K.H. Buetow, H.J.
Issaq, and T.D. Veenstra, Direct Ampholyte-free Liquid Phase Isoelectric Peptide Focusing,Application to the Human
Serum Proteome, Electrophoresis 25:128–
133 (2004).
 Hashimoto, K., K. Sato,Y. Nawa,
Y. Nakamura, and K. Ohtsuki, Development of a Large-scale (50 L) Apparatus
for Ampholyte-free Isoelectric Focusing(autofocusing) of Peptides in Enzymatic
Hydrolysate of Food Proteins, J. Agric.
Food Chem. 53:3801–3806 (2005).
 Hashimoto, K., K. Sato,Y. Nakamura, and K. Ohtsuki, Development ofContinuous Type Apparatus for Ampholyte-free Isoelectric Focusing (autofocusing) of Peptides in Protein Hydrolysates, J. Agric. Food Chem. 54:650–655 (2006).