Flavor Constituents in Savory Seafood: Dried Kelp (Kombu), … · savory seafood revealed that...

Aqua-BioScience Monographs, Vol. 10, No. 1, pp. 1–22 (2017) www.terrapub.co.jp/onlinemonographs/absm/

Received onNovember 10, 2015

Accepted on April 13, 2016Online published on

March 10, 2017

Keywords• dried kelp• garlic• onion• scallop• dried bonito• mannitol• glutamate• sweet amino acid• sulfur compound• glutathione• pyrazin• hydrogen sulfide• umami• flavor interaction• kokumi• deterioration odor

© 2017 TERRAPUB, Tokyo. All rights reserved.doi:10.5047/absm.2017.01001.0001

modalities are called “kokumi flavors” in Japan. Aromaor odors of foods are generated by volatile compounds.In the case of several foods, such as fruits, the wholearoma character can be reconstructed by a few kindsof volatile compounds. In many cases a lot of volatilecomponents, however, comprise whole flavor charac-teristics of foods. For the components easily disappearor change to other compounds by the reactions amongthem, it has been a big issue how to control unstablevolatile compounds generated after enzymatic or ther-mal reaction.

Consumers request various kinds of high qualityprocessed foods which are convenient, have attractivetastes or flavors, natural feeling, etc. To reply to thosedemands from industry, basic studies are inevitable to

AbstractOmission tests of the synthetic water extract of dried kelp (Kombu-dashi) revealed thatthe water solution prepared by mannitol, monosodium glutamate (MSG), potassium chlo-ride (KCl), and sodium chloride (NaCl) represented the whole taste characters of thesynthetic extract. Succeeding study suggested that the characteristic Kombu-dashi likekokumi flavors (continuity, mouthfulness, and thickness) were generated by flavor inter-action among mannitol, MSG, and KCl. Some studies of taste interactions among NaCl,KCl, sweet amino acids, and 5¢-inosinate (IMP) showed that NaCl strengthened umamiand sweetness of some amino acids and also suggested that IMP enhanced umami of Gly,L-Ser, and L-Ala synergistically. The sulfur-containing compounds in garlic and onion,such as alliin, S-propenyl-L-cysteinesulfoxide and reduced glutathione (GSH), had notastes by themselves but they gave rise to kokumi flavors in umami solution or soups.Scallop and beef contained a high amount of GSH and the peptide strengthened kokumiflavors of synthetic extracts of these foods. The examination of volatile compounds indried bonito by using GC-MS and GC-sniffing methods revealed that acetol and 2,3-pentandione reacted with amino acids or protein in boiled bonito meat, generated pyrazines.Straight-chain aldehydes might be involved in the deterioration of crushed dried bonito.Hydrogen sulfide, one of the key flavor components, decreased during preservation ofcrushed dried bonito catalytically by the complex of Fe3+ and histidine. This study, in-cluding new findings, would lead to improve the quality of processed foods and contrib-ute to future studies of taste phisiology and flavor chemistry.

Flavor Constituents in Savory Seafood: DriedKelp (Kombu), Scallop, and Dried Bonito(Katsuobushi)

Yoichi Ueda1* and Kenji Fukami2

1Japan Food Additives Association Kodenma-cho Shin Nihonbashi Building, 4-9, Nihonbashi-Kodenma-cho, Chuo-ku, Tokyo 103-0001, Japan2Ajinomoto Co., Inc., 1-15, Kyobashi 1-chome, Chuo-ku, Tokyo 104-8315, Japan*e-mail: [email protected]

1. Introduction

Various kinds of food flavoring materials are usedfor traditional dishes or cuisine in the world. Manystudies on flavors of foodstuffs have been done. Manyof their subjects were volatile odor components or non-volatile taste active compounds which have basic tastes(sweetness, sourness, saltiness, bitterness, or umami).On the other hand many foods have flavor charactersexpressed by terms such as complexity, body, continu-ity, mouthfulness, thickness, etc. It is difficult to de-fine these terms clearly, but these flavors sometimesgovern palatability of the foods. These flavor charac-teristics are presumed to be generated by the results ofinteractions among components in foods. These flavor

2 Y. Ueda and K. Fukami / Aqua-BioSci. Monogr. 10: 1–22, 2017

doi:10.5047/absm.2017.01001.0001 © 2017 TERRAPUB, Tokyo. All rights reserved.

develop new ingredients and technology. We have en-gaged in the research to get basic new knowledge onkokumi flavors and to control unstable volatile com-pounds for quality improvement of processed foods,by the combination of instrumental analyses and sen-sory evaluation. In this monograph, we will show threecategories of our studies on the flavor constituents inseafood and other foodstuffs. First of all, we will de-scribe the studies on the key non-volatile componentsof “Kombu-dashi”, popular Japanese soup stock pre-pared from dried kelp (Laminariaceae). The taste orflavor interaction between amino acids and other tasteactive compounds will also be shown. The next sec-

tion will deal with the flavor characteristics of glutath-ione contained in scallop and other food materials, fo-cusing on the flavor interaction with umami substancesor other components extractive from some foods. Thethermal degradation mechanism of glutathione will alsobe mentioned. The third section will give an overviewof the mechanisms of increase or decrease in key vola-tile flavor components of dried bonito.

2. Flavor interaction among active componentsin seafood

L-Glutamic acid was found in 1908 as the key taste

Component removed Effect on the synthetic extract

Sea urchin Snow crab Scallop Short-necked clam Dried skipjack

Glutamate Umami Ø Umami Ø Umami Ø Umami Ø Umami ØSweetness ≠ Sweetness Ø Sweetness Ø Sweetness Ø Sweetness Ø

Character Ø Palatability Ø Palatability Ø

AMP No effect Umami Ø Umami Ø Umami Ø No effect

Sweetness Ø Sweetness ØPalatability Ø Palatability Ø

IMP Umami Ø No effect No effect No effect Umami ØAftertaste Ø Sweetness Ø

Palatability Ø

Glycine Sweetness Ø Umami Ø Sweetness Ø Sweetness Ø No effect

Character Ø Sweetness Ø Palatability ØBitterness ≠

Alanine Sweetness ØBitterness ≠

Sweetness Ø Sweetness Ø No effect No effect

Arginine Umami ≠ Character Ø Character Ø Character Ø No effect

Sweetness ≠ Palatability Ø

Na+ NA Sweetness ØUmami Ø

Character Ø

Umami ØCharacter Ø

Palatability Ø

Sweetness ØUmami Ø

Sourness Ø

Sourness ØCharacter Ø

Palatability Ø

Cl- NA Palatability Ø Sweetness ØUmami Ø

Sweetness ØUmami Ø

Sourness ØCharacter Ø

Palatability Ø Palatability Ø Palatability Ø

Table 1. Role of each component on the flavor of synthetic seafood extracts. Cited from Trends in Food Science & Technol-ogy, December 1996 (Vol. 7), Fuke S and Ueda Y, Interactions between umami and other flavor characteristics, 407–411,Table 1, „ 1996, with permission from Elsevier.

“Character” refers to the specific characteristic flavor of the seafood. The descriptions used were: umami, bitterness, specificflavor, aftertaste, sourness, saltiness, palatability, no effect. Effects on saltiness are not shown. ≠ indicates flavor characteris-tic increased when component removed. Ø indicates flavor characteristic decreased when components removed. NA, Notanalyzed.

Y. Ueda and K. Fukami / Aqua-BioSci. Monogr. 10: 1–22, 2017 3


constituent of “Kombu-dashi”, Japanese common soupstock, prepared from dried kelp (Ikeda 1912). Afterindustrialization of its salt, monosodium glutamate(MSG), this taste active crystal has been used for manycuisines as an inevitable seasoning worldwide. Manystudies have been done on the flavor characteristics ofMSG. Especially the findings of synergistic effect be-tween glutamate and 5¢-ribonucleotides on umami tastewas a big event worthy of special mention (Kuninaka1964; Yamaguchi 1967). Glutamate does not only addumami to food but also enhances kokumi flavors suchas continuity, mouthfulness, and thickness of manyfoods (Yamaguchi and Kimizuka 1979; Yamaguchi1979, 1987). This phenomenon suggested that theflavor interaction between glutamate and other com-ponents would be developed in various foods.

Several studies about taste active components insavory seafood revealed that taste active compounds,such as amino acids, interact with other componentsand strengthen the whole taste and character (Fuke andUeda 1996). Methionine was identified as a key flavorcomponent of sea urchin (Komata 1964). This bitter

taste amino acid enhanced urchin-like taste and wholeflavors. Taste active components in snow crab wereinvestigated by using omission tests from syntheticextract made of commercial chemical compounds andarginine was determined to enhance flavors under thecondition of coexisting with glutamic acid, inosinicacid, adenyric acid and glycine (Hayashi et al. 1981).Glycogen was found to constitute characteristic flavorsof scallop (Watanabe et al. 1990).

2-1. Determination of non-volatile key flavor com-ponents in Kombu-dashi

As mentioned above, Kombu-dashi is used widelyfor Japanese cuisines. This common soup stock itselfhas characteristic aroma and complex tastes. Althoughthe key taste component was revealed to be monoso-dium glutamate, other constituents are necessary toreconstruct whole Kombu-dashi characters. We inves-tigated the other flavor active non-volatile componentsin Kombu-dashi. There are some kinds of dried kelp inJapan and it is believed that the quality of Kombu-dashidepends on cultivation location or rating (size, shape,etc.) of kelp.

At the beginning of our study we determined theauthentic extraction condition for preparation of stand-ard Kombu-dashi. We investigated the preparation con-dition, focusing on temperature, resources of water, andflavor characters, and we finally determined that thebest condition was the forty minutes extraction with

Fig. 1. Concept of flavor.

Compound Conc. (mg/100 ml)

Glu◊Na (MSG) 37

Asp◊Na 16

Lactic acid◊Na 0.4

Formic acid 0.3Malic acid 0.3Citric acid 1.1Succinic acid◊Na 0.2

Acetic acid 0.3Pyroglutamic acid 0.9KCl 255KI 12NaCl 135CaCl2 5MgCl2 16NaH2PO4 19Mannitol 1200Alginic acid◊Na 7

Table 2. Synthetic extract of Kombu-dashi. Unpublisheddata.

Prepared by mixing commercial chemical compounds tomatch the compositional profile as determined analytically.pH 6.3.

Fig. 2. Dried kelp (kombu). Provided from UMAMI Infor-mation Center, „ 2016.



distilled water at 50∞C. Dried Kombu (30 g) was ex-tracted with 1.0 L of distilled water under this condi-tion and filtered through a filter cloth.

We employed omission tests to investigate taste-ac-tive components in accordance with the establishedmethods (Konosu 1979; Hayashi et al. 1981; Watanabeet al. 1990). Synthetic reconstructed extract was pre-pared by mixing commercial chemical compoundsthrough the analytical data of the standard extraction(Table 2). The content of Cl occupied 90% of totalinorganic anion (Cl, PO4 and I). The synthetic extracthad kokumi flavors equal to the original extract. Aftera series of omission tests, we finally determined thefive compounds (mannitol, Glu, K+, Na+, and Cl–) aseffective. Table 3 shows composition of simplified syn-thetic essence which exhibited Kombu-dashi like tasteand kokumi flavors. In order to reconfirm the effec-tiveness of the synthetic essence, we further obtainedthe secondary extract from the Kombu residue afterpreparation of the original Kombu-dashi and added thefour chemicals (mannitol, MSG, KCl, and NaCl) to thesecondary extract according to the analytical data. Theresults of sensory examination showed that the stand-ard and secondary extracts were not discriminated sta-tistically by the highly trained panelists (Triangle dis-tinction test). We hence concluded that mannitol, Glu,K+, Na+, and Cl– were key taste components of Kombu-dashi.

2-2. Flavor interactions among mannitol, MSG,and KCl

It is well known that sodium and chloride ions aretaste active components in various kinds of seafood(see Table 1). It can be said that three other compounds,mannitol, Glu+, and K+, should generate characteristicKombu-dashi like kokumi flavors. We prepared the

model solution containing mannitol, MSG, and KClby eliminating NaCl from the simplified synthetic ex-tract. Although whole flavors of the model solutionwere weaker than those of simplified synthetic extract,it still had Kombu-dashi like kokumi flavors.

To clarify the flavor interactions among these threecompounds by sensory evaluations, we prepared vari-ous kinds of the 40 solutions which had different com-positions of these three chemicals but the total weightconcentrations were adjusted to almost equal levels ofthe simplified synthetic extract. The sensory testsshowed that Kombu-dashi like kokumi flavors shouldbe active under the restricted conditions (Fig. 3). Pro-posed formula of the condition is shown as follows:

Compounds Conc. (mg/100 ml)

Mannitol 1200MSG 37KCl 255NaCl 135

Fig. 3. Generation of Kombu-dashi like kokumi flavors bymixing of mannitol, MSG, and KCl (molar ratio). Four ex-pert panelists were employed for the judgements. Cited fromJpn. J. Taste Smell Res., 4(2), Ueda Y, The studies on theflavor characteristics “Koku” and “Atsumi” in some tastyfoodstuffs, 197–200, Fig. 1, „ 1997, with permission fromthe Japanese Association for the Study of Taste and Smell.

Fig. 4. A proposed flavor interaction among mannitol, MSG,KCl, and NaCl. Changes of flavor impression were profiledby four expert panelists.

Table 3. Simplified synthetic extract of Kombu-dashi com-posed of taste-active components. Unpublished data.

The solution was prepared by commercial chemical com-pounds. The key taste-active compounds were determinedby a series of omission tests of synthetic extract of Kombu-dashi (Table 2) by four expert panelists. Each compound wasomitted independently from the synthetic extract and the tastecharacter of every omitted solution was compared to thesynthetic solution. If the difference was recognized in thetwo solutions, we judged the eliminated component shouldbe contribute for the whole tastes or flavors.



Am

ino

acid

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lK

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Tab

le 4

. S

umm

ary

of th

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tast

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sol

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r K

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Mod

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pn.

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, The

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200

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The

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p <

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5.



[Condition under which three compounds generated“kokumi flavors” (by molar ratio)]1) [KCl]/[MSG] = 2.5–202) [mannitol]/([mannitol]+[KCl]+[MSG]) = 0.25–0.75.

In Japan dried Kombu products were generallyranked to low or high grade classes according to theirflavors. We analyzed contents of mannitol, Glu, and Kin several high grade kombu samples and demonstratedthat the high grade Kombu-dashi contained three keyflavor components well in accordance with the resultsin Fig. 3.

Figure 4 shows a conceptual diagram of the flavorinteractions of four compounds. KCl itself had bitter,astringent, and salty tastes. Bitter taste of KCl weredecreased by MSG. Kombu-dashi like kokumi flavorsgenerated under the condition mannitol, KCl, and MSGcoexisted. Mannitol itself had a sweet taste, but thissugar alcohol generated characteristic kokumi flavorsin the coexistence of MSG and KCl. It was the firstdescription about flavor characteristics of mannitol,suggesting that this sugar alcohol would be a usefulmaterial for quality improvement of processed foods.

Subsequently we compared the flavor characteristicsof three kinds of sweet sugar alcohols (sorbitol,maltitol, and xylitol) with that of mannitol. The threesugar alcohols had the flavor character similar withmannitol, suggesting that they will be applicable asfood additives to enhance kokumi flavors of processedfoods.

2-3. Taste interactions between amino acids andNaCl/KCl

Sodium and several amino acids had been known askey flavor components of savory seafood as shown inTable 1. Although several studies on the taste interac-tion of NaCl with other compounds were performed,the characteristics of KCl had not been studied much.We then studied the taste interactions between theseminerals and broad amino acids by sensory tests usinga highly trained panel.

NaCl strengthened sweetness of many amino acids(D-Ala, Gly, His, Hyp, Met, Pro, Ser, and Thr) andstrengthened umami of some amino acids (Asp·Na,Glu, Glu·Na, Gln, and Thr) as shown in Table 4. Theseresults supported the previous studies by omission tests(in Table 1). NaCl also diminished sourness and/or bit-terness of many amino acids. In addition, this studyalso confirmed the report by Ugawa et al. (1992) thatNaCl added sweetness to the solution of Gly, Ala, andSer. The addition effect of NaCl to amino acids withregards to sweetness, umami, sourness, and bitternessis supposed to be one of the reasons why NaCl is usedas an important seasoning ingredient.

KCl, a salty and bitter mineral, had not exhibitedremarkable addition effects to the tastes of broad amino

acids. The mineral did not affect sweetness of all theamino acids tested, but it enhanced umami of MSG,suggesting that KCl would be a useful ingredient un-der the combination with MSG.

2-4. Taste interactions between amino acids andIMP

Synergistic effect between MSG and 5¢-inosinemonophosphate (IMP) on umami taste is familiar. Driedbonito (katsuobushi) contains high amount of IMP andJapanese people traditionally use the soup stock pre-pared in combination of katsuobushi and kombu.Nowadays MSG and IMP are industrially produced andused for various kinds of foods in the world. We trustin the fact that people acquire flavor rich and savoryprocessed foods manufactured by using the synergisticeffect. On the other hand, many savory foods containamino acids other than glutamic acid and then we in-vestigated the flavor interaction of IMP with otheramino acids.

Low concentration of IMP (0.5 mM, approximate itsthreshold) affected the tastes of many amino acids ex-cept for some bitter taste amino acids (Table 5). Sev-eral sweet amino acids (Gly, L-Ser, and L-Ala) and theother amino acids showed significant umami enhance-ment. It is known that only acidic amino acids havebeen reported to enhance umami by addition of purine5¢-monophosphates (Yamaguchi et al. 1971; Furukawa1991), but our study showed that the addition of IMPaffected the tastes of various amino acids.

To examine the effects of IMP to three sweet aminoacids (Gly, L-Ser, and L-Ala) in detail, another experi-ment was carried out, using sweetness inhibitor ±2-(p-methoxyphenoxy) propanoic acid (PMP) and 80 ses-sions of triangle distinction tests by 4 expert panelmembers (Kawai et al. 2002). The result suggested thatthe enhanced taste characters of the three amino acidswere not sweetness but umami.

In order to reveal whether these enhancement effectswere synergistic or not, addition effects of IMP to fouramino acids (Gly, L-Ser, L-Ala, and D-Ala) were exam-ined by modulus-free magnitude estimation by 8 ex-pert panel members. Potentiation ratio (PR) values wereemployed to judge on the synergistic effect betweeneach amino acid and IMP (Fig. 5). The results showedthat PR values of Gly, L-Ser, and L-Ala were greaterthan 1, suggesting that the amino acids exhibitedumami taste enhancement synergistically with IMP,though D-Ala did not exhibit the effect.

This was the first report that these three sweet aminoacids had umami taste. We speculated that these aminoacids would bind to both sweet and umami receptors,and IMP would strengthen the affinities between theseamino acids and the umami receptor. Just at that time,the umami receptor (T1R1/T1R3) was found to respondto L-Glu synergistically with IMP, and respond to L-



Tab

le 5

. S

umm

ary

of t

he c

hang

es i

n ta

ste

qual

ity

of a

min

o ac

id s

olut

ions

by

the

addi

tion

of

IMP.

Mod

ifie

d fr

om C

hem

.Se

nses

, 27

, Kaw

ai e

t al

., T

aste

enh

ance

men

t be

twee

n va

riou

s am

ino

acid

s an

d IM

P, 7

39–7

45, T

able

1, „

200

2, w

ith

perm

is-

sion

fro

m O

xfor

d U

nive

rsit

y pr

ess.

Pai

rs o

f st

i mul

us s

olut

i ons

wi t

h an

d w

i tho

ut 0

.5 m

M I

MP

wer

e pr

esen

t ed

rand

oml y

to

10 s

ubj e

cts.

Ave

, av

erag

e. W

i lc,

Wi l

coxo

n. *

p <

0.0

5; *

*p <

0.0

1, s

t at i

sti c

all y

si g

nifi

cant

diff

eren

ces

base

d on

Wi l

coxo

n’s

sign

ed r

ank

t est

. w/ o

, wi t

hout

.

Am

ino

acid

Con

c.(m

M)

Sw

eetn

ess

Sal

tine

ssS

ourn

ess

Bit

tern

ess

Um

ami

Oth

er t

aste

Tot

al t

aste

Ave

.W

ilc.

Ave

.W

ilc.

Ave

Wil

c.A

ve.

Wil

c.A

ve.

Wil

c.A

ve.

Wil

c.A

ve.

Wil

c.

L-A

la2

50

.01

.33

*0

.01

00

.22

0.1

80

-0.3

30

.31

70

.00

0.3

17

1.3

3*

*0

.00

60

.00

0.3

17

1.4

4*

*0

.00

6

L-C

ys7

5.0

1.0

0*

0.0

14

0.0

01

.00

0-1

.26

0.2

06

-0.3

30

.38

00

.78

*0

.03

80

.89

*0

.03

80

.89

0.1

07

L-A

sp3

.00

.44

0.1

57

0.1

10

.65

5-1

.00

0.0

86

-0.2

20

.31

71

.89

**

0.0

04

-0.1

10

.65

5-0

.22

0.7

08

D-A

la2

50

.01

.33

**

0.0

09

0.0

00

.31

7-0

.56

0.4

08

0.1

10

.40

80

.33

0.1

02

0.0

00

.31

71

.00

*0

.01

3

L-G

lu1

.00

.00

1.0

00

0.7

80

.05

9-1

.56

*0

.02

0-0

.11

0.3

17

1.5

6*

0.0

20

-0.1

10

.65

50

.44

0.4

20

L-G

lu- L

-Glu

3.6

-0.1

10

.31

70

.00

1.0

00

-0.5

60

.38

60

.11

0.6

55

0.8

9*

0.0

38

0.2

20

.15

7-0

.56

0.3

86

L-P

he2

5.0

0.0

01

.00

00

.00

1.0

00

0.0

01

.00

0-0

.67

0.1

07

0.1

10

.65

50

.00

0.3

17

-0.6

70

.08

4

Gly

25

0.0

0.1

10

.63

30

.11

0.1

80

-0.3

30

.88

8-0

.22

1.0

00

1.4

4*

*0

.00

7-0

.22

0.5

64

1.2

2*

0.0

29

Gly

-Gly

60

.60

.22

0.3

17

0.0

01

.00

0-0

.78

0.0

53

-0.3

30

.25

7-0

.11

0.6

55

0.1

10

.31

7-1

.00

*0

.02

1

L-H

is5

0.0

1.0

0*

0.0

24

0.0

01

.00

00

.00

1.0

00

0.1

10

.77

30

.00

1.0

00

-0.2

20

.31

70

.89

*0

.04

6

4-O

H- L

-Pro

10

0.0

1.2

2*

0.0

18

0.0

00

.31

7-0

.11

1.0

00

-0.1

10

.89

00

.00

0.3

17

-0.1

11

.00

0-0

.22

0.7

15

L-I

lo4

0.0

-0.2

20

.41

40

.00

1.0

00

0.0

01

.00

00

.22

0.5

66

-0.1

10

.31

70

.22

0.3

17

0.1

10

.71

5

L-L

ys�H

CL

20

.0-0

.11

1.0

00

0.3

3*

0.0

46

0.3

30

.10

2-0

.22

0.6

55

0.1

10

.57

70

.00

0.3

17

0.4

40

.24

8

L-L

eu4

0.0

0.1

10

.31

70

.00

1.0

00

0.2

20

.31

7-0

.78

0.0

53

0.2

20

.31

70

.00

1.0

00

-0.7

80

.05

3

L-M

et5

0.0

0.3

30

.18

0-0

.11

0.3

17

-0.5

60

.05

9-0

.22

0.7

29

1.0

0*

0.0

24

0.2

20

.13

10

.44

0.3

57

L-A

sn5

0.0

-0.1

10

.31

70

.44

0.1

02

-1.4

4*

*0

.00

90

.11

0.3

17

1.7

8*

*0

.00

50

.11

0.3

17

0.6

70

.19

6

L-P

ro2

50

.00

.44

0.4

17

0.0

00

.31

7-0

.22

0.0

83

0.2

20

.74

80

.11

1.0

00

0.0

01

.00

00

.56

0.3

51

DL-p

yro

Glu

3.0

0.2

20

.31

7-0

.11

0.3

17

-0.2

20

.60

3-0

.11

0.3

17

0.0

01

.00

00

.22

0.3

17

-0.2

20

.60

3

L-G

ln2

50

.0-0

.44

0.2

01

0.0

00

.56

4-1

.11

*0

.01

4-0

.11

0.3

96

2.0

0*

0.0

11

0.0

01

.00

01

.22

0.0

80

L-A

rg1

0.0

1.1

1*

0.0

13

0.0

00

.31

70

.00

0.3

17

-0.4

40

.58

40

.00

0.3

17

0.0

00

.31

70

.11

0.7

20

L-S

er2

50

.00

.67

0.2

72

0.4

40

.27

6-1

.11

0.0

50

0.1

11

.00

02

.00

**

0.0

03

0.0

00

.65

51

.78

**

0.0

06

Suc

cini

c ac

id2

.00

.11

0.3

17

0.1

10

.31

7-1

.11

*0

.02

5-0

.22

0.4

14

0.0

01

.00

0-0

.22

0.3

34

-0.7

80

.14

9

L-T

hr2

50

.00

.56

0.2

70

0.0

00

.31

7-1

.22

*0

.02

0-0

.11

1.0

00

1.5

6*

*0

.00

60

.00

0.1

57

1.2

2*

0.0

36

L-V

al5

0.0

0.4

40

.15

70

.00

1.0

00

-0.2

20

.15

7-0

.44

0.4

65

0.0

01

.00

00

.22

0.3

17

-0.2

20

.60

3

L-T

rp1

0.0

0.2

20

.15

70

.00

1.0

00

-0.3

30

.18

00

.11

0.7

73

0.0

01

.00

00

.11

0.3

17

-0.1

10

.85

5

w/o

am

ino

acid

0.2

8*

0.0

25

0.0

60

.31

7-0

.22

*0

.04

6-0

.06

0.6

55

0.1

70

.31

70

.00

1.0

00

0.1

10

.56

4



Ser and L-Ala (Li et al. 2002; Nelson et al. 2002). Thestudies supported our speculation. As generally ac-cepted, glutamic acid and other amino acids, whichenhance umami taste perception, take a role of indica-tor for humankind to recognize protein and contrib-utes to human nutritional intake.

3. Flavor characteristics of glutathione in scallopand other foodstuff

Glutathione (g-L-glutamyl-L-cysteinylglycine, GSH)is a well known tripeptide and exists in a wide rangeof organisms and foodstuffs. The representative physi-ological activity of the peptide is detoxification ofmethylglyoxal, nitrobenzene derivatives, etc., owingto its reductive action. Although many studies on thebiological functions of the peptide have been done, lit-tle was known about its flavor characteristics. After aseries of studies on the flavor components in garlic andonion, it was revealed that GSH has a so-called kokumiflavor expressed by the terms of continuity,mouthfulness and thickness. At the beginning of thereview about GSH, the studies on flavor componentsof the Allium plants, which were the starting point ofthe study of GSH taste characteristic, will be discussed.

3-1. Flavor characteristics of sulfur-containingcomponents in garlic and onion

Garlic (Allium sativum) and onion (Allium cepa) be-long to the Allium genus. There are many studies aboutthe components in the plants from the perspective oftheir bioactivities or characteristic odor. The plants

have several specific sulfur-containing compounds. S-alkyl-L-cysteine sulfoxides are known as the popularprecursors of the specific odors through enzyme reac-tions. Once we cut these plant tissues, pungent and hottaste other than the odors are generated by results ofspecific enzymes such as alliinase.

Cooked garlic is used for various kinds of meals, likesoup, fried meat, grilled meat etc., all over the world.The characteristic additional flavor effects on the foodcan be expressed by terms such as continuity,mouthfulness, and thickness (kokumi flavors). Table6 shows the addition effects of water extract of garlicon the flavor profiles of soups. Key effective compo-nents were found out through both traditional columnchromatography and sensory tests. A cation exchangeresin absorbed fraction, contained sulfur-containing

Fig. 5. The potentiation ratio (PR) of amino acid-IMP solutions derived from mean taste intensities. The PR was calculated bydividing the taste intensity of the mixture by the sum of taste intensities of the individual components in the mixture. Thelarger PRs at low stimulus concentrations might have been due to quasi-zero ratings for both amino acids and IMP unmixedsolutions. Modified from Chem. Senses, 27, Kawai et al., Taste enhancement between various amino acids and IMP, 739–745,Fig. 2, „ 2002, with permission from Oxford University Press.

Fig. 6. Scallop, garlic, and onion.



amino acids and peptides, exhibited characteristickokumi flavors in soups. This fraction also generatedkokumi flavors when it was added to the umami solu-tion prepared from MSG and IMP (Ueda et al. 1990).This fraction contained S-allyl-L-cysteine sulfoxide (al-liin), S-methyl-L-cysteine sulfoxide (MeCSO), g-L-glutamyl-S-allyl-L-cysteine (GAC), g-L-glutamyl-S-al-lyl-L-cysteine sulfoxide (GACSO), and 3-(S)-methyl-1,4-thiazane-5-(R)-carboxylic acid (cycloalliin), iden-tified by using 1H-NMR, 13C-NMR, and FD-MS (Fig.7). As shown in Table 7, we determined some sulfur-containing amino acids and peptides as key kokumiconstituents of garlic. GSH, one of sulfur-containingcomponents in garlic, also exhibited strong kokumiflavors as alliin did.

Onion is also used for cuisine all over the world. Wesuccessfully isolated S-propenyl-L-cysteine sulfoxide(PeCSO) and g-L-glutamyl-S-propenyl-L-cysteinesulfoxide (g-Glu-PeCSO), representative sulfur-con-taining compounds in onion, by the same purificationmethods employed in the case of garlic. These isolatedsulfur-containing compounds exhibited kokumi flavorin the umami solution (Ueda et al. 1994).

We determined contents of these sulfur-containingcompounds in garlic and onion. Alliin, MeCSO,PeCSO, and g-Glu-PeCSO were analyzed by using anamino acid analyzer. GSH and cycloalliin wereanalyzed by these moments, using specific HPLC meth-

ods we established (Ueda et al. 1990; Kuroda et al.1997). The average content of alliin in 13 garlic sam-ples was 2515 mg (/100 g dry weight) and the contentwas as much as that of arginine (2184 mg), the mostprincipal component among amino acids, while aver-age GSH content was small (19 mg/100 g dry weight)(Ueda et al. 1991). In the case of onion, PeCSO (47.3–82.3 mg/100 g dry weight) and g-Glu-PeCSO (50.3–147.3 mg/dry weight) were major sulfur-containingamino acids, while GSH contents were relatively small(0.4–1.8 mg/100 g dry weight) (Ueda et al. 1994).These results suggested that these specific sulfur-con-taining amino acids and peptides would be kokumiflavor constituents in garlic and onion. The contribu-tion of GSH, common sulfur-containing peptide, wasseemed not so large for the whole flavors of the foods.

Garlic and onion were often used for cuisines by heatcooking methods, such as fry, roast, and boil. We wereinterested in thermal stability and degradation of al-liin, PeCSO, and g-Glu-PeCSO in water. Alliin wasvery stable in boiled water. PeCSO and g-Glu-PeCSOwere not so stable when they were heated for long time.PeCSO gradually changed to cycloalliin during heattreatment. g-Glu-PeCSO degraded to 5-oxo-2-pyrolidinecarboxylic acid (PCA) and PeCSO, and theresulting PeCSO was further converted to cycloalliin(Ueda et al. 1994).

3-2. Flavor charac teristics of glutathione inumami solution

As stated above GSH, known as the most populartripeptide in seafood and other foods, also exhibitedkokumi flavors in umami solution. GSH itself had sour-

Chinese soup Curry soup

AROMAWhole Aroma æ æ

BASIC TASTESaltiness æ æSweetness æ æSourness æ æBitterness æ æUmami æ æ

FLAVOR CHARACTERContinuity (0.84)* (0.84)**Mouthfulness (0.90)* (0.78)*Thickness (0.84)* (0.74)*

Component* Kokumi flavors** Other flavor

Alliin +++ Garlic-likeCycloaliin +MeCSO ++ Leak-likeGAC ++ Garlic-likeGACSO + Garlic-likeGlutathione +++Cys +Met +

Table 6. Addition effects of garlic extract on flavor profilesof soups. Modified from Biosci. Biotech. Biochem., 54(1),Ueda et al., Characteristic flavor constituents in water ex-tract of garlic, 163–169, Table I, „ 1990, with permissionfrom Japan Society for Bioscience, Biotechnology, andAgrochemistry.

The addition effects of garlic extract were evaluated by afive-point rating scale (–2 ~ +2). n = 19. *,**Stronger thancontrol significantly. *p < 0.01 and **p < 0.001 versus con-trol (Student’s t-test). —, not significant. ( ), Average score.

Table 7. Kokumi flavors of each sulfur-containing compo-nent. Cited from Biosci. Biotech. Biochem., 54(1), Ueda etal., Characteristic flavor constituents in water extract ofgarlic, 163–169, Table IV, „ 1990, with permission fromJapan Society for Bioscience, Biotechnology, andAgrochemistry.

Umami solution contained 0.05% MSG and 0.05% IMP.*0.2% (w/v) of each compound was added to the umamisolution. **Continuity, mouthfulness, and thickness in thesolution. +++, strongly recognized; ++, apparently recog-nized; +, recognized.



ness in a water solution because of its acidic nature.But in neutral pH condition GSH did not have any tastein water. We then examined effects of GSH on fourbasic taste perceptions (sweetness, saltiness, sourness,and umami) by addition tests. The results of sensoryexamination showed that GSH did not affect theintensities of each basic taste in the tested solutions(Ueda et al. 1997).

Although the threshold value of GSH in water was40 mg/100 ml in the sensory evaluation, the value de-creased to 10 mg/100 ml in the umami solution con-taining MSG and IMP (Table 8), suggesting that GSHhad an interaction with umami substances on its thresh-old. The results showed that kokumi flavors of thesedishes were strengthened by addition of 10 mg/100 gGSH in various kinds of cooked foods, such as Chi-nese soup, hamburger steak, shaomai, etc., suggestingthat the peptide would be a new promising food ingre-dient for processed foods.

3-3. Additional effects of glutathione in the syn-thetic beef and scallop extracts

There was not much knowledge about contents ofGSH in foodstuffs, and then we determined GSH con-tents in a broad range of foodstuffs. GSH was detectedin almost all the tested foods, 13–40 mg/100 g in beefmeats, 3–38 mg/100 g in wines and high levels of 9.6–25 mg/100 g in scallop (Ueda et al. 1997). Table 9showed the results of more specific analyses in fishesand other seafoods. GSH was detected at a lower levelin fishes, but adductor muscle of scallop contained

GSH at a high level concentration that exceeds itsthreshold value, proposing that GSH would be one ofthe flavor constituents in scallop.

GSH was detected in beef meats at relatively highconcentration. The synthetic beef extract was preparedby 36 commercial chemical compounds (amino acids,sugars, nucleotides, carbonic acids, salts, and others)according to analytical data of beef (Ueda et al. 1997).This solution had rather beef like taste though it didnot have any aroma. Effects of the addition of 20 mg/100 ml GSH to the synthetic extract were investigatedby sensory tests. As shown in Table 10, kokumi flavorswere significantly enhanced in the presence of GSH,

Fig. 7. Major sulfur-containing components found in garlic. MeCSO, S-methyl-L-cysteine sulfoxide; GAC, g-L-glutamyl-S-allyl-L-cysteine; GACSO, g-L-glutamyl-S-allyl-L-cysteine sulfoxide.

Solution Threshold value (w/v)

Water 0.04MSG 0.05% 0.04MSG 0.80% 0.02MSG 3.1% 0.01MSG 0.05 + IMP 0.05% 0.01

Table 8. Threshold values of glutathione in water and umamisolutions. Modified from Biosci. Biotech. Biochem., 61(12),Ueda et al., Flavor characteristics of glutathione in raw andcooked foodstuffs, 1977–1980, Table II, „ 1997, with per-mission from Japan Society for Bioscience, Biotechnology,and Agrochemistry.

The test solutions were adjusted to pH 7.0. Triangle distinc-tion tests were used to measure the threshold values (n = 20,p < 0.001).



Samples GSH (mg/100 g) GSSG* (mg/100 g)

FishesBigeye tuna Thunnus obesus 0.81 3.75Bluefin tuna Thunnus thynnus 5.46 ndSkipjack tuna Katsuwonus pelamis 2.58 4.13Chub mackerel Scomber japonicus 0.29 ndHorse mackerel Trachurus japoncus 2.48 ndSardine Sardinops melanosticus 6.51 1.06Yellow tail Seriola quinqueradiata 0.17 ndCoho salmon Onchorhynchus kisutch 9.22 0.38Flatfish Paralichthys olivaceus 2.57 nd

Other seafoodsNorthern green sea urchin Strongylocentrotus intermedius 7.36 3.24Black tiger prawn Penaeus mondon 0.19 ndNorthern shrimp Pandalus borealis 4.45 ndNeon flying squid Ommastrephes batrami nd ndJapanese flying squid Todarodes pacificus nd ndShort-neck clam Ruditapes philippinarum nd ndScallop Pactinopecten yessoensis 29.05 nd

Table 9. Reduced glutathione (GSH) and oxidized glutathione (GSSG) contents in seafood. Cited from Nippon Suisan Gakkaishi,64(4), Ueda et al., Contents of glutathione in seafoods and its flavor characteristics, 710–714, Table 1, „ 1998, with permis-sion from The Japanese Society of Fisheries Science.

*Concentration of GSSG were determined by the differences between the total amount of GSH + GSSG and the amount ofGSH. The total amount of GSH were determined after reduction with GSH reductase. nd, Not detected.

Fig. 8. Effects of GSH on the flavor of synthetic extract of scallop. Added GSH concentration was 29 mg/100 ml. The flavorof GSH-added synthetic scallop extract was evaluated by a five-point rating scale (–2 ~ +2, very weak~very strong), com-pared with that of GSH-free extract (n = 28). Each bar diagram includes a line indicating the 95% confidence level. Citedfrom Nippon Suisan Gakkaishi, 64(4), Ueda et al., Contents of glutathione in seafoods and its flavor characteristics, 710–714,Fig. 1, „ 1998, with permission from The Japanese Society of Fisheries Science.

suggesting that GSH would contribute to generationof kokumi flavors of beef. It was noteworthy that themeet-like flavor of the extract was also strengthenedtogether with kokumi flavors.

Konosu et al. (1988) and Watanabe et al. (1990) al-ready revealed some key flavor components of scal-lop, Gly, Ala, Arg, Glu, AMP, K+, Na+, and Cl–, by aseries of omission tests. We then employed the simpli-

fied synthetic extract containing (mg/100 ml) Gly 1925,Ala 256, Arg 323, Glu·Na·H2O 179, AMP·Na2 195,KOH 232, KCl 109, NaCl 71. The pH was adjusted to6.1 by HCl. GSH was added to the solution at the con-centration of 29 mg/100 ml. GSH significantly en-hanced kokumi flavors as well as sweetness and umami(Fig. 8), suggesting that GSH would be one of the flavorconstituents in scallop.



3-4. Thermal stability of glutathione

We investigated the heat stability of GSH in waterto reveal the possibility of the future application of thispeptide for food processing industry. At first we deter-

mined the degradation products of GSH. GSH washeated in hot water (98∞C, 5 hours) and three com-pounds were isolated from the mixture of the degrada-tion products. The main products were determined asoxidized glutathione (GSSG), PCA, andcyclocysteinylglycine (cyclo Cys-Gly) disulfide. PCAand cyclo Cys-Gly were derived from glutamyl resi-due and cysteinylglycine residue, respectively. Ap-proximately 90% of GSH remained under 30 min heat-ing (pH 5, 98∞C), though 80% of the peptide degradedafter heating for 5 hours (Fig. 9), suggesting that GSHcould be applied as useful materials for many proc-essed food products, except for the retort sterilizationfoods. In addition, it was the new finding that GSHchanged to two kinds of cyclic compounds in water(Fig. 10).

On the other hand GSH is known as a precursor ofreaction flavors when it was heated with reducing sugar(Zhang et al. 1988; Zhang and Ho 1989, 1991a, 1991b).We assured the generation of MFT (2-methyl-3-furanthiol) and FFT (2-furanmethanethiol), known asthe key flavor compounds of roasted meat or coffee,after the reaction of GSH and xylose. GSH could bethe multiple functional food ingredient as a kokumiflavor and a base of reaction flavor.

4. Generation and decreasing mechanism of keyvolatile flavor compounds in dried bonito

Dried bonito (katsuobushi) has been used for Japa-nese cuisine for a long time as the savory food mate-

Fig. 9. Degradation curve for GSH in aqueous solution (98∞C). �, GSH; �, GSSG; �, PCA. Modified from Biosci. Biotech.Biochem., 61(12), Ueda et al., Flavor characteristics of glutathione in raw and cooked foodstuffs, 1977–1980, Figure, „ 1997,with permission from Japan Society for Bioscience, Biotechnology, and Agrochemistry.

Umami solution Model beef extract

Whole aroma æ æBasic tastesa æ æContinuity 0.80b* 0.95**Mouthfulness 0.65* 0.85*Thickness 0.65* æMeat-like flavor æ 0.70**

Table 10. Addition effects of glutathione on flavor profilesof umami solution and model beef extract. Modified fromBiosci. Biotech. Biochem., 61(12), Ueda et al., Flavor char-acteristics of glutathione in raw and cooked foodstuffs, 1977–1980, Table IV, „ 1997, with permission from Japan Soci-ety for Bioscience, Biotechnology, and Agrochemistry.

Umami solution contained 0.05% MSG and 0.05% IMP. Themodel beef extract was prepared by 36 commercial chemi-cal compounds (amino acids, sugars, nucleotides, carbonicacids, salts, and others). GSH concentration was 0.02% w/v.Well-trained 20 panel members evaluated on a five-point rat-ing scale (–2 ~ +2). *,**Significantly stronger than control.*p < 0.01 and **p < 0.001 versus control (t-test). —, notsignificant. a saltiness, sweetness, bitterness, sourness, andumami. b Average score.



rial of soup stock (dashi). It is well-known that driedbonito contains a large amount of IMP and enhancessavory flavors of many kinds of dishes, and also itssavory odor makes various foods preferable. Althoughsome Japanese scientists investigated key volatile com-ponents (Sakakibara et al. 1990), the complete disso-lution of key compounds and their changes duringcooking had remained big issues for the prevention ofdeterioration of savory odors.

Ishiguro et al. (2001) studied the changes in volatilecompounds during smoking process and evaluation ofodor-effective constituents of dried bonito. They ex-amined the changes of major volatile compounds inthe production process (from boiled meats tokatsuobushi) by using gas chromatography-massspectrometry (GC-MS) and gas chromatography-olfac-tometry (GC-O). The study suggested that someethyldimethylpyrazines and phenols (guaiacol and 4-methylguaiacol) contributed to the developments ofroasty and smokey aromas, respectively, and the sulfur-containing compounds were also essential for wholearoma characters of dried bonito.

Taking these results into account we sequentiallyinvestigated generation and decrease mechanism ofsome key volatile compounds, such as pyrazine,phenols, and sulfur-compounds using GC-O methodsestablished by Ishiguro et al. (2001). Although AromaExtract Dilution Analysis (AEDA) method had beenemployed as a popular method for determination ofeffective aromatic components in many foods (Mistryet al. 1997), there still remained the possibility to over-look unstable or low boiling point components. Themethod of Ishiguro et al. seemed effective to examineodor active components in savory foods because wecould analyzed the amounts of volatile components ofthe comparable samples which had actual olfactoryodors (Fig. 12).

4-1. The generation of pyrazines and changes inthe amounts of volatile compounds in smokeduring preparation of dried bonito

The generation mechanisms of pyrazines andphenols, which had been already confirmed to be thekey flavor components of dried bonito, were examinedby GC-MS analysis of various volatile compounds. Atfirst the changes in the amounts of pyrazines was mea-sured in the model dried bonito prepared by heating ofboiled bonito with and without smoking. The amountsof 2,5-dimethylpyrazine, 2,6-dimethylpyrazine, 2-ethyl-6-methyl pyrazine, 2-ethyl-5-methyl pyrazine,and trimethylpyrazine in the dried bonito with smokewere larger than those in the dried bonito withoutsmoke. Both 3-ethyl-2,5-dimethylpyrazine and 2-ethyl-3,5-dimethylpyrazine were not detected in the modeldried bonito (Kawaguchi et al. 2001). We preparedcrushed dried bonito (crushed by a rotary cooking cut-ter) as model powder and reheated it with and withoutsmoke. Figure 13 shows the changes in the amountsof pyrazines in the model powders. Almost all the

Fig. 10. A proposed degradation mechanism of glutathione in water.

Fig. 11. Dried bonito (katsuobushi). Provided from UMAMIInformation Center, „ 2016.



generated during heat treatment of the boiled bonitomeat without smoking, but their amounts and varietieswere less than those in the ordinary dried bonito. Thissuggested that some unknown substances in smokecould be related to the generation of pyrazines.

Fig. 12. Analytical method of volatile compounds in dried bonito.

Fig. 13. Changes in the amounts of pyrazines in dried bonito prepared with and without smoking by heating the powder ofdried bonito at 70∞C for 4 hours. Modified from Nippon Shokuhin Kagaku Kogaku Kaishi, 48(12), Kawaguchi et al., Study onthe generation of pyrazines and change in amounts of volatile compounds in smoke during preparation of dried bonito, 899–905, Fig. 2, „ 2001, with permission from The Japanese Society for Food Science and Technology.

pyrazines were detected in the model powders and theiramounts increased by heating with smoke. 3-ethyl-2,5-dimethylpyrazine and 2-ethyl-3,5-dimethylpyrazinewere not detected in the model powder reheated with-out smoke. All the studies showed that pyrazines were



There had been many studies on the generationmechanism of pyrazines, focusing on Strecker degra-dation between a-dicarbonyl compounds and aminoacids (Hwang and Hartman 1994; Cerny and Grosch1994; Huang et al. 1996). In addition it was knownthat pyrazines were generated by the reaction betweena-ketoalcohol and ammonia (Rizzi 1988). We pre-sumed the generation mechanisms of pyrazines in themanufacture process of dried bonito to be as shown inFig. 14. We added acetol and 2,3-pentanedione to themodel powder and examined the addition effects onthe changes in the amounts of pyrazines. As shown inFig. 15, the amounts and varieties of pyrazines in-creased markedly in the heated model powder. Fromthe result we concluded that acetol and 2,3-pentandionereacted with amino acids or protein in boiled meat, andgenerated pyrazines.

Phenols were not detected in the dried bonito pre-pared by heating without smoking, proposing thatphenols in dried bonito would be mainly derived from

Fig. 14. A presumed mechanism of pyrazine generation indried bonito. Cited from Nippon Shokuhin Kagaku KogakuKaishi, 48(12), Kawaguchi et al., Study on the generationof pyrazines and change in amounts of volatile compoundsin smoke during preparation of dried bonito, 899–905, Fig.3, „ 2001, with permission from The Japanese Society forFood Science and Technology.

Fig. 15. Comparison of the amounts of pyrazines by heating the powder of dried bonito at 70∞C for 4 hours with and withoutacetol and 2,3-pentanedione. Cited from Nippon Shokuhin Kagaku Kogaku Kaishi, 48(12), Kawaguchi et al., Study on thegeneration of pyrazines and change in amounts of volatile compounds in smoke during preparation of dried bonito, 899–905,Fig. 4, „ 2001, with permission from the Japanese Society for Food Science and Technology.



smoke. The incubation of the mixture prepared fromcrushed dried bonito together with cellulose powder,which contained substances in smoke, resulted in de-creasing the amounts of furfural, 2-cyclopenten-1-oneand 5-methylfurfural. Possibly, these compounds wereconverted to non-volatile compounds in the produc-tion process of dried bonito (Kawaguchi et al. 2001).

4-2. Changes in the volatile compounds relatedto deterioration of crushed dried bonito dur-ing storage

So far it had been known that savory odor of driedbonito was stable before crushed. The odors dramati-cally change after crushing and savory odor is lost af-ter long preservation. Though repress of deterioration

Fig. 16. GC chromatograms of volatile compounds in crushed dried bonito kept under different conditions. Volatile com-pounds in the stored samples were absorbed on Tenax-TA Polymer and applied for analysis. 1, propanal; 2, acetone; 3, butanal;4, 2-butanone; 5, 2-methylbutanal; 6, 3-methylbutanal; 7, 2-ethylfuran; 8, pentanal; 9, 2,3-pentanedione; 10, hexanal; 11, 1-penten-3-ol. Cited from Nippon Shokuhin Kagaku Kogaku Kaishi, 49(5), Kawaguchi et al., Changes in the volatile com-pounds related to deterioration of crushed dried bonito during storage, 312–319, Fig. 1, „ 2002, with permission from TheJapanese Society for Food Science and Technology.

of fresh crushed bonito odor had been a big issue forprocessed food industry, the changes of the key vola-tile compounds during storage were not studied satis-fyingly.

We preserved crushed dried bonito samples underthree conditions (–20∞ for 24 days, 25∞C for 12 days,and 70∞C for 4 hrs) and examined their volatile com-pounds. The sample kept at –20∞C had strong acidicodor and the one kept at 70∞C had weak roast odortogether with weak acidic odor. The acidic odor of thesample kept at 25∞C was less than that of the samplekept at –20∞C. Figure 16 showed GC chromatogramof volatile compounds. The amounts of carbonyl com-pounds increased during storage in all samples but thevariation of carbonyl compounds depended on the con-ditions (Table 11). The amounts of straight-chain al-



No. -20∞C, 24 d 25∞C, 12 d 70∞C, 4 h

1 propanal2 acetone acetone3 butanal4 2-butanone 2-butanone5 2-methylbutanal 2-methylbutanal6 3-methylbutanal 3-methylbutanal7 2-ethylfuran 2-ethylfuran8 pentanal pentanal9 2,3-pentanedione 2,3-pentanedione

10 hexanal11 1-penten-3-ol 1-penten-3-ol 1-penten-3-ol

Table 11. Volatile compounds increased in crushed dried bonito during storage under different conditions. Cited from NipponShokuhin Kagaku Kogaku Kaishi, 49(5), Kawaguchi et al., Changes in the volatile compounds related to deterioration ofcrushed dried bonito during storage, 312–319, Table 2, „ 2002, with permission from The Japanese Society for Food Scienceand Technology.

dehydes such as pentanal and hexanal increased at–20∞C, though these aldehydes were not detected inthe samples kept at 70∞C. On the other hand, theamounts of branched chain aldehydes and ketones in-creased in the sample kept at 70∞C. These results sug-gested that degradation of fat (first reaction) and reac-tion among degradation products and other components(secondary reactions) would proceed simultaneouslyand their reaction velocities were different each other.When some of these compounds were added to crusheddried bonito and heated, hexanal and 2,3-pentadionedecreased in the greatest degree (Fig. 17). This sug-gested that straight-chain aldehydes and a-diketonesshould have high reactivity.

To investigate the generation mechanism of increasedcarbonyl volatile compounds, a series of experimentswere carried out using crushed dried bonito and labeledoxygen compounds. The labeled model head space gaswas prepared by mixing of 18O2 (17.6%) and N2(82.4%). Both of these crushed dried bonito samples(with ordinary head space gas and with the labeledmodel gas) were stored at –20∞C and the volatile com-pounds were analyzed by GC-MS (Fig. 18). The re-sults showed that 18O was incorporated to 1-penten-3-ol during storage. When water contained in crusheddried bonito was replaced to labeled water (H2

18O) andkept at –20∞, labeled oxygen was incorporated topropanal, butanal, pentanal, 2,3-pentadione, andhexanal, which increased during storage (Kawaguchiet al. 2002b). It was clarified that these principal car-bonyl compounds were derived from the head spacegas or water in dried bonito during storage. The addi-tion tests of these carbonyl compounds to crushed driedbonito samples showed that straight-chain aldehydeshad strong sensual addition effects (adding rancidodor), though branched chain aldehydes and ketonesdid not have remarkable addition effects (Kawaguchiet al. 2002b).

Fig. 17. Recovery of the amount of volatile compounds af-ter heating crushed dried bonito at 70∞C for 4 hours. Eachcompound (2 ml) was added to crushed dried bonito (20 g)before heating. Cited from Nippon Shokuhin Kagaku KogakuKaishi, 49(5), Kawaguchi et al., Changes in the volatile com-pounds related to deterioration of crushed dried bonito dur-ing Storage, 312–319, Fig. 2, „ 2002, with permission fromThe Japanese Society for Food Science and Technology.

All the results showed that acidic odor was gener-ated by formation of straight-chain aldehydes undercool condition (–20∞C), but acidic odor was diminishedaccording to decreases in straight-chain aldehydes un-der higher temperatures. These findings suggested thatstraight-chain aldehydes might be the cause of flavordeterioration in crushed dried bonito.



4-3. Changes in the volatile sulfur compounds incrushed dried bonito during storage

Sakakibara et al. (1988) studied the changes of vola-tile compounds in dried bonito during storage under30∞C. They speculated that decrease of sulfur-contain-ing compounds, such as methanethiol and dimethylsulfide, was the major deterioration factor of odors.

We investigated the changes in major volatile sulfur-containing compounds during storage under variousconditions by GC analyses. The static head space gastrapping method was employed in place of the purgeand trap method in order to avoid volatilization ofsulfur compounds. The GC-FPD analyses revealed theamounts of volatile sulfur compounds (hydrogensulfide, carbonyl sulfide, methanethiol, carbondisulfide, and dimethyl sulfide) during storage ofcrushed dried bonito. The sulfur compounds decreasedwith time and hydrogen sulfide decreased most rap-idly (Fig. 19) . Addition of hydrogen sulfide,

methanethiol, and dimethyl sulfide to the deterioratedcrushed dried bonito made the flavor as fresh as justafter crushing. These results suggested that sulfur com-pounds, such as hydrogen sulfide, should be the keyflavor components of crushed dried bonito.

4-4. The mechanism of decrease of hydrogensulfide in crushed dried bonito during stor-age

The volatile sulfur compounds were known to havelow threshold and important key flavor components ofmany foods, such as seafood. But little was knownabout the generation or decrease mechanism of hydro-gen sulfide (H2S) in dried bonito because its boilingpoint is very low and analysis method had not beenestablished.

We investigated the decrease mechanism of hydro-gen sulfide in crushed dried bonito. We prepared ether-extractive fraction and water-soluble fraction fromdried bonito, and examined reactivity of each fractionwith H2S. After preservation of the aqueous solutioncomposed of water-soluble fraction and H2S in theclosed vessel for a few days at 25∞C, H2S disappearedand generated the pale precipitate of sulfur (Kawaguchiet al. 2002a). We investigated the key water solublecomponents which caused the decrease in H2S, focus-ing on the coexist-effect of FeCl3 with amino acids or

Fig. 18. Mass spectra of 1-penten-3-ol formed during stor-age of crushed dried bonito with labeled or unlabeled oxy-gen. Cited from Nippon Shokuhin Kagaku Kogaku Kaishi,49(5), Kawaguchi et al., Changes in the volatile compoundsrelated to deterioration of crushed dried bonito during stor-age, 312–319, Fig. 3, „ 2002, with permission from TheJapanese Society for Food Science and Technology.

Fig. 19. Changes in the amount of volatile sulfur compoundsin crushed dried bonito during storage. COS, carbonylsulfide; DMS, dimethyl sulfide. Cited from Nippon ShokuhinKagaku Kogaku Kaishi, 49(2), Kawaguchi et al., The mecha-nism of decrease of hydrogen sulfide in crushed dried bonitoduring storage, 99–105, Fig. 2, „ 2002, with permission fromThe Japanese Society for Food Science and Technology.



organic acids. Referencing the analytical data by Fukeet al. (1989), we prepared model solutions containedmixture of amino acids or mixture of organic acids.When FeCl3 and amino acids coexisted, H2S apparentlydecreased and precipitate generated (Kawaguchi et al.2002a).

Then we prepared several water solutions contain-ing several amino acids and FeCl3. The reactivities ofamino acids with H2S under various pH conditions wereshown in Table 12. Among tested amino acids, the ef-fect by histidine was the largest. The result suggestedthat H2S in dried bonito would be oxidized to sulfurpowder catalytically by the complex of Fe3+ and histi-dine (Fig. 20).

5. Summary and future perspective

We successfully revealed the key flavor constituentsin Kombu-dashi by a series of omission tests. The mix-ture of mannitol, monosodium glutamate, potassiumchloride and sodium chloride exhibited Kombu-dashilike tastes and characteristic kokumi flavors after theinteractions among these compounds. We got the ba-sic knowledge leading to new applications of KCl andsugar alcohols for quality improvements of food. Thesefindings have already contributed for developments of

many seasoning products and processed foods in Ja-pan.

We also revealed that NaCl or IMP influenced ontaste characters of some sweet amino acids. The re-sults would catch interest of scientists who are engagedin studies on amino acids or taste physiology. Toda etal. (2013) proposed the molecular mechanism foramino acids and IMP recognition of T1R1/T1R3 (theumami receptor) by using chimeric human-mouse mu-tants of the receptor. In near future the synergistic ef-fects between these amino acids and IMP will be stud-ied in more detail by using modern molecular biologi-cal research method.

Yamaguchi and Takahashi (1984) studied the inter-action of MSG and NaCl on saltiness and palatabilityof a clear soup. They revealed that a high palatabilityscore was retained even though NaCl concentration wasreduced, as long as MSG was added at its optimal level.The study suggested that NaCl intake level could bereduced by using MSG. Our findings would also con-tribute to the development of low salt ingredients andreduction of sodium intake from processed foods bythe results of flavor interactions among MSG, aminoacids, and salts.

We found a new kind of non-volatile flavor compo-nents in foods, focusing on the flavor interaction with

+, Recognized. –, not recognized. 480 ml of 1.0 mM FeCl3 and 80 mg of each amino acid were dissolved in 40 ml of distilledwater. 5 ml of this solution and 2 ml of H2S were mixed in the closed vessel and stored at 25∞C. pH values of the solution 1~8were not adjusted. pH values of the solution 9~16 were adjusted at 5.8.

No. Amino acid pH Generation ofprecipitate

Decrease ofH2S

Browning ofsolution

Before adjustment After adjustment

1 Gly 5.4 - - -2 L-Ala 5.4 - - -3 L-Asp 3.1 - - -4 L-Glu 3.7 - - -5 L-His 7.7 + + +6 L-Arg◊HCl 5.4 - - -7 L-Lys◊HCl 5.3 - - -8 Tau 5.1 - - -9 Gly 5.4 5.8 - - -

10 L-Ala 5.4 5.8 - - -11 L-Asp 3.1 5.8 - - -12 L-Glu 3.7 5.8 - - -13 L-His 7.7 5.8 + + +14 L-Arg◊HCl 5.4 5.8 - - -15 L-Lys◊HCl 5.3 5.8 - - -16 Tau 5.1 5.8 - - -

Table 12. The reactivities of amino acids with H2S under various pH conditions. Cited from Nippon Shokuhin Kagaku Kogaku

Kaishi, 49(2), Kawaguchi et al., The mechanism of decrease of hydrogen sulfide in crushed dried bonito during storage, 99–105, Table 2, „ 2002, with permission from The Japanese Society for Food Science and Technology.



umami substances. The sulfur-containing compoundsin garlic and onion, such as alliin, S-propenyl-cysteinesulfoxide and GSH, had no tastes themselves but theygave rise to kokumi flavors in the umami solution orsoups. Scallop contained GSH in a high amount andthe peptide suggested to contribute to characteristickokumi flavors of the food. The research on the chemi-cal nature of the peptide revealed that it would be valu-able ingredient for food industry. On the bases of ourstudies, a novel kokumi peptide, g-L-glutamyl-L-valylglicine (g-Glu-Val-Gly, Fig. 21), was found outby using modern biological technique (Ohsu et al.2010). They succeeded in the cloning of sensoryreceptor of GSH and determined several compoundswhich combined to the receptor. g-Glu-Val-Gly exhib-its strong kokumi flavors in various foods and recentlyit was approved by Japanese government as a speci-fied food additive, expecting to be applied for proc-essed foods. Few studies have been done on kokumiflavors. The difference between GSH and g-Glu-Val-Gly will be an interesting subject for scientists in fu-ture.

The generation and decreasing mechanism of somekey volatile flavor components of dried bonito weredetermined by using GC-MS and GC-Sniffing meth-ods. We found the mechanism of generating pyrazines,the key components of savory odors of dried bonito.Pyrazines were presumed to be produced after the re-action between some smoke derived compounds, suchas acetol and 2,3-pentandione, and the other compo-nents in bonito meat. The studies on the changes ofaldehydes and ketones during storage under differenttemperatures suggested that the compounds were pro-duced by the complex reactions among low molecularcomponents other than degradation of fat. Straightchain aldehydes, such as hexanal and pentanal, mightbe involved in the deterioration of flavor in crushed

dried bonito. We examined the changes in the amountsof some volatile sulfur compounds, key compounds forfresh dried bonito odors, during storage of crusheddried bonito. All these compounds decreased muchduring storage and hydrogen sulfide almost disap-peared. The results of our study suggested that hydro-gen sulfide would be oxidized to sulfur by ferric ionand histidine in dried bonito. There are various kindsof processed foods which are made of dried bonito inJapan. Effective generation of fresh dried bonito odorsand prevention of odor deterioration during total pro-duction processes are essential issues for developmentof high quality products. We believe all the findings inthis study should contribute to the resolutions of theseissues and for future studies of volatile flavor compo-nents in foods.

AcknowledgmentsWe are grateful to Dr. S. Watabe for helpful advices. This

study was realized in cooperation with many persons ofAjinomoto Co., Inc.: especially Mr. M. Sakaguchi, Mr. T.Shimizu, Dr. A. Okiyama, Mr. N. Miyamura, Dr. H.Wakabayashi, Mr. M. Yonemitsu, Dr. M. Kawai, Dr. M.Kuroda, and Dr. H. Kawaguchi.

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Fig. 20. A presumed mechanism of oxidation of H2S in crushed dried bonito. Modified from Nippon Shokuhin Kagaku Kogaku

Kaishi, 49(2), Kawaguchi et al., The mechanism of decrease of hydrogen sulfide in crushed dried bonito during storage, 99–105, Fig. 4, „ 2002, with permission from The Japanese Society for Food Science and Technology.



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Flavor Constituents in Savory Seafood: Dried Kelp (Kombu), … · savory seafood revealed that...

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Transcript of Flavor Constituents in Savory Seafood: Dried Kelp (Kombu), … · savory seafood revealed that...