Unique characteristics of Japanese

fermented seafood

Masataka Satomi National Research Institute of Fisheries Science, Fisheries Research Agency,

Japan-Norway Marine Seminar

National Research Institute of Fisheries Science (NRIFS)

(1) investigations on fisheries resources, marine environment and stock enhancement in Japan (2) studies on food processing and preservation, and fisheries management and economy

・Located in Yokohama ・beside Tokyo Bay ・Appro.100 researchers

Masataka SATOMI (NRIFS) Food Hygiene and Management Research Group, Research Center for Biochemistry and Food Technology.

Japanese fermented seafood

Map of major production area of Japanese fermented seafood

Fish sauce（Akita：しょっつる）

Fish sauce (Ishikawa:いしる・よしる)

Fish sauce （Kagawa：イカナゴ醤油）

Izushi（Hokkaido：いずし）

Shiokara （Hokkaido・Tohoku:塩辛）

Kusaya（Izu islands：くさや）

Funazushi（Shiga）

Shiokara（Kochi：酒盗） Sukugarasu（Okinawa：スクガラス）

Katsuobushi （かつお節）

Fermentation types of Japanese fermented seafood

・salted seafood

・Kusaya

・Fish sauce

Fermented types period Fermented food

・Funazushi

・cured with bran

・pickled with rice

・Katsuobushi

2～3weeks

>100 years

>6 months

self-digestion Microorganisms

salted

pickled

others

1 year

>1 year

2～3weeks

△ ○ △

○ × ○

◎ ○ ○

△ ○ ○

× △ >6 months

Shiokara（salted and fermented seafood）

・squid, skipjack tuna, salmon kidney, and others ・unique taste is mainly induce by autolysis

Procedure of Shiokara production

10～20days

Salt conc. >10％

Liver Paste

body Slice

salt

mix Ripen

Katsuobushi （boiled, smoke-dried and molded skipjack tuna）

製造工程

焙乾（二番火）

荒

節

削

り 裸

節

あんじょう

小骨抜き・修繕

1日（10～20日繰り返す）

原料魚（カツオ等）

三枚おろし

かご立て

放

冷

骨抜き

かご離し

なまり節

煮

熟

頭・内臓・鰭除去

5～6時間

水切り焙乾（一番火）

日

乾

カビ払い落し

本枯れ節

カビ付け（一番カビ）

10～20日間 （４番カビまで繰り返す）

・boiled, smoke-dried and molded; >6months for production ・moisuture and lipid content are very low; solid and hard ・Umani taste and flavor is important for Japanese cuisine

Heshiko（cured mackerel in rice bran）

・Aging of salted mackerel with rice bran at ambient temperature for over 7 months. ・Strong flavor with mild sour taste is impressive ・Norwegian mackerel is used for Heshiko production

Salted in brine

Curing in rice bran

Fish sauce -try to reduce histamine-

Regulation of histamine Japan: no regulation Codex: 400ppm for fish sauce FDA: 50ppm for every food

Whole Fish Minced with Organs Mix with materials

Fig.1. Procedure for producing Fish Sauce

Fermentation for 6 month

ExtractHeat Filtration

Step 1 Step 2 Step 3

Step 4

Step 5Step 6Step 7

Products

Step 8

・Bacterial counts・Identification of Bacteria・Chemical compounds

Analysis of

・Fish meat・Koji (mould starter)・Salt (final 15%)

What is a Fish Sauce? -Fish sauce is common and traditional fermented condiment in Southeast and East Asia. -Many fish sauces are made of small seawater fish with long term fermentation -Many kinds of fish are used in the production of fish sauce; anchovies , squid and others.

・Hm concentration

・predominant bacteria

>2,000ppm

Tetragenococcus sp.

Tetragenococcus sp.

Undesired bacteria

異常発酵

正常発酵

Fig. Changes in bacterial counts during

fish sauce fermentation

Fig. Time course of Hm accumulation

in fish sauce mash

Hm accumulation in fish sauce

3

4

5

6

7

8

9

0 4 8 12 16 20 24

Fermentation period（weeks）

log

cfu

(vi

able

co

un

ts)

0

400

800

1200

1600

2000

0 4 8 12 16 20 24

Fermentation period （weeks）

His

tam

ine

co

nc.

（p

pm

)

Halophilic lactic acid bacteria

Undesired bacteria Desired fermentation

Hm accumulation

Histamine production

Histidine (His) One of the amino acid Scombroid Fish

Histamine (Hm) Heat resistance No odor Allergy like symtonms

Decarboxylation by microorganisms

CO2

Major Hm producers

・Gram negative bacteria (pyridoxal type)

－Enterobacteriaceae（Morganella sp.）

－Marine bacteria（Photobacterium spp.）

・Gram positive bacteria (pyruvoyl type)

－Lactic acid bacteria

－Clostridium perfringens

Post harvest

Marine environment Psychrophiles（can grow 4℃)

Wine, cheese, fermented sea foods

Bacterial histidine decarboxylase (HDC) produce Hm Scombroid fish contain large amount of His

Isolation of Hm producing bacteria

・Enrichment culture and limiting dilution methods with

histidine broth (1%, L-histidine in 10% NaCl-GYP broth)

・Suspects wereTetragenococcus halophilus

・Single copy of pyruvoyl hdc was coded on plasmid

0.02

Bacillus pumilus

Enterococcus ratti

Tetragenococcus muriaticus

Tetragenococcus koreensis

Tetragenococcus halophilus

new isolates (3 strains) 100

92

Tetragenococcus solitarius 97

100

Fig. Phylogenetic tree of the new isolates producing

Hm based on 16S rRNA gene sequence.

Fig. Microscopic observation

of the Hm producing bacteria

with gram stain (above) and

SEM observation (below).

Sequence analysis of hdc among lactic acid bacteria

E. aerogenes ATCC43176

R. planticola ATCC43176

M. morganii AM-15

V. anguillarum 775

T. muriaticus JCM10006T

T. halophilus H

Lactobacillus sakei LTH2076

Lb. hilgardii 0006

Lb. reuteri F275

C. perfringens 13

Lactobacillus 30a

Lb. buchneri B301

T. halophilus

0.1

% Similarity of nt.

with strain H

44.4

47.4

45.0

41.6

99.7

99.7

99.8

100

89.1

77.4

75.1

56.3

99.7

Protein

Function

HdcA

(pyridoxal phosphate

as cofactor)

HdcA

(pyruvoyl moiety

as prostetic group)

Oenococcus oeni 9204 99.4

% Similarity of aa

with strain H

16.7

21.4

22.0

22.5

99.4

99.1

99.4

100

97.8

78.2

79.1

43.5

99.0

98.4

Strains

Wine

cheese

Fish sauce

Was hdc able to transfer beyond genus?

Final goal of this study

To elucidate the mechanisms of hdc transfer beyond bacterial species

・collect various Hm producing bacteria

・make clear hdc coding regions →Plasmids or genome

・determine plasmid sequences coding hdc

→mobile genetic elements?

・sequence analysis for planking region of hdc

→transposones?

procedures

Grouping of plasmids

Isolation source number

2

1

4

4

8

Ⅰ

Ⅱ

Ⅲ

Ⅳ

Ⅴ

Ⅵ

RFLP Rep. strain

H

A

I

HO

RI

T. muriaticus 1

Fish sauce A (2004)

Fish sauce C (2006)

Fish sauce D (2006)

Squid sauce (1997)

Fish sauce B (2004)

Fish sauce C (2006)

Full sequences were determined

Same color indicate homolog; a.a., amino acid; met., metabolite; mod., modify; ND, not determined

Sugar met.

a.a. metabo.

HDC

DNA mod.

DNA mod.

DNA mod.

DNAmod. mob

a.a. metabo.

HDC

HDC

HDC

IS 30

IS 30

IS 30

IS 30

Tn

Tn

Tn

IS 200

IS 200

IS 200

IS 200

Tn 11

Tn 11

Tn 11

ND

ND

oriC

oriC

oriC

oriC

Strain H （Fish Sauce A）

29,924ｂｐ

32,502ｂｐ

36,642ｂｐ

21,516ｂｐ

Strain HO （Fish sauce C）

T. muriaticus （Fish Sauce E）

DNA mod. HDC IS 30 Tn

IS 200

Tn 11

ND oriC

Strain RI （Fish Sauce D）

21,230ｂｐ

ND

Genetic map of plasmids encoding hdc

Strain A （Fish Sauce B）

Summary of sequence analysis

Length (bp)

30K

33K

>25K

37K

22K

Ⅰ

Ⅱ

Ⅲ

Ⅳ

Ⅴ

Ⅵ

RFLP strain

H

A

I

HO

RI

T. muriaticus 22K

Tn oriT oriC

H

A

ND

A

RI

A

+

-

ND

-

-

-

ND: not determined

+

+

+

+

+

+

source

A

B

C

C

D

E

Strain H

Strain A

T. muriaticus JCM10006 T

Lb. reuteri

HdcP HdcA HdcRS

HdcP HdcA HdcB HdcRS

HdcB

NhaC ORF ORF

IS1216V IS30 IS200

HdcP HdcA HdcRS HdcB ORF IS30 IS200

HdcP HdcA HdcRS HdcB ORF IS30 IS200

> 99 % Fig. 3. Genetic organizations of bacterial histidine decarboxylase loci. The box indicates a set of genes that are more than 99% identical.

Further study

・Origin of plasmids harbored in T. halophilus strains

were different

・Conjugative elements was found only in pHDC-H

・ IS30 and IS200 were determined on the regions

upstream and downstream of hdc clusters

hdc cluster was transferred by transposones.

Need more analysis of plasmids and transposones

Lb. reuteri?

Effect of starter culture and sugar for inhibition of histamine accumulation

Fermentation period (days)

His

tam

ine

co

nte

nt

(pp

m)

Control (Fish meat) Starter + sugar lot A Starter + sugar lot B

Use of starter culture for fish sauce fermentation

➣Histamine producer in fish sauce was halophilic lactic acid bacteria, Tetragenococcus spp.

The genes related to histamine production are coded on mobile genetic element.

Tetragenococcus spp. are dominant bacteria in fish sauce mash and produce lactic acid.

➣Starter culture for fish sauce fermentation was developed.

It is important to make condition that starter culture is able to grow and produce lactic acid in fish sauce mash; supplemented glucose or sucrose to mash for supporting growth of starter.

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Summary