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.
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
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.
スターターが十分に活躍できる 環境づくりがポイント
Summary