Halophilic Bacteria Potential for Development of Thai Fish Sauce Industry

1
Sirilak Namwong1

12-18

: , ,
2553;29(4):470-477

Abstract
Thai fish sauce, a traditional seasoning with salty flavor and mild fishy smell, is a clear brown liquid derived from

fish fermented with salt in natural conditions for 12-18 months. During the fish sauce fermentation process,
proteinase-producing halophilic bacteria were isolated and their proteinases were characterized, in order to speed up
the degradation of fish protein during fermentation. Recently, acceleration of fish sauce fermentation was investigated
using the combination of halophilic bacteria as a starter culture and commercial proteinases. The fermentation
process was completed within 4 months and the fermented product comprised of overall sensory characteristics of
traditional fish sauce. Therefore, the acceleration of fish sauce fermentation may expand the growth of the fish sauce
industry that is limited by high capital investment of land and extremely long fermentation time, and increase the
export of Thai fish sauce worldwide.
Keywords: fish sauce, acceleration of fish sauce fermentation, halophilic bacteria.
J Sci Technol MSU 2010;29(4):470-477

, 10300.
Lecturer, Department of Biotechnology, Faculty of Sciences and Technology, Suan Sunandha Rajabhat University, Dusit District,
Bangkok, 10300. Thailand. E-mail : sirilak.na@ssru.ac.th. Received: 15 June 2009; Accepted: 22 September 2009.

1
1

Vol 29, No 4, Oct-Dec 2010

Halophilic Bacteria Potential for Development of Thai Fish Sauce Industry

12-18 1,2

(alcalase flavourzyme)

4
(22-29%)

20 mg/100 ml 5

( pH 5.6-6.3)1




6,7
(fish sauce)



..
2550
21.40

(82%) (3.5%)


5 8

471

Figure 1 (Stolephorus
indicus) 2:1 3:1

(anaerobic

facultative fermentation) 12-18

1,2
(Table 1)
(27-28%) (2.9-7.1%)

(glutamic acid)
(aspartic acid) 1 2 1
9
()
( ) ( )

()

(ripen)10

( 20 mg/100 ml) 5

Histidine
decarboxylase ( )

Fresh water fish or marine fish

Mixing with salt (2:1 3:1) (fish:salt, w/w)

Fermentation (12-18 months)

Filtering

Extract/unripened
Fish residue
fihs sauce

Ripening
Added saturated brine

Top-grade
Fermentation 1-4 months
Fish sauce

Filtering


Second-grad fish sauce

Figure 1 Traditional fish sauce production scheme.1

472

Namwong S

J Sci Technol MSU

Table 1 Chemical composition of fish sauce.1

Chemical compositions

(g/100 mll)

NaCl

22.5-29.9

Total amino acids

Total nitrogen

2.9-7.7
2.0

Glutamic acid (g/total nitrogen)

0.38-1.32

Total organic acid

0.21-2.33

Lactic acid

0.06-0.48

Succinic acid

0.02-0.18

Decarboxylation

11
(halophilic bacteria)

(halophilic bacteria)
(taxomony
of halophilic bacteria)

Namwong 12,13
Spread
plate JCM. no. 377 (10% NaCl) JCM. no.
168 (20% NaCl) JMC. no. 169 (25% NaCl)
100-250 g NaCl, 5 g casamino
acid, 5 g yeast extract, 1 g glutamic acid; 2 g KCl, 3 g
trisodium citrate, 20 g MgSO4.7H20, 36 mg FeCl2.4H20
FeSO4.4H20, 0.36 mg MnCl2.4H20 MnSO4,

20 g agar 2
14,15
1. (halophilic archaea)

15% (saturated NaCl)

20-25%

22-29% 1,2

Halobacterium salinarum (

), Halococcus thailandensis, Hcc.

Saccharolyticus Natrinema gari 12,16-8
2. (moderate halophilic
bacteria)

0.5-30%

3-15%

2
Halobacillus thailandensis, Lentibacillus juripiscarius,
Halobacillus spp. SR5-3, Staphylococcus spp. 1-1-5,
Virgibacillus spp. SK33 Virgibacillus spp. SK376,7,13,19

L. salicampi, L. halophilus, S. piscifermentans,

Chromohalobacter salexigens13,17,20,21

pH
(pH and thermal stability effect of NaCl

on stability)

Thongthai 22

(15%
) (85%

Vol 29, No 4, Oct-Dec 2010

Halophilic Bacteria Potential for Development of Thai Fish Sauce Industry

.. 2006-2008

Halobacillus spp. SR5-3

Halobacillus spp. SR5-3

5
Halobacillus spp. SR5-3
JCM no. 377

(JCM no. 377) Izotova 23
25%

(acidic amino acids)

Halobacillus spp. SR5-3 1% yeast extract,

0.32% aspartic acid, 0.32% glycine 2% ami ami
(

4%) JCM no. 377 (Figure 2)

Halobacillus
spp. SR5-3

J+2%A+1%Y+0.32%Asp+0.32%Gly
J+2%A+1%Y+0.5%Lactose
J+2%A+1%Y+0.5%Sucrose

473

Halobacillus spp. SR5-3

(stability) pH 5.0-9.0 30-50C

(effect of NaCl on activity)

Suc-Ala-Ala-Phe-Pro-MCA
(substrate) (Figure 3A)
(Figure 3B)

(conformational changes)
Suc-Ala-Ala-Phe-ProMCA (synthetic substrate)

24
(effect of NaCl on
stability)

20-30%

(Halobacillus spp. SR5-3)

J+2%A+1%Y+0.5%Starch
J+2%A+1%Y
J+1% Yeast cxtract (Y)
J+1% ami (A)
J+2% ami (A)
JCM No.377(J)
0 50 100 150 200 250 300 350 400 450

Proteinase activity (U/ml)

Figure 2 Effect of nitrogen and carbon sources to

proteinase activity of strain SR5-3. SR5-3
strain was cultivated in the JCM No. 377 with
the specified nutrient supplementation.6,17

12-18
1

Squid hepatopancrease (SHP)25
(trypsin chymotrpsin)26

(trypsin-like serine proteinase) 27

Namwong S

J Sci Technol MSU

100

100

80

80

Remaining activity (%)

Remaining activity (%)

474

60
40

60
40

20

20

10

15

20

NaCl (%, w/v)

25

30

(A)

10

15

20

NaCl (%, w/v)

25

30

35

(B)

Figure 3 Effect of NaCl concentration on the activity (A) and stability (B) of the purified proteinase. A, The activity of
the purified enzyme was measured at various NaCl concentration at pH 7.5 using casein () or Suc-Ala-Al
a-Pro-Phe-MCA () as substrate. B, The purified enzyme was incubated at various concentraions of N
aCl at 37C at pH 7.5 for 24h. Then remaining activity was measured using casein () or Suc-Ala-

Ala-Pro-Phe-MCA ().6,17
28


Yongsawatdigul 7
3
(Virgibacillus spp. SK33, Virgibacillus spp. SK37
Staphylococcus spp. SK-1-1-5)

(fish broth) (25%)

1:9

18 (0.25% alcalase 65 C

2h 0.5% flavourzyme 50C

4h) 35C 4 Table 2

( )

12-18

3
20 mg/
100ml5
Control (,
21.53 mg/100ml) Virgibacillus spp. SK33
(11.76 mg/100ml)

(overall acceptance)
Staphylococcus spp. 1-1-5
7

.. 2551

2,000-3,000 nm
29

Vol 29, No 4, Oct-Dec 2010

Halophilic Bacteria Potential for Development of Thai Fish Sauce Industry

475

Table 2 Chemical compositions of fish sauce and mean score of color, odor, flavor, and overall acceptance of fish
sauce samples inoculated with various strains of bacterial starter cultures.7
Fish sauce compositions

Fish sauce samples (4 months)

Control
SK1-1-5
SK33

SK37

714.1
9576
1791
1023
941

11.76

0.229
1.079
0.073
0.050

4.53b
4.24
3.94b
3.94b

713.3
9513
1932
1090
968

15.42

0.212
0.925
0.041
0.033

4.35b
3.59
3.71b
3.82b

Commercial
(12 )
-Amino acid contents (mM)
-
682.7
733.4
Total amino acids (mg/100 ml)
9039
8729
8928
Glutamic acid (mg/100 ml)
1856
1542
1796
Aspartic acid (mg/100 ml)
998
911
1006
Lysine (mg/100 ml)
932
847
912
Biogenic amine



histamine (mg/100 ml)
208
21.5
20.65



Volatile compounds
2-methylbutanal (meathy note)
0.324
0.152
0.179
2-propanone (cheesy note)
1.360
0.564
0.918
n-propanol
0.066
0.029
0.069
Butanoic acid
0.115
0.102
0.156


Attibutes*

b
-
5.29a
color
4.18
odor
4.24
-
4.24
a
-
3.53b
flavor
4.94
overall acceptance
4.82a
-
4.24ab
*Acceptance score: 7 = extremely like; 4 = neither like nor dislike; 1 = extremely dislike.
Different superscripts within a column indicate significant differences (p<0.05)

1,2
1)

2)

3)

6,7
Virgibacillus
spp. SK33, Virgibacillus spp. SK37 Staphylococcus
spp. SK1-1-5

4 7

476

Namwong S

37C27

(Virgibacillus spp. SK33)
(Staphylococcus spp. SK1-1-5)

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