International Journal of Food Microbiology 73 (2002) 61 – 70

www.elsevier.com/locate/ijfoodmicro

Fermentation and microf lora of plaa-som, a Thai fermented f ish

product prepared with different salt concentrations
Christine Paludan-Müller a,*, Mette Madsen b, Pairat Sophanodora c, Lone Gram a,
Peter Lange Møller b
a
Department of Seafood Research, Danish Institute for Fisheries Research, Technical University of Denmark, Building 221,
DK-2800 Kgs. Lyngby, Denmark
b
Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, Rolighedsvej 30,
DK -1958 Frederiksberg C, Denmark
c
Faculty of Agro-Industry, Prince of Songkhla University, Hat Yai, Songkhla 90110, Thailand
Received 4 February 2001; received in revised form 31 July 2001; accepted 21 September 2001

Abstract

Plaa-som is a Thai fermented fish product prepared from snakehead fish, salt, palm syrup and sometimes roasted rice. We
studied the effects of different salt concentrations on decrease in pH and on microflora composition during fermentation. Two
low-salt batches were prepared, containing 6% and 7% salt (w/w) as well as two high-salt batches, containing 9% and 11% salt.
pH decreased rapidly from 6 to 4.5 in low-salt batches, whereas in high-salt batches, a slow or no decrease in pH was found.
Lactic acid bacteria (LAB) and yeasts were isolated as the dominant microorganisms during fermentation. LAB counts
increased to 108 – 109 cfu g 1 and yeast counts to 107 – 5  107 cfu g 1 in all batches, except in the 11% salt batch, where
counts were 1 – 2 log lower. Phenotypic tests, ITS-PCR, carbohydrate fermentations and 16S rRNA gene sequencing identified
LAB isolates as Pediococcus pentosaceus, Lactobacillus alimentarius/farciminis, Weisella confusa, L. plantarum and
Lactococcus garviae. The latter species was only isolated from high-salt batches. Phenotypic characteristics, ITS-PCR and
carbohydrate assimilation identified 95% of the yeasts as Zygosaccharomyces rouxii. It is concluded that the fermentation of
plaa-som is delayed by a salt-level of 9% due to an inhibition of LAB growth. The growth of Z. rouxii has no influence on the
fermentation rate, but may contribute positively to the flavour development of the product. D 2002 Elsevier Science B.V. All
rights reserved.

Keywords: Fermented fish; NaCl; Lactic acid bacteria; Pediococcus pentosaceus; Yeasts; Zygosaccharomyces rouxii

1. Introduction preferences. Therefore, large differences exist in pro-

Thai fermented fish products are mostly produced
according to family tradition and local geographic
*
Corresponding author. Tel.: +45-4588-3322; fax: +45-4588-
4774.
E-mail address: cpm@dfu.min.dk (C. Paludan-Müller).
duction methods, use and proportions of raw materi-
als, and in the names used for the various products.
The fermented fish product plaa-som is typically
composed of freshwater fish, salt, boiled rice and
garlic (Adams, 1986), and is mainly produced in the
central and north-eastern part of Thailand. However,
in the Songkhla province in Southern Thailand, a local

0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 1 6 0 5 ( 0 1 ) 0 0 6 8 8 - 2
62 C. Paludan-Müller et al. / International Journal of Food Microbiology 73 (2002) 61–70
variety of plaa-som is produced, in which garlic and
boiled rice are replaced by palm syrup and from time
to time by roasted rice, thus resembling plaa-uan,
another type of Thai fermented fish (Pithakpol et al.,
1995).
Lactic acid bacteria (LAB) are found as the
dominant microorganisms in many fermented fish
products (Orillo and Pedersson, 1968; Saisithi et al.,
1986; Olympia et al., 1992; Østergaard et al., 1998).
The primary role of LAB is to ferment the available
carbohydrates and thereby cause a decrease in pH.
The combination of low pH and organic acids
(mainly lactic acid) is the main preservation factor
in fermented fish products. Generally, pH should be
below 5 – 4.5 in order to inhibit pathogenic and
spoilage bacteria (Owens and Mendoza, 1985). In
addition, salt and spices (such as garlic, pepper or
ginger) may add to the safety of products. Also, in
some products garlic may serve as a carbohydrate
source for the fermentation (Paludan-Müller et al.,
1999).
Yeasts have been isolated in high numbers from
several fermented fish products, although in particular
from products in which a mould or yeast starter
culture (angkak or khao-mark) is used (Arroyo et
al., 1978; Sakai et al., 1983; Adams et al., 1985).
Yet, the role of yeasts in the fermentation process has
not been elucidated. In som-fak, a Thai-fermented fish
product, growth of yeast is a sign of spoilage (Saisithi
et al., 1986).
The salt concentration may range from one to 10%
(w/w) in different types and batches of fermented fish
(Anonymous, 1982; Saisithi, 1987). This is likely to
have a pronounced influence on the microbial growth
and the rate of fermentation, and thereby on the
sensory quality and safety of the product. It is there-
fore of interest to identify the optimal salt concen-
tration, which does not inhibit the growth of the
fermenting microorganisms, and in addition contrib-
utes positively to the flavour and texture of the
product.
The LAB and yeast flora of plaa-som produced in
Songkhla was characterised in this study. Salt levels in
plaa-som ranged from approximately 6 –11% water-
phase salt. The aims were to identify the dominant
microbial species and to evaluate the effect of typical
variations in the salt content on the microflora com-
position and the rate of fermentation.
2. Materials and methods
2.1. Production of plaa-som
Two batches of the product were carried out by a
local producer in the Songkhla province, Southern
Thailand and brought to the laboratory. Another two
batches were carried out in the laboratory with the use
of ingredients bought from the same local producer.
The freshwater fish species, striped snake-head fish
(Channa striatus), was used as fish raw material. The
fish was degutted, cleaned, and the whole fish mixed
thoroughly in a brine of sea salt and palm syrup.
Powdered, roasted rice was added to one of the two
batches of plaa-som produced by the local producer
(low-salt batches) and in the laboratory (high-salt
batches), respectively. At the producer, the propor-
tions of the ingredients were approximately: 0.75 kg
fish; 100 g dried sea salt; 125 ml palm syrup with and
without 80 g roasted rice. In the laboratory, the
proportions were approximately: 0.6 kg fish; 100 g
dried sea salt; 125 ml palm syrup with and without 80
g roasted rice. The products were packed in plastic
bags and fermented at ambient temperature (30 –38

C) for 8 and 12 days for the low- and high-salt
batches, respectively. Samples were withdrawn on
days 0, 1, 2, 3, 4, 5, 8 and 12 for pH measurements
and microbiological analysis (microscopy and plate
counts). On day 0, the fish, palm syrup and roasted
rice were sampled for microbiological analysis.
2.2. Chemical analyses
NaCI and moisture were measured on days 0, 5 and
8 by the AOAC method (AOAC, 1990). pH was
measured in 1:1 dilution of the samples. The dry-
matter content of the palm syrup was determined by
drying 2 g of syrup for 12 –14 h at 102 –105
C. The
concentration of sucrose, fructose and glucose in palm
syrup was determined with an enzymatic kit (Cat. No.
716260, Boehringer Mannheim, Tutzing, Germany).
2.3. Microbiological analyses
For microbiological analysis, a 10-g sample (5 g
fish/5 g liquid) was homogenised with 90 ml of
physiological saline water for 30 s in a Seward sto-
macher 400. Phase contrast microscopy was made on
 C. Paludan-Müller et al. / International Journal of Food Microbiology 73 (2002) 61–70
the 10 1 dilution. Aerobic, mesophilic counts were
performed on Tryptone Soy Agar (TSA) plates (Oxoid
CM131, Basingstoke, UK), LAB on de Man, Rogosa
and Sharpe (MRS) agar plates (Oxoid, CM361) and
yeasts on Potato Dextrose Agar (PDA) agar plates
(Difco 0013-17-6, Detroit, MI, USA). Plates were
incubated at 30
C. TSA-plates aerobically for 2 days,
MRS-plates in covered glass jars with a burning candle
(microaerophilic) for 3 days and yeasts aerobically for
5– 7 days. Colonies were randomly isolated from the
highest dilution of MRS- and PDA-agar, isolating all
colonies from one section of the plate. From MRS-
agar, colonies were isolated every sampling day for the
first batch and until day 5 for the second batch. The
isolates were purified on MRS-agar plates at 30
C and
stored at 80
C in freezing media containing glyc-
erol (Gibson and Khoury, 1986). From PDA agar,
colonies were randomly isolated every sampling day
and purified on PDA-agar plates at 30
C. The isolates
were transferred to transport tubes containing PDA-
agar, incubated for a minimum of 24 h at 30
C before
storage at 4
C.
2.4. Phenotypic characterisation of LAB and yeasts
All LAB isolates were phenotypically character-
ised as described by Paludan-Müller et al. (1999). In
addition, the isolates were tested for their capacity to
ferment sucrose and starch, the main carbohydrate
components in plaa-som. The fermentation substrate
used was modified MRS-broth without glucose, with
chlorophenol red as indicator and pH adjusted to 7.
The carbohydrate sources were added individually in
final concentrations of 10% soluble potato starch
(Potato, soluble, Sigma, St. Louis, USA) or 1.3%
sucrose (Sigma) before sterilisation of the substrate
(115
C, 10 min.). The media were dispensed into 4-
ml tubes and inoculated with isolates grown on MRS-
agar plates. The tubes were covered with paraffin oil
and incubated at 30
C for up to 5 days.
Fifteen LAB isolates representing different ITS-
groups (see below) were further characterised by their
carbohydrate fermentation pattern by the API 50 CHL
system (bioMereux, Marcy-l’Etoile, France). The type
strain of Lactobacillus plantarum (DSM 20174) was
included as a positive control. The results were
analysed by the identification software APILAB Plus,
version 3.3.3. (bioMérieux).
63
In addition, the LAB isolates were tested for their
ability to grow in MRS-broth containing 5%, 8%, 10%
and 15% NaCl (w/v). The tubes were covered with
paraffin oil and incubated at 30
C. Growth was
determined by optical density at OD600 (Novaspec II,
Pharmacia LKB) for up to 7 days. All yeast isolates
were initially tested for colony and cell-morphology,
reproduction, nitrate assimilation (Kurtzman and Fell,
1998) and growth in MYGP broth (3.0 g malt extract
(Difco), 3.0 g yeast extract (Difco), 10.0 g glucose
(Merck, Darmstadt, Germany) and 5.0 g Bactopeptone
(Difco) per litre distilled water, pH = 5.6) at 25
C for 3
days. Sixty-six of these isolates were further tested for
growth on 50% and 60% (w/w) glucose media con-
taining: D-(+)-Glucose-monohydrate, Merck 108342;
3% malt extract, Difco 0186-17-7 and 3% (w/v) Agar,
Difco 0145-17-0. The plates were incubated at 25
C
for 7 days. The capacity of yeasts to assimilate carbo-
hydrates was examined by API ID 32 C test system
(bioMérieux). The tests were incubated at 25
C for 2–
7 days. The results were analysed by the identification
software APILAB Plus, version 3.2.2. (bioMérieux).
Yeast strains used as reference strains for the
characterisation of plaa-som yeast isolates included
Zygosaccharomyces rouxii (CBS-732) and Z. Bailii
(CBS-7555) (Centraalbureau voor Schimmelcultures,
Yeast division, Delft, Holland).
2.5. Differentiation at the species level of LAB and
yeasts by rDNA intergenic spacer (ITS) PCR analysis
The intergenic spacer region between the 18S and
28S rRNA genes and between 16S and 23S RNA
genes were used for the analysis of yeasts and LAB,
respectively.
LAB isolates were cultured in MRS-broth and 1 ml
harvested after overnight growth. The pellet was
resuspended in 400 ml TE-buffer (10 mM Tris, pH,
8.0, 1 mM EDTA) and 0.1 g acid-washed glass-
beads (150 – 212 mm, Sigma G-1145) was added. Cells
were mechanically disrupted by shaking four times
for 30 s, the samples left on ice in between each
mixing.
Yeast isolates were grown on PDA-agar and one
loopful of colony material was mixed with 200 ml of
sterile, deionised water. The cells were lysed by
heating in a microwave (900 W, 2 min) and stored
on ice until DNA amplification.
64 C. Paludan-Müller et al. / International Journal of Food Microbiology 73 (2002) 61–70
The PCR was performed in 50 ml reaction mixtures
containing 5 ml of 10  PCR buffer (Amersham Phar-
macia Biotech, Uppsala, Sweden), 0.2 mmol l 1 of
each of the four dNTP (Amersham Pharmacia Biotech),
2 mmol l 1 MgCl2, 0.5 mmol l 1 of each primer, 1%
(v/v) for mamide, 2.5 U of Taq polymerase (Amersham
Pharmacia Biotech), and 1 ml of the lysed cells. For
LAB, two primers were used: a fluorescein labelled
forward primer Cy5-16S-1500F; 5VAAG TCC TAA
CAA GGT A-3V (50 pmol/ml) and a reverse primer
23S-30R; 5VGCC ARG GCA TGG ACC-3V(50 pmol/
ml). Similarly, for yeasts, two primers were used: a
fluorescein labelled CY5-Y-ITS-1F forward primer;
5VTCC GTA GGT GAA CCT GCG G-3V(50 pmol/ml)
and a reverse primer Y-ITS-4R; 5VTCCTCCGCT TAT
TGA TAT GC-3V (50 pmol/ml) (T-A-G Copenhagen
APS, Denmark). The PCR-reaction was performed in
a thermocycler (TRIO-Thermoblockk, Biometra,
Göttingen, Germany) with heated lid (TRIO Heated
Lid, Biometra).
PCR conditions were for LAB an initial step of 94

C for 5 min, followed by amplification using the
following thermal profile: 10 cycles of 94, 48 and 72

C each for 30 s, 25 cycles of 94, 55 and 72
C each
for 30 s. Finally, the mixture was heated at 72
C for 7
min and subsequently cooled to 4
C until use.
PCR conditions were for yeasts an initial denatu-
ration step of 95
C for 5 min, followed by a total of
35 cycles of amplification using the thermal profile:
95, 55 and 72
C each for 30 s. Finally the mixture
was heated at 72
C for 7 min and subsequently
cooled to 4
C for up to 24 h.
2.6. Electrophoresis
Fragments amplified by the PCR reaction were
separated by gel electrophoresis on the automatic
DNA sequencer, ALFexpress (Amersham Pharmacia
Biotech). The electrophoresis was carried out on a
denaturing polyacrylamide gel composed of 6% Long
Ranger (FMC, Vallensbæk Strand, Denmark), 7 mol
l 1 urea and 0.6  TBE (1 M Tris-base, 0.83 M
Boric acid and 10 mM EDTA, Amersham Pharmacia
Biotech) and were cast with 0.3 mm spacers. The PCR
products were mixed with an equal volume of loading
dye (Amersham Pharmacia Biotech), denatured at
94
C for 2 min. A size marker, Cy5 sizer 50 – 500
(Amersham Pharmacia Biotech) was included. The
electrophoresis conditions were 700 V, 60 mA at
55
C for 200 min with 0.6  TBE as the running
buffer. Peak positions and intensity of the separated
ITS fragments were analysed by use of the computer
program Fragment Manager (Amersham Pharmacia
Biotech). Isolates with the same profiles of ITS frag-
ments were grouped manually.
2.7. Sequencing of 16S rRNA genes
DNA was extracted as described for the ITS
amplification (see above) and used for amplification
of a part of the 16S rRNA gene from basepair 968 –
1401. The PCR reaction was performed in a total
volume of 100 ml containing five-unit Taq-Polymerase
(Amersham Pharmacia Biotech), 10 ml of 10  PCR
buffer, 0.2 mM dNTP mix, 1.5 mM MgCl2 and 40
pmol of each the primers: 968F (5V-GAACGCGAA-
GAACCTTAC-3V) and 1401R (5V-GCGTGTGTA-
CAAGACCC-3V). PCR conditions were 94
C for 3
min followed by 30 cycles of 94
C for 30 s, 56
C for
30 s, 72
C for 60 s, and finally an elongation step of
72
C for 7 min. The amplified PCR fragments were
purified with a QIAquick PCR purification kit (QIA-
GEN, Hilden, Germany). The sequence reaction was
performed in both directions with Cy5 market pri-
mers, Cy5-968 and Cy5-1372, by a Thermosequanase
fluorescent labelled primer cycle sequencing kit
(Amersham Pharmacia Biotech) following the instruc-
tions given by the manufacturer. Fragments were
separated by gel electrophoresis on the automatic
DNA sequencer, ALFexpress (Amersham Pharmacia
Biotech). Searches for 16S rRNA sequences were
performed in the GenBank database by BLAST at
NCBI (www.ncbi.nlm.nih.gov).
3. Results
3.1. Chemical analyses
The NaCl% decreased in the syrup during fermen-
tation in all four batches. The addition of roasted rice
caused a slight decrease in NaCl%. In low-salt
batches, the salt concentration in syrup decreased
from 7.5% to 6.2% in plaa-som without rice and from
6.8% to 5.1% in plaa-som with rice (Table 1). This
corresponded to an increase of the NaCI concentration
 C. Paludan-Müller et al. / International Journal of Food Microbiology 73 (2002) 61–70 65

Table 1
Waterphase salt (w/w) in plaa-som
Batch of plaa-som %Salt (w/w)a at day Average, %
0 5 8
Low-salt with rice 6.8 5.8 5.1 6
without rice 7.5 7.1 6.2 7
High-salt with rice 9.8 9.1 8.9 9
without rice 11.5 10.5 10.5 11
a
Measured in the syrup.

in the fish soaked in the syrup from 2.4% to 5.2% and
4.2% after 8 days in plaa-som without rice and with
rice, respectively (results not shown). In the high-salt
batches, the salt concentration decreased from 11.5 to
10.5 and from 9.8 to 8.9 in batches without and with
rice, respectively (Table 1).
In low-salt batches, pH decreased to 5 in plaa-som
without rice during 5 days of fermentation, whereas in
plaa-som with rice, pH was 4.8 already after 3 days
(Fig. 1). In high-salt batches, pH was 6 until day 5 in
plaa-som with rice, followed by a slow decrease in pH
to 5 after 12 days. pH did not decrease below 6 in
plaa-som without rice (Fig. 1). pH of the palm syrup
was 5.1 and the syrup contained 45 g sucrose, 5 g
glucose and 1 g fructose per 100 g.
3.2. Microbiological changes
Initial counts of lactic acid bacteria (LAB) were
105 – 106 cfu g 1 (Fig. 2). In the low-salt batch with-
out rice, the LAB counts increased to 108 cfu g 1
within 3 days, whereas LAB counts in the low-salt
1
Fig. 2. Growth of LAB on MRS-agar (log cfu g ) during
fermentation of plaa-som. Key as in Fig. 1.
batch with rice increased to 109 cfu g 1 (Fig. 2). The
growth of LAB was slower in the high-salt batches of
plaa-som. In plaa-som with rice, counts did not reach
109 cfu g 1 until day 5 and in plaa-som without rice,
counts were approximately 107 cfu g 1 throughout
incubation (Fig. 2). Mesophilic, aerobic counts were
similar to the LAB counts except for the high-salt
batch without rice (11% salt), where counts were 1– 2
log units higher during the first 4 days of incubation
(results not shown).
Yeasts were found at an initial level of approxi-
mately 105 and 104 cfu g 1 in the low- and high-salt
batches of plaa-som (Fig. 3). Of the raw materials,
palm syrup contained the highest level of yeasts, 103 –
104 cfu g 1, whereas the highest level of LAB, 105 –
106 cfu g 1, was found on the fish (Table 2). The
yeast counts increased to 107 – 5  107 cfu g 1 in all
batches during the first 2 days except in the high-salt
batch of plaa-som without rice, where counts did not
exceed 106 cfu g 1 (Fig. 3).

Fig. 1. Changes in pH during fermentation of plaa-som with 6% salt

1
with rice (n), 7% salt without rice (5), 9% salt with rice (.) and Fig. 3. Growth of yeasts on PDA agar (log cfu g ) during
11% salt without rice (6) at ambient temperature (30 – 38
C). fermentation of plaa-som. Key as in Fig. 1.
66 C. Paludan-Müller et al. / International Journal of Food Microbiology 73 (2002) 61–70

Table 2 NaCl). However, microscopy of the high-salt batch

Counts of mesophilic, aerobic bacteria, lactic acid bacteria (LAB) without rice (11% NaCl) at the end of the storage
and yeasts of raw materials used for production of plaa-som
period (after day 5) indicated that also this batch had a
Parameter Counts (log cfu/g)
microflora dominated by tetrad-forming cocci. Group
Low-salt High-salt IV consisted of obligate or facultative heterofermenta-
Fish Syrup Rice Fish Syrup Rice tive LAB strains, which were isolated only from low-
Aerobic a
7.9 4.0 5.3 6.4 2.5 4.4 salt batches. In contrast, strains of group V were
LABb 5.8 2.5 3.6 5.2 2.9 3.4 isolated only from high-salt batches. These strains
Yeastsc 2.3 3.9 3.1 2.0 3.3 2.5 had the lowest capacity of all LAB strains to decrease
a
TSA-agar. pH in modified MRS-broth (final pH of 4.2 – 4.4).
b
MRS-agar. Group VI consisted of 13 strains, which were isolated
c
PDA-agar.
from MRS-agar, but found not to be LAB strains, since
they were Gram positive, catalase positive, oxidase
negative and with a final pH in modified MRS-broth of
3.3. Phenotypic and genotypic characterisation of 4.6 – 5.2 (Table 3). In addition, the strains formed
lactic acid bacteria small, orange colonies on MRS-agar and had a coccoid
cell morphology. The strains were tentatively identi-
A total number of 158 LAB strains was obtained fied as Staphylococcus spp.
from the four batches of plaa-som. Of these were 135 A total number of 74 strains representing all five
strains divided into six major groups with more than 10 groups of LAB were differentiated by ITS-PCR anal-
strains per group (Table 3). The remaining 23 strains ysis. The strains that were analysed from each group
were placed in 10 different groups with less than six had identical ITS-PCR profiles except group II, which
strains per group and not further characterised. Group I were separated into two subgroups (Table 4). A total of
consisted of Lactobacillus spp. with a final pH of 3.4– 15 strains from the ITS-PCR groups was selected for
3.6 in modified MRS-broth. No gas was produced identification by API 50 CHL (Table 4). Strains of
from glucose. These were isolated only from the low- group I, III and IV were identified as L. acidophilus
salt batches. Group II strains were isolated from both (91.3% ID), Pediococcus pentosaceus (96.9% ID) and
high and low-salt batches and differed primarily from Weisella confusa (99.4% ID). For group II, one sub-
group I strains by their capacity to ferment sucrose. group (A) was identified as L. plantarum (99.9% ID),
The largest group of strains, group III, consisted of whereas one subgroup (B) was unidentified by API 50
tetrad-forming cocci. These were isolated from both CHL. Group V was also unidentified. Of the strains
low-salt batches and the high-salt batch with rice (9% identified by API 50 CHL, nine were selected for

Table 3
Sources of isolation and phenotypic characteristics used to divide the major groups of bacteria isolated on MRS-agar from plaa-som
Group Batch of plaa-som Final pH in Cell shape No. of % Positive strains
MRS-broth isolates Gas from NH3 from Fermentation
glucose arginine Sucrose Starch
I Low-salt with and without rice 3.4 – 3.6 Rods/coccoid rods 27 0 100 0 0
II Low- and high-salt with and 3.5 – 3.6 Rods/coccoid rods 12 0 75 100 0
without rice
III Low-salt with and without rice, 3.6 – 3.7 Tetrad forming cocci 62 0 100 12 0
high-salt with rice
IV Low-salt with and without rice 3.9 – 4.0 Rods/coccoid rods 15 100 100 100 0
V High salt with and without rice 4.2 – 4.4 Rods/coccoid rods 19 0 100 100 0
VI Low-salt without rice, high-salt 4.6 – 5.2 Cocci 13a 0 0 0 0
with and without rice
a
Gram-positive, catalase positive cocci tentatively identified as staphylococci.
 C. Paludan-Müller et al. / International Journal of Food Microbiology 73 (2002) 61–70
Table 4
Differentiation of 74 LAB strains by ITS-PCR analysis and identification of representative strains by the use of carbohydrate fermentation
patterns (API 50 CH) and 16S rRNA gene sequencing
Groupa ITS-PCR analysis
Size (basepair) of No. of isolates
PCR-products
I 259 F 1.4; 461 F 2.8 20
II A: 110 F 0.6; 293 F 0.8; 523 F 1.7 A: 4
B: 259 F 1.4; 455 F 0.5 B: 4
III 316 F 1.2; 506 F 3.4 23
IV 332 F 0.7; 435 F 0.9; 527 F 1.2 14
V 443 F 3.3; 549 F 3.1 9 Unidentified
a
Division into groups by phenotypic tests (Table 3).
partial sequence analysis of 16S rRNA genes. Sequen-
ces were obtained for the region 968 – 1401 (Escher-
richia coli positions) of the 16S rRNA gene. The
sequence of each of the nine strains was used to search
the GenBank and EMBL databases. The groups I and
II (B) strains were related to L. alimentarius, L.
farciminis and L. kimchii (98 – 99% similarity). The
group II (A) strain was related to L. plantarum (100%
similarity), group III strains to P. pentosaceus (99%
similarity), the group IV strain to W. confusa (99%
similarity) and finally, the three unidentified group V
strains were related to Lactococcus garviae with
similarities of 98– 99%.
All 74 LAB strains differentiated by ITS-PCR
grew well in MRS-broth containing 5% (w/v) NaCl.
Twenty-nine of the strains grew in 8% NaCl and 22 in
10% NaCl. All strains that were able to grow in 10%
NaCl were characterised as Lactobacillus spp. (group
I or II, Table 3), and were isolated on day 5 or day 8
during fermentation (results not shown). All Lact.
garviae strains (group V) grew in 5%, but not in
8% NaCl, although they were isolated from high-salt
samples. All P. pentosaceus strains (group III) grew in
8% NaCl, but not in 10% NaCl. Staphylococcus spp.
(group VI, Table 3) grew in APT-broth containing
15% NaCl.
3.4. Characterisation of yeasts
A total number of 350 yeast strains was isolated.
Twenty-eight strains were obtained from raw materi-
als, 130 from low-salt batches and 192 from high-salt
batches. The initial characterisation revealed that more
than 95% of the isolates were very similar and
67
API 50 CHL identification 16S rRNA gene sequencing
Identification No. of isolates Identification No. of isolates
L. acidophilus 2 L. alimentarius/ 2
L. farciminis/L. kimchii
A: L. plantarum A: 1 A: L. plantarum A: 1
B: Unidentified B: 2 B: L. alimentarius B: 2
P. pentosaceus 2 P. pentosaceus 2
W. confusa 3 W. confusa 1
5 L. garviae 3
differed only by their colony morphology. These
strains formed round/oval cells arranged in small
clusters. They showed reproduction by multilateral
budding and produced gas in MYGP-broth. None of
the isolates assimilated nitrate. The remaining 4% of
yeast isolates constituted a diverse group, which were
isolated sporadically and will therefore not be further
described.
Sixty-six representative isolates (of the main group
of yeasts) were tested for osmotolerance by their
ability to grow on 50% and 60% (w/w) glucose
media. All isolates were able to grow on 50% (w/w)
glucose media and 65 isolates were able to grow on
60% (w/w) glucose media (Table 5). Thus, indicating
that the yeast isolates belong to the genera of salt-and
sugar-tolerant yeasts, Zygosaccharomyces (Kurtzman
and Fell, 1998).
The same 66 isolates were analysed by ITS-PCR.
Fifty-five of those had one PCR-product of approx-
imately 700 basepair (bp). The typestrains of Z. rouxii
(CBS-732) and Z. bailii (CBS-7555) had PCR-prod-
ucts of 680 and 726 bp, respectively. It was therefore
not possible to identify the plaa-som strains as either
Z. rouxii or Z. bailii based on the ITS-PCR analysis.
Twenty-eight of the 66 isolates were characterised
by their carbohydrate assimilation patterns. None of
the isolates assimilated sucrose, fructose or starch
(actidion in API ID 32 test kit) and they were able
to assimilate only four to seven of the 31 carbohydrate
substrates in the API-ID 32 C test (Table 5). All
isolates were identified as Zygosaccharomyces spp.
Comparison with the carbohydrate assimilation pat-
terns for Z. rouxii and Z. bailii indicated that the
isolates belonged to the Z. rouxii species (Table 5).
68 C. Paludan-Müller et al. / International Journal of Food Microbiology 73 (2002) 61–70

Table 5 The palm syrup added to plaa-som contained

Phenotypic characteristics of Z. rouxii (CBS 732) and Z. bailii (CBS approximately 1.25 M sucrose, 278 mM glucose and
7555) and of yeast strains isolated from raw materials and during
fermentation of plaa-som 56 mM fructose. These three sugars were the only
sugars detected among 13 sugars analysed for in
Phenotypic % Positive Reaction
characteristics a strains coconut palm syrup (Atputharajah et al., 1986).
Z. rouxii Z. bailii
Plaa-som contained 12 – 13% palm syrup and thus
Growth on 50% (w/w) 100 + approximately 150 mM sucrose, 34 mM glucose and
glucose medium
7 mM fructose. The content of mono- and di-saccha-
Growth on 60% (w/w) 98 +
glucose medium rides in plaa-som will probably enhance the fermenta-
Assimilation of b tion by LAB as compared to other fermented fish
Galactose 100 + + products, which contain more complex carbohydrates
Maltose 36 in the form of rice starch. Forty percent of the LAB
Sorbitol 100 + +
strains isolated from plaa-som fermented sucrose.
Glycerol 100 +
Palatinose 46 Moreover, the amount of excess sugar can be used
Mannitol 54 + + for yeast growth. The growth of yeasts and LAB in
Glucose 100 + + relation to the available carbohydrate substrates has
Esculine 36 + been extensively studied for soy sauce products. In
a
Sixty-six strains were tested for growth on glucose media, of Indonesian soy sauce (Kecap) that contains initial low
these were 28 strains tested for assimilation of carbohydrates. levels of glucose ( < 10 mM), no fermentable sugars
b
Tested by API-ID32C, the assimilation of all remaining
are left for yeast growth after LAB fermentation
carbohydrates was negative.
(Röling et al., 1994a,b). In Japanese soy sauce that
has a high initial concentration of glucose ( > 100
This was verified by the Centraalbureau voor Schim- mM), LAB initiates the fermentation followed by
melcultures (CBS), which identified three representa- alcoholic fermentation by osmotolerant yeasts such
tive strains as Z. rouxii by morphological and phy- as Z. rouxii (Sasaki and Yoshida, 1966). In plaa-som, a
siological tests. parallel growth of yeasts and LAB was found, which
may be explained by the lower salt content as com-
pared to Japanese soy sauce, which contains 15 –20%
4. Discussion NaCl. At such high salt concentrations, the growth of
Z. rouxii is inhibited until LAB have started to acidify
Growth of LAB to levels of 108 – 109 cfu g 1 is the brine and pH is below 5.5 (Yong and Wood, 1976).
required to obtain a sufficient pH decrease in plaa- The dominant LAB species isolated from plaa-som
som (Figs. 1 and 2) similar to other fermented fish was identified as P. pentosaceus (42% of isolated
products (Olympia et al., 1992; Østergaard et al., strains). This species is widely distributed in fer-
1998). Further, an optimal growth of LAB is depend- mented Thai products including low-salt fermented
ent on the salt concentration, which should not be fish (Tanasupawat and Daengsubha, 1983). In con-
higher than 6% or 7% (w/w) for the fermentation of trast, in fermented or hydrolysed fish products with
plaa-som to occur within 4 or 7 days. In contrast, an salt concentrations higher than 10 – 15% NaCl, such as
increase in the salt concentration to 9% did not affect plaa-ra, nam-budu (fish sauce) and nam-plaa (fish
the growth of yeasts, and these did not seem to have paste), Tetragenococcus halophilus is isolated (Tana-
any influence on the rate of fermentation. supawat and Daengsubha, 1983; Ito et al., 1985).
Addition of roasted rice to plaa-som increased the Strains closely related to the group of L. alimentarius,
rate of fermentation. This is most likely due to a L. farciminis and L. kimchii were also isolated from
dilution of the NaCl concentration, since none of the plaa-som. Those species are able grow in the presence
isolated LAB (or yeast) strains fermented starch. of 8– 10% (w/v) NaCl and were previously isolated
Furthermore, roasted rice improved the flavour of from Thai fermented fish (Tanasupawat et al., 1998)
the product as evaluated by a sensory panel (results and from fermented vegetables, the Korean kimchii
not shown). (Yoon et al., 2000). Lact. garviae, which was isolated
 C. Paludan-Müller et al. / International Journal of Food Microbiology 73 (2002) 61–70
only from high-salt batches of plaa-som, is a major
pathogen of fish (Eldar et al., 1996), but was also
isolated as the dominant member of the LAB popu-
lation from the intestines of healthy Thai common
carp (Cai et al., 1999).
The osmotolerant species Z. rouxii was dominating
the yeast flora in plaa-som. This is most likely due to
the use of palm syrup, from which Z. rouxii strains
were isolated (103 – 104 cfu g 1), and furthermore
due to the relatively high concentrations of salt (6 –
11%) used in the preparation of plaa-som. Z. rouxii is
the yeast species causing spoilage of most foods
containing high concentrations of sugar such as syr-
ups, honey, dried fruits, etc. (Fleet, 1992). In contrast,
Z. rouxii is valuable for the development of aroma in
the fermentation of soy sauce and miso paste (Yokot-
suka, 1986). However, the role of Z. rouxii in flavour
formation in plaa-som remains to be determined.
Identification of yeast isolates in this study was based
upon the use of ITS-PCR and phenotypic methods.
Another study has found the ITS-PCR method to be
useful for discrimination of different species of Zygo-
saccharomyces (James et al., 1996), but in our case
ITS-PCR could not differentiate between Z. rouxii and
Z. bailii. On the other hand, we found that the ITS-
PCR method was useful for the initial grouping of
LAB isolates for subsequent phenotypic character-
isation by the use of API 50 CHL. However, verifi-
cation by genotypic methods, e.g. by 16S rRNA gene
sequencing is required, since the number of LAB
species included in the API LAB plus database is
limited. Thus, neither of the species L. alimentarius,
L. farciminis, L. kimchii or Lact. garviae, which were
isolated from plaa-som, were present in the API LAB
plus database.
In high-salt batches of plaa-som, the growth of
LAB was inhibited, allowing growth of Staphylococ-
cus spp. These have previously been isolated from
Thai-fermented fish with salt concentrations above
5% (Tanasupawat et al., 1991; Tanasupawat et al.,
1992) and from Korean fermented (hydrolysed) fish
with salt concentrations ranging from 8% to 26%
NaCl (Um and Lee, 1996). pH of the Korean products
was in the range of 4.8– 6.6. Thus, at high concen-
trations of salt and absence of lactic fermentation,
there is a risk for growth of Staph. aureus, although
the presence of pathogenic strains have not been
reported in fermented fish products.
69
In conclusion, it was found that increasing salt
concentrations from 6% to 11% delayed or inhibited
LAB growth and thereby the fermentation process of
plaa-som. It is suggested that a maximum of 6 – 7% is
used in plaa-som and other fermented fish products in
order to facilitate a rapid growth of LAB and sub-
sequent decrease in pH to below 4.5. P. pentosaceus
and Z. rouxii were identified as the predominant LAB
and yeast species, respectively during fermentation of
plaa-som.
Acknowledgements
We would like to thank Jureerat Saetae and
Nongluk Kiattansakul, Prince of Songkla University,
for technical assistance, and Jessica Nakawuma, The
Royal Veterinary and Agricultural University, for help
with the ITS-PCR analysis.
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