Fermentation and microflora of plaa-som, a Thai fermented fish product prepared with different salt concentrations

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-Mu¨ller 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 Thai fermented fish products are mostly produced according to family tradition and local geographic *

Corresponding author. Tel.: +45-4588-3322; fax: +45-45884774. E-mail address: [email protected] (C. Paludan-Mu¨ller).

preferences. Therefore, large differences exist in production methods, use and proportions of raw materials, 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

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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-Mu¨ller 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 therefore of interest to identify the optimal salt concentration, which does not inhibit the growth of the fermenting microorganisms, and in addition contributes 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% waterphase 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 composition 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 proportions 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 drymatter 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 stomacher 400. Phase contrast microscopy was made on

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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 MRSagar, 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 glycerol (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 PDAagar, 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 characterised as described by Paludan-Mu¨ller 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 4ml tubes and inoculated with isolates grown on MRSagar plates. The tubes were covered with paraffin oil and incubated at 30
C for up to 5 days. Fifteen LAB isolates representing different ITSgroups (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. (bioMe´rieux).

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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 containing: 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 carbohydrates was examined by API ID 32 C test system (bioMe´rieux). 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. (bioMe´rieux). 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 glassbeads (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.

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The PCR was performed in 50 ml reaction mixtures containing 5 ml of 10  PCR buffer (Amersham Pharmacia 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, Go¨ttingen, 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 denaturation 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 fragments 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-GAACGCGAAGAACCTTAC-3V) and 1401R (5V-GCGTGTGTACAAGACCC-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 (QIAGEN, Hilden, Germany). The sequence reaction was performed in both directions with Cy5 market primers, Cy5-968 and Cy5-1372, by a Thermosequanase fluorescent labelled primer cycle sequencing kit (Amersham Pharmacia Biotech) following the instructions 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 fermentation 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

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Table 1 Waterphase salt (w/w) in plaa-som Batch of plaa-som

Low-salt High-salt a

with rice without rice with rice without rice

%Salt (w/w)a at day 0

5

8

6.8 7.5 9.8 11.5

5.8 7.1 9.1 10.5

5.1 6.2 8.9 10.5

Average, %

6 7 9 11

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 without rice, the LAB counts increased to 108 cfu g 1 within 3 days, whereas LAB counts in the low-salt

Fig. 1. Changes in pH during fermentation of plaa-som with 6% salt with rice (n), 7% salt without rice (5), 9% salt with rice (.) and 11% salt without rice (6) at ambient temperature (30 – 38
C).

Fig. 2. Growth of LAB on MRS-agar (log cfu g fermentation of plaa-som. Key as in Fig. 1.

1

) during

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 approximately 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. 3. Growth of yeasts on PDA agar (log cfu g fermentation of plaa-som. Key as in Fig. 1.

1

) during

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Table 2 Counts of mesophilic, aerobic bacteria, lactic acid bacteria (LAB) and yeasts of raw materials used for production of plaa-som Parameter

Counts (log cfu/g) Low-salt

a

Aerobic LABb Yeastsc a b c

High-salt

Fish

Syrup

Rice

Fish

Syrup

Rice

7.9 5.8 2.3

4.0 2.5 3.9

5.3 3.6 3.1

6.4 5.2 2.0

2.5 2.9 3.3

4.4 3.4 2.5

TSA-agar. MRS-agar. PDA-agar.

3.3. Phenotypic and genotypic characterisation of lactic acid bacteria A total number of 158 LAB strains was obtained from the four batches of plaa-som. Of these were 135 strains divided into six major groups with more than 10 strains per group (Table 3). The remaining 23 strains were placed in 10 different groups with less than six strains per group and not further characterised. Group I consisted of Lactobacillus spp. with a final pH of 3.4– 3.6 in modified MRS-broth. No gas was produced from glucose. These were isolated only from the lowsalt batches. Group II strains were isolated from both high and low-salt batches and differed primarily from group I strains by their capacity to ferment sucrose. The largest group of strains, group III, consisted of tetrad-forming cocci. These were isolated from both low-salt batches and the high-salt batch with rice (9%

NaCl). However, microscopy of the high-salt batch without rice (11% NaCl) at the end of the storage period (after day 5) indicated that also this batch had a microflora dominated by tetrad-forming cocci. Group IV consisted of obligate or facultative heterofermentative LAB strains, which were isolated only from lowsalt batches. In contrast, strains of group V were isolated only from high-salt batches. These strains had the lowest capacity of all LAB strains to decrease pH in modified MRS-broth (final pH of 4.2 – 4.4). Group VI consisted of 13 strains, which were isolated 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 4.6 – 5.2 (Table 3). In addition, the strains formed small, orange colonies on MRS-agar and had a coccoid cell morphology. The strains were tentatively identified as Staphylococcus spp. A total number of 74 strains representing all five groups of LAB were differentiated by ITS-PCR analysis. The strains that were analysed from each group had identical ITS-PCR profiles except group II, which were separated into two subgroups (Table 4). A total of 15 strains from the ITS-PCR groups was selected for identification by API 50 CHL (Table 4). Strains of group I, III and IV were identified as L. acidophilus (91.3% ID), Pediococcus pentosaceus (96.9% ID) and Weisella confusa (99.4% ID). For group II, one subgroup (A) was identified as L. plantarum (99.9% ID), whereas one subgroup (B) was unidentified by API 50 CHL. Group V was also unidentified. Of the strains 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

I II

Batch of plaa-som

Low-salt with and without rice Low- and high-salt with and without rice Low-salt with and without rice, high-salt with rice Low-salt with and without rice High salt with and without rice Low-salt without rice, high-salt with and without rice

III IV V VI a

Final pH in MRS-broth

Cell shape

No. of isolates

% Positive strains Gas from glucose

NH3 from arginine

Fermentation Sucrose

Starch

3.4 – 3.6 3.5 – 3.6

Rods/coccoid rods Rods/coccoid rods

27 12

0 0

100 75

0 100

0 0

3.6 – 3.7

Tetrad forming cocci

62

0

100

12

0

3.9 – 4.0 4.2 – 4.4 4.6 – 5.2

Rods/coccoid rods Rods/coccoid rods Cocci

15 19 13a

100 0 0

100 100 0

100 100 0

0 0 0

Gram-positive, catalase positive cocci tentatively identified as staphylococci.

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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

API 50 CHL identification

16S rRNA gene sequencing

Size (basepair) of PCR-products

No. of isolates

Identification

No. of isolates

Identification

No. of isolates

I

259 F 1.4; 461 F 2.8

20

L. acidophilus

2

2

II

A: 110 F 0.6; 293 F 0.8; 523 F 1.7 B: 259 F 1.4; 455 F 0.5 316 F 1.2; 506 F 3.4 332 F 0.7; 435 F 0.9; 527 F 1.2 443 F 3.3; 549 F 3.1

A: 4 B: 4 23 14 9

A: L. plantarum B: Unidentified P. pentosaceus W. confusa Unidentified

A: 1 B: 2 2 3 5

L. alimentarius/ L. farciminis/L. kimchii A: L. plantarum B: L. alimentarius P. pentosaceus W. confusa L. garviae

III IV V a

A: 1 B: 2 2 1 3

Division into groups by phenotypic tests (Table 3).

partial sequence analysis of 16S rRNA genes. Sequences were obtained for the region 968 – 1401 (Escherrichia 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 materials, 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

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 approximately 700 basepair (bp). The typestrains of Z. rouxii (CBS-732) and Z. bailii (CBS-7555) had PCR-products 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 patterns for Z. rouxii and Z. bailii indicated that the isolates belonged to the Z. rouxii species (Table 5).

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Table 5 Phenotypic characteristics of Z. rouxii (CBS 732) and Z. bailii (CBS 7555) and of yeast strains isolated from raw materials and during fermentation of plaa-som Phenotypic characteristics a

% Positive strains

Reaction

Growth on 50% (w/w) glucose medium Growth on 60% (w/w) glucose medium Assimilation of b Galactose Maltose Sorbitol Glycerol Palatinose Mannitol Glucose Esculine

100

+

98

+

100 36 100 100 46 54 100 36

+

+

+ +

+

+ + +

+ +

Z. rouxii

Z. bailii

a

Sixty-six strains were tested for growth on glucose media, of these were 28 strains tested for assimilation of carbohydrates. b Tested by API-ID32C, the assimilation of all remaining carbohydrates was negative.

This was verified by the Centraalbureau voor Schimmelcultures (CBS), which identified three representative strains as Z. rouxii by morphological and physiological tests.

4. Discussion Growth of LAB to levels of 108 – 109 cfu g 1 is required to obtain a sufficient pH decrease in plaasom (Figs. 1 and 2) similar to other fermented fish products (Olympia et al., 1992; Østergaard et al., 1998). Further, an optimal growth of LAB is dependent on the salt concentration, which should not be higher than 6% or 7% (w/w) for the fermentation of plaa-som to occur within 4 or 7 days. In contrast, an increase in the salt concentration to 9% did not affect the growth of yeasts, and these did not seem to have any influence on the rate of fermentation. Addition of roasted rice to plaa-som increased the rate of fermentation. This is most likely due to a dilution of the NaCl concentration, since none of the isolated LAB (or yeast) strains fermented starch. Furthermore, roasted rice improved the flavour of the product as evaluated by a sensory panel (results not shown).

The palm syrup added to plaa-som contained approximately 1.25 M sucrose, 278 mM glucose and 56 mM fructose. These three sugars were the only sugars detected among 13 sugars analysed for in coconut palm syrup (Atputharajah et al., 1986). Plaa-som contained 12 – 13% palm syrup and thus approximately 150 mM sucrose, 34 mM glucose and 7 mM fructose. The content of mono- and di-saccharides in plaa-som will probably enhance the fermentation by LAB as compared to other fermented fish products, which contain more complex carbohydrates in the form of rice starch. Forty percent of the LAB strains isolated from plaa-som fermented sucrose. Moreover, the amount of excess sugar can be used for yeast growth. The growth of yeasts and LAB in relation to the available carbohydrate substrates has been extensively studied for soy sauce products. In Indonesian soy sauce (Kecap) that contains initial low levels of glucose ( < 10 mM), no fermentable sugars are left for yeast growth after LAB fermentation (Ro¨ling et al., 1994a,b). In Japanese soy sauce that has a high initial concentration of glucose ( > 100 mM), LAB initiates the fermentation followed by alcoholic fermentation by osmotolerant yeasts such as Z. rouxii (Sasaki and Yoshida, 1966). In plaa-som, a parallel growth of yeasts and LAB was found, which may be explained by the lower salt content as compared to Japanese soy sauce, which contains 15 –20% NaCl. At such high salt concentrations, the growth of Z. rouxii is inhibited until LAB have started to acidify the brine and pH is below 5.5 (Yong and Wood, 1976). The dominant LAB species isolated from plaa-som was identified as P. pentosaceus (42% of isolated strains). This species is widely distributed in fermented Thai products including low-salt fermented fish (Tanasupawat and Daengsubha, 1983). In contrast, in fermented or hydrolysed fish products with salt concentrations higher than 10 – 15% NaCl, such as plaa-ra, nam-budu (fish sauce) and nam-plaa (fish paste), Tetragenococcus halophilus is isolated (Tanasupawat and Daengsubha, 1983; Ito et al., 1985). Strains closely related to the group of L. alimentarius, L. farciminis and L. kimchii were also isolated from plaa-som. Those species are able grow in the presence of 8– 10% (w/v) NaCl and were previously isolated from Thai fermented fish (Tanasupawat et al., 1998) and from fermented vegetables, the Korean kimchii (Yoon et al., 2000). Lact. garviae, which was isolated

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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 population 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 syrups, 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 (Yokotsuka, 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 Zygosaccharomyces (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 ITSPCR method was useful for the initial grouping of LAB isolates for subsequent phenotypic characterisation by the use of API 50 CHL. However, verification 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 Staphylococcus 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 concentrations 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.

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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 subsequent 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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