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Print 1998-09-24 Symp on Fish Utilization in Asia and Pacific.Tif THE MICROBIOLOGY OF LOW-SALT FERMENTED FISH PRODUCTS by CHRISTINE PALUDAN-M~LLER Danish Institute for Fisheries Research Department of Seafood Research Danish Technical University Building 221 DK-2800 Lyngby, Denmark 1 ABSTRACT i ! A wide range of fermented fish products is produced in South East Asia. The combination of ; acid production by lactic acid bacteria and addition of salt is used as a low-cost means of fish 1 preservation. Due to the high buffering capacity of fish, a high level of lactic acid bacteria with a capacity to degrade the added carbohydrates is important to obtain the rapid decrease in pH that is required for the production of acceptable and safe products. The salt concentration should be in the range of 3-5% water phase salt (wlw) in order not to inhibit the growth of lactic acid bacteria. Salmonella, Escherichia coli, Staphylococcus aureus and Clostridium botulinum types A and B are the pathogenic bacteria of particular concern in fermented fish products. Implementation of processing control, and preferably the controlled use lactic acid bacteria starter cultures, is essential to improve the safety of products. The possible use of low-value fish as raw material requires more knowledge of the influence of product composition, e.g. salt concentration, buffering capacity of different fish species and carbohydrate sources on the fermentation process. INTRODUCTION Traditionally, the term "fermented fish" covers both enzyme hydrolysed and microbial fermented products (Mackie et al., 1971). However, as suggested by Ozen and Mendoza (1985) only those products involving fermentative growth of microorganisms should be described as fermented. A clear distinction should be made between the microbial fermented products and the hydrolysed products, which are dependent on the activity of indigenous or added enzymes (Table 1). Of course, many products will involve both microbial fermentation and enzymatic activity in particular in the high-salt range of fermented products. This paper will discuss fermented fish products with a salt concentration lower than 8% (wfw). These products have not been studied to the same extent as the high-salt fermented fish and hydrolysed fish. However, health hazards due to a high salt intake limit the use of high-salt products in the daily diet. The production of fermented fish is still carried out at a household level or in small industrial scale with limited or no quality control. However, there is a potential for technological improvement and .also for the use of low-value marine fish in processing by increased knowledge of the fermentation process and by the application of lactic acid bacteria (LAB) starter cultures in production. The raw material used for processing of fermented fish is, with a few exceptions, freshwater fish. This is in contrast to the hydrolysed products, which often are prepared from marine fish species (Kungsuwan, 1998). According to the Fishery Statistical Record of Thailand 1994, issued by the Thai Department of Fisheries (DOF), total freshwater fish production in Thailand was 373,000 tomes of which 0.44% was used for fish sauce, 0.05% for fish paste and 5.26 % for hydrolysedlfermented fish. Plaa-ra is by far the most common product in Thailand. Plaa-ra (pla = fish and ra = strong smell) contains from 8-25% NaCl and is today the only hydrolysedlfermented fish product, which is exported and play a role in the national economy (Kungsuwan, A., 1998). The main preservation factors in fermented fish products are NaCl (reduced water activity) and acid (pH reduction). Others include spices (garlic) and possibly also inhibitory substances (bacteriocins) produced I by LAB. There have been a number of reviews dealing with the characteristics of fermented fish products in South East and East Asia (Adams et al., 1985; Ozen and Mendoza, 1985; Lee et al., 1993). In this paper, a brief overview is given of different types of fermented fish products (Table 2). The role of LAB ifi the fermentation process is discussep, focusing in particular on products from Thailand, such as som-fak andplaa- som. The main focus is the influence of preservation factors and product composition on the growth of LAB and thereby on the quality of products. Furthermore, the bacteriological risks associated with fermented fish products are listed and methods to reduce these risks suggested. INFLUENCE OF PRESERVATION FACTORS AND PRODUCT COMPOSITION ON LAB GROWTH Indigenous starter cultures Fermented fish products are produced by spontaneous fermentation or with the use of indigenous starter cultures (Table 2). These are not pure cultures as most often used in the West, but mixed cultures of moulds, yeast and bacteria growing on rice. The starters serve as a source of a variety of enzymes, including amylolytic enzymes, which degrade the starch and thereby provide fermentable carbohydrates for the growth of LAB in the subsequent fermentation process (Lotong, 1998). Japanese koji is steamed rice overgrown with Aspergillus oryzae, whereas angkak used in the Philippines, Thailand and Cambodia is steamed rice overgrown with the red mould Monascus purpureus. Look-pang, a starter used for the Thai plaa-chao, is in the form of dry powder as small flattened cakes. Rice flour makes up the greater part of the starter, but typically it also contains one or more vegetable components (garlic, ginger, pepper). A lower final pH than in spontaneously fermented products is in general obtained, when indig~ous starters, such as angkak, are used in the preparation of fermented fish (Sakai et al., 1983a). High amounts of glucose are produced from rice starch by amylolytic enzymes present in the starter and a rapid fermentation by LAB is therefore obtained. The microflora is composed of both LAB and yeasts, where the latter originate from the starter culture. Depending on the species, yeasts are likely to be positively involved in flavour formation as has been found for fish and soy sauce (Mongkolwai et al., 1997). In som-fak, which is prepared without indigenous starter culture, yeast are considered as qpoilage organisms (Saisithi et al., 1986). Salt levels Salting is the most important preservation factor in hydrolysed products, such as fish sauce and fish paste. In contrast, a rapid acid fermentation by LAB is essential for the effective inhibition of spoilage and pathogenic bacteria in the fermented products. However, the level of salting will also affect the growth and selection of micro-organisms during fermentation. In many products an initial salting of the fish is performed. This is done to cause an immediate inhibition of the natural spoilage flora on the fish before acid production and also to improve the texture of the final product. Water phase salt levels above 7% (wlw) were inhibitory to the LAB fermentation of Thai plaa-som (Table 3) (Paludan-Miiller et aL, 1998b). The product contained an initial high level of the yeast Zygosaccharomyces rouxii (approx. lo6 cfulg) originating from the palm syrup used in the preparation. Z. rouxii was isolated throughout fermentation irrespective of the NaCl concentration (Table 3). Tetragenococcus sp. and Lactobacillus sp. dominated the microflora (5x108 cfdg) in plaa-som with 5.8 or 7.1% salt (w/w). However, only in plaa-som with 5.8% salt (wlw) did pH decrease to below 4.5 within five days. The growth of Lactobacillus sp. was inhibited and the microflora dominated by Gram-positive, catalase positive cocci (Staphylococcus sp.) in plaa-som with salt concentrations above 9% (wlw) (Table 3). Thus, the concentration of water phase salt must be kept lower than 7% (wlw) in this type of product, in order to proliferate fermentation by LAB. Higher salt concentrations will increase the risk of growth of salt-tolerant, potentially pathogenic bacteria, e.g. S. aureus. The investigated product contained 10-13% palm syrup (w/w), which was composed of sucrose, fructose and glucose. This high content of mono- and di-saccharides will increase the rate of fermentation by LAB. Thus, an inhibitory effect of salt may be found at lower salt concentrations in other types of products, which have more complex carbohydrates (e.g. rice starch) as the only carbohydrate source. Staphylococcus spp. have previously been isolated from fermented fish products with water phase salt > 5% (wlw) (Tanasupawat et al., 1991 and 1992). In a study of the Filipino product balao balao, 3 % salt was found to be optimal for a rapid decrease in pH as compared to 6,9 and 12 % (Arroyo et al. 1978). Investigations of the effect of salt on the growth of Lactobacillus sp. isolated from fermented fish products revealed that most strains tolerated up to 6.5% NaCl (0stergaard et al., 1998b). However, 6 % NaCl inhibited the growth of the Gram-negative spoilage flora in som-fak prepared from Danish marine fish, but LAB was also inhibited and the fermentation failed (Paludan-Miiller, unpublished results). In conclusion, a water phase salt concentration in the range of 3-5% (wfw) is optimal in order to ensure the growth of LAB and thereby a rapid fermentation of the fish products. Organic acids The antimicrobial effects of organic acids are due both to the depression of pH below the growth range of spoilage and pathogenic bacteria and to metabolic inhibition by the undissociated acid molecules (Rowan et al., 1998). Lactic and citric acids have low pKa-values of 3.1 and microbial growth is only inhibited at low pH values. Acetic acid has a pKa value of 4.75 and is therefore a better inhibitor in a higher pH range (Eklund, 1989). At pH 5, approximately 35% of acetic acid and only 6% of lactic acid will be in the undissociated form (Silliker, 1980). It is suggested, that the growth of LAB producing mixtures of lactic and acetic acid in the beginning of food fermentation is important for the inhibition of competing spoilage and pathogenic bacteria and to obtain a rapid decrease in pH (Adams and Hall, 1988).
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