War/d Joufna/

of A4icroNo/ogy

& 8ioteclIno/ogy

11, 253-256

Review

Lactic acid bacteria Thailand S. Tanasupawat*

in fermented

foods in

and K. Komagata

Traditional fermented foods (fish, meat and vegetable products), produced by many different processes, are eaten in many parts of Thailand. Lactic acid bacteria are responsible for the souring and ripening of these foods. Homofermentative strains of Lucfobucihs penfosus, L. plunkrum and Pediococcus pentosuceus are dominant in foods with low salt concentrations whereas P. hulophilus strains are present in foods containing high salt. Strains of Lucfobucillus suke, other Lucfobucillus spp., l? ucidiluctici and P. uritiueequi are frequently found. HeterofermentaL. vuccinoskrcus, other Luctobucillus spp., and of tive strains of L. breuis, L. confusus, L. fermenhm, Leuconostoc spp. are distributed as minor bacteria and strains of Skzphyfococcus, Enkrococcus and HuZobucterium are occasionally isolated. Key words: Enferococcus, lactic acid bacteria, ,Lucfobucillus, Pediococcus, Stuphylococcus.

Numerous fermented products are found in Thailand. For example, rice wines, such as sa-to, num-khuo, kru-chue and ou, are produced, with a starter cake (loog-pung) (Lotong 1985), by Succhuromyces cerevisiue, Succhuromycopsis fibuligeru, Aspergillus oryzue, Amylomyces rotcrii, Rhizopus spp. and Mucor spp. (Sukhumavasi et ~1. 1975; Lotong 1985). Aspergillus ory,zze is also important for producing koji in the soy sauce fermentation (Lotong 1985). More than 20 kinds of fermented foods, including fish, meat, vegetables and fruits, are produced in Thailand (Saisithi ef ul. 1966; Sundhgul ef ul. 1975; Anon. 1981-1982; Tanasupawat & Daengsubha 1983; Uchimura ef ul. 1991; Tanasupawat ef ul. 1991, 1992a, b, c, 1993a, b; Thongthai ef ul. 1992). Although yeasts are found in these fermented products (Suzuki ef ul. 1987) it is lactic acid bacteria (LAB) which are responsible for their souring and ripening (Tanasupawat & Daengsubha 1983; Tanasupawat ef cd. 1992a, 1993a, b). This review is on the distribution and functions of LAB in traditional fermented foods in Thailand.

S. Tanasupawat is with the Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand; fax: 662 255 6227. K. Komagata is with the Department of Agricultural Chemistry, Tokyo University of Agriculture, Sakuragaoka l-l-l, Setagaya-ku, Tokyo 156, Japan. *Corresponding author. @ 7995 Rapid Communicatiorw

of Oxford

Traditional

Fermented

Foods in Thailand

Fermented fish products, which are high in protein and amino acids, are often used to complement the amino acids obtained from cereals. In Thailand such products include num-plu (fish sauce), a much-used by-product of fermented fish and bu-du, ku-pi, fui-plu and plu-ru, all of which contain > 8% salt. The pH of these products ranges from 4.7 to 6.6 or even 8 and the total bacterial counts range from 1.8 X 10’ to 8.9 X 10’ cells/g. l&g-chom, hoi-dong, plu-chuo, plu-pueng-dueng, plu-chom, plu-som and som-fug are also fermented fish but contain < 8% salt, have a pH between 3.9 and 6.1 and contain 2.4 X 105 to 6.0 X 10” bacterial cells/g. Fermented meat and fermented vegetables are low salted products (I% to 4% salt) with a pH between 3.8 and 4.6 and bacterial counts of 3.2 X 10’ to 5.3 X IO“’ cells/ g. Nhum (fermented pork or beef) and sui-krog-prieo (a sausage of sour pork or beef) are made by mincing or chopping the meat. Pickled, fermented vegetables include phuk-gurd-dong (black mustard leaves), phuk-koom-dong (leaves and flowers of Crufuevu eryfhrocurpu), phuk-num-dong (young Lusiu heferophyllu), phuk-siun-dong (young Gynundropsis penfaphyllu), suuerkruuf, fhuu-ngok-dong, horn-dong and nuw-mui-dong and fruits are also salted and fermented in brine. Fermented tea leaves (miung) contain tannin and have a pH between 4.1 and 4.6. Bacterial counts in fermented tea leaves range from 3.4 X IO3 to 1.4 X 106 cells/g.

Ltd World]oumnl of Microbiology 6 Biotechrw&y,

Vol II, 1995

253

S. Tanasupawat and K. Komagata Khaomak is sweetened glutinous rice fermented to give the special taste and flavour of alcohol and lactic acid. The pH of Khaomak is 4.0 to 5.8 and bacterial counts are I.2 x 10’ to 4.3 X 10“’ cells/g. Khanom-jeen (fermented rice noodle) is fermented rice flour (from non-glutinous rice) and contains 4.9 x 10’ to 5.0 x 10“’ bacterial cells/g. The use of these fermented products varies. Fermented fish (pla-ra) is either chopped, flavoured with vegetables such as chilli, citrus leaves and lemon grass, and eaten raw or cooked, alone or as an ingredient of more complex dishes. Som-fug and nham are simply eaten with chilli, lettuce or ginger whereas fermented fruits and vegetables are generally eaten as a side-dish. Phak-gard-dong is cooked and served as a soup or a side-dish, whereas phak-koomdong and phak-Sian-dong are always side-dishes. Miang is eaten as a snack and khaomak as dessert. Khanom-jeen is cooked and eaten with nam-ya, a red curry of fish cooked with red chilli paste and coconut milk, in a dish called khanom-jeen nam-ya. Khanom-jeen kaeng-kai (green curry with chicken) and khanom-jeen kaeng-nua (green curry with beef) are also popular dishes. The components and production areas of various kinds of fermented Thai foods are described by Sundhgul et al. (1975), Appleton et al. (19781980) and Anon. (1981-1982).

Production

of Fermented

Foods

Traditional fermented foods are produced by natural fermentations in processes which vary from the simple to the complex (Sundhgul et al. 1975; Appleton et al. 1978-1980; Anon. 1981-1982). In general, each fermentation takes place under conditions that the producers have found to be favourable for the appropriate growth and action of microorganisms. Most fermentation methods involve the addition of salt. Fermented fish, such as pla-ra, is produced from a variety of freshwater, brackish-water and seawater fish. The fish meat is mixed well with salt before roasted rice is added, and the mix is packed in a closed jar for at least 6 months. Som-fug (fish cake) is produced only from the skinned fillets of freshwater fish. These are minced with cooked rice, salt and garlic to form a sticky paste which is pressed tightly into a bowl, wrapped with banana leaves or packed in plastic bags, and fermented for a few days. Nham is produced from minced pork mixed with sliced pork skin, garlic, pepper, salt and a small amount of potassium nitrate. The mixture is wrapped with banana leaves or plastic sheets and fermented for a few days. Miang (fermented tea leaves) is produced in the northern part of Thailand. The steamed tea leaves are wrapped tightly in individual bundles and packed into containers (small baskets for young tea leaves and large underground cement wells for mature tea leaves). The tea leaves are pressed tightly, weighted down, covered with banana leaves or plastic sheets, and fermented for a week or many

months. Yeasts, moulds and lactic acid bacteria play a vital role in the fermentation of khaomak and lactic acid bacteria also contribute to the fermentation of khanom-jeen. A number of fermented foods are home-made but some are produced at commercial plants using natural fermentation.

Lactic Acid Bacteria

in Fermented

Foods

Lactic acid bacteria have been isolated from various fermented products (Tanasupawat & Daengsubha 1983; Tanasupawat et al. 1992a). Homofermentative strains of Lacfobacillus penfosus, L. planfarum, L. sake and other Lacfobacillus spp. occur in a variety of fermented Thai food (Uchimura et al. 1991; Tanasupawat efal. 1992a, 1993a). Heterofermentative strains of L. vaccinosfercus, L. fermenturn, L. &revis, L. confusus, other Lacfobacillus spp. and Leuconostoc spp. can also be found (Okada 1992; Tanasupawat et al. I993b) along with Pediococcus penfosaceus, P. acidilactici, P. urinaeequi and P. halophilus strains (Tanasupawat 8~ Daengsubha 1983; Tanasupawat 1993a; Potacharoen et al. 1994). Pediococcus halophilus is now included in the genus Tetragenococcus (Collins et al. 1990). Lacfobacillus penfosus strains were isolated in at least six samples out of 10 of nham and miang and P. halophilus strains occurred in almost all tested samples of pla-ra (unpublished work). Although yeasts and other bacteria are also seen, their numbers are much smaller than those of LAB. Other bacteria present include Halobacterium salinarium, Staphylococcus camosus, S. piscifermenfans, Enferococcus hirae, E. faecalis, E. casseliflavus and other Enterococcus spp. (Tanasupawat et al. 1991, 1992b, c; Thongthai ef al. 1992) (see Table 1). The predominant LAB (L. pentosus, L. planfarum, Pediococcus pentosaceus and P. halophilus) are clearly different from the L. acidophifus, L. casei, L. frulgaricus, Lacfococcus factis, and Lacfoc. cremoris which are used for and found in dairy products (Sanders 1991).

Functions

of Lactic Acid Bacteria

The growth and activity of microorganisms play an important role in controlling the whole environment and ecosystem. The type of bacterial flora that develops in each fermenting food depends on the water activity, pH, salt concentration, temperature and the composition of the food. Lactic acid bacteria are perhaps the most widespread and desirable microorganisms in food fermentations. They convert most available carbohydrates to lactic acid, with small amounts of acetic acid, resulting in a lowering of the pH. If the fermentation is prolonged, the environment will be changed to become more suitable for yeast growth (Campbell-Platt 1987). Lactobacillus penfosus and L. plantarum strains that contain meso-diaminopimelic acid in the cell wall are the predominant rod-shaped LAB in fermented Thai foods. Pediococcus

Lactic acid bacteria in fermented foods Table 1. Distribution fermented

foods

Fermented

product

of lactic in Thailand.

acid bacteria and aitled bacterta

Mam-p/a (fish sauce) &du (fermented fish in liquid form) Tai-p/a (fermented fish entrails) P/a-ra (fermented fish) Hoi-malangpu-dong (pickled mussel) Kung-chom (fermented shrimp) P/a-paeng-daeng (fermented fish) P/a-chom (fermented small fish) Ching-c!xmg (fermented small fish) P/a-chao (fermented fish) P/a-som (fermented fish) Sam-fug (fish cake) Sour fish spawn yearn (fermented pork) Sai-krog-p&o (sour pork sausage) Mum (sour beef sausage) Phak-gard-dong (pickled mustard) Phak-koom-dong {pickle) Phak-nam-dong (pickle) Phak-Can-dong (pickle) Sauerkraut Thus-ngok-dong (pickled bean sprouts) ffom-dong (pickled spring onion) Flaw-mai-dong (fermented bamboo shoot) Satoh-dong (seed pickle) Sour durian (fermented durian) Miang (fermented tea leaves) Khaomak (sweetened rice) Khanom-jeen (fermented rice noodle)

fn

Bacterial species found’ 12,14 12,16 12 12, 16, 18 12 12, 15, 16 12 12, 15, 19 12 4 1,4,11,19 1, 2, 3, 11 5 1, 2, 3, 8, 9, 10, II 1,2,4, 11.19 2, 20 1, 4, 11, 13, 19

2.8 2 I, 4, 5, 11 2 2,4 1>2,8 1, .5 6 1 1,7,8, 17,20 2, 11 2, 4, 10

* 1-Lacfobaciilus penfosus; 2-L. pfantarum; 3-L. sake; 4-l.. fermentum; 5-L. brevis; 6-L. confuses; 7-L. vaccinostercus; 8-LactobaciHus spp.; 9-Leuconostoc sp.; lC-Pediococcus aciditactici; 11-P. pentosaceus; 12-P. halophi@ 13-P. I,& naeequi; l~~aiobacferium salinarium; l!j--Staphyiococcus carnosus; 16-S. piscifermentans; 17-Enterococcus cassefiffavus; 18-E. faeca/is; lQ--E. hirae; 20--Enterococcus spp, petifusacew strains are the major coccal bacteria although l? hcdqhilus strains occur in products containing high concentrations of salt. Strains of L. sake, L. fermwfum, L. breDis, L co~~~s~s~ other Lacfobaciilus spp., ~ed~ococc~ aci~ilacfici, and Leuconosnoc spp. are the minor LAB in fermented Thai foods. Lactobacillus pentosus, L. plantarum, L. fermenturn and Pe~jococ~ p~fosaceus are widely distributed, but the LAB are not generally specific for one kind of fermented product though the concentration of salt influences the flora of LAB. The lactobacilli containing meso-diaminopimelic acid in the cell wall, however, are usually found in fermented tea leaves (miang). Such bacteria include L. pentosw, L, planfarum, L. vaccinosfercus and some other Lactobacillus spp. (Okada 1992; T~asupawat et al. 1992a). The tannin in miang may have an effect on the growth of these cultures.

~a~obacf~ium sal~~arium produces proteases, which are important for fish-sauce fermentation (Thongthai et al. 1992). Staphylococci from fermented fish, which are coagulase-negative and haemolysin-negative (Tanasupawat et al. 1991, 1992b), produce a small amount of lactic acid but do not seem to play a role in the fermentation and ripening of the fermented foods. Staphylococcus carnosus strains were originally found in dry sausage and in a starter for the production of sausages in Germany (Schleifer & Fisher 1982). Not only can they reduce nitrate but they can also destroy peroxidases during sausage fe~entation (Lucke 198.5). Enterococci, especially E. faecajis and ,E, faecium strains, are common organisms in the intestinal tract of man and other animals and it is difficult to keep them out of foods (Robert 1982). Their presence in fermented foods may indicate inadequate sanitary practices. In western and European countries (Rose 1982; Lucke 1985; CampbellPlatt 1987), a variety of LAB is invoIved in the making of bread, cheese, fermented milk, sausage and fermented vegetables. LAB have been employed empirically in these areas for a long time; the main LAB fe~entation leads to the production of lactic acid, which contributes to the preservation of the foods. Most of the meat, dairy and vegetable food fermentations in these areas are now based on the use of dried, freeze-dried or frozen starter cultures, In addition to lactic acid, some starter cultures produce antimicrobi~ properties (Gould 199%). Bacteriocins of several LAB, including nisin and lactacin, inhibit spoilage bacteria and pathogens such as Listeria monocyfogenes, .9aphylococcus aweus, ~~os~ri~~urn bofulinum and Bacillus spp. (Lewus et al. 1991). ~ctobacillus aci~ophil~, L. sake, L, bulgaricus, L, helveticus, L. curvatus, L. plantarum, Lactococcus lactis, Lactococcus cremoris, P. pentosaceus and Z? acidilacfici strains produce lactacin, sakacin, buIgaricin, helveticin, lactocin, plantaricin, nisin, diprococcin, lactostrepcin and pediocin (Gould 1991; Lewus et al. l991), Lactic acid bacteria are widely distributed and domin~t in many kinds of fermented foods in Thailand. They occur naturally with other microorganisms in mixed-culture fermentations. Recently* several researchers have tried to use LAB as starters in the fermentations of fermented pork and vegetables. This should lead to improved food fermentation and hygiene.

References Anon. 1981-1982 Report on Thai Tradifional Fermetited Food. Bangkok: National Research Council of Thailand. Appleton, J.M., McGowan, V. & Skerman, V.B.D. ~%‘&Kx%I Microorgun&tns and Man. University of Queensland, Brisbane: World Data Center (MIRCEN). Campbell-PIatt, G. 1987 Fermenfd Foods of fhe World, a Dicfionmy and Guide. London: Butte~orths. Collins, M.D., Williams, A.M. & Wallbanks, S. I990 The phylogeny of Aerococcm and Pediococcm as determined by 16s rRNA

255

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and K. fimagafa

sequence analysis: description of Tetrugenococcus gen. nov. FEMS Microbiology Letters 70, 2~~-262. Gould, G.W. 1991 Antimicrobial compounds. In Biotechnology und Food Ingredients, eds Goldberg, I. & Williams, R. pp. 461-482. New York: Van Nostrand Reinhold. Lewus, C.B., Kaiser, A. & Montville, T.J. 1991 Inhibition of food borne bacterial pathogens by bacteriocins from lactic acid bacteria isolated from meat. Applied und Enuironmentul Microbiology 5 7,1683-1688. Lotong, N. 1985 Koji. In Microbiology of Fermenfed Foods, Vol. 2, ed Wood, B.J.B. pp. 237-270. London: Elsevier Applied Science. Lucke, F.K. 1985 Fermented sausages. In Microbiology of Fermented Foods, Vol. 2, ed Wood, B.J.B. pp. 41-83. London: Elsevier Applied Science. Okada, S. (ed) 1992 NRK C&ogue of Struins, 2nd edn. Tokyo: Tokyo University of Agriculture. Potacharoen, W., Arunpairojana, V. & Somchai, P. 1994 Thuilund’s Luctic Acid Bucteriu Collection ut Bungkok MlRCEN. Bangkok: Thailand Institute of Scientific and Technological Research. Robert, D. 1982 Bacteria of public health significance. In Meut Microbiology, ed Brown, M.H. pp. 356-367. London: Applied Science. Rose, A.H. 1982 honornic Microbiology. London: Academic Press. Saisithi, P., Kasemsam, B., Liston, J. & Dollar, A.M. 1966 Microbiology and chemistry of fermented fish. ]ournul of Food Science 31, 105-110. Sanders, M.E. 1991 Mixed culture in dairy fermentations. In Mi.red Cultures in Biotechnology, eds Zeikus, J.G. & Johnson, E.A. pp. 105-133. New York: McGraw Hill. Schleifer, K.H. & Fisher, LJ. 1982 Description of a new species of the genus Stuphylococcus: Stuphylococcus curnosus. ln~ernu~ionul ]ournul of System&c Bucteriology 32, 153-156. Sukhumavasi, J., Kato, K. & Harada, T. I975 Glucoamylase of a strain of Succhuromycopsisfibuligeru isolated from mold bran (loogpung) of Thailand. ]ournul of FermenWon Technology 53, 559-565. Sundhgul, M., Daengsubha, W. & Suyanandana, P. 1975 Thailand’s traditional fermented food products: a brief description. Thui ]ournu~ of Agricuhurul Science 8, 205-219. Suzuki, M., Nakase, T., Daengsubha, W., Chaosangket, M., Suyanadana, P. & Komagata, K. 1987 Identification of yeasts isolated from fermented foods and related materials in Thailand. ]ournul of Gent-ml und Applied Microbiology 33, 205-220. Tanasupawat, S. & Daengsubha, W. 1983 F’ediococcus species and

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related bacteria found in fermented foods and related materials in Thailand. ]ournul of Generul und Applied Microbiology 29, 487-506. Tanasupawat, S., Ezaki, T., Suzuki, K., Okada, S., Komagata, K. & Kozaki, M. 1992a Characterization and identification of Luctobucillus pentosus and Lactobucillus plunturum strains from femented foods in Thailand. ]ournul of Generul und Applied Microbiology

38,121-134. Tanasupawat, S., Hashimoto, Y., Ezaki, T., Kozaki, M. & Komagata, K. 1991 Identification of Sfuphylococcus curnosw strains from fermented fish and soy sauce mash. ]ournul of Generul und Applied Microbiology 37, 479-494. Tanasupawat, S., Hashimoto, Y., Ezaki, T., Kozaki, M. & Komagata, K. 1992b Sfuphylococcus piscifermentuns sp. nav., from fermented fish in Thailand. In~emufionul ]ournul of Systemufic Bucteriology 42, 577-581. Tanasupawat, S., Okada, S., Kozaki, M. & Komagata, K. 1993a Characterization of F’ediococcus penfosuceus and Pediococcm ucidiluctici strains and replacement of the type strain of Z’. ucidiluctici with the proposed neotype DSM 20284, request for an opinion. ln~ernu~ioncd ]oumul of Systemutic Bucteriology 43, 860-863. Tanasupawat, S., Okada, S., Suzuki, K., Kozaki, M. & Komagata, K. 1992~ Identification of Enterococcus hirue, E. fueculis, E. fuecium, and E. cusselifluoxs strains from fermented foods. Bulletin of the Jupun Federution for Culture Collecfion 8, 86-94. Tanasupawat, S., Okada, S., Suzuki, K., Kozaki, M. & Komagata, K. 1993b Lactic acid bacteria, particularly heterofementative lactobacilli, found in fermented foods in Thailand. Bulletin of fhe Jzpun Federution for Culfure Collection 9, 65-78. Thongthai, C., McGenity, T.J., Suntinanalert, P. & Grant, W.D. 1992 Isolation and characterization of an extremely halophilic archaeobacterium from traditional fermented Thai fish sauce (num-plu). Letters in Applied Microbiology 14, 111-114. Uchimura, T., Takao, T., Kikuchi, K., Niimura, Y., Okada, S., Ohara, N., Daengsubha, W. & Kozaki, K. 1991 Identification of lactic acid bacteria isolated from fermented rice noodle ‘khunom-jeen’ of Thailand. (Studies on the red orange pigment producing lactic acid bacteria L662 in the fermented rice noodle ‘khunom-jeen’ of Thailand. Part I.) Nippon Shokuhin Kogyo Gukkuisshi 38, 465-475 (in Japanese). (Received 1994)

in revised

form 2.5 Ocfober

19%;

accepfed

I November

Lactic acid bacteria in fermented foods in Thailand.

Traditional fermented foods (fish, meat and vegetable products), produced by many different processes, are eaten in many parts of Thailand. Lactic aci...
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