16S rRNA gene sequence analysis of lactic acid bacteria isolated from fermented foods in Thailand

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INTRODUCTION

　The National Institute of Technology and Evaluation (NITE) of Japan and the National Center for Genetic Engineering and Biotechnology (BIOTEC) of Thailand concluded their memorandum of understanding in accordance with the Convention on Biological Diversity, which promotes cooperative researches on the conservation and the sustainable use of biological resources in Japan and Thailand for academic, industrial, and other purposes. It is

expected that the utilization and commercialization of biological resources will provide some benefits to both countries. The NITE Biological Resource Center (NBRC) and the BIOTEC Culture Collection (BCC) started joint research projects on bacteria, yeasts, and fungi to enrich the microbiological resources and to develop the knowledge and skills of the scientists in both collections. We began a col- laborative research focused on acetic acid bacteria in 2005 and published its results (Muramatsu et al., 2009). In this paper, we report our study on lactic acid bacteria that we started in 2008.

　Lactic acid bacteria have been used to ferment foods before the existence of microorganisms was

*Corresponding author

E-mail: [email protected] Accepted: January 18, 2012

16S rRNA gene sequence analysis of lactic acid bacteria isolated from fermented foods in Thailand

Mika Miyashita

^1）

*, Pattaraporn Yukphan

^2）

, Winai Chaipitakchonlatarn

^2）

, Taweesak Malimas

^2）

, Masako Sugimoto

^1）

, Mayumi Yoshino

^1）

,

Wanchern Potacharoen

^2）

, Somboon Tanasupawat

^3）

, Yasuyoshi Nakagawa

^1）

, Kanyawim Kirtikara

^2）

, Morakot Tanticharoen

^2）

and Ken-ichiro Suzuki

^1）

1）NITE Biological Resource Center (NBRC), National Institute of Technology and Evaluation (NITE) 2-5-8 Kazusakamatari, Kisarazu, Chiba 292-0818, Japan

2）BIOTEC Culture Collection (BCC), National Center for Genetic Engineering and Biotechnology (BIOTEC) Pathumthani 12120, Thailand

3）Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University Bangkok 10330, Thailand

　In our investigation of a variety of lactic acid bacteria in fermented foods in Thailand, we isolated 945 strains from 114 varieties of fermented foods from the northeastern, central, and southern parts of the northern Thailand. We determined their 16S ribosomal RNA (rRNA) gene sequences and selected 410 strains for further analyses based on differences of sequences and/or isolation source. The result of the 16S rRNA gene sequence analysis showed that the isolates were divided into 50 groups of six genera; Aerococcus, Enterococcus, Lactobacillus, Pediococcus, Tetragenococcus, and Weissella. Only one strain belonging to the genus Aerococcus showed 100% sequence similarity to A. viridans. Forty-six strains were assigned to the genus Enterococcus. They were divided into six groups to which they were closely related, namely, E. avium, E. faecalis, E. faecium, E. hirae, E. thailandicus, or E. gilvus/E. raffinosus. We sorted 309 strains belonging to the genus Lactobacillus into 34 sequence groups. Most of them showed a sequence that is 100% identical to those of known species. However, 12 strains of eight groups showed slightly lower similarity rates of lower than 99.0%, suggesting that they may be classified into novel species. Thirty-eight strains of three groups were classified in the genus Pediococcus. One group was closely related to P. acidilactici, and the other groups were closely related to P. pentosaceus. Four isolates were classified in Tetragenococcus halophilus. Twelve strains belonging to the genus Weissella were divided into five species, namely, W. cibaria, W. confusa, W. paramesenteroides, W. thailandensis, and W. viridescens. The strains isolated in this study will be available for public use in the BIOTEC Culture Collection and the NITE Biological Resource Center, with the numbers NBRC 107174 to 107352 and NBRC 108277 to 108549.

Key words: lactic acid bacteria, Thailand, fermented foods, 16S rRNA gene sequence

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even recognized. It is well known that they are ben- eficial for human health. Various fermented foods are eaten in Thailand, especially in the northeastern region of Thailand. These fermented foods are served as main dishes and also used as condiments.

They give a unique taste and flavor to Thai foods.

In our investigation of a variety of lactic acid bacteria in fermented Thai foods, we isolated more than 900 strains and performed phylogenetic analyses based on their 16S rRNA gene sequences.

MATERIALS AND METHODS Sampling and isolation

　The samples used in this study were collected from the local markets located in three regions; the northeastern, central, and southern parts of the northern Thailand. Sixty samples of fermented foods (mainly fermented freshwater fish) were collected from eight local markets located in eight provinces in the northeastern Thailand. Twenty-six samples of fermented freshwater fishes and crustaceans, one sample of fermented meat, and 27 samples of fermented plant materials such as vegetables, fruits, tea leaves, and soy beans, were collected from 31 dif- ferent shops in 12 local markets located in eight provinces in the central and southern parts of the northern Thailand. Because the sodium chloride (NaCl) concentration of the samples varied from 0 to 50.0%, we used the MRS medium supplemented with 0, 5, 10, or 15% of NaCl for the isolation of lactic acid bacteria. In addition, we used pH levels between 4.0 and 10.0 for the culture media because the pH levels of the samples ranged from 4.0 to 8.0.

The samples were diluted and inoculated on MRS agar plates and then incubated at 30℃ in anaerobic conditions created by the AnaeroPack-Anaero system (Mitsubishi Gas Chemical Company, Inc., Tokyo, Japan). The colonies that emerged were repeatedly transferred on new MRS agar plates until purifica- tion was achieved.

Polymerase chain reaction amplification, sequencing, and phylogenetic analysis of 16S rRNA genes 　The almost complete 16S rRNA gene sequences, ranging from positions 28 to 1494 on the Escherichia coli numbering system (Brosius et al., 1978), were determined according to previously published proce- dures (Nakagawa et al., 2002). The sequences obtained were aligned with those of the representa- tive members of the lactic acid bacteria using

Clustal_X (Thompson et al., 1997) and then modified manually by referring to the 16S rRNA secondary structure of E. coli (Gutell et al., 1994) and using the BioEdit sequence alignment editor (http://www.

mbio.ncsu.edu/BioEdit/bioedit.html). A phylogenetic tree was reconstructed using the neighbor-joining method (Saitou & Nei, 1987) and the Knuc values derived from the sequences (Kimura, 1980).

Alignment gaps and unidentified base positions were not taken into consideration in the calculations.

The topology of the tree was evaluated by using the bootstrap resampling method of Felsenstein (1985) with 1,000 replicates.

RESULTS AND DISCUSSION Phylogenetic positions of isolates

　Initially, 945 strains of lactic acid bacteria were isolated from 114 varieties of fermented foods collected in Thailand. We determined their 16S rRNA gene sequences and selected 410 strains for further studies. Five hundred thirty-five strains whose isolation source and 16S rRNA gene sequences were identical were excluded from the study because we considered them as overlapping strains. Four hundred ten isolates were classified in the genera Aerococcus (number of isolates, 1), Enterococcus (46), Lactobacillus (309), Pediococcus (38), Tetragenococcus (4), and Weissella (12). They were divided into 50 groups according to the differences in their 16S rRNA gene sequences, and the species closely related to these groups are listed in Table 1.

　Only one strain, NB527, belonging to the genus Aerococcus had a 16S rRNA gene sequence that coincided with the sequence of the type strain of Aerococcus viridans. Forty-six isolates belonged to the genus Enterococcus and were divided into six groups to which they were closely related, namely, E. avium, E. faecalis, E. faecium, E. hirae, E. thailandicus, or E. gilvus/E. raffinosus. They showed greater than 99.8% sequence similarity to each type strain.

　In the genus Lactobacillus, we grouped 309 strains and divided them into 34 sequence groups (Table 1).

Twenty-five groups comprising 297 strains showed high rates of 16S rRNA gene sequence similarity (higher than 99.1%) to the following known species:

L. acidipiscis (group L1), L. brevis (L3), L. collinoides (L4), L. curvatus (L6), L. farciminis (L7–L15), L. fermentum (L16), L. futsaii (L18, L19), L. namurensis (L21), L. nantensis (L23), L. pantheris (L24), L. para-

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Table 1 List of the sequence groups of the Thai isolates Genus Phylogenetic

group 　　　 Strains Closest species based on 16S rRNA gene sequence similarity rates (%)　

Aerococcus A1 NB527 A. viridans (100)

Enterococcus E1 NB1011 E. avium (99.8)

E2 NB248 and 13 strains E. faecalis (100) E3 NB211 and 16 strains E. faecium (99.8)

E4 NB860 E. gilvus/E. raffinosus (99.9)

E5 NB152 and 9 strains E. hirae (100)

E6 NB320 and 2 strains E. thailandicus (99.8)

Lactobacillus L1 NB80 and 23 strains L. acidipiscis (99.9)

L2 NB278 L. alimentarius (98.5)

L3 NB465 and 13 strains L. brevis (100)

L4 NB916 L. collinoides (100)

L5 NB606 L. crustorum (98.7)

L6 NB146 L. curvatus (99.8)

L7 NB145 and 12 strains L. farciminis (99.5) L8 NB85 and 4 strains L. farciminis (99.6) L9 NB676 and 2 strains L. farciminis (99.7)

L10 NB730 L. farciminis (99.9)

L11 NB08 and 3 strains L. farciminis (99.4) L12 NB742 and 1 strain L. farciminis (99.5)

L15 NB40 and 60 strains L. farciminis (100) L16 NB523 and 16 strains L. fermentum (100)

L17 NB844 and NB1184 L. futsaii (98.7)

L18 NB659 L. futsaii (99.2)

L19 NB46 and 4 strains L. futsaii (99.3) L20 NB61 and 33 strains L. futsaii (100) L21 NB490 and 4 strains L. namurensis (99.9)

L22 NB483 L. nantensis (98.9)

L23 NB522 and NB609 L. nantensis (99.4)

L24 NB655 L. pantheris (99.7)

L25 NB642 and 1 strain L. paracasei (100)

L26 NB1110 L. paralimentarius (99.8)

L27 NB53 and NB834 L. plantarum group (98.9)

L28 NB446 and NB702 L. plantarum group (98.7) L29 NB1140 and NB1183 L. plantarum group (99.9) L30 NB07 and 93 strains L. plantarum group (100)

L31 NB625 L. pobuzihii (100)

L32 NB1023 L. saerimneri (100)

L33 NB521 L. senioris (97.7)

L34 NB196 and NB468 L. versmoldensis (99.0)

Pediococcus P1 NB38 and 11 strains P. acidilactici (99.7)

P2 NB04 and 4 strains P. pentosaceus (99.7) P3 NB20 and 20 strains P. pentosaceus (99.8)

Tetragenococcus T1 NB636 and 3 strains T. halophilus (99.8)

Weissella W1 NB27 and 2 strains W. cibaria (100)

W2 NB546 W. confusa (100)

W3 NB442 and 1 strain W. paramesenteroides (100) W4 NB173 and 4 strains W. thailandensis (99.8)

W5 NB25 W. viridescens (99.9)

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Oenococcus oeniJCM 6125^T(AB022924) Lactobacillus gigeriorum202^T(FR681899) NB523 (L16)

Lactobacillus fermentumNBRC15885^T(AB626052) Lactobacillus ingluviei KR3^T(AF333975)

Lactobacillus mucosaeLactobacillus rhamnosusLactobacillus caseiLactobacillus zeae ATCC 15820NB642 (L25)Lactobacillus paracaseiNBRC 15883S32^Tsubsp.NBRC 3425(AF126738)paracasei^T^T(D86516)(AB626050)^T(AB626049)NBRC 15889^T(AB626054) Lactobacillus saniviriLactobacillus camelliae MCH3-1Lactobacillus pentosusLactobacillus plantarum subsp. plantarumLactobacillus plantarum subsp. argentratensisNB07 (L30)NB28 (L30)Lactobacillus paraplantarumNB1140 (L29)NB1183 (L29)Lactobacillus fuchuensis B5M10Lactobacillus fabifermentansLactobacillus manihotivorans OND 32Lactobacillus xiangfangensisNB146 (L6)Lactobacillus curvatusNBRC 15884Lactobacillus graminis DSM 20719NB702 (L28)NB446 (L28)NB834 (L27)NB53 (L27)Lactobacillus sakeisubsp. sakeiLactobacillus parabrevisLactobacillus versmoldensisLactobacillus koreensisLactobacillus odoratitofui YIT 11304NB468 (L34)NB196 (L34)Lactobacillus zymnaeLactobacillus hammesii TMW 1.1236Lactobacillus paucivorans 1_1424Lactobacillus acidifarinaeNB465 (L3)Lactobacillus brevisLactobacillus namurensisLactobacillus tucceriLactobacillus senmaizukei L13Lactobacillus similis JCM 2765NB490 (L21)Lactobacillus kimchicusLactobacillus spicheriLactobacillus rapi YIT 11204Lactobacillus hilgardii NRIC 1060NB521 (L33)NB916 (L4)Lactobacillus collinoidesLactobacillus paracollinoides DSM 15502Lactobacillus pantherisNB40 (L15)Lactobacillus farciminis NBRC 107150NB655 (L24)NB522 (L22)NB609 (L22)Lactobacillus crustorumLactobacillus kisonensis YIT 11168Lactobacillus paralimentariusLactobacillus kimchiiLactobacillus nantensisNB730 (L10)NB1006 (L13)NB85 (L8)NB145 (L7)NB08 (L11)NB742 (L12)NB348 (L14)NB1110 (L26)Lactobacillus bobaliusLactobacillus mindensisLactobacillus parabuchneri LMG 11457Lactobacillus diolivorans JKD6NB606 (L5)NB676 (L9)Lactobacillus kefiriLactobacillus seniorisLactobacillus nodensisNB46 (L19)Lactobacillus otakiensisLactobacillus sunkiiNB659 (L18)Lactobacillus buchneriNB278 (L2)NB483 (L22)Lactobacillus alimentarius NBRC 106464NB61 (L20)Lactobacillus futsaiiLactobacillus hayakitensis KBL13Lactobacillus parakefiri JCM 8573NB1023 (L32)Lactobacillus saerimneriNBRC 107826Lactobacillus thailandensis MCH5-2Lactobacillus aviarius subsp. aviariusNBRC 102162NB844 (L17)NB1184 (L17)Lactobacillus ceti 142-2NB80 (L1)YIT 12363Lactobacillus acidipiscis NBRC 102163NB625 (L31)Lactobacillus pobuzihiiNBRC 106467NBRC 107147NBRC 107157NBRC 107151CECT 5920DCY50NBRC 107154DSM 21115NBRC158883.1.1NBRC 107155YIT 11161NBRC 106466^T^TDCY51NBRC 106106YIT 12364^TNBRC 107156NBRC 106069NBRC 107149(AB602569)NBRC 107158YM 0097(AB257864)NBRC 107153NBRC 107765NBRC 107160NBRC 107159DSM 20017NBRC 107162(AB370875)NBRC 107764YIT 11163^T^T(HM443954)^T^T(FJ904277)^T^T^T^T(AM113778)(AB626060)^T^T(AB366389)^T(AM292799)(AB626051)(AB297927)NBRC 107152^T(AF000162)(AB282889)^T(EU678893)NBRC 15891(FN185731)^T^T^T(AJ576006)^T^T^T^T^T^T^T(AF264701)^T(AB626075)(AB626062)^T(HQ322270)^T(AB366385)(AB365975)(AB626071)(AB626053)(AB262962)(AB626065)(AB602570)^T^T(AJ632219)(AB626068)NBRC 106468NBRC 103219^T^T^T(AB626069)^T(AB366388)^T(AM113784)^T^T^T^T(AB626059)(AB267406)^T(AB366386)^T^T^T^T^T(AB626057)(AB626070)(AB370879)^T(AB626072)(AB626056)(AB626063)^T(AB626067)(AB626064)(AB626074)(AB626078)^T^T(AB626073)(AB626076)^T(AB626077)(AJ970317)(AJ786665)(AB257863)^T^T(AB626079)^T^T(AB626058)(AB626066)(AB626055)^T^T^T(AB626061)(AB326358)(AB326356)^T(AB326355) 0.01

977

1000

1000 1000

1000 874

950

1000

1000 901 999

705 863

9991000 1000 963 937 1000

997 962 886

1000 1000

1000 910

955

921929 987

1000 993

780 825

704 999

862 1000

997 782 708

822

L. buchnerigroup L. plantarum group L. alimentarius

-L. farciminis group

758 981

962 980 946 954

995 1000

988

Fig. 1 The neighbor-joining tree showing the phylogenetic positions of the isolates in the genus based on the 16S rRNA gene sequences. Bar, 0.01 Knuc. Bootstrap values higher than 700 are shown in 1,000 replicates.

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casei (L25), L. paralimentarius (L26), L. plantarum group (L29, L30), L. pobuzihii (L31), and L. saerimneri (L32). According to Stackebrandt and Ebers (2006), if the similarity rate between the 16S rRNA gene sequences of two organisms is lower than 98.7–

99.0%, they belong to separate species. We found that eight groups that included 12 strains showed rates of similarity to known species lower than 99.0%, as will be discussed in the rest of the para- graph. Based on the system of classification of Felis and Dellaglio (2007), five of the eight groups (groups L2, L5, L17, L22, and L34) belonged to L. alimentarius–L. farciminis group, two (L28 and L37) to L.

plantarum group, and one (L33) to L. buchneri group (Fig. 1). Isolates NB278 (group L2), NB606 (group L5), NB844 and NB1184 (group L17), NB483 (group L22), and NB196 and NB468 (group L34) were closely related to the following species based on the similarity of their 16S rRNA gene sequences: L. alimentarius (98.5%), L. crustorum (98.7%), L. futsaii (98.8%), L.

nantensis (98.9%), and L. versmoldensis (99.0%), respectively. The 16S rRNA gene sequences of the strains NB446 and NB702 (group L28) and NB53 and NB834 (group L37) were similar to the sequences of the type strains of the L. plantarum group by 98.7%

and 98.9%, respectively. The isolate NB521 (group L33) showed a 97.7% 16S rRNA gene sequence similarity to that of L. senioris. Because the similarity rates between the 16S rRNA gene sequences of the isolates and those of the strains considered closely related to them were lower than 99.0% (Table 1), the isolates may be classified in new species.

　Thirty-eight strains belonging to the genus Pediococcus were divided into two species. Twelve strains (group P1) were closely related to P. acidilactici and 26 strains (P2 and P3) to P. pentosaceus.

They showed higher than 99.7% 16S rRNA gene sequence similarity to each type strain.

　Four isolates were classified in Tetragenococcus halophilus. The similarity rate between the 16S rRNA gene sequences of these isolates and that of the type strain was 99.9%.

　Twelve strains belonging to the genus Weissella were divided into five species, namely, W. cibaria, W. confusa, W. paramesenteroides, W. thailandensis, and W. viridescens. The similarity rates between the 16S rRNA gene sequences of the isolates and those of the type strains were higher than 99.8%.

Distribution of lactic acid bacteria in fermented foods in Thailand

　We used 26 kinds of 114 fermented foods as sources for isolation. These sources and the number of isolates from each are recorded in Table 2. Most Enterococcus strains were isolated from fermented fishes and crustaceans, and only eight of the 46 isolates were from fermented plant materials.

Lactobacillus strains were found in all the fermented Thai food samples, except in pu-dong (fermented crab) and kapi (fermented shrimp paste). Most strains belonging to L. acidipiscis, L. farciminis, and L. futsaii were isolated from fermented fish. On the other hand, strains of the L. fermentum were mainly isolated from fermented plant materials. The strains of the L. plantarum group were widely distributed in fermented Thai foods, having been found in 21 of the 26 kinds of samples. Pediococcus pentosaceus strains were isolated from various fermented fishes and crab. Tetragenococcus halophilus strains were isolated from pla-ra, pla-jaw (fermented fish), and tuaw jeaw (fermented soybean). Among the 26 kinds of fermented Thai foods, pla-ra, pla-som, pla-jom (fermented fish), and tuaw jeaw contained diverse species. However, most fermented plant materials, except tuaw jeaw, do not contain Weissella and Tetragenococcus species.

　The NaCl concentration of the fermented foods collected from the central and southern parts of the northern Thailand ranged from 0 to 50%. Most fermented plant materials contained low amount of NaCl (lower than 6%). On the other hand, NaCl concentration of all fermented fishes, crustaceans, meat, and soybeans are high (10 to 50%) (Table 2).

Relationships between isolated species and NaCl concentration of the 54 samples collected from central and southern parts of the northern region are summarized in Table 3. A lot of fermented fishes (16 samples) and fermented shrimp (1), crab (1), and soy beans (5) contained more than 22% NaCl. We found various lactic acid bacteria species in these high NaCl concentration sources (Table 3). Most of them were isolated from the medium without NaCl. These results suggested that they can survive in more than 22% NaCl concentration, even if they cannot grow in such high NaCl concentration. Most of L.

fermentum were mainly isolated from the fermented plant materials of which NaCl concentrations were less than 6%. L. plantarum group were widely distributed in fermented foods, however more than half

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Table 2 Number of isolates of each species isolated from Thai fermented foods Meat Som-khai-pla (Fermented ﬁsh roe) Mum-khai-pla (Fermented ﬁsh roe) Mum-sai-pla

(Fermented ﬁsh intestines) Nham-pla

(Fermented ﬁsh sauce) Pla-ra sub (Chili paste made from fermented ﬁsh and seasoning) Pla-ra-taung

(Fermented fish with seasoning) Pla-ra (Fermented fish) Pla-som (Fermented fish) Pla-jom (Fermented fish) Pla jaw (Fermented fish) Kapi

(Fermented shrimp paste) Pu dong

(Fermented crab) Mum (Fermented meat) Hom dong (Fermented vegetable ) Hua chai pow chem (Fermented vegetable ) Noa mai (pai ruak) (Fermented vegetable ) Noa mai dong (Fermented vegetable ) Pak kard dong (Fermented vegetable ) Pak kum dong (Fermented vegetable ) Pak Sean dong (Fermented vegetable ) Pak Sean and hua plee dong (Fermented vegetable ) Pak Sean and Makhua dong (Fermented vegetable ) Ma kham dong (Fermented fruit) Ma yom dong (Fermented fruit) Miang (Fermented tea leaf) Tuaw jaew (Fermented soy beans)

214411291530000000000000000060 00000013443111111541112112654 ndandndndndnd15〜4410〜1913〜3630〜3848341961503〜63〜62.5422〜43.53018〜50 Aerococcus viridans11 Enterococcus avium11 Enterococcus faecalis1012114 Enterococcus faecium6231517 Enterococcus gilvus/raﬃnosus11 Enterococcus hirae531110 Enterococcus thailandicus213 Lactobacillus acidipiscis16241124 Lactobacillus brevis13123111114 Lactobacillus collinoides11 Lactobacillus curvatus11 Lactobacillus farciminis3245231199331241291 Lactobacillus fermentum21121321111117 Lactobacillus futsaii2222732240 Lactobacillus namurensis12115 Lactobacillus nantensis22 Lactobacillus pantheris11 Lactobacillus paracasei112 Lactobacillus paralimentarius11 Lactobacillus plantarum group12341111710391622442215696 Lactobacillus pobuzihii11 Lactobacillus saerimneri11 Pediococcus acidilactici1611312 Pediococcus pentosaceus11284311111226 Tetragenococcus halophilus1214 Weissella cibaria123 Weissella confusa11 Weissella paramesenteroides112 Weissella thailandensis12115 Weissella viridescens11 Novel species candidates Lactobacillus sp. NB278 (L2)11 Lactobacillus sp. NB606 (L5)11 Lactobacillus sp. NB844, NB1184 (L17)112 Lactobacillus sp. NB483 (L22)11 Lactobacillus sp. NB446, NB702 (L28)112 Lactobacillus sp. NB521 (L33)11 Lactobacillus sp. NB196, NB468 (L34)112 Lactobacillus sp. NB53, NB834 (L37)112 Total numbers of isolates7511102512567421312614332014456531927410

NaCl concentration (%) of samplesb

Closest relative

Isolation source Total Fishes and CrustaceansPlant materials No. of samples collected from Northeastern region No. of samples collected fromthecentral and southern parts of the northern region and: no data, bData were derived from the samples collected from the central and southern parts of the northern region.

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of them were isolated from the fermented plant materials with low NaCl concentration. It is suggested that these two species have a high association to low NaCl concentration of fermented plant materials.

E. faecalis, E. faecium, L. acidipiscis, L. farciminis, L. futsaii, P. acidilactici, P. pentosaceus, T. halophi-

lus, and W. thailandensis were frequently isolated from samples containing more than 10% NaCl (Table 3). It was reported that these species except for the Enterococcus species can grow in the presence of around 10% NaCl (Chao et al., 2012; Dicks et al., 2009;

Hammes & Hertel, 2009; Holzapfel et al., 2009;

Table 3 Relationships between isolated species and NaCl concentration range of the 54 samples collected from the central and southern parts of the northern region

Closest relative

Source

0 to 6â 10 to 19â 22 to 50â

F^ｂ P^ｃ F P F P

n=0 n=20 n=9 n=2 n=18 n=5

Aerococcus viridans 　　 1 　

Enterococcus avium 1 　　

Enterococcus faecalis 　 1 　 3 　

Enterococcus faecium 　 3 2 4 3

Enterococcus gilvus/raffinosus 　 1 　

Enterococcus hirae 1 2 　　

Enterococcus thailandicus 　 1 　　

Lactobacillus acidipiscis 1 4 　 10 1

Lactobacillus brevis 9 2 　 1 　

Lactobacillus collinoides 1 　　

Lactobacillus farciminis 7 17 1 16 2

Lactobacillus fermentum 11 1 2 2 1

Lactobacillus futsaii 2 2 　 10 　

Lactobacillus namurensis 　 3 　 1

Lactobacillus nantensis 2 　　

Lactobacillus pantheris 1 　　

Lactobacillus paracasei 1 　 1 　

Lactobacillus paralimentarius 1 　　

Lactobacillus plantarum group 38 10 6 5 　

Lactobacillus pobuzihii 　　 1 　

Lactobacillus saerimneri 1 　　

Pediococcus acidilactici 2 2 2 1

Pediococcus pentosaceus 3 3 　 3 2

Tetragenococcus halophilus 　　 3 1

Weissella confusa 　　 1

Weissella paramesenteroides 　 1 　

Weissella thailandensis 　　 3 　 1 　

Novel species candidates 　　　

Lactobacillus sp. NB606 (L5) 1 　　

Lactobacillus sp. NB844, NB1184 (L17) 　 1 1 　

Lactobacillus sp. NB702 (L28) 　 1 　　

Lactobacillus sp. NB521 (L33) 1 　　

Lactobacillus sp. NB834 (L37) 　　　 1 　　

Total numbers of isolates 0 84 54 17 63 13

aNaCl concentration (%) of samples, ^bSamples of fermented fishes, crustaceans, and meat, ^cSamples of fermented plant materials

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Tanasupawat et al., 2000). Of these species, E. faecalis, E. faecium, L. acidipiscis, L. farciminis, L. futsaii, and P. pentosaceus were mainly isolated from fermented fishes, as described above, but E. faecium, L. farciminis, P. acidilactici, and P. pentosaceus were also found in fermented vegetables and soybean to some extent. Cooked rice or roasted ground rice called kao-kouwa were added into most of pla-ra, and some of pla-jaw, pla-jom, and pla-som.

Therefore, we cannot conclude that the species mainly found in fermented fishes are associated to fishes. However, because these species tolerate to high NaCl concentration, they were frequently isolated from fermented fishes.

　L. plantarum and L. farciminis were popular in Thai fermented foods examined, and most of them appeared on the medium without NaCl. Frequency of appearance of these species could be suppressed by using the isolation medium containing high NaCl concentration. For example, four strains of L.

farciminis, five strains of L. plantarum group, and two strains of Enterococcus species were isolated from one fermented fish (Pla-jom containing 13%

NaCl, pH 4.5) by using the medium with 0 or 5 % NaCl. In contrast, when we apply the medium with 10 or 15% NaCl for the same source, the number of isolates of L. farciminis and L. plantarum groups is only one, respectively and we could obtain L. acidipiscis (1 strain), P. pentosaceus (1), W. thailandensis (2), and a novel species candidate (1). T. halophilus were isolated from sources containing high NaCl concentration (24–44%) with low pH (pH 5–5.5).

However, they were isolated only from medium adjusted to pH 10 with various NaCl concentrations (0, 5, or 15%). For isolation of T. halophilus known as halophilic and alkalophilic bacteria, use of alkaline condition seems effective, even if pH of isolation source was acidic. These results suggested that various NaCl concentration and pH should be used to isolate diverse lactic acid bacteria from fermented foods.

　As described previously, we isolated 945 strains of lactic acid bacteria from 114 varieties of Thai fermented foods and selected 410 strains for further studies. The isolates were classified into 50 groups of six genera. We found several candidate groups that will be classified in new species. We are investi- gating their taxonomic characteristics to confirm whether they constitute new taxa. The results of the investigation will be published elsewhere by the

BCC and the NBRC. Some species of lactic acid bacteria, for example, E. thailandicus, L. acidipiscis, and Weissella thailandensis, were first found in fermented foods in Thailand (Tanasupawat et al., 2000, 2008).

Because we isolated 32 strains belonging to the three species, distributed in 27 samples of fermented foods (data not shown), these species are considered common organisms in fermented Thai foods and cer- tainly play important roles during fermentation. As a result of this study, isolates were preserved in the BCC, which then deposited them in the NBRC to make available for the public. Accordingly, cooperative researches through culture collections from dif- ferent countries should be useful in enriching biological resources and in promoting their conservation and sustainable use.

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タイ産発酵食品から分離した乳酸菌の 16S rRNA 遺伝子解析

宮下美香^1），Pattaraporn Yukphan^2），Winai Chaipitakchonlatarn^2），Taweesak Malimas^2），杉本昌子^1），吉野真由美^1），Wanchern Potacharoen^2），Somboon Tanasupawat^3），

中川恭好^1），Kanyawim Kirtikara^2），Morakot Tanticharoen^2），鈴木健一朗^1）

1）独立行政法人製品評価技術基盤機構（NITE），バイオテクノロジーセンター，生物資源課（NBRC）

2）BIOTEC Culture Collection （BCC）, National Center for Genetic Engineering and Biotechnology（BIOTEC），

Thailand，^3）Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Thailand 　タイの発酵食品に存在する乳酸菌の多様性を調べるために，タイの東北部，中部，および北部南寄りの地域から 114 の様々な発酵食品を収集し，945 株の乳酸菌を分離した．更なる解析に供するため，決定した 16S rRNA 遺伝子配列と分離源情報に基づいて 410 株を選抜した．16S rRNA 遺伝子配列に基づく解析の結果，分離株は Aerococcus 属，Enterococcus 属，

Lactobacillus 属，Pediococcus 属，Tetragenococcus 属，Weissella 属の 6 属に近縁な 50 の配列グループに分かれた．

Aerococcus 属に近縁だった 1 株は，A. viridans の配列と 100% の相同性を示した．46 株が Enterococcus 属に含まれ，それぞれ E. avium，E. faecalis，E. faecium，E. hirae，E. thailandicus，E. gilvus/E. raffinosus に近縁な 6 グループに分かれた．

Lactobacillus 属に含まれた 309 株は 34 の配列グループに分かれた．殆どの分離株は既知種の配列と 100% の相同性を示したが，8 つのグループに属する 12 株は 99.0% 以下のやや低い相同値を示し，新種である可能性が示唆された．Pediococcus 属に含まれたのは 3 グループの 38 株で，その 1 つは P. acidilactici に，他の 2 グループは P. pentosaceus に近縁だった．4 株が Tetragenococcus halophilus に近縁だった．Weissella 属に含まれた 12 株は W. cibaria，W. confusa，W. paramesenteroides，W. thailandensis，W. viridescens の 5 種のいずれかに近縁だった．この研究で分離した株は，タイの BIOTEC culture collection（BCC）と NITE Biological Resource Center（NBRC）の両コレクションから入手することができる．

（担当編集委員：田中尚人）