Isolation and Identification of Lactic Acid Bacteria in Traditional Fermented Sushi, Funazushi, from Japan

Food Sci. Technol. Res., 18 (1), 77–82, 2012

Isolation and Identification of Lactic Acid Bacteria in Traditional Fermented Sushi,

Funazushi, from Japan

1 2 1 1* Harutoshi Tsuda , Kenzo Kubota , Teruki Matsumoto and Yoshiko Ishimi

1 National Institute of Health and Nutrition, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8063, Japan 2 Department of Biotechnology, Faculty of Life Sciences, Ritsumeikan University, 1-1-1, Nojihigashi, Kusatsu, Shiga 525-8577, Japan

Received June 21, 2011; Accepted September 8, 2011

The lactic acid bacterial flora in commercial and homemade Funazushi (fermented crucian carp and rice) were investigated. Funazushi is a fermented fish product that continues to be produced in the traditional style in Japan. Lactic acid bacteria in four commercial and five homemade Funazushi were enumerated. The viable counts of commercial samples ranged from 3.0 × 103 to 2.7 × 105 cfu/g, with an average of 2.4 × 104 cfu/g, while the viable counts of homemade samples ranged from 2.0 × 102 to 2.6 × 107 cfu/g, with an average of 1.3 × 105 cfu/g. Twenty-seven lactic acid bacteria isolates were obtained from the commercial samples, and identified as Streptococcus salivarius, Lactobacillus buchneri, and Lactobacillus parabuchneri. Forty-eight isolates were obtained from the homemade samples, and identified as Lactoba- cillus plantarum, Lb. buchneri, Lactobacillus alimentarius, Lactobacillus farciminis, Lactobacillus acidipiscis, and Lactobacillus casei. Lb. buchneri was the predominant species in commercial Funazushi, while Lb. plantarum and Lb. buchneri were the predominant species in the homemade products.

Keywords: lactic acid bacteria, microbial flora, fermented fish product, funazushi

Introduction rel, and 8. the fish is fermented under the pressure of stones Fermented fish products called “narezushi” have long for 1 year (Fujii, 2001). been produced in Japan. Narezushi is a general term for Lactic acid bacteria (LAB) are the main fermentative mi- fermented cured fish and boiled rice. The fish and rice are croorganisms in Funazushi. Determination of the microflora usually fermented in barrels, under the pressure of stones, for of Funazushi using culture-based methods and molecular more than one year (Fujii et al., 2011). Many kinds of nar- biological techniques has been reported (Fujii et al., 2008, ezushi, e.g., Funazushi, sabanarezushi, and izushi, are known Fujii et al., 2011, Isobe et al., 2002). These studies investi- in Japan, and Funazushi is thought to have remained true gated one or two samples; therefore, investigation of mul- to the traditional style (Fujii et al., 2008). Funazushi is pro- tiple Funazushi samples would be worthwhile. duced around Lake Biwa in Shiga Prefecture and is a unique Funazushi has been experimentally noted to have benefi- sour tasting product of lactic fermented funa (crucian carp, cial health effects, including the cure of diarrhea (Ohshima Carassius buergeri grandoculis) and rice. The production and Fujii, 1994). Furthermore, Funazushi may be a good process of commercial Funazushi is as follows: 1. the scales, source of useful microorganisms. In the present study, we gills, and internal organs of crucian carp are removed, 2. the isolated and identified the LAB in commercial and home- carp is cured, 3. the fish is fermented in barrels under the made Funazushi. A comparison of commercial and home- pressure of stones for 1 year (a few months in the homemade made products would help in understanding the LAB micro- process), 4. the salt is washed out from the fish, 5. salted rice flora of Funazushi. Furthermore, these data could be useful is stuffed into the fish abdominal cavity, 6. the carp and salt- not only for the development of more efficient fermentation ed rice are placed in barrels, 7. salt water is added to the bar- and preservation techniques for traditional fermented fish products, but also in the investigation of the source of the *To whom correspondence should be addressed. beneficial health effects of Funazushi. E-mail: [email protected] 78 H. Tsuda et al.

Materials and Methods Tween 80 1.0 g/L, and L-cysteine HCl monohydrate 0.1 g/L, Salinity of Funazushi Salinity of Funazushi samples BCP 60 mg/L, pH 6.8 ± 0.2). The test strain was subcultured was determined with an atomic absorption spectrometer in 5 mL TYLG broth at 30℃ for 24 - 48 h, and the culture (Spectra AA220, Varian Techtron Pty Ltd., Victoria, Austra- was centrifuged (1,000 × g, 10 min). The cells were washed lia) (AOAC 969.23). Samples were generated by mixing an with 5 mL sterile 0.85 % NaCl solution and 50 μL of this cell equal amount of fish muscle and rice. suspension was inoculated into each sugar medium, and in- Enumeration and isolation of lactic acid bacteria Nine cubated for 7 days. The color change of BCP in the medium Funazushi samples were used (Fig. 1). Four samples were by acid production was determined. purchased from four different companies in Shiga Prefecture, 16S rRNA gene analysis Total DNA was extracted from and five samples were home produced in Shiga Prefecture. bacterial strains (Reyes-Gavilan et al., 1992). The 16S rRNA All samples were brought to the laboratory in a cooler box gene was amplified by PCR using Takara EX Taq (Takara (approximately 10℃) and stored in the refrigerator (4℃) un- Bio, Shiga, Japan). The bacteria-specific primer sequences til analyzed. were 5′-GTTTGATCCTGGCTCA-3′ (10F) and 5′-TAC- Samples (1 g) were serially diluted in sterile 0.85% NaCl CAGGGTATCTAATCC-3′ (800R), and PCR was performed solution (10 mL), plated on Plate Count Agar with bromocre- for 30 cycles (30 sec 94℃, 60 sec 55℃ and 60 sec 70℃). sol purple (BCP agar, Nissui, Japan) and MRS agar (Oxoid, PCR products were purified with phenol extraction and etha- Hampshire, England) containing cycloheximide (10 mg/L), nol precipitation. The purified fragments were sequenced. and incubated at 20 and 37℃ for 3−5 days. Colonies were Sequencing reactions of the purified fragments were per- enumerated, and individual colonies were picked randomly formed in a BioRad DNA Engine Dyad PTC-220 Peltier and purified by streaking. Thermal Cycler using an ABI BigDye Terminator v3.1 The strains were incubated in TYLG broth (tryptone 10 Cycle Sequencing Kit with AmpliTaq DNA polymerase (FS g/L, yeast extract 5.0 g/L, lactose 5.0 g/L, glucose 5.0 g/L, enzyme, Applied Biosystems, CA, USA). The fluorescent- Tween 80 1.0 g/L, and L-cysteine HCl monohydrate 0.1 g/ labeled fragments were purified with an ethanol precipitation L, pH 6.8 ± 0.2) and stock cultures were maintained in 10% reconstituted skim milk at −20℃. Identification and characterization of lactic acid bacteria The isolated strains were identified based on their physiolog- ical and biochemical characteristics as described by de Vos et al. (2009) and Wood and Holzapfel (1995), as well as by 16S rRNA sequence analysis. The tests included Gram-staining, catalase test, growth temperature test, production of gas from glucose, the type of lactic acid isomer, NH3 production from arginine, salt tolerance and carbohydrate (22 sugars) fermentation. The growth at 10, 40, and 45℃ for lactococci and at 15 and 45℃ for lactobacilli in TYLG broth containing 0.006% BCP, incubated for 7 days, was determined. The production of gas from glucose was determined according to the descrip- tion of Harrigan and McCance (1966). The types of lactic acid isomers produced from glucose were assayed by high- pressure liquid chromatography on a Sumichiral OA-5000 column (Otsuka et al., 1994). Salt tolerance was determined in TYLG broth containing 6.5 and 10% NaCl (w/v) after 3 days incubation. The carbohydrate fermentation profile of all strains was determined as follows. Twenty-two sugars were used (Table 1). Individual sugars were prepared as 5.0% (w/v) solutions, except esculin (2.5% w/v), and the solutions were sterilized using a 0.22 μm filter (Sartorius, Minisart, Germa- Fig. 1. Map of Shiga prefecture, Japan, showing the sam- ny). Next, 0.5 mL of the sterile sugar filtrate was added to 4.5 pling locations. mL of basal medium (tryptone 10 g/L, yeast extract 5.0 g/L, △, commercial Funazushi; ○, homemade Funazushi. Lactic Acid Bacteria in Japanese Traditional Fermented Fish Products, Funazushi 79

Table 1. Taxonomic properties of lactic acid bacteria isolated from commercial Funazushi.

No. of isolates 8 17 2 Cell shape cocci rod rod Growth at 10℃ − 15℃ + + 40℃ + 45℃ − − −

NH3 from arginine − + + Gas from glucose − + + Lactic acid isomer L DL DL Growth at 6.5% NaCl − Acid detected (No. of + isolates) Amygdalin 2 0 0 D-Arabinose 0 17 2 D-Cellobiose 8 0 0 Esculin 8 0 0 D-Fructose 8 17 2 D-Galactose 2 15 2 D-Glucose 8 17 2 D-Lactose 2 6 0 D-Maltose 8 15 2 D-Mannitol 0 0 0 D-Mannose 8 0 0 D-Melezitose 0 0 2 D-Melibiose 0 16 2 Na-Gluconate 0 17 2 L-Raffinose 8 3 2 L-Rhamnose 0 0 0 D-Ribose 0 14 2 Salicin 8 0 0 D-Sorbitol 8 0 0 Sucrose 8 0 2 D-Trehalose 6 0 0 D-Xylose 0 11 0 16S rRNA sequence Streptococcus Lactobacillus Lactobacillus (No. of tested isolates) salivarius (3) buchneri (14) parabuchneri (1)

protocol. The samples were resuspended in distilled water 105 cfu (colony forming unit)/g, with an average of 2.4 × 104 and subjected to electrophoresis in an ABI 3730xl sequencer cfu/g. The viable counts of homemade samples ranged from (Applied Biosystems), and the obtained sequences were ana- 2.0 × 102 to 2.6 × 107 cfu/g, with an average of 1.3 × 105 cfu/g. lyzed using the BLAST search program (i). These values agreed with previous studies (Fujii et al., 2008, Fujii et al., 2011, Isobe et al., 2002). Results and Discussion Identification of lactic acid bacteria in commercial Funa- Salinity and enumeration of lactic acid bacteria Salin- zushi Twenty-seven LAB isolates were obtained from the ity in the commercial Funazushi ranged from 3.1 to 6.4 %, commercial Funazushi. Eight isolates were lactococci and with an average of 4.1 %, while salinity in the homemade 19 were lactobacilli. Table 1 shows the characteristics of the Funazushi ranged from 2.3 to 5.0 %, with an average of 4.0 %. isolated LAB. These values agreed with previous studies (Fujii et al., 2008, The 8 lactococci isolates were identified as Streptococ- Kubo et al., 2008). cus salivarius. These isolates exhibited growth at 40℃, but LAB in the commercial and homemade Funazushi was not 10℃, and no growth in the presence of 6.5% NaCl. They enumerated on BCP agar and MRS agar plates. The viable produced no NH3 from arginine and no gas from glucose. counts of commercial samples ranged from 3.0 × 103 to 2.7 × They fermented cellobiose, maltose, and salicin, but not 80 H. Tsuda et al. mannitol, melibiose, or ribose. The 16S rRNA sequences of species in this study. Lb. buchneri was isolated from Funa- 3 selected isolates showed 99% homology to Str. salivarius. zushi in a previous study (Isobe et al. 2002). Fujii et al. iso- The 19 lactobacilli isolates were identified as Lactobacil- lated Lactobacillus curvatus, Lactobacillus plantarum, and lus buchneri (17 isolates) and Lactobacillus parabuchneri Lactobacillus acetotolerans from Funazushi (2011). Howev- (2 isolates). The Lb. buchneri isolates exhibited growth at er, these species were not isolated from commercial products.

15℃, but not 45℃, and produced NH3 from arginine and This disagreement might be due to differences in the samples gas from glucose. They fermented arabinose, but not sucrose used and the fermentation period. It would be expected that or trehalose. The 16S rRNA sequences of 14 selected isolates these species would be identified with the isolation of greater showed 99% homology to Lb. buchneri. The Lb. parabu- colony numbers. This is the first report of Str. salivarius and chneri isolates exhibited growth at 15℃, but not 45℃, and Lb. parabuchneri isolated from Funazushi. produced NH3 from arginine and gas from glucose. They fer- Identification of lactic acid bacteria in homemade Fu- mented arabinose, raffinose, and sucrose, but not trehalose. nazushi Forty-eight LAB isolates were obtained from the The 16S rRNA sequence of 1 selected isolate showed 99% homemade Funazushi, and all isolates were lactobacilli. homology to Lb. parabuchneri. Table 2 shows the characteristics of LAB. Str. salivarius and Lb. parabuchneri were isolated from The forty-eight lactobacilli isolates were identified as Lb. one product, respectively. Lb. buchneri was isolated from all plantarum (19 isolates), Lb. buchneri (16 isolates), Lacto- Funazushi products, and was observed to be the dominant bacillus alimentarius (5 isolates), Lactobacillus farciminis

Table 2. Taxonomic properties of lactic acid bacteria isolated from homemade Funazushi.

No. of isolates 19 16 5 4 3 1 Cell shape rod rod rod rod rod rod Growth at 15°C + + + + + + 45°C − − − − − −

NH3 from arginine + + − Gas from glucose − + − − − − Lactic acid isomer DL DL L DL L L Growth at 10% NaCl + + + Acid detected (No. + isolates) Amygdalin 19 0 5 0 3 1 D-Arabinose 19 16 5 0 3 0 D-Cellobiose 19 0 5 4 3 1 Esculin 19 16 5 4 3 1 D-Fructose 19 16 5 4 3 1 D-Galactose 19 16 5 4 3 1 D-Glucose 19 16 5 4 3 1 D-Lactose 19 14 0 4 0 1 D-Maltose 19 16 5 4 3 1 D-Mannitol 19 10 0 0 3 1 D-Mannose 19 0 5 4 3 1 D-Melezitose 18 3 0 4 0 1 D-Melibiose 19 16 0 0 0 0 Na-Gluconate 19 16 0 0 3 1 L-Raffinose 15 11 0 0 0 0 L-Rhamnose 5 0 0 0 0 0 D-Ribose 19 7 5 0 3 1 Salicin 19 0 5 4 3 1 D-Sorbitol 19 0 0 0 0 1 Sucrose 19 4 5 0 0 1 D-Trehalose 19 0 5 4 3 1 D-Xylose 1 5 0 0 0 0 16S rRNA sequence Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus (No. of tested isolates) plantarum (5) buchneri (10) alimentarius (3) farciminis (1) acidipiscis (2) casei (1) Lactic Acid Bacteria in Japanese Traditional Fermented Fish Products, Funazushi 81

(4 isolates), Lactobacillus acidipiscis (3 isolates), and Lac- and 4.8 %) than the average. The reason for the low salinity tobacillus casei (1 isolate). All isolates exhibited no growth of the sample containing Lb. alimentarius was unclear. Fu- at 45℃. The Lb. plantarum isolates produced DL-lactic acid nazushi might formerly have been made with high salt con- and no gas from glucose. They fermented arabinose, melibi- centrations in the home, and the used barrel or residual sub- ose, ribose, and sorbitol. The 16S rRNA sequences of 5 se- stances might harbour bacteria that could serve as a starter lected isolates showed 99% homology to Lb. plantarum. The culture. The contribution of these species to the fermentation

Lb. buchneri isolates produced NH3 from arginine and gas of Funazushi remains to be investigated. from glucose. They fermented arabinose, but not trehalose. The LAB isolated from homemade Funazushi are simi- The 16S rRNA sequences of 10 selected isolates showed lar to those isolated from other narezushi i.e., aji-narezushi, 99% homology to Lb. buchneri. The Lb. alimentarius iso- aji-no-susu, and saba-narezushi (Fujii et al., 1992, Kuda et lates produced L-lactic acid and no gas from glucose. They al., 2009, An et al., 2010). These narezushi were made with exhibited growth in the presence of 10% NaCl. They fer- marline fish, aji (scad, Trachurus japonicus) and saba (mack- mented cellobiose, esculin, and ribose, but not mannitol or erel, Scomber japonicus) instead of funa, and were pickled in melezitose. The 16S rRNA sequences of 3 selected isolates vinegar after curing. The fermentation period was generally showed 99% homology to Lb. alimentarius. The Lb. farcimi- shorter than for Funazushi (one or several months). The sa- nis isolates produced NH3 from arginine, and produced DL- linity of these narezushi was approximately 6%, higher than lactic acid and no gas from glucose. They exhibited growth for Funazushi (Fujii, 2001, Kuda et al., 2009). These nar- in the presence of 10% NaCl. They fermented cellobiose, ezushi contained Lb. plantarum as the predominant bacteria, salicin, and trehalose, but not mannitol, melibiose, or raf- as well as Lb. alimentarius and Lb. acidipiscis, species that finose. The 16S rRNA sequence of 1 selected strain showed were also isolated from homemade Funazushi in the present 99% homology to Lb. farciminis. The Lb. acidipiscis isolates study. This similarity suggested that Lb. plantarum, Lb. ali- produced L-lactic acid and no gas from glucose. They exhib- mentarius, and Lb. acidipiscis might play an important role ited growth in the presence of 10% NaCl. They fermented in the high salinity narezushi products in Japan. amygdalin, mannitol, and ribose, but not sorbitol. The 16S Investigation of the symbiotic relationships among the rRNA sequences of 2 selected isolates showed 99% homolo- different LAB will enable the optimization of the technology gy to Lb. acidipiscis. The Lb. casei isolate produced L-lactic for Funazushi production. Future studies will examine the acid and no gas from glucose. This isolate fermented amyg- beneficial characteristics of LAB for human health. dalin, cellobiose, gluconate, mannitol, melezitose, ribose, and sorbitol, but not arabinose, melibiose, or raffinose. The References 16S rRNA sequence of the isolate showed 99% homology to AOAC Official Method 969.23. (2010). “Official methods of analy- Lb. casei. sis of AOAC international, 18th ed.” AOAC International, Mary- The LAB microflora of five homemade Funazushi land. showed a number of differences with each other. Lb. plan- An, C., Takahashi, H., Kimura, B. and Kuda, T. (2010). Compari- tarum and Lb. buchneri were the dominant species in the son of PCR-DGGE and PCR-SSCP analysis for bacterial flora of five homemade samples, and these species had been isolated Japanese traditional fermented fish products, aji-narezushi and from Funazushi in previous studies (Fujii et al., 2011, Isobe iwashi-nukazuke. J. Sci. Food Agric., 90, 1796-1801. et al., 2002). Lb. alimentarius, Lb. acidipiscis, Lb. farciminis, De Vos, P., Garrity, G.M., Jones, D., Krieg, N.R., Ludwig, W., and Lb. casei were isolated from one sample, respectively. Rainey, F.A., Schleifer, K.H. and Whitman, W.B. (2009). “Ber- Previous study has shown the isolation of Lb. alimentarius gey’s Manual of Systematic Bacteriology second edition volume and Lb. acidipiscis from Funazushi (Fujii et al., 2011). On three.” Springer, New York, pp. 464-735. the other hand, this is the first time that Lb. farciminis and Fujii, T., Sasaki, T. and Okuzumi, M. (1992). Chemical composition Lb. casei were isolated from Funazushi. and microbial flora of saba-narezushi (fermented mackerel with The homemade Funazushi had a wider diversity of lac- rice). Nippon Suisan Gakkaishi, 58, 891-894. tobacilli than the commercial products. Lb. buchneri is the Fujii, T. (2001). “Shiokara, Kusaya, Katsuobushi.” Koseisha, To- predominant species in both commercial and homemade kyo. (in Japanese). products. The halo-tolerant species Lb. alimentarius, Lb. Fujii, T., Nishi, T. and Okuzumi, M. (2008). Chemical composition farciminis, and Lb. acidipiscis were only isolated from the and microbial flora of Funazushi, crucian carp fermented with homemade samples. The sample containing Lb. alimentarius rice. Yamawaki Stud. Art. Sci., 46, 90-103 (in Japanese). showed lower salinity (2.3 %), and the samples containing Fujii, T., Watanabe, S., Horikoshi, M., Takahashi, H. and Kimura, Lb. farciminis and Lb. acidipiscis showed higher salinity (5.0 B. (2011). PCR-DGGE analysis of bacterial communities in Fu- 82 H. Tsuda et al.

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