National Institute of Health and Nutrition, Toyama, Shinjuku-ku, Tokyo , Japan

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1 Food Sci. Technol. Res., 18 (1), 77 82, 2012 Isolation and Identification of Lactic Acid Bacteria in Traditional Fermented Sushi, Funazushi, from Japan Harutoshi Tsuda 1, Kenzo Kubota 2, Teruki Matsumoto 1 and Yoshiko Ishimi 1* 1 National Institute of Health and Nutrition, Toyama, Shinjuku-ku, Tokyo , Japan 2 Department of Biotechnology, Faculty of Life Sciences, Ritsumeikan University, 1-1-1, Nojihigashi, Kusatsu, Shiga , 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 to cfu/g, with an average of cfu/g, while the viable counts of homemade samples ranged from to cfu/g, with an average of cfu/g. Twenty-seven lactic acid bacteria isolates were obtained from the commercial samples, and identified as Streptococcus salivarius, buchneri, and parabuchneri. Forty-eight isolates were obtained from the homemade samples, and identified as plantarum, Lb. buchneri, alimentarius, farciminis, acidipiscis, and 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 *To whom correspondence should be addressed. ishimi@nih.go.jp Introduction Fermented fish products called narezushi have long been produced in Japan. Narezushi is a general term for fermented cured fish and boiled rice. The fish and rice are usually fermented in barrels, under the pressure of stones, for more than one year (Fujii et al., 2011). Many kinds of narezushi, e.g., Funazushi, sabanarezushi, and izushi, are known in Japan, and Funazushi is thought to have remained true to the traditional style (Fujii et al., 2008). Funazushi is produced around Lake Biwa in Shiga Prefecture and is a unique sour tasting product of lactic fermented funa (crucian carp, Carassius buergeri grandoculis) and rice. The production process of commercial Funazushi is as follows: 1. the scales, gills, and internal organs of crucian carp are removed, 2. the carp is cured, 3. the fish is fermented in barrels under the pressure of stones for 1 year (a few months in the homemade process), 4. the salt is washed out from the fish, 5. salted rice is stuffed into the fish abdominal cavity, 6. the carp and salted rice are placed in barrels, 7. salt water is added to the barrel, and 8. the fish is fermented under the pressure of stones for 1 year (Fujii, 2001). Lactic acid bacteria (LAB) are the main fermentative microorganisms in Funazushi. Determination of the microflora of Funazushi using culture-based methods and molecular biological techniques has been reported (Fujii et al., 2008, Fujii et al., 2011, Isobe et al., 2002). These studies investigated one or two samples; therefore, investigation of multiple Funazushi samples would be worthwhile. Funazushi has been experimentally noted to have beneficial health effects, including the cure of diarrhea (Ohshima and Fujii, 1994). Furthermore, Funazushi may be a good source of useful microorganisms. In the present study, we isolated and identified the LAB in commercial and homemade Funazushi. A comparison of commercial and homemade products would help in understanding the LAB microflora of Funazushi. Furthermore, these data could be useful not only for the development of more efficient fermentation and preservation techniques for traditional fermented fish products, but also in the investigation of the source of the beneficial health effects of Funazushi.

2 78 Materials and Methods Salinity of Funazushi Salinity of Funazushi samples was determined with an atomic absorption spectrometer (Spectra AA220, Varian Techtron Pty Ltd., Victoria, Australia) (AOAC ). Samples were generated by mixing an equal amount of fish muscle and rice. Enumeration and isolation of lactic acid bacteria Nine Funazushi samples were used (Fig. 1). Four samples were purchased from four different companies in Shiga Prefecture, and five samples were home produced in Shiga Prefecture. All samples were brought to the laboratory in a cooler box (approximately 10 ) and stored in the refrigerator (4 ) until analyzed. Samples (1 g) were serially diluted in sterile 0.85% NaCl solution (10 ml), plated on Plate Count Agar with bromocresol purple (BCP agar, Nissui, Japan) and MRS agar (Oxoid, Hampshire, England) containing cycloheximide (10 mg/l), and incubated at 20 and 37 for 3 5 days. Colonies were enumerated, and individual colonies were picked randomly and purified by streaking. The strains were incubated in TYLG broth (tryptone 10 g/l, yeast extract 5.0 g/l, lactose 5.0 g/l, glucose 5.0 g/l, Tween g/l, and L-cysteine HCl monohydrate 0.1 g/ 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 physiological 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, NH 3 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 description of Harrigan and McCance (1966). The types of lactic acid isomers produced from glucose were assayed by highpressure 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, Germany). Next, 0.5 ml of the sterile sugar filtrate was added to 4.5 ml of basal medium (tryptone 10 g/l, yeast extract 5.0 g/l, H. Tsuda et al. Tween g/l, and L-cysteine HCl monohydrate 0.1 g/l, BCP 60 mg/l, ph 6.8 ± 0.2). The test strain was subcultured in 5 ml TYLG broth at 30 for h, and the culture was centrifuged (1,000 g, 10 min). The cells were washed with 5 ml sterile 0.85 % NaCl solution and 50 μl of this cell suspension was inoculated into each sugar medium, and incubated for 7 days. The color change of BCP in the medium by acid production was determined. 16S rrna gene analysis Total DNA was extracted from bacterial strains (Reyes-Gavilan et al., 1992). The 16S rrna gene was amplified by PCR using Takara EX Taq (Takara Bio, Shiga, Japan). The bacteria-specific primer sequences were 5 -GTTTGATCCTGGCTCA-3 (10F) and 5 -TAC- CAGGGTATCTAATCC-3 (800R), and PCR was performed for 30 cycles (30 sec 94, 60 sec 55 and 60 sec 70 ). PCR products were purified with phenol extraction and ethanol precipitation. The purified fragments were sequenced. Sequencing reactions of the purified fragments were performed in a BioRad DNA Engine Dyad PTC-220 Peltier Thermal Cycler using an ABI BigDye Terminator v3.1 Cycle Sequencing Kit with AmpliTaq DNA polymerase (FS enzyme, Applied Biosystems, CA, USA). The fluorescentlabeled fragments were purified with an ethanol precipitation Fig. 1. Map of Shiga prefecture, Japan, showing the sampling locations., commercial Funazushi;, homemade Funazushi.

3 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 Cell shape cocci rod rod Growth at NH 3 from arginine + + Gas from glucose + + Lactic acid isomer L DL DL Growth at 6.5% NaCl Acid detected (No. of + isolates) Amygdalin D-Arabinose D-Cellobiose Esculin D-Fructose D-Galactose D-Glucose D-Lactose D-Maltose D-Mannitol D-Mannose D-Melezitose D-Melibiose Na-Gluconate L-Raffinose L-Rhamnose D-Ribose Salicin D-Sorbitol Sucrose D-Trehalose D-Xylose S rrna sequence (No. of tested isolates) Streptococcus salivarius (3) buchneri (14) parabuchneri (1) protocol. The samples were resuspended in distilled water and subjected to electrophoresis in an ABI 3730xl sequencer (Applied Biosystems), and the obtained sequences were analyzed using the BLAST search program (i). Results and Discussion Salinity and enumeration of lactic acid bacteria Salinity in the commercial Funazushi ranged from 3.1 to 6.4 %, with an average of 4.1 %, while salinity in the homemade Funazushi ranged from 2.3 to 5.0 %, with an average of 4.0 %. These values agreed with previous studies (Fujii et al., 2008, Kubo et al., 2008). LAB in the commercial and homemade Funazushi was enumerated on BCP agar and MRS agar plates. The viable counts of commercial samples ranged from to cfu (colony forming unit)/g, with an average of cfu/g. The viable counts of homemade samples ranged from to cfu/g, with an average of cfu/g. These values agreed with previous studies (Fujii et al., 2008, Fujii et al., 2011, Isobe et al., 2002). Identification of lactic acid bacteria in commercial Funazushi Twenty-seven LAB isolates were obtained from the commercial Funazushi. Eight isolates were lactococci and 19 were lactobacilli. Table 1 shows the characteristics of the isolated LAB. The 8 lactococci isolates were identified as Streptococcus salivarius. These isolates exhibited growth at 40, but not 10, and no growth in the presence of 6.5% NaCl. They produced no NH 3 from arginine and no gas from glucose. They fermented cellobiose, maltose, and salicin, but not

4 80 mannitol, melibiose, or ribose. The 16S rrna sequences of 3 selected isolates showed 99% homology to Str. salivarius. The 19 lactobacilli isolates were identified as buchneri (17 isolates) and parabuchneri (2 isolates). The Lb. buchneri isolates exhibited growth at 15, but not 45, and produced NH 3 from arginine and gas from glucose. They fermented arabinose, but not sucrose or trehalose. The 16S rrna sequences of 14 selected isolates showed 99% homology to Lb. buchneri. The Lb. parabuchneri isolates exhibited growth at 15, but not 45, and produced NH 3 from arginine and gas from glucose. They fermented arabinose, raffinose, and sucrose, but not trehalose. The 16S rrna sequence of 1 selected isolate showed 99% homology to Lb. parabuchneri. Str. salivarius and Lb. parabuchneri were isolated from one product, respectively. Lb. buchneri was isolated from all Funazushi products, and was observed to be the dominant H. Tsuda et al. species in this study. Lb. buchneri was isolated from Funazushi in a previous study (Isobe et al. 2002). Fujii et al. isolated curvatus, plantarum, and acetotolerans from Funazushi (2011). However, these species were not isolated from commercial products. This disagreement might be due to differences in the samples used and the fermentation period. It would be expected that these species would be identified with the isolation of greater colony numbers. This is the first report of Str. salivarius and Lb. parabuchneri isolated from Funazushi. Identification of lactic acid bacteria in homemade Funazushi Forty-eight LAB isolates were obtained from the homemade Funazushi, and all isolates were lactobacilli. Table 2 shows the characteristics of LAB. The forty-eight lactobacilli isolates were identified as Lb. plantarum (19 isolates), Lb. buchneri (16 isolates), alimentarius (5 isolates), farciminis Table 2. Taxonomic properties of lactic acid bacteria isolated from homemade Funazushi. No. of isolates Cell shape rod rod rod rod rod rod Growth at 15 C C NH 3 from arginine + + Gas from glucose + Lactic acid isomer DL DL L DL L L Growth at 10% NaCl Acid detected (No. + isolates) Amygdalin D-Arabinose D-Cellobiose Esculin D-Fructose D-Galactose D-Glucose D-Lactose D-Maltose D-Mannitol D-Mannose D-Melezitose D-Melibiose Na-Gluconate L-Raffinose L-Rhamnose D-Ribose Salicin D-Sorbitol Sucrose D-Trehalose D-Xylose S rrna sequence (No. of tested isolates) plantarum (5) buchneri (10) alimentarius (3) farciminis (1) acidipiscis (2) casei (1)

5 Lactic Acid Bacteria in Japanese Traditional Fermented Fish Products, Funazushi 81 (4 isolates), acidipiscis (3 isolates), and casei (1 isolate). All isolates exhibited no growth at 45. The Lb. plantarum isolates produced DL-lactic acid and no gas from glucose. They fermented arabinose, melibiose, ribose, and sorbitol. The 16S rrna sequences of 5 selected isolates showed 99% homology to Lb. plantarum. The Lb. buchneri isolates produced NH 3 from arginine and gas from glucose. They fermented arabinose, but not trehalose. The 16S rrna sequences of 10 selected isolates showed 99% homology to Lb. buchneri. The Lb. alimentarius isolates produced L-lactic acid and no gas from glucose. They exhibited growth in the presence of 10% NaCl. They fermented cellobiose, esculin, and ribose, but not mannitol or melezitose. The 16S rrna sequences of 3 selected isolates showed 99% homology to Lb. alimentarius. The Lb. farciminis isolates produced NH 3 from arginine, and produced DLlactic acid and no gas from glucose. They exhibited growth in the presence of 10% NaCl. They fermented cellobiose, salicin, and trehalose, but not mannitol, melibiose, or raffinose. The 16S rrna sequence of 1 selected strain showed 99% homology to Lb. farciminis. The Lb. acidipiscis isolates produced L-lactic acid and no gas from glucose. They exhibited growth in the presence of 10% NaCl. They fermented amygdalin, mannitol, and ribose, but not sorbitol. The 16S rrna sequences of 2 selected isolates showed 99% homology to Lb. acidipiscis. The Lb. casei isolate produced L-lactic acid and no gas from glucose. This isolate fermented amygdalin, cellobiose, gluconate, mannitol, melezitose, ribose, and sorbitol, but not arabinose, melibiose, or raffinose. The 16S rrna sequence of the isolate showed 99% homology to Lb. casei. The LAB microflora of five homemade Funazushi showed a number of differences with each other. Lb. plantarum and Lb. buchneri were the dominant species in the five homemade samples, and these species had been isolated from Funazushi in previous studies (Fujii et al., 2011, Isobe et al., 2002). Lb. alimentarius, Lb. acidipiscis, Lb. farciminis, and Lb. casei were isolated from one sample, respectively. Previous study has shown the isolation of Lb. alimentarius and Lb. acidipiscis from Funazushi (Fujii et al., 2011). On the other hand, this is the first time that Lb. farciminis and Lb. casei were isolated from Funazushi. The homemade Funazushi had a wider diversity of lactobacilli than the commercial products. Lb. buchneri is the predominant species in both commercial and homemade products. The halo-tolerant species Lb. alimentarius, Lb. farciminis, and Lb. acidipiscis were only isolated from the homemade samples. The sample containing Lb. alimentarius showed lower salinity (2.3 %), and the samples containing Lb. farciminis and Lb. acidipiscis showed higher salinity (5.0 and 4.8 %) than the average. The reason for the low salinity of the sample containing Lb. alimentarius was unclear. Funazushi might formerly have been made with high salt concentrations in the home, and the used barrel or residual substances might harbour bacteria that could serve as a starter culture. The contribution of these species to the fermentation of Funazushi remains to be investigated. The LAB isolated from homemade Funazushi are similar to those isolated from other narezushi i.e., aji-narezushi, aji-no-susu, and saba-narezushi (Fujii et al., 1992, Kuda et al., 2009, An et al., 2010). These narezushi were made with marline fish, aji (scad, Trachurus japonicus) and saba (mackerel, Scomber japonicus) instead of funa, and were pickled in vinegar after curing. The fermentation period was generally shorter than for Funazushi (one or several months). The salinity of these narezushi was approximately 6%, higher than for Funazushi (Fujii, 2001, Kuda et al., 2009). These narezushi contained Lb. plantarum as the predominant bacteria, as well as Lb. alimentarius and Lb. acidipiscis, species that were also isolated from homemade Funazushi in the present study. This similarity suggested that Lb. plantarum, Lb. alimentarius, and Lb. acidipiscis might play an important role in the high salinity narezushi products in Japan. Investigation of the symbiotic relationships among the different LAB will enable the optimization of the technology for Funazushi production. Future studies will examine the beneficial characteristics of LAB for human health. References AOAC Official Method (2010). Official methods of analysis of AOAC international, 18 th ed. AOAC International, Maryland. An, C., Takahashi, H., Kimura, B. and Kuda, T. (2010). 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