Research Article

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Table 4. Results of PCR-SSCP analysis and identification of 16S rDNA
sequencing of aji-narezushi and iwashi-nukazuke
Sample Medium
No. of
isolates
Identification of 16S
rDNA sequencing
Aji-narezushi
As-1 MRS 7 Not amplified (yeasts)
MRS 2 Lactobacillus acidipiscis
MRS 1 Lactobacillus plantarum
GAM 2 Lactobacillus paralimentarius
GAM 7 Lactobacillus acidipiscis
GAM 1 Lactobacillus plantarum
As-2 MRS 8 Lactobacillus plantarum
MRS 2 Not amplified (yeasts)
GAM 3 Lactobacillus plantarum
GAM 7 Lactobacillus acidipiscis
As-3 MRS 10 Not amplified (yeasts)
GAM 2 Lactobacillus casei
GAM 7 Lactobacillus acidipiscis
GAM 1 Tetragenococcus halophilus
Iwashi-nukazuke
In-1 MRS-HS 10 Not amplified (yeasts)
GAM-HS 10 Tetragenococcus muriaticus
In-2 MRS-HS 10 Not amplified (yeasts)
GAM-HS 10 Not amplified (yeasts)
In-3 MRS-HS 10 Not amplified (yeasts)
GAM-HS 8 Tetragenococcus halophilus
GAM-HS 2 Tetragenococcus muriaticus
MRS, de Man, Rogosa and Sharpe agar; GAM, Gifu anaerobic medium
agar; HS, high salt (containing 100 g L −1 NaCl).
SSCP analysis. These agar plates were incubated under anaerobic
conditions.
As summarised in Table 4, the V3 region of some isolates from
MRS agar plates was not amplified. Although MRS agar is regarded
as a medium for LAB, the cells of the isolates were observed as
oval yeast shapes under a microscope. Particularly in the iwashinukazuke
samples, all isolates from MRS-HS agar were yeasts.
The diversity of bacterial flora was not very high in the ajinarezushi
samples. The predominant LAB isolated from all three
samples using GAM agar plates was Lb. acidipiscis. In the case
of samples As-2 and As-3, Lb. acidipiscis was also detected
as predominant by the non-culture-based PCR-DGGE analysis
(Table 3). On the other hand, Lb. versmoldensis, which was shown
to be predominant in sample As-1 by the PCR-DGGE analysis, was
not detected by the PCR-SSCP method. Kröckel et al. 24 reported
that Lb. versmoldensis grows better in MRS broth than on MRS agar
and that its colonies on MRS agar are small. Furthermore, a lag
phase of up to 4 days could be observed when it was transferred
from MRS agar to MRS broth. 24 It is considered that the growth
rate of Lb. versmoldensis is slower than that of other LAB such as Lb.
acidipiscis. These results suggest that isolation of Lb. versmoldensis
under cultivation method conditions is difficult and the population
of Lb. versmoldensis was not reflected in the viable cell counts in
Table 2.
Lactobacillus plantarum was detected in samples As-1 and As-2,
though this bacterium was not detected by the PCR-DGGE analysis.
The composition of GAM agar, which contains liver extract, may
be suitable for growth of Lb. plantarum. Lactobacillus plantarum
is isolated not only from fermented vegetables but also from
www.soci.org C An et al.
fermented meat and fish. 14 Furthermore, it is well known that
Lb. plantarum has beneficial activities in fermented foods, such
as high lactic acid production, acid tolerance and bacteriocin
production. 14 Other LAB species, Lb. casei and T. halophilus, were
isolated from sample As-3.
In iwashi-nukazuke sample In-1 the predominant bacterium was
identified as T. muriaticus, while no bacterial colony was detected
in sample In-2. These results are in agreement with those of the
PCR-DGGE analysis (Table 3). However, in the case of sample In-3
the predominant isolate was identified as T. halophillus, which was
not detected by the PCR-DGGE analysis.
As reported above, different results were obtained from the
non-culture-based PCR-DGGE method and the culture-based PCR-
SSCP method. It is considered that the PCR-DGGE method is more
useful than the PCR-SSCP method to determine the predominant
bacteria and check the growth of starter strains. However, a greater
variety of microflora was expressed in the PCR-SSCP method than
in the PCR-DGGE method. The non-bacterial (rice chloroplast)
band in the PCR-DGGE analysis may hide the bacterial band.
Therefore further study of the PCR-DGGE analysis using the V3 and
other LAB-specific regions is necessary. Furthermore, biochemical
investigation of the LAB strains isolated from aji-narezushi and
iwashi-nukazuke is now in progress.
CONCLUSIONS
We studied the bacterial flora of traditional fermented fish
products, aji-narezushi and iwashi-nukazuke, using non-culturebased
PCR-DGGE and culture-based PCR-SSCP methods. In the
PCR-DGGE analysis, Lb. acidipiscis and Lb. versmoldenis were
detected as the predominant bacteria in aji-narezushi. However,
Lb. versmoldensis could not be isolated using GAM and MRS agars.
The PCR-DGGE analysis showed that the predominant bacterium
in iwashi-nukazuke was T. muriaticus rather than T. halophilus.
Some of our results differed from those of previous studies using
cultivation methods. Further studies on the detection, isolation
and biochemical and fermentation properties of LAB, particularly
Lb. versmoldensis,inaji-narezushi are necessary.
ACKNOWLEDGEMENT
ThisstudywassupportedbyafundfromtheMinistryofAgriculture,
Forestry and Fisheries for research and development projects
promoting the new policies of Agriculture, Forestry and Fisheries
(No. 2041).
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www.interscience.wiley.com/jsfa c○ 2010 Society of Chemical Industry J Sci Food Agric 2010; 90: 1796–1801