マサバへしこの「米糠」の性状変化とアミラーゼ生産菌の検 索 · (dsc)...
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マサバへしこの「米糠」の性状変化とアミラーゼ生産菌の検索
誌名誌名 日本食品保蔵科学会誌
ISSNISSN 13441213
巻/号巻/号 452
掲載ページ掲載ページ p. 85-93
発行年月発行年月 2019年3月
農林水産省 農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat
( 33) Food Preservation Science VOL. 45 NO. 2 2019 (Research NoteJ 85
Characterization of Fermented Rice Bran from 'Heshiko' and Isolation of Amylase-Producing Bacteria
NAGAOKA Junko*1, WADA Kanae* 1, KATOH Miki*1, FUJITA Naoko* 2
,
TANAKA Naoto* 3, IRISAWA Tomohiro* 4 and AKUZAWA Sayuri*1
§
* 1 Faculty of Applied Bio-science, Tokyo· University of Agriculture,
1-1 - 1 Sakuragaoka, Setagaya-ku, Tokyo 156-852
* 2 Faculty of Bioresource Sciences, Tokyo Akita Prefectural University.,
241-438 Kaidobata-Nishi, Nakano, Shimoshinjo, Akita city, Akita 010-0195
* 3 Faculty of Life Sciences, Tokyo University of Agriculture,
1-1 -1 Sakuragaoka, Setagaya-ku, Tokyo 156-852
* 4 Faculty of Agriculture, Tokyo University of Agriculture,
1737 Funako, Atsugi city, Kanagawa 243-0034
In this study, we characterized the main properties of the fermented rice bran around mackerel in the Japanese product Heshiko. We found increases in the moisture, lipids, and ash contents after fermentation, which was attributed to the effects of the Shiejiru added for filtration during the manufacturing process. However, there is no change of the general composition of the fermented rice bran of Heshiko with respect to the shipping time. We further characterized the non-starch, starch, and water-soluble polysaccharide fractions to understand better the chemical changes occurring as a result of enzyme and microbial action during fermentation. For instance, SEM photographs of nonstarch and starch polysaccharides indicate the breakage of cell wall and surface of the starches. Furthermore, regarding the branch chain-length distribution pattern of. amylopectin, we observed a decrease in the short chains of DP 6-12 and an increase in other chains of DP 13-20 in the fermented rice bran. Finally, we isolated amylase-producing bacteria from the fermented rice bran, which were estimated as Bacillus amyloliquefaciens ( A 1) , Paenibacillus amylolyticus ( A 3) , Bacillus subtilis ( B 1 ) , Bacillus licheniformis (B 2), and Oceanobacillus picturae (B 3).
(Received Oct. 25, 2018 ; Accepted Nov. 29, 2018)
Key words : Heshiko, fermented rice bran, polysaccharide, amylase-producing bacteria
~~~. ~M*•· ~~r~•. 7~7-~~£ffi
Heshiko is a fermented seafood product in Japan
that is produced from the fermentation of fresh rice
bran using only a carbohydrate source. The Heshiko
manufacturing process involves washing mackerels
after removing the viscera, adding salt, and then
pressing the fish under stones for approximately
seven days. The mackerels are then separated from
the effused liquid, which is filtered out and boiled to
prepare Shiejiru. After immersion in Shiejiru, the
mackerels are washed again and preserved in a mixture of fresh rice bran, pepper, salt, and Shiejiru.
The stones are then placed on top of the mackerels
again, and the fish is left to spontaneously ferment
for one year at room temperature. During the
fermentation period, the powdery fresh rice bran
becomes very pasty and develops strong umami
§ Corresponding author, E-mail : [email protected]
and sour taste0, which is then consumed together
with the fish as Heshiko. Nukazuke is another type of
fermented food prepared using rice bran, but, in
this case, the rice bran is washed off of the
vegetables before they are served. Although rice
bran contains nutrients such as dietary fiber and
minerals') , it is also often used for non - dietary
applications such as to extract oil, feed livestock,
and improve the quality of the soil In fact, humans
rarely consume rice bran alone because of its
powdery texture. Thus, Heshiko is a unique food
product since it is eaten along with the fermented
rice bran, mainly owing to its pasty texture, making
it difficult to separate but easier to consume. One
likely explanation for this pasty texture is that the
components of the mackerel's body diffuse into the
86 Food Preservation Science VOL. 45 NO. 2 2019 ( 34)
rice bran during the fermentation period, and are
then degraded by enzymes produced by
microorganisms. However, the majority of studies on
Heshiko conducted to date have only focused on
changes in the components of the fish meat during
the fermentation period'H', along with changes in
the microbial layer and chemical components, such as peptides6,-s,, and no study has yet analyzed the
detailed properties of the rice bran itself after
fermentation. Therefore, the purpose of this study
was to compare the properties of fresh and
fermented rice bran in Heshiko. Specifically, we
analyzed the general composition, non-starch
polysaccharides, starch polysaccharides, and water
soluble polysaccharides. In addition, we hypothesized
that microbial strains with amylase activity are involved in changing the quality of the fermented
rice bran. To test this hypothesis, we isolated and
identified several amylase-producing bacteria present
in the fermented rice bran surrounding the
mackerel body. This study makes a significant
contribution to the literature because Heshiko is a unique type of fermented seafood product that is
eaten along with the rice bran used for
fermentation. Moreover, this is the first study to
report the potential of Oceanobacillus of assimilating
starch, providing new insight into the adaptations of
this genus in an environment similar to a marine
environment (i.e., containing fish and salt).
Materials and Methods
1 . Materials
The Heshiko used in this study was conducted
with products manufactured at the MA TSUDA
store in Fukui City, Fukui Prefecture, Japan. The
fresh rice bran sample was that used in the manufacturing of Heshiko at MA TSUDA ; two
separate rice bran products were tested, which were shipped in September 2009 (sample A) and
February 2010 ( sample B) , respectively. For the
purpose of collecting two kinds of scores clearly
differing in the start of production, samples shipped
with a period of six months were used. Fermented
rice bran was collected around the body of Heshiko.
2 . General components and sodium content of
the rice bran samples
The general components of samples A and B of
fresh rice bran were measured as previously
reported•'. In brief, lipids were measured with a
standard chloroform-methanol extraction method, ash
was measured from a direct ashing method, and the
carbohydrates content was determined by
subtracting the water, protein, lipid, and ash
contents from the total weight. The sodium content
was determined by atomic absorption spectroscopy
(Atomic absorption photometer AA 2400 : Shimadzu
Corp., Kyoto, Japan).
3 . Fractionation of polysaccharides from the rice
bran samples
To investigate the changes due to fermentation of the rice bran ( sample A ) , the polysaccharide
components of the rice bran were fractionated10'·
11'
into non-starchy polysaccharides (fraction I ) , starch
polysaccharides ( fraction II ) , and water-soluble
polysaccharides (fraction III), respectively, as shown
in Fig. 1 and Fig. 2. The morphology, thermal
properties, branch chain-length distribution of
amylopectin, and sugar composition of the different
fractions were then analyzed as described in the
following sections.
test tube r Sample* 1
r n-Hexane 280m€ centrifugation at 3,000 rpm for 10 min
residue supernatant J 60 % ethanol 600 m€
mixed at 5 °C more than 4 hours I
separate with 508 nylon mesh (200 µm)
residue I substances A J added water
heating at 105 °C. 20 min with autoclave
I centrifugation at 3,000 rpm, 10 min
O.lM Sodium acetate buffer (pH 5.0) supernatant
Glucoamylase *2 10 mg (400 U)
Pronase *3 2 m€ (1,600 PU)
incubation at 40 °C, 24 hr I
centrifugation at 3,000 rpm for 10 min
I I residue (Fr. I) I supernatant
Fig. 1 Flow chart of the separation of Fr. I with fresh
and fermented rice bran
* 1 fresh rice bran 32 g, fermented rice bran 64 g * 2 Glucoamylase from Rhizopus sp. (Wako). 40 units/mg * 3 PronaseE from Streptmyces griseus (Merckbiosciences).
4,000,000 Proteolytic Units/g, dilution 1 mg I 5 ml! water
( 35 ) (Research NoteJ Fermented Rice Bran in 'Heshiko' 87
substances A I I
centrifugation at 3,000 rpm for 10 min.
residue supernatant ~ 85 % methanol 80 m£
extracted fat at 75 °C for 1hr. ~ 100 % ethanol
concentrated at 50 °C I
centrifugation at 3,000 rpm for 10 min. ~ residue supernatant
~ ;:~ase " 2 m£ (1,600PU) incubation at 40 °C, 3 hr.
I centrifugation at 3,000 rpm for 10 min.
I I residue (Fr. II) I supernatant
Fig. 2 Flow chart of the separation of Fr. II and III with fresh and fermented rice bran
* 1 fresh rice bran 32 g, fermented rice bran 64 g * 2 Glucoamylase from Rhizopus sp. (W ako), 40 units/mg * 3 PronaseE from Streptmyces griseus (Merckbiosciences),
4,000,000 Proteolytic Units/g, dilution 1 mg/ 5 ml! water
4 . Morphology of fractions I and Il from each
rice bran sample
The morphology of fraction I ( non-starchy
polysaccharides ) and fraction 11 ( starchy
polysaccharides) was observed using scanning
electron microscopy (SEM; model 5600, Jeol Ltd.,
Tokyo, Japan). The samples were mounted on brass
disks with double-sided adhesive tape, coated with
gold, and then viewed at 15 kV.
5 . Thermal properties of fractions I and Il
The thermal properties of fractions I and 11
from the fresh and fermented rice bran were
analyzed by 6,100 differential scanning calorimetry
(DSC) (Seiko Denki Kogyo K. K., Tokyo, Japan).
Each sample was loaded into a 70 -µ.R, silver
chamber of the instrument to obtain 2. 4 mg of
fraction I and 12.0 mg of fraction 11 on a dry basis
in water ( the total weight of the solution was
40 mg). After allowing the sample to equilibrate at
5 °C for 24 h, the mixture was heated from 30 °C
to 150 °C at a rate of 2 °C/min. Water (40 mg)
was used as a reference. The DSC system was
calibrated using indium, and the thermal properties
of the individual samples were calculated. The onset
(To), peak (Tp), and conclusion (Tc) temperatures
of gelatinization were determined, and the enthalpy
change (~H) was calculated using Muse Ver. 5.9
software (SII NanoTechnology Inc., Chiba, Japan).
Data are presented as the average of three
replicates per sample.
6 . Branch chain-length distribution of amylopectin
of fraction Il
The chain-length distributions of each native and
enzyme-digested
were analyzed
according to the
starch granules from fraction 11
using capillary electrophoresis
methods described by O'SHEA and
MORELL1" and FUJITA et al .13) in a P / ACE MDQ
carbohydrate system (Beckman Coulter Inc., Tokyo,
Japan). Each sample was analyzed in duplicate.
7 . Sugar composition of fraction III
The sugar composition of fraction III ( water
soluble polysaccharides) was analyzed using high
performance amon exchange chromatography
( HP AEC) with a DX- 500 system ( Dionex, CA,
USA) equipped with an ED-40 pulsed amperometric
detector. A Dionex CarboPac P Al column (250 mm x
94 mm ID) with a guard column (25 mm x 93 mm ID)
was used to evaluate the oligosaccharide of each
sample. The samples were diluted with water and
injected into a 25 -µ.R, sample loop. We used an
aqueous solution of 15 mM sodium hydroxide, and
the pulse potentials and durations followed those
described by AKUZAWA and KAWABATA14l. The
standard ( Cosmo Bio Co. Ltd. , GlyScope
Monosaccharide mixture-11 ) was measured under
the same conditions, and the peak was identified
from the retention time of the obtained
chromatogram.
Peaks that could not be identified with the
standard substance were determined by lH- and 13
C-nuclear magnetic resonance (NMR) spectroscopy
on a JNM-ECS 400 system (JEOL Ltd.) using 5-mm
TH/FG probes for measurement of nuclides. The lH
NMR and 13 C-NMR chemical shift patterns were
estimated at room temperature (22 °C) by referring
to the National Institute of Advanced Industrial
Science and Technology database.
8 . Viable cells count in rice bran used for
Heshiko and screening of amylase-producing
bacterial strains
Serial dilutions within the rage of 10-10-• CFU/g
were plated in duplicate on medium I and medium
11 , which were both composed of ( per liter of
distilled water) 8.0 g nutrient broth, 50 g NaCl, and
12 g agar, except that medium 11 also contained 5.0
g soluble starch. The plates were incubated at 30 °C
for 72 h, and then colonies were counted.
88 Food Preservation Science VOL. 45 NO. 2 2019 ( 36 )
For screening amylase-producing bacterial strains,
approximately 2 g of sample A and B were
suspended in 12 me of sterilized water, respectively.
The dilution solution was plated on starch azure
agar (10 g soluble starch, 5 g yeast extract, 5 g
peptone, 1.0 g K2HPO., 0.2 g MgSO.·7H2O, 17 g agar,
and 2.0 g starch azure per liter of distilled water)
or on starch azure agar containing 5% NaCL The
plates were incubated at 30°C for 48 h, and colonies
with a halo formed around them were determined
to be amylase-producing bacteria. These colonies
were then preserved at - 80 °C as glycerol stocks
until subsequent analysis for identification.
9 . Phylogenetic analysis of the amylase-producing
bacterial strains based on 16 S rRNA gene
sequences
Chromosomal DNA was prepared from the
isolates obtained from sample A using the method
described by ZHU et al .15), which was then used as
template DNA for amplification of the 16S rRNA
gene sequence according to a previously described
method16). The closest recognized relatives of the
obtained isolates were determined based on
searching public databases such as GenBank/EMBL/
DDBJ. Furthermore, the sequences of closely related
species were also obtained from the public database and were aligned using CLUSTALX (version 2.1) 17
)
for phylogenetic analysis. A distance matrix was
obtained by the two-parameter KIMURA method18).
The robustness of the individual branches of the
tree was established by bootstrapping with 1,000 replicates1
'). The phylogenetic tree was
reconstructed using the neighbor-joining method.
Results and Discussion
1 . General components and sodium content of
rice bran samples
Table 1 shows the general components and
sodium content of each rice bran sample. Overall, the results matched those reported previously3
J,4J.
Moreover, there was an approximately 5 % difference in lipid, ash, and carbohydrate contents
between samples A and B that were shipped at
different times. However, it was considered that this
difference was due to individual mackerel effects,
and not to the general component of the fermented
rice bran of Heshiko according to shipping time. The
moisture, lipid, and ash contents increased
significantly in the fermented rice bran compared to
those of the fresh rice bran. The increase in
Table 1 Moisture content and general composition of each sample
fresh rice bran sample A sample B
moisture content 10.4 ± 0.2 41.2 ± 0.8 41.6 ± 1.6
( % dry basis)
protein 16.6±0.1 15.0±0.3 16.1 ± 0.3
lipid 25.8±0.8 43.5±0.3 39.5±0.4
ash 10.8±0.1 19.1 ± 0.4 16.0± 0.1
carbohydrate 46.8 22.4 28.4
Na 0.034 5.6 3.5
moisture was considered to be due to the addition
of Shiejiru used for separating the liquid from the
fish body during the manufacturing process.
Similarly, the increase in lipid was considered to be
due to the lipids eluted in Shiejiru and their
subsequent migration from the mackerel fish body
during the fermentation period.
2 . Physicochemical properties of fractions I , Il ,
and m ( 1 ) Morphology of fractions I and Il of each
rice bran sample SEM photographs of fraction
I ( non-starch polysaccharides) and fraction II
(starch polysaccharides) obtained by fractionation
from fresh and fermented rice bran are shown in
Fig. 3. In the fraction I , a cell wall honeycomb
structure was observed, and no starch granules
could be confirmed. This indicated that only the cell
wall components had been fractionated, and this
breakage of the cell wall was remarkably affected
by the enzymatic action during fermentation. By
contrast. starch granules and non-starchy substances
such as cell wall components were mixed in fraction
II . In addition, holes and breakage were observed
on the surface of the fermented rice bran starch granules20
J.zi). These observations suggested that the
starch granules and cell walls in rice bran were
hydrolyzed by enzymatic action throughout the
fermentation period.
( 2 ) Thermal properties of fractions I and Il
The DSC thermograms of fraction II are shown
in Fig. 4, and the To, Tp, Tc, and H values of
starch are shown in Table 2 . The DSC
thermograms of fraction I (data not shown) were
similar to those of fraction II , showing two peaks
around 80 °C and 112 °C as Tp. Furthermore, the
DSC thermograms showed the same peak temperature in the fresh and fermented rice bran.
The thermal transition of starch polysaccharides
( 37 ) (Research NoteJ Fermented Rice Bran in 'Heshiko' 89
fresh rice bran
fermented rice bran
Fig. 3 Scanning Electron Micrographs (SEM) of Fr. I and Fr. II separated fresh and fermented rice bran
Fresh rice bran
i 5 ~ 0
q:1 .......
"' (!)
::r::
l l 0.lmW
40 50 60 70 80 90 100 Temperature ('t)
Fig. 4 DSC thermograms of starch polysaccharide (Fr. II ) of the fresh and fermented rice bran
(fraction II ) was observed in the range of 58- 83
~C and no peak was apparent at temperatures
above 90 °C for melting of the amylose-lipid
complex. The t-.H of fraction II was calculated to
be 13.6 mJ / mg and 15.5 mJ / mg in the fresh and
fermented rice bran, respectively. This suggested
that the undigested components remaining as part
of the crystalline structure of the starch granules
during the fermentation process needed to melt.
result ing in the larger endothermic enthalpy value22>_
( 3 ) Branch chain-length distribution of
amylopectin of fraction Il The branch chain-
length distribution pattern of amylopectin from
fraction II of the fres h rice bran and fermented
rice bran showed a peak chain length of DP 11 - 12
and 41-43 for the short- and long-branch chains,
respectively (Fig. 5) ; a similar distribution pattern
was found for the fermented rice bran ( data not
shown).
However. there was a decrease in the short
chains of DP 6- 12 and an increase in other chains
of DP 13- 20 in the fermented rice bran. Percentage
comparison of each fraction calculated from the
differences in chain-length distribution patterns
(t-.molar%) between the fresh and fermented rice
bran showed that the chain length of DP 6-12 of
the A chains decreased by around 1.4% and the
chain length of DP 13- 20 of the B, chains increased
by around 1.2% after fermentation"> . Furthermore,
the amount of intermediate-sized fragments
decreased ( 21 < DP < 36 in particular) . These
findings suggested that the short chain of
amylopectin was hydrolyzed by enzymes during the
fermentation period, so that the proportion of the
medium chain relatively increased.
( 4) Sugar composition of fraction ill by HPAEC
Four peaks appeared on the HP AEC
chromatogram of the water-soluble polysaccharides
(fraction ill ) from the fresh rice bran ( data not
shown ) . Two of these peaks were identified as
glucose and sucrose based on the standard, and the
other two peaks were identified as glycerol and
ethanol from 1 H-NMR and 13 C-NMR. Many more
peaks were obtained in the fermented r ice bran.
including those corresponding to arabinose. galactose,
glucose, and xylose, accounting for the starch,
cellulose in the cell wall, and constituent sugars of
the matrix polysacclrnridesw.,s> . These results
revealed that both the starch and non-starch
polysaccharides were decomposed during the
fermentation ripening period.
90 Food Preservation Science VOL.45 NO.2 2019 ( 38 )
Table 2 Characteristics of fresh and fermented rice bran by DSC
Peak 1 Peak 2
Samples To Tp To t.H To Tp To L'.H (°C) (°C) (°C) (mJ/mg) (°C) (°C) (°C) (mJ/mg)
58.8 67.0 73.6 13.6 78.6 77.9 82.3 2.8 Fresh rice bran
(5.2) (1.3) (0.7) (2.3) (0.7) (0.4) (1.6) (0.2)
58.0 67.4 72.8 15.5 72.8 77.4 82.8 4.9 Fermented rice bran
(0.8) (0.2) (0.4) (2.2) (0.4) (0.3) (0.9) (0.5)
1 ) From the DSC thermograms obtained, the onset temperature (To), the peak temperature ( Tp) . the conclusion temperatures (Tc), and the enthalpy of the gelatinization (6.H) were calculated.
2 ) Average of data were calculated more than five samples, and each standard deviation was showed in parentheses.
8
6
~ ~ 4
~
2
- ; fresh rice bran
D ; fermented rice bran
QI I I I I IUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUQOCGoo~o~ 5 10 15 20 25 30 35 40 45 50 55 60 (DP)
Fig. 5 Chain-length distribution pattern of amylopectin of starch polysaccharide (Fr. II ) in fresh rice bran and fermented rice bran
3 . Viable cell count in rice bran and screening of
amylase-producing bacterial strains
The number of viable cells grown on medium I and medium II was 2.0 x 104 CFU/g and 1.1 x
106 CFU/g, respectively; this substantial increase in
bacteria in the starch-containing medium suggested
that starch-assimilation bacteria were present in the
rice bran of Heshiko.
There was no significant difference in the number
of colonies of amylase-producing bacterial strains
growing on the starch azure plate and the starch
azure plate containing 5% NaCL Ultimately, five amylase-producing strains (A-1, A-3, B-1, B-2, and
B-3) were obtained from the two media.
4 . Phylogenetic analysis of the amylase-producing
bacterial strains based on 16 S rRNA gene
sequences
Approximately 1,500 nucleotides of the 16S rRNA
gene sequences of the five isolates were determined,
and 1,400 -nucleotide sequences were used to
construct a phylogenetic tree. The public database
search of the closest recognized relatives of the isolates showed high sequence similarities ( >99.3%)
to Bacillus amyloliquefaciens ( A 1 ) , Paenibacillus
amylolyticus ( A 3 ) , Bacillus subtilis ( B 1 ) , Bacillus
licheniformis (B2), and Oceanobacillus picturae (B3).
The phylogenetic tree of the isolates based on 16S
rRNA gene sequences is shown in Fig. 6.
Several strains in the genera Bacillus'6' and
Paenibacillus"' are known to have starch-assimilation
ability. Thus, the isolates found in the fermented
nee bran samples might produce a glycoside
hydrolase such as amylase. However, to our
knowledge, this lS the first report of a starch-
assimilating strain in the genus Oceanobacillus.
Several species of this genus have been isolated
from sea-related environments, showing halophilic and alkalophilic physiological characteristics. These
characteristics suggest that strain B- 3 is adapted to
Heshiko rice bran, which contains similar components to sea water, including starch, fish, and
salt. Furthermore, it was confirmed that each of the
( 39) (Research Note) Fermented Rice Bran in 'Heshiko' 91
,-------Bacillus indicus Sd/3 (AY904033)
0.01 998 1,000
1,000 998
Bacillus atrophaeus JCM 9070 (AB021181) 796 Bacillus amyloliquefaciens Al
Bacillus amyloliquefaciens BCRC1160F (EF433406) Bacillus subtilis Bl
999 Bacillus subtilis subsp. subtilis IAM 12118 (AB042061) Bacillus licheniformis DSM 13 (X68461)
1,000 Bacillus licheniformis B2 987 Bacillus aerius 24K (AJ831843)
Bacillus aerophilus 28K (AJ831844) Bacillus pumilus NCDO 1766 (X60637)
1,000 Oceanobacillus picturae B3 Oceanobacillus Picturae LMG 19429T (AJ315060)
----Oceanobacillus profundus CL-MP28T (DQ386635)
981 '-------Oceanobacillus caseni S-lF (AB275883) ----Oceanobacillus iheyensis HTE83F (AB010863)
Oceanobacillus oncorhynchi subsp. oncrohynchi R-V (AB188089) Oceanobacillus oncorhynchi subsp. incaldanensis 20AGT (AJ640134)
'-------Oceanobacillus locisalsi CHL-2F (EU817570) '---------Oceanobacillus chironomi T3944DT (DQ298074)
,---------Paenibaillus macquariensis DSM 2T (AB073193)
759 976 ----Paenibaillus illinoisensis JCM 9907T (AB073192) .------1 Paenibaillus pabuli JCM 9074T (AB073191)
Paenibaillus amy/o/yticus A3 1,000 Paenibaillus amylolyticus NRRL NRS-290T (D85396)
,---------Paenibaillus glucanoliticus DSM5162T (AB073189)
996 Paenibaillus po/ymyxa DSM36T (AJ320493) Paenibaillus peoriae DSM8320T (AB073186)
,-----------Paenibaillus kobensis DSM10249T (AB073363) '-------Paenibaillus thiaminolyticus DSM7262T (AJ320490)
,------------Paenibaillus apiarius NRRL NRS.1438T (D49247)
891 1,000
'----------------Paenibaillus alginolyticus NBRC 15375T (AB680848) '----------------------------Alicyclobacillus acidocaldarius DSM 446T (X60742)
Fig. 6 Phylogenetic relationship of the isolates and closely related species based on the 16S rRNA gene
The tree was constructed by the neighbour-joining method. Alicyclobacillus acidocaldarius DSM 446T was used as an outgroup. Bootstrap percentages above 70 % are given at branching points. Bar indicates 1 % sequence divergence.
isolated strains was grown in a medium containing
only rice bran as a carbon source. In addition, as a
result of observing these rice bran by SEM, their
rice starch and cell wall were decomposed ( data
not shown) . Therefore, we can conclude from these
fact these isolated strains are involved in the
change in character of rice bran.
Conclusion
Fresh rice bran was degraded by starch, and the
cell walls were degraded by enzymes produced by
microorganisms during the Heshiko fermentation
period. Moreover, our results suggest that the
addition of moisture and lipids transferred from the
fish body of mackerel contribute to turning the rice
bran into a paste state so that it can be eaten
easily, which is mainly due to the pasty texture
formed around the mackerel.
Moreover, bacterial strains producing amylase
were isolated from fermented rice bran, and were
estimated as Bacillus amyloliquefaciens ( A 1 ) ,
Paenibacillus amylolyticus .(A 3), Bacillus subtilis (B 1),
Bacillus licheniformis (B 2), and Oceanobacillus picturae
(B 3). I would like to report on the properties of
Oceanobacillus picturae (B 3) in the next report.
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マサバヘしこの「米糠」の性状変化と
アミラーゼ生産菌の検索
長岡純子*'.和田佳苗*'.加藤美樹*'.藤田直子*'
田中尚人*3 • 入澤友啓*4. 阿久澤さゆり*'
* 1 東京農業大学応用生物科学部
(〒156-8502 東京都世田谷区桜ヶ丘 1-1-1)
* 2 秋田県立大学生物資源科学部
(〒010-0195 秋田市下新城中野字街道端西241-438)
* 3 東京農業大学生命科学部
(〒156-8502 東京都世田谷区桜ヶ丘 1-1-1)
*4 東京農業大学農学部
(〒243-0034 神奈川県厚木市船子1737)
マサバヘしこの米糠について,生の米糠と性状を比較
した。その結果,水分,脂質,および灰分は発酵後の米
糠で増加していたが,これは製造工程でくわえられたし
え汁によるものであると考えられた。また,米糠の一般
成分は製造時期による違いはほとんどみられなかった。
米糠の性状は, SEMでは,非澱粉性多糖では細胞壁や
澱粉粒の表面に損傷が観察された。また,アミロペクチ
ンのDP6 ~12の鎖長は減少し, DP13~20の鎖長は増加
していた。非澱粉性多糖 (Fr.I)および澱粉性多糖 (Fr.
II)水可溶性区分 (Fr.皿)の分析より,発酵期間中の
酵素による分解が推察された。
さらに,発酵後の魚体周辺の米糠より,アミラーゼ生
産菌株を分離同定した結果, Bacillusamyloliquefaciens
(A 1) , Paenibacillus amylolyticus (A 3) , Bacillus subtilis
(B 1) , Bacillus licheniformis (B 2) , Oceanobacillus
picturae (B 3) と推定された。
(平成30年10月25日受付,平成30年11月298受理)