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 : akuzawa@nodai.ac.jp

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）