Fermentation of Germinated- and Nongerminated-Yellow Soybean Chungkookjang Using Bacillus subtilis

Bacillus subtilis 에 의한 발아 및 미발아 황태 청국장 발효

이나리¹, 고태훈¹, 박성보¹, 이상미¹, 황대연¹, 김동섭¹, 박근태², 손홍주¹

*

1부산대학교생명자원과학대학

2부산대학교산학협력단

Received _: November 28, 2012 / Revised _: January 17, 2013 / Accepted _: February 5, 2013

서 론

콩은동양전통식품의주단백질공급원으로써오랜기간

동안널리이용되어왔다

^[9].

생활을해온우리나라는단백질공급원으로써콩발효식품 을많이섭취하여왔다

^.

^(38%),

^(30%),

^(18%)

^,

^B

^E

, saponin, isoflavone, phytic acid

^,

질환이나골다공증예방효과등이속속밝혀지고있다

^{[5, 14].}

한편

^,

물질을포함한다양한영양원의변화가발생한다

^{. Yoo[30]}

고함량

isoflavone

^,

^{, Kim}

^[10]

24

기간에따른품질변화를확인한바있다

^.

청국장은콩을원료로한우리나라의대표적인발효식품 이다

^.

^2-3

^,

Bacillus spp.

의해특유의맛과향

^,

^[26].

은연구자들의의해청국장은항산화능

^[17],

^[3],

^[31],

^[15]

^,

^.

그러나이러한많은기능과연구가있음에도불구하고아 직까지청국장제조는관습적인방법을통해생산되고있고 있는실정이다

^.

^,

청국장을제조할수없다는한계를가지고있다

^[12].

은제조방법

^,

^,

능성의차이가있다

^[7].

Fermentation of Germinated- and Nongerminated-Yellow Soybean Chungkookjang Using Bacillus subtilis. Lee, Na- Ri

, Tae-Hun Go

, Sung-Bo Park

, Sang-Mee Lee

, Dae-Youn Hwang

, Dong-Seob Kim

, Geun-Tae Park

, and Hong- Joo Son

*.

College of Natural Resources and Life Science, Pusan National University, Miryang 627-706, Korea,

Research and University-Industry Cooperation, Pusan National University, Busan 609-735, Korea

In order to investigate changes in quality and enzyme activity during Chungkookjang fermentation, germinated- and nongermi- nated yellow soybeans were fermented by Bacillus subtilis and traditional methods. When the soybean was soaked for 6 h and then watered for 4 days with 2 h-interval at 25

C, the highest germination rate was obtained. The germinated soybeans had a higher total isoflavone (988.4 µg/g) than that of the nongerminated soybeans (859.5 µg/g). Amino type nitrogen contents, pro- tease and amylase activities were higher in germinated soybean Chungkookjang, which was fermented with B. subtilis, than nongerminated soybean Chungkookjang, which was fermented with B. subtilis and traditional methods. Reducing sugar and amino type nitrogen contents, the number of viable cells and protease and amylase activities, were higher for Chungkookjang fermented with B. subtilis, than Chungkookjang fermented by traditional methods. ALP and SOD activities in the Chungkook- jang diet group were considerably higher than in the control group. AST activity in the germinated soybean Chungkookjang diet group was higher than in the nongerminated soybean Chungkookjang diet group. In conclusion, it is suggested that Chungk- ookjang prepared with germinated soybeans using B. subtilis D7 could be practically used as a functional product.

Keywords: Chungkookjang, germinated soybean, fermentation, B. subtilis

*Corresponding author

Tel _: +82-55-350-5544, Fax _: +82-55-350-5549 E-mail _: [email protected]

© 2013, The Korean Society for Microbiology and Biotechnology

는제조법의표준화와품질균일화가선행되어야할것이다

^.

^,

^.

^.

재료 및 방법

청국장 발효균주 및 원료콩

본실험에서청국장발효를위하여사용된균주는

^Lee

[18]

^,

Bacillus subtilis D7

^,

⁽

^, Glycine max L. Merr)

^.

콩의 발아조건 검토

원료콩

^{150 g}

^1-7

후

^{, 20-30}

^C

^2-6

^.

(germination rate)

^.

Germination rate (%) = (No. of germinated soybean / No. of total soybean)

¹⁰⁰

콩의 isoflavone 함량

콩의

isoflavone (Sigma, USA)

HPLC (PerkinElmer, USA)

^[29].

1 g

0.1 N HCl 2 ml

acetonitrile 10 ml

30

C

³

, Whatman no. 4 filter

paper

^.

^80%

methanol 10 ml

^{, 0.45}

m membrane filter

여과하여

^HPLC

^.

럼은 µ

-Bondapak C18

(Agilent Technologies, USA)

, UV detector

^{254 nm}

^.

20% methanol 100

⁵⁰

^60%

methanol 100

^,

^{1 ml/}

^.

Isoflavone

표준정량곡선으로부터계산하였다

^.

청국장 제조

Luria-Bertani

³⁰

^{C, 200 rpm}

²⁰

B. subtilis D7

^starter

다

^.

¹²¹

^C

³⁰

^,

^1%(v/w)

⁴⁰

^C

⁴⁸

^.

발효변수 조사

청국장과증류수를

1 : 6 (w/v)

였다

^.

^,

^.

B. subtilis D7

(100

C, 20

)

^.

생균수는청국장

^{1 g}

^{9 ml}

한후

^,

^.

dinitrosalicylic acid (DNS; Sigma, USA) method [20]

^.

아미노태질소는

^Formol

^[1]

^,

^.

Amino nitrogen (mg%/g) = [(A

^B)

^1.4

^F

^{100] /} sample (g)

A : 0.1 N NaOH

^(ml) B : 0.1 N NaOH

^blank

^(ml) F : 0.1 N NaOH

^factor

암모니아태질소는

Indophenol blue method [1]

^{, NH}

Cl

Indophenol

^.

Protease

^[24]

^.

0.6% Hammerstern casein (Calbiochem, Germany)

sodium phosphate buffer (pH 7.2)

.

^{1 ml}

^{1 ml}

³⁰

^{C, 10}

, 0.4 M trichloroacetic acid (TCA) 1 ml

^.

^,

UV/VIS spectrophotometer

²⁸⁰ nm

^.

^{1 unit}

^0.01/ml/

^.

α

^-Amylase

^[25]

^0.5%

soluble starch

(0.4 M acetate buffer, pH 4.8) 2 ml

^{1 ml}

³⁰

^{C, 10}

^{. 0.4 M}

TCA 1 ml

^,

전물을제거하였다

^.

^{1 ml}

^{[0.005% I}

+ 0.05% KI]

용액

^{10 ml}

^{. 600 nm}

^UV/VIS

spectrophotometer (Pharmacia Biotech, England)

^.

^{1 unit}

^blank

^10%

^.

^-Amylase

^[21]

^-amylase

친반응중지액

¹⁰⁰

^l

^DNS

^maltose

^.

^maltose

^.

^{1 unit}

^{1 mg}

^maltose

^.

청국장의 동물 식이실험

본연구는부산대학교동물실험윤리위원회

(PNU-IACAU)

.

8

ICR

^,

^(Korea)

^{. ICR}

(Purina Mills Inc, Korea)

^, 12

(08:00~20:00)

specified pathogen-free

⁽

²³

²

^C,

⁵⁰

^5%)

^{. ICR}

⁴

⁵

^,

⁽

^), B. subtilis D7-

^, B. subtilis D7-

^,

^-

^.

^.

^{20 g}

^{200 ml}

(200 rpm, 20

⁾

^,

⁽⁵⁸⁰

^{g, 20}

⁾

^,

^.

적으로얻어진추출물을증류수에

^{70 mg/ml}

하여존데를통하여

¹

¹

^{. 7}

^CO

^,

^.

간세포내

superoxide dismutase (SOD)

microplate WST-1 assay

^[33].

^{100 mg}

sucrose buffer 600

^l

^homo- genizer

^.

³⁰

^, 4

C, 5000 rpm

¹⁰

¹⁰⁰

^l

^.

WST working solution, enzyme working solution, ddH

O, dilution buffer

³⁷

^C

20

, microplate reader (Molecular Devices, USA)

^{450 nm}

^.

청국장섭취가혈청내유전자의발현에미치는영향을최 소화하기위하여

²⁴

^CO

^,

^.

³⁰

^,

^,

(HITACHI 7080 Automatic Analyzer, Japan)

ALP (alkaline phosphatase)

^Tietze

[28], AST (asparatate aminotrans- ferase)

ALT (alanine aminotransferase)

^Reitman

Frankel

^[23]

^.

통계처리

실험결과는

^mean

^SD

^,

SPSS (Statistical Package Inc., USA) version 18

one-way ANOVA

(Turkey method)

⁽ p < 0.05).

결과 및 고찰

콩의 발아

콩을

^1-7

^2-6

^{, 6}

²

⁴

안물을공급했을때발아율이가장높았다

(Table 1).

건에서온도에따른발아율을검토한결과

^{, 25}

^C

^72%

²⁰

^C

²⁵

^C

^.

^20-25

^C

¹⁰

^C, 18

C, 25

C

³⁵

^C

^2%

, 95%, 75%

^68%

의발아율을나타내었고

^{, 25}

^C

¹⁸

^C

^Park

^[22]

^.

콩의 isoflavone 함량

콩의발아전후

isoflavone

^,

(859.5

^g/g)

^(988.4

^g/g)

isoflavone

(Table 2).

isoflavone

^,

isoflavone

^[11]

^.

isoflavone

[11]. Isoflavone

genistin, daiszin, glycitin

genistein, daidzein, glycitein

^.

당류는청국장발효과정중에원료콩의전분질이

Bacillus

등의미생물이생산한효소의작용으로분해되어생성되며

^,

^.

^glucose

Table 1. Germination condition of yellow soybean.

Optimal soaking time

Optimal water supply

Temperature (

C)

Germination rate (%) 3-6 h Once per 2 h

for 4 days

20 25 30

69 ± 2

72 ± 3

60 ± 3

An asterisk (*) denotes statistical difference at p < 0.05.

환되는양을조사하기위하여발효경과에따른환원당의양 을측정하였고

^,

^{Fig. 1}

^. B.

subtilis D7-

³⁶

^{16.5 mg/g}

^, B. subtilis D7-

²⁴

^{16.4 mg/g}

^.

^-

³⁶

^{14.5 mg/g}

^.

^, B. subtilis D7

^,

^,

^.

²⁴

유는미생물의생육에따른

^amylase

보이며

^[21],

^-amylase

⁽

⁾

^{. Kwon}

^[13]

B. pumilus JB-1

^{8.5 mg/g}

^{16.5 mg/}

^.

청국장이가지는구수한맛의척도인아미노태질소는청 국장발효숙성중단백질이분해되어생성되는물질로서

^,

^.

에는청국장의아미노태질소의함량을

280 mg%/g

로규정하고있다

^{. Fig. 2}

에서발효시간경과에대체로비례하여아미노태질소의함

량이증가하였고

^,

²⁴

^.

⁴⁸

B. subtilis D7-

^, B. subtilis D7-

^,

^-

345 mg%/g, 238 mg%/g, 208 mg%/g

^.

^.

B. subtilis D7-

내었으나

B. subtilis D7-

값을나타내었다

^. B. subtilis D7-

^-

^,

238 mg%/g, 208 mg%/g

^{280 mg%}

^{. Eom}

^[4]

⁴⁸

^,

³⁶

^.

^.

^.

암모니아태질소는단백질이탈아민됨으로써생성되는데

^,

^.

^,

^{Fig. 3}

^.

^,

¹⁸

Fig. 1. Variation of reducing sugar in Chungkookjang during

fermentation.

●, germinated soybean; ○ non-germinated soybean; ▲ rice straw soybean.

Fig. 2. Variation of Amino type nitrogen in Chungkookjang during fermentation.

●, germinated soybean; ○ non-germinated soybean; ▲ rice straw soybean.

Table 2. Isoflavone contents of non-germinated and germinated soybean.

Non-germinated soybean (µg/g) Germinated soybean (µg/g)

Daidzin Glacitin Genistin Total Daidzin Glacitin Genistin Total

409.7 97.8 352.0 859.5 465.9 128.0 394.5 988.4

30

^{68 mg%/g}

^{287 mg%/g}

하였다가발효

⁴²

^{321 mg%/g}

었다

^.

⁴⁸

^{221 mg%/g}

^.

^.

, 5-124.1 mg%/g

^,

B.

subtilis

^.

함량만고려할때

^,

^24-36

는것이불쾌취가없는청국장제조에유리할것으로판단 된다

^.

^.

는부분에대해서연구할필요가있다고사료된다

^{. Youn}

[32]

B. subtilis

B. natto

⁴⁵

422 mg%, 356 mg%

^{, Eom}

^[4]

^,

⁴⁸

간에

^{162 mg%}

사하였다

^.

pH

^{Fig. 4}

성영역을나타내다가발효시간경과에따라알칼리영역으 로전환되었다

^.

^pH

^6.7-7.9

^{, pH}

^.

^pH

^Jeong

^[6]

^{pH 6.3}

⁷²

^{pH 8.2}

^{, Sung}

^[27]

^pH

^6.6-8.0

^pH

^{. Youn}

^[32]

^{pH 6.13}

^{pH 6.21}

의증자한대두에

Bacillus

⁴⁰

^C

⁴⁵

^{, pH}

^8.13

^8.68

^.

^pH

^.

청국장에서의생균수를측정한결과는

^{Fig. 5}

^. B. subtilis D7-

1.5 log CFU/g

9.8 log CFU/g

^. B. subtilis D7-

1.5-9.7 log CFU/g

^.

^.

^-

, 1.0-6.6 log CFU/g

^.

^, B. subtilis D7

Fig. 4. Variation of pH in Chungkookjang during fermentation.

●, germinated soybean; ○ non-germinated soybean; ▲ rice straw soybean.

Fig. 5. Variation of viable cell number in Chungkookjang dur- ing fermentation.

●, germinated soybean; ○ non-germinated soybean; ▲ rice straw soybean.

Fig. 3. Variation of Ammonia type nitrogen in Chungkookjang during fermentation.

●, germinated soybean; ○ non-germinated soybean; ▲ rice

straw soybean.

었다

^{. Cho}

^[2]

B. pumilus

3.0 log CFU/g

11.7 log CFU/g

^{, Jeong}

^[6]

²⁴

7 log CFU/g

9 log CFU/g

결과와유사하였다

^.

콩단백질을 가수분해하여구수한맛성분인 아미노산

^, polypeptide

^protease

결정짓는중요한인자중의하나이다

^{. Fig. 6}

같이청국장의

^protease

적으로증가하였다

^. B. subtilis D7-

^,

^42- 48

975-989 U/g

^. B. subtilis D7-

22-899 U/g

^,

^-

40-834 U/g

^.

^protease

⁴⁸

^Oh

^[21]

^Youn

^[32]

^.

청국장발효는단백질뿐만아니라전분의분해속도도중 요한인자인것으로알려져있다

^.

^-amylase

^{Fig. 7A}

B. subtilis D7-

^,

⁶

^{48 U/g}

¹⁸

^{64 U/g}

^. B. subtilis D7-

^,

⁶

^{24 U/g}

¹⁸

^{49 U/g}

^,

^.

^,

^.

^-

^{16-44 U/g}

^, B. subtilis D7

^{. Jeong}

^[6]

²⁴

α

^-amylase

과와유사하였다

^.

^Oh

^[21]

B. natto

^-amylase

^10-12 U/g

^-amylase

^(44- 64 U/g)

^{. Fig. 7B}

같이실험한모든청국장의β

^-amylase

됨에 따라증가하였다가감소하는패턴을보여주었다

^. B.

subtilis D7-

2.3-3.9 U/g, B. subtilis D7-

1.3-5.0 U/g

^.

^-

1.1-3.2 U/g

^-amylase

B. subtilis D7

^,

^.

^amylase

^,

^,

^-amylase

^{. Oh}

^[21]

¹²

²⁴

^,

^,

^-

β

^-amylase

여본실험결과와유사하였다

^.

청국장의 SOD 및 각종 효소 활성에 대한 효과

Superoxide dismutase (SOD)

Fig. 7. Variation of amylase activity in Chungkookjang during fermentation.

●, germinated soybean; ○ non-germinated soybean; ▲ rice straw soybean. (A) α-amylase, (B) β-amylase.

Fig. 6. Variation of protease activity in Chungkookjang during fermentation.

●, germinated soybean; ○ non-germinated soybean; ▲ rice

straw soybean.