Inhibition of Bacillus cereus in Doenjang Fermented with Multiple Starters Showing Inhibitory Activity against Pathogens

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유해균 억제능을 지닌 복합종균을 접종하여 제조한 된장에서 Bacillus cereus 억제

이재용¹, 심재민¹, 이강욱¹, 조계만², 김경민³, 신정혜³, 김정상⁴, 김정환^1,5*

1경상대학교대학원응용생명과학부(BK21 Plus)

2경남과학기술대학교식품과학부

3남해마늘연구소

4경북대학교식품공학부(BK21 Plus)

5경상대학교농업생명과학연구원

Received: June 14, 2016 / Revised: July 15, 2016 / Accepted: July 15, 2016

서 론

된장은각종식품들조리에조미를위해널리사용되는전

통대두발효식품으로여러식품들의풍미증진과함께아미

노산급원으로중요하게활용되어왔다^[3].또연구들을통

해된장은항산화^,항비만^,혈전용해능^,항암^,항고혈압등의 다양한기능성들을지닌것이 알려졌다[2, 5, 7, 8]. 하지만 최근된장을비롯한간장^,고추장등전통장류의소비는정 체기내지는쇠퇴기에접어들어서소비촉진을위한다양한 방안마련이요구되고있다^.청소년과외국인기호에맞는 다양한장류제품들개발과함께기능성이향상된고품질제 Inhibition of Bacillus cereus in Doenjang Fermented with Multiple Starters Showing Inhibitory Activity against Pathogens Jae Yong Lee¹, Jae Min Shim¹, Kang Wook Lee¹, Kye Man Cho², Gyoung Min Kim³, Jung-Hye Shin³, Jong-Sang Kim⁴, and

Jeong Hwan Kim^1,5*

1Division of Applied Life Science (BK21 Plus), Graduate School, Gyeongsang National University, Jinju 52828, Republic of Korea

2Department of Food Science, Gyeongnam National University of Science and Technology, Jinju 52725, Republic of Korea

3Namhae Garlic Research Institute, Namhae 52430, Republic of Korea

4School of Food Science and Biotechnology (BK21 Plus), Kyungpook National University, Daegu 41566, Republic of Korea

5Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea

Doenjang samples were prepared by inoculation of multiple starters consisting of two Bacillus spp., one yeast, and one fungus. Doenjang A was fermented with Bacillus amyloliquefaciens EMD17, B. amyloliquefaciens MJ1-4, Pichia farinosa SY80, and Rhizopus oryzae. Doenjang B and C were fermented with the same yeast and fungus but different Bacillus strains; namely, B. amyloliquefaciens EMD17 and B. subtilis CH3-5 for doenjang B, and B. amyloliquefaciens MJ1-4 and B. subtilis CH3-5 for doenjang C. Doenjang D was fermented with microorganisms present in rice straw (control). The doenjang samples were spiked with B.

cereus ATCC14579 at two different levels, 10⁴ CFU/g doenjang (I) and 10⁷ CFU/g doenjang (II). All eight doenjang samples were fermented for 70 days at 25°C. Growth of B. cereus was inhibited in doenjang A, B, and C, with the bacterial cell count after 70 days being less than the initial 10⁴ CFU/g added, whereas B.

cereus was not inhibited in doenjang D. Doenjang B showed the strongest inhibitory activity against B.

cereus, with a cell count of less than 10³ CFU/g after 42 days, even when B. cereus was initially added at 10⁷ CFU/g. Some properties of the doenjang samples, such as pH, TA, and amino-type nitrogen content, were similar to those of doenjang fermented with starters only. The results indicate that carefully selected starters can effectively prevent the growth of B. cereus during doenjang fermentation.

Keywords: Doenjang, multiple starters, microbial safety, Bacillus cereus

*Corresponding author

Tel: +82-55-772-1904, Fax: +82-55-772-1909 E-mail: [email protected]

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품개발이요구되며동시에장류의안전성제고를위한제 조방법개선도요구된다^.전통방식으로제조되는장류의경 우제조및발효과정에서주위환경에존재하는독소생성미 생물들오염가능성이지속적으로제기되어서안전성확보 는장류소비촉진과세계화를위해반드시해결해야할과 제이다^[18].장류의 대표적인유해균들로는^aflatoxin이나

ochratoxin 등진균독소를생성하는곰팡이들과설사형혹

은구토형독소를생성하는 Bacillus cereus를들수있다^[1,

12]. B. cereus의경우식품공전에는메주를제외한장류에

서¹⁰⁴^CFU/g이하로만검출되어야한다^[10].하지만실제 오염되는숫자는알려져있지않고위생적으로는허용치보 다적을수록바람직하다된장에서유해곰팡이나^{B. cereus} 증식을억제하는효과적방법중하나는이들을억제하는능

력을지닌^bacilli균주들을선발하여된장제조시종균으로

사용하는것이다^.유해균증식억제능력외에혈전용해능과 같은기능성을지닌균주들을종균으로함께사용한다면장

류안전성확보와함께기능성개선도기대할수있다^{[4, 6].}

저자들은이전연구에서복합종균을사용하여된장을제조 하고²⁰℃에서¹⁰주간숙성하면서품질변화를측정한결과

된장의안전성과기능성개선을보고한바있다^[5].본실험

에서는 Bacillus 2 균주와효모와곰팡이각각¹균주씩총 4종균주들로구성된복합종균을사용하여된장을제조하고 여기에^{B. cereus}를된장시료^{1 g}당각각¹⁰⁴과¹⁰⁷^CFU (colony forming unit)으로오염시켰다^.된장들을¹⁰주간²⁵℃ 에서숙성시키면서 B. cereus 균수를측정한결과종균에의 해효과적으로 B. cereus 증식이저해되었기에그결과를보 고한다^.

재료 및 방법

콩알메주를 이용한 된장 제조

콩⁽²⁰¹³년산^,함양농협구매^{) 300 g}을¹⁸시간²⁰℃에서 수침한 다음¹²¹℃에서¹⁵분간증자한 후식힌콩^{100 g} 씩에각각 Baillus 2 균주^,곰팡이^,효모를접종하여발효 시켰다. Bacillus는 B. amyloliquefaciens EMD17, B.

amyloliquefaciens MJ1-4, B. subtilis CH3-5 3 균주들중에 서²균주씩조합을달리하여콩^{100 g}에^{1% (v/w,}각균주 0.5%) 접종(5 log CFU/g) 한후³⁷℃^{, 45}시간발효시켰다^.곰 팡이는 Rhizopus oryzae를^{1% (v/w)}접종하여(6 log spores/g) 25℃에서⁴⁸시간두어포자증식을유도한후⁴⁵시간더발 효시켰다^.효모는 Pichia farinosa SY80 균주를^{1% (v/w)}접 종하여(5 log CFU/g) 30℃에서⁴⁵시간 발효시켰다^{. B.}

amyloliquefaciens EMD 17은 청국장에서 분리한 균주로 lipopeptides들을생산하여^{B. cereus}와 Listeria monocytogenes

증식을억제하고또 ochratoxin을생성하는 Penicillium 속 곰팡이와여러식물들에서병을유발하는병원성곰팡이들 4종을억제한다[16]. B. amyloliquefaciens MJ1-4는메주에 서분리된균으로 aflatoxin B1 (AFB1) 생산균인 Aspergillus와 ochratoxin 생성 Penicillium 증식을 억제한다^{[13]. B.}

subtilis CH3-5는청국장에서분리한균주로혈전용해능이

우수한균주이다[9]. R. oryzae와 P. farinosa SY80들은간장 에서분리되었고각각단백분해능과발효시향미증진특성 이우수하다^[6].

발효후콩들을섞은후⁵⁵℃에서⁴⁸시간건조시켜서콩 알메주를 얻었다^.콩알메주에 증자 후 냉각한 콩을 동량 (300 g) 첨가하고물과소금⁽정제염^,한주소금^,울산^,한국⁾을 첨가하고섞어서최종염도^12%인된장들을제조하였다^.콩 알메주제조와된장제조의상세한과정들은앞서보고한바 와같다^[5].된장시료들을정리하면다음과같다^.된장^{A (B.}

amyloliquefaciens EMD17, B. amyloliquefaciens MJ1-4, R. oryzae, P. farinosa SY80), 된장 B (B. amyloliquefaciens EMD17, B. subtilis CH3-5, R. oryzae, P. farinosa SY80), 된장 C (B. amyloliquefaciens MJ1-4, B. subtilis CH3-5, R. oryzae, P. farinosa SY80), 된장^{D (}볏짚을균원시료로 접종^).된장^D는콩알메주제조시증자한콩^{300 g}을잘게 썰은볏짚으로둘러싼후발효시켜얻은^,즉볏짚에존재하 는미생물들을접종하여얻은것이다^.된장을제조할때^LB 배지에서미리배양한 Bacillus cereus ATCC14579를^{4 log} CFU/g과 7 log CFU/g로첨가량을각각달리하여된장시료 들에첨가하여^,인위적으로오염시킨된장들을얻었다^{. B.}

cereus를 4 log CFU/g 접종한 된장들을 A-I, B-I, C-I, D-I 으로 7 log CFU/g 접종한된장들은 A-II, B-II, C-II, D-II로 명명하였다^.총⁸종류의된장시료들을²⁵℃에서⁷⁰일간숙 성하면서⁷일간격으로 B. cereus 생균수변화와품질변화 를조사하였다^.

된장 발효중 생균수 변화

된장시료^{10 g}을취해서 0.1% peptone 수^{90 ml}와섞은 후 stomacher (stomacher^®80, Seward, USA)를 사용하여 균질화하고단계희석하였다^.희석액^{0.1 ml}를^LB한천배지 에도말하여³⁷℃^{, 24}시간배양하여얻은균수에희석배율 을 곱하여^bacilli생균수를 구하였다. B. cereus는^MYP (mannitol-egg york-polymixin B) 선택배지를이용하여계수 하였다^{[14]. MYP}평판배지에도말하여³⁷℃에서배양후혼 탁한환을보이는분홍색집락을계수하고여기에희석배수 를곱하여 B. cereus 균수를측정하였다^.효모균수는희석액 0.1 ml씩을^YM한천배지(chloramphenicol, 10µ^g/ml)에도말 하여³⁰℃^{, 48}시간배양한후동일한방법으로계수하였다^.

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발효 중 된장시료들의 특성 변화

된장 시료의^pH는된장 시료 균질액을 pH meter (DP-

215M; DMS, Korea)를 사용하여 조사하였고 적정산도

(Titratable acidity)는시료액을^{pH 8.3}이될때까지^{0.1 N} NaOH로적정하고사용된^NaOH부피^(ml)로부터계산하여 이를 젖산^(%)으로 나타내었다^. 된장의 아미노태질소는 Formol 법을이용하여측정하였다^[11].

결과 및 고찰

된장 숙성중 생균수 변화

B. cereus 균주를오염시킨된장시료들을⁷⁰일동안숙성 하면서매주시료를취하여^Bacillus와효모생균수변화를 측정하였다^{(Fig. 1).}된장제조직후 Bacillus 총균수⁽종균⁺

B. cereus)는^8.35−8.66 log CFU/g이고숙성중서서히증가 하여³⁵−⁴⁹일사이^9.12−9.25 log CFU/g로최고점에도달하 고숙성종료시에는^8.82−9.07 log CFU/g을나타내었다^.동일 한 종균 조합들과 동일한 방법을 사용하여 제조하되^B.

cereus 오염은없는된장시료들발효중총^bacilli균수는숙 성¹⁴일이후큰변화없이⁹⁰일까지⁸−9 log CFU/g (결과미 제시⁾범위여서 B. cereus 오염이일어난본실험의총^bacilli 균수와차이가없었다^.

효모는된장제조직후^4.74−4.94 log CFU/g에서숙성중 서서히증가하여³⁵−⁵⁶일에^5.89−6.33 6 log CFU/g까지증 가하고 이후 완만히 감소하여숙성 종료시^5,61−^{6.28 log} CFU/g 범위였다^.된장^D-II의효모 수가⁵⁶일에^{6.33 log} CFU/g으로시료들중가장많고종료시에도 6.28 log CFU/g으 로타된장들보다많다(Fig. 1D). B. cereus를¹⁰⁴^CFU/g오

Fig. 1. Changes in the numbers of total bacilli (A, B) and yeasts (C, D) of doenjang samples spiked with B. cereus during fermen- tation. (A, C) B. cereus 10⁴ CFU/g; (B, D) B. cereus 10⁷ CFU/g. ●, doenjang A (B. amyloliquefaciens EMD17, B. amyloliquefaciens MJ1-4, R. oryzae, P. farinosa SY80); ○, doenjang B (B. amyloliquefaciens EMD17, B. subtilis CH3-5, R. oryzae, P. farinosa SY80); ▼, doenjang C (B. amyloliquefaciens MJ1-4, B. subtilis CH3-5, R. oryzae, P. farinosa SY80); △, doenjang D (rice straw was used as the source for micro- organisms).

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염시킨된장^A-I의숙성종료시효모수가 6.19 log CFU/g 으 로다음으로많았다^.반면된장^B-I과^B-II의효모수는숙성 종료시타된장들보다적었다^.

B. cereus를각각¹⁰⁴과¹⁰⁷^CFU/g으로오염시킨된장들 간에는총^bacilli와효모수는차이를보이지않았다^. B. cereus 생균수 변화

된장 A-I, B-I, C-I 제조직후 B. cereus 균수는접종량과

비슷한^4.08−4.58 log CFU/g이였다(Fig. 2A). 된장^D-I이 5.25 log CFU/g으로다른된장들보다많았다^.이는된장^D 는볏짚을균원시료로사용하였기에볏짚에존재하던^B.

cereus로인해타된장들보다많은것으로추정된다^{. A-II, B-} II, C-II 된장의 제조직후 B. cereus 균수는^7.79−^{7.86 log} CFU/g이지만^D-II는 8.26 log CFU/g으로많았다^{(Fig. 2B).}

B. cereus 균수는숙성이진행됨에따라된장종류에따라큰

차이를보였다^.된장^D-I은제조직후 5.25 log CFU/g에서 숙성중처음³⁵일은조금씩감소하다가이후서서히증가 하여숙성종료싯점에서는 5.22 log CFU/g으로제조직후 와비슷하였다^.된장^C-I은 B. cereus 균수가제조직후^4.58 log CFU/g에서숙성²¹일까지증가하여²¹일에최대치인 5.89 log CFU/g에달하고이후감소하여종료시에는^{3.43 log} CFU/g이였다^.된장^A-I의 B. cereus 균수는숙성중지속적 으로감소하여종료시 2.74 log CFU/g까지감소하였다^{. B.}

cereus 균수 감소가 가장 큰 된장은 B-I으로 제조 직후

4.56 log CFU/g에서숙성중균수가크게감소하여⁴²일에 0.85 log CFU/g까지감소하였다^{. 42}일이후서서히증가하 여숙성종료시에는 1.36 log CFU/g을나타내었다^.

B. cereus를 7 log CFU/g으로오염시킨된장들(A-II, B-II, C-II, D-II)의숙성중 B. cereus 균수는 4 log CFU/g으로오 염시킨된장들보다많게유지되었다^.된장^D-II는제조직후 8.26 log CFU/g에서숙성³⁵일에 5.07 log CFU/g까지감소 하고이후서서히증가하여숙성종료시에는 5.61 log CFU/g 으로된장시료들중가장많았다^.된장^C-II는제조직후^7.86 log CFU/g에서숙성²¹일까지비슷한숫자를유지하다가이 후감소하기시작하여숙성⁴²일에 4.89 log CFU/g 그리고 숙성종료시에는 4.47 log CFU/g이였다^.된장^A-II는제조 직후 7.79 log CFU/g이고숙성⁷일에 5.09 log CFU/g으로 급격히감소한후숙성⁴⁹일까지비슷한수준으로유지되다 가^,이후조금더감소하여숙성종료시 4.34 log CFU/g를 나타내었다^.된장^B-II는 7.85 log CFU/g로시작하여숙성 7일에 5.18 log CFU/g로급격히감소하고숙성³⁵일까지비 슷한숫자를유지하였다^.숙성⁴²일에 2.88 log CFU/g로감 소하며숙성종료시에는 2.56 log CFU/g을보였다^.된장^B- II의경우 B. cereus를 4 log CFU/g 보다^1,000배더오염시 켰지만숙성중 B. cereus 억제효과는여전히뛰어남을알수 있었다^.

B. cereus를 4 log CFU/g으로오염시킨된장들에서대조 구를제외한모든된장에서숙성기간대부분과숙성종료시

B. cereus 균수가식약처허용기준보다더적은것을확인하

였다^.된장^B1이가장효과적이고다음이^A-I이고^C-I은발 효⁴²일이후로기준을충족시켰다. B. cereus를 7 log CFU/g 수준으로오염시킨된장들의경우된장^B-II는숙성⁴²일이 후에는 B. cereus 균수가허용기준이하로감소되었다^.허용 Fig. 2. Changes in the number of B. cereus of doenjang samples

spiked with B. cereus during fermentation. (A) B. cereus 10⁴ CFU/g; (B) B. cereus 10⁷ CFU/g. ●, doenjang A (B. amyloliquefa- ciens EMD17, B. amyloliquefaciens MJ1-4, R. oryzae, P. farinosa SY80); ○, doenjang B (B. amyloliquefaciens EMD17, B. subtilis CH3- 5, R. oryzae, P. farinosa SY80); ▼, doenjang C (B. amyloliquefaciens MJ1-4, B. subtilis CH3-5, R. oryzae, P. farinosa SY80); △, doenjang D (rice straw was used as the source for microorganisms).

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기준이상이긴하지만^A-II그리고^C-II순서로저해효과를 보였다^.이상결과들에서 Bacillus 3 균주들중 B. cereus 저 해능이가장 큰균주는 B. amyloliquefaciens EMD17임을 알수있었다. B. amyloliquefaciens EMD17을단독으로혹 은타 Bacillus 균주와함께종균으로사용하여된장을제조 한다면 B. cereus 오염억제에효과적일것으로사료된다^.전 숙성기간을통하여곰팡이증식은관찰되지않았고이는된 장숙성기간이비교적짧아서곰팡이에비해생육속도가빠 른^bacilli와효모들로인해접종한 Rhizopus oryzae 생육이 억제된때문이라추정된다^.마찬가지로오염을일으키는다 른곰팡이들도된장시료들에서생육이억제돤것으로보인다^. pH 및 적정산도(titratable acidity) 변화

숙성중된장들의^pH와적정산도변화를측정하였다^(Fig.

3). 된장들(A-I, B-I, C-I, D-I)은제조직후 pH 6.62-6.77를

나타내었다(Fig. 3A). 된장 D-I (6.62)가가장낮고 C-I (6.77) 이가장높았다^.이후숙성⁷일까지^{pH 6.42}−^6.58로조금감 소하고⁷일에서¹⁴일사이^5.27−^5.38로급격히감소하였다^. 14일이후에는서서히감소하거나비슷한수준을유지하여 종료시^{pH 5.11}−^5.20을나타내었다^.된장 A-II, B-II, C-II, D-II들의^pH또한제조직후^{pH 6.47}−^6.78에서숙성¹⁴일 에^5.16−^5.45까지급격히감소하고^,이후로는비슷하거나조 금감소하여숙성종료시^{pH 4.99}−^5.15을나타내었다(Fig.

3B). B. cereus를인위적으로오염시킨된장들의전체적인

pH 변화는숙성중^pH가감소하다가일정한수준으로유지 되었으며된장 A-I, B-I, C-I, D-I와된장 A-II, B-II, C-II, D-

II가이런경향에서크게다르지않았고^,일반적인된장숙

성중^pH변화와도다르지않았다. B. cereus 오염없이제조 한된장들의제조직후^pH는^6.1−^6.3이고숙성⁷⁰일에^pH 5.7−^6.0이며볏짚을접종한된장의^pH가가장낮았다⁽결과

Fig. 3. Changes in pH (A, B) and TA (C, D) of doenjang samples spiked with B. cereus during fermentation. (A, C) B. cereus 10⁴ CFU/

g; (B, D) B. cereus 10⁷ CFU/g. ●, doenjang A (B. amyloliquefaciens EMD17, B. amyloliquefaciens MJ1-4, R. oryzae, P. farinosa SY80); ○, doen- jang B (B. amyloliquefaciens EMD17, B. subtilis CH3-5, R. oryzae, P. farinosa SY80); ▼, doenjang C (B. amyloliquefaciens MJ1-4, B. subtilis CH3-5, R. oryzae, P. farinosa SY80); △, doenjang D (rice straw was used as the source for microorganisms).

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미제시). B. cereus 오염된장들의^pH는미오염된장들보다 초기^pH값은높지만숙성중더많이감소하였다^.이는^B.

cereus가생육하면서생성하는유기산들때문이라추정된다^.

비슷한결과가종균접종된장들과볏짚접종된장들을²⁰℃ 에서⁷⁰일간숙성하면서^pH를측정한실험에서보고된바 있다^[5].

숙성중적정산도변화를보면된장 A-I, B-I, C-I, D-I은 제조직후^0.33−^0.41%에서숙성⁷일에^0.41−^0.49%로증가 하며¹⁴일에^1.13−^1.19%로급격히증가하였다(Fig. 3C). 14일 이후서서히증가하거나비슷한수준을유지하며숙성종료 시^1.20−^1.27%였다^. 된장^B-I이 가장 높고(1.38%) D-I (1.27%), A-I (1.23%) 그리고 C-I (1.20%) 순이였다^.된장^A- II, B-II, C-II, D-II들은제조직후^0.36−^0.50%에서숙성⁷일 에^0.45−^0.57%로증가하고¹⁴일에^0.97−^1.09%로급격히증 가하였다(Fig. 3D). 14일이후조금씩증가하거나비슷한수 준을유지하며종료시 1.13−1.28%를보였다. D-II가가장높 았고(1.28%) C-II (1.25%), A-II (1.19%), B-II (1.13%) 순서 였다^.된장의적정산도증가는^pH강하와대체로병행하여 일어나나특정된장에서^pH감소정도는적정산도증가정 도와정확히비례하지는않는다^.볏짚접종된장의^pH와적 정산도값들은종균접종된장들과비교할때각각유의적

으로낮고또높았다^[5].하지만본실험과동일한종균들과

방법들을사용하되^{B. cereus}는오염시키지않은된장들의

경우볏짚접종된장의^pH는종균접종된장들보다숙성기 간내내유의적으로낮았지만적정산도역시유의적으로타 된장들보다낮았다^[결과미제시^].볏짚을균원으로사용하 더라도볏짚종류에따라미생물균총에차이가있고이로 인해된장의균총이달라지고또산생성도달라지기때문 이라추정된다^.이런결과들은일정한품질을지닌된장을 얻기위해서는선발된종균을접종할필요성을획인시켜주 는것이라하겠다^.

아미노태 질소 함량 변화

B. cereus가오염된된장들의숙성중아미노태질소함량

을측정하였다^{(Fig. 4).}된장 A-I, B-I, C-I, D-I은제조직후 145.83 ±^7.89−^223.31±^{7.89 mg%}로나타났으며^,된장^B-I (223.31 ±^{7.89 mg%)}이가장높고된장 D-I (145.83 ±^7.89 mg%)이가장낮았다(Fig. 4A). 숙성중아미노태질소함량 은증가하였는데숙성¹⁴일까지^244.66±^13.59−^296.30±

8.02 mg% 빠르게증가하다가그이후로서서히증가하였다^.

숙성종료시된장 A-I (369.04 ±^{6.44 mg%)}이가장높고된 장 D-I (340.61 ±^{9.24 mg%)}이가장낮았다^.된장^{A-II, B-} II, C-II, D-II의경우 제조 직후^149.33±^8.08−^195.96± 7.89 mg%로된장^B-II가가장높고된장^D-II가가장낮았 으며숙성이진행됨에따라서서히증가하였다(Fig. 4B). 특

히된장^D-II는제조직후가장낮았지만숙성중증가하여

14일에^457.33±^{8.08 mg%}로가장높고숙성종료시^448.06± 6.20 mg%로된장들중가장높았다^.다음이 A-II (431.57 ± 16.56 mg%), A-I (369.04 ± 6.44 mg%) 된장들이였다^.이는 이전연구에서보고한종균접종된장들의²⁰℃^{, 70}일숙성 후아미노태질소함량들^(513.3±^8.1−^707.8±^{6.9 mg%)}보 다는낮지만본실험과동일한종균과방법들을사용하되^B.

cereus는오염시키지않은된장들과는비슷하였다^[5].단백

Fig. 4. Changes in the amino-type nitrogen of doenjang sam- ples spiked with B. cereus during fermentation. (A) B. cereus 10⁴ CFU/g; (B) B. cereus 10⁷ CFU/g. ●, doenjang A (B. amylolique- faciens EMD17, B. amyloliquefaciens MJ1-4, R. oryzae, P. farinosa SY80); ○, doenjang B (B. amyloliquefaciens EMD17, B. subtilis CH3-5, R. oryzae, P. farinosa SY80); ▼, doenjang C (B. amyloliquefa- ciens MJ1-4, B. subtilis CH3-5, R. oryzae, P. farinosa SY80); △, doen- jang D (rice straw was used as the source for microorganisms).

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분해활성이우수한 B. subtilis DJ1을접종한된장을²⁰℃에 서⁶⁰일간숙성시킨후아미노태질소함량은^{507 mg%}이고^[3]

Aspergillus oryzae를배양한소맥분코지를대두에접종하 고식염농도^12%로제조한된장을²⁰℃에서⁷⁰일발효시킨 경우아미노태질소함량이^{400 mg%}이였다는보고^[15]들을

볼때^{B. cereus}는숙성중대두단백질분해에부정적영향

을준것같지는않다^.이전식품공전상아미노태질소함량 규격은^{160 mg%}이상이였고^[3],전통재래된장의아미노테 질소함량은평균^{308.4 mg%}라는보고도있다^[17].

복합종균을접종한된장숙성중 B. cereus 증식은효과적 으로억제되었다^.종균접종된장들의경우 4 log CFU/g으 로오염시킨 B. cereus를효과적으로저해하여⁷⁰일후에는 식약처기준치이하로감소시켰다^.특히 B. amyloliquefaciens EMD17과 B. subtilis CH3-5를 접종한 된장^B는^{7 log} CFU/g로오염시킨 B. cereus 증식도효과적으로억제하였 다. 이결과들로볼때복합종균의사용은된장의안전성개 선에도움이줄것으로사료된다^.앞으로복합종균된장의 기능성과기호성에관한연구들이필요하다^.

Acknowledgments

This work was supported by a grant from IPET (High Value-added Food Technology Development Program, 2012, 112066-3), Ministry of Agriculture, Food and Rural Affairs, Republic of Korea. Lee JY, Shim JM, and Lee KW were supported by BK21 Plus program from Ministry of Education, Republic of Korea.

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