Isolation of the Alcohol-Tolerant Lactic Acid Bacteria Pediococcus acidilactici K3 and S1 and their Physiological Characterization

알코올 내성 젖산균 Pediococcus acidilactici K3와 S1의 분리 및 생리적 특성

장단비¹, 박슬기¹, 이현주¹, 표상은¹, 이한승^1,2*

1신라대학교의생명과학대학바이오식품소재학과

2신라대학교막걸리세계화연구소

Received: August 8, 2013 / Revised: September 25, 2013 / Accepted: October 25, 2013

서 론

젖산균

(lactic acid bacteria, LAB)

(probiotics)

^{[8, 14].}

^,

^,

^{[18, 19]}

^.

생물을제거하지않고그대로섭취하기때문에상당량의단 백질과식이섬유및당질이함유되어있고소량의비타민과 유기산및생리활성물질이포함되어있어영양과기능면에 서가치가높다

^[20].

전통적인막걸리발효는곰팡이

^,

^,

^.

^[17]

정장작용등

probiotics

악영향을주는양면성을갖고있다

^.

에악영향을주는주요요인중하나로추정된다

^[16].

막걸리발효후

^,

Isolation of the Alcohol-Tolerant Lactic Acid Bacteria Pediococcus acidilactici K3 and S1 and their Physiological Characterization. Jang, Danbie

, Seulki Park

, Hyunjoo Lee

, Sangeun Pyo

, and Han-Seung Lee

*.

Department of Bio-Food Materials, College of Medical and Life Sciences, Silla University, Busan 617-736, Korea,

Makgeolli Globalization Research Center, Silla University, Busan 617-736, Korea

Lactic acid bacteria (LAB) are a representative group of probiotics and used in many fermented foods and beverages. Several recent studies have shown that LAB are present in makgeolli which is a traditional Korean alcoholic beverage. However, most LAB are intolerant of more than 6% (v/v) alcohol concentrations. For this reason, alcohol-tolerant LAB are isolated from kimchi, makgeolli and nuruk using alcohol containing selective media. After being cultured in MRS broth containing 13% (v/v) alcohol, the two strains which showed the highest increasing O.D values, were finally selected. As results of 16S rRNA gene sequenc- ing and biochemical characterization using an API kit, the two species were identified as Pediococcus acidilactici K3 and S1. In addition, the identified two strains produced bacteriocins against Staphylococcus aureus. When compared with the P. acidilactici type strain, the two selected strains possessed two to three time higher growth on 12-13% (v/v) alcohol containing MRS broth.

The viability of P. acidilactici K3 and S1 when inoculated in makgeolli and stored at 10

C did not decrease through a period of one month indicating that the selected strains can be used for LAB containing makgeolli.

Keywords: Alcohol-tolerant, lactic acid bacteria, Pediococcus acidilactici, makgeolli

*Corresponding author

Tel: +82-51-999-6308, Fax: +82-51-999-5458 E-mail: [email protected]

© 2013, The Korean Society for Microbiology and Biotechnology

도는

^12%(

^v/v)

^.

^5%

^10%

하게된다

^{[3, 18].}

대신고두밥에백국균을접종하여당화하는입국방식을사

용하고있다

^[13].

용해서발효하는방법은발효공정을제어하고부발효나이 상발효가일어나지않도록조절하는데는용이하지만

^,

^.

따라서젖산균이풍부히함유된막걸리를개발하기위해 서는알코올에내성을가진젖산균주를탐색

^,

⁽

⁾

(

)

^.

^,

^.

재료 및 방법

알코올 내성 젖산균주의 1차 스크리닝

알코올내성젖산균을탐색하기위하여현재판매중인생 막걸리

^,

^,

^,

^.

bromocresol purple (BCP)

^,

^.

알코올 함유 배지에서의 생장 실험

분리해낸젖산균주를알코올

⁽

⁾

0%, 10%, 15%

함유된

^MRS

^{. MRS}

는알코올농도에따라들어가는알코올부피를제외한만 큼만들어가압멸균후냉각하고알코올을첨가하여혼합한 후제조하였다

^.

^MRS

10 ml

seed culture

^1%

^.

³⁷

^C

^{150 rpm}

²⁴

^OD

^{. O.D}

²⁴

spectrophotometer

^{600 nm}

^.

선별 균주의 분자생물학 및 생화학적 동정

10%

^15%

^O.D

⁵

^{MRS broth}

한 후

RBC Genomic DNA Extraction Kit (RBC Bioscience Co., Taiwan)

Genomic DNA

template

^PCR

^{. PCR}

27F primer

1492R primer

^{16S rRNA}

^DNA

진텍

(Daejeon, Korea)

BLAST

^.

의생화학적 동정을 위하여젖산균 동정용

^Kit

^{API 50}

CHL Medium (bioMerieux, France)

^.

알코올 내성 비교

최종선별한알코올내성젖산균

²

Pediococcus acidilactici DSM 20284

^.

0%, 6%, 12%

MRS

²⁴

간에한번씩시료채취한후

^,

^.

Bacteriocin Assay

Bacteriocin

well diffusion assay

. Nutrient agar

Staphylococcus aureus

^1%

^,

^tip

²

Pediococcus acidilactici DSM 20284

^,

^MRS

²

^.

막걸리의 저장 기간 중 젖산균의 안정성

시중에서 구입한 살균막걸리에 선별된 젖산균주

P.

acidilactici K3

^S1

⁵

¹⁰

CFU/ml

⁽⁴

^C),

⁽¹⁰

^C),

⁽²⁵

^C)

³⁰

플링을한후연속희석후

^BCP

타내는젖산균수를측정하였다

^.

결과 및 고찰

알코올 내성 젖산균주의 스크리닝 및 선별

다양한종류의김치

^,

⁴

⁵

으로부터젖산균선택배지인

^BCP

로니를생성하는총

⁵⁶

^.

⁵⁶

^10%

^15%

된

^MRS

^,

은

^{600 nm}

^(OD

)

^OD

가가장 많이증가한

⁵

(N3-2-2, S1, SN3, K3, K5)

¹

^{. 1}

⁵

^10%

MRS

올농도인

^13%

^MRS

⁴⁸

^{, K3}

^S1

^{(Fig. 1).}

선별 균주의 분자생물학 및 생화학적 동정

1

⁵

두균주

^(K3

^S1)

^{16S rRNA}

^PCR

Pediococcus acidilactici

99% identity

^{(Fig. 2)}

³

(N3-2-2, SN3, K3)

Pediococcus

^.

¹

⁵

Fig. 1. Growth of selected five lactic acid bacteria on MRS broth containing 10% (A) and 13% ethanol (B).

Five strains were designated as followings: N3-2-2, open square

; S1, closed circle

; SN3, open circle

; K3, closed square

; K5, open triangle

.

Fig. 2. Multiple alignment of partial 16S rRNA gene sequences of Pediococcus acidilactici DSM 20284 (Pac20284), K3, S1, and Pediococcus pentosaceus DSM 20336 (Ppe20336).

Open ovals designated the differences from P. acidilactici and P. pentosaceus.

Table 1. Biochemical properties of Pediococcus acidilactici DSM 20284 (Pa), S1, and K3.

Tube Test Active ingredients Pa S1 K3

0 CTRL CONTROL + + +

1 GLY GLYcerol − − −

2 ERY ERYthritol − − −

3 DARA D-ARAbinose − − −

4 LARA L-ARAbinose _{+ + +}

5 RIB D-RIBose _{+ + +}

6 DZYL D-XYLose _{+ + +}

7 LXYL L-XYLose − − −

8 ADO D-ADOnitol − − −

9 MDX Methyl- β D-Xylopyranoside − − −

10 GAL D-GALactose _{+ + +}

11 GLU D-GLUcose + + +

12 FRU D-FRUctose _{+ + +}

13 MNE D-MaNnosE _{+ + +}

14 SBE L-SorBosE − − −

15 RHA L-RHAmnose △ △ −

16 DUL DULcitol − − −

17 INO INOsitol − − −

18 MAN D-MANnitol − − −

19 SOR D-SORbitol − − −

20 MDM Methyl-α D-Mannopyranoside − − −

21 MDG Methyl- α D-Glucopyranoside − − −

22 NAG N-AcetylGlucosamine + + +

23 AMY AMYgdalin △ △ △

24 ARB ARButin _{+ + +}

25 ESC Esculin ferric citrate _{+ + +}

26 SAL SALicin _{+ + +}

27 CEL D-CELlobiose _{+ + +}

28 MAL D-MALtose _{+ + +}

29 LAC D-LACtose(bovine origin) △ − −

30 MEL D-MELibiose − − −

31 SAC D-SACcharose(sucrose) − − −

32 TRE D-TREhalose _{+ + +}

33 INU INUlin − − −

34 MLZ D-MeleZitose − − −

35 RAF D-RAFfinose − − −

36 AMD AmiDon(starch) − − −

37 GLYG GLYcoGen − − −

38 XLT XyLiTol − − −

39 GEN GENtiobiose △ △ △

40 TUR D-TURanose − − −

41 LYX D-LYXose − − −

42 TAG D-TAGatose + + +

43 DFUC D-FUCose − − −

44 LFUC L-FUCose − − −

45 DARL D-ARabitoL − − −

46 LARL L-ARabitoL − − −

47 GNT potassium GlucoNaTe △ △ −

48 2KG potassium 2-KetoGluconate − − −

49 5KG potassium 5-KetoGluconate − − −

Each symbol designated in the following meaning: +, fully growth; −, not growth; △ , slightly growth.

은모두생막걸리와시판누룩에서분리되었는데

^,

Lactobacillus

^{[7, 9]}

^{[11, 12]}

^DGGE

석을통해보고한바에따르면

Pediococcus

누룩과막걸리에상당수함유되어있는것으로알려지기시 작했으며

^,

Pediococcus

이보고되고있으나

[6, 21, 22]

전혀없는실정이다

^.

13%

^(K3

S1)

P. acidilactici DSM 20284

^{API 50}

CHL Medium kit

(Table 1),

Pediococcus pentosaceus

사한결과

^(99%)

^.

용가능성을보는생화학적테스트는정확한동정에한계가 있으며변종의경우에는차이를보일수있고

P. pentosaceus

P. acidilactici

[10]

^(K3

^S1)

^{16S rRNA}

P.

acidilactici DSM 20284

P. pentosaceus DSM 20336

준균주의

^{16S rRNA}

^.

과

^{(Fig. 2),}

P. acidilactici

P.

acidilactici K3

P. acidilactici S1

^.

본연구를통해선발한

P. acidilactici K3

^S1

P. acidilactici DSM 20284 (KCTC 13734)

알코올내성이뛰어난지확인하기위하여서로다른

⁴

(0%, 6%, 12%, 13%)

MRS

¹

^.

그결과

^{(Fig. 3),}

이지않았으나본연구를통해선별된두균주모두초기생 육속도가뛰어났으며알코올농도가높아질수록선별된두 균주가표준균주보다더잘생육하였다

^.

^13%

이포함된

^MRS

^K3

3

P.

acidilactici K3

^.

Oenococcus oeni

^13%

malolactic acid

향을주는것으로알려져있다

^[5].

선별한 알코올 내성 균주의 Bacteriocin 생산 여부 확인

P. acidilactici

(colonization)

probiotics

^{[4, 23]}

pediocin

[1, 2]. Pediococcus

산균이생산하는

bacteriocin

서주목을받고있고

^[15]

유익한역할을기대하고있기때문에

^,

알코올내성균주들도

bacteriocin

다

^.

S. aureus

bacteriocin assay

^{, K3}

^S1

bacteriocin

Fig. 3. Comparison of alcohol-tolerance of Pediococcus acidilactici DSM 20284 (

), K3 (

), and S1 (

) according to various

ethanol concentration (A, 0%; B, 6%; C, 12%; D, 13% v/v).

생성하여

S. aureus

^(Fig.

4).

probiotics

^.

젖산균을 첨가한 막걸리의 저장 기간 중 젖산균의 안정성 알코올내성젖산균이막걸리에서안정하게유지되는지확 인하기 위하여 저온 살균 막걸리에 선별된 젖산균주

P.

acidilactici K3

^S1

⁽⁴

^C),

⁽¹⁰

^C),

⁽²⁵

^C)

^{, 4}

^C

¹⁰

^C

³⁰

지초기접종한균수를일정하게유지하였고막걸리변패 온도인상온에서는젖산균이초기에급격히생육하다가후

기에감소하는패턴을보였다

^{(Fig. 5).}

통해선별된젖산균을후첨가하여막걸리를제조하는것이 가능함을시사하며

^,

^.

요 약

김치

^,

후다양한농도의알코올을함유한

^MRS

생육여부를조사하였다

^.

^13%(v/v)

^MRS

^OD

²

별하였고이두균주의

^{16S rRNA}

API kit

P. acidilactici

P. acidilactici K3

P. acidilactici S1

^.

P. acidilactici K3

^S1

bacteriocin

S. aureus

bacteriocin

^.

(P.

acidilactici DSM 20284)

^6%

이하의저농도알코올이포함된배지에서는균주생육에큰

차이가없었으나

^12%

표준균주보다최대

³

^.

P. acidilactici K3

^S1

⁽⁴

^C

¹⁰

^C)

Fig. 4. Bacteriocin production by well diffusion assay with

Staphylococcus aureus. B stands for MRS Broth as a nega- tive control and each well contains culture broth of K3, S1, and DSM 20284 (Pa).

Fig. 5. Cell viability of Pediococcus acidilactici K3 (

) and S1 (

) for 30 days on different temperatures (A, 4

C; B, 10

C; C, 25

C)

after adding to pasteurized makgeolli.

됨을확인하였다

^.

Acknowledgments

This work (Grant # 00041254) was supported by Business for Cooperative R&D between Industry, Academy, and Research Insti- tute funded Korea Small and Medium Business Administration in 2010.

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