A Study on Biological Activities of Fermented Jujube and Grape

대추 및 포도 발효물의 생리활성 효과에 관한 연구

박태순¹, 김동희¹, 권오준², 손준호¹*

1

(

)

2

(

)

Received: December 11, 2013 / Revised: March 18, 2014 / Accepted: March 24, 2014

서 론

최근건강에대한지대한관심과함께각종기능성식품 과원료등에대한연구가활발히진행되고있다

^.

^.

^[14].

특히전통적으로이용되어온발효는최근상당한관심을모 으고있는데

^,

분당이붙은고분자로구성되어있는경우가많아그럴경 우에는체내흡수에지장이있어발효를하면당이떨어져 나가면서저분자화되어체내흡수율이증가되고

^,

^,

^,

^,

^[17, 23, 29].

식품과한방재료로서널리사용되어온대추는갈매나무과

(Rhamnaceae)

은각종

sterol, alkaloid, saponin, vitamin, serotonin, organic acid, fatty acid, polyphenol, flavonoid

amino acid

보고되고있으며

[2, 19, 34],

용

^,

^,

^,

^,

^,

다

[17, 24].

검증결과미숙

^,

^,

^,

A Study on Biological Activities of Fermented Jujube and Grape

Tae-Soon Park

, Dong-Hee Kim

, O-Jun Kwon

, and Jun-Ho Son

*

1

Herbal Cosmetic Team, Korea Promotion Institute for Traditional Medicine Industry, Gyeongsan 710-260, Republic of Korea

2

Daegyeong Institute for Regional Program Evaluation, Gyeongsan 712-210, Republic of Korea

For the development of high value consumables utilizing jujube and grape, we investigated the biological activities of a variety of existing fermentation products of jujube and grape. The results revealed that ethanol fermentation products of jujube and grape had a higher antioxidative activity than acetic acid fermentation products. In addition, the ethanol fermentation products of jujube (JEF) had the highest antioxidative activity, with it being greater than that of the ethanol fermentation products of grape (GEF), the acetic acid fermentation products of jujube (JAF) and the acetic acid fermentation products of grape (GAF). As regards tests on whitening effects, JEF exhibited the highest tyrosinase inhibition effects amongst the test groups. However, when immunofluorecence was employed, JAF was seen to inhibit the expression of proteins related to the whitening effect. In the lipopolysaccharide-stimulated mode peritoneal macrophage model, all tested groups of fermentation products (JEF, GEF, JAF and GAF) suppressed nitric oxide production dose-dependently, with ethanol fermentation products demonstrating a higher nitric oxide expression inhibition effect than acetic acid fermentation products. When subjected to antibacterial activity tests, GAF exhibited antibacterial activity against all tested strains except Propionibacterium acnes. Both GAF and JEF revealed high antibacterial activity against Escherichia coli.

Keywords: Jujube, grape, fermentation, biological activities, antimicrobial activity

*Corresponding author

Tel: +82-53-810-0320, Fax: +82-53-810-0399 E-mail: [email protected]

© 2014, The Korean Society for Microbiology and Biotechnology

보고된바있다

^[8].

포도는갈매나무목

(Rhamnales)

^(Vitaceae)

^,

¹¹

^{, 700}

의포도가있다

^[12].

천연항산화제로서가치에대하여많은연구가진행되고있 다

. (+)-catechin, (

)-epicatechin, procyanidin, viviferin

resveratrol

polyphenol

성물질로알려져있으며

^,

^oral

tumor cell

, proanthocyanidin

^free radical

^,

polyphenolic fraction

antitumor

^,

resveratrol

[1, 20, 22, 28,

33].

리활성에관한 연구는아직 이루어지지않고있는실정이 다

^.

^.

재료 및 방법

알코올 발효 및 초산 발효 방법

본실험에사용된 대추

(Zizyphus jujuba Miller)

(Vitis vinifera L.)

^.

Saccharomyces klutveri DJ97 (KCTC 8842P)

⁽¹⁸

^brix)

^5%

³⁰

^C

⁴

올발효를수행하여알코올발효액이알코올도수가

^3-4%

및

^8-9

^brix

^.

Acetobacter pasteurianus KFC 819 (KCTC 10173BP)

³⁰

^C

¹⁰

^3-4%

^7-8

^brix

^.

재료 및 시약

생리활성 측정 실험에 사용된 시약인

1-1-diphenyl-2-

picryl-hydrazyl (DPPH), xanthine, xanthine oxidase, nitro blue tetrazolium (NBT), mushroom tyrosinase, tyrosine

L-3,4-dihydroxyphenyl-alanine (L-DOPA)

^griess reagent

Sigma Chemical Co. (St. Louis, MO, USA)

^,

^{RAW 264.7}

^B16F10

을

Korean Cell Line Bank (KCLB)

^.

Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum, penicillin/streptomycin, trypsin 250, 0.4% trypan blue stain

Gibco BRL Co.

(Grand Island, USA)

^{, 3-[4,5-} dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium-bromide (MTT)

Sigma Chemical Co. (St. Louis, MO, USA)

^.

Staphylococcus aureus KCTC 1621, Staphylococcus epidermidis KCTC 1917, Escherichia coli KCTC 1039

Propionibacterium acnes KCTC 3314

^.

nutrient broth, tryptic soy broth

Difco Lab. (Sparks, MD., USA)

Gifu anaerobic medium (GAM, Nissui Co. Japan)

^,

agar

^.

항균효과 측정

항균효과측정은

paper disc

^[6].

즉

^,

¹

^{10 ml}

^18-24

^{10 ml}

^{0.1 ml}

^3-6

¹

^{1 × 10}

^cells

^.

filter paper disc (8 mm, Tokyo, Japan)

0.05 ml/disc

³⁷

^C

18-24

^disc

clear zone (cm)

^.

세포 생존율 측정

대식세포

(RAW 264.7)

^(B16F10)

^(KCLB)

^,

^10%

fetal bovine serum

1% penicillin-streptomycin

^DMEM

^.

³⁷

^{C, 5% CO}

^,

Carmichael

^[5]

^.

^MTT

^formazan

전환되는 것을 측정하는 것으로

, RAW 264.7

B16F10

0.6~0.8 × 10

cells/ml

96 well plate

주하고대추및포도의 발효물을농도별

(0.01-5 mg/ml)

24

^{. Well}

²⁰

^l

^MTT

³⁷

^{C, 5% CO}

incubator

⁴

^,

microplate reader

^{540 nm}

^.

항산화 효과 측정

대추및포도의발효물들의항산화활성은

DPPH anion scavenging activity, superoxide anion radical scavenging activity, ABTS

· cation scavenging activity

^.

DPPH anion scavenging activity

96 well plate

^[3].

⁸⁰⁰

^l

^{0.15 mM}

DPPH

²⁰⁰

^l

³⁰

517 nm

^,

무첨가군의흡광도감소율로나타내었다

. Superoxide anion radical scavenging activity

^{, NBT}

^[15].

¹⁰⁰

^l

0.1 M potassium phosphate buffer (pH 7.5) 400

^l

xanthine (0.4 mM)

⁴⁰⁰

^l

NBT (0.24 mM)

^xanthine oxidase (0.2 U/ml) 100

^l

³⁷

^C

¹⁵

1 N HCl 1 ml

^,

superoxide anion radical

^{560 nm}

^.

^{, ABTS}

· cation scavenging activity

7 mM 2,2-azino-bis (3-ethyl-benthiazoline-6- sulfonic acid)

2.4 mM potassium persulfate

실온에서

²⁴

^ABTS

^·

ethanol

^ABTS

^{· 100}

^l

¹⁰⁰

^l

1

^{732 nm}

^[25].

미백효과 측정

Tyrosinase

^Yagi

^[31]

^.

0.175 M sodium phosphate buffer (pH 6.8) 500

^l

10 mM L-DOPA

²⁰⁰

^l

¹⁰⁰

^l

mushroom tyrosinase (110 U/ml) 200

^l

²⁵

^C

²

DOPA chrome

^{475 nm}

^.

Tyrosinase

광도감소율로나타내었다

^.

, immnunoflurecence

^B16F10

Lab-tek chamber

8 well

^{1 × 10}

²⁴

^,

시료를 농도별로 처리한다

^{. 24-48}

phosphate buffer saline (PBS)

³

0.5% triton X-100

¹⁰

^.

^PBS

³

¹

bovine serum albumin

¹

. Primary anti-body (tyroisnase 1:100, microphthalmia-associated transcription factor (MITF) 1:100)

⁴

^C

over night

^{. 1}

^PBS

³

alexa flour 568 goat anti- mouse IgG (1:1,000)

¹

^{. 40}

^DAPI

²⁰⁰

^l

^well

^. PBS

⁴

^chamber

^slide

mounting solution

^.

항염증 효과 측정

항염증효과측정은

nitric oxide (NO)

^NO

^nitrite

^griess reagent

. RAW 264.7

^DMEM

^{5 × 10}

^cells/ml

12 well plate

^{, 5% CO}

incubator

²⁴

^.

¹

^g/ml

lipopolysaccharide (LPS)

1

0.01-1 mg/ml

²⁴

^.

^griess

ELISA reader

^{540 nm}

^NO

^.

결과 및 고찰

항균효과 측정 결과

대추알코올및초산발효물과포도의알코올및초산발 효물의항균력을검토하기위하여피부상재균인

S. aureus, E. coli

S. epidermidis

P. acnes

clear zone

^{Table 1}

Table 1. Antimicrobial activity of various fermentation products on several microorganisms.

Strains JEF (mg/disc) GEF (mg/disc) JAF (mg/disc) GAF (mg/disc)

1.5 2.5 5 1.5 2.5 5 1.5 2.5 5 1.5 2.5 5

S. epidermidis -

- - - - - - - - - - 1.1 ± 0.21

S. aureus - - - - - - - - - - - 1.2 ± 0.10

E. coli - - - - - 1.3 ± 0.55 - 1.2 ± 0.07 1.7 ± 0.55 - 1.1 ± 0.06 1.5 ± 0.15

P. acnes - - - - - - - - - - - -

a

No inhibition.

b

Inbition zone diameter (cm), JEF: ethanol fermentation of jujube, GEF: ethanol fermentation of grape, JAF: acetic acid

fermentation of jujube, GAF: acetic acid fermentation of grape.

다

^.

P. acnes

^,

E.

coli

^.

, E. coli

^{5 mg/disc}

^{1.3 cm,} 1.5 cm

^,

^{2.5 mg/}

disc

^{5 mg/disc}

1.2 cm, 1.7 cm

^.

^,

^.

세포 생존율 측정 결과

세포 생존율을 확인하기 위한

^MTT

^{96 well}

plate

^,

ELISA reader (multiwell microplate reader)

되고있는방법중의한방법이다

^[21].

들이세포의증식에미치는영향에대해알아보기하여

^0.01-

5 mg/ml

⁴⁸

^{1 mg/}

ml

^90%

^{, 5 mg/}

ml

^{(Fig. 1).}

^NO

immunofluorecence

^{1 mg/}

ml

^.

대추및포도발효물들의항산화효과를측정한결과를

Table 2

^.

, DPPH anion scavenging activity (

⁾

^{5 mg/}

ml

^59%

^DPPH

능을나타내었으며

^,

^53%,

알코올발효물은

^37%,

^32%

을나타내어알코올발효물과초산발효물비교시알코올발 효물이높은

^DPPH

^.

^Hong

^[8]

의건대추의전자공여능측정결과

^{5 mg/ml}

^53.8%

.

, Krigaya [13]

Yamaguchi [32]

Fig. 1. Cell viability of various fermentation poducts. (A) macrophage (RAW 264.7), (B) melanoma cell (B16F10), JEF : ethanol fermentation of jujube, GEF : ethanol fermentation of grape, JAF : acetic acid fermentation of jujube, GAF : acetic acid fer- mentation of grape. Result are means ± S.D. of triplicate data.

Table 2. Antioxidant activity of various fermentation products.

Samples (mg/ml)

Antioxidant activity (%) DPPH

scavenging activity

Superoxide anion radical

scavenging activity

ABTS scavenging

activity JEF 1 33.8 ± 1.2 36.9 ± 1.9 58.1 ± 4.9

5 58.7 ± 2.4 76.4 ± 0.8 95.6 ± 1.6 GEF 1 29.6 ± 4.1 19.5 ± 3.9 23.8 ± 1.6 5 36.9 ± 1.7 61.5 ± 2.2 60.7 ± 5.0 JAF 1 24.1 ± 1.5 41.6 ± 6.7 38.6 ± 2.8 5 53.2 ± 0.4 69.9 ± 2.8 73.7 ± 1.9 GAF 1 9.5 ± 1.2 21.3 ± 4.1 18.5 ± 1.5 5 32.1 ± 0.4 57.8 ± 3.9 55.5 ± 1.4 Vit. C 1 83.8 ± 0.2 85.6 ± 7.2 98.8 ± 2.8 5 86.9 ± 0.1 96.2 ± 5.2 100 ± 1.2 JEF: ethanol fermentation of jujube, GEF: ethanol fermenta- tion of grape, JAF: acetic acid fermentation of jujube, GAF:

acetic acid fermentation of grape, Vit. C: ascorbic acid.

Result are means ± S.D. of triplicate data.

사료되며

^,

^.

^, superoxide anion radical scavenging activity

^{5 mg/ml}

70%,

^69%

^,

도의알코올및초산발효물은각각

^{62%, 58%}

나타내어대추의알코올발효물이가장높은소거능을나타 내었다

^.

^Hong

^[8]

^,

^,

xantnin oxidase

모든추출물

^{5 mg/ml}

저해능을나타내어발효물들이우수함을나타내었다

^.

^{, ABTS}

· cation scavenging activity

^ABTS

potassium persulfate

^ABTS

^{· radical}

^,

decolorization

, hydrogen donating antioxidant

chain breaking antioxidant

[30].

ABTS

5 mg/ml

^{61%, 56%}

^ABTS

^,

추의 발효물중알코올 발효의경우

^{5 mg/ml}

vitamin C

은소거능을나타내었다

^.

미백효과 측정 결과

Melanin

tyrosinase, tyrosinase related protein 1, tyrosinase related protein 2

³

^[11].

cyclic adenosine monophosphate / protein kinase A

^melanin

합성한다

^.

^,

keratinocyte

-melanocyte stimulating hormone, adrenocorticotropic hormone

melanocyte

cyclic adenosine monophosphate

^, protein kinase A

^MITF

^{[4, 27].}

^MITF

tyrosinase, tyrosinase related protein 1, tyrosinase related protein 2

^melanin

[26]. 3

tyrosinase

^tyrosin

^DOPA

DOPA-quinone

^melanin

생성에관여하는효소로자외선에의하여

melanocyte

사분열이일어나고이어서

melanocyte

^[7].

melanocyte

tyrosinase

melanin

게되어기미

^,

^.

Fig. 2. Tyrosinase inhibition rate of various fermentation products. JEF: ethanol fermentation of jujube, GEF: eth- anol fermentation of grape, JAF: acetic acid fermentation of jujube, GAF: acetic acid fermentation of grape, Vit. C:

ascorbic acid. Result are means ± S.D. of triplicate data.

Fig. 3. Reduced cellular expression of tyrosinase and

MITF using immunofluorescence assay. (A) control, (B) etha-

nol fermentation of jujube, (C) ethanol fermentation of grape, (D)

acetic acid fermentation of jujube, (E) acetic acid fermentation of

grape. B16F10 cells (1 × 10

cells) were seeded in serum free

medium for 1 h at room temperature. The cells were treated with

1 mg/ml of various fermentation products for 48 h.

는따라서

tyrosinase

^melanin

polymer

개발에있어서

tyrosinase

로인정되고있다

^{[9, 10].}

tyrosinase

^{5 mg/}

ml

^54%

저해활성을나타내었으며

^,

^{5 mg/ml}

^36%

tyrosinase

^.

효물은

^{5 mg/ml}

^26%

다

^{(Fig. 2).}

^melanin

^MITF

tyrosinase

(immunofluorescence)

^{Fig. 3}

^{. MITF}

, tyrosinase

물을

1 mg/ml

효물은대조군과비교시형광발현이줄어드는것을확인하 였다

^.

^,

^.

tyrosinase

^.

항염증효과 측정 결과

NO

^NO

^L-arginie

^,

^,

등여러가지생물학적인과정에관여하는것으로알려져있 으며농도에따라세포기능유지에중요한작용을하기도하 고세포독성을일으키기도한다

[16, 18]. RAW 264.7 cell

NO

¹⁰

^g/ml

LPS

생성되는

^NO

^{Fig. 4}

^.

LPS

^LPS

^NO

나타내었으며

^,

^NO

^.

^,

^{1 mg/ml}

^37%

^NO

^,

^35%

^NO

^.

이와같은상기의결과를종합해보면초산발효물보다알 코올발효물이우수한활성을나타내었으며

^,

^,

^,

.

^.

요 약

대추및포도를이용하여고부가가치성소재개발및특 화작물로서개발하기위하여이들을이용한알코올발효와 초산발효를한후발효물들의생리활성을검증하였다

^.

Fig. 4. Effects of various fermentation products on the production of nitric oxide in macrophage cell (RAW 264.7). nor: LPS not

induced group, con: LPS induced group, JEF: ethanol fermentation of jujube, GEF: ethanol fermentation of grape, JAF: acetic acid fer-

mentation of jujube, GAF: acetic acid fermentation of grape. Result are means ± S.D. of triplicate data.

한활성을나타내었으며

^,

^.

, immunofluorecence

^.

lipopolysaccharide

nitric oxide

^,

^.

Propionibacterium acnes

, Escherichia coli

^.

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