J Korean Soc Food Sci Nutr 한국식품영양과학회지 39(1), 25~3(21) DOI: 1.3746/jkfn.21.39.1.25 큰느타리버섯균사체로제조한발효두부추출물의면역활성 이상원 1 강종우 2 김재용 3 박경욱 2 박석규 2 주옥수 4 이성태 5 서권일 2 1 진주산업대학교미생물공학과, 2 순천대학교식품영양학과 3 경북대학교식품공학과, 4 진주산업대학교식품과학과 5 순천대학교생물학과 Immuno-Activities of Extracts of Tofu Fermented with Pleurotus eryngii Mycelia Sang-Won Lee 1, Jong-Woo Kang 2, Jae-Yong Kim 3, Kyung-Wuk Park 2, Seok-Kyu Park 2, Ok-Soo Joo 4, Sung-Tae Yee 5, and Kwon-Il Seo 2 1 Dept. of Microbiological Engineering, Jinju National University, Gyeongnam 66-758, Korea 2 Dept. of Food and Nutrition, Sunchon National University, Jeonnam 54-742, Korea 3 Dept. of Food Science and Technology, Kyungpook National University, Daegu 72-71, Korea 4 Dept. of Food Science, Jinju National University, Gyeongnam 66-758, Korea 5 Dept. of Biology, Sunchon National University, Jeonnam 54-742, Korea Abstract In order to improve the functional benefits and storage properties of soybean tofu, fermented tofu was developed using Pleurotus eryngii mycelia. The immune activities of water and methanol extracts of the tofu were investigated. The optimal medium for the growth of Pleurotus eryngii mycelia was PD broth medium and the optimal fermentation period for the tofu was 7 days. The water and methanol extracts of the fermented tofu induced the proliferation of spleen cells at above.1 μg/ml. The water extract increased IL-2, IFN-γ production, while the methanol extract increased IFN-γ synthesis. The water and methanol extracts of the fermented tofu induced the NO production in RAW264.7 macrophage cells at above 1 μg/ml and above 1 μg/ml concentration, respectively. The extracts also significantly increased the production of IL-6, TNF-α, IL-1β and GM-CSF in the cells. These results suggest that the tofu fermented with Pleurotus eryngii mycelia could be developed as a functional tofu. Key words: Pleurotus eryngii mycelia, fermented tofu, immuno-activity 서론두부는콩을물에담구어충분히수화시킨뒤물과함께마쇄하여끊인다음, 여과또는압착하여두유에적당량의응고제를첨가하여침전 응고된겔상태의식품이다 (1). 두부는수분함량이높은식품으로소화율이 95% 이상되며, 필수아미노산이다량함유되어있는양질의단백질식품이다 (2). 또한두부의재료인대두단백질은혈중콜레스테롤, 지질단백질 (LDL) 등의농도를감소시켜심혈관질환예방효과가있으며, 대두올리고당은장내비피스더스균증식촉진등의효과가있어기능성식품으로활용할가치가있다 (3). 하지만두부는수분함량이높고단백질등영양성분들이풍부하여쉽게부패하는단점이있다 (4). 최근에두부의품질특성을향상시키고저장성을증대시 키기위한방법으로다양한생리활성성분을함유하고있는천연소재를두부에첨가하여두부의기능성과저장성을향상시키는연구들이진행되고있다. 그대표적인예로두부의저장성을증대시키기위해두부에유기산이나키토산등을첨가하는방법 (5,6), 오미자즙및매실즙과같은천연응고제를사용하여식품의오염을억제한두부 (7), 인삼 (8), 녹차 (9), 해조류 (1), 클로렐라 (11), 허브 (12), 마늘 (13), 복분자 (2) 등을첨가하여저장성및기능성을향상시키는연구들이진행되고있으나, 그실효성에서문제점들이제기되어실용화가어려운실정이다. 한편식품의저장성을향상시키기위한다른방법으로발효를이용한연구들이보고되고있다 (14). 이는발효과정중에맛, 향뿐만아니라다양한생리활성물질들이생성되어저장성, 기호성뿐만아니라다양한기능성이함유된식품으로발전될수가있기때문에이에관한연구들이진행되고있다. Corresponding author. E-mail: seoki@sunchon.ac.kr Phone: 82-61-75-3655, Fax: 82-61-752-3657
26 이상원 강종우 김재용 박경욱 박석규 주옥수 이성태 서권일 대표적인두부발효식품은중국및대만의 Sufu, 인도네시아의 Taokon, 필리핀의 Tahuri, 태국의 Tauhu yee, 일본의 Tofuyo 등을들수있다 (14). 이것은두부에곰팡이를번식시켜분비되는효소에의해단백질이펩타이드및아미노산으로분해되어숙성이진행됨에따라치즈와유사한부드러운조직과풍미등의기호적가치를가지게된다 (15). 우리나라에서는간장, 된장및술등과같은발효식품에관한연구들이많이진행되고있으나, 두부발효식품에관한연구는발효두부의품질특성에관한연구만보고되고있으며 (15), 생리활성에관한체계적인연구는진행되지않고있다. 따라서본연구에서는저장성이높은고품질기능성두부의개발가능성을확인하고자생리활성물질을함유하고있는큰느타리버섯 (Pleurotus eryngii mycelia) 균사체를이용하여발효두부를제조하여면역활성을조사하였다. 재료및방법실험재료 24년가을에순천지역의농가에서수확한대두로직접가정에서제조한두부를구입하여발효두부제조에사용하였으며, 두부의발효를위한큰느타리버섯균사체는경남농업기술원에서분양받아사용하였다. 배지및균주의배양발효두부제조를위한큰느타리버섯균주는 MEA(malt extract 2%, dextrose 2%, peptone.1%, MgSO 4 7H 2O.5%, agar 2%), MPA(malt extract 2%, peptone.5%, agar 2%) 및 PDA(potato dextrose agar) 배지를사용하여검토하였다. 즉 PDA배지는박피한감자 2 g을 1 1 cm 정도로세저한후 1 L의증류수를첨가하여 1분동안끓여 3~4겹의가제로여과한여액을정확하게 1 L로정용한다음 dextrose와 agar를각각 2% 첨가하여제조하였다. 버섯균주의보존은이들평판배지에 3 o C, 6일동안배양하여 5 o C이하의냉장보관하면서 2주일이내에액체배양을위한 starter로사용하였다. 큰느타리버섯균사체액체배양은최적평판배지조성중 agar가제거된성분의배지를사용하여 3 o C, 12 rpm으로 7일동안배양하여 pellet이형성되었을때마그네틱스틱으로 pellet를완전히분쇄하여사용하였다. 큰느타리버섯균사체발효두부의제조및추출큰느타리버섯균사체를이용한발효두부의제조는두부를적당한크기로세절 (1 1 5.5 cm) 한후스테인리스용기 (22 16 6 cm) 에담아 125 o C, 15분동안멸균시킨다음, 미리배양하여 pellet를마쇄한큰느타리버섯균사체의배양용액에두부조각을침지시키는방법으로종균을접종하였다. 종균을접종한두부는 3 o C 항온기에옮겨 7일동안배양하면서큰느타리버섯의균사가두부의표면을완전히피복하도록생육시켜발효두부를제조하였으며, 발효가종료된발 효두부는즉시동결건조시켜분쇄하였다. 동결건조된발효두부 5 g을시료의중량대비 1배의메탄올및물을첨가하여메탄올은 6 o C, 물은 1 o C에서 3회 3시간씩환류추출한후 rotary vacuum evaporator로완전감압농축한후면역활성측정시료로사용하였다. 비장세포증식생후 8주된생쥐 (BALB/c) 암컷에서분리한비장세포에서 spleenocyte를분리하여비장세포를 96 well plate에넣고여기에큰느타리버섯균사체발효두부물및메탄올추출물을농도별로첨가하여배양한다음비장세포증식정도를측정하였다. 비장세포증식측정은배양 48시간후, Cell titer 96 R solution을첨가하여 37 o C, 5% CO 2 incubator에서 4~8 시간동안배양한다음 Microplate reader(titer-tek Multiscan Plus, Helsinci, Finland) 를사용하여 49 nm에서 O.D. 값을측정하였다 (16). 일산화질소측정안정된일산화질소 (nitric oxide) 산화물인 NO 2- (nitrite) 는 Greiss 반응을이용하여측정하였다 (17). RAW 264.7( 대식세포 ) 에큰느타리버섯균사체발효두부의물및메탄올추출물을농도별로첨가하여 48시간배양한다음배양상층액을회수하여 96 well plate에 1 μl씩넣고 Greiss 시약 (.1% N-1-naphthyl-1-ethylendiamine in H 2O:1% sulfanilamide in 5% H 3PO 4) 을동량첨가하여 1분간반응시킨후, Microplate reader(titertek Multiscan Plus) 로 54 nm에서흡광도를측정하였다. Nitrite의농도는 sodium nitrite를이용하여 32 μm까지 2배씩희석하여얻은표준곡선과비교하여계산하였다. 사이토카인분비량측정비장세포또는대식세포주 (RAW264.7) 가분비하는사이토카인농도측정은 ELISA법을이용하여결정하였다. 즉, flat-bottomed micro well plate에 goat anti-mouse cytokine 항체 (1차항체 ) 를 coating buffer를이용하여 4 o C에서 overnight incubation한후, 3% BSA용액으로 2시간동안상온에서 blocking하였다. 실험에서채취한배양상층액을, plate에각각넣어서, 37 o C에서 2시간 incubation 시킨후, biotinylated anti-cytokine 항체 (2차항체 ) 를첨가하였다. 그리고 avidin-conjugated alkaline phosphate를적당량가하고, 37 o C에서 2시간 incubation 시키고, 기질로 p-nitrophenyl phosphate를넣은후 Microplate reader(titertek Multiscan Plus) 를사용하여 41 nm와 45 nm에서흡광도값으로측정하여나타내었다. 이때각사이토카인의측정한계는 1 pg/ml이었다 (18). 통계처리실험결과는평균 (mean)± 표준편차 (SD) 로나타내었고, Student t-test를이용하여통계처리한후 p<.5 수준에서
큰느타리버섯균사체로제조한발효두부추출물의면역활성 27 유의성을검정하였다. 결과및고찰 큰느타리버섯균사체를이용한발효두부의제조 큰느타리버섯균사체의생육을위한최적배지를검토하기위하여 PDA배지외 2종의평판배지에대한버섯균사체의생육정도를관찰하여 Table 1에나타내었다. 즉이들평판배지에서큰느타리버섯의균사생육은모두왕성하게나타났으나 MEA와 MPA 평판배지에서보다 PDA 평판배지에서의균사생육이더양호하게나타나큰느타리버섯의생육배지로는 PDA배지가적당한것으로생각되었다. 따라서발효두부제조를위하여큰느타리버섯균사체의생육을위한최적배지로는 PD broth배지를사용하였다. 큰느타리버섯균사체의배양용액및포자현탁액을두부에접종한후 3 o C의항온기에서 7일동안배양하여제조한발효두부의성상은 Fig. 1과같다. 즉, 큰느타리버섯균사체를접종한두부에서는배양 2일째부터흰색의균사가성장하기시작하여배양 5일째부터는두부표면전체가솜털모양의균사덩어리로뒤덮였다. 이와같이균사가뒤덮인상태 ( 배양 7일째두부 ) 의두부를수직으로절단하여두부내부로의균사침투정도를관찰한결과두부표면으로부터약 3~5 mm 정도의깊이까지만큰느타리버섯의균사가두부내부로침투하여성장하는것을관찰할 Table 1. Growth effect of Pleurotus eryngii mycelia by several media Media Mycelia (diameter, mm) 2 day 4 day 6 day 8 day MEA 1) MPA 2) PDA 3) 13.2 14.3 13.8 24. 24.5 25.7 36.7 39.4 43.8 5.3 56.2 63.8 1) MEA: malt extract 2%, dextrose 2%, peptone.1%, MgSO 4 7H 2O.5%, agar 2%. 2) MPA: malt extract 2%, peptone.5%, agar 2%. 3) PDA: potato 2%, dextrose 2%, agar 2%. Fig. 1. Photograph of soybean tofu fermented with Pleurotus eryngii mycelia. 수있었다. 하지만이상태에서배양 1일째까지계속배양하였을때버섯의균사가두부내부로의침투는더일어나지않고오히려부분적으로다른세균등에의한오염이발생하였다. 따라서발효두부제조를위한두부의발효기간은 7일정도가적당한것으로판단되었다. Lee 등 (19) 은홍삼추출물을첨가하지않은대조군은저장후 4일에부패가시작되었으나, 홍삼추출물을.5% 와 2% 첨가한두부의부패는 6일이후에시작되었다고보고하였다. 또한본연구에서도두부를 37 o C에서 4일동안저장하였을때 1일째부터두부의색깔변화, 이취발생및세균수증가등의부패가시작됨을확인하였다. 따라서큰느타리버섯균사체를접종한두부의발효기간 7일동안세균등의오염이관찰되지않았으므로발효두부의저장성이일반두부보다향상될것으로생각되며, 앞으로본연구의결과를토대로큰느타리버섯균사체발효두부의품질특성및저장성에관한구체적인연구들이더진행되어야할것으로생각된다. 비장세포증식유도생쥐의비장에서분리한세포에큰느타리버섯균사체발효두부의물및메탄올추출물을.1,.1, 1, 1 및 1 μg/ ml 농도로첨가하고 48시간배양한후비장세포의증식반응을측정하였다. 즉, B세포의증식을유도하는 LPS와 T세포의증식을유도하는 α-cd3에의해서비장세포의증식을유도하였으며, 큰느타리버섯균사체발효두부추출물을첨가하지않은대조군 (.7) 에비해물및메탄올추출물을첨가한실험군에서는저농도.1 μg/ml 이상에서비장세포의증식반응이증가하는것으로나타났으나 (Fig. 2), 본연구진의이전연구에서는큰느타리버섯균사체물추출물 1 μ g/ml 농도에서도대조군에비하여유의적으로비장세포의증식을유도하지못하였다 (2). 따라서큰느타리버섯균사체발효두부추출물이동일한농도 (1 μg/ml) 에서큰느타리버섯균사체추출물보다비장세포증식을높게유도한이유는발효두부제조시생성되는기능성물질들의상승효과에기인한것으로생각되며, 추후더구체적인연구가진행되어야할것으로판단된다. 한편, Hong 등 (21) 은콩발효식품인청국장물추출물이마우스비장세포의증식을유도한다고보고한바있으며, Kang 등 (22) 은큰느타리버섯조다당체추출물이 3 μg/ml 농도이상에서비장세포의증식을유도한다고보고하였다. 비장세포사이토카인생산유도비장세포에대한독성이없는최대농도의큰느타리버섯균사체발효두부물및메탄올추출물 (1 μg/ml) 을첨가하여 24시간배양한다음에상층액을분리하여 IL-2, IFN-γ, IL-6 및 TNF-α의사이토카인이분비되는지 ELISA 방법으로측정하였다 (Table 2). 그결과큰느타리버섯균사체발효두부물추출물은대조군에비하여 IL-2, IFN-γ의사이토
28 이상원 강종우 김재용 박경욱 박석규 주옥수 이성태 서권일 (A) OD at 49 nm 1.8 1.6 1.4 1.2 1.8.6.4.2 Contorl LPS α-cd3.1.1 1 1 1 (B) OD at 49 nm 1.8 1.6 1.4 1.2 1.8.6.4.2 Contorl LPS α-cd3.1.1 1 1 1 Concentration (µg/ml) Concentration (µg/ml) Fig. 2. Effects of the extracts of tofu fermented with Pleurotus eryngii mycelia on the growth of spleen cells. (A) Water extract. (B) Methanol extract. Data values were expressed as mean±sd of triplicate determinations. Significant differences were compared with the control group at p<.5 by Student's t-test. Table 2. Effects of the extracts of tofu fermented with Pleurotus eryngii mycelia on the cytokines of spleen cells Cytokines, pg/ml IL 1) -2 INF 2) -γ IL-6 TNF 3) -α Control 1.2±. 16.±8.2 15.1±. 248.3±.7 LPS (1 μg/ml) 4) <1 855.6±8.1 2,266.5±16. 1,6.6±4.7 α-cd 5) 3 (.1 μg/ml) 616.7±13.1 12,987.6±8.6 1,75.5±21.2 625.±68.3 TEFPM 6) Water extract 11.±1. 59.2±7. 129.3±1.1 148.3±29. Methanol extract 1±. 33.±5.3 116.±27.1 133.8±3.9 1) IL: Interleukin. 2) INF: Interferon. 3) TNF-α: Tumor necrosis factor. 4) LPS: Lipopolysaccharide. 5) CD: Cluster of differentiation. 6) TEFPM: Tofu extracts fermented with Pleurotus eryngii mycelia. Data values were expressed as mean±sd of triplicate determinations. Significant differences were compared with the control group at p<.5 by Student's t-test. 카인분비량을증가시켰으며, 메탄올추출물은 IFN-γ의분비량만증가시켰다. 즉 IL-2는보조 T세포와세포독성 T세포의증식을유도하는사이토카인이며 (23), IFN-γ는내재면역반응에관여하는대식세포를활성화시키는중요한역할을한다 (24). Kang 등 (22) 은큰느타리버섯조다당체추출물이비장세포의사이토카인중 IL-6, IFN-γ의생산을증가시켜비장세포중에서 T세포의증식은유도하지않고, B세포의증식을유도한다고보고하였다. 한편본연구의이전연구결과에의하면큰느타리버섯균사체는 IL-2, IFN-γ와같은비장세포싸이토카인의분비량을유의적으로증가시키지못하였다. 따라서본연구결과및이전의연구결과를종합하여볼때큰느타리버섯균사체가두부에서발효되는과정에서새로운활성물질들이생성되는것으로생각되며, 이에대한심도있는연구들은차후에더진행되어져야할것으로생각된다. 대식세포일산화질소생산유도대식세포주 (RAW264.7) 에큰느타리버섯균사체의물및메탄올추출물을.1, 1 및 1 μg/ml의농도로처리하여 48 시간배양한후배양액중대식세포가생산한 NO의산화형태인 NO - 2 농도를측정하였다. 그결과물추출물은대조군 에비하여대식세포의일산화질소생산을 1 μg/ml 농도이상에서유의적으로증가시키는것을알수있었다. 한편메탄올추출물은 1 μg/ml 농도이상에서그생산을증가시켰다 (Fig. 3). Kim 등 (25) 은오미자발효액과오미자추출물 ( 물및에탄올 ) 의대식세포일산화질소생성을측정한결과발효액에서그생성이가장높음을알수있었으며, 발효액과추출물의함유성분을 HPLC로분석한결과용매추출물에서확인할수없었던새로운물질들이생성되었다고보고하였다. 따라서본연구의큰느타리버섯균사체발효두부의물및메탄올추출물에서대식세포의일산화질소생산증가는발효과정에서생성된대사산물이유효성분으로작용한결과로생각되며, 앞으로큰느타리버섯균사체발효두부의면역활성을나타내는생리활성성분의기작과이활성물질들에대한연구가필요할것으로생각된다. 대식세포사이토카인생산유도큰느타리버섯균사체발효두부의물및메탄올추출물에의해분비되는대식세포의사이토카인의종류 (IL-6, TNFα, IL-1β 및 GM-CSF) 와분비량을알아보기위하여대식세포주에일산화질소생산을최대로유도하는농도 (1 μg/ ml) 의물추출물과메탄올추출물을첨가하여 24시간배양한후, 상층액을회수하여상층액에포함된사이토카인의
큰느타리버섯균사체로제조한발효두부추출물의면역활성 29 (A) NO2- Concentration (µm) 12 1 8 6 4 2 Control 1 1.1 1 1 LPS Concentration (µg/ml) TEFPM (B) NO2- Concentration (µm) 12 1 8 6 4 2 Control 1 1.1 1 1 LPS Concentraiton (µg/ml) Fig. 3. Effects of the extracts of tofu fermented with Pleurotus eryngii mycelia on the production of nitric oxide in macrophage cells. (A) Water extract. (B) Methanol extract. Data values were expressed as mean±sd of triplicate determinations. Significant differences were compared with the control group at p<.5 by Student's t-test. TEFPM Table 3. Effects of the extracts of tofu fermented with Pleurotus eryngii mycelia on the cytokines in macrophage cells Cytokines, pg/ml IL 1) -6 TNF 2) -α IL-1β GM-CSF 1) Control 18.6±1.6 1993.7±93.6 <7.8 <1 LPS 4) (1 μg/ml) 34884.±678.8 54975.±424.2 46.1±1.2 859.5±1.6 TEFPM 5) Water extract 16248.±141.4 39487.5±1856.1 13.9±.6 381.±35.3 Methanol extract 7587.±31.8 3465.±1484.9 <7.8 154.5±3.5 1) IL: Interleukin. 2) TNF-α: Tumor necrosis factor. 3) GM-CSF: Granulocyte-macrophage stimulating factor. 4) LPS: Lipopolysaccharide. 5) TEFPM: Tofu extracts fermented with Pleurotus eryngii mycelia. Data values were expressed as mean±sd of triplicate determinations. Significant differences were compared with the control group at p<.5 by Student's t-test. 양을측정하였다. 즉대식세포를활성화시키는물질인 LPS 는많은양의 IL-6, TNF-α, IL-1β 및 GM-CSF 생산을유도하는것을확인하였다. 큰느타리버섯균사체발효두부의물추출물은대조군에비해 IL-6, TNF-α, IL-1β 및 GM-CSF 분비량을유의하게증가시키는것으로나타났다 (Table 3). 한편메탄올추출물은 IL-1β를제외한나머지사이토카인의분비량을유의하게증가시키는것으로나타났다 (Table 3). 따라서큰느타리버섯균사체발효두부추출물은대식세포를활성화시켜다양한사이토카인을분비하여면역활성을유도하는것을알수있었다. 요약 일산화질소생산을 1 μg/ml 농도이상에서유의적으로증가시키는것을알수있었으며, 메탄올추출물은 1 μg/ml 농도이상에서그생산을증가시켰다. 발효두부추출물들은대식세포가분비하는 IL-6, TNF-α, IL-1β 및 GM-CSF 분비량을유의하게증가시켰다. 따라서큰느타리버섯균사체로발효한두부는기능성두부로개발이가능하리라생각된다. 감사의글본연구는 24년도농림기술관리센터 (ARPC) 연구비지원에의해수행된결과의일부이며, 이에감사드립니다 ( 과제번호 :24152). 두부의기능성및저장성을향상시킬목적으로큰느타리버섯균사체를이용한발효두부를제조하여물과메탄올로추출하여면역세포활성에미치는효과를조사하였다. 큰느타리버섯균사체를배양하기위한최적배지는 PD broth 배지인것을확인하였으며, 큰타리버섯균사체를이용한두부의최적발효기간은 7일정도가적당하였다. 큰느타리버섯균사체를이용하여발효한두부의물및메탄올추출물은.1 μg/ml 농도이상에서비장세포의증식을유도하였으며, 이들추출물은 IL-6, IFN-γ 분비를유도하는것으로나타났다. 발효두부물추출물은대조군에비해대식세포의 문 1. Shin YM, Kwon OY, Kang SY, Park JY, Park HS, Kim MR. 25. Physicochemical characteristics of tofu added with spinach juice. J Chungnam Home Economics 18: 61-66. 2. Oh SW, Lee YC, Hong HD. 22. Effects on the shelf-life of tofu with ethanol extracts of Rubus coreanus miquel, Therminalia chebula Retz and Rhus javanica. Korean J Food Sci Technol 34: 746-749. 3. Kim JS, Choi SY. 28. Quality characteristics of soybean curd with Omija extract. Korean J Food Nutr 21: 43-5. 4. Han MR, Kim MH. 27. Quality characteristics and stor- 헌
3 이상원 강종우 김재용 박경욱 박석규 주옥수 이성태 서권일 age improvement studies of Rubus coreanus added soybean curd. Food Engineering Progress 11: 167-174. 5. Chun KH, Kim BY, Son TI, Han YT. 1997. The extension of tofu shelf-life with water soluble degraded chitosan as immersion solution. Korean J Food Sci Technol 29: 476-481. 6. Lee KS, Kim DH, Baek SH, Choun SH. 199. Effects of coagulants and soaking solutions of tofu (soybean curd) on extending its shelf-life. Korean J Food Sci Technol 22: 116-122. 7. Jung GT, Ju IO, Choi JS, Hong JS. 2. Preparation and shelf-life of soybean curd coagulated by fruit juice of Schizandra chinensis ruprecht (omija) and Prunus mume (maesil). Korea J Food Sci Technol 32: 187-192. 8. Kim KT, Im JS, Kim SS. 1996. A study of the physical and sensory characteristics of ginseng soybean curd prepared with various coagulants. Korean J Food Sci Technol 28: 965-969. 9. Jung JY, Cho EJ. 22. The effect of green tea powder levels on storage characteristics of tofu. Korean J Soc Food Cookery Sci 18: 129-135. 1. Kim DH, Lim MS, Kim YO. 1996. Effect of seaweeds addition on the physicochemical characteristics of soybean curd. J Korean Soc Food Nutr 25: 249-254. 11. Kim SS, Park MK, Oh NS, Kim DC, Han MS, In MJ. 23. Studies on quality characteristics and shelf-life of chlorella soybean curd (tofu). J Korean Soc Agric Chem Biotechnol 46: 12-15. 12. Jeon MK, Kim MR. 26. Quality characteristics of tofu prepared with herbs. Korean J Food Cookery Sci 22: 3-36. 13. Park YJ, Oh NS, Han MS, Park MK, In MJ. 24. Effects of coagulants on the yield and textural properties of soybean curd (tofu) containing garlic. J Korean Soc Appl Biol Chem 47: 37-372. 14. Kim JH, Lee WJ, Cho YW, Kim KY. 29. Storage-life and palatability extension of Betula platyphylla sap using lactic acid bacteria fermentation. J Korean Soc Food Sci Nutr 38: 787-794. 15. Lee SH, Kim YT, Shon MY, Sung CK, Park SK. 21. Quality properties of fermented tofu prepared with different molds and coagulants. J Korean Soc Food Sci Nutr 3: 617-622. 16. Jeung YR, Ha MH, Kim SH, Jo SK, Byun MW, Cho HW, Seo KI, Yee ST. 2. Immunosupporessive effects of herbal plant extracts alloantigen reactive cell proliferation and cytotoxicity. J Korean Soc Food Sci Nutr 29: 1133-1138. 17. Kondo Y, Takano F. 1994. Nitric oxide production in mouse peritoneal macrophage enhanced with glycyrrhizin. Biol Pharm Bull 17: 759-761. 18. Kato T, Morokata T, Igarashi O, Yee ST, Inobe M, Uede T, Azuma M, Okumura K, Nariuchi H. 1997. Costimulatory effects of IL-2 on the activation of naive, memory CD4 T cells, and Th1 clone. Cell Immunol 176: 5-58. 19. Lee JS, Kim GN, Jang HD. 28. Effects of red ginseng extract on storage and antioxidant activity of tofu. J Korean Soc Food Sci Nutr 37: 1497-156. 2. Seo KI. 26. Commercialization and development of direct edible functional fermented tofu using biotechnology. Final Report of Reserch. Ministry for Food, Agriculture, Forestry and Fisheries, Seoul, Korea. p 42. 21. Hong SW, Kim JY, Lee BK, Chung KS. 26. The bacterial biological response modifier enriched Chungkookjang fermentation. J Korean Food Sci Technol 38: 548-553. 22. Kang HI, Kim JY, Kwang DM, Seo KI, Cho YS, Lee SD, Yee ST. 24. Effect of the crude polysaccharide of Pleurotus eryngii on the activation of immune cells. J Korean Soc Food Sci Nutr 33: 192-197. 23. Jain J, Loh C, Rao A. 1995. Transcriptional regulation of the IL-2 gene. Curr Opin Immunol 7: 333-342. 24 Osmond DG, Rolink A, Melchers F. 1998. Murine B lymphopoiesis: towards a unified model. Immunol Today 19: 65-68. 25. Kim CH, Kwon MC, Kim HS, Bae GJ, Ahn JH, Choi GP, Choi YB, Ko JR, Lee HY. 27. Enhancement of immune activities of Kadsura Japonica Dunal. through conventional fermentation process. Korean J Medicinal Crop Sci 15: 162-169. (29 년 8 월 13 일접수 ; 29 년 12 월 3 일채택 )