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1 농업생명과학연구 Journal of Agriculture & Life Science Vol. 42, No. 2, pp (2008) 기능성올리고당첨가가 Bifidobacterium 균에미치는영향 노효영장금일이강휘김광엽 * 충북대학교식품공학과 접수일(2008년 1월 21 일), 수정일(2008년 5월 23 일), 게재확정일(2008년 5월 23 일) Effects of functional oligosaccharides for physiological characteristics of Bifidobacterium spp. Hyo-Young Roh Keum-Il Jang Kang-Whi Lee Kwang-Yup Kim * Department of Food Science and Technology, Chungbuk National University, Chungbuk , Korea Received : January 21, 2008, Revised : May 23, 2008, Accepted : May 23, 2008 초록국내에서도소득증대와시장확대등에따라기능성식품에대한의존도가증대되고있다. 특히유아용및노인용식품제조에적합한유산균제제의개발과함께선발된유산균의생육을위한특정생장촉진물질의탐색이필요한실정이다. 따라서본연구에서는국내두유수유아의분변으로부터분리, 선발된 Bifidobacterium 균을생균제로서의능력을향상시키기위해 Bifidobacterium 균의성장요인으로알려져있고, 인체에유용한기능성올리고당의첨가에따른생육특성을조사하였다. 선발된균주는기능성올리고당이첨가된배지에서증식이더빠르게나타났고, 그중에서도 GOS가 5% 첨가된배지에서가장빠르게증식하였다. 또한, 기능성올리고당을첨가한경우 α -galactosidase와 β-galactosidase 의활성이더좋아졌고, 균체의세포막지방산조성을비교해본결과 Oleic acid (C 18:1 ω9 C ) 함량또한월등히높게나타났다. ph 변화와총산도실험을수행한결과, 기능성올리고당을첨가하지않은배지에서 ph 5.79의 0.873% 를나타낸반면, 5% GOS 가첨가된배지에서는 ph 3.6의 1.188%, 3% IMO 는 ph 3.69 의 1.134%, 그리고 FOS는 ph 3.71 의 1.098% 등으로, 현저히낮은 ph를나타냈다. 내담즙성의실험결과, 기능성올리고당중 5% GOS 가가장높은나타냈으나, 첨가하지않은경우보다는낮은수치를나타냈다. 병원성균성장저해실험을한결과, 5% GOS 를첨가한배지에서자란 Bif. breve PBH-30이 E. coli O157, Salmonella typhimurium, Staphylococcus aureus 등과같은병원성균을더효과적으로저해하였으며, 특히 E. coli O157 의생육이효과적으로저해되었다. In vitro 부착능을실험한결과, 대부분의 Bif. breve PBH-30이 Caco-2 cell 의세포외막의굴곡을따라부착되었음을확인하였다. 검색어 - 기능성올리고당, Bif. breve PBH-30, GOS, FOS, IMO ABSTRACT This study was performed to find the changes of growth properties of Bif. breve PBH-30 selected from korean infant by adding growth promotive factors such as galactooligosaccharides, fructooligosaccharides, and isomaltooligosaccharides. Bif. breve PBH-30 showed faster growth in TPY - containing functional oligosaccharides than TPY - (control ). The medium with 5% GOS showed the fastest growth and 3% IMO showed faster growth than 5% FOS. Bif. breve PBH-30 cultivated in TPY - containing functional oligosaccharides showed higher - activies of α-galactosidase and β-galactosidase than TPY (control ). Also activities of α-galactosidase and β-galactosidase were higher in TPY - than with functional oligosaccharides except 3% IMO. In the analysis of fatty acid, the content of C 18:1 ω9 C was 31.53% in TPY, 38.41% in TPY -, 55.50% in TPY -, 50.95% in TPY - containing 5% FOS, and 45.75% in TPY - containing 3% IMO. Bif. breve PBH-30 cultivated in the media that containing functional oligosaccharides showed remarkably low ph than TPY -. Also the total acidity of Bif. breve PBH-30 was 0.873% in TPY -, 1.188% in TPY - containing 5% GOS, 1.134% in TPY - containing 3% IMO, and 1.098% in TPY - containing 5% FOS. Although Bif. breve PBH-30 cultivated in TPY -, affected significantly than with other functional oligosaccharides, growth in TPY - (control ) supported highest bile tolerance. TPY - containing 5% GOS was more effective than TPY - in growth inhibition of pathogens such as E. coli O157, Salmonella typhimurium, and Staphylococcus aureus. Adherences of Bif. breve PBH-30 to Caco-2 cells were observed at most of the Caco-2 cell surfaces using confocal scanning microscope. Key words : functional oligosaccharides, Bif. breve PBH-30, GOS,FOS,IMO * 교신저자(Corresponding author) : 김광엽(Kwang-Yup Kim) Tel. : Fax. : kimky@chungbuk.ac.kr

2 38 J. of Agriculture & Life Science 42(2), June 2008 I. 서론 생균제(probiotic) 는미생물에의해생산된대사산물로, 다른미생물의생육을촉진하는물질이라고 Lilly와 Stillwell 에의해정의되었다(Lilly & Stillwell, 1965). 그후로생균제에대한많은정의가내려졌지만그중대표적인것은 Fuller 에의한정의로써, 생균제란살아있는미생물제제로, 숙주동물의장내균총을개선하여좋은영향을미치는것이라하였다 (Fulluer, 1989; Ouwehand et al., 1999). 최근에는그이용범위가가축에서인간에게로확대되면서그정의도넓어지고있다(Salminen et al., 1999). 알려진생균제의효과로는장내균총안정화, 병원성균의생육저해, 유당과민성증상완화, 면역조절, rotavirus diarrhoea 지속기간의단축, 분변박테리아효소의활성감소, 방광암의재발가능성축소, 혈청콜레스테롤, 저혈압등의감소등이있다 (Fulluer, 1989; Salminen et al., 1999; Hilton et al., 1992; Takiguchi et al.,1997; Goldin & Borbach, 1977; Matszaki et al., 1996; Gillialand & Walker, 1990). 일반적으로생균제로사용되는균주로는 Lactobacillus, Bifidobacterium, Enterococcus, Leuconostoc, Pediococcus, Propionibacterium, Bacillus, Yeast 등이동물용생균제로사용되고있다 (Shin et al., 1999; Clark et al., 1993; Kirjavainen et al., 2001). 생균제용박테리아의선발기준으로는사람기원, 내산성ㆍ내담즙성, 항생물질에대한저항성, 인체세포에의부착성, 병원성균에대한대항성, 인체무해성등이고려되어야한다 (Jung, 2001; Fernades et al., 1987; Canzi, 2000). Bifidobacteria는 Tissier(1899) 에의해처음발견되었으며그특성으로는비운동성, 그람양성, 절대혐기성, 무포자간균으로(Mitsuoka, 1990), 인체또는동물의장내에거하며숙주의건강에메우유익한균으로알려져있다 (Hoover, 1993; Yaeshima, 1996; Holzapfel et al.,1998; Alander et al., 1999). Prebiotic은체내에서잘소화되지않는식품구성성분으로서, 위장관상부에서가수분해나흡수가이루어져서는안되며, 제한된특정공생박테리아의생육또는활성에자극을주어결국에는장내균총을숙주의건강에유익한방향으로하는물질이다 (Tuohy et al., 2003; Collins & Gibson, 1999). 대부분의 올리고당들이며그종류로는 Inulin, prebiotic들은기능성 Galactooligosaccharides (GOS), Lactulose, Isomaltooligosaccharides(IMO), Fructooligosaccharides(FOS), Lactosucrose, Soybean oligosaccharides, Xylooligosaccharides, Gentiooligosaccharide 등이사용되고있다 (Rastall & Vatsala, 2002). 본연구에서는기능성올리고당이 Bif. breve PBH-30 의생육에미치는영향을분석하고, 또한기능성올리고당의영향을받은균체의 Caco-2 cell에대한부착도를확인하여 prebiotic으로서의기능성올리고당의효과를확인하고자하였다. Ⅱ. 재료및방법 2.1 Bacterial strains Probiotic으로서사용된유산균은두유수유아의분변에서분리된 Bifidobacteriaum breve PBH-30을사용하였다. 병원성균성장저해실험을하기위하여 E. coli O157:H7 (USDA, human isolate), Salmonella typhimurium KCTC 2515, 그리고 Staphylococcus aureus KCTC 1916 을사용하였다. 2.2 Culture media Bifidobacteriaum breve PBH-30의 배양을 위하여 TPY (Trypticase Pytone Yeast extract medium, Bergey`s manual) 에서 glucose를 제외한 TPY - (enrichment medium) 배지와 BS (selective medium) 가사용되었다. 장내위해세균의배양을위하여 TSB (Trypticase Soy Broth, BBL) 과 EMB agar, SS agar, 그리고 MSA agar (Difco, USA) 가사용되었다. 2.3 Functional Oligosaccharides Prebiotic으로서의기능성올리고당은삼양제넥스에서생산된 Fructo-oligosaccharide (FOS, purity 55%), Galacto-oligosaccharide (GOS, purity 50%), 그리고 Isomalto-oligosaccharide (IMO, purity 50%) 를사용하였다. 2.4 Screening step Growth of Bif. breve PBH-30 with prebiotics TPY - (control ) 과 GOS, FOS, IMO를각각 1%, 3%, 그리고 5% 씩함유된배지( 총10 종) 가사용되었다. 각각의배지에 Bif. breve PBH-30 (1x10 5 CFU/mL) 를접종하여 37, 42시간동안혐기배양하면서 0, 10, 18, 32, 48 시간대에 TPY agar를사용하여생장활성을관찰하였다 Enzymatic activities TPY - (control ) 과 GOS, FOS, IMO를각각 5%, 5%, 그리고 3% 씩함유된 TPY - 배지에 Bif. breve PBH-30를 37 에서 48 시간동안혐기배양한후,

3 Roh, Jang, Lee, Kim 39 : Effects of functional oligosaccharides for physiological characteristics of Bifidobacterium spp. API-ZYM kit 를이용하여효소의활성도를측정하였다. 배양액을원심분리하여침전된침전물을 2mL의희석액을이용하여 5-McFarland 의탁도로조정하였다. 그후 65μl의각각의표본을 tray에분주하여 37, 8시간동안혐기배양한후 ZYM A 시약과 ZYM B 시약을한방울씩적하하고 10분간상온에방치하여얻은결과를 API-ZYM kit manual 과비교하였다 Analysis of Fatty acid (GC-MIS, MIDI) TPY - (control ) 과 GOS, FOS, IMO를각각 5%, 5%, 그리고 3% 씩함유된 TPY - 배지에 Bif. breve PBH-30를 37 에서 48시간동안혐기배양한후 Sherlock Microbial Identification system (MIDI, Inc., HP 6890 Series GC System, USA) 을이용하여각각의지방산을분석하였다 Acid production Acid production은 ph 측정과총산도의측정으로규명하였다. TPY - (control ) 과 GOS, FOS, IMO 를각각 5%, 5%, 그리고 3% 씩함유된 TPY - 배지에 Bif. breve PBH-30를 37 에서 20시간동안혐기배양하는동안각각의시간대(0, 3, 6, 15, 20hrs) 에서의배양액의 ph 측정과총산도를구하였다. Total activity(%) = (amount (ml) of 0.1N-NaOH factor of 0.1N-NaOH) / amount of sample (ml) 100 Growth rate of Bif. breve PBH-30 with prebiotics such as GOS, FOS, and IMO Bile tolerance TPY - (control ) 과 GOS, FOS, IMO를각각 5%, 5%, 그리고 3% 씩함유된 TPY - 배지에 0.3% oxgall(bole, Sigma) 을첨가하고 Bif. breve PBH-30 를접종하여 37 에서 24 시간동안혐기배양한후, 균의활성도를측정하기위하여 TPY agar에도말하여시간대(0, 3, 6, 12, 24hrs) 별로생성된집락의수를측정하였다 (Fig. 1). 2.5 Inhibition on pathogen growth Bif. breve PBH-30의병원성균성장저해능을확인하기위하여유해장내세균인 E. coli O157, Sal. typhimurium, 그리고 Staphy. aureus 과함께 5% GOS가첨가된 TPY - 에서혐기적조건으로 37, 12 시간동안배양하면서각시간대(0, 3, 6, 12 hrs) 별로각균의선택배지에도말하여집락의형성을통하여그활성도를알아보았다. 2.6 In vitro adherence assay Hemocytometer 를이용하여농축 Caco-2 cell line 을계측하였다. 약 1.0x10 5 cells/ml 의 cell을 4-well chamber slide에옮겨완전한 monolayer를형성할때까지 37 에서배양(5% CO2-95% air atmosphere) 한후 PBS 용액(pH7.4) 으로 2 회세척한다. 그후 0.5mL DMEM 을첨가하고 0.5mL Bif. breve PBH-30 (1.0x10 9 CFU/mL) 를접종하여 37 에서 3시간동안배양한다. Live/Dead Baclight Bacterial Viability probe처리를한후 PBS 5mL로 5 회세척하고, Confocal Laser Scanning Microscope 를이용하여 Bif. breve PBH-30 의부착능을측정하였다. Enzymatic activites : API-ZYM kit Ⅲ. 결과및고찰 Analysis of fatty acid : MIDI Acid production ph measurement Total acidity Bile tolerance Prebiotic selection Fig. 1. Overall procedure of selection for prebiotics. 3.1 Growth of Bif. breve PBH-30 with functional oligosaccharides(gos, FOS, IMO) 유도기에서 Bif. breve PBH-30은 TPY - (control ) 에비하여기능성올리고당을첨가한 TPY - 에서더빠른성장을나타냈다. 배양 15시간후에는 TPY - (control ) 은느린성장세를계속유지하지만기능성올리고당을첨가한 TPY - 의경우는이미정지기를지나사멸기에진입하였다. 기능성올리고당의농도별실험에서는 5% FOS, 5% GOS, 그리고 3% IMO 에서더높은성장률을나타내었다(Fig. 2, 3, 4). 또한기능성올리고당들간의성장률비교실험에서 는 5% FOS 가가장높을성장률을보였다(Fig. 5).

4 40 J. of Agriculture & Life Science 42(2), June 2008 Fig. 2. Growth of Bif. breve PBH-30 with FOS (a) TPY -, (b) FOS 1%, (c) FOS 3%, (d) FOS 5% Fig. 5. Comparison of growth rates at the presence of different oligosaccharides (a) TPY -, (b) FOS 5%, (c) GOS 5%, (d) IMO 3% Fig. 3. Growth of Bif. breve PBH-30 with GOS (a) TPY -, (b) GOS 1%, (c) GOS 3%, (d) GOS 5% 3.2 Enzymatic activities Bif. breve PBH-30 의 α-galactosidase와 -galactosidase의활성도에서기능성올리고당을첨가한 TPY - 가 control 에비하여높게나타났다. 또한 α-glucosidase와 β-glucosidase의활성도역시 3% IMO를제외한모든기능성올리고당을첨가한배양액에서자란 Bif. breve PBH-30에서높게나타났다. 위의결과로미루어기능성올리고당의첨가는유당과민성증상의감소에효과가있을것으로판단된다 (Table 1). β Fig. 4. Growth of Bif. breve PBH-30 with IMO (a) TPY -, (b) IMO 1%, (c) IMO 3%, (d) IMO 5% 3.3 Fatty acid composition 미생물의지방산조성은온도, ph, 배지의조성등, 여러요인에의하여바뀔수있다(Tohnsson, 1995; Grogan & Cronan, 1997). 특히 Oleic acid (C 18:1 ω9 C ) 는성장촉진요소로작용한다고알려져있다(Nikkila, 1996). 본실험에서지방산분석결과, TPY 에서생육한 Bif. breve PBH-30의 C 18:1 ω9 C 함량은 31.53%, TPY - 는 38.41%, 5% GOS를첨가한 TPY - 는 55.50%, 5% FOS를첨가한 TPY - 는 50.95%, 그리고 3% IMO를첨가한 TPY - 는 45.75% 로나타나 5% GOS를함유한배지에서가장높은함량을보였다 (Table 2).

5 Roh, Jang, Lee, Kim 41 : Effects of functional oligosaccharides for physiological characteristics of Bifidobacterium spp. Table 1. Measurement of enzymatic activities Bi. breve PBH-30 Enzyme TYP - 5% GOS a 3% IMO b 5% FOS c Control Alkaline phosphatase Esterase(C4) Esterase Lipase(C8) Lipase(C14) Leucine arylamidase Valine arylamidase Crystine arylamidase Trpsin α-chymotrypsin Acid phospatase Naphtol-AS-BI-phosphohydrolase α-galactosidase β-galactosidase β-glucuronidase α-glucosidase β-glucosidase N-acetyl-β-glucosaminidase α-mannosidase α-fucosidase : A value ranging from 0 to 5 is assigned to the color standard, 0 represent a negative reaction: 5 represent a maximum intensity reaction. Values 1-4 represent intermediate reaction. The approximate activity may be estimate from color strength: 1 corresponds to 5 nanomoles. 2 to 10nanomoles. 3 to 20 nanomoles. 4 to 30 nanomoles. 5 to nanomoles. a,tpy -. b,tpy - containing 3% IMO. c,tpy - containing 5% FOS. Table 2. Fatty acid composition of Bif. breve PBH-30 cultured in TPY, TPY - and TPY - added prebiotics for48hrat37 Fatty acid composition(%) Strain Medium 14:1 14:0 16:0 ω5 C ISO 17:1 17:1 17:0 a ω8 C ω5 C 18:1 18:1 18:0 ω9 C ω7 C TPY Bif. breve PBH-30 TPY b ND ND ND TPY - +GOS 5% ND ND ND ND TPY - +FOS 5% ND 0.78 ND TPY - +IMO 3% 1.30 ND ND 0.77 ND a This position of the double can be located by counting from methyl( ) end the carbon chain. ω b ND : not detected

42 J. of Agriculture & Life Science 42(2), June 2008 3.4 Acid production Bif. breve PBH-30을기능성올리고당을함유한배지에서배양했을때의 ph 를측정한결과, TPY - (control) 에비하여현저히낮은 ph 를나타냈다. TPY - 는 ph 5.79 를나타낸반면, 5% GOS는 ph 3.

6 42 J. of Agriculture & Life Science 42(2), June Acid production Bif. breve PBH-30을기능성올리고당을함유한배지에서배양했을때의 ph 를측정한결과, TPY - (control) 에비하여현저히낮은 ph 를나타냈다. TPY - 는 ph 5.79 를나타낸반면, 5% GOS는 ph 3.6, 5% FOS는 ph 3.71, 그리고 3% IMO에서는 ph 3.69 를나타냈다(Fig. 6). functional oilgosaccharides (a) TPY -, (b) GOS 5%, (c) IMO 3%, (d) FOS 5% 3.5 Bile tolerance 5% GOS를첨가한 TPY - 에서생육한경우가 다른기능성올리고당을첨가한 TPY - 에서보다는 높은내담즙성을보였지만, 올리고당을첨가하지않은 TPY - (control) 에서가장높은내담즙성을나타냈 다 (Fig. 8). Fig. 6. ph values of Bif. breve PBH-30 with different functional oligosaccharides (a) TPY -, (b) GOS 5%, (c) IMO 3%, (d) FOS 5% 총산도는 TPY - 은 0.873% 였으나, 5% GOS 는 1.188%, 5% FOS는 1.098%, 그리고 3% IMO는 1.134% 로역시기능성올리고당을함유한배지에서더높은총산도를보였다 (Fig. 7). Fig. 8. Bile tolerance of Bif. breve PBH-30 in TPY - with and without functional oligosaccharides (a) TPY -, (b) FOS 5%, (c) GOS 5%, (d) IMO 3% Fig. 7. Total acidity of Bif. breve PBH-30 with different 3.6 Inhibition on pathogen growth 5% GOS를첨가한 TPY - 와 TPY - (control) 에서생육한 Bif. breve PBH-30의장내병원성균선장저해실험을한결과, 기능성올리고당을첨가한배지에서 E.coli O157, Sal. typhimurium, 그리고 Staphy. aureus 모두에서더높은생장저해도를보였다. 특히, E. coli O157의경우 TPY - (control) 에서는 3.3 x 10 8 CFU/mL의성장을나타냈지만 5% GOS를첨가한 TPY - 에서는 2.2 x 10 7 CFU/mL의성장도를보여위해균생육저해의효과가있는것으로판단된다 (Fig. 9, 10, 11).

7 Roh, Jang, Lee, Kim 43 : Effects of functional oligosaccharides for physiological characteristics of Bifidobacterium spp. (b) Control of Sal. typhimurium in TPY - (c) Mixed culture of Bif. breve PBH-30 and Sal. typhimurium in TPY - (d) Control of Bif. breve PBH-30 in TPY - (e) Control of Sal. typhimurium in TPY - (f) Mixed culture of Bif. breve PBH-30 and Sal. typhimurium in TPY - Fig. 9. Inhibition of E. coli O157 by Bif. breve PBH-30 in TPY - and in TPY - (a) Control of Bif. breve PBH-30 in TPY - (b) Control of E. coli O157 in TPY - (c) Mixed culture of Bif. breve PBH-30 and E. coli O157 in TPY - (d) Control of Bif. breve PBH-30 in TPY - (e) Control of E. coli O157 in TPY - (f) Mixed culture of Bif. breve PBH-30 and E. coli O157 in TPY - Fig. 11. Inhibition of Staphy. aureus by Bif. breve PBH-30 with in TPY - and in TPY - containing GOS 5% (a) Control of Bif. breve PBH-30 in TPY - (b) Control of Staphy. aureus in TPY - (c) Mixed culture of Bif. breve PBH-30 and Staphy. aureus in TPY - (d) Control of Bif. breve PBH-30 in TPY - (e) Control of Staphy. aureus in TPY - (f) Mixed culture of Bif. breve PBH-30 and Staphy. aureus in TPY - Fig. 10. Inhibition of Sal. typhimurium by Bif. breve PBH-30 in TPY - and in TPY - (a) Control of Bif. breve PBH-30 in TPY In vitro adherence assay Bif. breve PBH-30의 Caco-2 cell의부착을확인하기위하여 CLSM을이용하여그형태를관찰한결과대부분의균들이 cell에부착한것을확인할수있었다 (Fig. 12).

8 44 J. of Agriculture & Life Science 42(2), June 2008 Fig. 12. Adherence images of Bif. breve PBH-30 to Caco-2 cells using confocal laser scanning microscopy (magnification 40 10) adherence images(a, B) and no adherence image(c) Ⅳ. 감사의글 이논문은 2006년도충북대학교학술연구지원사업의 연구비 지원에 의하여 연구되었음 (This work was supported by Chungbuk National University Grant in 2006) Literature cited Alander, M., De Smet, I., Nollet, L., Verstraete, W., and Mattila Sandholm, T The effect of probiotic strains on the microbiota of the simulator of the human intestinal microbial ecosystem. Int. J. Food Microbiol. 46: Canzi, E., Raffaella, Z., Paola, C., Alberto, C., Gian, F. G., Carla, O., Monica, S., and Annamaria, F Modulation by lactic acid bacteria of the intestinal ecosystem and plasma cholesterol in rabbits fed a casein diet. Nutr. Res. 20(9): Clark, P.A., Cotton, L.N., Martin, J.H Selection of bifidobacteria for use as dietary adjuncts in cultured dairy foods: Ⅱ-Tolerance to simulated ph of human stomachs. Cult. J. Dairy Prod. 28: Collins M. D. and Gibson G. R Probiotics, prebiotics, and synbiotics: approaches for modulating the microbial ecology of the gut. Am. J. Clin Nutr. 69: 1052S-7S. Dennis W. Grogan and Jhon E. Cronan, Jr Cyclopropane ring formation on membrane lipids of bacteria. Microbiol Mol. Biol. Rev. 61(4): Fernades, C. F., Shahani, K. M., and Amer. M. A., Therapeutic role of dietary lactobacilli and lactobacillic fermented dairy products. FEMS Microbiol. Letter. 46: Fulluer, R Probiotics in man and animals. J. Appl. Bacteriol. 66: Gillialand, S. E., and Walker, D. K Factors to consider when selecting a culture of Lactobacillus acidophilus as a dietary adjunct to produce a hypocholesterolemic effect in humans. J. Dairy. Sci. 73: Goldin, B.R., and Borbach, S. D Alterations in fecal microflora enzymes related to diet, age, lactobacillus supplements, and dimethylhydrazine. Cance. 40: Hilton, E., Henry, D.I., Phyllis, A., Kenneth, F., and Michael, T. B Ingestion of yoghurt containing Lactobacillus acidophilus as prophylaxis for candidal vaginitis. Ann. Int. Med. 116: Holzapfel, W.H., Haberer, P., Snel, J., Schillinger, U., and Huis in't Vele, H Overviex of

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untitled 3. 농업환경연구과 과제구분 기본연구 수행시기 전반기 연구과제 및 세부과제 수행 기간 소 속 책임자 농가에 적합한 부식성곤충 대량 사육기술 개발 12~ 13 농업환경연구과 곤충팀 이영혜 1) 부식성 곤충 먹이 제조 기술 개발 12~ 13 농업환경연구과 곤충팀 이영혜 색인용어 부식성곤충, 장수풍뎅이, 계통, 먹이제조 ABSTRACT In first check,