Korean J. Food Sci. An. Vol. 33, No. 6, pp. 772~780(2013) DOI http://dx.do.org/10.5851/kosfa.2013.33.6.772 ARTICLE 계절에따라여러지역의원유에서분리된내냉성미생물의효소활성 신용국 1 오남수 1 이현아 1 남명수 * 충남대학교농업생명과학대학동물바이오시스템과학과, 1 서울우유협동조합중앙연구소 Enzyme Activity of Isolated Psychrotrophic Bacteria from Raw Milk of Different Regions on Season Yong Kook Shin 1, Nam Su Oh 1, Hyun Ah Lee 1, and Myoung Soo Nam* Division of Animal Science & Resources, Chungnam National University, Daejeon 305-764, Korea 1 R&D Center, Seoul Dairy Cooperative, Ansan 425-839, Korea Abstract The aim of this study was to investigate the effect of season and location on activities of enzyme produced by psychrotrophic bacteria isolated from raw milk located in Kyunggi region of South Korea. Agar diffusion and colorimetric methods were used for the lipase and protease activities of psychrotrophic bacteria. Intensities of dark blue and transparent ring around colony were compared for activity measurement. Nutrient agar with 1% skim milk added was employed for measuing protease activity. 14 strains of Arthrobacter russicus with lipase activity and 19 strains of Chryserobacterium shigense with protease activities were found to be present. It was found that Acinetobacter genomospecies 10 (match %: 99.90) isolated from B region in fall was the most lipolytic species, whereas Serratia liquefaciens (match %: 99.39) isolated from the same region in spring was the most proteolytic species. Growth curve of Acinetobacte and Serratia liquefaciens was a typical sigmoidal form. Lipase activity increased with incubation time, but its activity began to drop at stationary to motality phase. Optimum condition for incubation time, ph and temperature for extracellular lipase from Acinetobacter genomospecies 10 (match %: 99.90) was 12 h, 8.5, and 45 o C, respectively. Extracellular protease from Serratia liquefaciens (match %:99.39) had the same optimum incubation time and ph as extracellular lipase, but optimum temperature was 35 o C. Key words: psychrotrophic bacteria, lipolytic species, proteolytic species 서 원유내의내냉성미생물은냉장저장중증식하여균체외로내열성효소를생산하며, 이는대부분미생물의생장곡선중정지기 (stationary phase) 에서생성된다 (McKellar, 1989). 이러한효소는주로지방분해효소나단백질분해효소로서저온살균또는초고온살균과같은시유의살균과정이후에도균체는사멸되지만효소의활성은잔존하여유제품의품질과수율의저하를유도하게된다 (Cousin, 1982; McKellar, 1989; Witter, 1961). 대표적인내냉성미생물인 Pseudomonas 균종으로부터분비되는단백질분해효소는 1 몰당하나의아연원자와최대 8개까지의칼슘원자를포함하는 metalloenzyme이며, 대부분이 κ-, α s1 - 및 β-casein을 *Corresponding author: Myoung Soo Nam, Division of Animal Science & Resources, Chungnam National University, Daejeon 305-764, Korea. Tel: 82-42-821-5782, Fax: 82-42-823-2766, E-mail: namsoo@cnu.ac.kr 론 분해하여우유응고활성을가지고있는반면유청단백질에서는낮은활성을나타내는것으로보고되었다. 또한 Pseudomonas 균종으로부터분비되는지방분해효소는유지방을포함한지용성의기질에활성을나타내는반면, 이로부터분비되는 esterase의경우균체내에존재하여유지방의가수분해에유의적인영향을주지않는것으로보고되었다 (McKellar, 1989). 지방분해효소의기질특이성은상반된연구결과가보고 (Cousin, 1982; McKellar, 1989) 되었는데내냉성미생물의지방분해효소가위치특이성이없는 lipoprotein lipase와같은역할을한다고보고한반면, Makhzoum 등 (1996) 은 triglyceride의 sn-1과 sn-3에위치하는지방산에효소활성의특이성이있는것으로보고하였다. 본연구는원유에서분리한내냉성미생물이생산하는단백질분해효소와지방분해효소의활성을측정하여효소활성이높은균주를선발하고이로부터생산된효소의생화학적특성과조효소의활성을조사하여원유의품질에영향을미치는기초자료를제공하는데있다. 772
Enzyme Activity on Psychrotrophic Bacteria by Season and Region 773 재료및방법 원유의시료채취원유의시료는 Shin(2013) 의보고에따라지역별로채취하여실험에사용하였다. 내냉성미생물의동정내냉성미생물은 Shin(2013) 의보고에따라동정하여실험에사용하였다. 지방분해효소와단백질분해효소의활성측정및균주선발내냉성미생물이생산하는효소활성은 agar 확산법과 colorimetric 법으로측정하였다. 지방분해효소의 agar 확산법은 Sprit blue agar(becton, Dickson and Company, USA) 를이용하여 colony 주변의진한푸른색의환을확인하여활성정도를비교하였으며, 단백질분해효소의경우 1% skim milk(becton, Dickson and Company, USA) 를첨가한 nutrient agar(becton, Dickson and Company, USA) 에서투명한환을확인함으로써활성정도를비교하였다 (Ramesh and Sudesh, 2007). Agar 확산법으로지방분해효소와단백질분해효소활성이높은균을선발하여각각 colorimetric 법으로활성정도를비교하였다. 지방분해효소의 colorimetric 법은 Shamsher 등 (2005) 의방법을변형하여수행하였다. 0.05 M Tris buffer(ph 8.5) 2.85 ml에 75 µl의 20 mm p-nitrophenyl palmitate(p-npp) stock solution과 enzyme solution 75 µl을혼합하여 45 o C의항온수조에서 20분동안반응시켰다. 반응이끝난후, ethanol과 acetone을 1:1의비율로혼합하여 -20 o C에서보관한 solution을 1mL 첨가하여반응을완전히종료시켰다. 이반응물을 0.45 µm syringe filter로여과시켜 410 nm에서 UV spectrophotometer로 p-nitrophenol (p-np) 의농도를측정하여지방분해효소활성을비교하였다. 단백질분해효소의 colorimetric 법은 Hull(1947) 의방법을변형하여수행하였다. 5 ml의 10% skim milk와 1 ml의 enzyme solution을 30 o C 항온수조에서 2시간동안반응시킨후, 0.7 N trichloroacetic acid 10 ml을가하여혼합한후 10분동안상온에방치하였다. Whatman filter paper No. 2(Whatman, UK) 를이용하여여과한여과액 2.5 ml와 sodium carbonate reagent 5 ml를혼합하여 1.5 ml의 Folin's reagent와반응시켰다. 10,000 rpm/min에서 5분동안원심분리시켜상징액을 650 nm에서흡광도를측정하여생성된 tyrosine의양으로단백질분해효소활성을측정하였고, 생성된 tyrosine의양은 µg/ml로나타내었으며, tyrosine의양으로작성한표준곡선에따라활성을비교하였으며, 지방분해효소활성과단백질분해효소활성이가장높은균주를선발하였다. 선발된균주의생물학적특성및조건별조효소의활성비교효소활성이높은균주로선발된균은생물학적특성을측정하기위해 1 colony를 3mL의 nutrient broth(becton, Dickson and Company, USA) 에접종하여 12시간동안배양한후, 99 ml의 nutrient broth에배양액 1mL을계대배양하여 0시간부터배양시간별로 600 nm에서흡광도를측정하여생장곡선을작성하였으며, 효소는 ph, 반응온도및배양시간에따라활성을비교하였다. ph에따른효소활성은 ph 2.5 에서 ph 13.5 사이에서측정하여비교하였고, 반응온도에따른효소활성은 5 o C에서 75 o C 사이에서측정하여활성을비교하였다. 통계분석본실험은 3반복으로수행하였으며실험결과는 SAS 프로그램 (2010) 을이용하여분산분석을실시하고 Duncan의다중검정법을통해 5% 수준에서처리구평균값간의유의성을검증하였다. 결과및고찰 단백질분해효소및지방분해효소활성균주의분리동정한 psychrotrophic bacteria (Shin et al., 2013) 를계절별로분류하고지방분해효소활성과단백질분해효소활성을 agar 확산법으로측정하여그결과를육안으로관찰한뒤, 분해능이높은순서대로 +++, ++, + 로표시하고활성이없는균주의경우 Ο으로표시하였다 (Table 1). 분리된균주일부는지방분해효소활성또는단백질분해효소활성을나타내지않았고, 지방분해효소활성을나타내는균주모두가또한단백질분해효소활성을나타내지도않았다. Table 1에나타낸것처럼지방분해효소의활성이강력한균주 (+++) 는 Arthrobacter russicus를포함한 14개균주, 단백질분해효소의활성이강력한균주 (+++) 는 Chryseobacterium shigense를포함한 19개균주로확인되었다. 지방분해효소와단백질분해효소의활성 Table 1에나타낸바와같이 Agar 확산법으로지방분해효소와단백질분해효소활성정도를측정하여높은활성을나타낸 24개의균주을선발한후, colorimetric 법으로효소활성을측정하였다. 지방분해효소활성측정결과는 Fig. 1 에나타난바와같이가을철 B 지역에서공급받은원유로부터분리한 20번균주 (B20_F, Acinetobacter genomospecies 10, match %: 99.90) 가 3.41 µg/ml의 p-np를생성하면서가장높은활성을나타내었고, 그다음으로 2.21 µg/ml을생성한 D 지역의겨울철원유로부터분리한 3번균주 (D3_W, Psychrobacter maritimus, match %: 99.90) 가높은지방분해효소활성을나타내었다 (p<0.05). 반면에가을철 D지역원
774 Korean J. Food Sci. An., Vol. 33, No. 6 (2013) Table 1. Lipase and protease activities of isolated bacteria over four seasons Season Closest relative in MicroSeq Library Lipase Activity Protease Activity Chryseobacterium shigense +++ Rahnella aquatilis Acinetobacter haemolyticus Sphingobacterium faecium + Pseudomonas poae Rhodococcus baikonurensis Acinetobacter genomospecies 10 Curtobacterium flaccumfaciens ++ Rhodococcus erythropolis DSM=43933 + Chryseobacterium scophthalmum ++ Pseudomonas marginalis +++ Arthrobacter russicus +++ Microbacterium liquefaciens + Yersinia kristensenii Obesumbacterium proteus Janthinobacterium lividum Raoultella omithinolytica + Spring Acinetobacter genomospecies 3 ++ Pedobacter heparinus Acinetobacter Iwoffii +++ Acinetobacter baumannii +++ Serratia grimesii + Yersinia aldovae + Pseudomonas asplenii Serratia liquefaciens +++ Pseudomonas fluorescens ATCC=13525 +++ Pseudomonas fluorescens A(bt) ATCC=17554 ++ Psychrobacter faecalis Raoultella planticola Pseudomonas mucidolens ++ Pseudomonas fragi Psychrobacter maritimus ++ Pseudomonas brenneri ++ Pseudomonas viridiflava Carnobacterium maltaromaticum ++ Pseudomonas brenneri ++ Pseudomonas asplenii Sphingobacterium faecium Chryseobacterium indologenes Pseudomonas mucidolens ++ Kluyvera cryocrescens + Enterobacter amnigenus + Leclercia adecarboxylata +++ Chryseobacterium indoltheticum ++ Summer Pseudomonas fluorescens A(bt) ATCC=17554 +++ Pseudomonas fragi Azorhizophilus paspali +++ Pseudomonas chlororaphis +++ Acinetobacter Iwoffii + Raoultella planticola Pantoea agglomerans ATCC=27155 Hafnia alvei + Exiguobacterium acetylicum + +++ Acinetobacter genomospecies 9 ++
Enzyme Activity on Psychrotrophic Bacteria by Season and Region 775 Table 1. Lipase and protease activities of isolated bacteria over four seasons (Continued) Season Closest relative in MicroSeq Library Lipase Activity Protease Activity Acinetobacter genomospecies 11 ++ Summer Acinetobacter genomospecies 10 +++ Acinetobacter johnsonii +++ Acinetobacter johnsonii +++ Acinetobacter genomospecies 9 ++ Acinetobacter genomospecies 10 +++ Acinetobacter genomospecies 11 ++ Hafnia alvei + Exiguobacterium oxidotolerans +++ +++ Chryseobacterium indoltheticum ++ Enterobacter amnigenus + Pantoea agglomerans ATCC=27155 Pseudomonas mucidolens ++ Acinetobacter haemolyticus Janthinobacterium lividum ATCC=12473 Escherichia coli ATCC=35382 Pseudomonas lundensis + Sphingobacterium faecium Carnobacterium maltaromaticum ATCC=35586 + Rhodococcus baikonurensis Fall Chryseobacterium indologenes Chryseobacterium wanjuense Chryseobacterium shigense Chryseobacterium gleum ++ Acinetobacter baumannii ++ Stenotrophomonas rhizophila ++ Gordonia terrae ++ Pseudomonas asplenii + Rahnella aquatilis Raoultella terrigena + Pseudomonas poae ++ Acinetobacter genomospecies 3 Pseudomonas fulva + + Pseudomonas brenneri ++ Brevundimonas diminuta Pantoea dispersa +++ Microbacterium liquefaciens +++ Serratia quinivorans + +++ Acinetobacter johnsonii +++ Sphingobacterium faecium Serratia fonticola Chryseobacterium joostei Chryseobacterium shigense +++ Serratia liquefaciens Acinetobacter genomospecies 9 Winter Acinetobacter genomospecies 11 ++ Acinetobacter haemolyticus Ï Pseudomonas extramorientalis ++ +++ Acinetobacter genomospecies 10 +++ Flavobacterium saccharophilum + Rhodococcus baikonurensis Stenotrophomonas rhizophila +++ Rhodococcus erythropolis ATCC=4277 Janthinobacterium lividum ATCC=12473
776 Korean J. Food Sci. An., Vol. 33, No. 6 (2013) Table 1. Lipase and protease activities of isolated bacteria over four seasons (Continued) Season Closest relative in MicroSeq Library Lipase Activity Protease Activity Pantoea agglomerans ATCC=27155 Pseudomonas lundensis Pseudomonas brenneri +++ Pseudomonas fluorescens A(bt) ATCC=17554 +++ Arthrobacter sulfureus ATCC=19098 Chryseobacterim balustinum +++ Pseudomonas marginalis + ++ Arthrobacter russicus +++ Psychrobacter faecalis + Hafnia alvei Pedobacter heparinus Winter Psychrobacter maritimus +++ Lactococcus raffinolactis + Pseudomonas veronii + Serratia grimesii ++ +++ Acinetobacter baumannii Pseudomonas mucidolens ++ Chryseobacterium scophthalmum Yersinia intermedia Arthrobacter pascens Serratia plymuthica +++ Lactococcus lactis cremoris ATCC=19257 Streptococcus parauberis Fig. 1. Lipase activities of isolated bacteria from different location over four seasons. A~H: different locations, F: fall, W: winter, S: summer, s: spring. Significant differences indicated by different letters were analyzed using Duncan's multiple range test (p<0.05). 유의 17번균주 (D17_F, Lactococcus raffinolactis, match %: 99.94), 가을철 H 지역원유의 20번균주 (H20_F, Exiguobacterium oxidotolerans, match % : 96.79), 가을철 A 지역원유의 7번균주 (A7_F, Acinetobacter johnsonii, match %: 99.58), 겨울철 E 지역원유의 7번균주 (E7_W, Arthrobacter russicus, march %: 93.19) 의경우에는지방분해효소활성이거의나타나지않았다 (p<0.05). Elionora와 Malka(2007) 에따르면, Bacillales 강에속하는 Enterococcus, Leuconostoc, Streptococcus, Lactococcus 속의경우지방분해효소또는단백질분해효소의활성이거의나타나지않으며, Alphaproteobacteria 강에속하는 Brevundimonas 속, Betaproteobacteria 강에속하는 Delftia 속, Gammaproteobacteria 강에속하는 Pseudomonas 속과 Acinetobacter 속, Flavobacteria 강에속하는 Chryseobacterium 속, Sphingobacteria 강에속하는 Sphingobacterium 속이가장대표적인지방분해효소활성을갖는내냉성미생물이라고보고하였다. Table 1에서도지방분해효소활성이높은미생물로 Gammaproteobacteria 강에속하는 Acinetobacter 속, Psychrobacter 속이선발되었으며, Bacillales 강에속하는 Lactococcus 속과 Exiguobacterium 속, Gammaproteobacteria 강에속하는 Acinetobacter
Enzyme Activity on Psychrotrophic Bacteria by Season and Region 777 Fig. 2. Protease activities of isolated bacteria from different location over four seasons. A~C, E~H: different locations, W: winter, S: summer, s: spring, F: fall. Significant differences indicated by different letters were analyzed using Duncan's multiple range test (p<0.05). 속, Actinobacteridae 강에속하는 Arthrobacter 속의경우지방분해효소활성이거의나타나지않았다. 이와같이 Gammaproteobacteria 강에속하는 Acinetobacter 속이지만 Acinetobacter genomospecies 10(B20_F, match %: 99.90) 의경우지방분해효소활성이가장높게측정된반면, Acinetobacter johnsonii(a7_f, match %:99.58) 의경우는지방분해효소활성이거의없는것으로보아같은속의종간에도효소활성정도에는차이가있는것으로판단되며, 이는 Elionora와 Malka(2007) 가보고한내용과도일치하였다. Acinetobacter 속에속하는균종은그램음성구균으로여러병원성감염을유발하며균혈증, 요로감염, 뇌막염의원인이되고있다 (Constantinju et al., 2004). Acinetobacter johnsonii(genomospecies 7) 를비롯하여 Acinetobacter lwoffii(genomospecies 8) 등현재까지 32종의균종이보고되었고, 특히 Acinetobacter baumannii에의한감염이 90% 이상을차지하고있다 (Bergogne-Bérézin and Towner, 1996). 단백질분해효소활성측정은 colorimetric 방법으로반응시킨후 18개균주의단백질분해효소활성을비교한결과는 Fig. 2와같다. 이중가장높은활성을나타낸균주는봄철 B 지역에서공급받은 1번균주 (B1_s, Serratia liquefaciens, match %: 99.39) 로 24.17 µg/ml의 tyrosine을생성하였다. 겨울철 F 지역의원유에서분리한 13번균주 (F13_W, Serratia plymuthica, match %: 89.53) 가 17.92 µg/ml의 tyrosine을생성하여그다음으로높은단백질분해효소활성을나타내었다. 이와반면에겨울철 H 지역의원유에서분리한 14번균주 (H14_W, Pseudomonas extremorientalis, match %: 99.82), 겨울철 C 지역의원유에서분리한 11번균주 (C11_W, Pseudomonas fluorescens A(bt) ATCC=17554, match %: 100.00), 13번균주 (C13_W, Pseudomonas brenneri, match %: 100.00), 여름철 G 지역의원유에서분리한 7번균주 (G7_S, Pseudomonas chlororaphis, match %: 99.82) 와 8번균주 (G8_S, Azorhizophilus paspali, match %: 99.96) 의경 우활성이나타나지않았다. Elionora와 Malk(2007) 의보고에서는단백질분해효소활성이높은내냉성미생물로 Flavobacteria 강에속하는 Chryseobacterium 속과 Actinobacteria 강에속하는 Microbacterium 속을대표적인균으로명시하였다. 본연구에서는 Gammaproteobacteria 강에속하는 Serratia 속이가장높은단백질분해효소활성을나타내었고, Pseudomonas 속과 Azorhizophilus 속의경우단백질분해효소활성이거의나타나지않았다. Morales(2003) 등의연구에서도 Serratia 속이단백질분해효소활성이매우높은균주라고보고하였다. 본연구에서단백질분해효소활성이가장높게나타난균주인 Serratia liquefaciens는그램음성균으로 Enterobacteriaceae 과에속한다. 지방분해효소와단백질분해효소활성이높은균주의성장과조효소의특성지방분해효소활성이가장높게나타난균주인 Acinetobacter geno- mospecies 10(match %: 99.90) 의생장곡선은 Fig. 3(A) 와같다. 배양시작후 2시간까지균수가서서히증가하는유도기 (lag phase) 였으며, 그이후로 6시간까지급격하게균수가증가하는지수기 (exponential phase) 를거쳐, 21시간까지정지기 (stationary phase) 를거친후, 21시간이지난후부터는균수가감소하는사멸기 (death phase) 로접어들었다. 이는미생물의생장곡선의전형적인시그모이드 (sigmoid) 곡선의형태를보여주었다. 단백질분해효소활성이가장높게나타난봄철 B 지역에서공급받은 1번균주 (Serratia liquefaciens, match %: 99.39) 의생장곡선은 Fig. 3(B) 와같다. 배양을시작한지 3시간동안유도기 (lag phase) 를유지하다가, 배양 8시간까지급격하게균수가증가하여지수기 (exponential phase) 를거쳐, 24 시간까지균수가거의일정하게유지되는정지기 (stationary phase) 에도달한후부터는균수가급격하게감소하는사멸기 (death phase) 로접어들었으며, 이는앞의 Acinetobacter
778 Korean J. Food Sci. An., Vol. 33, No. 6 (2013) Fig. 3. Growth curve of psychrotrophic bacteria at 30 o C. (A) Acinetobacter genomospecies 10, (B) Serratia liquefaciens. Fig. 4. Changes in lipase activities during incubation time. Significant differences indicated by different letters were analyzed using Duncan's multiple range test (p<0.05). (A) Acinetobacter genomospecies 10 (lipase), (B) Serratia liquefaciens (protease). genomospecies 10(match %: 99.90) 과같이전형적인생장곡선형태인시그모이드 (sigmoid) 곡선임을확인하였다. 지방분해효소활성균주로부터분리한조효소의특성을조사하기위하여배양시간별로지방분해효소활성을측정한결과, 유도기에속하는배양 1시간후부터지수기를지나정지기의중반정도, 즉배양한지 12시간까지지속적으로지방분해효소활성이증가하다가배양 12시간후에지방분해효소활성이최대를나타내었다 (Fig. 4A). 하지만그이후로정지기후반부에서부터사멸기에도달하기까지는배양시간이증가할수록활성이감소하는것을확인할수있었다. 이균주가생산한조효소의특성은배양시간별로단백질분해효소의활성을측정한결과, 배양 7시간까지는활성을나타내지않다가 9.5시간부터활성이점진적으로증가하여 12시간후에가장높은활성을나타내었고, 18시간후에는다시감소하는양상을나타내었다 (Fig. 4B). 지방분해효소의 ph에따른활성정도를비교한결과, ph 8.5에서최대활성을나타내었으며 ph가증가할수록다시 감소하는추세를보였다 (Fig. 5A). ph에의한효소활성은 ph 2.5에서가장낮은활성을보였으며, ph 8.5까지는 ph가높아질수록활성이점차증가하다가 ph 8.5에서가장높은활성을나타내었으며, ph 13.5로증가할수록다시감소하는경향을나타내었다 (Fig. 5B). 온도에따른활성은 45 o C에서 2.76 µg의 p-np를생성하면서가장높은활성을나타내었고, 그이상의온도에서는다시활성이감소하는경향을나타내었다 (Fig. 6, A). Suzuki 등 (2001) 은반응온도별 Acinetobacter sp. strain no.6의지방분해효소활성을측정하였는데, 이균주의경우 20 o C에서가장높은분해활성을나타내어본실험과상이한결과를나타내었다. Kojima와 Shimizu(2003) 는 Pseudomonas fluorescens HU380의지방분해효소활성을측정하였는데, 반응최적조건은 ph 8.5, 45 o C로나타났으며, 이는본연구의 Acinetobacter genomospecies 10(match %: 99.90) 의결과와동일한것을알수있었다. 원유에서분리한내냉성미생물인 Pseudomonas sp. YJ103의지방분해효소활성을온도
Enzyme Activity on Psychrotrophic Bacteria by Season and Region 779 Fig. 5. Effect of ph on lipase activities at 45 C (A) and 30 C (B). Significant differences indicated by different letters were analyzed using Duncan's multiple range test (p<0.05). (A) Acinetobacter genomospecies 10 (lipase), (B) Serratia liquefaciens (protease). 별, ph별로비교측정한 Kim 등 (1997) 의보고에서는 30 o C 와 ph 7.5에서활성이가장높은것으로나타났다. 반응온도에따른효소활성은 5 o C에서 35 o C까지는온도가증가할수록활성이높아져 35 o C에서최고에도달했고, 75 o C 까지는온도가높아질수록감소하다가 75 o C에서는활성이거의나타나지않았다 (Fig. 6B). Lee(1993) 는원유에서분리한내냉성미생물중 Pseudomonas sp. K101의단백질분해효소활성을측정한결과, 최적활성온도와 ph는각각 30 o C와 ph 7.5로보고하였는데본실험과유사한결과를나타내었다. 요 지방분해효소활성이가장높은균은가을철에집유한 B 지역의원유로부터분리한 Acinetobacter genomospecies 10 (match %: 99.90) 이었으며, 단백질분해효소활성이가장높은균은봄철에 B 지역의원유로부터분리한 Serratia liquefaciens(match %: 99.39) 였다. Acinetobacter genomospecies 10(match %:99.90) 는전형적인생장곡선의형태인시그모이드곡선의형태를나타냈으며, 배양시간별조효소활성은배양시간이증가할수록지방분해효소활성이증가하다가정지기후반부에서부터사멸기에도달하기까지는배양시간이길수록활성이감소하였다. ph에따른지방분해효소활성은 ph 8.5에서가장높았고온도는 45 o C에서가장 약 Fig. 6. Effects of temperature on lipase activities at ph 8.5. Significant differences indicated by different letters were analyzed using Duncan's multiple range test (p< 0.05). (A) Acinetobacter genomospecies 10 (lipase), (B) Serratia liquefaciens (protease). 높은활성을나타내었다. Serratia liquefaciens(match %: 99.39) 의생장곡선은전형적인시그모이드형태를나타내었고, 단백질분해효소활성은배양 12시간후에가장높았고, 18시간후에는다시감소하는양상을나타냈다. 단백질분해효소의 ph 영향은 2.5에서활성이가장낮았고 ph 8.5 에서가장높은활성을나타내었다. 또한반응온도에의한효소활성은 35 o C에서가장높았다. 참고문헌 1. Bergogne-Bérézin, E. and Towner, K. (1996) Acinetobacter spp. as nosocomial pathogens: Microbiological, clinical, and epidemiological features. Clin. Microbiol. Rev. 9, 148-165. 2. Constantinju, S., Romaniuc, A., Smaranda Iancu, L., Filimon, R., and Taraşi, I. (2004) Cultural and biochemical characteristics of Acinetobacter spp. strains isolated from hospital units. J. Prev. Med.12(3-4), 35-42. 3. Cousin, M. A. (1982) Presence and activity of psychrotrophic microorganisms in milk and dairy product, a review. J. Food Prot. 45, 172-178. 4. Elionora, H. and Malka, H. (2007) Culturable psychrotrophic bacterial communities in raw milk and their proteolytic and lypolytic traits. Appl. Environ. Microbiol. 73, 7162-7168. 5. Hull, M. E. (1947) Studies on milk proteins, II. Colorimetric
780 Korean J. Food Sci. An., Vol. 33, No. 6 (2013) determination of the partial hydrolysis of the proteins in milk. J. Dairy Sci. 30, 881-884. 6. Kim, J. W., Shim, Y. S., and Yoon, S. S. (1997) Isolation and purification of a lipase from Pseudomonas sp. YJ103 isolated from raw milk. Korean J. Dairy Sci. 19, 17-24. 7. Kojima, Y. and Shimizu, S. (2003) Purification and characterization of the lipase from Pseudomonas fluorescens HU380. J. Biosci. Bioeng. 96, 219-226. 8. Lee, Y. Y. (1993) Purification and properties of protease produced from psychrotrophic bacterium, Pseudomonas sp. K101. Korean J. Environ. Biol. 11, 82-90. 9. Makhzoum, A., Owusu-Apenten, R. K., and Knapp, J. S. (1996) Purification and properties of lipase from Pseudomonas fluorescens strain 2D. Int. Dairy J. 6, 459-472. 10. McKellar, R. C. (1989) Enzymes of psychrotrophs in raw food. CRC Press, pp. 102-118. 11. Morales, P., Fernández-García, E., and Nuñez, M. (2003) Caseinolysis in cheese by Enterobacteriaceae strains of dairy origin. Lett. Appl. Microbiol. 37, 410-414. 12. Ramesh, C. K. and Sudesh, K. Y. (2007) Isolation of a psychrotrophic Exiguobacterium sp. SKPB5(MTCC 7803) and characterization of its alkaline protease. Curr. Microbiol. 54, 224-229. 13. Shamsher, S. K., Rajeev, K. K., Arshad, J., Reena, G., and Swapandeep, S. C. (2005) Methods for inhibition of residual lipase activity in colorimetric assay: A comparative study. Indian J. Biochem. Biol. 42, 233-237. 14. Shin, Y. K., Lee, H. A., Oh, N. S., and Nam, M. S. (2013) Seasonal, regional distribution and identification of psychrotrophic bacteria in milk. CNU J. Agri. Sci. 40, 27-34. 15. Suzuki, T., Nakayama, T., Kurihara, T., Nishino, T., and Esaki, N. (2001) Cold-active lipolytic activity of psychrotrophic Acinetobacter sp. strain No.6. J. Biosci. Bioeng. 92, 144-148. 16. Witter, L. D. (1961) Psychrophilic bacteria. A review. J. Dairy Sci. 44, 983-1015. (Received 2013.3.5/Revised 2013.11.12/Accepted 2013.11.14)