90 Journal of Agriculture & Life Science 48(2) membranaceus Bunge), Zingiberis rhizoma crudus (Zingiberis officinale Roscoe), Ziziphi fructus (Ziziphu

농업생명과학연구 48(2) pp.89-100 Journal of Agriculture & Life Science 48(2) pp.89-100 http://dx.doi.org/10.14397/jals.2014.48.2.89 한약추출부산물이 in vitro 반추위발효특성에미치는영향 안승규 1 구영민 1 고건희 1 이신자 2 문여황 3 이상석 4 김진욱 2 이성실 2* 1 ( 재 ) 산청한방약초연구소, 2 경상대학교응용생명과학부 (BK21 program) 농업생명과학연구원 3 경남과학기술대학교동물생명과학과, 4 순천대학교동물자원과학과 접수일 (2014 년 1 월 10 일 ), 수정일 (2014 년 3 월 6 일 ), 게재확정일 (2014 년 3 월 7 일 ) Effects of Herbal Medicine By-products on Rumen Fermentation Characteristics In vitro Seung Kyu Ahn 1 Young-Min Goo 1 Keon Hee Ko 1 Shin Ja Lee 2 Yea Hwang Moon 3 Sang Suk Lee 4 Jin Wook Kim 2 Sung Sill Lee 2* 1 Sancheong oriental medicinal herb institute, Sancheong, Korea. 2 Division of Applied Life Science (BK21 program) & Institute of Agriculture and Life Science (IALS), Gyeongsang National University, Jinju, Korea. 3 Department of animal Science & Biotechnology, Gyeongnam National University of Science and Technology, Jinju, Korea. 4 Department of animal Science & Technology, Sunchon National University, Sunchon, Korea. Received: JAN. 10. 2014, Revised: MAR. 6. 2014, Accepted: MAR. 7. 2014 초록 한약추출부산물의영양가치및사료가치를평가하기위해 in vitro 반추위분해시험을하였다. 시험에사용한 10 가지한약추출부산물은인삼 (Ginseng radix alba), 백복령 (Hoelen), 감초 (Glycyrrhizae radix), 당귀 (Angelicae gigantis radix), 천궁 (Cnidii rhizoma), 작약 (Paeoniae radix), 황기 (Astragali radix), 생강 (Zingiberis rhizoma crudus), 대추 (Ziziphi fructus), 진피 (Aurantii nobilis pericarpium) 로한약재중빈번하게사용하는약재이다. In vitro 시험을위해 50 ml serum bottle 에혐기상태로 McDougall's buffer 와반추위액을 2:1 비율로 15 ml 을넣었다. 한약추출부산물은 timothy(300 mg) 를기질로 3%, 5% 수준으로첨가하였다. Serum bottle 은배양시간대별 (6, 12, 24, 48, 및 72 h) 로 39 에서 120 rpm 으로진탕배양하였다. 총가스발생량은한약추출부산물 3% 첨가구에서는발효 24 시간대에서대조구가생강 (Zingiberis rhizoma crudus) 구에비해많았으며, 발효 72 시간대에서도대조구가가장많았고, 진피 (Aurantii nobilis pericarpium) 구에서적었다 (P<0.05). 한약추출부산물 5% 첨가구에서는 48 시간대가스발생량이진피 (Aurantii nobilis pericarpium) 구보다대조구에서많았으며, 72 시간발효구에서는황기 (Astragali radix) 구에서가장적었다 (P<0.05). 미생물성장률은한약추출부산물 5% 첨가구의 48 시간대를제외한모든배양에서유의적으로증가하였다 (P<0.05). VFA 농도는배양시간의따라증가되는경향을보였으며, 배양초기 (6 h) 에서한약추출부산물의총 VFA 농도는일부한약추출부산물처리 ( 작약, 황기, 진피 ) 를제외하고유의적으로증가하였다 (P<0.05). 위의결과로한약추출부산물은미생물성장률및발효성상개선을증진시킬수있으며, 결론적으로조사료에대한한약추출부산물 5% 대체가가능할것으로예상된다. 검색어 - 한약추출부산물, 총가스, 미생물성장률, VFA ABSTRACT An in vitro ruminal degradation trial was conducted to evaluate the nutritive values of herbal medicine by-products (HMB). Major ten materials for preparing oriental herbal medicine, Ginseng radix alba (Panax ginseng C. A. Meyer.), Hoelen (Poria cocos Wolf), Glycyrrhizae radix (Glycyrrhiza uralensis Fisch.), Angelicae gigantis radix (Angrlica gigas Nakai), Cnidii rhizoma (Cnidium officinale Makino), Paeoniae radix (Paeonia lactiflora Pall.), Astragali radix (Astragalus * Corresponding author: Sung Sill Lee Tel.: +82-55-772-1883 Fax: +82-55-772-1889 E-mail: lss@gnu.ac.kr

90 Journal of Agriculture & Life Science 48(2) membranaceus Bunge), Zingiberis rhizoma crudus (Zingiberis officinale Roscoe), Ziziphi fructus (Ziziphus jujuba var. inermis Mill.), and Aurantii nobilis pericarpium (Citrus unshiu Marcow.) were used in the experiment. The 15 ml of mixture contained McDougall s buffer and rumen fluid in the ratio 2 to 1, was dispensed anaerobically into 50 ml serum bottles. The HMB was added at the level of 3% or 5% against 300 mg of timothy as a substrate (v/w). The serum bottles were incubated at 39, 120 rpm for 6, 12, 24, 48 and 72 h with triplicate. Total gas production (55 values) did not differ (P<0.05) between all HMB and control in both two additional levels during whole incubation time, whereas only 4 values including 172.96 (3% Zingiberis rhizoma crudus in 24 h), 232.31 (3% Aurantii nobilis pericarpium in 72 h), 209.87 (5% Aurantii nobilis pericarpium in 48 h) and 188.12 (5% Astragali radix in 72 h) were significantly lower than other 51 values. The microbial growth rate on all incubation except for 48 h incubation tended to be increased by addition of 5% HMB with the statistical differences (P<0.05). The VFA concentration tended to be increased gradually with the increasing incubation times, At the initial stage (6 h) of incubation, total VFA concentration of HMB was increased with the significant differences (P<0.05) except for few HMB treatment (Paeoniae radix, Astragali radix, Aurantii nobilis pericarpium). It is concluded that herbal medicine by-products (HMB) can increase microbial growth rate and improve ruminal fermentation. Therefore, the 5% substitution of HMB for roughage are highly recommended to be used in practice. Key words - Herbal medicine by-products, Total gas, Microbial growth rate, VFA Ⅰ. 서론 일반적인한약추출부산물은대부분식물성이지만종류가다양하고한약재를탕제후버린약재로처방전의종류, 수거시기및장소에따라변동이심하게나타난다. 이러한한약추출부산물의경우일부작물농가의사용외에는전량폐기처분되고있는실정으로한약추출부산물을반추가축의사료첨가제및대체사료로사용할수있다면, 농후사료와조사료의대부분을수입에의존하는우리나라에서사료비절감과한약추출부산물폐기처분에큰도움이될것이라예상된다 (Lee et al., 201. 또한많은연구진들이한약추출부산물에대한연구를진행해왔다 (Choi et al., 1996; Jin et al., 1999; Kim et al., 2002). 이러한한약추출부산물은사료비절감등의장점도있지만, 한약추출부산물자체가아닌가공처리등을함으로서추가비용이발생 하는단점이있다. Sung(1997) 은식물성한약재로부터액을추출한한약추출부산물을펠렛으로만든후화학적성분을분석하였을때조단백질함량이 11.4%, NDF 함량이 50.0% 및 TDN(total digestible nutrients) 함량이 61.7% 라고보고하였으며, Kim et al.(1994) 은건조인삼박을반추가축용사료로써의이용성을볼때알팔파건초보다는다소떨어지는것으로나타났으나, 특히단백질및가소화영양소함량이높아반추가축용사료로의이용성이높은것으로보고하였다. 또한한약추출부산물은유기물함량이높아사료와퇴비및토양미생물제제의원료로서도이용가치가높다고하였다 (Joo et al., 2005). 본시험에서는탕제원이나한의원에서많이사용되는한약재를선정하고선정된한약추출부산물이 in vitro 반추위발효특성에미치는영향을조사하여한약추출부산물이부존자원으로써의사용가능여부와그에따른효과를구명하기위하여수행하였다.

Ahn et al : Effects of Herbal Medicine By-products on Rumen Fermentation Characteristics In vitro 91 Ⅱ. 재료및방법 2.1 공시재료공시재료는한약처방전이나한약재료로서가장빈번하게사용하는 10가지약재, 인삼 (Ginseng radix alba), 백복령 (Hoelen), 감초 (Glycyrrhizae radix), 당귀 (Angelicae gigantis radix), 천궁 (Cnidii rhizoma), 작약 (Paeoniae radix), 황기 (Astragali radix), 생강 (Zingiberis rhizoma crudus), 대추 (Ziziphi fructus), 진피 (Aurantii nobilis pericarpium) 를경남생약협동조합에서구입하여사용하였다. 구입한한약재는 80 에서 8시간열수추출후 65 의건조기에서 3일간건조시킨다음, 1 mm screen이부착된분쇄기 (hammer mill) 를이용하여분쇄한후분석시료로사용하였다. 공시재료의화학적조성은 Table 1에서보는바와같다. 2.2 반추위액의준비반추위액은조사료 (timothy) 와농후사료 (6:4) 를 섭취하는반추위누관이장착된 32개월령한우암소로부터사료급여 1시간후에채취하여 4겹의 cheese cloth로여과하고 1시간정치후진공펌프로사료입자를제거한후상등액을채취하여사용하였다. 2.3 시험설계및시험방법공시재료무첨가구와첨가수준에따라기본기질의 3% 및 5% 로하여총 21개처리구를두었으며, 처리구와배양시간대별로각각 3반복을두어대조구를제외하고총 300개의시험관으로수행하였다. In vitro 시험방법은 50 ml serum bottle에혐기상태로 McDougall, s buffer(1948) 10 ml과기본기질로분쇄한 timothy 건초 0.3 g, 위액 5 ml, 그리고각처리구에따라기질의 3% 와 5% 수준으로한약추출부산물을넣고, butyl rubber stopper 및 aluminum cap으로밀봉한후, 39 의 shaking incubator(jeio Tech, SI-900R; 120 rpm) 에서배양시켰다. Table 1. Chemical composition of herbal medicine by-products Moisture Crude protein Ether extracts Crude fiber Crude ash NDF ADF 2) % -----------------------------------%, DM------------------------------------ Ginseng radix alba 9.66 11.20 0.71 5.84 3.23 83.41 62.62 Hoelen 6.75 2.02 0.59 9.26 0.54 57.68 12.00 Glycyrrhizae radix 5.59 9.11 2.50 30.33 2.56 62.30 19.47 Angelicae gigantis radix 9.10 19.71 2.83 5.76 3.31 42.56 35.47 Cnidii rhizoma 7.62 15.00 9.75 11.66 6.09 12.13 14.11 Paeoniae radix 7.45 7.16 0.21 7.25 3.71 74.93 35.51 Astragali radix 5.88 10.66 0.81 28.39 2.14 75.53 15.40 Zingiberis rhizoma crudus 8.63 7.92 1.55 5.71 3.51 50.35 24.33 Ziziphi fructus 9.79 7.96 1.14 18.94 2.06 48.52 32.70 Aurantii nobilis pericarpium 7.33 9.32 1.22 14.88 3.94 46.94 12.75 2) NDF: Neutral detergent fiber. ADF: Acid detergent fiber.

92 Journal of Agriculture & Life Science 48(2) 2.4 조사항목및방법 2.4.1 ph의변화측정가스발생량측정후 Weaton decapper를이용하여 serum bottle의 aluminum seal 및 butyl rubber stopper 를제거한후배양액의 ph를 ph meter(mettler Toledo, MP230) 를이용하여측정하였다. 2.4.2 총가스발생량측정 In vitro 배양시간에따라 serum bottle 을 shaking incubator 에서꺼낸후, Theodorou et al.(1994) 의방법으로 serum bottle의 head space에존재하는가스발생량을 detachable pressure transducer 및 digital read-out voltmeter(laurel Electronics, Inc., CA, USA) 를사용하여측정하였다. 2.4.3 미생물성장률측정 ph 측정후, 배양액 1.5 ml를 Eppendorf tube에채취하여사료입자를제거하기위해 3,000 rpm에서 3분간원심분리하였다. 상등액을취하여 14,000 rpm에서다시 3분간원심분리하여미생물 pellet 을침전시킨후, 상등액은제거하고 pellet 에 sodium phosphate buffer(ph 6.5) 를 1 ml 첨가하여 vortex 로교반시키는과정을 3회반복후 ELISA reader(spectra Max reader M5, Molecular Devices, USA) 를이용하여 550 nm에서 OD(optical density) 값을구하여미생물성장률을측정하였다. 2.4.4 암모니아 (NH 3-N) 측정 Chaney 와 Marbach(1962) 의방법에의해 phenol color reagent와 alkali-hypochlorite reagent로위액중의암모니아를발색한후 ELISA reader (Spectra Max reader M5, Molecular Devices, USA) 를이용하여 630 nm에서 OD 값을구하여측정하였다. 2.4.5 휘발성지방산 (VFA) 농도측정휘발성지방산의농도는 Erwins et al.(196 의 방법으로분석하였다. 각배양시간별배양후배양액 1 ml을채취하여발효를중지시키기위해 HgCl 2 10 μl과시료중단백질침전을위해 25% H 3PO 4 용액 200 μl를첨가하고, 내부표시물질로 Pivalic acid 용액 40 μl를첨가한후완전히혼합한다음, 원심분리 (12,000 rpm, 5 min) 후상층액을취하여 2 ml 시료병에옮겨담고분석시까지냉장보관하였다. 분석은 Gas Chromatography(Agilent 7890A, USA) 를이용하여분석하였다. 2.5 통계처리통계처리는 SAS package program(1996) 의 General Linear Model(GLM) procedure 에따라처리하였으며, 각처리구간의유의성검증을위해분산분석을실시후, Duncan`s multiple range tests(duncan, 1955) 로 5% 수준 (P<0.05) 에서유의성을검정하였다. Ⅲ. 결과및고찰 3.1 ph의변화한약추출부산물첨가에따른 in vitro 배양시간대별 ph의변화는 Table 2에서와같다. In vitro 배양액의 ph는한약추출부산물 3% 첨가구에서발효 12시간대에감초구가인삼구에비해유의적으로높았으나 (P<0.05), 타처리구간에는차이가없었다. 한약추출부산물 5% 첨가구에서는 6시간대진피구가인삼구나작약구, 그리고당귀구에비해높았으나 (P<0.05), 타처리구간에는차이가없었다. 72시간발효구에서는감초구가타처리구에비해상대적으로높았다 (P<0.05). 전처리구에서발효시간이경과됨에따라점차감소하였으며, 한약추출부산물첨가수준에따른영향은크지않았다. Kim et al.(1994) 은건조인삼박의건초대체수준에따른제 1위액의 ph변화는배양개시직후의 7.10에서배양 48시간째 5.45까지배양시간이경과함에따라감소하는경향을보였다고하였다. 본시험에서의결과는평균 ph

Ahn et al : Effects of Herbal Medicine By-products on Rumen Fermentation Characteristics In vitro 93 Table 2. Effects of supplemental herbal medicine by-products on ph value in vitro Control 7.12 6.97 ab 6.57 6.44 6.38 7.10 ab 6.94 6.55 6.40 6.42 ab Ginseng radix alba 6.92 6.78 b 6.59 6.27 6.25 6.98 b 6.93 6.49 6.22 6.24 abc Hoelen 7.07 6.94 ab 6.68 6.35 6.28 7.13 ab 6.96 6.52 6.29 6.16 bc Glycyrrhizae radix 7.03 6.99 a 6.64 6.33 6.26 7.08 ab 6.82 6.52 6.24 6.47 a Angelicae gigantis radix 7.08 6.96 ab 6.59 6.40 6.32 7.03 b 6.91 6.49 6.40 6.15 bc Cnidii rhizoma 7.06 6.95 ab 6.62 6.37 6.26 7.09 ab 6.88 6.48 6.34 6.10 c Paeoniae radix 7.13 6.95 ab 6.61 6.31 6.45 6.99 b 6.88 6.48 6.44 6.03 c Astragali radix 7.08 6.93 ab 6.61 6.39 6.27 7.17 ab 6.91 6.47 6.40 6.09 c Zingiberis rhizoma crudus 7.06 6.92 ab 6.58 6.27 6.40 7.22 ab 6.91 6.52 6.31 6.10 c Ziziphi fructus 7.02 6.93 ab 6.55 6.36 6.37 7.25 ab 6.88 6.46 6.40 6.11 c Aurantii nobilis pericarpium 7.04 6.93 ab 6.58 6.25 6.23 7.35 a 6.87 6.52 6.50 6.16 bc SEM 0.12 0.10 0.08 0.14 0.22 0.14 0.09 0.08 0.16 0.16 a,b,c Table 3. Effects of supplemental herbal medicine by-products on total gas emission in vitro (ml/g DM) Control 150.3 149.8 199.5 a 248.8 267.4 a 142.7 148.4 196.7 260.5 a 250.7 a Ginseng radix alba 137.6 126.2 193.4 ab 228.8 245.4 ab 145.0 136.0 192.6 232.3 ab 231.7 ab Hoelen 115.9 138.2 181.3 ab 219.4 245.3 ab 142.2 136.6 179.1 226.5 ab 247.0 a Glycyrrhizae radix 144.0 127.4 189.9 ab 229.5 248.7 ab 142.1 139.4 183.7 237.3 ab 222.3 ab Angelicae gigantis radix 146.9 122.9 185.1 ab 223.9 244.4 ab 130.3 141.9 182.3 223.2 ab 236.8 ab Cnidii rhizoma 143.2 143.2 177.6 ab 234.6 261.0 ab 145.8 142.0 188.1 234.7 ab 247.3 a Paeoniae radix 145.7 142.8 197.0 ab 235.0 253.2 ab 143.4 142.1 183.1 225.1 ab 247.8 a Astragali radix 146.7 142.9 185.6 ab 227.4 243.4 ab 136.0 141.2 186.5 225.6 ab 188.1 b Zingiberis rhizoma crudus 144.5 143.4 173.0 b 237.0 243.6 ab 133.5 141.7 184.3 241.7 ab 231.5 ab Ziziphi fructus 148.6 144.4 197.8 ab 230.0 261.8 ab 132.0 141.0 185.6 221.3 ab 239.2 ab Aurantii nobilis pericarpium 146.7 128.9 190.0 ab 240.6 232.3 b 135.6 140.5 181.6 209.9 b 227.8 ab SEM 27.64 33.29 12.93 21.39 15.92 27.13 26.07 17.52 22.76 30.26 a,b

94 Journal of Agriculture & Life Science 48(2) Table 4. Effects of supplemental herbal medicine by-products on microbial growth rate in vitro (OD at 550nm) Control 0.12 a 0.21 b 0.42 0.61 ab 0.54 0.10 cde 0.25 bcd 0.37 abc 0.67 0.43 b Ginseng radix alba 0.07 ab 0.40 a 0.42 0.62 ab 0.66 0.16 a 0.21 d 0.34 bc 0.71 0.53 ab Hoelen 0.06 b 0.36 a 0.39 0.51 b 0.59 0.12 bcd 0.23 cd 0.33 bc 0.70 0.50 ab Glycyrrhizae radix 0.08 ab 0.39 a 0.41 0.70 a 0.58 0.12 abc 0.24 bcd 0.32 c 0.68 0.44 ab Angelicae gigantis radix 0.10 ab 0.43 a 0.40 0.61 ab 0.64 0.15 ab 0.23 cd 0.40 ab 0.68 0.53 ab Cnidii rhizoma 0.08 ab 0.41 a 0.39 0.58 ab 0.56 0.11 bcd 0.30 ab 0.42 a 0.60 0.51 ab Paeoniae radix 0.10 ab 0.41 a 0.50 0.60 ab 0.63 0.10 cde 0.26 bcd 0.40 ab 0.56 0.56 a Astragali radix 0.09 ab 0.25 b 0.38 0.60 ab 0.56 0.07 e 0.28 abc 0.38 abc 0.58 0.48 ab Zingiberis rhizoma crudus 0.11 ab 0.25 b 0.37 0.60 ab 0.59 0.09 cde 0.25 bcd 0.37 abc 0.57 0.50 ab Ziziphi fructus 0.13 a 0.29 b 0.41 0.50 b 0.50 0.11 bcde 0.31 a 0.34 bc 0.60 0.48 ab Aurantii nobilis pericarpium 0.12 a 0.26 b 0.43 0.63 ab 0.58 0.08 de 0.29 ab 0.31 c 0.55 0.50 ab SEM 0.03 0.04 0.06 0.07 0.08 0.02 0.03 0.04 0.09 0.06 a,b,c,d,e 6.03~7.35 의범위로일반적인반추위내적정 ph를벗어나지않았다. 이는한약추출부산물첨가가반추위 ph에영향을미치지않음을알수있었다. 3.2 총가스발생량한약추출부산물첨가에따른 in vitro 배양시간대별총가스발생량은 Table 3에서와같다. 총가스발생량은한약추출부산물 3% 첨가구에서는발효 24시간대에대조구에서가장많았고, 생강구에서가장낮았다. 발효 72시간대에서도대조구가가장많았고, 진피구에서가장적었다 (P<0.05). 한약추출부산물 5% 첨가구에서는 48시간대에서총가스발생량은대조구에서가장많았고, 진피구에서가장낮았다. 72시간발효구에서는황기구에서가장낮았다 (P<0.05). 전처리구에서발효시간이경과됨에따라점차증가하였으며, 한약추출부산물첨가수준이 5% 대비 3% 에서많은편이었다. In vitro 가스발 생량을조사하는것은조사료의발효특성즉, 분해율을평가하는간접적지표로알려져있고 (Theodorou et al., 1994, 1998), 기질의발효정도에따라가스발생량이변화하여 (Beuvink et al., 1992) 건물분해율과가스발생량의상관관계가높은것으로알려져있다 (Kim et al., 201. 본시험에서의결과는 3%, 5% 첨가구모두에서대조구가유의적으로높았지만, 한약추출부산물처리구들과유의적으로큰차이를보이지않았다 (P<0.05). 전체적으로가스발생량에큰차이를보이지않은것은건물소화가잘이루어졌다는것을의미하며, 가스가소량줄어든것은환경적인측면에서효과가있을것으로생각된다. 3.3 미생물성장률한약추출부산물첨가에따른 in vitro 배양시간대별미생물성장률변화는 Table 4에서와같다. 미

Ahn et al : Effects of Herbal Medicine By-products on Rumen Fermentation Characteristics In vitro 95 Table 5. Effects of supplemental herbal medicine by-products on ammonia concentration in vitro (mg/100ml) Control 6.60 a 6.21 9.94 a 11.49 b 16.06 ab 6.54 a 6.32 ab 9.64 12.76 14.22 bc Ginseng radix alba 5.59 ab 5.64 8.86 ab 9.51 b 15.21 ab 6.41 ab 6.00 b 9.00 9.74 13.60 c Hoelen 5.51 ab 5.76 8.25 b 10.22 b 15.72 ab 5.88 ab 6.30 ab 9.37 10.20 13.76 c Glycyrrhizae radix 6.12 ab 6.21 9.22 ab 9.53 b 14.92 ab 5.82 ab 6.42 ab 8.59 10.79 15.71 abc Angelicae gigantis radix 6.61 a 6.52 8.85 ab 11.47 b 15.77 ab 6.24 ab 7.11 a 8.71 11.28 16.31 abc Cnidii rhizoma 5.26 b 6.32 8.92 ab 18.70 a 18.60 a 6.08 ab 6.59 ab 9.08 10.89 16.53 abc Paeoniae radix 6.08 ab 5.89 9.25 ab 10.15 b 14.64 ab 6.88 a 6.45 ab 8.82 13.37 20.05 ab Astragali radix 6.06 ab 6.30 8.50 b 11.56 b 13.43 b 5.43 b 6.23 ab 8.83 11.23 21.02 a Zingiberis rhizoma crudus 6.18 ab 6.23 9.10 ab 9.33 b 14.35 ab 5.41 b 6.56 ab 8.79 13.45 19.94 ab Ziziphi fructus 5.84 ab 5.93 10.01 a 9.42 b 18.91 a 6.08 ab 6.21 b 9.10 12.58 19.84 ab Aurantii nobilis pericarpium 5.70 ab 6.68 8.63 b 10.74 b 15.74 ab 6.65 a 6.54 ab 8.66 13.31 17.13 abc SEM 0.57 0.63 0.66 1.36 2.59 0.55 0.46 0.89 2.15 3.02 a,b,c 생물성장률은한약추출부산물 3% 첨가구에서 48 시간대감초구에서가장높았으며, 백복령구와대추구에서가장낮았다 (P<0.05). 한약추출부산물 5% 첨가구에서 24시간대천궁구가가장높았으며, 감초구와진피구에서가장낮았다 (P<0.05). 72시간발효구에서는작약구에서가장높았으며, 대조구에서가장낮았다 (P<0.05). 반추위미생물성장의경우, 가스발생량과밀접한관계가있으므로 (Holdman et al., 1977; Parodi, 1994) 가스발생량이많은첨가구가반추위미생물성장률이높을것이다. 본시험에서의가스발생량은 3%, 5% 첨가구에서 48시간대유사한경향을나타났는데, 이는반추위미생물성장률과일치한다고할수있겠다. 그러나 Shin et al.(2007) 은생강추출액의첨가가병원성균뿐만아니라반추위박테리아및프로토조아를억제한다고보고하였으나본시험에사용한생강추출부산물은생강추출액과는달리반추위미생물성장률에영향을미치지않음을알수있었다. 3.4 암모니아농도한약추출부산물첨가에따른 in vitro 배양시간대별암모니아발생량에미치는영향은 Table 5에서와같다. 암모니아발생량은한약추출부산물 3% 첨가구에서발효 72시간대천궁구와대추구에서가장높았으며, 황기구에서가장낮았다 (P<0.05). 한약추출부산물 5% 첨가구에서발효 72시간대황기구가가장높았으며, 인삼구와백복령구에서가장낮았다 (P<0.05). 반추위미생물단백질합성효율을위한적정암모니아수준은 100 ml당 5~8 mg 수준으로보고가되고있으나 (Allison, 1969; Sarter and Slyter, 1974; Stern and Hoover, 1979), 본시험에서는 12시간이후에는암모니아발생량이 8.63~ 18.91 mg으로적정수준보다높게나타나는것으로확인되었다. 이는시간의증가에따라축적된양의수치로시간의증가에따라암모니아가증가한것으로생각된다. Ok et al.(201 은 saponin 함유식물추출물중오가피와유카추출물처리구는대조구와

96 Journal of Agriculture & Life Science 48(2) Table 6. Effects of supplemental herbal medicine by-products on total VFA concentration(mm) in vitro Control 49.38 d 54.75 ab 86.69 100.5 94.32 b 48.99 b 54.99 ab 63.00 d 109.3 92.27 b Ginseng radix alba 59.20 a 51.33 b 84.34 108.1 99.00 b 51.22 ab 53.76 ab 93.45 a 101.7 111.1 a Hoelen 56.42 ab 52.10 b 74.72 106.2 101.4 b 52.00 ab 52.85 b 84.55 ab 101.1 106.7 ab Glycyrrhizae radix 54.46 bc 52.36 b 77.80 103.0 93.00 b 50.11 ab 57.07 ab 68.67 cd 103.3 96.82 ab Angelicae gigantis radix 54.23 bc 53.19 b 77.39 96.78 90.81 b 53.76 a 56.90 ab 80.01 abc 99.10 108.6 a Cnidii rhizoma 52.93 bcd 52.36 b 86.83 100.6 130.7 a 52.73 ab 57.28 ab 75.62 bcd 98.88 109.0 a Paeoniae radix 52.11 cd 54.16 ab 99.93 98.23 97.66 b 53.14 ab 58.65 a 75.47 bcd 92.38 106.1 ab Astragali radix 52.01 cd 63.40 a 77.40 97.03 88.61 b 50.41 ab 57.74 ab 80.79 abc 97.57 108.9 a Zingiberis rhizoma crudus 53.15 bcd 52.45 b 76.31 98.49 89.80 b 50.53 ab 58.84 a 71.44 bcd 108.7 109.0 a Ziziphi fructus 52.73 bcd 53.82 b 97.97 101.1 98.25 b 51.28 ab 58.60 a 70.40 bcd 99.18 110.7 a Aurantii nobilis pericarpium 51.48 cd 54.11 ab 96.03 105.2 103.4 ab 50.45 ab 55.99 ab 63.15 d 89.44 110.3 a SEM 2.21 5.12 13.95 7.08 16.48 2.27 2.73 7.09 12.52 8.34 a,b,c,d Table 7. Effects of supplemental herbal medicine by-products on acetic acid concentration(mm) in vitro Control 24.51 d 27.97 50.74 56.61 51.38 ab 24.01 b 27.98 abc 41.12 de 61.09 50.08 b Ginseng radix alba 31.24 a 25.93 49.60 61.02 57.14 ab 26.03 ab 27.42 bc 55.62 a 55.39 60.88 a Hoelen 29.64 ab 26.40 44.01 60.92 56.61 ab 26.73 ab 26.72 c 52.85 ab 56.36 58.44 ab Glycyrrhizae radix 28.40 bc 26.61 47.91 58.38 51.15 ab 25.11 ab 29.94 abc 47.33 bcd 57.68 53.52 ab Angelicae gigantis radix 28.03 bc 26.78 46.02 55.11 49.96 ab 27.83 a 29.93 abc 50.40 abc 57.16 59.52 a Cnidii rhizoma 27.52 bc 26.25 53.10 57.52 53.05 ab 27.17 ab 30.37 abc 44.62 cd 55.14 58.46 ab Paeoniae radix 27.03 bcd 27.43 57.77 54.78 52.60 ab 27.08 ab 31.01 ab 44.73 cd 53.01 55.44 ab Astragali radix 26.85 bcd 27.75 47.39 53.93 46.94 b 24.88 ab 30.51 abc 47.20 bcd 55.64 57.04 ab Zingiberis rhizoma crudus 27.68 bc 26.40 47.30 54.41 48.19 ab 24.91 ab 31.58 a 41.25 de 62.41 57.90 ab Ziziphi fructus 26.97 bcd 27.33 59.05 56.29 53.70 ab 25.45 ab 31.12 ab 41.41 de 56.93 57.65 ab Aurantii nobilis pericarpium 25.95 cd 27.80 58.13 58.44 59.79 a 24.80 ab 29.56 abc 37.18 e 51.19 60.23 a SEM 1.54 1.13 8.16 3.74 6.44 1.71 1.98 3.14 6.57 4.28 a,b,c,d,e

Ahn et al : Effects of Herbal Medicine By-products on Rumen Fermentation Characteristics In vitro 97 Table 8. Effects of supplemental herbal medicine by-products on propionic acid concentration(mm) in vitro Control 13.05 d 14.10 b 23.03 ab 30.72 26.13 13.13 14.05 14.46 bc 33.37 25.99 c Ginseng radix alba 14.68 a 13.27 b 21.74 ab 33.45 29.27 13.26 13.76 24.25 a 33.22 35.95 ab Hoelen 14.13 ab 13.45 b 18.09 ab 31.22 30.72 13.32 13.62 17.99 bc 31.36 33.42 abc Glycyrrhizae radix 13.79 bcd 13.31 b 17.12 ab 31.45 26.07 13.15 14.22 15.69 bc 32.32 27.72 bc Angelicae gigantis radix 13.91 bc 13.89 b 18.58 ab 29.03 26.20 13.70 14.18 17.16 bc 29.26 34.46 abc Cnidii rhizoma 13.37 bcd 13.70 b 19.18 ab 29.83 28.70 13.49 14.17 18.63 abc 30.92 36.09 ab Paeoniae radix 13.27 bcd 14.10 b 28.14 a 30.60 29.33 13.71 14.53 17.41 bc 26.34 35.55 ab Astragali radix 13.33 bcd 23.06 a 16.39 ab 30.20 26.62 13.42 14.33 20.44 ab 28.83 36.26 ab Zingiberis rhizoma crudus 13.49 bcd 13.69 b 15.63 b 31.14 24.47 13.46 14.40 17.79 bc 31.49 36.01 ab Ziziphi fructus 13.62 bcd 13.93 b 24.69 ab 31.81 26.21 13.61 14.53 16.48 bc 28.95 37.52 a Aurantii nobilis pericarpium 13.47 bcd 13.90 b 23.84 ab 33.19 28.26 13.46 13.81 14.07 c 25.21 33.03 abc SEM 0.43 4.75 5.61 2.70 6.20 0.36 0.52 3.03 5.66 4.52 a,b,c,d Table 9. Effects of supplemental herbal medicine by-products on the rate of acetate to propionate in vitro Control 1.88 1.98 2.20 1.84 1.97 1.83 1.99 2.84 1.83 1.93 Ginseng radix alba 2.13 1.95 2.28 1.82 1.95 1.96 1.99 2.29 1.67 1.69 Hoelen 2.10 1.96 2.43 1.95 1.84 2.01 1.96 2.94 1.80 1.75 Glycyrrhizae radix 2.06 2.00 2.80 1.86 1.96 1.91 2.11 3.02 1.78 1.93 Angelicae gigantis radix 2.02 1.93 2.48 1.90 1.91 2.03 2.11 2.94 1.95 1.73 Cnidii rhizoma 2.06 1.92 2.77 1.93 1.85 2.01 2.14 2.39 1.78 1.62 Paeoniae radix 2.04 1.95 2.05 1.79 1.79 1.98 2.13 2.57 2.01 1.56 Astragali radix 2.01 1.20 2.89 1.79 1.76 1.85 2.13 2.31 1.93 1.57 Zingiberis rhizoma crudus 2.05 1.93 3.03 1.75 1.97 1.85 2.19 2.32 1.98 1.61 Ziziphi fructus 1.98 1.96 2.39 1.77 2.05 1.87 2.14 2.51 1.97 1.54 Aurantii nobilis pericarpium 1.93 2.00 2.44 1.76 2.12 1.84 2.14 2.64 2.03 1.82

98 Journal of Agriculture & Life Science 48(2) 암모니아농도가비슷한경향을나타내었지만, 인삼과차나무에서는대조구보다암모니아농도가낮았다고보고하였다. 이에본시험에서는한약추출부산물처리구와대조구간의결과가일정하게나타나지않았는데이는한약추출부산물에는한약재추출물과달리 saponin 등의생리활성물질이반추위프로토조아의수를감소시킬정도의함유량을가지고있지않는것으로생각된다. 3.5 VFA 분석한약추출부산물첨가에따른총 VFA 농도 (Table 6) 는 3% 첨가구에서발효 72시간대천궁구에서가장높았다 (P<0.05). 한약추출부산물 5% 첨가구에서발효 72시간대대조구에서가장낮았다 (P<0.05). 총 VFA 농도는배양시간이증가함에따라전체적으로증가하는경향을보였으며, 24시간이후급격하게증가하였다. Acetic acid 농도 (Table 7) 는한약추출부산물 3% 첨가구에서발효 72시간대진피구에서가장높았으며, 황기구에서가장낮았다 (P<0.05). 한약추출부산물 5% 첨가구에서발효 72시간대인삼구, 진피구및당귀구에서가장높았으며, 대조구에서가장낮았다 (P<0.05). Propionic acid 농도 (Table 8) 는한약추출부산물 3% 첨가구에서발효 24시간대작약구에서가장높았으며, 생강구에서가장낮았다 (P<0.05). Acetic acid(a) 와 Propionic acid(p) 의비율 (A/P) 은한약추출부산물 3%, 5% 첨가구모두대조구와처리구간의유사한경향을보였다 (Table 9). Wina et al.(2005) 은반추위발효에서 saponin 의주된효과는 propionate 의농도를증가시키는것으로 propionate 의농도증가에의해프로토조아의주요최종산물인 acetate 의농도를감소시키고, Moon(2009) 은감초추출물의첨가는발효시간의경과에따른 VFA 농도변화는 acetate 가발효시간이지속됨에따라줄어드는경향이라고하였다. 하지만본시험에서는 VFA 농도변화는대조구와차이가없었는데이는한약재추출물의첨가와는달리한약추출부산물의첨가는 VFA 농도와암모니아발생에영향을미치지않는것으로생각된다. Ⅳ. 감사의글 본논문은농촌진흥청 ( 과제번호 PJ00928903) Development and evaluation of databases needed for applying CNCPS to predict energy and protein utilization in Korean lactating dairy cattle 의지원과농림수산식품부지정공모과제 Researches on feed additive for reducing the production of methane by ruminants 의지원을받았으며, 안승규의박사학위논문을이용하여작성한논문입니다.» Literature cited Allison, M. J. 1969. Biosynthesis of amino acids by ruminal microorganisms. J. Anim. Sci. 29: 797-807. Beuvink, J. M., S. F. Spoelstra and R. J. Hogendorp. 1992. An automated method for measuring the time course of gas production of feedstuffs incubated with buffered rumen fluid. Neth. J. Agri. Sci. 40: 401-407. Chaney, A. L. and E. P. Marbach. 1962. Modified reagents for determination of urea and ammonia. Clin. Chem. 8: 130-132. Choi, J. H., D. W. Kim, Y. S. Moon, and D. S. Chang. 1996. Feeding Effect of oriental medicine on the Functional properties of pig meat. J. Korean Soc. Food Nutr. 25(: 110-117. Duncan, D. B. 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Erwin, E. S., G. J. Marco and E. M. Emery. 1961. Volatile fatty acid analysis of blood and rumen fluid by gas chromatography. J. Dairy Sci. 44: 1768-1771. Holdeman, L. V., E. P. Cato and W. E. C. Moore. 1977. Anaerobe laboratory manual. Vol. 124. Blacksburg: Virginia Polytechnic Institute and State

Ahn et al : Effects of Herbal Medicine By-products on Rumen Fermentation Characteristics In vitro 99 University. Jin, S. K. Y. M. Song, T. S. Park, J. I. Lee, S. T. Joo, and G. B. Park. 1999. Effects of Feeding Medicinal Herba Residue on Growth Performance, Carcass Quality and Production Cost in Finishing Pigs. Kor. J. Anim. Sci. 41(3): 365-374. Joo, G. J., Y. M. Kim, J. W. Cheol, O. S. Lee, J. W. Kim, J. H. So, Y. Y. Kwak, J. J. Lee, J. H. Kim and I. K. Rhee. 2005. Development of Microbial Inoculant using By-product of Oriental Herbal Medicine. J. Korean Soc. Appl. Biol. Chem. 48(3): 201-206. Kim, B. K., I. U. Hwing, S. S. Kang, S. H. Shin, S. C. Woo, Y. J. Kim, and Y. H. Hwang. 2002. Effects of Dietary Panax Ginseng, Dioscorea Japonica and Oriental Medicine Refuse on Productivi. J. Anim. Sci. & Technol.(Kor). 44: 297-304. Kim, H. S., J. K. Park, H. Y. Kim, S. B. Kim, S. H. Yang, C. H. Kim, and J. H. Ahn. 2002. Effects of Dietary Herbaceous Peat on In Vitro Fermentation and Milk Production in Dairy Cows. Journal of The Korean Society of Grassland and Forage Science. 31: 177-190. Kim, K. H., H. J. Kim, J. W. Joo, S. R. Lee, D. S. Kim, and W. J. Maeng. 1994. Effects of Substitution level of ginseng meal for alfalfa hay on the ruminal fermentation characteristics In Vitro. Kor. J. Anim. Nutr. Food. 18(6): 481-490. Lee S. J, N. H. Shin, J. J. An, G. M. Chu, Y. H. Moon, S. S. Lee. 2011. Effects of Replacing Rice Straw with By-products of Medical Herbs on the in vitro Fermentation Characteristics. Journal of Agriculture & Life Science 45(3): 69-79. McDougall, E. I. 1948. The composition and output of sheep`s saliva. Biochem. J. 43: 99. Moon, Y. H. 2009. Effect of Herbal Extracts on the Ruminal Dry Matter Digestibility, Volatile Fatty Acid Production and Growth Rate of Microbes in vitro. Journal of Agriculture & Life Science. 43(6): 67-75. Ok, J. U., Y. C. Back, K. H. Kim, S. C. Lee, Y. J. Seol, K. Y. Lee, C. W. Choi, C. O. Jeon, S. S. Lee, S. Sill. Lee, and Y. K. Oh. 2011. Effects of Saponin Contained Plant Extracts on Ruminal Fermentation Characteristics and Methane Production. J. Anim. Sci. & Technol.(Kor). 53(2): 147-154. Parodi, P. W. 1994. Conjugated linoleic acid: An anticarcinogenic fatty acid present in milk fat. Aust. J. Dairy Technol. 49: 93-97. Sarter, L. D. and L. L. Slyter. 1974. Effect of ammonia concentration on rumen microbial protein production in vitro. Brit. J. Nutr. 32: 199-208. SAS. 1996. SAS user's guide: Release 6.12 edition. SAS Inst. Inc., Cary, NC, USA. Shin, S. W., S. J. Lee, J. U. Ok, S. M. Lee, J. H. Lim, K. H. Kim, Y. H. Moon, and S. S. Lee. 2007. Effects of Biologically Active Materals Prepared for Several Minerals and Plants on the Growth of Rumen Microbe. Journal of Life Science. 17(1: 1555-1561. Stern, M. D. and W. H. Hoover. 1979. Method of determining and factors affecting rumen microbial protein synthesis: a Review. J. Anim. Sci. 49: 1590-1603. Sung, K. I., 1997. Feeding value of pellet of by-products of oriental medicines and silage of soybean curd residue as feed resources in ruminant. Korean Journal Of Animal Nutrition & Feedstuffs. 21(6): 511-518. Theodorou, M. K., B. A. Williams, M. S. Dhanoa, A. B. McAllan, and J. France. 1994. A simple gas production method using a pressure transducer to determine the fermentation kinetics of ruminant

100 Journal of Agriculture & Life Science 48(2) feeds. Anim. Feed Sci. Technol. 48: 185-197. Theodorou, M. K., R. S.Lowman, Z. S. Davies, D. Cuddeford, and E. Owen. 1998. Principles of techniques that rely on gas measurement in ruminant nutrition. In: In vitro techniques for measuring nutrient supply to ruminants. British Society of Animal Science Occassional Publication No. 22, Reading: pp55-63. Wina, E., S. Muetzel, and K. Becker. 2005. The Impact of Saponins or Saponin-Containing Plant Materials on Ruminant Production A Review. Journal of agricultural and food chemistry. 53: 8093-8105.