农业部饲料工业中心MAFIC

Alternativesofantibioticsforpigs猪用抗生素的替代DefaLi

李德发MinistryofAgricultureFeedIndustryCentre

农业部饲料工业中心2006年11月30日于北京猪营养

营养需要与代谢分子生物学WhooperatesMAFICResearch植物提取物免疫学微生物学分析化学营养与免疫基因工程菌细胞生物学

分子营养与肉品质团队研究Antibiotics抗生素Residue残留Resistance耐药性Harmfultoanimalandhumanhealth影响动物和人类的健康Reducetheuseofantibiotics减少抗生素的使用EU欧盟China中国Currentsituationandfuturedevelopment现状与展望Alternativesofantibiotics

抗生素替代Antimicrobialpeptides

抗菌肽

Herbalextracts

天然提取物Enzymepreparations

酶制剂Probiotics

益生素Probiotics

益生素Lactobacillus乳酸杆菌Mechanism机理研究Application应用研究绿色荧光蛋白(GFP)标记蛋白组学粘附位点无抗发酵饲料蛋白差异复合发酵培养悉生动物模型信号传导作用机制Targetingdistribution

目的细菌在肠道中的定位分布情况，其与肠道菌系之间的关系Specificpasswayandfunction

具体作用途径与功能的研究Experimentalmodelof

gnotobiotic

animals

悉生动物实验模型悉生动物（已知菌动物）空白组益生菌致病菌益生菌+致病菌Developmentofantibiotics-freefermentedfeed

无抗发酵饲料的开发

Fermentativerespiratorymembrane发酵呼吸膜Singlewayvalve

单向阀门CO2venting,preventingairtoenter

排出袋内微生物产生的CO2，阻止大气进入A,E.coliK88

大肠杆菌K88B,E.coliK99

大肠杆菌K99C,S.typhimurium

鼠伤寒沙门氏菌D,Staph.Aureus

金黄色葡萄球菌Int.J.Gener.Mol.Microbiol.,X.H.Guo,DefaLi,2006InhibitoryactivityofBacillusspp.139againstpathogenicbacteria,butnotLactobacillus外源芽孢杆菌可抑制有害菌，对乳酸菌无影响Multi-strainscombination,creatinganaerobicconditionautomatically多菌菌种种组组合合，，自自身身创创造造厌厌氧氧环环境境Yeast酵母母菌菌Bacillusspp.芽胞胞杆杆菌菌好氧氧兼性性兼性性Lactobacillusreproductioninanaerobiccondition乳酸酸杆杆菌菌厌氧氧发发酵酵Oligosaccharide寡糖NSPenzyme非淀粉多糖酶Activator激活剂

刺激ActivateInoculating,packing,thenfermentation先接接种种、、包包装装，，后后发发酵酵接菌激活剂原料发酵轻工业副副产物：：玉米浆浆、果渣渣Effectoffermentationtimeonenergyandgossypol能量与游游离棉酚酚随发酵酵时间的的变化Decreasethecontentofgossypol降低游离离棉酚的的含量Fermentationtime(h)EnergyGossypolGossypol（mg/kg）Energy（kcal/g）Effectsofantibiotics-freefermentedfeedongrowthperformanceinpigs无抗发酵酵饲料对对猪生产产性能的的影响P>0.05RFIDsystemRFID追溯溯系统基因芯片片Genechip：Theonlineexaminationtechniqueofantibiotics基因芯片片：快速速检测分分割肉中中的抗生生素含量量（20-100ppb）Proteomics蛋白质组组学ProteomicsanalysisofinteractionbetweenLactobacillusandintestine乳酸杆菌菌和肠道道细胞相相互作用用的蛋白白质组学学研究J.Nutr.,J.J.Wang,DefaLi,2006蛋白质组组学双向电泳泳样品制备备数据库比比对鉴定定蛋白相互作用用机制生物功能能差异蛋白白质谱分析析图像分析析胶内酶切切蛋白Lactobacillus乳酸杆菌菌Intestinalepithelialcells肠道上皮皮细胞等点聚焦焦SDSExperimentaldesign试验设计计IntestinalcellsAdheredornotLactobacillusfermentumI5007cellsCulturemedium,37ºC37ºC温箱4hRabbitjejunum兔子空肠肠Two-dimensionalgelelectrophoresis双向电泳泳37ºC1hMassspectrometry质谱Differentialproteins差异蛋白白Lactobacillusinrabbitjejunumculture兔子空肠肠培养条条件下乳乳酸杆菌菌表达差差异的蛋蛋白Electrophoresis,F.Yang,DefaLi,2006Glycosidehydrolase糖苷水解酶（粘蛋白的分解酶）降解肠粘膜表达的粘蛋白有利于乳酸杆菌粘附定植Fructose-6-phosphatePhosphoketolase(F6PPK)6-磷酸酸果糖磷磷酸酮酶酶Lactatedehydrogenase乳酸脱氢氢酶Dihydrolipomidedehydrogenase二氢硫辛辛酸脱氢氢酶（丙酮酸酸脱氢酶酶系）糖酵解三羧酸循循环磷酸戊糖糖途径减少氧自自由基产生生NADPH生成增加加有利于乳乳酸杆菌菌存活IntestinalcellsafterLactobacillusadhesion肠细胞被被乳酸杆杆菌粘附附条件下下表达差差异的蛋蛋白Electrophoresis,F.Yang,DefaLi,2006Voltage-dependentanionchannel1电压依从从性阴离离子通道道1Glutathionetransferase谷胱甘肽肽转移酶酶Heatshockproteingp96热应激蛋蛋白gp96调控膜通通透性改善机体体免疫减少氧化化应激改善肠道道完整性性及其健康康Synbiotics合生素Functionalmechanismofsynbiotics合生素的的作用机机理Synbiotics合生素Bacillussubtilis枯草芽孢孢杆菌Astragalus,aloepolysaccharide,etc.黄芪多糖糖，芦荟荟多糖等等Immunity免疫力Antibacterialsubstance抑菌物质质Organicacid有机酸Health动物健康康Bacteriophage产生抗菌菌素挥发性脂脂肪酸：：乙酸、、丙酸、、丁酸Toleranceoftheintestinalenvironment耐受肠道道内环境境Bacillussubtilis枯草芽孢孢杆菌ZnO氧化锌Hypothesis假设Intestinefunction肠道功能能Entericnervoussystem肠神经系系统Feedintake采食量Diarrhea腹泻生产性能能GrowthperformanceGastrointestinalhormones胃肠激素素Mastcellmediators肥大细胞胞介质IntestinalIGF-1system小肠IGF-1系统Hormoneoneating采食相关关激素ZnOEffectsofdietarysupplementof3000mg/kgZnOontheexpressionoffundicglandGhrelingeneinpiglets日粮中添添加3000mg/kg氧氧化锌对对断奶仔仔猪胃底底腺Ghrelin基基因表达达的影响响P=0.008IncreasetheexpressionoftheGhrelingene显著提高高断奶仔仔猪胃底底腺Ghrelin基基因的表表达,提提高采采食量Improvetheintestinalfunction药理剂量量氧化锌锌促进仔仔猪肠道道健康Thesmallintestinalmorphologyofweanedpigsreceivingadietwithorwithoutsupplementationof3000mg/kgZn日粮中添添加3000mg/kg氧氧化锌ZnO对对仔猪小小肠形态态的影响J.Nutr.,X.L.Li,DefaLi,2006ItemsZnSO4ZnOgroupSEMPVillusheight,µm

341387140.036Cryptdepth,µm

19417980.248IncreasetheexpressionofIGF-1gene提高小肠肠IGF-1基因因的表达达通过IGF-1的旁分分泌作用用促进仔仔猪肠道道健康和和生长Effectsofdietarysupplementof3000mg/kgZnOontheexpressionofsmall-intestinalmucosaIGF-1geneinpiglets日粮中添添加3000mg/kg氧氧化锌对对仔猪小小肠粘膜膜IGF-1基基因表达达的影响响P=0.012P=0.041ablibitum

Pair-fedJ.Nutr.,X.L.Li,DefaLi,2006Histamineconcentrationsinthesmallintestineofweanlingpigletsfeddietscontainingnormalandhigh-levelsofZnO高锌日粮粮对断奶奶仔猪小小肠组胺胺浓度的的影响Decreasethereleaseofthehistamineinthesmallintestine高锌显著著降低断断奶仔猪猪小肠组组胺的释释放稳定小肠肠通透性性P=0.005P=0.049P=0.049DuodenumJejunumIleumThenumbersofmastcells(cell/cm2)inthesmallintestineofweanlingpigletsfeddietscontainingnormalorhigh-levelsofzincoxide高锌日粮粮对断奶奶仔猪小小肠肥大大细胞数数量的影影响Decreasethenumbersofthemastcellsinthesmallintestine高锌显著著降低断断奶仔猪猪小肠各各层肥大大细胞数数改善小肠肠功能LocationControlTreatmentP

Duodenummucosa74.9±21.936.7±8.9<0.01Jejunummucosa99.7±13.148.8±13.2<0.01Ileummucosa81.4±19.555.1±3.4<0.01mRNAandproteinlevelsforthestemcellfactor(SCF)geneinthemidjejunumofweanlingpigletsfeddietscontainingnormalandhigh-levelsofZnO高锌对空空肠中段段干细胞胞因子表表达（基基因与蛋蛋白）的的影响DecreasetheexpressionofSCF高锌显著著降低空空肠干细细胞因子子的表达达调控肥大大细胞数数量及介介质释放放P=0.016ProteinNormalZnOHighZnOP=0.024mRNANormalZnOHighZnOTheeffectsofdietaryzinconthechangesinIsc(ΔΔIscZn)andinG(ΔGZn)日粮锌对对短路电电流和电电导率改改变的影影响Comp.Biochem.Physiol.,Z.K.Feng,2006Znimprovestightjunctionpermeability高锌降低低断奶仔仔猪小肠肠粘膜跨跨上皮电电位差维持肠道道紧密连连接的完完整性ItemsDietsSEMPZn2500DietZnDiet×Zn-Zn+ZnΔIscZn(µm/cm2)9.012.45.10.450.230.84ΔGZn(ms/cm2)1.3a0.4bc0.30.03<0.0010.82Enzymepreparations酶制剂Complexenzyme-mutationandselection复合酶———诱变变选育Aspergillussulphureus-MAFIC001可高产复复合酶的的硫色曲曲霉及其其诱变筛筛育方法法和固态态发酵制制备饲用用复合酶酶的应用用Highyieldofcomplexenzyme:xylanase,-glucanase,pectinase,etc.高产复合合酶：木木聚糖酶酶、-葡聚糖糖酶、果果胶酶等等Industrialproduction工业化生生产，商商业利润润超过1000万元Aspergillusniger-MAFIC005饲用非淀淀粉多糖糖酶高产产菌种的的选育及及其固态态发酵生生产技术术Highyieldofcomplexenzyme:xylanase,-glucanase,pectinase,etc.高产复复合酶酶：木木聚糖糖酶、、葡聚聚糖酶酶、果果胶酶酶等Existingquestionsandthemethodstoincreasetheyieldof-mannanase存在问问题及及提高高-甘露露聚糖糖酶产产量的的方法法Lowyieldandenzymaticactivity,difficultformassiveapplicationof-mannanase产量低低，酶酶活力力不高高，使使-甘露露聚糖糖酶不不能大大量推推广使使用ScreeninghighyieldingA.sulphureusMAFIC001-mannanasestrainsbyamutanttreatment诱变选选育高高产硫硫色曲曲霉MAFIC001-甘露露聚糖糖酶菌菌种Modifying-mannanasegenesequenceaccordingtocodonbias根据密密码子子偏好好性，，优化化-甘露露聚糖糖酶基基因序序列-mannanase-AspergillussulplhureusMAFIC001-甘露露聚糖糖酶－－硫色色曲霉霉MAFIC001RT-PCRandrapidamplificationofcDNAends(RACE)techniqueRT-PCR和和cDNA末端端快速速扩增增技术术PartialsequenceC-terminaldomainN-terminaldomain5’-RACE

(5’-fullRACECoreSet)3’-RACE

(3’-fullRACECoreSet)Primer1：5'-TTCAACGACGTCAACAC-3'Primer2：5'-A（T/C）CCAGCC（G/A）TTGCCCCA-3'Primer1andPrimer2usedtoamplifythepartialsequenceof-mannanase设计引引物Primer1和和Primer2扩增增-甘露露聚糖糖酶部部分序序列ObtaintheN-terminalandC-terminalof-mannanasegenesequenceusingRACEtechnique使用RACE技技术获获得-甘露露聚糖糖酶的的N-端和和C-端序序列1375bp947bp1152bpJBiotechnol.,X.L.Chen,DefaLi,2006Thefirsttimeclonethefull-lengthgenesequenceofA.sulphureusMAFIC001-mannanase(GenBankaccessionno.DQ328335)首次克克隆了了硫色色曲霉霉MAFIC001β-甘露露聚糖糖酶全全基因因序列列Modifyingthemature-mannanasegenesequencebycodonbias密码子子优化化改造造-甘露露聚糖糖酶成成熟编编码基基因序序列ConstructingP.pastorisexpressionvectorpPICZαA,highdensityfermentationofrecombinant-mannanasein10Lfermenter构建毕毕赤酵酵母表表达载载体pPICZαA，，实现现重组组-甘露露聚糖糖酶在10L发发酵罐罐中高高密度度发酵酵Pichiapastorisexpressionvector毕赤酵酵母表表达载载体

AFermentationdevice（（10L）发酵装装置（10升））SDSofexpressed-mannanaseinfermentorwithdifferentinductiontimes发酵罐罐中-甘露聚聚糖酶酶在不不同时时间的的表达达量Highestenzymaticactivity(最高酶酶活力力)：1100U/mLPurity(纯度)：96％Highestproteinexpressionlevel(最高蛋蛋白浓浓度)：3g/LAccumulationof-mannanaseactivitywithdifferentinductiontimesforfermentation发酵中中-甘露聚聚糖酶酶活性性随诱诱导时间的的积累累1100U/mLJAM,X.L.Chen,DefaLi,200635kDa45kDa66.2kDa116kDaM12345670h,12h,24h,48h,72h,96h,120h48kDa3.3fold3.9fold-mannanasecharacterization:anextremelybroadpH(2.2-8.0)range,optimumpHis2.4酶学特特性：：pH（2.2-8.0）范范围宽宽，最最适pH2.4JAM,X.L.Chen,DefaLi,2006-mannanaseproteinexpressionlevel-甘露露聚糖糖酶蛋蛋白表达水水平-mannanaseactivity-甘露聚糖糖酶活力Furtherresearch进一步的研研究Pilotscale,mass-industrialproduction中试，实现现工业化生生产Determinetheefficacybyanimalfeedingtrials动物试验验验证其效果果NationalfeedengineeringtechnologyresearchcenterNankoufermentationbase国家饲料工工程技术研研究中心南南口发酵实实验基地EnzymeMicrob.Tech.,P.Deng,DefaLi,2006Xylanase木聚糖酶AspergillusnigerMAFIC005Xylanasegene克隆黑曲霉霉MAFIC005木聚糖酶酶xynB基因，GenBank登录号号为DQ174549实现了在大大肠杆菌和和毕赤酵母母中的表达达酶学性质表表明，与动动物胃肠道道环境相适适应AspergillussulphureusMAFIC001Xylanasegene克隆硫色曲曲霉MAFIC001木聚糖糖酶xynA和xynB基因，GenBank登录号分别别为DQ355509和DQ168666实现了在大大肠杆菌和和毕赤酵母母中的表达达酶学性质表表明，与动动物胃肠道道环境相适适应+ModifiedxynA改造xynApGAPZαA载体ConstitutiveexpressioninPichiapastoris毕赤酵母中中的组成型型表达Aspergillussulphureus硫色曲霉Thermostabilityofthemodifiedrecombinantxylanase改造的重组组木聚糖酶酶热稳定性性研究Enzymaticactivity:120U/mL最高酶活力力达120U/mLProteinconcentration:3.78mg/mL蛋白浓度为为3.78mg/mLGoodstability:80ºCfor90min热稳定性好好，80ººC保持90minHerbalextracts(Astragaluspolysaccharide,APS)天然提取物物（黄芪多多糖）Hypothesis假设InflammationreactionofLPSLPS炎性性反应DisorderofGH/IGF-IaxisGH/IGF-I轴异常常Immunesuppress(cyclophosphamide)免疫抑制（（环磷酰胺胺，CY））Immuneenhancement免疫增强Immunetype免疫类型APS黄芪多糖Inflammationfactorsecretion炎性因子分分泌-？+--？-？++？-Cellculture细胞培养？(LinearP<0.05)(LinearP<0.05)EffectsofAPSonratioofCD4+/CD8+andLymphocyteproliferationinpigletsAPS对仔仔猪CD4+/CD8+比值和淋巴巴细胞增殖殖的影响Anim.Sci.,S.L.Yuan,X.S.Piao,DefaLi,2006Improvecellimmunefunction,theoptimalAPSlevelwas500mg/kgAPS提高高断奶仔猪猪的细胞免免疫功能，，增强其对对入侵微生生物和疾病病的抵抗力，，促进仔猪猪健康和生生长APS在断断奶仔猪日日粮中的最最适添加量量为500mg/kgCD4+/CD8+淋巴细胞增增殖EffectsofdietarysupplementofAPSonserumcytokinesinweanedpigsreceivedcyclophosphamide(CY)APS（注注射环磷酰酰胺,CY）对断断奶仔猪血血清细胞因因子的影响响(pg/mL)Anim.Sci.,S.L.Yuan,X.S.Piao,DefaLi,2006－CY＋CY－APS＋APS－APS＋APSd14

IL-255.479.850.677.8IFN-γ133.3160.1111.7138.9ReverseCY-inducedcellimmunesuppressandhumourimmunesuppress环磷酰胺免免疫抑制模模型研究发发现，APS可促进进猪的正常常细胞免疫，也可逆逆转CY造造成的免疫疫抑制本研究建立立了一种评评价APS对仔猪营营养和免疫疫作用的有有用模型EffectsofAPSonserumindexinpigletsAPS对对仔猪血血清指标标的影响响RecoverthenormalfunctionofGH/IGF-Iaxis炎性细胞胞因子过过度分泌泌以及GH/IGF-I轴的的异常，，导致断断奶仔猪猪发生免疫疫炎症和和生长速速度下降降APS通通过降低低炎性细细胞因子子、GH和皮质质醇的含含量，增增加抗炎炎性因子的合合成，恢恢复GH/IGF-I轴的正正常而发发挥其免免疫抗炎炎作用Anim.Sci.,S.L.Yuan,X.S.Piao,DefaLi,2006

－LPS

＋LPS－APS＋APS－APS＋APSd14

Cortisol115.3113.0155.5136.0GH3.823.834.563.94IGF-I235.6240.0215.9224.9bIncreaseNOandiNOSactivityoftheperipheralbloodlymphocytesinpigletsAPS显显著增加加仔猪外外周血淋淋巴细胞胞NO和和iNOS的活活性，并并呈剂量量依赖性性EffectsofAPSonNOandiNOSactivityinpigletsAPS对对仔猪NO和iNOS的影响响J.Anim.Sci.,X.F.Mao,DefaLi,2005*****(P<0.05)*******(P<0.05)EffectsofAPSondifferentcytokinesAPS对对不同细细胞因子子的影响响IncreasetheIL-2andIFN-γsecretionT细胞可可能是APS直直接作用用的靶细细胞之一一APS可可在体外外促进细细胞因子子IL-2和IFN-γ的分分泌，这这可能是APS的的免疫调调节作用用之一Anim.Sci.,S.L.Yuan,X.S.Piao,DefaLi,2006J.Anim.Sci.,X.F.Mao,DefaLi,2005MechanismofimmunefunctionofAPSAPS的的免疫作作用机理理APSiNOSNO细胞因子子表达淋巴细胞胞增殖+改善动物物免疫IL-2etc.IFN-γ++ReceptorGeneknockout基因敲除除Signaltransductionpathway信号传导导通路TLRsToll样受体Antimicrobialpeptides

(AMPs)抗菌肽AMPs抗菌肽Abundantanddiversegroupsofmoleculesthatareen