Alternativesofantibioticsforpigs豬用抗生素的替代DefaLi

MinistryofAgricultureFeedIndustryCentre

農(nóng)業(yè)部飼料工業(yè)中心2006年11月30日于北京豬營養(yǎng)

營養(yǎng)需要與代謝分子生物學(xué)WhooperatesMAFICResearch植物提取物免疫學(xué)微生物學(xué)分析化學(xué)營養(yǎng)與免疫基因工程菌細(xì)胞生物學(xué)

分子營養(yǎng)與肉品質(zhì)團隊研究Antibiotics抗生素Residue殘留Resistance耐藥性Harmfultoanimalandhumanhealth影響動物和人類的健康Reducetheuseofantibiotics減少抗生素的使用EU歐盟China中國Currentsituationandfuturedevelopment現(xiàn)狀與展望Probiotics

益生素Lactobacillus乳酸桿菌Mechanism機理研究Application應(yīng)用研究綠色熒光蛋白(GFP)標(biāo)記蛋白組學(xué)粘附位點無抗發(fā)酵飼料蛋白差異復(fù)合發(fā)酵培養(yǎng)悉生動物模型信號傳導(dǎo)作用機制Targetingdistribution

目的細(xì)菌在腸道中的定位分布情況，其與腸道菌系之間的關(guān)系Specificpasswayandfunction

具體作用途徑與功能的研究Experimentalmodelof

gnotobiotic

animals

悉生動物實驗?zāi)Ｐ拖ど鷦游铮ㄒ阎鷦游铮┛瞻捉M益生菌致病菌益生菌+致病菌Fermentativerespiratorymembrane發(fā)酵呼吸膜Singlewayvalve

單向閥門

CO2venting,preventingairtoenter

排出袋內(nèi)微生物產(chǎn)生的CO2，阻止大氣進入A,E.coliK88

大腸桿菌K88B,E.coliK99

大腸桿菌K99C,S.typhimurium

鼠傷寒沙門氏菌D,Staph.Aureus

金黃色葡萄球菌Int.J.Gener.Mol.Microbiol.,X.H.Guo,DefaLi,2006InhibitoryactivityofBacillusspp.139againstpathogenicbacteria,butnotLactobacillus外源芽孢桿菌可抑制有害菌，對乳酸菌無影響Multi-strainscombination,creatinganaerobicconditionautomatically多菌種組合，自身創(chuàng)造厭氧環(huán)境Yeast酵母菌Bacillusspp.芽胞桿菌好氧兼性兼性Lactobacillusreproduction

in

anaerobiccondition乳酸桿菌厭氧發(fā)酵Oligosaccharide寡糖NSPenzyme非淀粉多糖酶Activator激活劑

刺激ActivateEffectoffermentationtimeonenergyandgossypol能量與游離棉酚隨發(fā)酵時間的變化Decreasethecontentofgossypol

降低游離棉酚的含量Fermentationtime(h)EnergyGossypolGossypol（mg/kg）Energy（kcal/g）Effectsofantibiotics-freefermentedfeedongrowthperformanceinpigs無抗發(fā)酵飼料對豬生產(chǎn)性能的影響P>0.05RFID

systemRFID追溯系統(tǒng)

基因芯片Genechip

：Theonlineexaminationtechniqueofantibiotics

基因芯片：快速檢測分割肉中的抗生素含量（20-100ppb）ProteomicsanalysisofinteractionbetweenLactobacillusandintestine

乳酸桿菌和腸道細(xì)胞相互作用的蛋白質(zhì)組學(xué)研究J.Nutr.,J.J.Wang,DefaLi,2006蛋白質(zhì)組學(xué)雙向電泳樣品制備數(shù)據(jù)庫比對鑒定蛋白相互作用機制生物功能差異蛋白質(zhì)譜分析圖像分析膠內(nèi)酶切蛋白Lactobacillus乳酸桿菌Intestinalepithelialcells腸道上皮細(xì)胞等點聚焦SDSExperimentaldesign

試驗設(shè)計IntestinalcellsAdheredornotLactobacillusfermentumI5007cellsCulturemedium,37oC

37oC溫箱4hRabbitjejunum兔子空腸Two-dimensionalgelelectrophoresis

雙向電泳37oC1hMassspectrometry

質(zhì)譜Differentialproteins差異蛋白Lactobacillusin

rabbitjejunumculture兔子空腸培養(yǎng)條件下乳酸桿菌表達(dá)差異的蛋白Electrophoresis,

F.Yang,DefaLi,2006Glycosidehydrolase糖苷水解酶（粘蛋白的分解酶）降解腸粘膜表達(dá)的粘蛋白有利于乳酸桿菌粘附定植Fructose-6-phosphatePhosphoketolase(F6PPK)

6-磷酸果糖磷酸酮酶Lactatedehydrogenase乳酸脫氫酶Dihydrolipomidedehydrogenase二氫硫辛酸脫氫酶（丙酮酸脫氫酶系）糖酵解三羧酸循環(huán)磷酸戊糖途徑減少氧自由基產(chǎn)生NADPH生成增加有利于乳酸桿菌存活

Synbiotics

合生素

Functionalmechanismof

synbiotics

合生素的作用機理Synbiotics

合生素Bacillussubtilis枯草芽孢桿菌Astragalus,aloepolysaccharide,etc.黃芪多糖，蘆薈多糖等Immunity免疫力Antibacterialsubstance

抑菌物質(zhì)Organicacid有機酸Health動物健康Bacteriophage產(chǎn)生抗菌素?fù)]發(fā)性脂肪酸：乙酸、丙酸、丁酸Toleranceoftheintestinalenvironment

耐受腸道內(nèi)環(huán)境Bacillussubtilis枯草芽孢桿菌Hypothesis

假設(shè)Intestinefunction腸道功能Entericnervoussystem腸神經(jīng)系統(tǒng)Feedintake采食量Diarrhea腹瀉生產(chǎn)性能GrowthperformanceGastrointestinalhormones胃腸激素Mastcellmediators肥大細(xì)胞介質(zhì)IntestinalIGF-1system小腸IGF-1系統(tǒng)

Hormoneoneating采食相關(guān)激素ZnOEffectsofdietarysupplementof3000mg/kgZnOontheexpressionoffundicglandGhrelingeneinpiglets

日糧中添加3000mg/kg氧化鋅對斷奶仔豬胃底腺Ghrelin基因表達(dá)的影響P=0.008IncreasetheexpressionoftheGhrelingene

顯著提高斷奶仔豬胃底腺Ghrelin基因的表達(dá),提高采食量Histamineconcentrationsinthesmallintestineofweanlingpigletsfeddietscontainingnormalandhigh-levelsofZnO

高鋅日糧對斷奶仔豬小腸組胺濃度的影響Decreasethereleaseofthehistamineinthesmallintestine

高鋅顯著降低斷奶仔豬小腸組胺的釋放穩(wěn)定小腸通透性P=0.005P=0.049P=0.049DuodenumJejunumIleumThenumbersofmastcells(cell/cm2)inthesmallintestineofweanlingpigletsfeddietscontainingnormalorhigh-levelsofzincoxide高鋅日糧對斷奶仔豬小腸肥大細(xì)胞數(shù)量的影響Decreasethenumbersofthemastcellsinthesmallintestine

高鋅顯著降低斷奶仔豬小腸各層肥大細(xì)胞數(shù)改善小腸功能LocationControlTreatmentP

Duodenummucosa74.9±21.936.7±8.9<0.01Jejunummucosa99.7±13.148.8±13.2<0.01Ileummucosa81.4±19.555.1±3.4<0.01

mRNAandproteinlevelsforthestemcellfactor(SCF)geneinthemidjejunumofweanlingpigletsfeddietscontainingnormalandhigh-levelsofZnO高鋅對空腸中段干細(xì)胞因子表達(dá)（基因與蛋白）的影響DecreasetheexpressionofSCF高鋅顯著降低空腸干細(xì)胞因子的表達(dá)調(diào)控肥大細(xì)胞數(shù)量及介質(zhì)釋放P=0.016ProteinNormalZnOHighZnOP=0.024mRNANormalZnOHighZnOTheeffectsofdietaryzinconthechangesinIsc(ΔIscZn)

andinG(ΔGZn)

日糧鋅對短路電流和電導(dǎo)率改變的影響Comp.Biochem.Physiol.,Z.K.Feng,2006Znimprovestightjunctionpermeability高鋅降低斷奶仔豬小腸粘膜跨上皮電位差維持腸道緊密連接的完整性ItemsDietsSEMPZn2500DietZnDiet×Zn-Zn+ZnΔIscZn(μm/cm2)9.012.45.10.450.230.84ΔGZn(ms/cm2)1.3a0.4bc0.30.03<0.0010.82Enzymepreparations

酶制劑Complexenzyme-mutationandselection復(fù)合酶——誘變選育Aspergillussulphureus-MAFIC001

可高產(chǎn)復(fù)合酶的硫色曲霉及其誘變篩育方法和固態(tài)發(fā)酵制備飼用復(fù)合酶的應(yīng)用Highyieldofcomplexenzyme:xylanase,-glucanase,

pectinase,etc.

高產(chǎn)復(fù)合酶：木聚糖酶、-葡聚糖酶、果膠酶等Industrialproduction

工業(yè)化生產(chǎn)，商業(yè)利潤超過1000萬元Aspergillusniger-MAFIC005

飼用非淀粉多糖酶高產(chǎn)菌種的選育及其固態(tài)發(fā)酵生產(chǎn)技術(shù)Highyieldofcomplexenzyme:xylanase,-glucanase,

pectinase,etc.

高產(chǎn)復(fù)合酶：木聚糖酶、葡聚糖酶、果膠酶等Existingquestionsandthemethodstoincreasetheyieldof-mannanase存在問題及提高-甘露聚糖酶產(chǎn)量的方法Lowyieldandenzymaticactivity,difficultformassiveapplicationof-mannanase

產(chǎn)量低，酶活力不高，使-甘露聚糖酶不能大量推廣使用ScreeninghighyieldingA.sulphureusMAFIC001

-mannanase

strainsbyamutanttreatment

誘變選育高產(chǎn)硫色曲霉MAFIC001-甘露聚糖酶菌種Modifying-mannanasegenesequenceaccordingtocodonbias

根據(jù)密碼子偏好性，優(yōu)化-甘露聚糖酶基因序列-mannanase-AspergillussulplhureusMAFIC001-甘露聚糖酶－硫色曲霉MAFIC001RT-PCRandrapidamplificationofcDNAends(RACE)techniqueRT-PCR和cDNA末端快速擴增技術(shù)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

設(shè)計引物Primer1和Primer2擴增-甘露聚糖酶部分序列ObtaintheN-terminalandC-terminalof-mannanase

genesequenceusingRACEtechnique

使用RACE技術(shù)獲得-甘露聚糖酶的N-端和C-端序列1375bp947bp1152bpJBiotechnol.,X.L.Chen,DefaLi,2006Thefirsttimeclonethefull-lengthgenesequenceofA.sulphureusMAFIC001-mannanase(GenBankaccessionno.DQ328335)首次克隆了硫色曲霉MAFIC001β-甘露聚糖酶全基因序列

Modifyingthemature-mannanasegenesequencebycodonbias

密碼子優(yōu)化改造-甘露聚糖酶成熟編碼基因序列ConstructingP.pastorisexpressionvectorpPICZαA,highdensityfermentationofrecombinant-mannanasein10Lfermenter

構(gòu)建畢赤酵母表達(dá)載體pPICZαA，實現(xiàn)重組-甘露聚糖酶在10L發(fā)酵罐中高密度發(fā)酵Pichiapastorisexpressionvector畢赤酵母表達(dá)載體

AFermentationdevice（10L）發(fā)酵裝置（10升）SDSofexpressed-mannanaseinfermentorwithdifferentinductiontimes

發(fā)酵罐中-甘露聚糖酶在不同時間的表達(dá)量Highestenzymaticactivity(最高酶活力)：1100U/mLPurity

(純度)：96％Highestproteinexpressionlevel(最高蛋白濃度)：3g/LAccumulationof-mannanaseactivitywithdifferentinductiontimesforfermentation發(fā)酵中-甘露聚糖酶活性隨誘導(dǎo)時間的積累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

酶學(xué)特性：pH（2.2-8.0）范圍寬，最適pH2.4JAM,X.L.Chen,DefaLi,2006-mannanase

proteinexpressionlevel-甘露聚糖酶蛋白表達(dá)水平-mannanaseactivity-甘露聚糖酶活力Furtherresearch進一步的研究Pilotscale,mass-industrialproduction

中試，實現(xiàn)工業(yè)化生產(chǎn)

Determinetheefficacybyanimalfeedingtrials

動物試驗驗證其效果NationalfeedengineeringtechnologyresearchcenterNankoufermentationbase國家飼料工程技術(shù)研究中心南口發(fā)酵實驗基地EnzymeMicrob.Tech.,P.Deng,DefaLi,2006Xylanase木聚糖酶Aspergillus

nigerMAFIC005Xylanasegene

克隆黑曲霉MAFIC005木聚糖酶xynB基因，GenBank登錄號為

DQ174549

實現(xiàn)了在大腸桿菌和畢赤酵母中的表達(dá)酶學(xué)性質(zhì)表明，與動物胃腸道環(huán)境相適應(yīng)Aspergillus

sulphureusMAFIC001Xylanasegene

克隆硫色曲霉MAFIC001木聚糖酶xynA和xynB基因，GenBank

登錄號分別為DQ355509和DQ168666

實現(xiàn)了在大腸桿菌和畢赤酵母中的表達(dá)酶學(xué)性質(zhì)表明，與動物胃腸道環(huán)境相適應(yīng)

+ModifiedxynA改造xynA

pGAPZαA載體ConstitutiveexpressioninPichiapastoris畢赤酵母中的組成型表達(dá)Aspergillussulphureus硫色曲霉Thermostabilityofthemodifiedrecombinantxylanase改造的重組木聚糖酶熱穩(wěn)定性研究

Enzymaticactivity:120U/mL

最高酶活力達(dá)120U/mL

Proteinconcentration:3.78mg/mL

蛋白濃度為3.78mg/mLGoodstability:

80oCfor90min

熱穩(wěn)定性好，80oC保持90minHerbalextracts

(Astragaluspolysaccharide,APS)

天然提取物（黃芪多糖）Hypothesis假設(shè)InflammationreactionofLPSLPS炎性反應(yīng)DisorderofGH/IGF-IaxisGH/IGF-I軸異常Immunesuppress(cyclophosphamide)

免疫抑制（環(huán)磷酰胺，CY）Immuneenhancement免疫增強Immunetype免疫類型APS黃芪多糖Inflammationfactorsecretion炎性因子分泌-？+--？-？++？-Cellculture細(xì)胞培養(yǎng)？(LinearP<0.05)(LinearP<0.05)EffectsofAPSonratioofCD4+/CD8+andLymphocyteproliferationinpiglets

APS對仔豬CD4+/CD8+比值和淋巴細(xì)胞增殖的影響Anim.Sci.,S.L.Yuan,X.S.Piao,DefaLi,2006Improvecellimmunefunction,theoptimalAPSlevelwas500mg/kg

APS提高斷奶仔豬的細(xì)胞免疫功能，增強其對入侵微生物和疾病的抵抗力，促進仔豬健康和生長

APS在斷奶仔豬日糧中的最適添加量為500mg/kgCD4+/CD8+淋巴細(xì)胞增殖EffectsofdietarysupplementofAPSonserumcytokinesinweanedpigsreceivedcyclophosphamide(CY)

APS（注射環(huán)磷酰胺,CY）對斷奶仔豬血清細(xì)胞因子的影響(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-inducedcellimmunesuppressandhumour

immunesuppress環(huán)磷酰胺免疫抑制模型研究發(fā)現(xiàn)，APS可促進豬的正常細(xì)胞免疫，也可逆轉(zhuǎn)CY造成的免疫抑制本研究建立了一種評價APS對仔豬營養(yǎng)和免疫作用的有用模型EffectsofAPSonserumindexinpiglets

APS對仔豬血清指標(biāo)的影響RecoverthenormalfunctionofGH/IGF-Iaxis炎性細(xì)胞因子過度分泌以及GH/IGF-I軸的異常，導(dǎo)致斷奶仔豬發(fā)生免疫炎癥和生長速度下降A(chǔ)PS通過降低炎性細(xì)胞因子、GH和皮質(zhì)醇的含量，增加抗炎性因子的合成，恢復(fù)GH/IGF-I軸的正常而發(fā)揮其免疫抗炎作用

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-I235.6240.0215.9224.9bIncreaseNOandiNOSactivityofthe

peripheralbloodlymphocytes

inpigletsAPS顯著增加仔豬外周血淋巴細(xì)胞NO和iNOS的活性，并呈劑量依賴性EffectsofAPSonNOandiNOSactivityinpiglets

APS對仔豬NO和iNOS的影響J.Anim.Sci.,X.F.Mao,DefaLi,2005*****(P<0.05)*******(P<0.05)EffectsofAPSondifferentcytokines

APS對不同細(xì)胞因子的影響

IncreasetheIL-2andIFN-γsecretionT細(xì)胞可能是APS直接作用的靶細(xì)胞之一APS可在體外促進細(xì)胞因子IL-2和IFN-γ的分泌，這可能是APS的免疫調(diào)節(jié)作用之一Anim.Sci.,S.L.Yuan,X.S.Piao,DefaLi,2006J.Anim.Sci.,X.F.Mao,DefaLi,2005MechanismofimmunefunctionofAPSAPS的免疫作用機理APSiNOSNO細(xì)胞因子表達(dá)

淋巴細(xì)胞增殖+改善動物免疫IL-2etc.IFN-γ++ReceptorGeneknockout

基因敲除Signaltransductionpathway信號傳導(dǎo)通路TLRsToll樣受體Antimicrobialpeptides

(AMPs)

抗菌肽AMPs

抗菌肽Abundantanddiversegroupsofmoleculesthatareencodedbydefinitegenes

特定編碼基因產(chǎn)生的一類小分子