猪用抗生素的替代

1、会计学1猪用抗生素的替代猪用抗生素的替代猪营养营养需要与代谢分子生物学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Harmful to animal and human health影响动物和人类的健康Reduce the use of antibiotics减少抗生素的使用 EU欧盟China中国Current situation and future development 现状与展望Alternatives of antibiotics 抗生素替代uAntimicrobia

2、l peptides 抗菌肽 u Herbal extracts 天然提取物u Enzyme preparations 酶制剂u Probiotics 益生素Probiotics益生素益生素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Experimental model of gnotobiotic animals悉生动物实验模型空白组Development of antibiotics-free fermented feed无抗发酵饲料的开发无抗

3、发酵饲料的开发Fermentative respiratory membrane发酵呼吸膜 Single way valve 单向阀门 CO2 venting, preventing air to enter 排出袋内微生物产生的CO2，阻止大气进入A, E. coli K88 大肠杆菌K88B, E. coli K99 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, Defa Li, 2006Inhibitory activity of Ba

4、cillus spp. 139 against pathogenic bacteria, but not Lactobacillus外源芽孢杆菌可抑制有害菌，对乳酸菌无影响Multi-strains combination, creating anaerobic condition automatically 多菌种组合，自身创造厌氧环境Yeast酵母菌Bacillus spp.芽胞杆菌好氧兼性兼性Lactobacillus reproduction in anaerobic condition 乳酸杆菌厌氧发酵Oligosaccharide寡糖NSP enzyme非淀粉多糖酶Activato

5、r激活剂 刺激ActivateInoculating, packing, then fermentation先接种、包装，后发酵接菌激活剂原料发酵轻工业副产物：玉米浆、果渣Effect of fermentation time on energy and gossypol能量与游离棉酚随发酵时间的变化 Decrease the content of gossypol 降低游离棉酚的含量Fermentation time (h)EnergyGossypolGossypol（mg/kg）Energy（kcal/g）Effects of antibiotics-free fermented feed

6、 on growth performance in pigs无抗发酵饲料对猪生产性能的影响P 0.05RFID systemRFID 追溯系统 基因芯片uGene chip ：The on line examination technique of antibiotics 基因芯片：快速检测分割肉中的抗生素含量（20-100 ppb）Proteomics 蛋白质组学Proteomics analysis of interaction between Lactobacillus and intestine乳酸杆菌和肠道细胞相互作用的蛋白质组学研究J. Nutr., J. J. Wang, Def

7、a Li, 2006蛋白质组学双向电泳样品制备数据库比对鉴定蛋白相互作用机制生物功能差异蛋白质谱分析图像分析胶内酶切蛋白Lactobacillus乳酸杆菌Intestinal epithelial cells肠道上皮细胞等点聚焦SDS-PAGEExperimental design试验设计试验设计Intestinal cellsAdhered or notLactobacillus fermentum I5007 cellsCulture medium, 37C 37C温箱4 hRabbit jejunum兔子空肠lTwo-dimensional gel electrophoresis 双向电

8、泳37C 1 hlMass spectrometry 质谱Differential proteins差异蛋白Lactobacillus in rabbit jejunum culture兔子空肠培养条件下乳酸杆菌表达差异的蛋白Electrophoresis, F. Yang, Defa Li, 2006Glycoside hydrolase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-phosphate Phosphoketolase (F6PPK) 6-磷酸果糖磷酸酮酶Lactate dehydrogenase乳酸脱氢酶Dihydrolipom

9、ide dehydrogenase二氢硫辛酸脱氢酶（丙酮酸脱氢酶系）糖酵解三羧酸循环磷酸戊糖途径减少氧自由基产生NADPH生成增加有利于乳酸杆菌存活Intestinal cells after Lactobacillus adhesion肠细胞被乳酸杆菌粘附条件下表达差异的蛋白Electrophoresis, F. Yang, Defa Li, 2006Voltage-dependent anion channel 1 电压依从性阴离子通道1Glutathione transferase 谷胱甘肽转移酶Heat shock protein gp96 热应激蛋白gp96调控膜通透性改善机体免疫减

10、少氧化应激改善肠道完整性及其健康Synbiotics 合生素Functional mechanism of synbiotics 合生素的作用机理Synbiotics 合生素Bacillus subtilis枯草芽孢杆菌Astragalus, aloe polysaccharide, etc.黄芪多糖，芦荟多糖等Immunity免疫力Antibacterial substance 抑菌物质Organic acid有机酸Health动物健康Bacillus subtilis枯草芽孢杆菌ZnO氧化锌氧化锌Hypothesis假设假设Intestine function肠道功能Enteric ner

11、vous system 肠神经系统Feed intake采食量Diarrhea腹泻生产性能Growth performanceGastrointestinal hormones胃肠激素Mast cell mediators肥大细胞介质Intestinal IGF-1 system小肠IGF-1系统 Hormone on eating采食相关激素ZnOEffects of dietary supplement of 3000 mg/kg ZnO on the expression of fundic gland Ghrelin gene in piglets日粮中添加日粮中添加3000 mg/k

12、g3000 mg/kg氧化锌对断奶仔猪胃底腺氧化锌对断奶仔猪胃底腺GhrelinGhrelin基因表达的影响基因表达的影响P = 0.008Increase the expression of the Ghrelin gene 显著提高断奶仔猪胃底腺Ghrelin基因的表达, 提高采食量 Improve the intestinal function 药理剂量氧化锌促进仔猪肠道健康The small intestinal morphology of weaned pigs receiving a diet with or without supplementation of 3000 mg/k

13、g Zn 日粮中添加3000 mg/kg氧化锌ZnO对仔猪小肠形态的影响J. Nutr., X. L. Li, Defa Li, 2006ItemsZnSO4ZnO groupSEMPVillus height, m 341 387 14 0.036Crypt depth, m 194 179 8 0.248 Increase the expression of IGF-1 gene 提高小肠 IGF-1 基因的表达 通过IGF-1的旁分泌作用促进仔猪肠道健康和生长Effects of dietary supplement of 3000 mg/kg ZnO on the expression

14、 of small-intestinal mucosa IGF-1 gene in piglets日粮中添加3000 mg/kg氧化锌对仔猪小肠粘膜IGF-1基因表达的影响P = 0.012P = 0.041ab libitum Pair-fedJ. Nutr., X. L. Li, Defa Li, 2006Histamine concentrations in the small intestine of weanling piglets fed diets containing normal and high-levels of ZnO高锌日粮对断奶仔猪小肠组胺浓度的影响高锌日粮对断奶仔

15、猪小肠组胺浓度的影响uDecrease the release of the histamine in the small intestine 高锌显著降低断奶仔猪小肠组胺的释放稳定小肠通透性P = 0.005P = 0.049P = 0.049Duodenum Jejunum Ileum The numbers of mast cells (cell/cm2) in the small intestine of weanling piglets fed diets containing normal or high-levels of zinc oxide高锌日粮对断奶仔猪小肠肥大细胞数量的

16、影响 Decrease the numbers of the mast cells in the small intestine 高锌显著降低断奶仔猪小肠各层肥大细胞数改善小肠功能LocationControlTreatmentP Duodenum mucosa74.9 21.936.7 8.90.01Jejunum mucosa99.7 13.1 48.8 13.20.01Ileum mucosa81.4 19.555.1 3.40.01mRNA and protein levels for the stem cell factor (SCF) gene in the mid jejunum

17、 of weanling piglets fed diets containing normal and high-levels of ZnO高锌对空肠中段干细胞因子表达（基因与蛋白）的影响 Decrease the expression of SCF 高锌显著降低空肠干细胞因子的表达 调控肥大细胞数量及介质释放P = 0.016ProteinNormal ZnOHigh ZnOP = 0.024mRNANormal ZnOHigh ZnOThe effects of dietary zinc on the changes in Isc (IscZn) and in G (GZn)日粮锌对短路

18、电流和电导率改变的影响日粮锌对短路电流和电导率改变的影响Comp. Biochem. Physiol., Z. K. Feng, 2006 Zn improves tight junction permeabilityl 高锌降低断奶仔猪小肠粘膜跨上皮电位差l 维持肠道紧密连接的完整性ItemsDietsSEMPZn2500DietZnDiet Zn- Zn+ ZnIscZn (m/cm2)9.012.45.10.450.230.84GZn (ms/cm2)1.3a 0.4bc0.30.03 0.0010.82Enzyme preparations酶制剂酶制剂Complex enzyme-mu

19、tation and selection复合酶诱变选育 Aspergillus sulphureus-MAFIC001 可高产复合酶的硫色曲霉及其诱变筛育方法和固态发酵制备饲用复合酶的应用 High yield of complex enzyme: xylanase, -glucanase, pectinase, etc. 高产复合酶：木聚糖酶、 -葡聚糖酶、果胶酶等 Industrial production 工业化生产，商业利润超过1000万元 Aspergillus niger-MAFIC005 饲用非淀粉多糖酶高产菌种的选育及其固态发酵生产技术 High yield of comple

20、x enzyme: xylanase, -glucanase, pectinase, etc. 高产复合酶：木聚糖酶、葡聚糖酶、果胶酶等Existing questions and the methods to increase the yield of -mannanase存在问题及提高-甘露聚糖酶产量的方法 Low yield and enzymatic activity, difficult for massive application of -mannanase 产量低，酶活力不高，使-甘露聚糖酶不能大量推广使用uScreening high yielding A. sulphure

21、us MAFIC001 -mannanase strains by a mutant treatment 诱变选育高产硫色曲霉MAFIC001 -甘露聚糖酶菌种uModifying -mannanase gene sequence according to codon bias 根据密码子偏好性，优化-甘露聚糖酶基因序列-mannanase-Aspergillus sulplhureus MAFIC001-甘露聚糖酶硫色曲霉MAFIC001RT-PCR and rapid amplification of cDNA ends (RACE) techniqueRT-PCR和cDNA末端快速扩增技

22、术Partial sequenceC-terminal domain N-terminal domain 5-RACE (5-full RACE Core Set)3-RACE (3-full RACE Core Set)Primer 1：5-TTCAACGACGTCAACAC -3Primer 2：5-A（T/C）CCAGCC（G/A）TTGCCCCA-3 Primer 1 and Primer 2 used to amplify the partial sequence of -mannanase 设计引物Primer 1和Primer 2扩增-甘露聚糖酶部分序列 Obtain the N

23、-terminal and C-terminal of -mannanase gene sequence using RACE technique 使用RACE技术获得-甘露聚糖酶的N-端和C-端序列1 3 7 5 bp 947 bp1152 bpJ Biotechnol., X. L. Chen, Defa Li, 2006The first time clone the full-length gene sequence of A. sulphureus MAFIC001 -mannanase (GenBank accession no. DQ328335)首次克隆了硫色曲霉MAFIC00

24、1-甘露聚糖酶全基因序列 Modifying the mature -mannanase gene sequence by codon bias 密码子优化改造-甘露聚糖酶成熟编码基因序列Constructing P. pastoris expression vector pPICZA, high density fermentation of recombinant -mannanase in 10 L fermenter 构建毕赤酵母表达载体pPICZA，实现重组-甘露聚糖 酶在10 L发酵罐中高密度发酵Pichia pastoris expression vector毕赤酵母表达载体 A

25、Fermentation device （10 L）发酵装置（10升）SDS-PAGE of expressed -mannanase in fermentor with different induction times 发酵罐中-甘露聚糖酶在不同时间的表达量 Highest enzymatic activity (最高酶活力)：1100 U/mL Purity (纯度)：96 Highest protein expression level (最高蛋白浓度)：3 g/LAccumulation of -mannanaseactivity with different induction t

26、imes for fermentation发酵中-甘露聚糖酶活性随诱导时间的积累 1100 U/mLJAM, X. L. Chen, Defa Li, 200635 kDa 45 kDa66.2kDa 116 kDa M 1 2 3 4 5 6 7 0 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h48 kDa3.3 fold3.9 fold-mannanase characterization: an extremely broad pH (2.2-8.0) range, optimum pH is 2.4 酶学特性：pH（2.2-8.0）范围宽，最适pH 2.

27、4JAM, X. L. Chen, Defa Li, 2006-mannanase protein expression level-甘露聚糖酶蛋白表达水平-mannanase activity-甘露聚糖酶活力Further research进一步的研究 Pilot scale, mass-industrial production 中试，实现工业化生产 Determine the efficacy by animal feeding trials 动物试验验证其效果National feed engineering technology research center Nankou ferm

28、entation base国家饲料工程技术研究中心南口发酵实验基地Enzyme Microb. Tech., P. Deng, Defa Li, 2006Xylanase木聚糖酶Aspergillus niger MAFIC005 Xylanase gene l 克隆黑曲霉MAFIC005木聚糖酶xynB基因，GenBank登录号为 DQ174549l 实现了在大肠杆菌和毕赤酵母中的表达l 酶学性质表明，与动物胃肠道环境相适应 Aspergillus sulphureus MAFIC001 Xylanase gene l 克隆硫色曲霉MAFIC001木聚糖酶xynA和xynB基因，GenBan

29、k 登录号分别为DQ355509和DQ168666l 实现了在大肠杆菌和毕赤酵母中的表达l 酶学性质表明，与动物胃肠道环境相适应 +Modified xynA改造xynA pGAPZA载体Constitutive expression in Pichia pastoris毕赤酵母中的组成型表达Aspergillus sulphureus硫色曲霉Thermostability of the modified recombinant xylanase改造的重组木聚糖酶热稳定性研究l Enzymatic activity: 120 U/mL 最高酶活力达120 U/mL l Protein conc

30、entration: 3.78 mg/mL 蛋白浓度为3.78 mg/mLl Good stability: 80C for 90 min 热稳定性好，80C保持90 minHerbal extracts(Astragalus polysaccharide, APS)天然提取物（黄芪多糖）天然提取物（黄芪多糖）Hypothesis 假设假设Inflammation reaction of LPS LPS炎性反应Disorder of GH/IGF-I axisGH / IGF-I轴异常Immune suppress (cyclophosphamide) 免疫抑制（环磷酰胺，CY ）Immune

31、 enhancement免疫增强Immune type免疫类型APS黄芪多糖Inflammation factor secretion炎性因子分泌-？+-？-？+？-Cell culture细胞培养？(Linear P 0.05)(Linear P 0.05)Effects of APS on ratio of CD4+/CD8+ and Lymphocyte proliferation in pigletsAPS对仔猪CD4+/CD8+比值和淋巴细胞增殖的影响Anim. Sci., S. L. Yuan, X. S. Piao, Defa Li, 2006uImprove cell immu

32、ne function, the optimal APS level was 500 mg/kgAPS提高断奶仔猪的细胞免疫功能，增强其对入侵微生物和疾病 的抵抗力，促进仔猪健康和生长 APS在断奶仔猪日粮中的最适添加量为500 mg/kg CD4+/CD8+淋巴细胞增殖Effects of dietary supplement of APS on serum cytokines in weaned pigs received cyclophosphamide (CY)APSAPS（注射环磷酰胺（注射环磷酰胺, CY, CY）对断奶仔猪血清细胞因子的影）对断奶仔猪血清细胞因子的影响响 (pg/

33、mL)(pg/mL)Anim. Sci., S. L. Yuan, X. S. Piao, Defa Li, 2006 CY CYAPSAPSAPSAPSd 14 IL-2 55.4 79.8 50.6 77.8 IFN- 133.3160.1111.7138.9Reverse CY-induced cell immune suppress and humour immune suppressl环磷酰胺免疫抑制模型研究发现，APS可促进猪的正常细胞免 疫，也可逆转CY造成的免疫抑制l本研究建立了一种评价APS对仔猪营养和免疫作用的有用模型Effects of APS on serum inde

34、x in pigletsAPS对仔猪血清指标的影响 Recover the normal function of GH/IGF-I axisv 炎性细胞因子过度分泌以及GH/IGF-I轴的异常，导致断奶仔猪 发生免疫炎症和生长速度下降v APS通过降低炎性细胞因子、GH和皮质醇的含量，增加抗炎性 因子的合成，恢复GH/IGF-I轴的正常而发挥其免疫抗炎作用 Anim. Sci., S. L. Yuan, X. S. Piao, Defa Li, 2006 LPS LPSAPSAPSAPSAPSd 14 Cortisol 115.3113.0155.5136.0 GH 3.82 3.83 4.5

35、6 3.94 IGF-I 235.6240.0215.9224.9bIncrease NO and iNOS activity of the peripheral blood lymphocytes in pigletsn APS显著增加仔猪外周血淋巴细胞NO和iNOS的活性，并呈剂量依赖性Effects of APS on NO and iNOS activity in piglets APSAPS对仔猪对仔猪NONO和和iNOSiNOS的影响的影响J. Anim. Sci., X. F. Mao, Defa Li, 2005* (P 0.05)*(P 0.05)Effects of APS

36、 on different cytokines APSAPS对不同细胞因子的影响对不同细胞因子的影响Increase the IL-2 and IFN- secretionvT细胞可能是APS直接作用的靶细胞之一vAPS可在体外促进细胞因子IL-2和IFN-的分泌，这 可能是APS的免疫调节作用之一Anim. Sci., S. L. Yuan, X. S. Piao, Defa Li, 2006J. Anim. Sci., X. F. Mao, Defa Li, 2005Mechanism of immune function of APSAPS的免疫作用机理APSiNOSNO细胞因子表达 淋

37、巴细胞增殖+改善动物免疫IL-2 etc.IFN-+ReceptorGene knockout 基因敲除Signal transduction pathway 信号传导通路TLRsToll 样受体Antimicrobial peptides(AMPs)抗菌肽抗菌肽AMPs抗菌肽抗菌肽1: control with no insert; 2-6: positive recombinants induced 72 h1：对照；2-6：重组菌诱导72 h的正对照组（抗菌肽）Process biochemistry, J. Zhang, 2006 Antibacterial assay抑菌分析E. c

38、oli K12D31AMPs-sensitive strain抗菌肽敏感菌株Current situation and existing problems现状及存在问题现状及存在问题Key points for product development产品研发关键点Development work 开展工作enzyme preparations, herbal extracts, probiotics, antimicrobial peptides酶制剂，天然提取物，益生素，抗菌肽Recombinant DNA technique重组DNA技术Microbiology微生物学Antibiotic

39、s 抗生素Extraction 提取Conclusions 结论Animal products绿色畜产品再次感谢各位对饲料工业中心十年来的关心与支持！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Proteomics 蛋白质组学Experimental design试验设计试验设计Intestinal cellsAdhered or notLactobacillus fermentum I5007 cellsCulture medium, 37C 37C温箱4 hRabbit jejunum兔子空肠lTwo-dimensional gel electrophoresis 双向电泳37C 1 hlMass spectrometry 质谱Differential proteins差异蛋白 Improve the intestinal function 药理剂量氧化锌促进仔猪肠道健康The small intestinal morphology of weaned pigs rece