短芒大麦草青贮微生物特性研究及优良乳酸菌筛选

以短芒大麦草为研究对象,利用传统培养法从叶围和青贮发酵体系中分离出乳酸菌、大肠杆菌、好氧细菌、酵母菌和霉菌,并计数;结合细菌形态学、生理生化特征及16S rDNA序列分析鉴定分离出的乳酸菌菌株;通过研究乳酸菌的生长曲线、产酸特性及耐酸性,筛选优质乳酸菌。以期探明短芒大麦草叶围及青贮发酵体系中微生物菌群特性及青贮料中乳酸菌多样性,筛选出具有促发酵效果的乳酸菌菌株,为有益微生物饲料研发奠定基础。试验结果表明,短芒大麦草经青贮发酵后各微生物菌群数量发生不同程度变化,乳酸菌数量由0 cfu/g FM增加到4.00×10⁸ cfu/g FM,酵母菌数量由8.50×10⁵ cfu/g FM增加到1.02×10⁸ cfu/g FM,而好氧细菌、大肠杆菌和霉菌数量变化不明显;从短芒大麦草青贮发酵体系分离得到4株乳酸菌,经鉴定Lx36为Lactobacillus pentosus,Lx37为Lactobacillus brevis,Lx53为Pediococcus pentosaceus,Lx54为Lactobacillus parabuchneri;筛选得到1株益于青贮的乳酸菌株Lx36,约在20 h后进入稳定生长期,OD值达到4.21,且发酵12 h的pH仅为4.08,并可以在pH=3.0环境条件下生长。综上所述,青贮发酵是体系中各种微生物相互作用的过程,微生物菌群的数量及变化直接影响青贮饲料发酵品质。短芒大麦草青贮饲料中乳酸菌种类较丰富,筛选得到的戊糖乳杆菌繁殖速度快、产酸能力强同时表现出了较强的耐酸性,具有潜在的生产应用价值,适宜用作促发酵的青贮添加剂菌种。     

运用OD值对常见畜禽腹泻病原菌计数的研究

本研究就畜禽常见腹泻病原菌的生长特性、细菌悬液浊度和细菌计数之间的关系进行探讨，通过对常见畜禽腹泻病原菌大肠杆菌(O8型和O139型)、鸡白痢沙门菌、鼠伤寒沙门菌和鸡伤寒沙门菌不同培养时间段吸光度(optical density,OD)值的测定，结合平板活菌计数法来确定这5种细菌的生长曲线，并对细菌浊度和细菌计数相关性进行统计分析，发现极显著相关(P <0.01)，建立了5种细菌在生长期和稳定期的OD值-细菌数对数的曲线回归方程，O8型大肠杆菌：y=2.55x+6.72(r²=0.96);O139型大肠杆菌：y=1.45x+8.77(r²=0.95)；鸡伤寒沙门菌：y=4.50x+7.02(r²=0.95)；鼠伤寒沙门菌：y=5.59x+7.67(r²=0.97)；鸡白痢沙门菌：y=9.72x+6.60(r²=0.98)。利用该回归方程可快速准确地确定菌液浓度，为探讨细菌的培养特性、致病机制及动物实验中攻毒剂量的确定等相关实验提供参考依据。     

一株解淀粉芽孢杆菌的特性及抑菌活性研究

试验旨在对一株牛瘤胃源解淀粉芽孢杆菌的生长曲线、耐酸性和耐胆酸盐性等特性进行分析，同时对该菌发酵产物的抑菌活性和抑菌活性稳定性进行测定。采用生长速率法测定该菌生长曲线，活菌计数法测定耐酸性和耐胆酸盐性等特性，牛津杯法测定抑菌活性和抑菌活性稳定性。结果显示，该菌生长4 h时进入对数生长期，20 h后生长趋于稳定，进入生长稳定期；同时该菌表现出较好的耐酸性，在pH 3.0~7.0时存活率为23.8%~90.6%；有较好的耐胆酸盐性，在0.1%~0.4%的胆酸盐中存活率为64.2%~83.6%。以大肠杆菌（Escherichia coli）、副溶血弧菌（Vibrio parahemolyticus）、藤黄八叠球菌（Sarcina lutea）和产气杆菌（Aerogenes）等细菌和黑曲霉（Aspergillus niger）、黑根霉（Rhizopus nigricans）等霉菌作指示菌进行抑菌试验发现，该菌的发酵产物具有抑菌活性，同时其抑菌活性物质的耐热性和耐酸性较好。本试验通过对该解淀粉芽孢杆菌的特性进行研究发现，该菌具有耐酸、耐胆酸盐等特性；其抑菌活性及抑菌稳定性较好，具有耐酸碱及耐高温特性。     

禽大肠杆菌病的临诊表现与防治方案

感染大肠杆菌的鸡只，由于大肠杆菌在其体内滋生繁殖，消耗机体大量的能量，鸡只便会出现眼扁、嘴长、消瘦、怕冷。同时细菌代谢排出的大量毒素进入血液循环，导致菌毒血症，这时如果细菌伴随体液进入关节腔便会引起鸡体关节肿大；细菌在血液内进一步繁殖会消耗大量的氧，引起血液含氧量低、粘稠度高、运行缓慢，临诊观察表现为精神沉郁、呆立、肌肉发绀。     

地衣芽孢杆菌M109高密度发酵条件的优化

试验旨在筛选一株地衣芽孢杆菌M109(Bacillus licheniformis M109)进行培养基和发酵条件的优化。采用单因素试验方法筛选出最佳碳源和氮源的培养基,并利用正交试验方法确定其最佳的碳氮比。为了继续提高地衣芽孢杆菌M109的发酵水平,研究其在发酵过程中的生长曲线、pH和溶氧(DO)水平的变化曲线,通过对发酵过程中生长参数的测定,优化了地衣芽孢杆菌M109最佳的接种量和最适pH。结果发现,溶氧限制是地衣芽孢杆菌M109生长的关键因素;在限定通风量的条件下,通过调节搅拌转速的方法来提高培养基中的溶氧水平,提高发酵密度;通过流加培养基的方法也能提高地衣芽孢杆菌M109的发酵水平。因此,通过对培养基和发酵条件的优化,使地衣芽孢杆菌M109的发酵水平由最初的1.0×10⁹ CFU/mL提高到1.2×10¹⁰ CFU/mL,芽孢形成率为88%。     

青贮饲料添加剂及其研究应用现状

青贮添加剂的主要作用是能够增加青贮饲料的营养物质含量，促进乳酸菌的发酵作用，抑制有害细菌和霉菌的生长，减少青贮饲料氧和水分的含量，防止腐烂等作用。目前，常用的青贮饲料添加剂可归纳为四大类即发酵促进剂、发酵抑制剂、防腐剂和营养性添加剂。     

鼠李糖乳杆菌对三种细菌生长的抑制作用

为了研究鼠李糖乳杆菌对大肠杆菌、沙门氏菌和产气荚膜梭菌的抑菌作用,试验采用牛津杯法检测了鼠李糖乳杆菌与三种细菌共培养的细菌数、抑菌圈直径,并测定了细菌生长曲线。结果表明:共培养后三种细菌的数量均显著低于对照组(P0.05);鼠李糖乳杆菌发酵上清液和全菌裂解液与三种细菌共培养后,细菌对数生长期明显滞后或未出现对数生长期;鼠李糖乳杆菌发酵上清液和全菌裂解液对三种细菌的抑菌圈直径均显著大于对照组(P0.05),全菌裂解液对大肠杆菌和产气荚膜梭菌的抑菌圈直径显著大于上清液(P0.05)。说明鼠李糖乳杆菌对大肠杆菌、沙门氏菌和产气荚膜梭菌的生长具有明显的抑制作用,其中对产气荚膜梭菌的抑制效果最强,全菌裂解液的抑菌效果优于上清液。     

运用OD值法进行细菌计数的研究

本试验通过细菌不同稀释度菌液ＯＤ值的测定，与平板计数结果相结合，建立了以下四种常用试验菌ＯＤ值──细菌数回归方程：①大肠杆菌；②金黄色葡萄球菌③枯草杆菌；④多杀性巴氏杆菌结果证明用ＯＤ值进行细菌计数具有迅速、准确等优点。     

功能菌株及其复合菌剂发酵配合饲料的研究

发酵饲料富含有益微生物、消化酶及功能物质，已广泛应用于畜牧行业，应用效果良好。微生物种类及功能是影响微生物发酵饲料发酵效果的关键因素之一。为研究不同功能菌株在发酵过程中的作用，以pH、酸溶蛋白含量、抗原蛋白和抑菌活性为指标，对不同菌株及其复合菌剂固态发酵鸡配合饲料进行评价。结果表明：FM-20菌株产酸性物质能力较强，能显著降低饲料pH(P<0.05);FM-18菌株发酵产酸溶蛋白最快；JM-1菌株抑制大肠杆菌能力较强。经复配后，各菌株产生一定协同发酵作用，酸溶蛋白含量达到35.98%,β-伴大豆球蛋白降解明显，对大肠杆菌和沙门氏菌抑制活性显著（P<0.05），对大肠杆菌的抑菌面积达到1.18 cm2。试验为功能菌株及其复合菌剂在发酵配合饲料中的应用提供了相应的基础。     

7种抗菌药物对金黄色葡萄球菌及大肠杆菌体外抗生素后效应的研究

采用稀释法去除抗生素，用菌落计数测定细菌生长曲线的方法测定环丙沙星、洛美沙星、诺氟沙星、恩诺沙星、庆大霉素、青霉素及氨苄西林对金葡球菌及大肠杆菌的抗生素后效应(PAE)。结果：几种药物在各浓度组对金黄色葡萄球菌均呈现明显的PAE，氟喹诺酮类药物及庆大霉素对大肠杆菌产生较长的PAE，而β-内酰胺类药物作用后的PAE则很短。PAE的存在提示：在兽医临床设计给药方案时，适当延长给药间隔时间，减少给药次数，仍能维持抗菌效果。     

Isolation and Identification of E. coli in the Microbial-fermentation Bed of Piggery

To investigate the distribution and characteristic of E. coli in the microbial-fermentation bed of piggery, litters with different levels of fermentation were used for the isolation, identification and drug sensitivity test of E. coli, to analyze the pathogenicity and drug susceptibility of E. coli in the microbial-fermentation bed of piggery. The results showed that, E. coli could be isolated only from litters with low degree of fermentation (0<△E≤7.63), and totally 41 strains were isolated from litters with 10^-5 dilution, but none from litters with higher degree of fermentation (△E>7.63). The number of E. coli reduced along with the fermentation of litters. Among the 41 strains of E. coli, 15 were intestinal pathogenic strains, 9 of which had astA gene (22%), and 2 strains had sepA gene (4.8%). 11 strains were resistant to doxycycline, accounted for 27% of the total.The two strains containing sepA gene had strong resistance to doxycycline. In conclusion, the microbial-fermentation bed fermentation bed had a inhibitory effect on E. coli which promoted the application of fermentation bed and reusing of litters.     

副产物乳酸对猪链球菌疫苗株ST171发酵的影响

为提高猪链球菌(S.suis)疫苗的高密度发酵效果,本研究对S.suis疫苗株(ST171)的发酵过程进行检测,利用外源添加试验研究乳酸对ST171发酵的影响,并通过对发酵工艺的优化减少乳酸的生成.结果表明,ST171发酵液中积累有大量对其生长及活菌数量有不利影响的代谢副产物乳酸.经优化确定ST171发酵的最优工艺参数为:溶氧水平20％,初始葡萄糖浓度10g/L,残糖浓度1g/L,最大比生长速率小于0.7 h-1.在优化条件下,发酵过程中乳酸生成量与原工艺相比降低了41.41％,菌体生物量和活菌数量分别提高了70.58％、129.09％,取得了良好的发酵效果.     

微生物发酵床猪舍不同发酵等级垫料中大肠杆菌的分离鉴定

为了研究微生物发酵床不同发酵等级垫料中大肠杆菌的分布及其特性,试验采集了微生物发酵床猪舍不同发酵程度的垫料,进行大肠杆菌的分离鉴定和药敏试验,分析了发酵床中大肠杆菌的致病性和抗生素抗性。试验结果表明,在10^-5稀释倍数下,仅在垫料发酵级别为一级的垫料中,即垫料使用时间较短、发酵程度较低（0<△E≤7.63）的条件下,分离到41株大肠杆菌;而在发酵程度较高的垫料中,即垫料发酵程度二级以上（△E>7.63）的环境中未分离到大肠杆菌。随着发酵的进行,发酵床中大肠杆菌数量逐渐减少。41株大肠杆菌中含有致泻性大肠杆菌15株,其中具有热稳定肠毒素（astA）基因的大肠杆菌9株,占总数的22%,含耐药性因子（sepA）基因的大肠杆菌2株,占总数的4.8%;耐强力霉素的大肠杆菌11株,占总数的27%。含有sepA基因的大肠杆菌具有强耐药性。从以上结果来看,微生物发酵床对猪舍大肠杆菌具有抑制作用,可为发酵床的推广应用及垫料再利用提供一定依据。     

鸭大肠杆菌制苗用基础种子的建立及高密度发酵培养试验研究

从定型的优势血清型菌株中选择了2株,制备了基础种子,并对基础种子第1代、第10代和第13代的传代细菌的培养特性、免疫原性以及毒力进行了测定。结果证明,鸭大肠杆菌GX-5株和GX-21株的基础种子传至13代,其培养特性、免疫原性和毒力均不发生变化,表明这两个分离株是良好的疫苗生产菌株,为建立种子批提供了依据。本试验还进行了鸭大肠杆菌高密度发酵试验,获得了鸭大肠杆菌在马丁肉汤培养基中的最佳发酵条件,即发酵温度37.5 ℃,pH 7.4,初糖浓度10 g/L,补糖量为10 g/L,接种量为4%,通气量为1 L/L·min,搅拌转速为200 r/min,培养12 h后得到的发酵菌液浓度最高,D_{600 nm}值达到30.32。本研究为鸭大肠杆菌抗原的规模化生产奠定了基础,也为新疫苗的研制和开发提供了基础材料。     

In vitro and in vivo characterization of avian Escherichia coli. II. Factors associated with pathogenicity

The pathogenicity of 197 Escherichia coli isolates obtained from clinically affected commercially grown broiler chickens and normal hatchery chicks was assessed by inoculating day-old broilers intratracheally. The degree of pathogenicity (high, intermediate, low) was judged according to mortality and lesions occurring within 7 days following inoculation. Serotype, metabolic activity, motility, and in vitro antibiotic sensitivity of each isolate were evaluated and related to pathogenicity. Seventy-five of the isolates of high to intermediate pathogenicity belonged to serogroup O2, O78, or O35. In addition, 51 pathogenic E. coli isolates could not be serotyped, and several had multiple serotypes. Most isolates had similar metabolic activity, as determined by amino acid decarboxylation and carbohydrate fermentation, regardless of pathogenicity. An exception was the fermentation of adonitol, which occurred more frequently with the highly pathogenic strains. Motility and in vitro antibiotic sensitivity were not related to pathogenicity. An age-associated resistance to intratracheal E. coli administration occurred by 15 days of age in uncompromised birds. Relative susceptibility of birds older than 2 weeks to intratracheal and/or intravenous E. coli inoculation could be increased by prior exposure to pathogenic reovirus 1733, adenovirus 3167, or infectious bursal disease virus (IBDV). Birds infected with IBDV at 3 weeks failed to clear apathogenic and pathogenic E. coli from circulating blood.     

发酵鸡粪中病原微生物检测方法的探讨

鸡粪发酵后作为再生饲料或有机肥已广泛使用,其中病原微生物检测尤显重要,为此本研究对发酵前后的鸡粪样品中主要病原菌大肠杆菌和沙门氏菌检测方法进行了探讨。结果表明,鸡粪样品中大肠杆菌CFU(菌落形成单位)由发酵前3.67×107个/g减少至6.33×105个/g,经发酵处理的鸡粪样品均未检出沙门氏菌,两者与发酵前样品均存在极显著差异(P0.01),说明所建立的方法可行。     

Influence of processed grains on fecal pH, starch concentration, and shedding of Escherichia coli O157 in feedlot cattle

Manipulation of cattle diets has been proposed as a possible preharvest control measure for Escherichia coli O157. Altering hindgut fermentation through diet changes may be a means to reduce fecal shedding of E. coli O157. In Exp. 1, the objective was to determine whether fecal shedding of E. coli O157 was related to fecal starch concentration. Beginning on d 20, and every week thereafter until d 61, steers in 54 pens (6 to 7 steers per pen) were sampled (n = 122) by fecal collection and rectoanal mucosal swabs (RAMS) for E. coli O157 and fecal starch concentration determinations. Escherichia coli O157 prevalence was 3.3% in fecal samples, 4.1% as measured by RAMS, and 4.9% by fecal or RAMS samples. Steers positive for E. coli O157 contained 21% more (P < 0.05) fecal starch than steers that were negative for E. coli O157. In Exp. 2, we attempted to alter the concentration of starch escaping rumen fermentation by feeding finishing diets based on steam-flaked corn (SFC) and dry-rolled corn (DRC) to 30 heifers prescreened for being culture positive for fecal E. coli O157. Beginning on d 13, heifers were sampled (feces and RAMS) weekly to monitor fecal pH and starch concentration, and prevalence of E. coli O157. Prevalence of E. coli O157 remained above 30% for the first 13 d, but declined (P < 0.05) over the entire 7-wk period. Based on RAMS, the prevalence of E. coli O157 tended to be greater (P = 0.08) for heifers fed SFC than for those fed the DRC diet. After d 20, heifers fed DRC had greater (P < 0.05) fecal starch and lower (P < 0.05) fecal pH than heifers fed SFC. Fecal pH was negatively correlated (r = - 0.34; P < 0.05; n = 143) with fecal starch concentration. Fecal starch concentration and pH were not different (P > 0.05) for heifers that were positive or negative for E. coli O157. Our data suggest that fecal shedding of E. coli O157 was not related to fecal pH or starch concentration in cattle fed grain-based diets.     

化学添加剂对有氧胁迫下湿贮玉米发酵品质和有氧稳定性的影响

为研究苯甲酸钠、山梨酸钾和亚硝酸钠组成的化学添加剂对有氧胁迫处理的湿贮玉米发酵品质和有氧稳定性的影响。以35%和50%含水量的湿贮玉米为原料，设置对照组(无添加)、添加组1(2mL·kg^-1)、添加组2(4mL·kg^-1)在湿贮期设置不同时期有氧胁迫处理分别为:前期有氧胁迫处理(1~7d，Early air stress，EAS)、后期有氧胁迫处理(22~28 d，Later air stress，LAS)、全程有氧胁迫处理(1~28d，Full air stress，FAS)，28d后分析发酵品质、有氧稳定性以及有氧暴露7d后的发酵品质。结果表明：与对照组相比，添加组2的有机酸浓度最高，干物质损失最少，而且有氧稳定性最高。4mL·kg^-1的化学添加剂还显著抑制了酵母菌和大肠杆菌的生长。因此，化学添加剂明显消除了氧气的负面影响，并延长了有氧胁迫条件下的有氧稳定时间。     

短芽孢杆菌高产短杆菌素的分段供氧发酵条件优化

本研究通过优化溶氧条件，对短芽孢杆菌（BacillusBrevis）（ATCC8185）进行分段培养，提高发酵液中短杆菌素产量。以析因试验确定菌体生成阶段培养条件为装液量35mL／100mL，摇床转速120r／min；以二次饱和D-最优设计试验确定短杆菌素合成阶段培养条件为：培养36h后静置培养。通过对溶氧条件的优化，发酵液中短杆菌素的产量达到616．93＋9．72μg／mL，比优化前提高了89％。