新型饲料添加剂──功能性低聚糖

１低聚糖的功能性质低聚糖是由２个一１０个糖单基组成的低聚物，单基之间不同以及单基间不同的连接方式从而形成了形形色色的低聚糖，不同结构的低聚糖具有不同的性质。本文探讨的是对双歧杆菌的生长有促进作用的低聚糖，又称为双歧杆菌增殖因子。由于这类低聚糖大多属于难消化或非消化性糖，如异麦芽寡糖、大豆低聚糖、低聚果糖、半乳寡糖、乳果寡糖、低聚木糖等，其本身并不对动物机体产生多大的影响，其效应主要是由于对动物机体中的双歧杆菌有增殖作用，从而增加了双歧杆菌的生长速率，相应地在动物肠道总菌量不变的情况下，使肠道中的…     

由耐热木聚糖酶从硬木和谷物木聚糖中生成低聚木糖作为短乳杆菌和青春双歧杆菌的碳源

为了比较农业生产型林业中的木聚糖,使用木聚糖RmXyn10A的两种变体,只有源自海洋红嗜热盐菌的全长酶和催化模块,对硬木(桦木)木聚糖和谷物(黑麦)阿拉伯木聚糖进行水解。用含有水解产物的低聚木糖(XOS)作为碳源,培养4种精选的细菌菌种,结果显示了短乳杆菌DSMZ 1264和青春双歧杆菌ATCC 15703出现选择性生长。这两种菌株被证实可利用低聚木糖组分(DP 2-5);而来自黑麦阿拉伯木聚糖水解产物的假定阿拉伯低聚木糖仅能被青春双歧杆菌利用。大肠杆菌不能生长,但能在单糖阿拉伯糖和木糖上生长。研究还表明,小片球菌(Pediococcus parvulus)2.6株既不能利用木糖,也不能利用低聚木糖进行生长。总之,RmXyn10A或其催化模块证实可用于硬木木聚糖和谷物阿拉伯木聚糖的高温水解,产生XOS,因此能在功能性食品中充当益生元。     

绿色饲料添加剂——低聚木糖

低聚糖，亦称寡糖，是由2～8个单糖分子通过糖苷键构成的聚合物，并根据糖苷键不同有不同的名称，如低聚木糖、低聚果糖和低聚甘露糖等。低聚糖作为双歧杆菌生长促进因子，是20世纪70年代日本东京大学光罔教授发现的。因其具有独特的生理功能，而广泛用于食品和饲料生产中，并为各国政府批准，承认其可促进双歧杆菌生长，有良好的整肠功效。     

影响绿色饲料添加剂寡聚糖使用效果的因素

1寡聚糖的种类 寡聚糖的种类不同,所产生的作用效果不同。如不同来源的寡聚糖对双歧杆菌的影响状况是不同的,其中甘露寡糖不能作为双歧杆菌的增殖因子．其应用于饲料主要是起吸附有害菌、毒素和刺激动物机体免疫系统的功能,而其他大多数寡聚糖对免疫系统的影响并未得到肯定。     

双歧杆菌代谢母乳寡糖机制的研究进展

母乳寡糖(human milk oligosaccharides,HMO)结构复杂,其能够选择性地促进母乳喂养婴儿肠道中益生菌双歧杆菌的生长。婴儿肠道相关双歧杆菌具有糖苷酶等分子工具,使其能够代谢HMO,且代谢过程具有菌株特异性。本文总结了HMO的结构特点,探讨了婴儿肠道相关双歧杆菌代谢HMO的机制,重点分析了双歧杆菌代谢HMO的种内差异以及代谢过程涉及到的分子工具的研究进展。     

苦豆籽粕-两歧双歧杆菌合生元对鸡肠道致病菌体外生长的影响

为了研究苦豆籽粕-两歧双歧杆菌合生元对鸡肠道微生物区系的影响,试验研究了0.5%、1%、1.5%、2%不同比例液体合生元对鸡白痢沙门杆菌和大肠杆菌体外生长的影响。结果表明:苦豆籽粕-两歧双歧杆菌合生元对鸡肠道致病菌的抑菌作用明显,苦豆籽粕-两歧双歧杆菌合生元对鸡大肠杆菌的体外生长抑制作用与对照组对比差异极显著(P0.01);苦豆籽粕-两歧双歧杆菌合生元对鸡白痢沙门杆菌的抑制效果与对照组相比差异也极显著(P0.01)。     

酵母细胞壁多糖提取纯化鉴定技术及其应用研究进展

酵母细胞壁多糖的主要活性成分是β-葡聚糖和甘露聚糖,在动物机体内与肠道受体竞争吸附病原菌以及在肠道降解提供能量,改善动物肠道环境;也会利用其结构与Fe~(2+)离子螯合抑制羟自由基的产生以及防止脂质过氧化连锁反应等,提高抗氧化能力;其特有的化学位点会通过多种分子间作用力吸附霉菌毒素;β-葡聚糖和甘露聚糖通过作用于巨噬细胞调节多种细胞因子,通过多个途径增强机体的特异性和非特异性免疫力。目前在养殖业中酵母细胞壁多糖可用于替代金霉素、硫酸黏杆菌素等抗生素控制鸡的坏死性肠炎、减少水产动物细菌感染、降低猪、牛等幼畜腹泻率、预防各种老化性疾病以及促进动物生长发育。对酵母细胞壁多糖现有的提取方法进行比较,提取酵母细胞壁β-葡聚糖,选用超声辅助酶解产率最高、效果最好;用低浓度的氢氧化钠(NaOH)溶液提取甘露聚糖以及酶碱法提取酵母细胞壁多糖效果最好。分离纯化技术包括有脱蛋白、脱色、单一多糖的分离,将其分离纯化效果分别比较,除去酵母细胞壁多糖中蛋白质的最佳方法是Sevage法;脱除色素的最佳方法是颗粒活性炭吸附法;单一多糖分离纯化采用DEAE-纤维素A52阴离子交换柱和Sephadex G-100柱联用的方法最佳。酵母细胞壁多糖结构鉴定一般采用高效液相色谱法(HPLC)测定多糖的单糖组成,高效凝胶渗透色谱测定多糖的均匀性和分子质量,傅立叶变换红外光谱(FT-IR)、气相色谱-质谱(GC-MS)和核磁共振(NMR)测定多糖的精细结构。文章通过对酵母细胞壁多糖的作用及应用、提取、分离纯化和鉴定技术进行阐述,为酵母细胞壁多糖作为抗生素替代品在畜牧业生产中的推广应用提供理论依据,为抗生素替代品的研发提供新的思路。     

蜡样芽孢杆菌W-3木聚糖酶xynA基因原核表达及其酶学性质研究

【目的】克隆蜡样芽孢杆菌(Bacillus cereus)W-3菌株的木聚糖酶基因xynA并进行生物信息学分析,探究其异源表达及酶学特性。【方法】采用同源扩增法克隆xynA基因,构建原核表达载体pCola-xynA,转化大肠杆菌BL21(DE3)感受态细胞进行异源表达,通过镍柱亲和层析分离纯化并通过SDS-PAGE鉴定重组蛋白,利用在线软件对xynA蛋白进行生物信息学分析。以榉木木聚糖为底物探究xynA在不同温度、不同pH条件下的酶学性质。【结果】xynA基因全长642 bp,含1个完整的开放阅读框,编码213个氨基酸。序列比对结果显示,xynA和解淀粉芽孢杆菌(Bacillus methylotrophicus)亚种FZB42木聚糖酶(AJD80562.1)相似性为96%,和类芽孢杆菌(Paenibacillus macerans)NBRC15307木聚糖酶(AAZ17386.1)及高地芽孢杆菌(Bacillus altitudinis)同家族木聚糖酶(WP-007407578.1)相似性为94%。xynA蛋白N-端含1个信号肽,理论等电点为9.42,分子质量为23.3 ku,蛋白结...     

大熊猫源双歧杆菌分离株的培养特性及药敏试验

双歧杆菌的培养特性和药敏试验是研究双歧杆菌微生态制剂的基础。从大熊猫新鲜粪便中分离的两株双歧杆菌进行细菌培养、药敏试验及耐温驯化等研究发现,改良肉汤培养基是双歧杆菌大熊猫分离株体外增殖理想的培养基;2个双歧杆菌大熊猫分离株均对红霉素、氯霉素等敏感,对青霉素等产生较高的耐药性;在较高温度下双歧杆菌大熊猫分离株能迅速地增殖,代谢能力大为提高,并与其他乳酸菌协同发酵,抑制致病菌的生长。双歧杆菌大熊猫分离株在体外生长良好,不是广谱的耐药菌株。     

低聚木糖的特性及其在乳制品中的应用

低聚木糖又称木寡糖,是指2～7个木糖分子以β-1,4糖苷键连接而成的功能性聚合糖。它在国外被誉为超强双歧杆菌增殖因子,其增殖功能约是其它低聚糖的20倍,且对双歧杆菌有高效选择性增殖效果。与其它低聚糖相比,即使微量地摄入,也可增加体内双歧杆菌的数量。当前,用低聚糖作为优良的双歧因子来源,不仅     

In vivo morphological and antigenic characteristics of Photobacterium damselae subsp. piscicida

The present study was conducted to examine the morphology and antigenicity of Photobacterium damselae subsp. piscicida by culturing the bacterium in vivo in the peritoneal cavity of sea bass (Dicentrarchus labrax) within dialysis bags with either a low molecular weight (LMW) cut-off of 25 kDa or a high molecular weight (HMW) cut-off of 300 kDa. Differences were observed in the growth rate between the bacteria cultured in vivo or in vitro. Bacteria cultured in vivo were smaller and produced a capsular layer, which was more prominent in bacteria cultured in the HMW bag. Antigenicity was examined by Western blot analysis using sera from sea bass injected with live Ph. d. subsp. piscicida. The sera recognised bands at 45 and 20 kDa in bacteria cultured in vivo in the LMW bag. Bacteria cultured in vivo in the HMW bag did not express the 45 kDa band when whole cell extracts were examined, although the antigen was present in their extracellular products. In addition, these bacteria had a band at 18 kDa rather than 20 kDa. Differences in glycoprotein were also evident between bacteria cultured in vitro and in vivo. Bacteria cultured in vitro in LMW and HMW bags displayed a single 26 kDa band. Bacteria cultured in the LMW bag in vivo displayed bands at 26 and 27 kDa, while bacteria cultured in vivo in the HMW bag possessed only the 27 kDa band. These bands may represent sialic acid. The significance of the changes observed in the bacterium''s structure and antigenicity when cultured in vivo is discussed.     

Diagnostic relevance of qualitative proteinuria evaluated by use of sodium dodecyl sulfate-agarose gel electrophoresis and comparison with renal histologic findings in dogs

OBJECTIVE: To evaluate results of SDS-agarose gel electrophoresis (AGE) of urinary proteins for use in defining glomerular and tubulointerstitial derangements, investigate patterns of high-molecular-weight (HMW) proteins for differentiating among glomerular disorders, and assess low-molecular-weight (LMW) proteins as markers of severity of tubulointerstitial disease in dogs. ANIMALS: 49 dogs with increased serum creatinine concentrations or abnormal renal protein loss. PROCEDURE: Urinary proteins were examined by use of SDS-AGE and differentiated on the basis of molecular weight. The HMW proteins (> or = 69 kd) were considered indicative of glomerular origin, whereas LMW proteins (< 69 kd) were of tubular origin. Renal specimens were examined by use of light microscopy. Glomerular and tubulointerstitial lesions were differentiated by use of the classification for the World Health Organization and semiquantitative grading, respectively. RESULTS: Sensitivity of SDS-AGE was 100% for detection of glomerular lesions and 92.6% for tubulointerstitial lesions; specificity was 40% and 62.5%, respectively. Although HMW urinary proteins were not significantly associated with the type of glomerular lesion, LMW urinary proteins were significantly associated with the grade of tubulointerstitial damage. Detection of 12- or 15-kd proteins or both was highly indicative of a severe tubulointerstitial lesion. CONCLUSIONS AND CLINICAL RELEVANCE: SDS-AGE of urinary proteins in dogs represents a noninvasive test with high sensitivity for identifying glomerular and tubulointerstitial damage, but low specificity limits its validity as a stand-alone test to differentiate between glomerular and tubulointerstitial lesions. The test is particularly useful for identifying dogs with advanced tubulointerstitial disease but cannot be used to characterize glomerular disorders.     

Purification of carbonic anhydrase isozyme VI (CA-VI) from swine saliva.

Salivary or secreted carbonic anhydrase (CA), which constitutes a new class of CA, designated CA-VI, was isolated. Swine CA-VI purified from swine saliva by inhibitor-affinity chromatography and ion exchange chromatography had a specific activity of 5,468 units/mg. The molecular weight was 250,000, as determined by gel filtration under non-denaturing conditions, and the subunit molecular weight was found to be 37,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that swine CA-VI consists of 7 subunits. The treatment of the enzyme with endo-N-acetylglucosaminidase F reduced its subunit molecular weight from 37,000 to 35,000 and 32,000. We raised a rabbit antibody against purfied swine CA-VI. Double immunodiffusion showed that anti-swine CA-VI serum reacted with swine CA-VI and swine saliva, but not with hemolysate (containing CA-I and CA-Il) or muscle extracts (containing CA-III). The concentration of CA-VI in swine saliva, measured using single radial immunodiffusion, was 0.027 +/- 0.017 mg/mg total protein.     

牛白细胞髓过氧化物酶的分离纯化

应用分子筛层析(Sephadex G-200凝胶)和亲和层析(ConA-Sephrose 4B)从奶牛全血中分离纯化髓过氧化物酶(myeloperoxidase,MPO)。分离出的MPO活性为0.068 U/mL,分子质量为64.7、47.9、13.4 ku,纯度为94.2%,蛋白质浓度为147.3 μg/mL。本研究建立了MPO分离纯化方法,对于深入研究MPO具有重要意义。     

新型鸭呼肠孤病毒σB蛋白的原核表达及其多克隆抗体制备

为制备新型鸭呼肠孤病毒（new-type duck reovirus,NDRV）XX株σB蛋白的多克隆抗体,试验经RT-PCR扩增NDRV XX株σB基因编码序列,构建原核表达质粒pET-32a（+）-σB,将其转化至大肠杆菌BL21（DE3）感受态细胞后,经IPTG诱导获得His-σB重组蛋白。SDS-PAGE显示成功表达出约55 ku的融合蛋白,主要以包涵体形式存在,其表达时的最佳诱导时间、IPTG诱导浓度分别为3 h和0.25 mmol/L。经Ni²⁺柱亲和层析纯化获得可溶性重组蛋白,将蛋白经Western blotting和蛋白质谱鉴定为高纯度的σB重组蛋白,将纯化后σB重组蛋白按合理免疫程序免疫家兔,获得多抗隆抗体经Western blotting分析显示出特异性的反应。本试验结果为NDRV σB蛋白功能的深入研究及基因工程疫苗的研发奠定了基础。     

Purification and partial characterization of canine pepsinogen A and B

OBJECTIVE: To purify and partially characterize various isoforms of canine pepsinogen (PG) from gastric mucosa. SAMPLE POPULATION: Stomachs obtained from 6 euthanatized dogs. PROCEDURE: Mucosa was scraped from canine stomachs, and a crude mucosal extract was prepared and further purified by use of weak anion-exchange chromatography, hydroxyapatite chromatography, size-exclusion chromatography, and strong anion-exchange chromatography. Pepsinogens were characterized by estimation of molecular weights, estimation of their isoelectric points (IEPs), and N-terminal amino acid sequencing. RESULTS: Two different groups of canine PG were identified after the final strong anion-exchange chromatography: PG A and PG B. Pepsinogens differed in their molecular weights and IER Pepsinogen B appeared to be a dimer with a molecular weight of approximately 34,100 and an IEP of 4.9. Pepsinogen A separated into several isoforms. Molecular weights for the various isoforms of PG A ranged from 34,200 to 42,100, and their IEPs ranged from 4.0 to < 3.0.The N-terminal amino acid sequence for the first 25 amino acid residues for PG A and B had good homology with the amino acid sequences for these proteins in other species. CONCLUSIONS AND CLINICAL RELEVANCE: Canine PG B and several isoforms of canine PG A have been purified. Availability of these PGs will facilitate development of immunoassays to measure PG in canine serum as a potential diagnostic marker for gastric disorders in dogs.     

中国斗鸡高分子质量DNA的高效制备

采集斗鸡翅静脉血液,加ACD抗凝,离心收集血细胞,经细胞计数后,用低融点琼脂糖包埋血细胞,蛋白酶K消化,经脉冲电泳分离,获得了较纯净的720 kb以上的DNA.对大片段DNA进行限制性酶切,回收特定大小的DNA片段.利用该方法制备的高分子质量DNA,经酶切、回收的特定大小DNA片段可用于后续细菌人工染色体(BAC)文库的构建和基因组分析.     

The antigenic activity of chromatographic fractions of a saline extract of Taenia saginata (Goeze, 1782) proglottides

A saline extract of a homogenate of Taenia saginata proglottides was partially purified by gel filtration chromatography on Sephadex G200 or Sepharose 4B and by ion exchange chromatography on DEAE cellulose. Gel filtration produced two distinct fractions with different antigenic properties. The first was of molecular weight of approximately 1,000,000 and contained a high level of activity in the haemagglutination inhibition test. The second fraction of molecular weigh of approximately 100,000 contained most of the immuno-precipitin activity. Other fractions had little or no antigenic activity. Eight fractions were obtained by DEAE cellulose chromatography, of which 4 had detectable antigenic activity. Subsequent rechromatography of fractions obtained by gel filtration on DEAE cellulose produced relatively pure fractions of high antigenic activity, from which small molecular weight contaminants had been removed.     

Isolation of the fifth component of the bovine complement system

Bovine C5 has been isolated from fresh bovine serum by a five-step procedure: polyethylene glycol precipitation, sequential ion-exchange chromatography on DEAE-Sephacel and CM-Sephadex, hydroxylapatite chromatography, and affinity chromatography. The purified C5 was a protein of apparent molecular weight 202,000 +/- 9,000 composed of two chains: an alpha-chain of molecular weight 127,000 +/- 5,000 and a beta-chain of molecular weight 74,000 +/- 2,000. The alpha-chain was cleaved by Sepharose-CVF.Bb (a cobra venom factor (CVF)-induced C3/C5 alternative pathway convertase) in the absence of any C3 or C3b. The monocarboxylic acid form of K-76, a sesquiterpene compound isolated from the culture filtrates of Stachybotris complementi, inhibited the alternative pathway of bovine serum, and the inhibited hemolytic activity was restored, in a dose dependent manner, by bovine C5. This provided the basis for a C5 functional assay throughout the purification procedure. The purified C5 showed species specificity and was functionally distinct from bovine C3.     

Beaded agarose affinity chromatography of bovine fibroblast interferon

Bovine fibroblast interferon (BoF-IFN), produced in bovine embryonic kidney cell cultures by priming and infection with bluetongue virus, was partially purified by controlled pore glass chromatography. The partially purified BoF-IFN then was subjected to beaded agarose affinity chromatography in 2 distinct fractions--1 after the addition of 1M NaCl and the other one after the addition of 1.5M NaCl containing 50% ethylene glycol. Analysis of fractions by sodium dodecyl sulfate/polyacrylamide-gel electrophoresis revealed a broad molecular weight range (14,900 to 27,900) for IFN eluted by 1M NaCl, and 2 discrete molecular weight ranges (16,000 to 19,500 and 28,300 to 34,000) for IFN eluted by 1.5M NaCl containing 50% ethylene glycol. The specific activity of the IFN eluted with 1.5M NaCl containing ethylene glycol was 2.85 X 10(6) U/mg of protein, compared with 5.7 X 10(5) U/mg of protein in the controlled pore glass-purified IFN.