小菌虫肠道可培养细菌的分离鉴定 及产消化酶活性分析

摘要：目的 分离小菌虫肠道可培养细菌,并研究其产消化酶活性,探讨肠道细菌对小菌虫消化食物的影响。方法 采用传统细菌分离培养方法分离小菌虫肠道细菌,利用16S rDNA序列进行细菌分子鉴定;利用筛选培养基鉴别各细菌的产蛋白酶、脂肪酶、淀粉酶和纤维素酶活性。结果 在小菌虫肠道中分离到4种可培养细菌,分别是枯草芽胞杆菌（Bacillus subtilis）、肺炎克雷伯菌（Klebsiella pneumoniae）、Pseudocitrobacter faecalis和芽胞杆菌（Bacillus sp.）。其中,2种芽胞杆菌属细菌有产消化酶活性。枯草芽胞杆菌有产纤维素酶、淀粉酶和蛋白酶活性;芽胞杆菌仅有产蛋白酶活性,但产酶能力低于枯草芽胞杆菌。结论 小菌虫肠道细菌中可培养细菌结构简单,但其中的芽胞杆菌属细菌有产纤维素酶、淀粉酶和蛋白酶能力,说明小菌虫肠道中的2种芽胞杆菌属细菌可能有协助小菌虫进行食物消化的功能。     

芽胞杆菌抗菌活性和纤维素酶活性的测定

目的筛选鸡用益生菌。方法通过牛津杯法、混菌法和DNS分光光度法对从鸡肠道分离的10株芽胞杆菌进行抑菌效果及纤维素酶活性测定。结果编号为Y1、Y5、Y7的3株芽胞杆菌对测试菌具有较好抑菌效果,编号为Y2、Y4、Y5的3株芽胞杆菌具有较高的产酶活性。结论从分离的10株芽胞杆菌中筛选出1株（编号为Y5）兼具较好抑菌效果和较高产酶活性的地衣芽胞杆菌,具有作为饲用益生菌开发的潜能。     

地衣芽胞杆菌活菌制剂的临床应用进展

整肠生是应用二十多年的地衣芽胞杆菌活菌制剂,已有大量的研究报道其临床有效性和安全性。地衣芽胞杆菌大量分泌种类丰富的消化酶,通过抑制肠道有害菌生长和促进有益菌增殖调节肠道菌群,并产生杆菌肽、地衣素和乙酸等生物活性物质发挥益生作用。本文总结了益生菌的益生特点,并重点分析了芽胞杆菌的益生特点,归纳了整肠生的作用机制与临床研究现状,揭示了其在肝脏疾病、溃疡性结肠炎、肠易激综合征、幽门螺旋杆菌感染以及病毒感染等疾病中的治疗作用,并对整肠生未来的临床应用方向进行了展望。     

枯草杆菌的芽胞在肉鸡肠道中的生活状态和分布

目的探讨枯草芽胞杆菌的芽胞在肉鸡肠道中的生活状态和分布。方法以20日龄AA肉鸡为研究对象,饲喂枯草芽胞菌剂,分别测定鸡粪中芽胞数量和鸡肠道不同部位的活菌数量。结果饲喂3h后,鸡粪中开始检测到芽胞的存在,24h达到最高值,直至饲喂120h后,肠道内的芽胞基本排除。排出芽胞总量为饲喂芽胞总数的3．0倍左右,同时研究还表明：芽胞在实验肉鸡的十二指肠2内开始萌发,并进行了繁殖,在小肠的后端,即小肠3和小肠4,活菌数量达到高峰。结论部分芽胞进入小肠后即可开始萌发,并进行生长繁殖,而且在肠道内有短暂滞留。     

解淀粉芽胞杆菌分离鉴定及培养优化的研究进展

解淀粉芽胞杆菌(Bacillus amyloliquefaciens)是芽胞杆菌属的一个种,广泛存在于自然界,具有丰富的生境多样性,其芽胞具有抗逆性,可抵抗不良环境,同时能产生多种抗菌物质,如脂肽、抗菌蛋白和多种酶类,能抗植物病原真菌和有害细菌,该菌能形成生物膜,定植于植物根系,促进植物生长,因此对该菌进行分离鉴定以及培养优化具有重要意义。截至目前,已经从各种生境中分离、鉴定了多种有应用价值的解淀粉芽胞杆菌,通过不同策略对该菌的培养基和培养条件进行优化,使该菌相关功能得以提高。本文综述了近年来对解淀粉芽胞杆菌的生境多样性、分离鉴定及培养基和培养条件优化的策略,简要归纳了国内外关于解淀粉芽胞杆菌重要的工业和商业产品,为更好地研究和应用解淀粉芽胞杆菌提供必要的参考和借鉴。     

地衣芽胞杆菌对白色念珠菌等的拮抗作用

目的了解地衣芽胞杆菌在试管内与阴道正常菌群共生关系的情况。方法将地衣芽胞杆菌菌液分别与葡萄球菌、大肠埃希菌、白色念珠菌、德氏乳杆菌混合培养,定量计数各菌在不同时间内单独培养和混合培养时各菌的活菌数。结果地衣芽胞杆菌生长不受金黄色葡萄球菌、白色念珠菌和大肠埃希菌的影响,金黄色葡萄球菌和白色念珠菌在有地衣芽胞杆菌存在的情况下,其生长受到明显的抑制（P〈0.05）;乳杆菌在12-48 h内,有显著的抑制地衣芽胞杆菌生长的作用,而乳杆菌的生长不受地衣芽胞杆菌的存在与否而正常生长。结论地衣芽胞杆菌对金黄色葡萄球菌及白色念珠菌在体外具有明显的拮抗作用,地衣芽胞杆菌对大肠埃希菌、乳杆菌无明显的体外拮抗作用。     

枯草芽胞杆菌活菌制剂促进烧伤创面愈合的实验研究

目的　探讨枯草芽胞杆菌活菌制剂对烧伤创面愈合的影响。方法　通过大鼠深Ⅱ°烧伤模型,使用枯草芽胞杆菌活菌制剂,观察烧伤创面愈合过程中,成纤维细胞增殖周期的变化,以及测定羟脯氨酸（OHP）的含量,同时记录烧伤创面愈合时间,从而评价该制剂对烧伤创面愈合的影响。结果　应用枯草芽胞杆菌活菌制剂可促进成纤维细胞分裂、增殖,胶原含量提高,创面愈合时间明显缩短。结论　枯草芽胞杆菌活菌制剂具有促进烧伤创面愈合的作用。     

冷榨花生粕蛋白胨质量分析及其在枯草芽胞杆菌增殖中的应用

为降低微生物培养基制备成本和实现冷榨花生粕蛋白质的深加工,本试验以双酶协同酶解（胃蛋白酶+碱性蛋白酶）冷榨花生粕蛋白质制备植物蛋白胨,从一般性状检查和化学指标两个方面对冷榨花生粕蛋白胨进行了质量分析。参考《微生物培养基的制造与应用》和《中国生物制品主要原辅材料质控标准》对蛋白胨进行了质量评价,并以枯草芽胞杆菌为试验菌进行了冷榨花生粕蛋白胨对细菌的增殖培养试验以评价其质量优劣。结果表明,双酶协同酶解制备得到的冷榨花生粕蛋白胨各项指标均满足生化蛋白胨的质量标准,与标准蛋白胨（胰蛋白胨）的对照试验发现两者差异不显著（p>0.05）,冷榨花生粕蛋白胨可完全替代胰蛋白胨用于枯草芽胞杆菌的增殖培养。     

鸡源益生芽胞杆菌的筛选

根据菌体对低pH和高胆盐的耐受性、产酶特性和对病原菌的抑菌效果,对家鸡肠道中的芽胞杆菌进行了筛选。结果表明,有4株芽胞杆菌(A2、C5、C19和D8)能抑制金黄色葡萄球菌(Staphylococcus aureus)的生长。这4株菌在pH 3.0和高胆盐条件下的存活率都达到60%以上,产淀粉酶和蛋白酶的能力也较好,表明这些菌株具有作为益生菌的开发潜力。经形态学和生理生化反应鉴定,初步确定A2和D8为地衣芽胞杆菌(Bacillus licheniform is),C5和C19为蜡样芽胞杆菌(Bacillus cereus)。     

牛乳铁蛋白肽(LfcinB)在苏云金芽胞杆菌中的融合表达

牛乳铁蛋白肽（bovine lactoferrincin,LfcinB）来源于牛乳铁蛋白（bovine lactoferrin）,是目前已知所有乳铁蛋白肽中活性最高的。前期研究表明,可以通过大肠杆菌表达体系和毕赤酵母表达体系表达有活性的LfcinB,但得到的产物难以纯化,产量也不理想,所以研究构建新型的LfcinB表达体系有很重要的意义。苏云金芽胞杆菌（Bacillus thuringiensis, Bt）能在芽胞形成的同时产生一种δ-内毒素组装成的杀虫晶体,在发酵完成后细胞裂解,将芽胞和晶体释放到培养基中。基于Bt的这种优势,将LfcinB与分子量为35kDa的Cry60Ba晶体蛋白作融合表达。通过PCR扩增和酶切连接,将LfcinB基因连接到 cry60Ba 的下游,构建融合基因并转入无晶体Bt菌株中表达,得到了工程菌4Q7/pPFT60Ba-LfcinB。利用GYS培养发酵48h,再经过超声破碎、包涵体纯化,SDS-PAGE分析表明,工程菌表达了一条38kDa左右的蛋白质条带。将纯化的融合蛋白盐酸水解后进行SDS-PAGE分析,得到近似于3kDa的蛋白质条带,挖取目的条带进行胶内酶解和质谱分析,质谱结果显示,挖取的目的条带样品为LfcinB蛋白的特异性肽段,说明在酸水解过后,融合蛋白Cry60Ba-LfcinB中的Cry60Ba和LfcinB分离开,得到单独的LfcinB蛋白。由此可见,利用晶体蛋白在苏云金芽胞杆菌中进行融合表达可能作为一种新型有效的LfcinB的高效表达体系,为采用基因工程的方法大量生产具有活性的牛乳铁蛋白肽LfcinB蛋白奠定基础。     

两个大豆GmSBP基因的特征、亚细胞定位及对非生物胁迫的响应

我国南方春大豆种子发育过程中,常处于高温、高湿季节,加之种子本身富含蛋白(约40%)和脂肪(约20%),导致南方春大豆种子易劣变。本项目组前期差异蛋白质组学研究发现蔗糖结合蛋白在高温高湿胁迫168 h时在种子田间劣变抗性品种湘豆3号发育种子中呈下调表达。为进一步从分子水平了解Gm SBP基因表达以及响应高温高湿胁迫的特性,本研究利用RT-PCR技术从大豆扩增出两个Gm SBP基因(Gm SBP2和Gm SBPL)。两个基因编码的蛋白均为亲水性,不完整的膜蛋白。荧光定量PCR分析表明:在高温高湿条件下,种子田间劣变不抗品种宁镇1号和抗性品种湘豆3号发育种子中Gm SBP2和Gm SBPL基因表达量均受高温高湿胁迫影响,也会导致种子中蔗糖和可溶性糖含量变化。在籽粒发育过程中,Gm SBP2和Gm SBPL基因表达量在花后30 d左右达到最高,对应时期的蔗糖和可溶性糖含量也达到最大值。组织特异性显示Gm SBP和Gm SBPL基因在不同组织间存在差异表达。亚细胞定位结果表明Gm SBP2和Gm SBPL蛋白均定位在细胞膜和细胞质中。以上结果表明Gm SBP2和Gm SBPL基因可能参与了植物非生物胁迫的应答过程,这将从一个侧面丰富我们对大豆种子田间劣变性和劣变抗性的认识。     

Selected prebiotics support the growth of probiotic mono-cultures in vitro

The aim of the present study was to identify suitable prebiotics for synergistic combinations with representative probiotics from the lactobacilli and bifidobacterium genera. Eleven different types of commercially available carbohydrates and compounds were screened in basal media for their effects on the growth of the probiotic cultures Lactobacillus acidophilus LAFTI L10 (L10), Bifidobacterium animalis lactis LAFTI B94 (B94) and Lactobacillus casei LAFTI L26 (L26). Growth of these probiotics was investigated by measuring optical density (OD) levels over a period of 48 h. The results of this study showed that all three probiotic strains were able to utilize a range of carbohydrates. Maximal growth of L10 was observed in basal medium supplemented with soybean oligosaccharide (SOS), followed by fructooligosaccharide (FOS) and inulin, while maximal growth of B94 was observed in basal medium supplemented with SOS, followed by raffinose, FOS, beta-glucan hydrolysate, inulin and Fibregum TAN. L26 was shown to exhibit maximal growth in basal medium supplemented with FOS followed by inulin, SOS, beta-glucan hydrolysate and beta-glucan concentrate. The results of this study indicate that several carbohydrates/compounds can enhance the growth of these representative probiotic strains in vitro.     

Enzymatic Removal of Phenol and Chlorophenols Using Soybean Seed Hulls

Soybean peroxidase (SBP), (EC 1.11.1.7) can be readily extracted from soybean seed hulls. This study reports on the direct use of soybean seed‐hull extracts for the bioremediation of phenolic wastes. The crude SBP extract from the hulls, like pure soybean peroxidase, is catalytically active in a broad range of pH and temperatures. As SBP is gradually released into the aqueous solution from seed hulls, the direct use of soybean seed hulls can reduce SBP inactivation by H₂O₂ and enhance the utilization efficiency of SBP through the slow release of the enzyme from the seed hulls. However, large doses of soybean seed hulls were found to be ineffective in phenol removal. Gradual additions of H₂O₂ in combination with the SBP released from the hulls were applied to optimize the bioremediation. Since the crude extract contains a mixture of multiple soybean proteins, soybean seed hull slurry required a higher concentration of H₂O₂ to remove the phenolic substrates than did the purified enzyme. Under the experimental conditions, 80 % of phenol (10.6 mM), 96 % of 2‐chlorophenol (3.9 mM), 95 % of 2,4‐dichlorophenol (3.1 mM), and 94 % of mixed phenol and chlorophenols were removed using soybean seed hulls in a single batch reactor. These results demonstrate that soybean seed hulls, compared to purified SBP, may be a more cost‐effective alternative in the enzymatic removal of phenolic compounds through polymerization reactions.     

早期应用微生态制剂对极低出生体重儿影响的临床观察

目的观察早期应用微生态制剂对极低出生体重儿黄疸、喂养、生长以及免疫功能的影响。方法我院NICU收治的生后24h内极低出生体重儿84例,随机分为观察组46例,对照组38例。观察组生后4h内开始口服或鼻饲胃管服用妈咪爱0.5g,2次/d,连用14d。观察两组达到高胆红素血症标准的人数,以及生后24h、5d总胆红素值;喂养不耐受人数;生后5d生理性体重下降及1个月体重增长情况;生后1个月免疫学指标。并加以对比。结果24h内血清胆红素比较无统计学意义(P0.05),治疗5d后观察组血清总胆红素明显低于对照组(P0.05),观察组出现高胆红素血症人数明显低于对照组(P0.05);观察组喂养不耐受出现例数明显低于对照组(P0.01);观察组生后5d体重下降的克数低于对照组(P0.05),1个月时体重增长克数高于对照组(P0.01);观察组IgA水平较对照组明显增加(P0.05),但IgG及IgM含量与对照组比较差异无统计学意义(P0.05)。结论早期应用微生态制剂能降低血清胆红素水平,对预防极低出生体重儿高胆红素血症有一定作用;能提高喂养的耐受性,有利于患儿生长发育;对极低出生体重儿的肠道功能及免疫功能有明显影响,能增加免疫球蛋白IgA水平,从而促进体液免疫的发展。     

益生菌预防自发性腹膜炎再发的临床研究

目的探讨益生菌药物对自发性腹膜炎（SBP）再发的预防效果。方法51例肝硬化并SBP治疗后,在综合护肝治疗的基础上随机分为3组：A组18例,除外综合治疗外无其他用药;B组20例,予益生菌口服;C组13例,予喹诺酮类药物口服。观察其自发性腹膜炎再发生率。结果A组与B组比较,SBP再发生率高（x2=3．99,P〈o．05）。A组与B、C组比较,SBP再发生率高,差异有统计学意义（x2=5．26,P〈0．05）。B组与C组比较,x2=0．001,P〉0．05,两治疗组间差异无统计学意义。结论益生菌对预防自发性腹膜炎再发有效。     

Soybean peroxidase propeptides are functional signal peptides and increase the yield of a foreign protein

Elements that contribute to the high, stable yield of soybean peroxidase (SBP) in soybean seed coats can be exploited in the development of this tissue as a protein production platform. SBP contains an N-terminal and a C-terminal propeptide that are predicted to direct vacuolar targeting; this may be one factor that contributes to its high yield and stability. We characterized the function of the SBP propeptides and investigated their ability to increase the yield of a foreign protein in a heterologous plant system. SBP propeptides are functional signal peptides capable of directing vacuolar transport in Arabidopsis. The use of these propeptides as well as an endoplasmic reticulum (ER)-retention signal to direct a foreign protein to the apoplast, ER, or vacuole can significantly increase yield and will therefore be useful for the development of the seed coat as a protein production platform. We also demonstrate that growth conditions may have a significant impact on the yield of a foreign protein and that this may be subcellular compartment-specific.     

Citric acid improves growth performance and phosphorus digestibility in Beluga (Huso huso) fed diets where soybean meal partly replaced fish meal

A 2 × 3 factorial experiment was conducted to evaluate the effect of partial substitution of dietary fish meal with soybean meal and citric acid (CA) supplementation on growth, food utilization, muscle composition and nutrient digestibility of Beluga, Huso huso. Three isonitrogenic and isoenergetic diets, as SBM₁ (soybean meal protein (SBP):fishmeal protein (FP) = 1:3), SBM₂ (SBP:FP = 2:3) and SBM₃ (SBP:FP = 1:1) containing two levels of CA (0 and 30 g kg⁻¹) were fed to triplicate groups of fish for 8 weeks. The results revealed that adding CA increased (P<0.05) the weight gain (WG), specific growth rate (SGR) and protein efficiency ratio (PER) and decreased (P<0.05) food conversion rate (FCR) whereas partial substitution of fishmeal with soybean meal decreased (P<0.05) growth performance. Partial replacement of fishmeal with SBM (P=0.021) as well as CA supplementation (P=0.019) and their interaction (P<0.001) affected hepatosomatic index (HSI). No differences (P>0.05) were observed for viscerosomatic index (VSI) among treatments (P>0.05). No differences (P>0.05) were detected in moisture, protein of muscle sample among treatments, but lipid content was reduced (P<0.05) while ash content increased (P=0.035) by CA. Soybean meal decreased (P<0.05) nutrient digestibility, whereas CA improved apparent protein and phosphorus (P) digestibility (P=0.011 and P<0.001 respectively). No interaction (P<0.05) between levels of SBM and CA was found on these parameters. Results of the present study indicate that Beluga has a limited ability to utilize SBM as a protein source in practical diets whereas CA can improve growth and nutrient utilization in Beluga.     

地衣芽胞杆菌治疗肝硬化并自发性细菌性腹膜炎的临床疗效研究

目的观察益生菌治疗肝硬化自发性细菌性腹膜炎（SBP）患者的效果。方法将60例肝硬化自发性细菌性腹膜炎随机分成2组,对照组30例,予抗感染、保肝、降门脉高压、利尿、营养支持治疗;治疗组30例,在对照组的综合治疗基础上加用地衣芽胞杆菌胶囊,每日3次,每次2粒,连服21d后观察感染控制及肝功能变化情况。结果治疗组总有效率明显高于对照组,除了腹痛、腹部压痛无差异外,发热、腹泻消失、腹水常规、肝功能改善方面与对照组比较差异存在统计学意义（P〈0．05）。结论益生菌对肝硬化并发自发性腹膜炎有一定治疗效果。     

Evaluation of Probiotic Diversity from Soybean (<Emphasis Type="Italic">Glycine max</Emphasis>) Seeds and Sprouts Using Illumina-Based Sequencing Method

There is increasing interest in the use of plant probiotics as environmental-friendly and healthy biofertilizers. The study aimed at selecting for novel probiotic candidates of soybean (Glycine max). The bacteriome and mycobiome of soybean sprouts and seeds were analyzed by Illumina-based sequencing. Seeds contained more diverse bacteria than those in sprouts. The seeds contained similar fungal diversity with sprouts. Total 15 bacterial OTUs and 4 fungal OTUs were detected in seeds and sprouts simultaneously, suggesting that the sprouts contained bacterial and fungal taxa transmitted from seeds. The Halothiobacillus was the most dominant bacterial genus observed and coexisted in seeds and sprouts. The OTUs belonged to Ascomycota were the most dominant fungal taxa observed in seeds and sprouts. Halothiobacillus was firstly identified as endophytic probiotics of soybean. The results suggested that sprouts might contain diverse plant probiotics of mature plants and Illumina-based sequencing can be used to screen for probiotic candidates.     

A comparative study of free and immobilized soybean and horseradish peroxidases for 4-chlorophenol removal: protective effects of immobilization

Horseradish peroxidase (HRP) and soybean peroxidase (SBP) were covalently immobilized onto aldehyde glass through their amine groups. The activity yield and the protein content for the immobilized SBP were higher than for the immobilized HRP. When free and immobilized peroxidases were tested for their ability to remove 4-chlorophenol from aqueous solutions, the removal percentages were higher with immobilized HRP than with free HRP, whereas immobilized SBP needs more enzyme to reach the same conversion than free enzyme. In the present paper the two immobilized derivatives are compared. It was found that at an immobilized enzyme concentration in the reactor of 15 mg l(-1), SBP removed 5% more of 4-chlorophenol than HRP, and that a shorter treatment was necessary. Since immobilized SBP was less susceptible to inactivation than HRP and provided higher 4-chlorophenol elimination, this derivative was chosen for further inactivation studies. The protective effect of the immobilization against the enzyme inactivation by hydrogen peroxide was demonstrated.