聚合酶链式反应技术快速检测化妆品中大肠杆菌的研究

利用聚合酶链式反应(PCR)技术建立了一种快速检测化妆品中大肠杆菌的方法.根据大肠杆菌的ITS保守序列设计特异性引物,确定引物的特异性;比较3种不同提取大肠杆菌DNA的方法,确定煮沸法为最优方法;模拟染菌的化妆品样品,对其进行增菌,并作为模板进行PCR扩增.结果表明,此方法可以在原样品活菌浓度约8×100 cfu/mL时通过12h增菌后检测出来,总的检测时间可以控制在24 h内.     

水力空化对水中微生物的灭活作用及特性

在以文丘里管为空化发生器的水力空化装置中,对含有大肠杆菌的水体进行灭菌处理。通过检测大肠杆菌的灭菌率,考察了水力空化对水中微生物的灭活效果。分析了水流空化数、文丘里管入口压力、空化作用时间、文丘里管结构特性等参数对灭菌效果的影响。结果表明,水力空化的能量效应对水中微生物能够产生灭活作用,实现对含菌污水的灭菌消毒处理。提高文丘里管入口压力、增加空化处理时间、优化空化器结构设计均有利于增强空化灭菌效果。     

Microbiological controls and control points in a hake fillets manufacturing process for exportation

Indicator and foodborne pathogen microorganisms in the "for export" hake fillets manufacturing were investigated in this study. Critical control points were identified and prevention activities and control were proposed during seafood elaboration process. 45 samples of hake from sequential processing operation stages, 15 ice samples and 12 water samples from utensil washing, were collected. The samples were analyzed for their content of aerobic mesophilic bacteria, psychrotrophic bacteria, enterobacteria, total and fecal coliform bacteria, Staphylococcus aureus and the presence of Escherichia coli, Salmonella and Shigella. The analysis of the samples collected from the factory revealed that the amounts of aerobic mesophile bacteria increased during manual filleting and packaging, in comparison with raw material. Psychrotrophic bacteria were the predominant microorganisms, specially in hake samples. In addition, high levels of enterobacteria, which do not occur normally in fish, were detected in raw hake samples. Staphylococcus aureus, Escherichia coli, Salmonella and Shigella, were not isolated from any samples in this study. The goal of this work is to establish microbiological risks in the hake fillets manufacturing process and, therefore to make possible corrective and control actions to assure the quality and safety of seafood.     

乳糖诱导重组人睫状神经营养因子在大肠杆菌中的可溶性表达

目的利用乳糖替代IPTG,诱导重组人睫状神经营养因子(Recombinanthumanciliaryneurotrophicfactor,rhC-NTF)在原核工程菌BL-TCS中可溶性表达,并选择最适的诱导表达条件。方法在不同的温度下,利用不同浓度的乳糖及0·5mmol/L的IPTG,诱导含有人CNTF基因的工程菌9h,比较菌体生长、乳糖消耗以及目标蛋白的表达规律。结果乳糖组的菌体A600值均高于IPTG组。乳糖诱导产生的rhCNTF主要以可溶形式表达,各浓度乳糖的诱导效果均能接近或超过IPTG的效果。以2%乳糖在25℃下诱导6h,可以达到比较好的诱导效果。结论乳糖可取代IPTG诱导人CNTF基因表达,并获得良好效果。     

Glycopolymer N-halamine-modified biochars with high specificity for Escherichia coli eradication

Antibiotic-resistant bacteria contamination in environments imposes great threats to human life health.This research aims to develop novel targeted antibacterial biochars for achieving high selectivity to kill pathogenic Escherichia coli (E.coli).The glycopolymer N-halamine-modified biochars (i.e.,BCPMA-Cl)were synthesized by the modification of biochars with poly[2-(methacrylamido) glucopyranose-co-acrylamide](P(MAG-co-AM),followed by chlorination treatment.Based on the results of FTIR,turbidity,XPS,and UV-vis,BCPMA-Cl was successfully synthesized and demonstrated to be able to eliminate Staphylococcus aureus (S.aureus) and E.coli.Especially,BCPMA-Cl possessed extremely potent to specific-killing 104 CFU·ml-1 of E.coli with lower hemolytic activity (＜5％).Additionally,the antibacterial mechanisms of BCPMA-Cl against bacteria were contact-killing and release-killing contributed by active chlorine (i.e.,Cl+).Therefore,this work provided a cost-effective and facile approach for preparation of functional biochars used for bacteria-specific therapeutic applications via livestock pollutants as well as showing a promising strategy to avoid bacterial resistance.     

Evaluation of the effect of probiotic cultures added to commercial yogurt over a known population of Listeria monocytogenes and Escherichia coli O157:H7

The effect of probiotic cultures over Listeria monocytogenes and Escherichia coli O157:H7 during yogurt storage was evaluated. Two different yogurt brands, one with additional probiotic cultures (Lactobacillus casei and L. acidophilus) were inoculated with known populations (106 UFC/g) of either L. monocytogenes or E. coli O157:H7 in three different times and stored for 32 days at 5 degrees C. Every four days the count of lactic bacteria, the added pathogens and pH was evaluated, according to the methodology described in the Bacteriological Analytical Manual. The pH and lactic bacteria population were constant during the testing period. Yogurt with additional probiotic cultures reduced the population of L. monocytogenes in 8 days, the population of E. coli O157:H7 in 16; yogurt with no additional probiotics took 20 days to reduce L. monocytogenes to non-detectable levels and even after 28 days of storage, E. coli O157:H7 was cultured. In this work, the beneficial effects of additional probiotic cultures in yogurt is confirmed again.     

鹿茸促菌作用的量热法研究

用热活性检测仪测定了鹿茸对大肠杆菌,金黄色葡萄球菌起促进代谢作用的热功率－时间曲线,并根据曲线建立了细菌促进生长的实验模型,计算了生长速率常数和最佳促菌浓度     

重组人aFGF工程菌发酵条件的优化

目的优化重组人酸性成纤维细胞生长因子(rhaFGF)工程菌的发酵条件。方法采用分批补料方式,在菌体快速生长阶段,通过变速流加碳源和氮源,控制流加比例,调节pH值和溶解氧(DO)含量,实现工程菌的高密度发酵及目的蛋白的高效表达。结果当碳源与氮源流加比例为3:1时,菌体湿重可达145g/L,干重达36g/L,目的蛋白表达量达28.9%;DO控制在20%时,全菌体和裂解液中目的蛋白的表达量均最高,分别可达29.11%和44.28%。结论已初步优化了rhaFGF工程菌的发酵条件。     

樟树叶片提取物抑菌作用研究

通过制备樟树叶片提取物及该提取物对霉菌与细菌的抑制作用的研究，结果表明樟树叶片（脂溶性）抑菌物比（水溶性）抑菌物的抑菌效果强，且（脂溶性）抑菌化合物对霉菌（青霉、曲霉、根霉）的抑制效果比细菌（大肠杆菌、枯草芽孢杆菌）强。     

牙膏抑菌效果分析研究

初步研究牙膏对大肠杆菌、金黄色葡萄球菌、绿脓杆菌三种比较常见致病菌的抑制效果,检验牙膏抑菌合格与否。用"抗菌有效性"方法,研究LP、MP、HP、LP-HE、LP-BA五种牙膏抑菌效果。LP牙膏中三种细菌浓度维持在106cfu/mL以上,其余四种牙膏在第14天后没有细菌生长,其中MP在4个周期5次检验中都没有细菌生长,LP-BA有类似抑菌效果,但第2次检验时,金黄色葡萄球菌和大肠杆菌的浓度分别为4×104cfu/mL和35×104cfu/mL,HP与LP-H有类似抑菌效果,在第2次检验时仅大肠杆菌生长,细菌浓度分别为1×106cfu/mL和2×106cfu/mL。LP牙膏不能对实验细菌起到抑制作用,而其余四种牙膏能有效的抑制细菌增长。     

季铵盐改性蒙脱土的抗菌活性及抗菌机理

采用离子交换法将不同季钱盐插层到钠基蒙脱土中制备了改性蒙脱土.改性蒙脱土对革兰氏阳性菌(S.aereus)和革兰氏阴性菌(E coli)均有很强的抗菌作用,并且随着季铵盐在蒙脱土中的质量分数增加其抗菌活性增强.不同季铵盐改性蒙脱土的抗菌活性不同,其中双季铵盐改性蒙脱土的抗菌活性最好,对S.aereus和E coli的最小抑菌浓度分别为6.25 mg/L和12.5 mg/L.用扫描电镜对与细菌接触不同时间后的改性蒙脱土进行观察,结果表明:细菌先吸附到改性蒙脱土的表面,然后慢慢的变形死亡.同时,对在0.9%的生理盐水中浸泡不同时间后的改性蒙脱土进行X射线衍射和热重分析.结果表明:随着浸泡时间的增加,蒙脱土中的有机物质量分数及层间距均逐渐减少,说明季铵盐能从蒙脱土的层间解吸出来,并进入溶液中直接杀死细菌.因而,改性蒙脱土的抗菌活性是吸附与释放到溶液中的季铵盐离子协同作用的结果.     

Monolithic Ceramic Foams for Ultrafast Photocatalytic Inactivation of Bacteria

Palladium-modified nitrogen-doped titanium dioxide (TiON/PdO) foams were synthesized by a sol–gel process on a polyurethane foam template. The TiON/PdO foam was tested for microbial killing using Escherichia coli cells as a target. Under visible-light illumination, the TiON/PdO foam displayed a strong antimicrobial effect on the bacteria cells in water. The antimicrobial effect was found to be dependent on the palladium content and the calcination temperature. In a flow-through dynamic photoreactor, the new photocatalyst efficiently inactivated E. coli within a short contact time (<1 min), the shortest ever reported for the photocatalytic killing of bacteria. The strong antimicrobial functions of the TiON/PdO foam were related to charge trapping by PdO and the high contact efficiency of the foam structure.     

糖基双子阳离子表面活性剂杀菌性能

利用抑菌圈法对糖基双子阳离子表面活性剂(SGCS)和糖基阳离子表面活性剂(SCS)对大肠杆菌(E.coli)和金黄色葡萄球菌的抑菌性能进行了评价。SGCS的抑菌性能优于SCS;抑菌圈随着SGCS碳链增长先增大后变小,C_(12)-SGCS具有较佳的抑菌性能。C_(12)-SGCS和C_(12)-SCS对硫酸盐还原菌(SRB)、铁细菌(FEB)、腐生菌菌群(TGB)、E.coli和禾谷镰刀菌的杀菌性能表明,C_(12)-SGCS的杀菌性能优于C_(12)-SCS,当C_(12)-SGCS的质量浓度为15 mg/L时对SRB,FEB和TGB的杀菌率均超过99%。SGCS抑菌杀菌更广谱高效,具有良好的应用前景。     

重组Echistatin融合基因工程菌高密度发酵工艺优化

目的优化大肠杆菌表达重组蛇毒锯鳞蝰素(Echistatin,Ecs)融合基因工程菌的发酵工艺。方法在15L发酵罐内,研究培养基、培养条件和诱导时间对工程菌生长和目的蛋白表达的影响,并考察工程菌中重组质粒的遗传稳定性。结果工程菌在pH7·4的2×YT培养基中诱导4h,菌体湿重可达75g/L,目的蛋白表达量约占总蛋白的35%,所构建的重组质粒在BL21宿主菌中传代稳定。结论优化了Ecs融合基因工程菌的发酵和表达条件,为规模化生产奠定了基础。     

重组人成纤维细胞生长因子-21的表达及纯化工艺的优化

目的优化重组人成纤维细胞生长因子-21(SUMO-rhFGF-21)融合表达工程菌的表达条件及目的蛋白纯化工艺。方法对工程菌的诱导温度、时间及诱导剂浓度进行优化,并进行罐发酵,对菌体裂解液进行离子交换层析、Ni离子亲和层析及分子筛层析等。纯化产物经SDS-PAGE和HPLC鉴定纯度。结果在诱导温度为37℃,诱导时间为4h,IPTG浓度为0.5mmol/L时,目的蛋白的表达量最高,达20.5%;罐发酵收菌量可达66g/L,目的蛋白的表达量达20.2%;纯化后的rhFGF-21纯度达96%以上。结论优化了rhFGF-21的表达条件及纯化工艺,为rhFGF-21用于新药开发奠定了基础。     

稳定性固体二氧化氯消毒剂杀菌效果的研究

为了定量研究稳定性固体二氧化氯的杀菌效果,采用五步碘量法对实验室制取的稳定性固体二氧化氯消毒剂进行了有效成分的测定,并采用悬液定量杀菌法对其杀菌力做了系统的研究。实验选用大肠杆菌和金黄色葡萄球菌分别作为革兰氏阳性菌和革兰氏阴性菌的代表菌种,用含50、100、200mg/L的二氧化氯消毒剂的水溶液对实验菌作用1、3、5min,实验结果平均杀灭对数值均≥4.00。可以表明,该粉状消毒剂杀菌效果理想,对革兰氏阳性菌和革兰氏阴性菌均有良好的杀灭效果。     

基因工程菌株BLG8900的遗传稳定性

目的研究基因工程菌株BLG8900的遗传稳定性。方法在有选择压力(加氨苄西林)的条件下,测定pYC2质粒在大肠杆菌BLG8900株[含有质粒pYC2的BL21(DE3)]中的遗传稳定性。结果工程菌连续传10、25、50和100代后质粒具有良好的稳定性,而且经BamHⅠ/EcoRⅠ双酶切,酶切图谱相同。第100代菌株重组质粒的GnRH/TRS序列与原代菌株相同。原代与第10、25、50和100代菌株经IPTG诱导表达,GST-GnRH/TRS融合蛋白表达水平、菌体蛋白的SDS-PAGE图谱鉴定无明显差异。Western blot表明各代菌株表达产物均具有GnRH抗原特异性。结论质粒pYC2在大肠杆菌BL21(DE3)宿主菌中有较好的遗传稳定性。     

生化黄腐酸与常见抑菌药品的抑菌效果比较

通过生化黄腐酸对金黄色葡萄球菌、大肠杆菌、苏云金杆菌、枯草杆菌的抑制作用和苯酚、高锰酸钾、乙酰螺旋霉素片及头孢拉定胶囊4种药品对上述4种菌的抑菌效果的比较,发现生化黄腐酸对上述4种菌均有明显的抑制作用,但其抑菌效果比其他4种药品弱。     

Characterization of microbial population present in the edible seaweed, Monostroma undulatum, Wittrock

The microbiological quality of Monostroma undulatum, Wittrock from the Southern Argentinean coast, was studied for its application for human food. Also the diversity and function of the native bacterial population to this green seaweed was analyzed. Samples were collected in Puerto Deseado, province of Santa Cruz, Southern Argentina (47 degrees 45'L.S., 65 degrees 55'L.W). The samples were analyzed for the presence of psycotrophic heterotrophic bacteria, marine heterotrophic bacteria, low nutritional request bacteria (LNRB), marine low nutritional request bacteria (LNRB marine), Vibrio spp, total and thermotolerant colifom bacteria, anaerobic sulfite reducing bacteria, yeasts and moulds. The isolates were identified using standard techniques based on morphologic, physiologic and metabolic characteristics. Among the gram-negative bacteria isolated, the predominant genera belonged to Vibrio (20%), E. coli inactiva (18%), Flavobacterium (11%), Flexibacter (9%), Moraxella (9%), Alcaligenes/Pseudomonas group (9%), Aeromonas (2%), Acinetobacter (2%). Cotophaga (2%), Photobacterium (2%), Ps/Caulobacter/Alteromonas/Spirillum group (2), The main genus of gram-positive bacteria was Staphylococcus. Human pathogenic bacteria were not detected. Fecal contamination indicator bacteria were not isolated from fresh seaweed and seawater. These results showed an adequate microbiological quality of seaweed acceptable for human food. The bacterial population associated to Monostroma undulatum, consisted of gram-negative, marine and psycotrophic microorganisms, including vibrios and enterobacteria as their main components. Also the identified bacteria showed a great capacity to hydrolyze different substrates and so they might contribute to the balance of this marine ecosystem.     

铁载体受体蛋白IroN的原核表达及其对肠道外致病性大肠杆菌感染小鼠的免疫保护作用

目的原核表达重组铁载体受体蛋白IroN,并探讨其对小鼠肠道外致病性大肠杆菌群(Extraintestinal pathogenic E.coli,ExPEC)感染的免疫保护作用。方法从E.coliJ96基因组DNA中扩增IroN基因,克隆入原核表达载体pET-32a(+)中,构建原核表达质粒pET-32a-IroN,转化E.coliBL21(DE3),IPTG诱导表达。表达的重组IroN蛋白纯化后,经皮下免疫BALB/c小鼠,采用ELISA法检测小鼠血清和尿液中的抗体水平;以E.coliJ96菌株分别经腹腔和尿道途径攻击免疫小鼠,计算保护率并计数细菌数。结果重组IroN蛋白的表达量为32%,纯化后纯度超过85%,且具有良好的反应原性。重组IroN蛋白经皮下免疫小鼠,可诱导小鼠血清产生高水平的特异性IgG抗体,在尿液中未检测到特异性IgA抗体。重组IroN蛋白对腹腔攻击ExPEC感染小鼠的保护率为81.8%(P<0.05);重组IroN蛋白免疫的小鼠经尿道攻击ExPEC后,肾脏分离的菌落数比对照组(PBS免疫)降低了100倍以上(P<0.05),但膀胱和尿液中的菌落数两组差异无统计学意义(P>0.05)。结论已原核表达并纯化了IroN蛋白,其对ExPEC感染小鼠的腹腔和肾脏具有显著的保护作用,但对膀胱感染无保护作用。