双歧番茄醋的实验研究

目的以番茄为主要原料,对双歧杆菌和醋酸杆菌共同发酵研制双歧番茄醋的工艺进行研究。方法通过正交试验筛选最适工艺。结果双歧番茄醋的最终醋酸度为27 g/L,含双歧杆菌总菌为1.9×1011CFU/mL,5 d内双歧杆菌活菌为5.5×107CFU/mL。双歧番茄醋棕黄色,光泽度好,有成熟番茄香味,入口酸甜适中。结论双歧杆菌与醋酸杆菌在可以番茄汁中共生,此方法制备双歧番茄醋可行。     

大肠杆菌-双歧杆菌穿梭表达载体的构建及内皮抑素基因的表达

目的:构建大肠杆菌-长双歧杆菌穿梭表达载体,并通过此载体使人内皮抑素基因在大肠杆菌和长双歧杆菌中得到表达。方法:以质粒pDG7、pBCSK( )、pET-9C为基础,构建大肠杆菌-长双歧杆菌穿梭表达载体pET-1128,并将人内皮抑素基因插入到新构建的表达载体中,分别转化大肠杆菌BL21(DE3)和长双歧杆菌NQ-1501。诱导表达,表达产物经SDS-PAGE和WesternBlot鉴定。结果:成功构建了大肠杆菌-双歧杆菌穿梭载体,人内皮抑素基因在大肠杆菌和长双歧杆菌中均可表达。结论:构建的穿梭载体为今后用双歧杆菌作为生理菌载体进行肿瘤的基因治疗奠定了基础。     

两歧双歧杆菌86321生长特性的研究

目的研究两歧双歧杆菌86321的生长特性,为该菌生理功能研究和高效发酵剂的研制提供理论依据。方法通过生长曲线、产酸量、最适厌氧方式、最适pH、最适培养温度及最适接种量等一系列实验,对两歧双歧杆菌86321进行生长特性的研究。结果两歧双歧杆菌86321在BL培养基中培养时间可缩短至16 h,最高活菌数的lg值达到9.5;其最适厌氧方式为自然厌氧法或密封法,装液量视实际情况而定;在pH7.08.0生长良好,最适初始pH为8.0;在3742℃生长良好,最适温度为37℃;综合总菌量和生产成本,确定最适接种量为7%（v/v）。结论用BL培养基可以大大提高两歧双歧杆菌86321的产量。细菌产量的高低和发酵速度的快慢与菌种活力、厌氧方式、培养温度及pH等因素密切相关。     

水貂粪便中双歧杆菌的分离与鉴定

目的运用TPY培养基,从健康水貂的粪便中分离培养、筛选出2个肠道菌株。方法细菌培养、菌落形态观察、染色镜检、分离纯化、生化试验和药敏试验。结果分离培养出的2株菌株为双歧杆菌,其中1株为长双歧杆菌,另1株为青春双歧杆菌;双歧杆菌对氯霉素极其敏感,对阿米卡星耐药。结论本实验为毛皮特种经济动物微生态制剂的研究工作奠定了基础。     

保健食品双歧杆菌和乳酸菌计数方法的优化

目的通过稀释液和培养基的选择对保健食品双歧杆菌和乳酸菌计数方法进行优化。方法对2个品种6个批次的益生菌类保健食品,分别采用2种稀释液和3种培养基进行双歧杆菌和乳酸菌计数,并比较结果。结果选择CYS缓冲液为最优稀释液,M-TOS培养基为双歧杆菌计数的最优培养基,BBL培养基为乳酸菌总数计数的最优培养基。结论优化方法所得菌数均明显优于国标方法,可以用于保健食品的双歧杆菌和乳酸菌计数。     

一株双歧杆菌质粒聚合酶基因的PCR扩增和鉴定

目的PCR扩增人双歧杆菌天然质粒的聚合酶基因。方法用改良型MRS双歧杆菌选择培养基,从人新鲜粪便分离长双歧杆菌,PCR扩增长双歧杆菌质粒聚合酶(Bifidobacterium plasmid polymerase,BPP)基因,对BPP基因检测阳性的PCR产物通过序列分析,进行鉴定。结果人长双歧杆菌天然质粒的聚合酶基因PCR扩增后,经1.0%琼脂糖凝胶电泳,测得BPP基因的相对分子质量约为1.9 kb。通过BLAST序列比对分析与GenBank中相应基因同源性为96%。结论成功克隆了1株双歧杆菌天然质粒的聚合酶基因,为构建与双歧杆菌宿主质粒相适应的载体奠定了基础。     

耐氧双歧杆菌rDNA ISR序列测定及特征分析

目的 对4种耐氧双歧杆菌16S-23S rDNA ISR序列进行克隆和序列分析。方法采用凝胶分离PCR产物的方法。结果长双歧杆菌和短双歧杆菌16S-23S rDNA ISR序列一致,全长568个碱基对;青春双歧杆菌与婴儿双歧杆菌序列一致,全长510个碱基对。通过19种双歧杆菌和4种耐氧双歧杆菌ISR序列分析表明,该序列中含有一个保守的特征序列,可作为双歧杆菌属的分子标记,此外还含有3个基因高变区,可用双歧杆菌种间分子鉴定的基础。结论对23种双歧杆菌聚类分析表明,耐氧双歧杆菌ISR序列已发生改变,为进一步研究双歧杆菌在有氧条件下进化机制奠定了基础。     

多菌种酸奶中活性乳酸菌的计数方法研究

利用4种选择性培养基MRS、MRS-山梨醇、MRS-5．2、Elliker,采用平板涂布法和倾注接种法。在蜡烛缸法（缺氧）、封口膜法（微氧）和供氧条件下对4种市售多菌种酸奶中保加利亚乳杆菌、嗜热链球菌、嗜酸乳杆菌、双歧杆菌进行选择性计数方法研究。结果表明：蜡烛缸法较能反映乳酸菌活菌数量的实际情况,封口膜法次之;在不同培养条件下4种选择性培养基对于乳酸菌活菌的计数都是灵敏的;而涂布法和倾注法接种活菌数有显著差异,倾注法要优于涂布法。     

破伤风杆菌产毒培养基主要原材料的替代研究

目的探索用干粉状的肝浸粉、胰酪胨、月示胨代替传统破伤风杆菌产毒培养基中使用的猪肝水透析外液、胰酶酪蛋白消化液、浓胨水,以满足规模化生产的需求。方法与传统的破伤风杆菌产毒培养基同时用于破伤风类毒素的生产过程,由小批量生产逐步转化到规模化批量生产,经多批次试验,以检测不同组分制备的培养基各生化指标和接种细菌后培养的产毒水平进行比对。先确定肝浸粉可以取代猪肝水透析外液,在此基础上筛选出代替浓胨水的月示胨,再以待用的胰酪胨配合肝浸粉、月示胨制备多批次不同生产量的破伤风杆菌产毒培养基。结果经过多批次小批量扩大到批量规模化生产的试验,用肝浸粉、胰酪胨、月示胨配制的破伤风杆菌产毒培养基与传统工艺制备的破伤风杆菌产毒培养基相比较,无统计学意义(P0.05)。结论肝浸粉、胰酪胨、月示胨可以替代传统破伤风杆菌产毒培养基中的猪肝水透析外液、胰酶酪蛋白消化液、浓胨水,适用于破伤风类毒素的规模化生产。     

长双歧杆菌NCC2705株果糖结合蛋白BL0033基因的克隆及其在大肠杆菌中的表达

目的：克隆长双歧杆菌NCC2705株果糖结合蛋白BL0033的基因,利用大肠杆菌表达GST-BL0033融合蛋白并纯化。方法：以长双歧杆菌NCC2705株基因组为模板,PCR扩增BL0033基因,并将其插入pGEX-4T-1表达载体,转化至大肠杆菌DH5α;提取质粒,经PCR、质粒双酶切及测序鉴定后,转入大肠杆菌BL21,并对表达条件进行摸索;用谷胱甘肽-Sepharose4B树脂对可溶性GST-BL0033融合蛋白进行纯化。结果：PCR扩增的BL0033基因长度接近1000bp,与预期值一致;重组菌在IPTG浓度为0.05mmoL/L的条件下,于16℃诱导过夜后,SDS-PAGE分析可见可溶性表达条带,相对分子质量约60×103,与预期值一致;亲和纯化后,SDS-PAGE结果显示单一的表达条带。结论：克隆了BL0033蛋白的基因,并表达纯化了融合蛋白GST-BL0033,为进一步研究长双歧杆菌NCC2705株BL0033蛋白功能奠定了基础。     

Optimization of culture conditions for a synthetic gene expression in Escherichia coli using response surface methodology: the case of human interferon beta

A human interferon beta (hINF-beta) synthetic gene was optimized and expressed in Escherichia coli BL21-SI using a vector with the T7 promoter. To determine the best culture conditions such as culture medium, temperature, cell density and inducer concentration, we used the response surface methodology and a Box-Behnken design to get the highest hINF-beta production. The maximum hINF-beta production of 61 mg l(-1) was attained using minimum medium and the following predicted optimal conditions: temperature of 32.5 degrees C, cell density of 0.64, and inducer concentration of 0.30 M NaCl. This is the first report showing the successful performance of the BL21-SI system in a minimum medium. The response surface methodology is effective for the optimization of recombinant protein production using synthetic genes.     

Monoxenic maintenance and reproduction of root-knot nematode (Meloidogyne hapla) on multiple-species in vitro root culture systems

The ability of tomato root tips transformed with Agrobacterium rhizogenes and non-transformed onion and dandelion root tips to regenerate and provide suitable monoxenic hosts for the maintenance of root-knot nematode (Meloidogyne hapla) was investigated. Populations of M. hapla were successfully established on all root culture systems examined, but the onion root culture was overall the most effective method for long-term maintenance and reproduction of M. hapla. The use of a pre-induction medium containing the hormone !-naphthaleneacetic acid was necessary for the production of onion root culture systems but was not required for the establishment of tomato or dandelion root cultures. Differences in nematode reproduction were observed on tomato when multiple populations of M. hapla were used for inoculations.     

Production of cutinase by Thermomonospora fusca ATCC 27730

Ten strains belonging to various Thermomonospora species were tested for their ability to hydrolyse the insoluble plant polyester cutin. One strain, the thermophile T . fusca ATCC 27730, was found to produce a highly inducible cutinase when grown in broth medium containing purified apple cv. Golden Delicious cutin. Apple pomace, tomato peel, potato suberin and commercial cork were also shown to induce cutinase production. Addition of glucose to the culture medium either at the beginning of fermentation or after 2 days of incubation in the presence of apple cutin led to repression of cutinase production. The cutinase was active against a wide range of cutins, including those isolated from other apple cultivars as well as tomato, cucumber, grapefruit, and green pepper. Cutinase activity in the induced culture supernatant fluids exhibited a half-life of over 60 min at 70 °C and a pH optimum of 11·0. Some potential applications for cutinases are discussed.     

乳糖诱导高分子量重组蛛丝蛋白发酵培养基优化

在M9培养基的基础上,以乳糖为诱导剂,对基因重组蛛丝蛋白工程菌pNSR32/BL21(DE3)的发酵培养基进行了优化。利用单因子实验和正交试验优化出表达高分子量重组蛛丝蛋白的最优培养基配方,结果表明：优化的碳源为0.3%的甘油,氮源为3%的酵母膏、0.75％的蛋白胨和0.05％(NH4)2SO4及少量的无机盐。优化培养基有利于菌体的生长和目的蛋白的表达,表达重组蛛丝蛋白达总蛋白量的20%。     

镉污染地区番茄品种的筛选及其抗氧化能力

为了筛选可供利用的番茄污染安全品种(Pollution-safe cultivar,PSC),减少镉(Cd)污染地区食品安全隐患,通过土培及水培试验研究了南方地区常见不同番茄(Solanumlycopersicum)品种对Cd的积累差异.首先利用土培试验在2.94 mg/kg Cd胁迫下从25个番茄品种中筛选出高低积累...     

野葛糖基转移酶PIUGT2蛋白原核表达及其适宜条件探讨

通过单因素试验分别研究温度和IPTG浓度对重组大肠杆菌BL21(DE3)/pProEXHTa-PIUGT2诱导表达野葛糖基转移酶PlUGT2蛋白量的影响。利用150 ml LB液体培养基培养重组大肠杆菌BL21(DE3),并优化发酵条件。结果表明,在温度20℃和IPTG浓度为0.75 mmol/L条件下,PIUGT2蛋白表达量最高。     

Colonization of cellulose membranes by species of Verticillium

Verticillium dahliae Kleb. (from Brussels sprouts), non-pathogenic to tomato, and V. tricorpus Issac, a virulent pathogen to tomato, differed in their ability to colonize samples of commercial Cellophane, their rate of colonization being dependent upon external sources of carbohydrate. Cellophane and filter paper were both utilized in culture as the sole carbon source and it appears that both fungi have identical cellulase enzyme systems. Prolonged growth of the isolates is liquid medium containing sucrose as the carbon source resulted in lysis of the mycelium, which coincided with the production of polysaccharide material in culture.     

Infection of Cultured Thin Cell Layer Roots of Lycopersicon esculentum by Meloidogyne incognita

A new aseptic culture system for studying interactions between tomato (Lycopersicon esculentum) and Meloidogyne incognita is described. Epidermal thin cell layer explants from peduncles of tomato produced up to 20 adventitious roots per culture in 4-9 days on Murashige &Scoog medium plus kinetin and indole acetic acid. Rooted cultures were transferred to Gamborg''s B-5 medium and inoculated with infective second-stage juveniles. Gall formation was apparent 5 days after inoculation and egg production by mature females occurred within 25 days at 25 C in the susceptible genotypes Rutgers and Red Alert. Resistant genotypes LA655, LA656, and LA1022 exhibited a characteristic hypersensitive response. This system provides large numbers of cultured root tips for studies on the molecular basis of the host-parasite relationship.     

Analysis of specific factors generating 2-cell block in AKR mouse embryos

The phenomenon of the developmental arrest at the 2-cell stage of 1-cell embryos from some mouse strains during in vitro culture is known as the 2-cell block. We investigated the specific factors involved in the 2-cell block of AKR embryos by means of a modified culture system, the production of reconstructed embryos by pronuclear exchange and a cross experiment. In a culture medium with phosphate, 94.6% of 1-cell embryos from the C57BL mouse strain developed to the blastocyst stage, but 95.7% of embryos from the AKR mouse strain showed 2-cell block. Phosphate-free culture medium rescued the 2-cell block of AKR embryos and accelerated the first cell cycle of the embryos. Co-culture with BRL cells and a BRL-conditioned medium fractionated below 30 kDa also rescued the 2-cell block of AKR embryos. Examinations of in vitro development of reconstructed embryos and of embryos from F1 females between AKR and C57BL strains clearly demonstrated that the AKR cytoplast caused the 2-cell block. In the backcrossed female progeny between (AKR x C57BL) F1 males and AKR females, about three-quarters of the embryos were of the 2-cell blocking phenotype and about one-quarter were of the non-blocking phenotype. These results suggest that two genes are responsible for the 2-cell block of AKR embryos.