高产脂微生物—深黄被孢霉M018变株的选育及其油脂合成条件的研究

以深黄被孢霉Ａｓ３．３４１０为出发菌株，经紫外线ＵＶ）、硫酸二乙酯（ＤＥＳ）和亚硝基胍（ＮＴＧ）复合诱变处理，选衣成功高产脂深黄被孢霉Ｍ０１８变株，其摇瓶培养，菌体油脂含量达６５．６％，经出发菌株提高１３３％、６０ｍ＾３罐三级发酵培养菌体油脂含量高达７９．２％，生物量达３７．８ｇ／Ｌ。气相色谱分析表达变株Ｍ０１８γ－亚麻酸的含量比出发菌株提高了５３％，连续传代试验表明Ｍ０１８是一稳定的变株。该变株     

螺旋霉素高产菌种的推理选育

根据螺旋霉素的生物合成途径对螺旋霉素产生菌产二素链霉菌进行推理选育，并获得了高产菌种。首先在紫外线诱变的基础上，筛选豆油耐性变株，得到菌株ＸＣ２－３７，其发酵效价较出发菌株ＸＣ１－２９提高９％。通过培养基优化，菌株ＸＣ２－３７的发酵效价提高６１．８％。再以紫外线处理菌株ＸＣ２－３７，并筛选缬氨酸诱导变株，得到菌株ＸＣ３－１１，其发酵效价较菌株ＸＣ２－３７提高２０％。菌株ＸＣ３－１１经自然分离得到菌株ＸＣ４－１８，发酵效价达到４５００ｕ／ｍｌ，为原始出发菌株ＸＣ１－２９的２．２５倍。传代试验表明菌株ＸＣ４－１８的高产遗传特性稳定。菌株ＸＣ４－１８应用于５０ｍ３发酵罐进行工业化生产，与原始出发菌株ＸＣ１－２９相比，发酵效价和发酵指数分别提高８４．９％和４３．６％。     

吉他霉素高产菌株的选育

以吉他霉素产生菌Ｒ－０１－１０５菌株为出发菌株，经紫外线、Ｃｏ６０、色氨酸、异亮氨酸和２－ＤＯＧ复合处理，获得４株高产突变株，其中Ｔ－１１－１２０的摇瓶效价（１５３０２ｕ／ｍｌ）比出发菌株（８５００ｕ／ｍｌ）提高８０．１％，在１０吨发酵罐上连续８批验证，平均发酵效价１２３８８ｕ／ｍｌ，比原生产水平（７４００ｕ／ｍｌ）提高６７．４％，并且质检全部合格，对生产实际具有重要意义     

林可霉素高产菌种的选育

以林可霉素产生菌82－7^#为出发菌株,采用氯化锂、紫外线（uV)、甲基磺酸乙酯（EMS）三重复合诱变处理、筛选耐自身产物的高产菌种,从中获得65－12^#菌株。其摇瓶效价较出发菌株提高25％,发酵培养基优化组合后,其摇瓶效价提高28％。应用于30吨发酵罐生产,其发酵指数较出发菌株提高16．8％。     

Avermectin产生菌异亮氨酸诱导变种的选育

用紫外线处理ａｖｅｒｍｅｃｔｉｎ（ＡＶＭ）产生菌ＳｔｒｅｐｔｏｍｙｃｅｓａｖｅｒｍｉｔｉｌｉｓＸＣ１－２５，并在含有Ｌ－异亮氨酸（Ｌ－Ｉｌｅ）的平板培养基上筛选Ｌ－Ｉｌｅ诱导变种。结果表明：Ｉｌｅｉ变株发酵产生的ＡＶＭＢ１ａ效价高于自然分离株与紫外线诱变株，其中Ｉｌｅｉ变株ＸＣ２－２６的ＡＶＭＢ１ａ效价较亲株提高２２％。该变株经自然分离获得菌株ＸＣ３－８，其ＡＶＭＢ１ａ效价比出发菌株提高５０％以上，传代试验表明菌株ＸＣ３－８的形态和高产性能稳定，它在７ｍ３发酵罐生产试验，产生ＡＶＭＢ１ａ效价与发酵指数均比出发菌株提高５６％。     

青霉素生产菌原生质体诱变选育

以青霉素生产菌丝状产黄青霉ＪＳ９４－１８为出发菌株制备菌丝原生质体·并对原生质体进行了紫外诱变，筛选到变异株ＪＳ９４－１８－５８，该菌株摇瓶发酵效价比生产菌株提高８．６３％，５０吨发酵罐试生产平均发酵效价比生产菌株提高１０．４０％。     

氮离子注入土霉素产生菌诱变高产菌株的研究

以土霉索生产菌株48。为出发菌株，采用氮离子注入诱变处理，经摇瓶筛选，得到土霉素02—2-44^#菌株。该菌株特性优良，经57m^3发酵罐试验。150批平均发酵效价高于对照菌株（1158．6μg／ml），提高3．42％。发酵总亿及发酵指数分别提高3.17％和3．99％。     

万古霉素产生菌的选育

以东方拟无枝酸菌（Amycolatopsisoriental）05—12为出发菌株,经紫外线（15W。253．7nm,距离18cm,照射5m）诱变处理,在含庆大霉素1000μg·mL^-1的浓度梯度平板上筛选庆大霉素抗性变株,得到一株万古霉素高产交株（Amyeolatopsisoriental）06-4。其摇瓶效价较出发菌株提高23％．对万古霉素的抗性提高200％。传代试验表明该变株的高产性能遗传特性较稳定。用正交试验法对万古霉素发酵培养基进行了优化,与原发酵培养基相比,万古霉素的摇瓶发酵效价提高了13．4%。     

万古霉素产生菌的选育与发酵培养基优化

万古霉素产生菌东方拟无枝酸菌（Amycolatopsis orientalis）5－13经紫外线（30W，253.7nm，距离30cm，照射50s）诱变后，在含万古霉素1000μg/ml的浓度梯度平板上筛选万古霉素抗性变株，得到一株万古霉素抗性变株A.orientalis 6-21，其摇瓶效价较出发菌株提高23％，对万古霉素的抗性提高200％。传代试验表明该变株的高产性能遗传特性较稳定。用正交试验法对万古霉素发酵培养基进行了优化，与原发酵培养基相比，万古霉素的摇瓶发酵效价提高了14.3%。     

复合诱变选育多黏菌素E高产菌株

以多黏芽胞杆菌（Bacillus polymyxa）090918为出发菌株,经60-Co-r射线照射（剂量为22Gy）、甲磺酸乙酯（EMS）诱变处理、多粘菌素E耐受等处理,得到一株多粘菌素E突变株120116,其摇瓶发酵效价较出发菌株提高26％。     

应用Stainer培养基与TFF系统制备白喉类毒素

目的  验证WHO推荐的Stainer培养基与切向流过滤(TFF)系统能否制备符合我国现行规程要求的精制白喉类毒素.方法   用PW8菌株、Stainer培养基进行了三批白喉杆菌产毒培养,用TFF微孔过滤和超滤系统分别进行了澄清与浓缩.毒素经甲醛脱毒后,用TFF超滤系统对类毒素进行了超滤精制,得到三批精制白喉类毒素,按我国现行规程主要技术指标进行评价  .结果   产毒效价分别为180Lf/ml、210Lf/ml和155Lf/ml.精制类毒素纯度分别为1900Lf/mgPN、1860Lf/mgPN、1760Lf/mgPN.毒性试验和毒性逆转试验全部合格.结论   应用Stainer培养基与TFF系统可获得符合我国现行规程要求的精制白喉类毒素.     

Protective effects of lactoferrin against intestinal mucosal damage induced by lipopolysaccharide in human intestinal Caco-2 cells

Indirect evidence suggests that lactoferrin (Lf), a major iron-binding protein in human milk, induces enterocyte growth and proliferation, depending on its concentration and affects the function and permeability of the intestinal mucosa. The bacterial endotoxin (lipopolysaccharide, LPS) is known to cause mucosal hyperpermeability in vivo. However, protective effects of Lf against LPS-mediated intestinal mucosal damage and barrier function in epithelial cells are not yet fully clarified. The aim of this study was to investigate whether Lf can reduce the cellular injury and alter epithelial hyperpermeability caused by LPS in human intestinal Caco-2 cells. When cell viability was measured by a WST-1 assay (tetrazolium salt-based assay), the protective effects against LPS-induced damage to Caco-2 cells were observed at doses of 800 and 1000 microg/ml Lf. The barrier function of Caco-2 monolayer tight junctions was assessed by measuring transepithelial electrical resistance (TEER) and permeability of FITC-labeled dextran 4000 (FD-4). The treatment of Caco-2 cells with Lf at doses of 400 and 1000 microg/ml significantly increased TEER as compared to treatment with LPS alone for 2 h (p<0.05). Further, at doses of 400 and 1000 microg/ml, Lf inhibited the enhancement of LPS-mediated permeability in Caco-2 cell monolayer. The results of this study suggest that Lf may have protective effects against LPS-mediated intestinal mucosal damage and impairment of barrier function in intestinal epithelial cells.     

Design and Fungicidal Activity of Mucoadhesive Lactoferrin Tablets for the Treatment of Oropharyngeal Candidosis

Lactoferrin (Lf) is a potential drug candidate for the treatment of oropharyngeal Candida infections. However, for an effective therapeutic treatment an appropriate dosage form is required. Therefore a mucoadhesive tablet for buccal application was developed. Tablets of sufficient strength could be produced on high speed tabletting machines, but they could only be obtained when the protein contained at least 7% moisture. The tablet contained sodium alginate both for its release-controlling properties as well as for its mucoadhesive properties. Furthermore, phosphate buffer was added to keep the pH of the saliva in the mouth within the range of 6.5 to 7.5. In this pH range, Lf has shown to have its highest activity against Candida growth inhibition. The tablet formulation containing Lf had the same antifungal properties as compared with Lf alone, because in most cases identical inhibitory concentrations were observed against several clinical isolates of Candida albicans and Candida glabrata. In human volunteers the tablets, containing 250 mg Lf and placed in each pouch, were able to keep the Lf concentration in the saliva at effective levels for at least 2 hr, while the pH of the saliva remained within the desired range. We concluded that the developed mucoadhesive tablet can improve the therapeutic efficacy of Lf and that it is suitable for further clinical research.     

Effect of lactoferrin- and transferrin-conjugated polymersomes in brain targeting： in vitro and in vivo evaluations

Aim:

To evaluate the effect of lactoferrin (Lf) and transferrin (Tf) in brain targeting.

Methods:

Polymersomes (PSs), employed as vectors, were conjugated with Lf or Tf and were characterized by morphology, particle size, zeta potential, and surface densities of the Lf or Tf molecules. In vitro uptake of Lf-PS and Tf-PS by bEnd.3 cells was investigated using coumarin-6 as a fluorescent probe. In vivo tissue distribution and pharmacokinetics of ¹²⁵I-Lf-PS and ¹²⁵I-Tf-PS were also examined.

Results:

The mean particle size of PS, Lf-PS, and Tf-PS was around 150 nm and the zeta potential of the PSs was about -20 mV. Less than 0.12% of the coumarin was released from coumarin-6-loaded PS in 84 h indicating that coumarin-6 was an accurate probe for the PSs'' behavior in vitro. It was shown that the uptake of Lf-PS and Tf-PS by bEnd.3 cells was time-, temperature-, and concentration-dependent. Both Lf and Tf could increase the cell uptake of PSs at 37°C, but the uptake of Tf-PS was significantly greater than that of Lf-PS. In vivo tissue distribution and pharmacokinetics in mice revealed higher brain uptake and distribution of Tf-PS than Lf-PS, which was in accordance with in vitro uptake results. The drug targeting index (DTI) of Tf-PS with regard to Lf-PS was 1.51.

Conclusion:

Using a PS as the delivery vector and bEnd.3 cells as the model of the blood-brain barrier (BBB), Tf was more effective than Lf in brain targeting.     

Design and fungicidal activity of mucoadhesive lactoferrin tablets for the treatment of oropharyngeal candidosis

Lactoferrin (Lf) is a potential drug candidate for the treatment of oropharyngeal Candida infections. However, for an effective therapeutic treatment an appropriate dosage form is required. Therefore a mucoadhesive tablet for buccal application was developed. Tablets of sufficient strength could be produced on high speed tabletting machines, but they could only be obtained when the protein contained at least 7% moisture. The tablet contained sodium alginate both for its release-controlling properties as well as for its mucoadhesive properties. Furthermore, phosphate buffer was added to keep the pH of the saliva in the mouth within the range of 6.5 to 7.5. In this pH range, Lf has shown to have its highest activity against Candida growth inhibition. The tablet formulation containing Lf had the same antifungal properties as compared with Lf alone, because in most cases identical inhibitory concentrations were observed against several clinical isolates of Candida albicans and Candida glabrata . In human volunteers the tablets, containing 250 mg Lf and placed in each pouch, were able to keep the Lf concentration in the saliva at effective levels for at least 2 hr, while the pH of the saliva remained within the desired range. We concluded that the developed mucoadhesive tablet can improve the therapeutic efficacy of Lf and that it is suitable for further clinical research.     

Hepatocellular carcinoma targeting effect of PEGylated liposomes modified with lactoferrin

A hepatocellular carcinoma targeting lactoferrin (Lf) modified PEGylated liposome system was developed for improving drug efficacies to hepatic cancer cells. In this present work, PEGylated liposomes (PLS) were successfully prepared by the thin film hydration method combined with peglipid post insertion. Lf was covalently conjugated to the distal end of DSPE-PEG2000-COOH lipid by amide bound and loaded onto PEGylated liposomes surface as the targeting ligand. To confirm the targeting efficacies to hepatic cancer, coumarin-6 and DiR were encapsulated as fluorescent probes. The confocal microscopy and flow cytometry demonstrated that Lf conjugated PEGylated liposomes (Lf-PLS) were efficiently associated by HepG2 cells, while limited interaction was found for liposomes modified with a negative control protein. A similar pharmacokinetic behavior was observed in pharmacokinetics study of the liposomal formulations. Meanwhile, the in vivo imaging of liposomes in HepG2 tumor bearing mice indicated that Lf-PLS achieved more accumulation in tumor compared with PLS without Lf conjugated. The significant in vitro and in vivo results suggested that Lf-PLS might be a promising drug delivery system for hepatocellular carcinoma therapy with low toxicity.     

Inhibition of HSV cell-to-cell spread by lactoferrin and lactoferricin

The milk protein lactoferrin (Lf) has multiple functions, including immune stimulation and antiviral activity towards herpes simplex virus 1 and 2 (HSV-1 and HSV-2); antiviral activity has also been reported for the N-terminal pepsin-derived fragment lactoferricin (Lfcin). The anti-HSV mode of action of Lf and Lfcin is assumed to involve, in part, their interaction with the cell surface glycosaminoglycan heparan sulfate, thereby blocking of viral entry. In this study we investigated the ability of human and bovine Lf and Lfcin to inhibit viral cell-to-cell spread as well as the involvement of cell surface glycosaminoglycans during viral cell-to-cell spread. Lf and Lfcin from both human and bovine origin, inhibited cell-to-cell spread of both HSV-1 and HSV-2. Inhibition of cell-to-cell spread by bovine Lfcin involved cell surface chondroitin sulfate. Based on transmission electron microscopy studies, human Lfcin, like bovine Lfcin, was randomly distributed intracellularly, thus differences in their antiviral activity could not be explained by differences in their distribution. In contrast, the cellular localization of iron-saturated (holo)-Lf appeared to differ from that of apo-Lf, indicating that holo- and apo-Lf may exhibit different antiviral mechanisms.     

Comparison of anticancer activity between lactoferrin nanoliposome and lactoferrin in Caco-2 cells in vitro

The anticancer activities of Lactoferrin (Lf) and Lf nanoliposomes in Caco-2 cells were observed in this study, and mitochondrial function (MTT assay), count kit-8(CCK-8), detection of intracellular reactive oxygen species (ROS) and apoptosis induction (AO/EB staining) assays were used to evaluate the anticancer activity. MTT results demonstrated that Lf nanoliposomes and Lf reduced the mitochondrial activity of cells in a manner of dose and time effect, and the viabilities of Caco-2 cell were significantly decreased in vitro following exposure to Lf nanoliposomes at the concentrations of 5 and 10 mg/mL. LDH leakage and ROS significantly increased in cells exposed to Lf nanoliposomes (⩾5 mg/mL), while Lf induced ROS only at higher doses (10 mg/mL). CCK-8 evaluation of cell proliferation and AO/EB double staining supported the anti-proliferative effects of Lf liposomes. Our findings demonstrated that the presence of Lf nanoliposome is more significant than Lf in inhibiting human tumor cells proliferation. Therefore, it can be concluded that Lf nanoliposomes are a potential therapeutic modality in the management of tumors.     

Enhanced immune response with a combination of alum and biodegradable nanoparticles containing tetanus toxoid

The purpose of the study was to determine the synergistic adjuvant effect of sustained release biodegradable nanoparticles in combination with alum. Nanoparticles containing tetanus toxoid (TT) were formulated using a biodegradable polymer, polylactic polyglycolic acid co-polymer (50:50, molecular weight 100 000). The immunization studies were carried out subcutaneously in rats. The results were expressed as mean serum anti-TT IgG levels. The nanoparticles demonstrated a TT loading of 4% w/w with mean particle diameter of 238 #45 31 nm. The TT encapsulated in nanoparticles was released slowly under in vitro conditions, with 67.5% cumulative release occurring in 20 days. A single injection of TT-nanoparticles (TT dose=10 Lf) mixed with TT-Alum (TT dose =5 Lf) induced a four-fold greater mean serum anti-TT IgG response than a single injection of TT nanoparticles alone (TT dose=15 Lf) (2235 #45 310 vs. 539 #45 49 #119 g/ml, mean #45 sem, p => 0.001). In addition, the mean immune response induced with the single injection of combination of nanoparticles and alum was comparable to the two injections of TT-alum alone (5 Lf each dose) given at 3 week intervals IgG = 1998 #45 333 #119 g/ ml). Furthermore, the combination induced a peak immune response (IgG = 4215 #45 546 #119 g/ml) as early as the first time point at 3 weeks post-immunization. In the case of a TT-alum alone, the animals showed a weaker immune response at 3 weeks and required a second dose of TT-alum to enhance the antibody response. The data thus suggest that the combination of TTnanoparticles and TT-alum acts as a much better adjuvant than nanoparticles or alum alone. A rapid induction of immune response is useful to curb the spread of communicable diseases in the case of an outbreak.     

A novel bovine lactoferrin peptide,FKCRRWQWRM, suppresses Candida cell growth and activates neutrophils

Abstract: To identify potent new antifungal agents, the Candida cell growth inhibitory activities of six lactoferrin (Lf) peptides consisting of 6–25 amino acid residues (peptide 1, FKCRRWQWRMKKLGAPSITCVRRAF = lactoferricin B; peptide 2, FKCRRWQWRM; peptide 2′, FKARRWQWRM; peptide 3, GAPSITCVRRAF; peptide 4, RRWQWR; and peptide 5, RWQWRM) were examined. Of these, peptide 2 strongly suppressed the multiplication of Candida cells, but other peptides showed only weak activities. In two strains of C. albicans, the minimum inhibitory concentration 100 of peptide 2 (17.3 ± 2.2 µm and 17.5 ± 2.4 µm ) was close to that of miconazole (13.0 ± 1.7 µm and 13.1 ± 1.6 µm ) but markedly different from that of amphotericin B (0.52 ± 0.09 µm and 0.56 ± 0.11 µm ). The suppression of Candida cell growth was additively increased by a combination of peptide 2 with amphotericin B and miconazole. Peptides 1, 3, 4 and 5 and Lf suppressed iron uptake by Candida cells, inversely correlated with their Candida cell growth inhibition activities. However, iron uptake was not inhibited by peptide 2. In addition, peptide 2 upregulated Candida cell killing activity of polymorphonuclear leukocytes (PMN) increasing their superoxide generation, protein kinase C activity, p38 MAPK activity and the expression of p47^phox. These results indicated that the main antimicrobial activity of the Lf peptides is dependent on the N‐terminal half of Lf and that the PMN upregulatory activity of peptide 2 and additive function of peptide 2 with antifungal drugs are useful for prophylaxis and control of candidiasis.