响应面法优化阿卡波糖发酵培养基

目的利用响应面法对阿卡波糖发酵培养基进行优化。方法以HPLC法检测发酵样品中的阿卡波糖峰面积,并以阿卡波糖发酵单位为指标,通过Plackett-Burman(PB)实验,筛选出阿卡波糖发酵的主要影响因素,进而进行最陡爬坡实验,最后通过Box-Behnken设计实验,利用Design-Ex-pert 7.1.6 Trail软件进行回归分析,确定主要影响因素的最佳浓度,得到阿卡波糖优化发酵培养基组成。结果确定了优化发酵培养基组成(g.L-1):麦芽糖80.0、葡萄糖23.0、黄豆饼粉24.7、玉米浆1.5、谷氨酸1.0、磷酸二氢钾1.5、三氯化铁1.0、氯化钙3.0、碳酸钙4.0。验证实验表明在优化培养基条件下阿卡波糖发酵单位为4 015 mg.L-1,与预测值4 047 mg.L-1接近,比优化前提高了14.3%。结论对阿卡波糖发酵培养基采用响应面Box-Behnken设计,可以有效地对发酵培养基进行优化。     

24孔板优化林肯链霉菌发酵培养基

目的 优化林肯链霉菌发酵培养基。方法 采用24孔板,对现有的两种发酵培养基中各因素进行考察,筛选出较优发酵培养基;在此发酵培养基基础上,采用L25(56)正交设计方法,对可溶性淀粉、葡萄糖、豆饼粉、硫酸铵、玉米浆和磷酸氢二钾这6个可能影响林可霉素发酵水平的因素进行效应评价。结果 得到最佳发酵培养基(g/L)：可溶性淀粉30,葡萄糖105,豆饼粉22.5,玉米浆1,氯化钠2.25,碳酸钙6,硫酸铵2.65,硝酸钾1,磷酸氢二钾0.5。结论 对优化后的发酵培养基进行孔板发酵验证,林可霉素的平均发酵产量达到2288μg/mL,比未优化组提高了42.03%。     

林可霉素高产菌株SL-42发酵培养基优化研究

目的优化培养基组成、碳氮比和接种量,提高林可霉素发酵水平。方法设计正交实验,通过独特的摇瓶取样和补料,监控碳氮比(C/N),并进行50L发酵实验及其条件的优化选择。结果对于菌株9229#、SL369和SL-42,发酵条件优化后,发酵单位分别地提高了7.1%、7.7%、8.2%。结论实验验证了我们的菌种培养优化方案成效明显,可以放大推广。     

壳二孢氯素产生菌的鉴定及发酵培养基优化

目的对壳二孢氯素产生菌F05Z0761进行菌种鉴定,并进行培养基优化,提高其发酵单位。方法首先通过形态学特征进行鉴定,然后通过发酵培养基筛选实验并采用Plackett-Burman实验、最陡爬坡实验、中心组合实验设计等方法提高发酵单位。结果 F05Z0761经形态学鉴定显示该菌株属于镰刀菌属（Fusarium）,并得到了优化后的发酵培养基配方即:淀粉1.8%、葡萄糖2.5%、棉籽粉1.9%、热榨黄豆饼粉0.8%和KH₂PO₄0.2%,发酵单位较对照提高了4.38倍。结论由镰刀菌属产生壳二孢氯素在国内还未见报道,并将响应面实验设计应用于该菌株的发酵培养基优化中。     

抗MRSA小链霉菌NIM521发酵工艺的优化

小链霉菌NIM521能产生对耐甲氧西林金黄色葡萄球菌(MRSA)有强烈抑制作用的活性物质.利用单因素多浓度和正交实验法对其发酵培养基组分及发酵条件进行了优化,并在优化后的条件下进行了20 L罐发酵放大实验.实验表明该菌的最适培养条件为:培养基含麦芽汁9%、蛋白胨6%、L-甲硫氨酸0.02%、维生素C 0.02%;培养温度30℃、转速250 r/min、接种量15%、发酵时间6 d.优化发酵条件下,20 L发酵罐发酵5 d时发酵液抑菌圈直径可达37.5 mm.     

表面活性剂对天然红色素发酵的影响

探讨了表面活性剂的添加对天然红色素灵菌红素发酵的影响,并在此基础上对发酵培养基进行优化,达到提高色素产量的目的。以发酵液中灵菌红素的OD535为指标,通过对单因素实验和正交实验进行分析,优化培养基条件。结果表明:在吐温-80、十二烷基磺酸钠和二甲亚砜3种表面活性剂中,添加二甲亚砜的效果最佳,其最佳添加浓度为0.1 g/L,最佳添加时间为发酵44 h,产量与对照组相比提高近1倍;通过正交实验进一步优化发酵培养基,发现在发酵培养基中添加二甲亚砜0.1 g/L、脯氨酸0.1 g/L、甘氨酸1 g/L、组氨酸0.5 g/L,灵菌红素产量为对照组的2.38倍,优化效果最佳。因此,在灵菌红素发酵中后期添加适量的表面活性剂有助于灵菌红素产量的提高。     

肠膜明串珠菌发酵草鱼骨酶解液工艺条件的优化

目的采用乳酸菌发酵的方法对鱼骨中的钙进行转化,并优化出最优发酵工艺。方法采用泡菜汤汁中分离出的肠膜明串珠菌对草鱼骨风味蛋白酶酶解液进行发酵,以发酵液游离钙含量为筛选指标,对鱼骨含量、碳源种类、碳源含量、发酵起始pH、不同无机盐种类进行单因素实验,初步确定了各因素最优水平,在鱼骨含量、碳源含量、发酵起始pH单因素结果为零水平的基础上,零水平重复实验3次,进行三因素三水平响应面实验。结果与结论响应面实验确定肠膜明串珠菌发酵草鱼骨酶解液的最优工艺条件为鱼骨含量81.25mg/mL,碳源含量7mg/mL,发酵初始pH=5.75,预测发酵液中游离钙含量为5.56mg/g鱼骨,在优化出的条件下进行验证实验,得到的游离钙含量为5.57±0.13mg/g鱼骨。     

酵母菌乳杆菌共培养产谷胱甘肽条件的研究

通过酿酒酵母和干酪乳杆菌共培养,在单因素实验的基础上,考察Plackett-Burman实验、正交实验,优化酵母菌和乳杆菌共培养的条件,以提高酵母菌产谷胱甘肽的产量。首先进行酵母菌和乳杆菌共培养的单因素实验,挑选出乳糖浓度、发酵培养基初始pH值、共培养温度、转速、乳杆菌接种量、接种时间、发酵时间,这7个对谷胱甘肽产量有影响的因素。利用分析软件Minitab16,对这7个因素进行Plackett-Burman实验,筛选出3个影响较为显著的因素。利用分析软件SPSS 19,再对这3个因素进行正交实验,得出最佳发酵条件。在最佳发酵条件下,谷胱甘肽产量比优化前提高了35.14%;相同条件下,比单菌培养提高了33.35%。得到最佳发酵条件为:乳糖浓度40 g/L,发酵培养基初始pH值为6.4,共培养温度为36℃,转速为200 r/min,乳杆菌接种量为8%,乳杆菌接种时间为8 h,发酵时间为24 h。酵母菌乳杆菌共培养产谷胱甘肽的方法具有可行性,并且利用Plackett-Burman设计和正交设计可有效优化发酵条件。     

响应面法优化金霉素发酵培养基

目的采用响应面法对金色链霉菌发酵产金霉素培养基进行优化。方法首先采用Plackett-Burman实验筛选出影响金色链霉菌发酵产生金霉素的主要影响因素,然后通过最速上升实验逼近最大值响应区域,最后采用Box-Behnken设计响应面方法确定主要影响因素的浓度,得到金霉素优化发酵培养基组成。结果玉米淀粉、酵母粉、(NH4)2SO4为对金霉素产量有显著影响的主要因素,最佳水平为:玉米淀粉125.10 g.L-1、酵母粉4.03 g.L-1、(NH4)2SO44.87 g.L-1。在优化发酵培养基条件下,金霉素发酵单位为22.813 g.L-1,与预测值接近,较优化前发酵单位19.476 g.L-1提高了17%。结论响应面实验设计和分析方法能够有效地对金霉素发酵培养基进行优化。     

南极放线菌XE发酵条件的优化及抑菌物质性质的初步研究

目的 优化南极放线菌XE发酵工艺,提高抑菌活性物质的产量并对菌株产生的活性代谢产物性质进行了初步研究。方法 以发酵液抑菌活性为指标,采用单因素实验和正交实验对放线菌XE发酵培养基和发酵条件进行优化;观察pH值、温度、储藏温度对抑菌物质稳定性的影响;了解抑菌物质的极性及疏水特性。结果 获得了南极放线菌XE的最佳发酵培养基：可溶性淀粉30g/L,黄豆10g/L,海水晶35g/L,KON₃ 1.5g/L;最佳发酵条件：温度28℃,起始pH8,接种量2%,摇床转速110rpm,发酵时间3天;抑菌物质热稳定性好,-20℃储藏活性基本不变,在强碱环境中失活,不耐强碱,活性物质极性较小。结论 通过优化工艺实验,确定了菌株XE最佳发酵培养基和发酵条件,抑菌物质热稳定性好,在-20℃储藏活性基本不变,极性较小,不耐强碱。     

铵离子在麦白霉素生物合成中的调节作用

本文报道用生米卡链霉菌（Ｓ．ｍｙｃａｒｏｆａｃｉｅｒｓ）四川变种的突变株研究铵离子在麦白霉素生物合成中的调节作用。在合成培养基和复合培养基中加入不同浓度的铵离子，对麦白霉素的生物合成显示不同程度的抑制作用，同时表明铵离子抑制分支氨基酸的降解是它调节麦白霉素生物合成的主要调节位点。在合成培养基中，铵离子对抗生素生物合成的调节作用似乎与培养基中糖浓度有关。     

Quantitative determination of clopidogrel active metabolite in human plasma by LC-MS/MS

A quantitative method for the determination of clopidogrel active metabolite (AM) in human plasma was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Clopidogrel AM contains a thiol group, thus requiring stabilization in biological samples. The alkylating reagent 2-bromo-3'-methoxyacetophenone was used to stabilize clopidogrel AM in blood. An analog of the derivatized clopidogrel AM was used as the internal standard (IS). The derivatized samples were subjected to solid-phase extraction with a C2 disk plate and the overall procedure exhibited good reaction (more than 90%) and recovery efficiencies (from 85% to 105%). The derivative of clopidogrel AM (MP-AM) and IS were separated on an ODS column and quantified by tandem mass spectrometry with electrospray ionization. No significant endogenous peaks corresponding to MP-AM or IS were detected in blank human plasma samples, and no significant matrix effect was observed for MP-AM and IS in human plasma samples (from 102% to 121%). The calibration curve ranged from 0.5 to 250 ng/mL with good linearity, and extended by validation of a 50-fold dilution. In the intra- and inter-assay reproducibility tests, the accuracy and precision were within 12% relative error and 6% coefficient of variation, respectively. The derivatized MP-AM was stable in human plasma for 4 months at -80 degrees C. The validated method was successfully used to analyze clinical samples and determine the pharmacokinetics of clopidogrel AM.     

THE THERMAL POTENTIATION OF ACETAMINOPHEN-INHIBITED PMN OXIDATIVE METABOLISM IN VITRO

The effect of high temperatures (39, 41, and 43 °C) on acetaminophen (AM-) induced inhibition of the oxidative respiratory burst of polymorphonuclear leukocytes (PMNs) in vitro has been examined. Whole blood or isolated human PMNs were exposed to various temperatures in vitro in the presence or absence of AM for 0–90 min. Phagocyte membrane-bound NADPH oxidase was studied using the luminol chemiluminescence (CL) response and the superoxide dismutase inhibitable reduction of ferricytochrome C. The NADPH oxidase was stimulated by phorbol myristate acetate (PMA). The results showed that high temperatures (39–43 °C) potentiate the AM inhibitory effect on CL peak response of phagocytes in a temperature-dependent manner. Furthermore, the inhibition of superoxide (O⁻₂) production induced by AM was potentiated by incubating the cells at 39 or 43 °C at different time intervals. These studies suggest that high temperatures significantly potentiate the AM inhibitory effect on oxidative metabolism of PMNs in vitro. These actions of AM may influence the outcome in patients with infectious febrile conditions.     

海洋细菌201721抗菌物质的发酵优化与性质初步研究

目的海洋细菌201 72l能产生对大肠杆菌有强烈抑制作用的活性物质,本文对其发酵条件进行优化,并对发酵产物性质进行研究。方法采用Plackett-Burman设计筛选出培养基pH、蛋白胨的量和培养温度等关键因素;继而采用响应曲面法关键因素进行优化。结果pH 6.3,蛋白胨的量8.2g,温度31.6℃为发酵的最优奈件,发酵所产生的抗菌活性物质极性中等.具有较好的热稳定性和酸碱稳定性。结论本研究所采用的条件优化方法.适用于菌株发酵条件的优化。     

Cannabinoid receptor antagonists AM251 and AM630 activate TRPA1 in sensory neurons

Cannabinoid receptor antagonists have been utilized extensively in vivo as well as in vitro, but their selectivity has not been fully examined. We investigated activation of sensory neurons by two cannabinoid antagonists - AM251 and AM630. AM251 and AM630 activated trigeminal (TG) sensory neurons in a concentration-dependent fashion (threshold 1 μM). AM251 and AM630 responses are mediated by the TRPA1 channel in a majority (90-95%) of small-to-medium TG sensory neurons. AM630 (1-100 μM), but not AM251, was a significantly more potent agonist in cells co-expressing both TRPA1 and TRPV1 channels. We next evaluated AM630 and AM251 effects on TRPV1- and TRPA1-mediated responses in TG neurons. Capsaicin (CAP) effects were inhibited by pre-treatment with AM630, but not AM251. Mustard oil (MO) and WIN55,212-2 (WIN) TRPA1 mediated responses were also inhibited by pre-treatment with AM630, but not AM251 (25 uM each). Co-treatment of neurons with WIN and either AM630 or AM251 had opposite effects: AM630 sensitized WIN responses, whereas AM251 inhibited WIN responses. WIN-induced inhibition of CAP responses in sensory neurons was reversed by AM630 pre-treatment and AM251 co-treatment (25 μM each), as these conditions inhibit WIN responses. Hindpaw injections of AM630 and AM251 did not produce nocifensive behaviors. However, both compounds modulated CAP-induced thermal hyperalgesia in wild-type mice and rats, but not TRPA1 null-mutant mice. AMs also partially regulate WIN inhibition of CAP-induced thermal hyperalgesia in a TRPA1-dependent fashion. In summary, these findings demonstrate alternative targets for the cannabinoid antagonists, AM251 and AM630, in peripheral antihyperalgesia which involve certain TRP channels.     

Anandamide transport inhibitor AM404 and structurally related compounds inhibit synaptic transmission between rat hippocampal neurons in culture independent of cannabinoid CB1 receptors

N-(hydroxyphenyl)-arachidonamide (AM404) is an inhibitor of endocannabinoid transport. We examined the effects of AM404 on glutamatergic synaptic transmission using network-driven increases in intracellular Ca2+ concentration ([Ca2+] spikes) as an assay. At a concentration of 1 microM AM404 inhibited [Ca2+]i spiking by 73+/-8%. The cannabinoid CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A), the vanilloid VR1 receptor antagonist capsazepine (CPZ), and treatment with pertussis toxin failed to block AM404-mediated inhibition. AM404 (3 microM) inhibited action-potential-evoked Ca2+ influx by 58+/-3% but failed to affect calcium influx evoked by depolarization with 30 mM K+, suggesting that the inhibition of electrically evoked [Ca2+]i increases and that [Ca2+]i spiking was due to inhibition of Na+ channels. Palmitoylethanolamide (PMEA), capsaicin (CAP) and (5Z,8Z,11Z,14Z)-N-(4-hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide (VDM11), compounds structurally similar to AM404, inhibited [Ca2+]i spiking by 34+/-10%, 42+/-18% and 67+/-12%, respectively. Thus, AM404 and related compounds inhibit depolarization-induced Ca2+ influx independent of cannabinoid receptors, suggesting caution when using these agents as pharmacological probes to study synaptic transmission.     

Synthesis of a novel polymeric magnetic solid phase extraction adsorbent for selective extraction of amphetamine from urine samples coupled with high performance liquid chromatography

A novel pH‐responsive block copolymer (Poly ethylene glycol‐b‐poly (N,N‐dimethylaminoethylmethacrylate‐co‐maleic acid) was designed for the decoration and stabilization of magnetic nanoparticles (MNPs) as an efficient magnetic nano adsorbent for extraction of amphetamine (AM) from biological urine samples to be determined by high performance liquid chromatography–ultra violet detector (HPLC–UV). Full characterization of the synthesized polymeric magnetic nanoparticles (PMNPs) were followed by various techniques like Fourier transform infrared (FT–IR) spectroscopy, powder x‐ray diffraction (XRD), scanning electron microscopy (SEM), and vibrating sample magnetometer (VSM). Important extraction parameters including pH, amount of sample volume, amount of adsorbent, type and amount of extraction organic solvent, time of extraction and desorption, agitation rate (rpm), and ionic strength of the extraction medium were studied and optimized. Under optimized extraction conditions, good linearity was observed in the concentration range of 30–2000 ng/mL for AM. The amount of the q_e was calculated as 0.18 (mg/g). The method was applied in determination of AM from positive urine samples with the recovery of 99.84%. Results indicated that the proposed method could be applied in clinical and forensic laboratories for simple, selective, and fast determination of AM from urine samples.     

Amikacin-loaded vascular prosthesis as an effective drug carrier

Strong covalent immobilization of amikacin on Uni-Graft((R)) DV straight vascular prostheses made of gelatine-sealed poly(ethylene terephthalate) fibres was performed according to procedure described in the Polish Patent No. P-358934. The concentrations of amikacin in sample solutions were estimated either by HPLC or by UV spectroscopy method previously optimized for amikacin measurements. A high correlation was found between these two methods. It was found that the antibiotic was bound in mixed-type way via three types of interactions: strong covalent bonds (dominating amount: 81.84%) and weak interactions: physical adsorption and ionic bonds (18.19%). Even when total amount of physically and ionically attached drug has been released, the remaining covalently bound amount still locally protected the prostheses in vitro against bacteria. The release test was conducted in PBS at pH 7.4 at 37 degrees C and showed that about 15% of total drug amount was eluted from the matrix during the first 7 days of shaking, then no more antibiotic was released. It suggested that about 85% of amikacin attached to prosthesis modified in mixed-type mode was bound via covalent interactions. A bacterial inhibition test on Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853 showed inhibition of growth for all strains at low inoculum concentrations up to 30 days as well as high inoculum concentration for E. coli. At high concentrations of S. aureus and P. aeruginosa, the modified prostheses showed slight bacteriostatic effect since 10th day of experiment. Amikacin-modified vascular prostheses might therefore be protected against bacterial infection locally, without long-lasting drug release to human system.     

Pharmacodynamics, Pharmacokinetics, and Safety of AM211: A Novel and Potent Antagonist of the Prostaglandin D2 Receptor Type 2

The prostaglandin D(2) receptor type 2 (DP2) and its ligand, PGD(2), have been implicated in the development of asthma and other inflammatory diseases. The authors evaluated the pharmacodynamics, pharmacokinetics and safety of [2'-(3-benzyl-1-ethyl-ureidomethyl)-6-methoxy-4'-trifluoromethyl-biphenyl-3-yl]-acetic acid sodium salt (AM211), a novel and potent DP2 antagonist, in healthy participants. Single and multiple doses of AM211 demonstrated dose-dependent inhibition of eosinophil shape change in blood with near-complete inhibition observed at trough after dosing 200 mg once daily for 7 days. Maximum plasma concentrations and exposures of AM211 increased in a greater-than-dose-proportional manner after single and multiple dosing. After multiple dosing, the exposures on day 7 were higher than on day 1 with accumulation ratio values ranging from 1.4 to 1.5. Mean terminal half-life values ranged from 14 to 25 hours across the dose range of 100 to 600 mg. AM211 was well tolerated at all doses in both the single- and multiple-dose cohorts. These data support additional clinical studies to evaluate AM211 in asthma and other inflammatory diseases.     

The phytocannabinoid Delta(9)-tetrahydrocannabivarin modulates inhibitory neurotransmission in the cerebellum

BACKGROUND AND PURPOSE: The phytocannabinoid Delta(9)-tetrahydrocannabivarin (Delta(9)-THCV) has been reported to exhibit a diverse pharmacology; here, we investigate functional effects of Delta(9)-THCV, extracted from Cannabis sativa, using electrophysiological techniques to define its mechanism of action in the CNS. EXPERIMENTAL APPROACH: Effects of Delta(9)-THCV and synthetic cannabinoid agents on inhibitory neurotransmission at interneurone-Purkinje cell (IN-PC) synapses were correlated with effects on spontaneous PC output using single-cell and multi-electrode array (MEA) electrophysiological recordings respectively, in mouse cerebellar brain slices in vitro. KEY RESULTS: The cannabinoid receptor agonist WIN 55,212-2 (WIN55) decreased miniature inhibitory postsynaptic current (mIPSC) frequency at IN-PC synapses. WIN55-induced inhibition was reversed by Delta(9)-THCV, and also by the CB(1) receptor antagonist AM251; Delta(9)-THCV or AM251 acted to increase mIPSC frequency beyond basal values. When applied alone, Delta(9)-THCV, AM251 or rimonabant increased mIPSC frequency. Pre-incubation with Delta(9)-THCV blocked WIN55-induced inhibition. In MEA recordings, WIN55 increased PC spike firing rate; Delta(9)-THCV and AM251 acted in the opposite direction to decrease spike firing. The effects of Delta(9)-THCV and WIN55 were attenuated by the GABA(A) receptor antagonist bicuculline methiodide. CONCLUSIONS AND IMPLICATIONS: We show for the first time that Delta(9)-THCV acts as a functional CB(1) receptor antagonist in the CNS to modulate inhibitory neurotransmission at IN-PC synapses and spontaneous PC output. Delta(9)-THCV- and AM251-induced increases in mIPSC frequency beyond basal levels were consistent with basal CB(1) receptor activity. WIN55-induced increases in PC spike firing rate were consistent with synaptic disinhibition; whilst Delta(9)-THCV- and AM251-induced decreases in spike firing suggest a mechanism of PC inhibition.