天然红色素灵菌红素的急性经口毒性和遗传毒性

灵菌红素属于天然色素,具有离体抑制肿瘤和癌症细胞增殖、抑制微生物生长以及抗疟疾和免疫抑制等功效,具有潜在的巨大应用价值。缺少急性经口毒性和遗传毒性实验的研究限制了灵菌红素在食品工业领域的应用。本研究依据食品安全国家标准进行了灵菌红素的小鼠急性经口毒性实验、哺乳动物红细胞微核实验、小鼠精子畸形实验和细菌回复突变实验。结果表明,灵菌红素急性经口半数致死剂量LD_(50)10 g/kg(以体质量计),3项遗传毒性实验结果为阴性,灵菌红素对小鼠的急性经口毒性属无毒级,对正常细胞无毒,对动物生殖系统没有毒害。     

粘质沙雷氏菌高产发酵灵菌红素的工艺优化及抗病毒活性

为提高粘质沙雷氏菌S3菌株的灵菌红素产率,本研究对粘质沙雷氏菌S3发酵过程中重要的发酵条件参数和关键组分进行逐项优化,并验证了发酵液灵菌红素提取物对本氏烟草病毒的抑制效果。结果表明,最佳培养基配方为：丙三醇0.5%,蛋白胨1.5%,氯化钠0.5%,氯化钾0.25%;最佳发酵条件为：接种量为10%,装液量为60%~70%,pH恒定为6.0,培养温度为28℃,振摇速度160r/min,发酵周期为60h。在该发酵条件下,既有利于粘质沙雷氏菌菌体生长,又能够使灵菌红素的产量达到最大化。按照该优化条件进行发酵后,其灵菌红素提取物溶液喷施于接种TMV、CMV和PVY的本氏烟,烟叶中TMV、CMV和PVY病毒量分别为空白对照的12.61%、29.41%和17.69%,对病毒的抑制效果优于氨基寡糖素。试验所得的发酵制备条件能够提高灵菌红素产量,提取物具备显著抗病毒特性,具有产业化开发潜力和经济应用价值。     

Substrate Flexibility of the Flavin-Dependent Dihydropyrrole Oxidases PigB and HapB Involved in Antibiotic Prodigiosin Biosynthesis

In the biosynthesis of the tripyrrolic pigment prodigiosin, PigB is a predicted flavin-dependent oxidase responsible for the formation of 2-methyl-3-amylpyrrole (MAP) from a dihydropyrrole. To prove which dihydropyrrole is the true intermediate, both possibilities, 5-methyl-4-pentyl-3,4-dihydro-2H-pyrrole ( 5 a , resulting from transamination of the aldehyde of 3-acetyloctanal) and 2-methyl-3-pentyl-3,4-dihydro-2H-pyrrole ( 6 , resulting from transamination of the ketone), were synthesised. Only 5 a restored pigment production in a strain of Serratia sp. ATCC 39006 blocked earlier in MAP biosynthesis. PigB is membrane-associated and inactive when its transmembrane domain was deleted, but HapB, its homologue in Hahella chejuensis, lacks the transmembrane domain and is active in solution. Two colourimetric assays for PigB and HapB were developed, and the HapB-catalysed reaction was kinetically characterised. Ten analogues of 5 a were synthesised, varying in the C2 and C3 side chains, and tested as substrates of HapB in vitro and for restoration of pigment production in Serratia ΔpigD in vivo. All lengths of side chain tested at C3 were accepted, but only short side chains at C2 were accepted. The knowledge that 5 a is an intermediate in prodigiosin biosynthesis and the ease of synthesis of analogues of 5 a makes a range of prodigiosin analogues readily available by mutasynthesis.     

灵菌红素的研究进展

灵菌红素最初由于其多功能生物活性而在医药领域备受关注。除在医药领域应用外,灵菌红素作为微生物代谢产生的色素在食品领域也有很大的应用潜力。灵菌红素还可以作为除藻剂和抗紫外物质应用在环境和化妆品领域,作为太阳能电池中的光敏剂代替化学敏化剂来开发染料敏化太阳能电池。该文就灵菌红素的应用、发酵工艺、生物合成途径以及生产菌株的改造进行综述,以期为灵菌红素的发展及应用提供参考。     

灵菌红素的研究进展

灵菌红素是一类含甲氧基吡咯骨架结构的天然色素,是一些放线菌、沙雷菌及其他细菌的次级代谢产物,具有免疫抑制、抗细菌、抗真菌和抗疟疾等多种生物活性。最近研究表明灵菌红素有很强的抗肿瘤及免疫抑制活性,因而成为研究热点。现对灵菌红素的结构、生物活性、生产现状及发展趋势作一综述。     

灵菌红素对人胰腺癌细胞增殖抑制的实验研究

目的 观察从生物工程技术得到的灵菌红素对人胰腺癌8898 细胞增殖抑制的药效学作用, 并探讨其作用的部分机理。 方法 用MTT 法测定8898细胞的存活率和抑制率, 用流式细胞仪分析细胞的周期变化和凋亡细胞的百分比, 用HPLC 检测人胰腺癌8898 细胞内灵菌红素的浓度, 用琼脂糖凝胶电泳对DNA 段片化进行分析。 结果 灵菌红素5 mg·L^-1的培养液中8898 细胞出现凋亡早期的形态学特征, 半数抑制浓度(IC₅₀) 为30 mg·L^-1。而3T3 细胞却未显示出该作用。研究表明灵菌红素抑制细胞的增殖, 与抑制S 期细胞的DNA 复制及调控细胞的增殖周期相关。同时, 细胞凋亡的产生与实验药物呈正相关。HPLC 结果显示, 灵菌红素的药理学作用与细胞内药物浓度相关。 结论 灵菌红素能有效的进入细胞内, 抑制细胞的增殖, 其机理与诱导细胞凋亡和调控细胞的增殖周期相关。     

粘质沙雷氏菌产灵菌红素的碳源分析、菌种诱变及动力学

使用流式细胞仪研究了不同碳源对粘质沙雷氏菌ZSG新陈代谢的影响,发现不同碳源导致ZSG的DNA含量、细胞内部颗粒密度、表面粗糙度和细胞大小在发酵过程中呈现有差异的变化。对ZSG进行复合诱变筛得一株稳定突变菌株ZSG7,在250 ml摇瓶和5 L发酵罐中进行发酵,其灵菌红素（PG）产量比出发菌株分别提高了62.5%和269%。对ZSG7进行发酵培养基优化后PG产量比优化前提高了100%。对ZSG7发酵进行溶氧分段控制模式调控后PG产量比DO调控前提高了30.9%。对ZSG7发酵进行恒定pH调控后比pH调控前提高了35.9%。对ZSG7发酵进行补料组分优化后比补料前提高了47.6%。基于Logistic方程和Luedeking-Piret方程建立了恒定pH7分批发酵和补料分批发酵的菌体生长模型和PG合成模型。拟合模型参数后,模型可以合理地描述恒定pH7分批发酵和补料分批发酵的过程。     

Enhanced production of prodigiosin-like pigment from Serratia marcescens SMdeltaR by medium improvement and oil-supplementation strategies

Serratia marcescens SMdeltaR, an SpnR-defective isogenic mutant of S. marcescens SS-1, was used to produce a prodigiosin-like pigment (PLP). Luria-Bertani (LB) broth, frequently used for prodigiosin biosynthesis with S. marcescens strains, was modified by increasing the concentrations of tryptone and yeast extract while completely removing NaCl from the medium. The resulting modified LB (MLB) medium achieved an almost 3.0-fold increase in PLP yield (152 mg l(-1)) when compared with the original LB broth. The addition of vegetable oils (2-6% [v/v]) to the fermentation broth markedly enhanced PLP production. PLP yields of 525, 579, and 790 mg l(-1) were obtained when the MLB medium was supplemented with 4% soybean oil, 4% olive oil and 6% sunflower oil, respectively. PLP production was found to be positively correlated with extracellular surface emulsification activity, suggesting a link between the PLP production and the presence of biosurfactant. This work shows that the optimal medium for PLP yield was sunflower oil (6%)-supplemented MLB medium, which resulted in an approximately 14-fold higher PLP yield than that in LB broth. Mass spectrometry and NMR analysis indicated that the PLP product is a prodigiosin derivative, called undecylprodigiosin.     

粘质沙雷氏菌代谢产物灵菌红素的鉴定

从柠檬酸厂糖化车间酸性土壤中筛选得到一株产红色素的粘质沙雷氏菌（Serratia marcescens）ZSG,菌株发酵液经酸性甲醇萃取、浓缩、硅胶柱层析、薄层色谱和柱色谱等分离纯化后,得到灵菌红素纯品,并采用紫外可见吸收光谱、红外光谱、液质联用分析对其结构进行了表征。     

Ultrafiltration of Coagulation-Pretreated Serratia marcescens Fermentation Broth: Flux Characteristics and Prodigiosin Recovery

The dead-end ultrafiltration (UF) of coagulation-pretreated fermentation broth of Serratia marcescens SMΔR for prodigiosin recovery was studied. Experiments were performed using different types (regenerated cellulose, YM; polyethersulfone, PES) and molecular weight cut-offs (MWCOs, 1–10 kDa) of the membranes, feed concentrations of prodigiosin (300–1000 mg/L), applied pressures (68.9–206.8 kPa), and stirring speeds (200–400 rpm). With the same MWCO, the YM membrane had a higher retention of prodigiosin and a lower flux than the PES membrane. A two-fold concentration of prodigiosin was observed in the retentate using a 1-kDa YM membrane compared to the concentration in the permeate using a 10-kDa YM membrane. In addition, the extent of membrane fouling was quantitatively analyzed in terms of the modified fouling index. Flux decline in the present batch UF process was mainly due to cake layer formation and partly due to pore blocking. A two-stage UF process was proposed for this purpose, with 81% recovery yield and four-fold concentration.