Ways to optimize the technological process of oleandomycin reextraction]

Dependence of the oleandomycin distribution coefficient on pH of the acqueous phase and temperature in the system of butylacetate extract-water acidified with orthophosphoric acid was studied. With a purpose of intensification of the process of oleandomycin reextraction, decreasing the antibiotic inactivation and evaporation of the organic solvent it was proposed to perfom oleandomycin extraction at pH 4.0--5.0 accompanied by simultaneous decreasing of the temperature.     

Kinetics of thermal inactivation of oleandomycin in aqueous and native alkaline solutions

The process of oleandomycin inactivation in aqueous alkaline solutions with their heating was studied by using the microbiological method of the antibiotic content assay. The initial specific rate of inactivation of crystalline oleandomycin in buffer solutions and oleandomycin in the fermentation broth filtrate was evaluated. It was shown that the inactivation was retarded by the reaction products and the components of the fermentation broth filtrate. The production rate of oleandomycin anhydro derivatives amounting to 3-40 per cent of the total mass of the inactivation product was estimated by UV spectrophotometry.     

Cyclic adenosine-3',5-monophosphate in Streptomyces antibioticus and its possible role in regulating oleandomycin biosynthesis and the growth of the culture

When glucose is substituted for sucrose in the fermentation medium for Streptomyces antibioticus, the pH of the cultural broth becomes more acidic, the rate of protein synthesis in the mycelium rises, and the rate of oleandomycin synthesis decreases abruptly. The dynamics of cAMP (cyclic monophosphate) accumulation was studied in the process of biosynthesis by the culture in different media. Most of the synthesized cAMP (80-90%) was shown to be excreted into the medium. Glucose stimulates cAMP synthesis and excretion from the mycelium by a factor of 1.5-3. No distinct correlation was found between cAMP content in S. antibioticus cells and the level of oleandomycin biosynthesis. A correlation between changes in the concentration of exocellular cAMP and protein synthesis in the mycelium suggests that the excreted cAMP may be involved in regulating the growth of the culture producing the antibiotic.     

Oleandomycin and its natural and synthetic analogs. II. Structure and properties of oleandomycin B, a new compound of the oleandomycin group

The revealed regularities of mass spectroscopic disintegration of oleandomycin and its derivatives made it possible to determine analytic criteria for identification of compounds related by their structure to oleandomycin. Analysis of the extracts from oleandomycin fermentation broth filtrates on the basis of the selected group of diagnostic ions showed that along with the main antibiotic there formed during the biosynthesis oleandomycin B, a structurally close minor component. The structure of the substance was assigned and its physico-chemical and biological properties were studied.     

猪苓发酵液抑菌活性物质的性质研究

研究猪苓发酵液中抑菌活性成分的性质,以期将为猪苓的药用机理的研究提供部分理论依据。以大肠埃希菌、枯草芽胞杆菌、金黄色葡萄球菌、热带假丝酵母、酿酒酵母、小麦赤霉为靶标菌,研究发酵液的抑菌作用;用不同极性有机溶剂萃取活性物质,检测萃取液抑菌效果;并探索了不同温度和pH下对发酵液活性的影响;利用捷克八溶剂系统和紫外扫描对物质类型进行了初步确定。结果表明：猪苓发酵液对细菌有抑菌活性;活性物质对酸碱敏感且热不稳定,可被乙酸乙酯、三氯甲烷、正丁醇等有机溶剂萃取,随萃取极性增大萃取能力增强;紫外扫描其萃取浓缩液,表明在λ210nm处有一典型吸收峰,与酯肽类抗菌素的紫外图谱相似;捷克八溶剂系统纸层析结果显示抑菌物质为非水溶性Ⅱ型抗生素。猪芩发酵液中存在抗生素类物质,但稳定性差。在猪苓的以多糖为主要药用成分的应用过程中不发挥重要作用。     

Adsorption of the antibiotic, nisin, to Streptococcus lactis cells

Nizin is produced by Str. lactis, strain MSU. During biosynthesis it is excreted into the fermentation broth and gradually adsorbed on the organism cells. This was confirmed by experiments with an inactive variant of Str. lactis IIa. The cells of this culture adsorbed nizin from "active" fermentation broth. Adsorption of nizin depended on pH of the medium; at pH 2,3 the cells did not adsorbe the antibiotic and at pH 6.6 the amount of the antibiotic adsorbed by the cells was maximum.     

土传真菌病害拮抗菌的筛选及其生防效果研究

为了获得对主要粮食作物水稻、小麦和经济作物大豆常见土传病害具有防治效果的生防菌,本研究从土壤中筛选到一株对所选6种病原真菌均有较好拮抗效果的菌株。基于形态学、生理生化特性及16S rDNA序列分析进行菌种鉴定,利用菌丝生长速率法对其无菌滤液和挥发性气体的抑菌效果进行验证,同时研究了其无菌滤液的热稳定性、酸碱稳定性和对蛋白酶K的稳定性。结果表明:本研究筛选到一株贝莱斯芽孢杆菌Bacillus velezensis CX-2,其无菌滤液对6种病原真菌的抑菌率在71%~95%之间,对小麦纹枯病菌、小麦全蚀病菌的抑菌率分别高达90.13%、94.34%;挥发性气体对6种病原菌的抑菌率在45%~80%之间;无菌滤液中的抗菌活性物质具有较强的热稳定性和对蛋白酶K的稳定性;无菌滤液pH在5~9之间时具有稳定的抑菌活性。该菌株可作为作物土传真菌病害生防菌剂较为理想的功能菌株。     

南极沉积物来源抗菌细菌的筛选及抑菌物质的鉴定

【背景】科学研究表明,由于南极环境条件特殊,微生物资源丰富,有望筛选出功效显著的抗菌微生物。【目的】以黄瓜枯萎病致病菌木贼镰刀菌(Fusarium equiseti)为指示菌,从南极沉积物中分离筛选具有拮抗作用的细菌菌株并对其抑菌物质进行初步鉴定。【方法】应用平板对峙法和琼脂扩散法分别对样品和发酵液进行初筛和复筛,筛选出对F. equiseti抑菌效果最强的菌株,基于形态学、生理生化、分子生物学分析,对该菌株进行鉴定。之后,对目标菌株发酵上清中的抑菌物质进行抑菌谱研究,并对其抑菌成分进行温度和pH的稳定性检测,通过硫酸铵沉淀的方法初步鉴定发酵液中的抑菌物质。【结果】从南极沉积物样品中共分离纯化出62株细菌,有5株具有较好的抑菌效果,其中抑菌效果最强的菌株鉴定为枯草芽孢杆菌斯氏亚种(Bacillus subtilis subsp.spizizenii),命名为JYM35。抑菌谱检测结果显示,菌株JYM35对丝瓜枯萎病致病菌层生镰刀菌(Fusarium proliferatum)和辣椒枯萎病致病菌F. equiseti具有较强的抑菌效果,对长豆褐腐病致病菌笄霉属(Choanephora)有较明显的拮抗作用,对水产致病菌副溶血弧菌(Vibrio parahaemolyticus)和溶藻弧菌(V.alginolyticus)也有一定的拮抗作用。菌株JYM35发酵上清中所含的抑菌物质热稳定性强且耐碱不耐酸,硫酸铵沉淀后可初步确定其抑菌物质隶属蛋白类。【结论】菌株JYM35是一株产蛋白类活性物质的广谱型抑菌菌株,对枯萎病致病菌的拮抗作用最强。因此具有较好的开发利用价值。     

Oleandomycin content of mycelium and fermentation solution during cultivation of Streptomyces antibioticus

The time-course of the oleandomycin content in the mycelium and fermentation broth-filtrate was studied by the microbiological assay at different periods of cultivation of strains 471 and 961 in fermenters and flasks containing a rich soybean-corn medium. It was shown that centrifugation of the mycelium over the sucrose density gradient induced a 25-80 per cent decrease in its moist weight at the expense of removal of the admixture components of the rich medium. Addition of glucose (2 per cent) to the culture-grown in a lactose medium by the 72nd hour of fermentation had no effect on further increase of the cell biomass. However, it lowered the content of the mycelium-fixed and excreted antibiotic at all the subsequent fermentation periods. The content of oleandomycin in the untreated mycelium was only 0.36 per cent of its content in the fermentation broth filtrate. After centrifugation of the mycelium over the sucrose density gradient and its intensive washing with distilled water the content of the mycelium-fixed antibiotic decreased still more. The time-course of the content of the mycelium-fixed and excreted oleandomycin was characterized by the presence of two activity peaks; by the 80-110th and by the 140-170th hour of cultivation.     

海洋链霉菌GB-2发酵产物的抗细菌活性及性质研究

从连云港海域潮间带采集的样品中筛选得到一株产高活性抗细菌物质的链霉菌GB-2。该菌的发酵产物对蜡样芽孢杆菌AS1.1846、金黄色葡萄球菌ATCC25923及6株耐药性金黄色葡萄球菌等11株革兰氏阳性菌,大肠杆菌AS1.487、荧光假单胞菌AS1.1802等4株革兰氏阴性菌有显著拮抗作用。纸层析对抗细菌物质分析结果表明,菌株GB-2所产抗细菌物质是中性的水溶性物质,其产生与海水的存在有显著相关性。发酵液稳定性研究表明,该物质在121℃,pH1和pH12条件下抑菌活性均不变;紫外线照射也不影响其抑细菌活性。显示菌株GB-2产物在生防、食品及医药方面潜在的应用价值。     

贝莱斯芽胞杆菌HN-Q-8菌株发酵液稳定性测定及抑菌活性成分分析

【背景】贝莱斯芽胞杆菌(Bacillus velezensis) HN-Q-8菌株是本实验室前期获得的能有效拮抗立枯丝核菌(Rhizoctonia solani)的生防细菌。【目的】明确贝莱斯芽胞杆菌HN-Q-8菌株的抑菌活性物质。【方法】采用菌丝生长速率法检测其发酵液对5种马铃薯病原菌的拮抗能力以及稳定性,利用基质辅助激光解吸电离飞行时间质谱技术对HN-Q-8菌株活性物质进行鉴定。【结果】HN-Q-8菌株对立枯丝核菌(Rhizoctonia solani)、尖镰孢菌(Fusarium oxysporum)、茄链格孢(Alternaria solani)和致病疫霉(Phytophthora infestans)具有良好的抑菌活性,抑菌率可达50%-90%。发酵液分别经紫外照射35min、自然光照射10 h以及100°C高温处理后,相对抑菌率分别为74%、92%和98%;发酵液的抑菌活性不受胰蛋白酶和蛋白酶K的影响;但不耐强酸、强碱,其适宜pH为4.0-10.0,表明HN-Q-8菌株活性物质具有良好的稳定性。活性成分能使立枯丝核菌菌丝形态畸形扭曲,从而抑制立枯丝核菌的生长,经鉴定活性物质为丰原素(fengycin)和表面活性素(surfactin)。【结论】贝莱斯芽胞杆菌HN-Q-8菌株发酵液具有较好的稳定性和较强的抑菌活性,具有良好的开发价值和应用前景。     

内生黑曲霉JS-1对稻瘟病的拮抗作用研究

利用平板对峙法和牛津杯法,从疏花水柏枝、金银花、秋华柳的内生菌中,筛选出1株对稻瘟病菌具有很强抑制作用的菌株JS-1。经生理生化实验和18S rDNAITS序列分析,确定该菌株为黑曲霉(Aspergillus niger)。实验结果表明,JS-1发酵液作用稻瘟病菌后,稻瘟病菌的菌丝变细,分支减少,菌丝基质颜色变浅,作用72 h后干重显著降低。进一步实验表明,该菌产生的活性物质位于其发酵液的乙酸乙酯酯相部位,对稻瘟病病菌抑制率高达96.1%。大田实验数据（天然接种圃）显示,添加该物质后,丰两优4号（中感）和广陆矮4号（易感）叶瘟病情指数分别只有16.25%和32.48%,对稻瘟病的防治取得了很好的效果,说明该菌株具有开发成高效生物农药的巨大潜能。     

青海湖M1菌发酵液体外抗菌活性及稳定性的初步研究

从青海湖中分离得到一株霉菌M1,采用平板抑菌法进行体外抗菌作用以及pH值、温度因素对其抗菌活性的影响。结果表明：该菌株发酵液对大肠杆菌、枯草芽孢杆菌及金黄色葡萄球菌都有明显的抑菌效果。M1菌株发酵液的抑菌活性对热稳定性较差,在pH值3～7的条件下抑菌效果最佳,遗传稳定性也不高。     

杀线虫芽孢杆菌发酵条件优化及大孔树脂筛选

芽孢杆菌 SMrs28分离自草原毒草瑞香狼毒根际土壤,其代谢产物有明显的线虫触杀活性。为了确定该菌株的最优发酵条件及初步纯化活性成分的树脂类型,采用单因素试验对发酵条件进行优化,通过静态吸附试验对大孔树脂类型进行筛选。结果表明: SMrs28菌株的最优发酵条件为:以葡萄糖和酵母粉作为最佳碳源和氮源,发酵时间48 h,接种量10%,温度28 ℃,转速180 r·min^-1,150 mL的三角瓶装液量30 mL,初始pH 7.2。静态吸附试验表明,大孔吸附树脂D101对发酵液中活性成分的吸附和解吸效果显著优于XAD-4、HP20和AB-8,其解吸液的杀线虫活性明显提高。发酵条件的优化和大孔吸附树脂的筛选,明显提高了发酵液的杀线虫活性,为进一步分离纯化该菌株的活性成分奠定了基础,为微生物杀线虫剂的开发和应用提供了理论依据。     

土壤拮抗放线菌的分离筛选及其抑菌物质特性的研究

目的从土壤中筛选拮抗能力强且抑菌特性稳定的放线菌菌株。方法采用双层琼脂法筛选出4株拮抗放线菌菌株,然后采用杯碟法测这4株菌株发酵液提取物的抗菌谱、最小抑菌浓度、热稳定性和酸碱稳定性。结果 4株菌株发酵液提取物都能抑制金黄色葡萄球菌、大肠埃希菌、铜绿假单胞菌和白色念珠菌的生长。以金黄色葡萄球菌为指示菌测发酵液提取物的最小抑制浓度,6#和9#拮抗作用较强,发酵液提取物稀释0.125mg/ml仍有抑菌作用。6#菌株在100℃处理30min后仍有40%的抑菌活性。6#菌株发酵液提取物在碱性环境条件下比在酸性环境条件下稳定。结论 4株菌株中6#菌株发酵液提取物具有拮抗能力强、最小抑菌浓度低和在碱性条件下活性较稳定的特点。     

波赛链霉菌JMC 06001抑菌活性物质的特性研究

使用藤黄八叠球菌（Sarcina lutea）、枯草芽胞杆菌（Bacillus subtilis）、大肠埃希菌（Escherichia coli）、黑曲霉（Asperillus niger）、产气肠杆菌（Enterobacter aerogenes）、变形杆菌（Proteus vulgaris）、白色念珠菌（Candidaalbicans）、青霉（Penicilliumsp.）、铜绿假单胞菌（P.aeruginosa）9种指示菌,采用杯碟法检测抑菌活性。研究了波赛链霉菌JMC 06001菌株（Streptomyces peucetiusJMC 06001）发酵液和菌丝体中活性物质的抑菌谱,对由该菌株产生的抑菌物质的温度、pH值、抗紫外线方面的稳定性进行了考察。利用硅胶柱层析和薄层层析对抑菌物质的分离也做了初步的研究。结果表明,该菌株的发酵产物对部分革兰阳性菌、革兰阴性菌和部分真菌有较为明显的抑制作用。稳定性试验结果显示,该菌株产生抑菌活性物质的最适温度为40℃左右,最佳pH值在7.0左右,具有较好的热稳定性和较宽的pH作用范围,抗紫外线辐射能力较强。通过硅胶柱层析分离和薄层色谱检测,显示该菌次生代谢产物中的抑菌活性物质包含了多种成分。     

Rice straw fermentation using lactic acid bacteria

To efficiently utilize rice straw and lessen its disposal problem on the environment, a lactic acid bacteria community, SFC-2 was developed from natural fermentation products of rice straw by continuous enrichment with the MRS-S broth (MRS broth with sucrose), and used to accelerate the fermentation of air-dried straws. The SFC-2 could rapidly lower the pH of the broth and produce high levels of lactic acid. Using a combination of plate isolation, denaturing gradient gel electrophoresis (DGGE) and 16S rDNA sequencing, the microbial composition of the SFC-2 was classified into Lactobacillus, mainly comprised of L. fermentum, L. plantarum and L. paracacei. An evaluation of the fermentation effect of SFC-2 on rice straw showed that it lowered the pH and significantly (P<0.05) increased lactic acid concentration in the straw. Further analysis with DGGE indicated that L. plantarum, L. fermentum and L. paracasei were the dominant species during fermentation.     

生防菌Ⅲ-61抗真菌活性产物的摇瓶发酵

对生防链霉菌Ⅲ-61产生抗真菌活性物质的摇瓶发酵工艺进行了研究。利用正交试验设计优化了发酵培养基组分,其最适配方为黄豆粉1．5％,蛋白胨0．3％,蔗糖1．0％,淀粉1．3％,磷酸二氢钾0．02％,硫酸镁0．025％,氯化钠0．5％,配咸水溶液,调pH至7～7．4,加碳酸钙1％。通过单因素试验,筛选获得了最优培养条件组合：液体种龄24h,接种量5％～10％,500mL摇瓶培养基装量为80mL,摇床转速240r／min,培养温度31℃,发酵周期96～120h。此优化的发酵培养基与发酵条件的组合昕得菌株Ⅲ-61发酵液对主要靶标黄瓜灰霉病菌的抑菌圈直径达49．5mm,较优化前提高了45．59％。     

核桃黑斑病拮抗放线菌WMF106发酵条件优化和抑菌物质稳定性

[背景]暗蓝色链霉菌WMF106对核桃黑斑病病原菌野油菜黄单胞菌(Xanthomonas campestris pv.campestris)有较好的抑制作用,优化其发酵培养基与发酵条件将为核桃黑斑病生防菌剂的制备与应用提供参考.[目的]优化核桃黑斑病生防菌株WMF106的发酵条件,测定其抑菌物质稳定性,并通过田间防效测...     

Biosynthesis of Streptomycin

A method for the accumulation of the streptomycin precursor (L) in the culture broth of Streptomyces griseus was developed and the precursor was successfully isolated from the broth.

When the microorganism was cultured under shaking in the glucose-meat extract-peptone medium (0.5% glucose, 0.2% yeast extract, 0.2% meat extract, 0.4% peptone, 0.5% sodium chloride, 0.025% magnesium sulfate, pH 7.0), the accumulation of the precursor in the broth was induced by the addition of supplementary glucose (e.g., 2 g glucose per 100 ml broth) 24 hr after inoculation followed by further cultivation for 48 hr. Increased accumulation of L component was obtained merely by increasing glucose content in the culture medium (e.g., 5% glucose-containing medium in the above-indicated one) instead of glucose supplement on the way of fermentation. For the accumulation of a large amount of L component in a culture broth, it looked to be necessary for pH value of the broth to be maintained between 6 and 7 during fermentation.

L component was isolated from the culture broth by adsorption on Amberlite IRC-50 and elution with 2% NaCl solution. The L component was separated on this column from contaminated streptomycin which requires 5% NaCl solution to be eluted. The L component in the 2% NaCl eluate was adsorbed on active carbon at neutral or slightly alkaline pH and eluted with 95% methanol at acidic pH, Partially purified L component precipitated as hydrochloride by addition of acetone to the methanol extract which had been concentrated in vacuo.