Potential of selected lactic acid bacteria to produce food compatible antifungal metabolites

The aim of this study was to assess the potential of lactic acid bacteria to inhibit the outgrowth of some common food-spoiling fungi. Culture supernatants of 17 lactic acid bacterial strains as well as of three commercial probiotic cultures were evaluated for antifungal activity using an agar-diffusion method. The method parameters were chosen in order to reveal compounds for potential use in food (bio)preservation. Thirteen strains showed antifungal activity of which five strains were very promising: Lactobacillus acidophilus LMG 9433, L. amylovorus DSM 20532, L. brevis LMG 6906, L. coryniformis subsp. coryniformis LMG 9196 and L. plantarum LMG 6907. Four of these five strains were further examined; it was found that the produced antifungal metabolites were pH-dependent. The exact chemical nature of these substances has not been revealed yet.     

纳豆芽孢杆菌Bna05菌株产抗霉脂肽的鉴定

【目的】分析枯草芽孢杆菌纳豆菌亚种Bna05菌株代谢产物中脂肽类物质的存在情况,并探讨它们在抗霉功能中所发挥的作用。【方法】利用特异性引物对Bna05菌株进行脂肽合成酶类基因片段扩增、测序和BLAST比对分析;通过平板抑菌圈区域取样法获得Bna05菌株的高抗霉活性代谢产物,对该产物进行反相高效液相色谱(RP-HPLC)分离;用琼脂微稀释法测定分离物的抗霉活性,并对活性分离物进行质谱鉴定。【结果】Bna05菌株含有sfp和srf AA基因,未检测到itu C、itu D、fen D、fen ACE、bym B、bym C基因;RP-HPLC分离得到3组抗霉活性物质F_2、F_3和F_4,F_2中未检测到脂肽类物质,从F_3和F_4中分别鉴定出两类Surfactin同系物:V_7-surfactin和I/L_7-surfactin。两类Surfactin分别与F_2组合使用时,均表现出抗霉协同作用;此外,与Surfactin单独使用相比,两类Surfactin混合物与F_2组合后的协同抗霉活性得到进一步增强。【结论】Bna05菌株所产脂肽类物质主要是V_7-surfactin和I/L_7-surfactin,Surfactin与Bna05菌株所产其它活性物质之间存在抗霉协同作用,而V_7-surfactin和I/L_7-surfactin的同时存在,对于增强这种协同抗霉作用是有利的。     

铜绿假单胞菌生防菌株抑菌代谢产物及其生防应用研究进展

微生物次生代谢产物的研究对开发微生物源农药具有重要意义。近年来一系列根际来源的铜绿假单胞菌被分离和鉴定,因其产生抑菌次生代谢产物,具有很好的生物防治效果。本文将系统综述铜绿假单胞菌生防菌株的种类及其抑菌代谢产物的多样性,并进一步介绍铜绿假单胞菌生防菌株的抑菌代谢产物合成机制及其遗传改造,简要讨论铜绿假单胞菌生防菌株抑菌代谢产物在生物防治上的应用和前景。     

Production of antifungal metabolites by the ectomycorrhizal fungusPisolithus tinctorius strain SMF

Summary An ectomycorrhizal fungus,Pisolithus tinctorius strain SMF, isolated from a basidiocarp removed from the roots of a recently fallen old growth fir in the Smoky Mountains of Tennessee, was characterized for its in vitro production of antifungal metabolites. On solid mediumP. tinctorius SMF strongly inhibited growth of strains ofFusarium solani, Geotrichum candidum, Phanerochaete chrysosporium, andVerticillium dahliae, all species known to be plant pathogens. Evidence from paired colony growth inhibition studies on agar plates indicated that production of antifungal agents byP. tinctorius SMF may be enhanced by close physical contact with other fungi. The antifungal activity ofP. tinctorius SMF was much greater than that of several culture collection strains ofP. tinctorius. The culture collection strains either showed no or very limited activity. The antifungal activity was associated with an apparently inducible metabolism ofP. tinctorius SMF and with the production of darkly colored water soluble phenolic metabolites. Small scale fermentation studies showed that the phenolics are readily producible by submerged culture fermentation. This is the first report of submerged culture production of antifungal metabolites by an ectomycorrhizal fungus.     

In vitro liquefaction of human blood clots by metabolites of bacillus subtilis

Summary The influence exerted by some cultural conditions on the ability of metabolites of Bacillus subtilis to liquefy the human blood clots was studied. Crude enzyme preparations capable of liquefying the clots were isolated from the culture solutions. The aminoacids produced during the liquefaction process could be traced. Both of the enzyme preparation and the metabolism solution induced no obvious toxic effects when injected into veins of rabbits.     

One-step synthesis of carbohydrate esters as antibacterial and antifungal agents

Carbohydrate esters are biodegradable, and the degraded adducts are naturally occurring carbohydrates and fatty acids which are environmentally friendly and non-toxic to human. A simple one-step regioselective acylation of mono-carbohydrates has been developed that leads to the synthesis of a wide range of carbohydrate esters. Screening of these acylated carbohydrates revealed that several compounds were active against a panel of bacteria and fungi, including Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Candida albicans, Cryptococcus neoformans, Aspergillus flavus and Fusarium graminearum. Unlike prior studies on carbohydrate esters that focus only on antibacterial applications, our compounds are found to be active against both bacteria and fungi. Furthermore, the synthetic methodology is suitable to scale-up production for a variety of acylated carbohydrates. The identified lead compound, MAN014, can be used as an antimicrobial in applications such as food processing and preservation and for treatment of bacterial and fungal diseases in animals and plants.     

枯草芽孢杆菌fmb60菌株非核糖体肽类化合物对铜绿微囊藻生长的抑制作用

铜绿微囊藻是一种分布广泛的水华微藻,可对人类健康和生态环境造成严重危害,而枯草芽孢杆菌作为一种生防微生物可通过非核糖体肽合成酶合成多种生物活性物质.因此,枯草芽孢杆菌fmb60非核糖体肽(Non-ribosomal peptide,NRP)类产物对铜绿微囊藻生长抑制作用的研究在水华治理方面具有重要的意义.利用基因组挖掘...     

Optimization of cultural conditions for the production of antifungal chitinase by Streptomyces sporovirgulis

Actinomycetes were screened from soil in the centre of Poland on chitin medium. Amongst 30 isolated strains one with high activity of chitinase was selected. It was identified as Streptomyces sporovirgulis. Chitinase activity was detected from the second day of cultivation, then increased gradually and reached maximum after 4 days. The maximum chitinase production was observed at pH 8.0 and 25–30°C in the medium with sodium caseinate and asparagine as carbon and nitrogen sources and with shrimp shell waste as inducer of enzyme. Chitinase of S. sporovirgulis was purified from a culture medium by fractionation with ammonium sulphate as well as by chitin affinity chromatography. The molecular weight of the enzyme was 27 kDa. The optimum temperature and pH for the chitinase were 40°C and pH 8.0. The enzyme activity was characterised by high stability at the temperatures between 35 and 40°C after 240 min of preincubation. The activity of the enzyme was strongly inhibited in the presence of Pb²⁺, Hg²⁺ and stabilized by the ions Mg²⁺. Purified chitinase from S. sporovirgulis inhibited growth of fungal phytopathogen Alternaria alternata. Additionally, the crude chitinase inhibited the growth of potential phytopathogens such as Penicillium purpurogenum and Penillium sp.     

Design,synthesis and antifungal activity of novel fenfuram-diarylamine hybrids

Ten novel fenfuram-diarylamine hybrids were designed and synthesized. And their antifungal activities against four phytopathogenic fungi have been evaluated in vitro and most of the compounds demonstrated a significant antifungal activities against Rhizoctonia solani and Sclerotinia sclerotiorum. Compound 5e exhibited the most potent antifungal activity against R. solani with an EC₅₀ value of 0.037 mg/L, far superior to the commercially available fungicide boscalid (EC₅₀ = 1.71 mg/L) and lead fungicide fenfuram (EC₅₀ = 6.18 mg/L). Furthermore, scanning electron microscopy images showed that the mycelia on treated media grew abnormally with tenuous, wizened and overlapping colonies compared to the negative control. Molecular docking studies revealed that compound 5e featured a higher affinity for succinate dehydrogenase (SDH) than fenfuram. Furthermore, it was shown that the 3-chlorophenyl group in compound 5e formed a CH-π interaction with B/Trp-206 and a Cl-π interaction with D/Tyr-128, rendering compound 5e more active than fenfuram against SDH.     

The influence of dissolved oxygen tension on the synthesis of the antibiotic difficidin by bacillus subtilis

The antibiotic, difficidin, and its hydroxylated derivative oxydifficidin, were synthesized by cultures of Bacillus subtilis grown on a complex medium. Maximum titers of about 200 and 130 mg/L, respectively, were obtained. In fermentations where the dissolved oxygen tension (DOT) was controlled, the maximum specific growth rate was only reduced below 5% air saturation. DOT had little effect on the volumetric rateof synthesis of oxydifficidin but greatly influenced the rate for difficidin, which was reduced at DOT values below 40% air saturation. (c) 1994 John Wiley & Sons, Inc.     

Co-culture of Aspergillus sydowii and Bacillus subtilis induces the production of antibacterial metabolites

The co-culture strategy, which mimics natural ecology by constructing an artificial microbial community, is a useful tool to activate the biosynthetic gene clusters to generate new compounds. However, without optimization of fermentation conditions, the antagonism between the microbes often interferes with the production of secondary metabolites. In this study, the fermentation conditions of co-culture of Aspergillus sydowii and Bacillus subtilis were optimized by response surface methodology to increase the production of active metabolites against Staphylococcus aureus. After optimization, the inhibitory rate of the co-culture extract was 74.62%, which was 29.20% higher than that of the initial conditions. Meanwhile, a total of 15 newly biosynthesized metabolites were detected only in optimized co-culture, occupying 13.2% of all detected metabolites. The structures of the 12 metabolites with high variable importance in projection score were elucidated by the established LC-MS/MS approach integrated with various metabonomic tools. Among them, 7 metabolites were newly induced and the content of other 5 metabolites increased by 1.1–2.4 folds in optimized co-culture. The bioassay of metabolites in co-culture against S. aureus indicated that compounds (?)- (7S)- 10-hydroxysydonic acid, serine sydonate and macrolactin U’ contributed much to the increment of antibacterial activity. This study demonstrated that optimizing the fermentation conditions of co-culture was beneficial to changing the metabolite profile and effective to induce the biosynthesis of active metabolites.     

Design,synthesis and antifungal evaluation of borrelidin derivatives

Borrelidin, a nitrile containing 18-membered polyketide macrolide, display potent antifungal activity. In this study, a library of borrelidin derivatives were synthesized. Their structures were elucidated by detailed spectroscopic data analysis. The antifungal activity and cytotoxicity of these target compounds were evaluated by broth microdilution and 3-(4,5-dimethylthiazol-2-yl)-3,5-phenytetrazoliumromide (MTT) methods. Among forty-seven prepared analogues, compound 3b had the inhibitory effect on Candida albicans and Candida parapsilosis (MIC: 50 and 12.5?μg/mL, respectively). Furthermore, compounds 4n and 4r presented better antifungal activity against Aspergillus fumigatus with 12.5?μg/mL MIC value, which were insensitive to borrelidin. Preliminary structure-activity relationships (SAR) revealed that the ester analogues containing fragment -OCH₂CH₂N- had an important effect on the antifungal activity. Meanwhile, the molecular docking study indicated the carboxyl substituents in BN could provide extra interaction with pathogenic fungal threonyl-tRNA synthetase (ThrRS).     

Observations from the UK Supra-Regional Assay Service laboratory for the measurement of Vitamin D metabolites

As a Supra-Regional Assay Service (SAS) laboratory, we receive samples from all over the UK. Of these some are sent frozen and others by post or courier. We have examined transport and storage conditions to see whether they affect the measurement of Vitamin D metabolites and potentially contribute to the variation in measurement of 25-hydroxyvitamin D (25OHD) seen in the DEQAS scheme. We have also examined the samples received during 2005. We found that different transport and storage conditions did not contribute significantly to the normal variation seen in measuring Vitamin D metabolites (CV% (±S.E.) for stored versus assay controls: 5.1 ± 0.06% versus 4.5 ± 0.04% for 25OHD and 10.8 ± 1.0% versus 12.3 ± 1.0% for 1,25D). A review of the service showed a 240% increase in samples received over the last 5 years. Despite an increased awareness of the need to measure Vitamin D status, in this cross-section of patient samples 92% of Asian and 86% of white patients were found to be Vitamin D-insufficient (<30 ng/ml) and 27% of Asian and 14% of white patients were profoundly deficient (<5 ng/ml) and at risk of bone disease.     

Novel hybrids of fluconazole and furanones: design, synthesis and antifungal activity

During our efforts to develop new antifungal agents, a number of hybrid molecules containing furanones and fluconazole pharmacophores were designed and synthesized. The new chemical entities thus synthesized were tested for their potential as antifungal agents against various fungal strains and it was observed that the compounds with general structure 7 were potent inhibitors of Candida albicans ATCC 24433, Candida glabrata ATCC 90030, Candida tropicalis ATCC 750 and Candida neoformans ATCC 34664 while the fluconazole analogues 12 exhibited antifungal activity against Candida albicans ATCC 24433 and Candida glabrata ATCC 90030. The structure-activity relationship for these compounds is discussed. The synthetic strategies used in the present work have potential to prepare a large number of compounds for further refinement of structures to obtain molecules suitable for development as antifungal drugs.     

Major secondary metabolites produced by two commercial Trichoderma strains active against different phytopathogens

AIMS: Trichoderma harzianum strains T22 and T39 are two micro-organisms used as active agents in a variety of commercial biopesticides and biofertilizers and widely applied amongst field and greenhouse crops. The production, isolation, biological and chemical characterization of the main secondary metabolites produced by these strains are investigated. METHODS AND RESULTS: Of the three major compounds produced by strain T22, one is a new azaphilone that shows marked in vitro inhibition of Rhizoctonia solani, Pythium ultimum and Gaeumannomyces graminis var. tritici. In turn, filtrates from strain T39 were demonstrated to contain two compounds previously isolated from other T. harzianum strains and a new butenolide. The production of the isolated metabolites was also monitored by liquid chromatography/mass spectrometry during in vitro interaction with R. solani. CONCLUSIONS: This paper reports the isolation and characterization of the main secondary metabolites obtained from culture filtrates of two T. harzianum strains and their production during antagonistic interaction with the pathogen R. solani. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first work on secondary metabolites produced by the commercially applied strains T22 and T39. Our results provide a better understanding of the metabolism of these fungi, which are both widely used as biopesticides and/or biofertilizers in biocontrol.     

枯草芽孢杆菌抗菌蛋白X98Ⅲ的纯化与性质

枯草芽孢杆菌（Bacillussubtilis）BS－98是一株能强烈抑制苹果轮纹病菌（Physalosporapiricola）等植物病原真菌的拮抗菌株。BS－98菌株培养液经硫酸铵分级盐析、SephadexG－100柱层析和DEAE－纤维素（DE32）柱层析后分离纯化出一种抗菌蛋白,命名为X98Ⅲ。蛋白电泳分析结果表明,此蛋白分子量为59000,等电点为4．50．醋酸纤维膜电泳后经特异染色证明X98Ⅲ含糖及胀。用DNS法测其含糖量为6％。此蛋白对热稳定,对蛋白酶部分敏感。氨基酸组分分析表明,该蛋白含11种不同氨基酸,尤富含谷氨酸和半胱氨酸等,而缺少天冬氨酸等。纯化后的X98Ⅲ对苹果轮纹病菌、芦笋茎枯病菌等有很强的抑制作用。X98Ⅲ的抑菌机理主要是溶解细胞壁,造成菌丝畸形、孢子不发芽或发芽异常。     

Design,synthesis and antifungal activity evaluation of isocryptolepine derivatives

In this paper, the nitrogen atom was inserted into the anthracycline system of the isocryptolepine nucleus to obtain the “Aza”-type structure benzo[4,5]imidazo[1,2-c] quinazoline. A series of “Aza”-type derivatives were designed, synthesized and evaluated for their antifungal activity against six plant fungi in vitro. Among all derivatives, compounds A-0, B-1 and B-2 showed significant antifungal activity against B. cinerea with the EC₅₀ values of 2.72 μg/mL, 5.90 μg/mL and 4.00 μg/mL, respectively. Compound A-2 had the highest activity against M. oryzae with the EC₅₀ values of 8.81 μg/mL, and compound A-1 demonstrated the most control efficacy against R. solani (EC₅₀, 6.27 μg/mL). Moreover, compound A-0 was selected to investigate the in vivo tests against B. cinerea and the results indicated that the preventative efficacy of it up to 72.80% at 100 μg/mL. Preliminary mechanism studies revealed that after treatment with A-0 at 5 µg/mL, the B. cinerea mycelia appeared curved, collapsed and the cell membrane integrity may be damaged. The reactive oxygen species production, mitochondrial membrane potential and nuclear morphometry of mycelia have been changed, and the membrane function and cell proliferation of mycelia were destroyed. Compounds A-0, A-1, B-1 and B-2 presented weaker toxicities against two cells lines than isocryptolepine. This study lays the foundation for the future development of isocryptolepine derivatives as environmentally friendly and safe agricultural fungicides.     

Green synthesis and characterization of nanosilver derived from extracellular metabolites of potent Bacillus subtilis for antifungal and eco-friendly action against phytopathogen

BioMetals - The potent antagonist Bacillus isolated from the soil rhizosphere elucidated the highest antagonism against the phytopathogen Fusarium oxysporum f. sp. cumini and was identified as...