Controllable preparation of chitosan oligosaccharides via a recombinant chitosanase from marine Streptomyces lydicus S1 and its potential application on preservation of pre-packaged tofu.

Chitosan oligosaccharides (COSs) are widely applied in many areas due to its various biological activities. Controllable preparation of COSs with desired degree of polymerization (DP) via suitable chitosanase is of great value. Herein, a novel glycoside hydrolase (GH) family 46 chitosanase (SlCsn46) from marine Streptomyces lydicus S1 was prepared, characterized and used to controllably produce COSs with different DP. The specific activity of purified recombinant SlCsn46 was 1,008.5 U/mg. The optimal temperature and pH of purified SlCsn46 were 50°C and 6.0, respectively. Metal ions Mn2+ could improve the stability of SlCsn46. Additionally, SlCsn46 can efficiently hydrolyze 2% and 4% colloidal chitosan to prepare COSs with DP 2-4, 2-5, and 2-6 by adjusting the amount of SlCsn46 added. Moreover, COSs with DP 2-4, 2-5, and 2-6 exhibited potential application value for prolonging the shelf-life of pre-packaged Tofu. The water-holding capacity (WHC), sensorial properties, total viable count (TVC), pH and total volatile base nitrogen (TVB-N) of pre-packed tofu incorporated with 4 mg/mL COSs with DP 2-4, 2-5, and 2-6 were better than those of the control during 15 days of storage at 10°C. Thus, the controllable hydrolysis strategy provides an effective method to prepare COSs with desired DP and its potential application on preservation of pre-packed tofu.

Antimicrobial Mechanism and Secondary Metabolite Profiles of Biocontrol Agent Streptomyces lydicus M01 Based on Ultra-High-Performance Liquid Chromatography Connected to a Quadrupole Time-of-Flight Mass Spectrometer Analysis and Genome Sequencing.

Streptomyces lydicus was used as biopesticide for crop protection in agriculture, however, the antimicrobial mechanism remains unclear and no systematic research on the secondary metabolites of S. lydicus has been reported. In this study, the extract of S. lydicus M01 culture was used to treat plant pathogen Alternaria alternata and morphological changes in the plasma membrane and cell wall of hyphae and conidia were observed. Fluorescence microscopy combined with different dyes showed that the accumulation of reactive oxygen species and cell death were also induced. To investigate the secondary metabolites in the culture filtrate, an online detection strategy of ultra-high-performance liquid chromatography connected to a quadrupole time-of-flight mass spectrometer (UPLC-Q-TOF-MS) was used for identification. The results revealed an excess of 120 metabolites, mainly consisted of fungicides, antibacterial agents, herbicides, insecticides, and plant growth regulators, such as IAA. Among which the five dominant components were oxadixyl, chloreturon, S-metolachlor, fentrazamide, and bucarpolate. On the other hand, the complete genome of S. lydicus M01 was sequenced and a number of key function gene clusters that contribute to the biosynthesis of active secondary metabolites were revealed. This is the first systematic characterization of S. lydicus secondary metabolites, and these results offer novel and valuable evidence for a comprehensive understanding of the biocontrol agent S. lydicus and its application in agriculture.

Review of the existing maximum residue levels for Streptomyces lydicus strain WYEC 108 according to Article 12 of Regulation (EC) No 396/2005.

According to Article 12 of Regulation (EC) No 396/2005, EFSA has reviewed the maximum residue levels (MRLs) currently established at European level for the pesticide active substance Streptomyces lydicus strain WYEC 108. Considering the information provided by Member States, neither EU uses nor import tolerances are currently authorised for Streptomyces lydicus strain WYEC 108 within the EU. Furthermore, no MRLs are established by the Codex Alimentarius Commission (codex maximum residue limits) for this active substance. Therefore, residues of Streptomyces lydicus strain WYEC 108 are not expected to occur in any plant or animal commodity and therefore a consumer risk assessment is not required. Nevertheless, the available information allowed EFSA to propose a marker residue definition noting that methods for identification and quantification of Streptomyces lydicus strain WYEC 108 are available; however, a method for enforcement fully validated at a specific limit of quantification (LOQ) was not provided. An inclusion of Streptomyces lydicus strain WYEC 108 in Annex IV of (EC) No 396/2005 is not recommended.

Streptomyces lydicus M01 Regulates Soil Microbial Community and Alleviates Foliar Disease Caused by Alternaria alternata on Cucumbers.

Due to the adverse effect on the environment caused by excessive use of chemical fertilizers, the development of sustainable agriculture attracts a growing demand of biological based fertilizers composed of living microorganisms. In this study, an Actinobacteria Streptomyces lydicus M01 was isolated from the rhizosphere soil of Pyrus calleryana. This strain effectively promoted the plant growth and suppressed a foliar disease caused by Alternaria alternata on cucumbers. S. lydicus M01 exhibited growth promoting characteristics such as phosphate solubilization, IAA secretion, siderophore and ACC deaminase production. Through Illumina sequencing of the 16S rRNA gene and ITS gene of the soil microbes, we found that the application of S. lydicus M01 altered the composition of the microbial community by promoting beneficial groups, including bacteria genera Pseudarthrobacter, Sphingomonas, Rhodanobacter, and Pseudomonas, fungi genera Fusicolla, Humicola, Solicoccozyma, and Paraphaeosphaeria. Most of these bacteria and eukaryotes exhibit positive effects on growth promotion, such as nutrient accumulation, auxin secretion, abiotic stress alleviation, biological control, or bioremediation. Furthermore, studies on the reactive oxygen species (ROS) level and antioxidants of cucumber leaves revealed that S. lydicus M01 treatment reduced the ROS accumulation and increased the activities of antioxidases related with ROS scavenging, which indicated an enhanced disease resistance of cucumbers under biotic stress. Thus, our results suggest that the application of S. lydicus M01 can systemically affect plant microbiome interactions and represent a promising sustainable solution to improve agricultural production instead of chemical fertilizers.

Identification of an antifungal metabolite produced by a potential biocontrol Actinomyces strain A01 / Identificação de um metabólito antifúngico produzido pela cepa Actinomyces A01

Actinomyces strain A01 was isolated from soil of a vegetable field in the suburb of Beijing, China. According to the morphological, cultural, physiological and biochemical characteristics, and 16S rDNA sequence analysis, strain A01 was identified as Streptomyces lydicus. In the antimicrobial spectrum test strain A01 presented a stable and strong inhibitory activity against several plant pathogenic fungi such as Fusarium oxysporum, Botrytis cinerea, Monilinia laxa, etc. However, no antibacterial activity was found. In pot experiments in greenhouse, the development of tomato gray mold was markedly suppressed by treatment with the fermentation broth of the strain A01, and the control efficacy was higher than those of Pyrimethanil and Polyoxin. A main antifungal compound (purity 99.503 percent) was obtained from the fermentation broth of strain A01 using column chromatography and HPLC. The chemical structural analysis with UV, IR, MS, and NMR confirmed that the compound produced by the strain A01 is natamycin, a polyene antibiotic produced by S. chattanovgensis, S. natalensis, and S. gilvosporeus, widely used as a natural biological preservative for food according to previous reports. The present study revealed a new producing strain of natamycin and its potential application as a biological control agent for fungal plant diseases.

A cepa Actinomyces A01 foi isolada do solo de um campo agrícola no subúrbio de Beijing, China. De acordo com as características morfológicas, culturais, fisiológicas e bioquímicas, e análise da sequência 16S rDNA , a cepa A01 foi identificada como Streptomyces lydicus. Nos testes de espectro antimicrobiano, a cepa A01 apresentou atividade inibitória intensa e estável contra vários fungos patogênicos para plantas, como Fusarium oxysporum, Botrytis cinerea, Monilia laxa, etc. Entretanto, não foi encontrada atividade antibacteriana. Em experimentos em estufas, o desenvolvimento do fungo cinza do tomate foi fortemente inibido pelo tratamento com o caldo de fermentação da cepa A01, com eficiência superior à do pyremethanil e polyoxin. Por cromatografia em coluna e HPLC, obteve-se um composto fúngico (pureza 99,503 por cento), cuja análise estrutural por UV, IR, MS e NMR revelou ser natamicina, um antibiótico polienico produzido por S. chattanovgensis, S. natalensis e S.gilvosporeus, empregado como conservador biológico natural em alimentos. O presente estudo relata a detecção de uma nova cepa produtora de natamicina e sua aplicação potencial como um agente de controle biológico de doenças fúngicas em plantas.

Novel process for the simultaneous extraction and degumming of banana fibers under solid-state cultivation

Various process parameters for the production of polygalacturonase by Streptomyces lydicus under solid-state fermentation were optimized. The optimum particle size of wheat bran for polygalacturonase production was in the range of 500-1000 µm. Initial moisture content of 70 percent was found to be the optimum for enzyme production. The most suitable inoculum size was 1.25 x 10(5) CFU/mL and the optimum incubation temperature was 30ºC. Addition of carbon sources resulted in 37 percent increase in enzyme yield (425 U/g), whereas no significant enhancement was obtained on nitrogen supplementation. Maximum enzyme yield was recorded at 72 h. When compared to the initial production medium (108.5 U/g), the enzyme yield was 3.9 fold after optimization. Solid-state fermentation was effectively employed to develop a novel process for the simultaneous extraction and degumming of banana fibers. Streptomyces lydicus was allowed to grow on wheat bran medium in which banana leaf sheath pieces were incorporated and the fiber bundles were separated after a two-step fermentative process.

Vários parâmetros de processo de produção de poligalacturonase por Streptomyces lydicus por fermentação em estado sólido foram otimizados. O tamanho ótimo de partícula de farelo de trigo para a produção de poligalacturonase esteve na faixa de 500 a 1000 mm. O teor inicial de umidade de 70 por cento foi o melhor para a produção da enzima. O inóculo inicial mais adequado foi de 1,25 x 10(5) UFC/mL e a temperatura ótima de incubação foi 30ºC. A adição de fontes de carbono resultou em aumento de 37 por cento no rendimento da enzima (425U/g), enquanto que a suplementação com nitrogênio não melhorou o rendimento. O rendimento máximo da enzima foi obtido em 72h. A otimização resultou em um aumento de 3,9 vezes na quantidade de enzima produzida inicialmente (108,5U/g). A fermentação em estado-sólido foi eficiente para o desenvolvimento de um novo processo de extração e simultânea degomagem. Streptomyces lydicus foi cultivado em meio de farelo de trigo acrescentado de fragmentos de folhas de banana, sendo os feixes de fibras separados após um processo de fermentação em dois passos.

Novel process for the simultaneous extraction and degumming of banana fibers under solid-state cultivation.

Various process parameters for the production of polygalacturonase by Streptomyces lydicus under solid-state fermentation were optimized. The optimum particle size of wheat bran for polygalacturonase production was in the range of 500-1000 µm. Initial moisture content of 70% was found to be the optimum for enzyme production. The most suitable inoculum size was 1.25 × 10(5) CFU/mL and the optimum incubation temperature was 30°C. Addition of carbon sources resulted in 37% increase in enzyme yield (425 U/g), whereas no significant enhancement was obtained on nitrogen supplementation. Maximum enzyme yield was recorded at 72 h. When compared to the initial production medium (108.5 U/g), the enzyme yield was 3.9 fold after optimization. Solid-state fermentation was effectively employed to develop a novel process for the simultaneous extraction and degumming of banana fibers. Streptomyces lydicus was allowed to grow on wheat bran medium in which banana leaf sheath pieces were incorporated and the fiber bundles were separated after a two-step fermentative process.

Identification of an antifungal metabolite produced by a potential biocontrol Actinomyces strain A01.

Actinomyces strain A01 was isolated from soil of a vegetable field in the suburb of Beijing, China. According to the morphological, cultural, physiological and biochemical characteristics, and 16S rDNA sequence analysis, strain A01 was identified as Streptomyces lydicus. In the antimicrobial spectrum test strain A01 presented a stable and strong inhibitory activity against several plant pathogenic fungi such as Fusarium oxysporum, Botrytis cinerea, Monilinia laxa, etc. However, no antibacterial activity was found. In pot experiments in greenhouse, the development of tomato gray mold was markedly suppressed by treatment with the fermentation broth of the strain A01, and the control efficacy was higher than those of Pyrimethanil and Polyoxin. A main antifungal compound (purity 99.503%) was obtained from the fermentation broth of strain A01 using column chromatography and HPLC. The chemical structural analysis with U V, IR, MS, and NMR confirmed that the compound produced by the strain A01 is natamycin, a polyene antibiotic produced by S. chattanovgensis, S. natalensis, and S. gilvosporeus, widely used as a natural biological preservative for food according to previous reports. The present study revealed a new producing strain of natamycin and its potential application as a biological control agent for fungal plant diseases.

Breeding of High Streptolydigin-Producing Strain by Complex Mutagenesis and Resistance Selection / 微生物学通报

A high Streptolydigin-producing strain Streptomyces lydicus AS 4.2501-L8 with genetic stability was obtained from the original strain S. lydicus AS 4.2501-P28 by mutagenesis with UV and acridine orange, followed by resistance selection. Its yield of Streptolydigin was 177.0 ?g/mL, 96.2% higher than the original strain.