The inhibition mechanism of co-cultured probiotics on biofilm formation of Klebsiella pneumoniae.

AIMS: Klebsiella pneumoniae, an important opportunistic pathogen of nosocomial inflection, is known for its ability to form biofilm. The purpose of the current study is to assess how co- or mono-cultured probiotics affect K. pneumoniae's ability to produce biofilms and investigate the potential mechanisms by using a polyester nonwoven chemostat and a Caco-2 cell line. METHODS AND RESULTS: Compared with pure cultures of Lactobacillus rhamnosus and Lactobacillus sake, the formation of K. pneumoniae biofilm was remarkably inhibited by the mixture of L. rhamnosus, L. sake, and Bacillus subtilis at a ratio of 5:5:1 by means of qPCR and FISH assays. In addition, Lactobacillus in combination with B. subtilis could considerably reduce the adherence of K. pneumoniae to Caco-2 cells by using inhibition, competition, and displacement assays. According to the RT-PCR assay, the adsorption of K. pneumoniae to Caco-2 cells was effectively inhibited by the co-cultured probiotics, leading to significant reduction in the expression of proinflammatory cytokines induced by K. pneumoniae. Furthermore, the HPLC and RT-PCR analyses showed that the co-cultured probiotics were able to successfully prevent the expression of the biofilm-related genes of K. pneumoniae by secreting plenty of organic acids as well as the second signal molecule (c-di-GMP), resulting in inhibition on biofilm formation. CONCLUSION: Co-culture of L. sake, L. rhamnosus, and B. subtilis at a ratio of 5:5:1 could exert an antagonistic effect on the colonization of pathogenic K. pneumoniae by down-regulating the expression of biofilm-related genes. At the same time, the co-cultured probiotics could effectively inhibit the adhesion of K. pneumoniae to Caco-2 cells and block the expression of proinflammatory cytokines induced by K. pneumoniae.

Synergistic Effect of Flavonoids and Metformin on Protection of the Methylglyoxal-Induced Damage in PC-12 Neuroblastoma Cells: Structure-Activity Relationship and Potential Target.

Methylglyoxal-induced ROS elevation is the primary cause of neuronal damage. Metformin is a traditional hypoglycemic drug that has been reported to be beneficial to the nervous system. In this study, flavonoids were found to enhance the protective effect of metformin when added at a molar concentration of 0.5%. The structure-activity relationship (SAR) analysis indicated that ortho- substitution in the B ring, and the absence of double bonds between the 2 and 3 position combined with the gallate substitution with R configuration at the 3 position in the C ring played crucial roles in the synergistic effects, which could be beneficial for designing a combination of the compounds. Additionally, the mechanism study revealed that a typical flavonoid, EGCG, enhanced ROS scavenging and anti-apoptotic ability via the BCL2/Bax/Cyto C/Caspase-3 pathway, and synergistically inhibited the expression of GSK-3ß, BACE-1, and APP in PC-12 cells when used in combination with metformin. The dose of metformin used in the combination was only 1/4 of the conventional dose when used alone. These results suggested that ROS-mediated apoptosis and the pathways related to amyloid plaques (Aß) formation can be the targets for the synergistic neuroprotective effects of flavonoids and metformin.

Chemical Epigenetic Regulation Secondary Metabolites Derived from Aspergillus sydowii DL1045 with Inhibitory Activities for Protein Tyrosine Phosphatases.

Protein tyrosine phosphatases (PTPs) are ubiquitous in living organisms and are promising drug targets for cancer, diabetes/obesity, and autoimmune disorders. In this study, a histone deacetylase inhibitor called suberoylanilide hydroxamic acid (SAHA) was added to a culture of marine fungi (Aspergillus sydowii DL1045) to identify potential drug candidates related to PTP inhibition. Then, the profile of the induced metabolites was characterized using an integrated metabolomics strategy. In total, 46% of the total SMs were regulated secondary metabolites (SMs), among which 20 newly biosynthesized metabolites (10% of the total SMs) were identified only in chemical epigenetic regulation (CER) broth. One was identified as a novel compound, and fourteen compounds were identified from Aspergillus sydowii first. SAHA derivatives were also biotransformed by A. sydowii DL1045, and five of these derivatives were identified. Based on the bioassay, some of the newly synthesized metabolites exhibited inhibitory effects on PTPs. The novel compound sydowimide A (A11) inhibited Src homology region 2 domain-containing phosphatase-1 (SHP1), T-cell protein tyrosine phosphatase (TCPTP) and leukocyte common antigen (CD45), with IC50 values of 1.5, 2.4 and 18.83 µM, respectively. Diorcinol (A3) displayed the strongest inhibitory effect on SHP1, with an IC50 value of 0.96 µM. The structure-activity relationship analysis and docking studies of A3 analogs indicated that the substitution of the carboxyl group reduced the activity of A3. Research has demonstrated that CER positively impacts changes in the secondary metabolic patterns of A. sydowii DL1045. The compounds produced through this approach will provide valuable insights for the creation and advancement of novel drug candidates related to PTP inhibition.

Biosynthetic Profile in the Co-culture of Aspergillus sydowii and Bacillus subtilis to Produce Novel Benzoic Derivatives.

Microbial co-culture simulates the natural ecosystem through the combination of artificial microbes. This approach has been widely applied in the study of activating silent genes to reveal novel secondary metabolites. However, there are still challenges in determining the biosynthetic pathways. In this study, the effects of microbial co-culture on the morphology of the microbes were verified by the morphological observation. Subsequently, through the strategy combining substrate feeding, stable isotope labeling, and gene expression analysis, the biosynthetic pathways of five benzoic acid derivatives N1-N4 and N7 were demonstrated: the secondary metabolite 10-deoxygerfelin of A. sydowii acted as an inducer to induce B. subtilis to produce benzoic acid, which was further converted into 3-OH-benzoic acid by A. sydowii. Subsequently, A. sydowii used 3-OH-benzoic acid as the substrate to synthesize the new compound N2, and then N1, N3, N4, and N7 were biosynthesized upon the upregulation of hydrolase, hydroxylase, and acyltransferase during co-culture. The plate zone analysis suggested that the biosynthesis of the newly induced compounds N1-N4 was mainly attributed to A. sydowii, and both A. sydowii and B. subtilis were indispensable for the biosynthesis of N7. This study provides an important basis for a better understanding of the interactions among microorganisms, providing new ideas for studying the biosynthetic pathways of the newly induced secondary metabolites in co-culture.

Two-Step Enzymolysis of Antarctic Krill for Simultaneous Preparation of Value-Added Oil and Enzymolysate.

Antarctic krill is a crucial marine resource containing plenty of high-valued nutrients. However, krill oil as a single product has been developed by the current solvent extraction with high cost. From the perspective of comprehensive utilization of Antarctic krill, this study proposed a novel two-step enzymolysis-assisted extraction in attempt to produce value-added oil and enzymolysate simultaneously. After two-step chitinase/protease hydrolysis, the lipid yield increased from 2.09% to 4.18%, reaching 112% of Soxhlet extraction. The method greatly improved the yields of main components while reducing the impurity content without further refining. After optimization, the oil contained 246.05 mg/g of phospholipid, 80.96 mg/g of free eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and 0.82 mg/g of astaxanthin. The by-product enzymolysate was abundant in water-soluble proteins (34.35 mg/g), oligopeptides (13.92 mg/g), amino acids (34.24 mg/g), and carbohydrates (5.79 mg/g), which was a good source of functional nutrients. In addition, both oil and enzymolysate showed high antioxidant capacity. This novel method could simultaneously provide oil and enzymolysate amounting for 58.61% of dried krill.

Protective Effect of Flavonoids against Methylglyoxal-Induced Oxidative Stress in PC-12 Neuroblastoma Cells and Its Structure-Activity Relationships.

Methylglyoxal-induced oxidative stress and cytotoxicity are the main factors causing neuronal death-related, diabetically induced memory impairment. Antioxidant and anti-apoptotic therapy are potential intervention strategies. In this study, 25 flavonoids with different substructures were assayed for protecting PC-12 cells from methylglyoxal-induced damage. A structure-activity relationship (SAR) analysis indicated that the absence of the double bond at C-2 and C-3, substitutions of the gallate group at the 3 position, the pyrogallol group at the B-ring, and the R configuration of the 3 position enhanced the protection of flavan-3-ols, and a hydroxyl substitution at the 4' and meta-positions were important for the protection of flavonol. These SARs were further confirmed by molecular docking using the active site of the Keap1-Nrf2 complex as the receptor. The mechanistic study demonstrated that EGCG with the lowest EC50 protected the PC-12 cells from methylglyoxal-induced damage by reducing oxidative stress via the Nrf2/Keap1/HO-1 and Bcl-2/Bax signaling pathways. These results suggested that flavan-3-ols might be a potential dietary supplement for protection against diabetic encephalopathy.

Mulberry Leaf Extract Improves Metabolic Syndrome by Alleviating Lipid Accumulation In Vitro and In Vivo.

Metabolic syndrome (MS) is a metabolic disease with multiple complications. Mulberry leaf extract (MLE) is rich in flavonoids and has great potential in alleviating glucose and lipid metabolism disorders. This study evaluated the effect and mechanism of MLE on the alleviation of MS. The components of the MLE were analyzed, and then the regulation of lipid metabolism by MLE in vitro and in vivo was determined. In a hepatocyte model of oleic acid-induced lipid accumulation, it was found that MLE alleviated lipid accumulation and decreased the expression of genes involved in lipogenesis. Furthermore, MLE improved obesity, insulin resistance, plasma lipid profile, and liver function in MS mice after a 15-week intervention. MLE decreased the expression of SREBP1, ACC, and FAS through the AMPK signaling pathway to inhibit lipid synthesis and increase the level of CPT1A to promote lipid decomposition to achieve its hypolipidemic effect. Meanwhile, MLE was also shown to affect the composition of the gut microbiota and the production of short-chain fatty acids, which contributed to the alleviation of lipid accumulation. Our results suggest that MLE can improve MS by improving lipid metabolism through multiple mechanisms and can be developed into dietary supplements for the improvement of MS.

Metabolomic Strategy to Characterize the Profile of Secondary Metabolites in Aspergillus aculeatus DL1011 Regulated by Chemical Epigenetic Agents.

Chemical epigenetic regulation (CER) is an effective method to activate the silent pathway of fungal secondary metabolite synthesis. However, conventional methods for CER study are laborious and time-consuming. In the meantime, the overall profile of the secondary metabolites in the fungi treated by the CER reagent is not well characterized. In this study, suberohydroxamic acid (SBHA), a histone deacetylase inhibitor, was added to a culture of Aspergillus aculeatus DL1011 and a new strategy based on LC-MS/MS analysis integrated with various metabolomic tools (MetaboAnalyst, MS-DIAL, SIRIUS and GNPS) was developed to characterize the profile of induced metabolites. As a result, 13.6%, 29.5% and 27.2% of metabolites were identified as newly biosynthesized, increasing and decreasing in abundance by CER, respectively. The structures of the 18 newly induced secondary metabolites were further identified by the new strategy to demonstrate that 72.2% of them (1 novel compound and 12 known compounds) were first discovered in A. aculeatus upon SBHA treatment. The accuracy of the new approach was confirmed by purification and NMR data analysis of major newly biosynthesized secondary metabolites. The bioassay showed that the newly biosynthesized compounds, roseopurpurin analogues, showed selective activities against DPPH scavenging, cytotoxicity and SHP1 inhibition. Our research demonstrated that CER was beneficial for changing the secondary metabolic profile of fungi and was an effective means of increasing the diversity of active metabolites. Our work also supplied a metabolomic strategy to characterize the profile changes and determine the newly induced compounds in the secondary metabolites of fungi treated with the chemical epigenetic regulator.

Regulation of c-di-GMP in Biofilm Formation of Klebsiella pneumoniae in Response to Antibiotics and Probiotic Supernatant in a Chemostat System.

The resistance of bacteria to antibiotics is a major public health issue. Klebsiella pneumoniae is a type exemplification of multi-resistant enterobacteria. Its high biofilm forming capacity is a major factor in the recurrent infection of the intestinal tract. In this study, the intrinsic mechanism of secondary growth of K. pneumoniae in response to antibiotics and the inhibition effect of probiotic supernatant on biofilm formation after antibiotic treatment were investigated in a polyester nonwoven chemostat bioreactor. The experimental results showed that the c-di-GMP content in the cells increased after treatment with levofloxacin, leading to the formation of a thick biofilm due to an increase in the production of extracellular polymer substance (EPS) and type 3 fimbriae. Biofilm prevents the mass transfer of levofloxacin and protects K. pneumoniae cells from being killed by levofloxacin. Under suitable conditions, K. pneumoniae cells on the biofilm enter into the suspension for secondary growth. Moreover, the inhibition of probiotic supernatant on the biofilm formation was mainly due to the reduced expression of yfiN and mrkJ genes, and the decreased concentration of c-di-GMP in cells, as well as the less secretion of EPS. At the same time, the decrease in the concentration of c-di-GMP also reduced the expression of the mrkABCDF gene and prevented the synthesis of the type 3 fimbriae. The results would help to understand the mechanism of antibiotic resistance of pathogenic bacteria and to provide evidence to address this problem through the use of probiotics.

Role of c-di-GMP in improving stress resistance of alginate-chitosan microencapsulated Bacillus subtilis cells in simulated digestive fluids.

OBJECTIVES: Probiotics (Bacillus subtilis 04178) were entrapped in alginate-chitosan microcapsules by high-voltage electrostatic process. The encapsulation pattern was established as entrapped low density cells with culture (ELDCwc). The performance of ELDCwc cells was investigated against stress environments of simulated digestive fluids. RESULTS: After incubation in simulated gastric (pH 2.5) and intestinal fluids (4% bile salt) for 2 h, the survival rate of ELDCwc cells (18.19% and 27.54%) was significantly higher than that of the free cells (0.0000009% and 0.0005%). The reason why B. subtilis embedded in microcapsules can resist the stress environments was that the mass production of extracellular proteins and polysaccharides prompted B. subtilis to form cell aggregates. The production of extracellular proteins and polysaccharides were regulated by the concentration of c-di-GMP and the expression of ydaJKLMN operon, abbA, sinI, slrA, slrB, abrR and sinR. CONCLUSIONS: c-di-GMP is important for the production of extracellular polymer substance to enhance probiotic viability in stress environments.

Prophages contribute to genome plasticity of Klebsiella pneumoniae and may involve the chromosomal integration of ARGs in CG258.

Klebsiella pneumoniae is an important multidrug-resistant pathogen carrying prophages. Here we explore the contribution of prophages to bacterial evolution and fitness in silico. This study showed prophages contribute to remarkable genome plasticity of K. pneumoniae. The strains of CG258 possess several conserved prophages including the couple of P2-P4 prophages. CRISPR-Cas system has limited impact on the presence of prophages. The strong MLST-depended distribution of CRISPR-Cas and prophages and the high proportion of strains with self-targeting spacers may be the causes. Four core ARGs (blaSHV, fosA and oqxAB) were detected on almost all the chromosomes, but the acquired ARGs were only found in CG258 and CRISPR-positive strains. The factors influencing the chromosomal integration of ARGs in CG258 and CRISPR-positive strains may be different. In CG258, prophages may involve the chromosomal integration of ARGs. For CRISPR-positive strains, the immunity of CRISPR-Cas systems against invading ARG-bearing mobile genetic elements may accelerate the process.

An aptly industrialized bioprocess for lactic acid production from corn stover using thermotolerant microbial consortia.

Chemical pretreatment of lignocellulosic biomass is a critical step in the conversion of lignocellulose to biofuels and biochemical. The main drawback of this pretreatment process is the formation of inhibitors which exhibit combined toxicity to microorganisms and result to low product concentrations and yields. In this study, the selection of microbial consortia by enrichment on hydrolysate of H2SO4-pretreated corn stover (pre-CS) without detoxification has been investigated as an efficient way to develop new strategies for lignocellulose utilization. The analysis of cattle stomach-dervied microbial consortia domesticated to degrade hydrolysate of pre-CS to produce lactic acid (LA) at different temperatures was investigated. Bacterial 16S rRNA gene amplicon sequencing analyses indicated that the three microbial consortia were taxonomically distinct and Enterococcus became dominant at high temperature. The highest glucose consumption rate was observed at 45 °C, while the three microbial consortia showed similar consumption rates of xylose and arabinose. The selected microbial consortia DUT37, DUT45 and DUT47 showed preferable resistances to inhibitors in hydrolysate of pre-CS and abilities of xylose utilization. A batch simultaneous saccharification and fermentation (SSF) process was developed by microbial consortium DUT47 at 47 °C to produce LA from pre-CS under non-detoxified and non-sterile conditions. The LA concentration and yield were 43.73 g/L and 0.50 g/g-corn stover (CS), respectively. Microbial consortium DUT47 has been shown to be suitable for LA production from H2SO4-pretreated corn stover without detoxification due to its thermophilic growth characteristics, robust tolerance of inhibitors, and the simultaneous utilization of glucose and xylose.

High-efficient L-lactic acid production from inedible starchy biomass by one-step open fermentation using thermotolerant Lactobacillus rhamnosus DUT1908.

The purpose of this study was to establish a simplified operational process for lactic acid (LA) production from inedible starchy biomass by open fermentation using thermotolerant Lactobacillus rhamnosus DUT1908. One step simultaneous liquefaction, saccharification and fermentation (SLSF) was proposed to produce LA using aging paddy rice with hull (APRH) as feedstock. First, a robust microbial strain was obtained by adaptive laboratory evolution under high temperature stress. As a result, L. rhamnosus DUT1908 showed high thermotolerance up to 50 °C and high efficiency of substrate utilization. Then, the performance of this thermotolerant L-lactic acid producing strain was demonstrated. Finally, various fermentation strategies were compared for LA production from APRH, including simultaneous saccharification and fermentation (SSF) and SLSF. In one-step open SLSF process, 107.8 g/L lactic acid was obtained with a productivity of 3.4 g/(L.h) and a yield to theoretical glucose of 0.89 g/g. This is the highest yield and productivity of lactic acid reported on starchy residues, and provides an efficient route for the development of high value-added products.

Sequential fed-batch fermentation of 1,3-propanediol from glycerol by Clostridium butyricum DL07.

The demand for 1,3-propanediol (1,3-PDO) has increased sharply due to its role as a monomer for the synthesis of polytrimethylene terephthalate (PTT). Although Clostridium butyricum is considered to be one of the most promising bioproducers for 1,3-PDO, its low productivity hinders its application on industrial scale because of the longer time needed for anaerobic cultivation. In this study, an excellent C. butyricum (DL07) strain was obtained with high-level titer and productivity of 1,3-PDO, i.e., 104.8 g/L and 3.38 g/(L•h) vs. 94.2 g/L and 3.04 g/(L•h) using pure or crude glycerol as substrate in fed-batch fermentation, respectively. Furthermore, a novel sequential fed-batch fermentation was investigated, in which the next bioreactor was inoculated by C. butyricum DL07 cells growing at exponential phase in the prior bioreactor. It could run steadily for at least eight cycles. The average concentration of 1,3-PDO in eight cycles was 85 g/L with the average productivity of 3.1 g/(L•h). The sequential fed-batch fermentation could achieve semi-continuous production of 1,3-PDO with higher productivity than repeated fed-batch fermentation and would greatly contribute to the industrial production of 1,3-PDO by C. butyricum. KEY POINTS: • A novel C. butyricum strain was screened to produce 104.8 g/L 1,3-PDO from glycerol. • Corn steep liquor powder was used as a cheap nitrogen source for 1,3-PDO production. • A sequential fed-batch fermentation process was established for 1,3-PDO production. • An automatic glycerol feeding strategy was applied in the production of 1,3-PDO.

Simultaneous liquefaction, saccharification, and fermentation of L-lactic acid using aging paddy rice with hull by an isolated thermotolerant Enterococcus faecalis DUT1805.

Simultaneous liquefaction, saccharification, and fermentation (SLSF) has attracted much attention for the production of bio-based chemicals, including L-lactic acid, due to its high efficiency and low cost. In this study, a lactic acid-producing bacterium with high tolerance of temperature up to 55 °C was isolated and characterized as Enterococcus faecalis DUT1805. Various strategies of stepwise controlled temperature were proposed and investigated for glucose utilization. The results indicated that E. faecalis DUT 1805 exhibited an optimal temperature at 50 °C, which could achieve temperature compatibility of enzyme, saccharification, and fermentation, and decrease the possibility of contamination by the other microorganisms during the large-scale fermentation. To reduce the cost of raw material and operation for lactic acid production, aging paddy rice with hull (APRH) was used in L-lactic acid production by simultaneous liquefaction, saccharification, and fermentation (SLSF). An open SLSF operation at 50 °C and pH 6.5, and 17% (w/v) solid loading in 5 L bioreactors was demonstrated with the lactic acid titer, yield, and productivity of 73.75 g/L, 87% to initial starch, and 2.17 g/(L h), respectively.

Effect of atmospheric cold plasma treatment on antioxidant activities and reactive oxygen species production in postharvest blueberries during storage.

BACKGROUND: Blueberry is universally acknowledged as a kind of berry rich in antioxidants. Cold plasma, an emerging non-thermal treatment technology, has been proved to be able to maintain or improve the antioxidant level while inactivating the microorganisms on the surface of fruits and vegetables. Postharvest blueberries were treated with atmospheric cold plasma (ACP; 12 kV, 5 kHz) for 0 s (Control), 30 s (ACP-30), 60 s (ACP-60), and 90 s (ACP-90) in this study, and the effects of ACP on the antimicrobial properties, antioxidant activities, and reactive oxygen species (ROS) production were investigated during storage at 4 ± 1 °C for 40 days. RESULTS: Total aerobic bacteria and mold populations on ACP-treated blueberries decreased significantly in a time-dependent manner (P < 0.05), and decreased by 0.34-1.24 and 0.57-0.87 log10 CFU g-1 respectively on ACP-60-treated blueberries during storage. The decay rate of blueberries was decreased by 5.8-11.7% and the decrease of blueberry firmness was slowed down by ACP-60. But the total phenol, anthocyanin, and ascorbic acid contents increased, and superoxide dismutase, catalase, and peroxidase activities were enhanced in ACP-treated blueberries. The free radical scavenging activity and total antioxidant capacity (T-AOC) were enhanced. Hydrogen peroxide (H2 O2 ) and superoxide anion (O2 - ) production rates declined by 27.3% and 41.3% at day 40 of storage, respectively. CONCLUSION: It is suggested that ACP may be a promising non-thermal treatment technology for postharvest sterilization and preservation of blueberry under suitable conditions. © 2020 Society of Chemical Industry.

Efficient Genome Engineering of a Virulent Klebsiella Bacteriophage Using CRISPR-Cas9.

Klebsiella pneumoniae is one of the most common nosocomial opportunistic pathogens and usually exhibits multiple-drug resistance. Phage therapy, a potential therapeutic to replace or supplement antibiotics, has attracted much attention. However, very few Klebsiella phages have been well characterized because of the lack of efficient genome-editing tools. Here, Cas9 from Streptococcus pyogenes and a single guide RNA (sgRNA) were used to modify a virulent Klebsiella bacteriophage, phiKpS2. We first evaluated the distribution of sgRNA activity in phages and proved that it is largely inconsistent with the predicted activity from current models trained on eukaryotic cell data sets. A simple CRISPR-based phage genome-editing procedure was developed based on the discovery that homologous arms as short as 30 to 60 bp were sufficient to introduce point mutation, gene deletion, and swap. We also demonstrated that weak sgRNAs could be used for precise phage genome editing but failed to select random recombinants, possibly because inefficient cleavage can be tolerated through continuous repair by homologous recombination with the uncut genomes. Small frameshift deletion was proved to be an efficient way to evaluate the essentiality of phage genes. By using the abovementioned strategies, a putative promoter and nine genes of phiKpS2 were successfully deleted. Interestingly, the holin gene can be deleted with little effect on phiKpS2 infection, but the reason is not yet clear. This study established an efficient, time-saving, and cost-effective procedure for phage genome editing, which is expected to significantly promote the development of bacteriophage therapy.IMPORTANCE In the present study, we have addressed efficient, time-saving, and cost-effective CRISPR-based phage genome editing of Klebsiella phage, which has the potential to significantly expand our knowledge of phage-host interactions and to promote applications of phage therapy. The distribution of sgRNA activity was first evaluated in phages. Short homologous arms were proven to be enough to introduce point mutation, small frameshift deletion, gene deletion, and swap into phages, and weak sgRNAs were proven useful for precise phage genome editing but failed to select random recombinants, all of which makes the CRISPR-based phage genome-editing method easier to use.

Isolation and characterization of a bacteriophage and its potential to disrupt multi-drug resistant Pseudomonas aeruginosa biofilms.

A lytic Pseudomonas aeruginosa bacteriophage, vB_PaeM_LS1, was isolated and characterized herein. To examine the eligibility of bacteriophage vB_PaeM_LS1 as a therapeutic bacteriophage, we analysed its genome and compared it to similar bacteriophages. Genome of bacteriophage vB_PaeM_LS1 consisted of a linear, double-stranded DNA molecule 66,095 bp in length and with 55.7% G + C content. Neighbor-joining analysis of the large subunit terminase showed that bacteriophage vB_PaeM_LS1 had similarity to the Pbunavirus genus. The potential of the lytic bacteriophage to disrupt Pseudomonas aeruginosa biofilms was assessed by scanning electron microscopy and bacterial counts. This study revealed that the bacteriophage vB_PaeM_LS1 with its lytic effect showed a high potential impact on the inhibition of the growth of Pseudomonas aeruginosa biofilm formation.

High production of optically pure (3R)-acetoin by a newly isolated marine strain of Bacillus subtilis CGMCC 13141.

Acetoin is one of the bio-based platform chemicals and its optically pure isomers are important potential intermediates and precursors in the synthesis of novel optically active materials. (3R)-acetoin could be synthesized via enzymatic catalysis, whole-cell catalysis and fermentation. In this study a marine strain of Bacillus subtilis was isolated to produce optically pure (3R)-acetoin with glucose as carbon source. The effects of nutrients on the formation of (3R)-acetoin and conversion of glucose to (3R)-acetoin were evaluated by Plackett-Burman design, and the fermentation medium was optimized by central composite design. The impact of oxygen supply on the production of (3R)-acetoin was studied at different aeration rates. Under the optimal conditions, 83.7 g/L (3R)-acetoin with an optical purity of 99.4% was achieved by fed-batch fermentation, and the conversion of glucose to (3R)-acetoin was 91.5% of the theoretical value. The results indicate the industrial potential of this strain for (3R)-acetoin production via fermentation.

Preparative Purification of Total Flavonoids from Sophora tonkinensis Gagnep. by Macroporous Resin Column Chromatography and Comparative Analysis of Flavonoid Profiles by HPLC-PAD.

For the full development and utilization of Sophora tonkinensis Gagnep., this study was primarily intended to established a simple and efficient approach for the preparative purification of total flavonoids from S. tonkinensis by macroporous resin column chromatography (MRCC). The adsorption and desorption characteristics of the total flavonoids on ten macroporous resins were first studied, and AB-8 resin was chosen as the most suitable, and the adsorption data were best fitted to the pseudo-second-order kinetics model and Langmuir isotherm model. Furthermore, the technological parameters for the purification of the total flavonoids were optimized using column chromatography. After a sample one-step purification procedure, the content of the total flavonoids increased by about 4.76-fold from 12.14% to 57.82%, with a recovery yield of 84.93%. In addition, the comparative analysis of the flavonoid extracts before and after purification was performed by high-performance liquid chromatography coupled with photodiode-array detection (HPLC-PAD). The results showed that the contents of six major flavonoids in the purified product were all higher than before the purification. Therefore, the AB-8 MRCC established in this work was a promising method for the industrial-scale purification of the total flavonoids from S. tonkinensis.