[Advances of studies on culture and product functions of Euglena gracilis].

Euglena gracilis is a unicellular eukaryote between animal and plant cells, which is widely distributed in nature. E. gracilis has both plant and animal characteristics, and can grow photoautotrophically, heterotrophically and mixotrophically. E. gracilis also features on abundant and various cellular composition. Recently, extensive researches on unique cellular components of E. gracilis have revealed its application in the field of medicine, food, and feedstuff, in terms of improving immunity, fighting inflammation, and lowering uric acid levels. The application prospects of paramylon in biomedical area were also discovered. As food ingredients, food additives, feedstuffs and cosmetic ingredients, E. gracilis has been certified domestically and overseas. A series of products have been developed overseas, especially in Japan. However, the research and development of E. gracilis are still in its infancy in China, and there is huge space for development. At present, the research and potential application of cultivation and product functions of E. gracilis have been rarely reviewed. This review systematically examines both the domestic and abroad research of cultivation and production of E. gracilis, as well as the biological activity of E. gracilis powder and paramylon. The existing problems in the application, exploitation, and possible development direction of E. gracilis in the future are prospected. This review might be useful for establishing and optimizing large-scale and efficient heterotrophic technology, as well as developing related products of E. gracilis with specific functions.

Magnolol, a Neolignan-like Drug, Inhibits Porcine Epidemic Diarrhea Virus Replication in Cultured Cells.

Porcine epidemic diarrhea virus (PEDV) is a destructive pathogen that continues to adversely affect the swine industry worldwide due to a current lack of vaccines and drugs capable of effective disease control. In the present study, the neolignan-like drug, magnolol (MAG), was tested for its ability to inhibit a Vero-cell adapted PEDV strain DR13att. Our data revealed that MAG exhibited anti-PEDV activity in vitro, with IC50 and CC50 values of 28.21 µM and 57.28 µM, respectively. MAG was an efficient inhibitor of viral replication, and repression of viral proliferation was strongest when the host cells were exposed to MAG and the virus at the same time. Although our data indicate that MAG has the potential to be a useful PEDV control agent, in vivo testing of the drug, using animal hosts, is required.

Bis-Benzylisoquinoline Alkaloids Inhibit Porcine Epidemic Diarrhea Virus In Vitro and In Vivo.

Porcine epidemic diarrhea virus (PEDV) belongs to the genus Alphacoronavirus of the family Coronaviridae that causes severe diarrhea and high mortality in neonatal suckling piglets. Currently, there is no effective medication against this pathogen. Cepharanthine (CEP), tetrandrine (TET), and fangchinoline (FAN) are natural bis-benzylisoquinoline alkaloids with anti-inflammatory, antitumor, and antiviral properties. Here, we first found that CEP, TET, and FAN had anti-PEDV activity with IC50 values of 2.53, 3.50, and 6.69 µM, respectively. The compounds could block all the processes of viral cycles, but early application of the compounds before or during virus infection was advantageous over application at a late stage of virus replication. FAN performed inhibitory function more efficiently through interfering with the virus entry and attachment processes or through attenuating the virus directly. CEP had a more notable effect on virus entry. With the highest SI index of 11.8 among the three compounds, CEP was chosen to carry out animal experiments. CEP in a safe dosage of 11.1 mg/kg of body weight could reduce viral load and pathological change of piglet intestinal tracts caused by PEDV field strain challenge, indicating that CEP efficiently inhibited PEDV infection in vivo. All of these results demonstrated that the compounds of bis-benzylisoquinoline alkaloids could inhibit PEDV proliferation efficiently and had the potential of being developed for PED prevention and treatment.

l-Arginine and l-Lysine Alleviate Myosin from Oxidation: Their Role in Maintaining Myosin's Emulsifying Properties.

This study investigated the alleviative effects of l-arginine and l-lysine on the emulsifying properties and structural changes of myosin under hydroxyl radical (·OH) stress. The results showed that ·OH decreased the emulsifying activity index and emulsifying stability index but increased the creaming index and droplet size of a soybean oil-myosin emulsion (SOME). Confocal laser scanning microscopy demonstrated that ·OH caused larger and more inhomogeneous SOME droplets. l-Arginine and l-lysine effectively alleviated ·OH-induced destructive effects on the emulsifying properties of myosin. In addition, ·OH increased the extent of protein carbonylation and dityrosine formation, surface hydrophobicity, and ß-sheet content, but decreased the tryptophan fluorescence intensity, solubility, total sulfhydryl, and α-helix content of myosin. Although l-lysine increased dityrosine fluorescence intensity, l-arginine and l-lysine effectively alleviated the aforementioned structural changes of myosin. Therefore, l-arginine and l-lysine could mitigate ·OH-induced structural changes of myosin, which enabled myosin to maintain its emulsifying capacity under oxidative stress.

L-arginine and L-lysine degrade troponin-T, and L-arginine dissociates actomyosin: Their roles in improving the tenderness of chicken breast.

This work investigated the effects of L-arginine (Arg) and L-lysine (Lys) on the tenderness of chicken breast and explored the possible mechanisms underlying this effect for the first time. The results showed that both Arg and Lys decreased the shear force and increased the pH value, sarcomere length and myofibrillar fragmentation index as well as degraded the troponin-T by keeping calpain activity in chicken breast. In addition, Arg effectively reduced Ca2+/Mg2+-ATPase activities and promoted actomyosin dissociation. These results indicated that both Arg and Lys could enhance the tenderness of chicken breast, and it could also explain why Arg was more effective than Lys in improving the tenderness of chicken breast. These results will help facilitate the development of industrial-scale methods for improving the tenderness of meat products.

Reduction and coordination properties of l-Lysine/l-arginine/l-cysteine for the improvement of the color of cured sausage.

This study investigated how l-lysine/l-arginine/l-cysteine improved cured sausage color. Visible detection showed that Mb(Fe2+)NO did not form when MetMb(Fe3+) was treated with a combination of l-lysine and NaNO2, l-arginine and NaNO2, or l-cysteine and NaNO2. Visible spectra and electron paramagnetic resonance (EPR) detection together indicated formation of Mb(Fe2+)O2 when MetMb(Fe3+) was treated with l-lysine, l-arginine, or l-cysteine; Mb(Fe2+)NO was formed when MetMb(Fe3+) was treated with a combination of l-lysine and NO, l-arginine and NO, or l-cysteine and NO. Visible, EPR, and fourier transform infrared results suggested formation of a complex of Mb(Fe2+) and l-cysteine by the coordination of sulfydryl and ferrous porphyrin. Therefore, l-lysine, l-arginine, or l-cysteine can promote the formation of Mb(Fe2+)NO by reducing MetMb(Fe3+) to Mb(Fe2+)O2, and l-cysteine promotes formation of a complex of Mb(Fe2+) and l-cysteine via coordination of sulfydryl and ferrous porphyrin, which may improve cured sausage color.

Pleiotropic regulation of a glucose-specific PTS in Clostridium acetobutylicum for high-efficient butanol production from corn stover without detoxification.

BACKGROUND: Corn stover (CS) is evaluated as the most favorable candidate feedstock for butanol production via microbial acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum. By independent acid pretreatment and enzymatic hydrolysis, fermentable sugars (mainly glucose and xylose) were released, of which glucose was naturally utilized as the most preferred carbon source by C. acetobutylicum. However, the ABE fermentation using corn stover hydrolysate (CSH) without detoxification is typically limited to poor sugars utilization, butanol production and productivity. In the presence of pretreatment-derived inhibitors, the intracellular ATP and NADH, as important factors involved in cell growth, solventogenesis initiation and stress response, are exceedingly challenged owing to disrupted glucose phosphotransferase system (PTS). Therefore, there is a necessity to develop effective engineering approaches to overcome these limitations for high-efficient butanol production from CSH without detoxification. RESULTS: PTS-engineered C. acetobutylicum strains were constructed via overexpression and knockout of gene glcG encoding glucose-specific PTS IICBA, which pleiotropically regulated glucose utilization, cell growth, solventogenesis and inhibitors tolerance. The PTSGlcG-overexpressing strain exhibited high fermentation efficiency, wherein butanol production and productivity was 11.1 g/L and 0.31 g/L/h, compared to those of 11.0 g/L and 0.15 g/L/h with the PTSGlcG-deficient strain. During CSH culture without detoxification, the PTSGlcG-overexpressing strain exhibited desirable inhibitors tolerance and solventogenesis with butanol production of 10.0 g/L, increased by 300% and 400% compared to those of 2.5 and 2.0 g/L with the control and PTSGlcG-deficient strains, respectively. As a result of extra glucose and 10 g/L CaCO3 addition into CSH, butanol production and productivity were further maximized to 12.5 g/L and 0.39 g/L/h. These validated improvements on the PTSGlcG-overexpressing strain were ascribed to not only efficient glucose transport but also its cascading effects on intracellular ATP and NADH generation, solventogenesis initiation and inhibitors tolerance at the exponential growth phase. CONCLUSION: The PTSGluG regulation could be an effective engineering approach for high-efficient ABE fermentation from lignocellulosic hydrolysates without detoxification or wastewater generation, providing fundamental information for economically sustainable butanol production with high productivity.

The impact of Paenibacillus polymyxa HY96-2 luxS on biofilm formation and control of tomato bacterial wilt.

The focus of this study was to investigate the effects of luxS, a key regulatory gene of the autoinducer-2 (AI-2) quorum sensing (QS) system, on the biofilm formation and biocontrol efficacy against Ralstonia solanacearum by Paenibacillus polymyxa HY96-2. luxS mutants were constructed and assayed for biofilm formation of the wild-type (WT) strain and luxS mutants of P. polymyxa HY96-2 in vitro and in vivo. The results showed that luxS positively regulated the biofilm formation of HY96-2. Greenhouse experiments of tomato bacterial wilt found that from the early stage to late stage postinoculation, the biocontrol efficacy of the luxS deletion strain was the lowest with 50.70 ± 1.39% in the late stage. However, the luxS overexpression strain had the highest biocontrol efficacy with 75.66 ± 1.94% in the late stage. The complementation of luxS could restore the biocontrol efficacy of the luxS deletion strain with 69.84 ± 1.09% in the late stage, which was higher than that of the WT strain with 65.94 ± 2.73%. Therefore, we deduced that luxS could promote the biofilm formation of P. polymyxa HY96-2 and further promoted its biocontrol efficacy against R. solanacearum.

Dolyemycins A and B, two novel cyclopeptides isolated from Streptomyces griseus subsp. griseus HYS31.

Two novel cyclopeptides with special skeleton, namely, dolyemycins A (1) and B (2) were isolated from Streptomyces griseus subsp. griseus HYS31 by bio-guided isolation. Their structures were elucidated by detailed analysis of spectroscopic data. These two compounds were cyclopeptides containing eleven amino acids including five unusual amino acids (hydroxyglycine, 3-hydroxyleucine, 3-phenylserine, ß-hydroxy-O-methyltyrosine, 2,3-diaminobutyric acid) in both of them and an extra nonprotein amino acids (3-methylaspartic acid) in Dolyemycin B only. Dolyemycins A and B performed antiproliferative activity against human lung cancer A549 cells with IC50 values of 1.0 and 1.2 µM, respectively.

Complete Genome Sequence of Industrial Biocontrol Strain Paenibacillus polymyxa HY96-2 and Further Analysis of Its Biocontrol Mechanism.

Paenibacillus polymyxa (formerly known as Bacillus polymyxa) has been extensively studied for agricultural applications as a plant-growth-promoting rhizobacterium and is also an important biocontrol agent. Our team has developed the P. polymyxa strain HY96-2 from the tomato rhizosphere as the first microbial biopesticide based on P. polymyxa for controlling plant diseases around the world, leading to the commercialization of this microbial biopesticide in China. However, further research is essential for understanding its precise biocontrol mechanisms. In this paper, we report the complete genome sequence of HY96-2 and the results of a comparative genomic analysis between different P. polymyxa strains. The complete genome size of HY96-2 was found to be 5.75 Mb and 5207 coding sequences were predicted. HY96-2 was compared with seven other P. polymyxa strains for which complete genome sequences have been published, using phylogenetic tree, pan-genome, and nucleic acid co-linearity analysis. In addition, the genes and gene clusters involved in biofilm formation, antibiotic synthesis, and systemic resistance inducer production were compared between strain HY96-2 and two other strains, namely, SC2 and E681. The results revealed that all three of the P. polymyxa strains have the ability to control plant diseases via the mechanisms of colonization (biofilm formation), antagonism (antibiotic production), and induced resistance (systemic resistance inducer production). However, the variation of the corresponding genes or gene clusters between the three strains may lead to different antimicrobial spectra and biocontrol efficacies. Two possible pathways of biofilm formation in P. polymyxa were reported for the first time after searching the KEGG database. This study provides a scientific basis for the further optimization of the field applications and quality standards of industrial microbial biopesticides based on HY96-2. It may also serve as a reference for studying the differences in antimicrobial spectra and biocontrol capability between different biocontrol agents.

Samholides, Swinholide-Related Metabolites from a Marine Cyanobacterium cf. Phormidium sp.

Cancer cell cytotoxicity was used to guide the isolation of nine new swinholide-related compounds, named samholides A-I (1-9), from an American Samoan marine cyanobacterium cf. Phormidium sp. Their structures were determined by extensive analysis of 1D and 2D NMR spectroscopic data. The new compounds share an unusual 20-demethyl 44-membered lactone ring composed of two monomers, and they demonstrate structural diversity arising from geometric isomerization of double bonds, sugar units with unique glyceryl moieties and varied methylation patterns. All of the new samholides were potently active against the H-460 human lung cancer cell line with IC50 values ranging from 170 to 910 nM. The isolation of these new swinholide-related compounds from a marine cyanobacterium reinvigorates questions concerning the evolution and biosynthetic origin of these natural products.

[Bioassay-guided isolation of functional components from hot water extract of Chlorella pyrenoidosa].

The main functional ingredients of hot water extract of Chlorella pyrenoidosa (CPE) were investigated through a bioassay-guided fractionation based on free radical scavenging and macrophage proliferation effects. The main functional ingredients of CPE were polysaccharides (PS) that were isolated by high pressure extraction, Sevag method, ethanol precipitation and ultrafiltration separation. Crude polysaccharides were further separated and purified by ion exchange chromatography DEAE52 and size exclusion chromatography Sephadex G-100. The purified fractions were analyzed by gel permeation chromatography. Molecular weights of the purified fractions PS-1-4-2, PS-1-3-2 and PS-2-3-3 were 3.97×104, 2.28×104 and 4.1×10³ Da, respectively. Bioassay-guided fractionation results indicated that CPE could remove free radicals and promote Ana-1 cells proliferation, mainly due to its various components working together. The components of free radicals scavenging mainly concentrated in PS-1-3, PS-1-4, PS-2-3 and PS-2-4. The components of Ana-1 proliferation mainly concentrated in PS-1-3, PS-1-4 and PS-2-3. This study established the activity screening method of main functional component from CPE, and got three new functional ingredients. It can be used to guide the development of high value products, further promote the industrialization process of microalgae energy, and realize microalgae 'high value products, microalgae energy and microalgae carbon' integration of exemplary role.

Ghanamycins A and B, two novel γ-butyrolactones from marine-derived streptomyces ghanaensis TXC6-16.

Two novel γ-butyrolactones ghanamycins A (1) and B (2) were isolated from the fermentation broth of marine-derived Streptomyces ghanaensis TXC6-16. Their structures were elucidated by spectroscopic analysis. These two novel compounds exhibited antimicrobial activities against some phytopathogens. The minimum IC (MIC) of 2 against Pseudomonas syringae and Erwinia sp. were 50 µg ml-1.

[Pilot-scale purification of lipopeptide from marine-derived Bacillus marinus].

This research was aimed at establishing the pilot-scale purification technology of lipopeptide from marine-derived Bacillus marinus. We studied lipopeptide surfactivity interferences on scale-up unit technologies including acid precipitation, methanol extraction, solvent precipitation, salting out, extraction, silica gel column chromatography and HZ806 macroporous absorption resin column chromatography. Then, the unit technologies were combined in a certain order, to remove the impurities gradually, and to gain purified lipopeptide finally, with high recovery rate throughout the whole process. The novel pilot-scale purification technology could effectively isolate and purify lipopeptide with 87.51% to 100% purity in hectograms from 1 ton of Bacillus marinus B-9987 fermentation broth with more than 81.73% recovery rate. The first practical hectogram production of highly purified lipopeptide derived from Bacillus marinus was achieved. With this new purification method, using complex media became possible in fermentation process to reduce the fermentation cost and scale-up the purification for lipopeptide production. For practicability and economy, foaming problem resulting from massive water evaporation was avoided in this technology.

Genomic Foundation of Starch-to-Lipid Switch in Oleaginous Chlorella spp.

The ability to rapidly switch the intracellular energy storage form from starch to lipids is an advantageous trait for microalgae feedstock. To probe this mechanism, we sequenced the 56.8-Mbp genome of Chlorella pyrenoidosa FACHB-9, an industrial production strain for protein, starch, and lipids. The genome exhibits positive selection and gene family expansion in lipid and carbohydrate metabolism and genes related to cell cycle and stress response. Moreover, 10 lipid metabolism genes might be originated from bacteria via horizontal gene transfer. Transcriptomic dynamics tracked via messenger RNA sequencing over six time points during metabolic switch from starch-rich heterotrophy to lipid-rich photoautotrophy revealed that under heterotrophy, genes most strongly expressed were from the tricarboxylic acid cycle, respiratory chain, oxidative phosphorylation, gluconeogenesis, glyoxylate cycle, and amino acid metabolisms, whereas those most down-regulated were from fatty acid and oxidative pentose phosphate metabolism. The shift from heterotrophy into photoautotrophy highlights up-regulation of genes from carbon fixation, photosynthesis, fatty acid biosynthesis, the oxidative pentose phosphate pathway, and starch catabolism, which resulted in a marked redirection of metabolism, where the primary carbon source of glycine is no longer supplied to cell building blocks by the tricarboxylic acid cycle and gluconeogenesis, whereas carbon skeletons from photosynthesis and starch degradation may be directly channeled into fatty acid and protein biosynthesis. By establishing the first genetic transformation in industrial oleaginous C. pyrenoidosa, we further showed that overexpression of an NAD(H) kinase from Arabidopsis (Arabidopsis thaliana) increased cellular lipid content by 110.4%, yet without reducing growth rate. These findings provide a foundation for exploiting the metabolic switch in microalgae for improved photosynthetic production of food and fuels.

[Research status and prospect on hot water extract of Chlorella: the high value-added bioactive substance from Chlorella].

Chlorella is nutritious and has been used as a functional food much earlier than the other microalgae. C. pyrenoidosa, the potential microalgae which is currently cultured and developed for the new strategic industry of biofuels production and biological CO2 fixation, is a new resource food announced by the Ministry of Health of the People's Republic of China late 2012. Accumulation of high value-added substances in C. pyrenoidosa during the cultivation for lipid makes it possible to reduce the costs for C. pyrenoidosa-based biofuels production. Among these potential substances, hot water extract of Chlorella (CE), commercially known as "Chlorella growth factor", is the unique one that makes Chlorella more precious than the other algae, and the market price of CE is high. It is believed that CE is effective in growth promotion and immunoregulation. However, there is no systematic analysis on the research status of CE and its bioactivity. The present report summarized recent research progress of CE and its bioactivity. Generally, besides the main effect on immunoregulation and tumor inhibition, CE was efficient in improving metabolic syndrome, scavenging for free radicals, protecting against ultraviolet damage, chelating heavy metals, and protecting liver and bowel. Several major challenges in CE research as well as its prospects were also analysed in the present report.

A comparison on the preparation of hot water extracts from Chlorella pyrenoidosa (CPEs) and radical scavenging and macrophage activation effects of CPEs.

Development of hot water extracts of Chlorella pyrenoidosa (CPE) may help in the reduction of the cost involved in C. pyrenoidosa-based biofuel production. The current extraction conditions of CPE greatly vary and the CPE-related research are developing rather slowly. To find an effective preparation method, in the present study, we have prepared CPEs using high pressures, ultrasonication, and enzymolysis, and we have compared the yields, general ingredients, and bioactivities of these products. The yields of the high pressure and enzymolysis methods were more than 25%. CPE-a, which has been prepared under an extra 0.1 MPa pressure in an autoclave, was rich in protein, and it had the strongest absorbance at 260 and 280 nm, whereas CPE-c and CPE-d, which were prepared via the enzymolysis of cellulase and pectinase, had higher sugar content. The CPEs had no inhibitory influence on the formation of advanced glycation end products, and their metal chelating activities were poor. However, all the products had significant positive effects on in vitro free radical scavenging and macrophage growth promotion. Hydroxyl radicals were scavenged in a concentration-dependent manner by CPE-a, and the cultured macrophage Ana-1 proliferated to 162.98% of the control when CPE-a was administrated at 200 µg mL(-1). Furthermore, phagocytic activity and intracellular nitric oxide levels of Ana-1 were significantly enhanced with the administration of CPE-a. In brief, our results suggest that hot water extraction with high pressure is an effective method for preparing high value-added bio-products from C. pyrenoidosa, which have strong potential for use in free radical scavenging and macrophage activation.

New indolocarbazoles from a mutant strain of the marine-derived actinomycete Streptomyces fradiae 007M135.

Fradcarbazoles A-C (1-3), three new indolocarbazoles, were isolated from a mutant strain of the marine-derived actinomycete Streptomyces fradiae 007M135. Their structures were established by spectroscopic analysis, quantum chemical calculation, CD spectra, and chemical transformation. Fradcarbazole A (1) possessed a unique skeleton consisting of a staurosporine core, a thiazole ring, and an indole fragment. Compounds 1-3 displayed significant cytotoxicity against HL-60, K562, A-549, and BEL-7402 cell lines and inhibitory effects on the kinase PKC-α with IC(50) values of 0.001-4.58 µM.

Two new antifungal cyclic lipopeptides from Bacillus marinus B-9987.

Two new cyclic lipopeptides maribasins A (1) and B (2) were isolated from the fermentation broth of the marine microorganism Bacillus marinus B-9987 isolated from Suaeda salsa in Bohai coastline of P. R. China. Both structures were established to be cyclo (D-Pro-L-Gln-L-Asn-L-Ser-D-Asn¹-D-Tyr-D-Asn²-D-ß-aminoisopentadecanoic acid) (1) and cyclo (D-Pro-L-Gln-L-Asn-L-Ser-D-Asn¹-D-Tyr-D-Asn²-D-ß-aminoanteisopentadecanoic acid) (2) by spectroscopic analysis and exhibited broad-spectrum activity against phytopathogens by the antifungal bioassay.