Combined effect of Photorhabdus luminescens and Bacillus thuringiensis subsp. aizawai on Plutella xylostella.

In this study, we evaluated a new biopesticide containing different combinations of Photorhabdus luminescens (ATCC 29,999; Pl) and Bacillus thuringiensis subsp. aizawai (Bt) to leverage their insecticidal activity against Plutella xylostella. Mixtures containing proteins of various sizes were assayed to determine which combination of the two bacteria would yield the maximum insecticidal activity. A histopathologic slide revealed vacuole formations and rifts near the apical membrane (a symptom of Bt) and severe thinning of the intestinal wall (a symptom of Pl). When the two bacteria were cultured separately and then mixed, the insecticidal activity of the treatment reached 83.33% ± 8.82%. The insecticidal activity was elevated and significantly accelerated when Bt was mixed with both the Pl supernatant and the isolated protein with a molecular mass [Formula: see text] 100 kDa of Pl. These results highlight the potential of Pl as a potent bioinsecticide to economically and sustainably control Pl. xylostella and other lepidopteran pests. KEY POINTS: • Growth inhibition by Bacillus thuringiensis exerted a significant effect on insecticidal activity. • Large Photorhabdus luminescens proteins can accelerate the synergistic insecticidal effect on Plutella xylostella.

Insecticidal Activity of Photorhabdus luminescens 0805-P2R Against Plutella xylostella.

Photorhabdus luminescens is an entomopathogenic rod-shaped bacterium infected with insect nematodes of the Heterorhabditidae family. It kills insects through the secretion of high molecular weight toxin complexes. In this study, Plutella xylostella larvae were orally administered P. luminescens for bioassay. After incubation in Luria-Bertani (LB) medium for a sufficiently long period, the mortality rates of P. xylostella observed after diluting the fermentation broth 50 times and diluting the supernatant 5 times were 18.89% and 91.11%, respectively. Retentates measuring more than 70 kDa showed 88% mortality after ultrafiltration (UF) membrane treatment. Thus, the supernatant of P. luminescens had insecticidal activity, and the main insecticidal toxin complexes had a molecular weight exceeding 70 kDa. The L9 (34) Taguchi orthogonal experimental optimized medium mode-predicted insecticidal activity levels were 84% and 119% in the 50-fold diluted fermentation broth and 5-fold diluted supernatant, respectively. Moreover, the insecticidal activity was improved to 92.2% in the 100-fold diluted fermentation broth and to 97.8% in the 10-fold diluted supernatant in the experiments. All combinations tested showed clear indications of lethality, including swelling, vesicle formation, cytoplasm vacuolization, and brush border membrane lysis. Thus, these results promote the use of P. luminescens 0805-P2R as a potent biopesticide to effectively control P. xylostella.

Production, extraction and stabilization of lutein from microalga Chlorella sorokiniana MB-1.

The efficiencies of extraction and preservation of lutein from microalgae are critical for the success of its commercialization. In this study, lutein was produced by Chlorella sorokiniana MB-1 via semi-batch mixotrophic cultivation. The microalgal biomass with a lutein content of 5.21mg/g was pretreated by bead-beating and high pressure cell disruption methods, and the lutein content was harvested by a reduced pressure extraction method. The effect of pretreatment, pressure, solvent type, extraction time and temperature on lutein recovery was investigated. Using high pressure pretreatment followed by extraction with tetrahydrofuran (THF) as solvent resulted in high lutein recovery efficiencies of 87.0% (20min) and 99.5% (40min) at 850mbar and 25°C. In contrast, using ethanol as the solvent, 86.2% lutein recovery was achieved under 450mbar, 35°C and 40min extraction. The extracted lutein was stabilized in olive oil or sunflower oil with half-lives of 53.1 and 63.8days, respectively.

Stress alters the expression of aquaporins in cultured rat intestinal epithelial cells.

Aquaporins (AQPs) are widely-expressed small water channel proteins that provide the major route for water transport across plasma membranes in various cell types. Although the quantity of water transported in the intestinal tract is second only to that in the kidney, the precise role of AQPs in this organ remains largely uncertain. The present study reports the effects of hypertonic stress and ischemia/reperfusion injury on the expression of AQPs in intestinal epithelial cells. Cultured rat intestinal epithelial cells were incubated in 300 mM mannitol-containing, hypertonic culture medium or subjected to simulated ischemia/reperfusion treatment. The cell viability was evaluated by MTT assay, and the expression of AQPs was determined by semi-quantitative reverse transcription polymerase chain reaction and western blotting. Despite reduced viability, the cells exposed to hypertonic stress for 16 h demonstrated enhanced expression of AQP1 mRNA and protein. AQP9 and glycosylated AQP11 proteins were also markedly upregulated. Ischemia alone did not affect the cell viability, but subsequent reperfusion significantly reduced viability. The mRNA expression levels of all the tested AQPs were not altered by ischemia alone or by ischemia/reperfusion; however, AQP8 protein was markedly reduced by ischemic injury. In addition, treatment with ischemia alone eradicated the normally-expressed, non-glycosylated AQP11 protein whilst inducing pronounced expression of the glycosylated form. These observations may indicate that AQPs function in the intestinal epithelia in response to stress.

A Systematic Review of the Mysterious Caterpillar Fungus Ophiocordyceps sinensis in Dong-ChongXiaCao ( Dong Chóng Xià Cǎo) and Related Bioactive Ingredients.

The caterpillar fungus Ophiocordyceps sinensis (syn.Cordyceps sinensis), which was originally used in traditional Tibetan and Chinese medicine, is called either "yartsa gunbu" or "DongChongXiaCao ( Dong Chóng Xià Cǎo)" ("winter worm-summer grass"), respectively. The extremely high price of DongChongXiaCao, approximately USD $20,000 to 40,000 per kg, has led to it being regarded as "soft gold" in China. The multi-fungi hypothesis has been proposed for DongChongXiaCao; however, Hirsutella sinensis is the anamorph of O. sinensis. In Chinese, the meaning of "DongChongXiaCao" is different for O. sinensis, Cordyceps spp., and Cordyceps sp. Over 30 bioactivities, such as immunomodulatory, antitumor, anti-inflammatory, and antioxidant activities, have been reported for wild DongChongXiaCao and for the mycelia and culture supernatants of O. sinensis. These bioactivities derive from over 20 bioactive ingredients, mainly extracellular polysaccharides, intracellular polysaccharides, cordycepin, adenosine, mannitol, and sterols. Other bioactive components have been found as well, including two peptides (cordymin and myriocin), melanin, lovastatin, γ-aminobutyric acid, and cordysinins. Recently, the bioactivities of O. sinensis were described, and they include antiarteriosclerosis, antidepression, and antiosteoporosis activities, photoprotection, prevention and treatment of bowel injury, promotion of endurance capacity, and learning-memory improvement. H. sinensis has the ability to accelerate leukocyte recovery, stimulate lymphocyte proliferation, antidiabetes, and improve kidney injury. Starting January 1(st), 2013, regulation will dictate that one fungus can only have one name, which will end the system of using separate names for anamorphs. The anamorph name "H. sinensis" has changed by the International Code of Nomenclature for algae, fungi, and plants to O. sinensis.

Purification and properties of an insecticidal metalloprotease produced by Photorhabdus luminescens strain 0805-P5G, the entomopathogenic nematode symbiont.

A total of 13 Photorhabdus luminescens strains were screened for proteolytic activity. The P. luminescens strain 0805-P5G had the highest activity on both skim milk and gelatin plates. The protease was purified to electrophoretical homogeneity by using a two-step column chromatographic procedure. It had a molecular weight of 51.8 kDa, as determined by MALDI-TOF mass spectrometry. The optimum pH, temperature, as well as pH and thermal stabilities were 8, 60 °C, 5-10, and 14-60 °C, respectively. It was completely inhibited by EDTA and 1,10-phenanthroline. Bioassay of the purified protease against Galleria mellonella by injection showed high insecticidal activity. The protease also showed high oral toxicity to the diamondback moth (Plutella xylostella) of a Taiwan field-collected strain, but low toxicity to an American strain. To our knowledge, this is the first report to demonstrate that the purified protease of P. luminescens has direct toxicity to P. xylostella and biopesticide potentiality.

Cloning and characterization of ß-agarase AgaYT from Flammeovirga yaeyamensis strain YT.

A bacterium with potent agar-degrading capability was isolated from the surface of a red algae, Gracilaria tenuistipitata. Based on phenotypic characteristics, 16S rDNA gene sequence and a phylogenetic analysis, this bacterium was identified and named as Flammeovirga yaeyamensis strain YT. PCR using homology-based degenerate primers was employed to clone any agarase gene belonging to GH16 family encoded in F. yaeyamensis strain YT. The resolved 1512 nucleotides revealed that the cloned gene, namely AgaYT, encodes a protein of 503 amino acids comprising a signal peptide, a glycosyl hydrolase catalytic module and a C-terminal domain with an unknown function. The recombinant protein r-AgaYT is an endo-type ß-agarase hydrolyzing agarose to yield neoagarobiose and neoagarotetraose as the main hydrolytic products. The specific activity of r-AgaYT was determined about 178.6 U mg(-1) at 40°C and pH 8.0.

orf4 of the Bacillus cereus sigB gene cluster encodes a general stress-inducible Dps-like bacterioferritin.

The function of orf4 in the sigB cluster in Bacillus cereus ATCC 14579 remains to be explored. Amino-acid sequence analysis has revealed that Orf4 is homologous with bacterioferritins and Dps. In this study, we generated an orf4-null mutant and produced recombinant protein rOrf4 to establish the role of orf4. In vitro, the purified rOrf4 was found to exist in two distinct forms, a dimeric form and a polymer form, through size exclusion analysis. The latter form exhibited a unique filament structure, in contrast to the typical spherical tetracosamer structure of bacterioferritins; the former can be induced to form rOrf4 polymers immediately after the addition of FeCl(2). Catalysis of the oxidation of ferrous irons by ferroxidase activity was detected with rOrf4, and the mineralized irons were subsequently sequestered only in the rOrf4 polymer. Moreover, rOrf4 exerted DNA-protective activity against oxidative damage via DNA binding in a nonspecific manner, as is seen with Dps. In vivo, deletion of orf4 had no effect on activation of the alternative sigma factor sigma(B), and therefore, orf4 is not associated with sigma(B) regulation; however, orf4 can be significantly upregulated upon environmental stress but not H(2)O(2) treatment. B. cereus strains with constitutive Orf4 expression exhibited a viability higher than that of the orf4-null mutant, under specific oxidative stress or heat shock. Taken together, these results suggest that Orf4 functions as a Dps-like bacterioferritin in response to environmental stress and can provide cell protection from oxidative damage through iron sequestration and DNA binding.

Study of mycelial growth and bioactive polysaccharide production in batch and fed-batch culture of Grifola frondosa.

The fermentation of Grifola frondosa was investigated in the shake flasks and a 5-L jar fermenter in batch and fed-batch modes. In the shake-flask experiments, the preferable mycelial growth and exopolysaccharide (EPS) production was observed at relatively low pH; maltose and glucose were preferred carbon sources for high mycelial production. The EPS was doubled after 13 d of cultivation when glucose was increased from 2% to 4%. Yeast extract (YE) (0.4%) in combination with corn steep powder (CSP) (0.6%) and YE (0.8%) in combination with CSP (1.2%) were preferred nitrogen sources for high mycelial production and EPS production, respectively. All plant oils tested significantly stimulate cell growth of G. frondosa but they failed to enhance EPS production. The EPS products usually consisted of two fractions of different molecular sizes varied by the plant oils used. The fed-batch fermentation by glucose feeding was performed when the glucose concentration in the medium was lower than 0.5% (5g/L), which greatly enhanced the accumulation of mycelial biomass and EPS; the mycelial biomass and EPS were 3.97g/L and 1.04g/L before glucose feeding, which reached 8.23g/L and 3.88g/L at 13 d of cultivation. In contrast, the mycelial biomass and EPS in the batch fermentation were 6.7g/L and 3.3g/L at 13 d of cultivation.

selective production and characterization of levan by Bacillus subtilis (Natto) Takahashi.

To meet the industrial need of an efficient microbial method for increased levan production, Bacillus subtilis (natto) Takahashi, a commercial natto starter for preparing fermented soybeans (natto), was used to produce levan. After cultivation for 21 h, 40-50 mg of levan mL(-1) was produced in medium containing 20% (w/w) sucrose, which was approximately 50% yield on available fructose. The product consisted of two fractions with different molecular masses (1794 and 11 kDa), which were easily separated by fractionation using an ethanol gradient. The products were well characterized by GPC, 13C NMR, and 1H NMR. The various sugars and concentrations, initial pH, fermentation temperature, and agitation speed affected the levan production by B. subtilis (natto) Takahashi. Takahashi strain is the most efficient levan-producing strain among all of the B. subtilis strains tested and, as previously reported, it produced the highest yield of levan in the least time (21 h) under the common cultivation condition.

Medium optimization for polysaccharide production of Cordyceps sinensis.

As a potential anticarcinogenic agent, polysaccharides from Cordyceps sinensis have been demonstrated to possess strong antioxidation activity. The aim of the present research was to study the optimal medium to produce polysaccharides of C. sinensis by using response surface methodology (RSM). The composition of optimized medium for polysaccharide production calculated from the regression model of RSM was 6.17% sucrose, 0.53% corn steep powder, 0.5% (NH4)2HPO4, and 0.15% KH2PO4 at pH 4.44, with a predicted maximum polysaccharide production of 3.17 g/L. When applying this optimal medium, the maximum polysaccharide production was 3.05 and 3.21 g/L in a shake flask and a 5-L jar fermentor, respectively. When the pH was controlled at a higher level such as pH 5.0, both cell growth and polysaccharide production were inhibited. A low pH of 2.85 was required for maximum production of polysaccharides.

Reusing soy residue for the solid-state fermentation of Ganoderma lucidum.

Lingzhi has been a popular oriental medicine used to treat various human diseases. Soy residue from the waste of tofu manufacturing was used to culture Ganoderma lucidum in solid-state fermentation. The solid-state fermentation was conducted in three types of containers: test tube, 500-ml flask, and sterilize-able polypropylene plastic bag. The highest rate of mycelial growth of 6 mm/day was observed in the medium of carbon/nitrogen (C/N) ratio of 80 using test tubes. However, a growth rate of 7.5 mm/day was found at the C/N ratio of 70-80 in the 500-ml flasks. In the tests using plastic bags, the fruiting bodies were fully developed only for the C/N ratios of 70 and 80. The components of fruiting bodies obtained from different media were also analyzed and compared. The contents of ash, polysaccharides, and crude protein of fruiting bodies were found higher in the media of C/N ratio of 80.