Supercooling Pretreatment Improves the Shelf-Life of Freeze-Dried Leuconostoc mesenteroides WiKim32.

Storage stability of freeze-dried lactic acid bacteria is a critical factor for their cost-effectiveness. Long-term storage of lactic acid bacteria enables microbial industry to reduce distribution costs. Herein, we investigated the effect of cold adaptation under supercooling conditions at -5°C on the viability of Leuconostoc mesenteroides WiKim32 during the freeze-drying process and subsequent storage. Cold adaptation increased the thickness of exopolysaccharides (EPS) and improved the viability of freeze-dried Leu. mesenteroides WiKim32. Compared to non-adapted cells, cold-adapted cells showed a 35.4% increase in EPS thickness under supercooling conditions. The viability of EPS-hydrolyzed cells was lower than that of untreated cells, implying that EPS plays a role in protection during the freeze-drying process. Cold adaptation increased the storage stability of freeze-dried Leu. mesenteroides WiKim32. Fifty-six days after storage, the highest viability (71.3%) was achieved with cold adaptation at -5°C. When EPS-containing broth was added prior to the freeze-drying process, the viability further increased to 82.7%. These results imply that cold adaptation by supercooling pretreatment would be a good strategy for the long-term storage of Leu. mesenteroides WiKim32.

Production, Characterization, and Antioxidant Activities of an Exopolysaccharide Extracted from Spent Media Wastewater after Leuconostoc mesenteroides WiKim32 Fermentation.

Bacterial exopolysaccharides (EPSs) are important alternatives to plant polysaccharides in fermented products and exhibit antioxidant activity, which is particularly desirable for functional foods. This study evaluated the use of spent media wastewater (SMW) derived from kimchi fermentation for the production of an EPS and analyzed the characterization and antioxidant activity of the resulting EPS. The EPS concentration and conversion yields of sequential purification were 7.7-9.0 g/L and 38.6-45.1%, respectively. Fourier transform infrared spectra and NMR spectra indicated that the EPS was a linear glucan with α-(1 → 6) linkages. The EPS also exhibited thermal tolerance to high temperatures. In vitro antioxidant activity analyses indicated the scavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, thiobarbituric acid reactance (TBAR), and ferric ion reducing antioxidant power (FRAP) values of 71.6-79.1, 28.2-33.0%, and 0.04-0.05 mM FeCl3, respectively. These results reveal that the EPS extracted from SMW has potential as a thermally tolerant, nontoxic, and natural antioxidant for industrial applications.

Mechanisms of Insecticidal Action of Metarhizium anisopliae on Adult Japanese Pine Sawyer Beetles (Monochamus alternatus).

Pine wilt disease, caused by Bursaphelenchus xylophilus (pine wood nematode), leads to severe environmental and economic damage. Here, we report the results of experiments on the biological control of pine wilt disease through termination of the insect vector of the nematode and the mechanism of the insecticidal action of Metarhizium anisopliae JEF-279 against Monochamus alternatus (Japanese pine sawyer). A combined treatment with a fungal conidia suspension and a fungal protease-containing culture filtrate caused 75.8% mortality of the insect vector. Additionally, the presence of destruxins was confirmed in the dead Japanese pine sawyer adults, and half of the 10 protein spots in proteomic analysis were identified as an actin related to muscle contraction. Based on proteomic and microscopic analyses, the infection cycle of the Japanese pine sawyer by M. anisopliae JEF-279 was inferred to proceed in the following sequence: (1) host adhesion and germination, (2) epicuticle degradation, (3) growth as blastospore, (4) killing by various fungal toxins (insecticidal metabolites), (5) immune response as defense mechanism, and (6) hyphal extrusion and conidiation. Consequently, the combined fungal conidia suspension and protease-containing culture filtrate treatment may be applied as an insecticidal agent, and flaccid paralysis is likely a major mechanism underlying the insecticidal action of M. anisopliae JEF-279 on host insects.

Coffee residue as a valorization bio-agent for shelf-life extension of lactic acid bacteria under cryopreservation.

The present work describes the feasibility of coffee residue extracts as cryoprotective agents in the storage stability of freeze-dried lactic acid bacteria. Coffee residue extracts were extracted from coffee residue, produced after coffee extraction for coffee powder and instant coffee preparation, using an autoclave. Leuconostoc mesenteroides WiKim32 was selected to evaluate the ability of coffee residue extracts to protect bacteria during freeze-dried storage. The storage stability of freeze-dried Leu. mesenteroides WiKim32 with coffee residue extracts was comparable to those with commercial cryoprotective agents. Coffee residue extracts contributed to storage stability immediately after freeze-drying (61.2%) and subsequent storage (48.7%). Our data indicate that the protective effect of the coffee residue extracts is associated with ions, carbohydrates, and phenolic compounds. Coffee residue extracts are feasible materials, which can reduce the storage and distribution costs compared to commercial agents currently available.

Advanced strategy to produce insecticidal destruxins from lignocellulosic biomass Miscanthus.

BACKGROUND: Biorefineries are widely recognized as the most feasible solution to the problem of achieving environmental sustainability along with economic growth. Furthermore, pine wilt disease has caused severe environmental and economic damage worldwide to date. Herein, a highly efficient, advanced process for producing destruxins (DTXs) from Miscanthus (MCT) is reported, along with an application strategy. RESULTS: The acetic acid-sodium chlorite pretreatment of MCT (AASC-MCT) is found to improve the monosaccharide production. Through biocatalytic conversion processes (simultaneous saccharification and cultivation), Metarhizium anisopliae JEF-279 can efficiently produce DTXs from 1% (w/v) AASC-MCT, i.e., DTX E (334.8 mg/L), A (288.8 mg/L), and B (48.6 mg/L). Monochamus alternatus (MA, Japanese pine sawyer) is known to act as a mediator transferring Bursaphelenchus xylophilus to pinewood. As B. xylophilus is associated with the occurrence of pine wilt disease, biological control of MA is a major strategy or controlling this disease. In this study, upon the application of a mixture of DTXs and protease-containing culture filtrate (PCF), complete mortality of MA is observed after a 5-day incubation. The MA immune system response is believed to cause an overexpression of actin and tropomyosin as a defense mechanism against the flaccid paralysis induced by the DTXs and PCF treatment. CONCLUSIONS: These results suggest that MCT can be used as a major feedstock in the biorefinery industry and that DTXs can be applied as an insecticide for biological control of pine wilt disease via MA termination.

Biorefining Process of Carbohydrate Feedstock (Agricultural Onion Waste) to Acetic Acid.

The biorefining of agricultural waste into green chemicals has clear potential for improving global environmental sustainability. In this study, we evaluated the potential of acetic acid production from carbohydrate feedstock (onion waste, OW) as a more environmentally friendly source than feedstock produced from natural gas. In particular, OW is an ideal feedstock for the biorefining process as it contains a sufficient amount of carbohydrates (69.7%). Five days of the simultaneous saccharification and two-step fermentation (SSTF) process produced acetic acid from OW more efficiently than the simultaneous saccharification and cofermentation (SSCF) process. SSTF produced 19.3 g/L acetic acid and recorded the highest conversion yield (90.5%) from OW (6% substrate loading, w/v). These results suggested that acetic acid can be efficiently and sustainably produced from OW by the SSTF process.

Effective approach to organic acid production from agricultural kimchi cabbage waste and its potential application.

The biotransformation of agricultural waste into valuable chemicals represents a promising approach in the field of biorefining. Herein, a general but highly efficient and robust process is reported for the production of organic acid from kimchi cabbage waste using lactic acid bacteria. The organic acid produced was tested for efficacy as a biological control agent. Lactobacillus sakei WiKim31 and L. curvatus WiKim38 could efficiently produce organic acids including lactic acid (12.1 and 12.7 g/L), fumaric acid (7.4 and 7.1 g/L), and acetic acid (4.5 and 4.6 g/L) from kimchi cabbage waste (3% substrate loading, w/v) by simultaneous saccharification and fermentation processes for 48 h, and the culture filtrate induced complete mortality of J2s Meloidogyne incognita at 2.5% concentration. These results suggested that lactic acid bacteria L. sakei WiKim31 and L. curvatus WiKim38 can efficiently produce organic acids, and the culture filtrate can be applied as a microbial nematicide.

Onion skin waste as a valorization resource for the by-products quercetin and biosugar.

Onion skin waste (OSW), which is produced from processed onions, is a major industrial waste. We evaluated the use of OSW for biosugar and quercetin production. The carbohydrate content of OSW was analyzed, and the optimal conversion conditions were evaluated by varying enzyme mixtures and loading volumes for biosugar production and quercetin extraction. The enzymatic conversion rate of OSW to biosugar was 98.5% at 0.72 mg of cellulase, 0.16 mg of pectinase, and 1.0mg of xylanase per gram of dry OSW. Quercetin extraction also increased by 1.61-fold after complete enzymatic hydrolysis. In addition, the newly developed nano-matrix (terpyridine-immobilized silica-coated magnetic nanoparticles-zinc (TSMNP-Zn matrix) was utilized to separate quercetin from OSW extracts. The nano-matrix facilitated easy separation and purification of quercetin. Using the TSMNP-Zn matrix the quercetin was approximately 90% absorbed. In addition, the recovery yield of quercetin was approximately 75% after treatment with ethylenediaminetetraacetic acid.

Bioethanol production from the nutrient stress-induced microalga Chlorella vulgaris by enzymatic hydrolysis and immobilized yeast fermentation.

The microalga Chlorella vulgaris is a potential feedstock for bioenergy due to its rapid growth, carbon dioxide fixation efficiency, and high accumulation of lipids and carbohydrates. In particular, the carbohydrates in microalgae make them a candidate for bioethanol feedstock. In this study, nutrient stress cultivation was employed to enhance the carbohydrate content of C. vulgaris. Nitrogen limitation increased the carbohydrate content to 22.4% from the normal content of 16.0% on dry weight basis. In addition, several pretreatment methods and enzymes were investigated to increase saccharification yields. Bead-beating pretreatment increased hydrolysis by 25% compared with the processes lacking pretreatment. In the enzymatic hydrolysis process, the pectinase enzyme group was superior for releasing fermentable sugars from carbohydrates in microalgae. In particular, pectinase from Aspergillus aculeatus displayed a 79% saccharification yield after 72h at 50°C. Using continuous immobilized yeast fermentation, microalgal hydrolysate was converted into ethanol at a yield of 89%.

Bioethanol production from rice straw by popping pretreatment.

BACKGROUND: Rice straw has considerable potential as a raw material for bioethanol production. Popping pretreatment of rice straw prior to downstream enzymatic hydrolysis and fermentation was found to increase cellulose to glucose conversion efficiency. The aim of this study was to investigate the influence of popping pretreatment and determine the optimal enzyme loading using a surface response design. RESULTS: The optimal doses of cellulase and xylanase enzymes were 23 FPU and 62 IU/g biomass, respectively. Using the optimized enzyme condition and popping pretreatment of rice straw (15% substrate loading, w/v), a sugar recovery of 0.567 g/g biomass (glucose; 0.394 g/g) was obtained in 48 h, which was significantly higher than that from untreated rice straw (total sugar recovery; 0.270 g/g biomass). Fermentation of the hydrolyzates by Saccharomyces cerevisiae resulted in 0.172 g ethanol/g biomass after 24 h, equivalent to 80.9% of the maximum theoretical yield (based on the amount of glucose in raw material). Changes in the chemical composition and surface area of rice straw were also investigated before and after popping pretreatment. The results showed little or no difference in chemical composition between the pretreated rice straw and the control. However, the surface area of pretreated rice straw increased twofold compared to the control. CONCLUSION: Popping pretreatment of rice straw can effectively improve downstream saccharification and fermentation, important for bioethanol production.

Heterologous expression of endo-1,4-ß-xylanase A from Schizophyllum commune in Pichia pastoris and functional characterization of the recombinant enzyme.

Endo-1,4-ß-xylanase A (XynA) from Schizophyllum commune was cloned into pPCZαA and expressed in Pichia pastoris GS115. The open reading frame of the xynA gene is composed of 684 bp, encoding 278 amino acids with a molecular weight of 26 kDa. Based on sequence similarity, XynA belongs to the CAZy glycoside hydrolase family 11. The optimal activity of XynA was at pH 5 and 50 °C on beechwood xylan. Under these conditions, the K(m), V(max) and specific activity of XynA were 5768 units mg(-1), 4 mg ml(-1) and 9000 µmol min(-1)mg(-1), respectively. XynA activity was enhanced in the presence of cations, such as K(+), Na(+), Li(2+), Cd(2+), and Co(2+). However, in the presence of EDTA, Hg(2+) and Fe(3+), xylanase activity was significantly inhibited. This enzyme effectively degraded approximately 45% of unsubstituted xylans in the cell wall from poplar stems. The high level of XynA activity might increase the yield of enzyme hydrolysis from biomass. Thus, XynA could be used as a major component of a lignocellulosic degrading enzyme cocktail.

Conversion of coffee residue waste into bioethanol with using popping pretreatment.

Coffee residue waste (CRW), which is produced after coffee extraction for coffee powder and instant coffee preparation, is a primary industrial waste. In this study, the use of CRW for bioethanol production was evaluated. The carbohydrate content of CRW was analyzed for fermentable sugars such as glucose, galactose, and mannose, which can be fermented by Saccharomyces cerevisiae. Pretreatment at a pressure of 1.47 MPa for 10 min with popping pretreatment was required to increase enzymatic hydrolysis. CRW was well hydrolyzed following popping pretreatment at 1.47 MPa. The enzymatic conversion rate of CRW to fermentable sugars was 85.6%. Ethanol concentration and yield (based on sugar content) following enzymatic hydrolysis after simultaneous saccharification and fermentation were 15.3g/L and 87.2%, respectively.

Engineering of the catalytic site of xylose isomerase to enhance bioconversion of a non-preferential substrate.

Mutation in active site would either completely eliminate enzyme activity or may result in an active site with altered substrate-binding properties. The enzyme xylose isomerase (XI) is sterospecific for the α-pyranose and α-fructofuranose anomers and metal ions (M1 and M2) play a pivotal role in the catalytic action of this enzyme. Mutations were created at the M2 site of XI of Thermus thermophilus by replacing D254 and D256 with arginine. Mutants D254R and a double mutant (D254R/D256R) showed complete loss of activity while D256R showed an increase in the specificity on D-lyxose, L-arabinose and D-mannose which are non-preferential substrates for XI. Both wild type (WT) and D256R showed higher activity at pH 7.0 and 85°C with an increase in metal requirement. The catalytic efficiency Kcat/Km (S(-1) mM(-1)) of D256R for D-lyxose, L-arabinose and D-mannose were 0.17, 0.09 and 0.15 which are higher than WT XI of T.thermophilus. The altered catalytic activity for D256R could be explained by the possible role of arginine in catalytic reaction or the changes in a substrate orientation site. However, both the theories are only assumptions and have to be addressed with crystal study of D256R.

Pervaporative separation of bioethanol using a polydimethylsiloxane/polyetherimide composite hollow-fiber membrane.

Pervaporation is one of the most promising separation processes for the purification of ethanol. In this study, a composite hollow-fiber membrane with a thin polydimethylsiloxane (PDMS) active layer on a polyetherimide (PEI) macroporous support was used for pervaporative separation of ethanol produced by Saccharomyces cerevisiae from glucose fermentation broth. The pervaporation performance for ethanol/water binary mixtures was strongly dependent on the feed concentration and operating temperature for ethanol concentrations of 1-10%. The composite hollow-fiber membrane was stable over the long-term (about 160 days) with an ethanol permeation flux of 60-62 g/m(2)h and a separation factor of 7-9. In comparison with published results for PDMS composite membranes, the PDMS/PEI hollow-fiber composite membrane had relatively good pervaporation performance with a total flux of 231-252 g/m(2)h.

Phenanthroline-based magnetic nanoparticles as a general agent to bind histidine-tagged proteins.

We herein report the preparation of PSMN (phenanthroline-based silica coated magnetic nanoparticles), and their applications for protein purification as selective magnetic probes of histidine-tagged proteins in cell lysates. This simple system serves as a useful alternative to existing protocols for his-tagged protein separation and as a versatile agent for transporting and anchoring proteins.

Enhanced production of bioethanol and ultrastructural characteristics of reused Saccharomyces cerevisiae immobilized calcium alginate beads.

Yeast immobilized on alginate beads produced a higher ethanol yield more rapidly than did free yeast cells under the same batch-fermentation conditions. The optimal fermentation conditions were 30°C, pH 5.0, and 10% initial glucose concentration with 2% sodium alginate beads. The fermentation time using reused alginate beads was 10-14 h, whereas fresh beads took 24h, and free cells took 36 h. All bead samples resulted in nearly a 100% ethanol yield, whereas the free cells resulted in an 88% yield. Transmission electron microscopy (TEM) showed that the shortened time and higher yield with the reused beads was due to a higher yeast population per bead as well as a higher porosity. The ultrastructure of calcium alginate beads and the alginate matrix structure known as the "egg-box" model were observed using TEM.

Arabidopsis thaliana Rubisco small subunit transit peptide increases the accumulation of Thermotoga maritima endoglucanase Cel5A in chloroplasts of transgenic tobacco plants.

Over the past decade various approaches have been used to increase the expression level of recombinant proteins in plants. One successful approach has been to target proteins to specific subcellular sites/compartments of plant cells, such as the chloroplast. In the study reported here, hyperthermostable endoglucanase Cel5A was targeted into the chloroplasts of tobacco plants via the N-terminal transit peptide of nuclear-encoded plastid proteins. The expression levels of Cel5A transgenic lines were then determined using three distinct transit peptides, namely, the light-harvesting chlorophyll a/b-binding protein (CAB), Rubisco small subunit (RS), and Rubisco activase (RA). RS:Cel5A transgenic lines produced highly stable active enzymes, and the protein accumulation of these transgenic lines was up to 5.2% of the total soluble protein in the crude leaf extract, remaining stable throughout the life cycle of the tobacco plant. Transmission election microscopy analysis showed that efficient targeting of Cel5A protein was under the control of the transit peptide.

Inhibition of hepatitis B virus by an aqueous extract of Agrimonia eupatoria L.

Inhibition of HBsAg release against hepatitis B virus (HBV) was investigated in an aqueous extract prepared from the aerial parts (stems and leaves) of Agrimonia eupatoria. The inhibitory effect on HBsAg secretion was footed using aqueous extracts of Agrimonia eupatoria at four different temperatures (37 degrees C 45 degrees C, 55 degrees C and 60 degrees C), and the extract prepared at 60 degrees C was found to have the greatest effect. The inhibitory activity of Agrimonia eupatoria extracts on HBsAg secretion varied over the growing season and was the highest at mid-July. This inhibitory activity was also shown with the aqueous extracts of two other species of the genus Agrimonia: A. pilosa and A. coreana pilosella. These results suggest that some plants of the genus Agrimonia contain potential antiviral activity against HBV.