Earticle

Synthetic resins were screened for their ability to selectively absorb n-butanol from an aqueous solution containing the various concentrations of mixed solvents(acetonebutanol-ethanol) and CGM medium. Hydrophobicity and narrow pore size of the resins were critical for the selective absorption of n-butanol. Fermentation experiments were carried out to test effect of the resins on butanol production by Clostridium acetobutylicum GSC-PI. It was found that this process using the resins enhanced the fermentation rate and productivity through in-situ recovery of produced n-butanol when volume ratio of the resins to the fermentation broth of 2/10 was used. About 60~70g/L glucose could be consumed in a batch fermentation and thus at maximum only 26g/L of total solvents concentration could be achieved in a traditional acetone-butanol-ethanol fermentation process. However, this extractive fermentation system could synthesized overall solvents to the concentration of 44.3 g/L from 120g/L of glucose with the productivity and the yield of 1.1 g/L/h and 37%, respectively, in CGM medium. When compared with the traditional batch fermentation, this fermentation system increased the final solvent concentration by 70% and the productivity by 110%. Retrieval of n-butanol from resins through elution with tetrahydrofuran was demonstrated with the recovery yield of 95% or more. The adsorption resin was used more than 7 times without significant loss of adsorptive capability in this system. This work was supported by the Energy Efficiency & Resources of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) grant funded by the Korea government Ministry of Knowledge Economy.

Xylose reductase (XR) is a key enzyme in xylitol production, catalyzing the reduction of D-xylose to xylitol. However, the XRs in most yeast and filamentous fungi have evolved to have broad substrate specificity for efficient reduction of a number of pentoses and hexoses. The promiscuous property of these enzymes could adversely affect the biological production of xylitol. The hemicelluloses, major resources of xylose, also contain abundant L-arabinose and it can be reduced to arabitol, an undesirable byproduct. Arabitol, epimer of xylitol, is difficult to remove, and requires high cost in downstream process. Candida tropicalis XR also have a higher activity for L-arabinose than D-xylose, and this substrate specificity causes the formation of an arabitol byproduct. Neurospora crassa XR (NcXR) has significantly higher activity toward D-xylose than L-arabinose. However, because C. tropicalis and N. crassa have different patterns of codon usage, all NcXR codons were changed into preferred codons in C. tropicalis. In order to change substrate specificity of optimized NcXR (NXRG), point mutations were introduced by site-directed mutagenesis. Expression cassettes harboring mutated NXRGs were integrated into the genome of XYL1-disrupted C. tropicalis. The resulting recombinant yeasts showed changed substrate specificity toward xylose and arabinose. In xylitol fermentation, they showed lower arabitol/xylitol ratio than that of the parental strain.

Many yeasts and fungi can assimilate L-arabinose via a complex pathway consisting of two reduction, two oxidation and phosphorylation. Unspecific aldose reductase catalyzes the reduction of L-arabinose to L-arabitol, and L-arabitol dehydrogenase (LAD) oxidizes L-arabitol to L-xylulose. This is subsequently reduced to xylitol by L-xylulose reductase (LXR). After that, xylitol dehydrogenase (XDH) catalyzes the oxidation of xylitol to D-xylulose. D-xylulose is converted to D-xylulose 5-phosphate by xylulose kinase (XK) and then enters the pentose phosphate pathway (PPP). However, Candida tropicalis ATCC 20336 cannot grow on media containing L-arabinose as the sole carbon, which indicates that there is not L-arabinose metabolic pathway in this strain. For consumption of L-arabinose via PPP, bacterial L-arabinose metabolic pathway was introduced to C. tropicalis. Bacillus licheniformis L-arabinose isomerase (araA, AI), E. coli L-ribulokinase (araB, RK) and L-ribulose-5-phosphate 4-epimerase (araD, RPE) were selected and backtranslated using the preferred codons in C. tropicalis. Codon-optimized genes were synthesized and expressed in C. tropicalis. The resulting recombinant yeast expressed araBAD successfully and survived utilizing L-arabinose as a sole carbon source.

The type III secretion system (T3SS) is a tool for bacteria to export proteins from the cytoplasm, through the membranes to the extracellular environment. The system is made up of more than 20 different proteins, about half of which are conserved (similarity in sequence) in most type III systems. This review would summarize the typical features of this system and discuss the potential in utilizing the T3SS to export recombinant proteins into the external environment as well as applicable capability in protein production technology by this novel apparatus. [This work was supported by National Research Foundation of Korea Grant funded by the Korean Government(KRF-2008-313-D00304)]

Xylose reductase (XR) is a key enzyme in xylitol production, catalyzing the reduction of D-xylose to xylitol. However, most XRs from yeasts containing Candida tropicalis have a higher activity for L-arabinose than D-xylose, and this broad substrate acceptance causes the formation of an arabitol byproduct. To disrupt endogenous XR activity, four strands of the XR gene were deleted completely from C. tropicalis BSXDH-3 (a XYL2-disrupted mutant). The resulting strain, C. tropicalis K1, showed no XR activity. Neurospora crassa XR (NcXR) was codon-optimized for heterologous expression in C. tropicalis and point-mutated for decrease of catalytic efficiency toward L-arabinose. XR activity for L-arabinose was not detected in engineered NcXRexpressing yeast, C. tropicalis KNV. In addition, codon-optimized Bacillus licheniformis L-arabinose isomerase (araA, AI), E. coli L-ribulokinase (araB, RK) and L-ribulose-5-phosphate 4-epimerase (araD, RPE) were introduced sequentially to KNV. The resulting C. tropicalis expressed these genes functionally and utilized L-arabinose for supporting metabolism. In xylitol fermentation using xylose and arabinose as substrates, xylose was converted with nearly theoretical maximum yield of 100 % and no arabitol was formed.

Previously we successfully identified a previously-unknown novel pleiotrophic global down-regulator named a whiB-like gene A (wblA ,SCO3579) using a Streptomyces interspecies DNA microarray analysis [1], followed by additional identification of another antibiotic down-regulator SCO1712 which encodes a putative tetR-family transcriptional regulator [2]. Interestingly, wblA is not necessarily required for SCO1712 to function as an antibiotic down-regulator from RT-PCR analysis results, implying that SCO1712 is more likely to encode a wblA-independent antibiotic down-regulator. S. coelicolor M145ΔwblAΔSCO1712 double knockout mutant strain exhibited the highest antibiotic actinorhodin volumetric productivity comparing with the S. coelicolor M145 wild type and S. coelicolor M145ΔwblA strains. To generate a regulatory network-optimized Streptomyces strain for high expression of foreign gene cluster, the entire actinorhodin biosynthetic gene cluster was deleted from the chromosome of this double knockout S. coelicolor M145ΔwblAΔSCO1712 mutant strain, followed by the heterologous expression of several foreign gene clusters. The more detail results will be discussed.

Synthesis of intelligent genetic network in bacteria is one of the great applications of synthetic biology. Here we show the assembly of an intelligent system that can trigger bacteria to sense and respond to the removal of heavy metals such as zinc and copper by engineering of two component systems. This bacterial system can sense zinc and copper using OmpR and NtrC family of two component sensor system and remove metals contaminants through cell surface display system. The genetic circuit of HydHG and CusSR were fused to cell surface display system for the expression of chimeric protein namely OmpC with heavy metal binding peptide. Our Intelligent bacterial system targets the specific heavy metal and triggers a cell surface display system in response to the heavy metal added to the bacterial population which is potentially used in the field of biosensor and bioremediation. [This work was supported by the Korean Systems Biology Research Project (20100002164) of the Ministry of Education, Science and Technology (MEST) through the National Research Foundation of Korea]

It is well known fact that E.coli adapts to several environmental changes for its survival. These microbial cells contain receptors in the cytoplasmic membrane for the detection of environmental signals called two component signaling system. The intelligent behavior of E. coli can be reprogrammed by the alteration of genetic parts, namely sensors, genetic circuits and actuators in the two component signaling system. Sensors transmit information to genetic circuits and it drives actuators in order to change the behavior of the microbial cell. Now day’s researchers, combine genetic parts which is encoded on the DNA to create biological programs that command cells to perform a multiple tasks. Normally in metal efflux system in bacteria that can sense copper in the environment by NtrC family of two component system. These two-component system (TCS) consists of a membrane bound, periplasmic sensor protein-histidine kinase, CusS receives the environmental signal from copper by an extracellular sensory domain called sensor. This sensor passes the information to a corresponding cytoplasmic response regulator, CusR, which has DNA – binding domains directly regulate the gene expression of genetic circuit namely cusC for metal efflux system. [This work was supported by the R&D Program of MKE/KEIT (10033199)]

2,3-Butanediol (2,3-BDO) is a four-carbon chemical intermediate with industrial potentials for the production of synthetic rubber, plastic, antifreezing agent and octane enhancer. Traditional and chemical synthesis of 2,3-BDO has faced the drawback of the petroleum deficiency and environmental pollution that allows the development of microbial production of 2,3-BDO from renewable biomass. A potential microorganism for 2,3-BDO production, Klebsiella oxytoca is a gram negative bacteria grown in both aerobic and anaerobic conditions. In anaerobic or micro-aerobic condition, K. oxytoca generally produces 2,3-BDO from glucose as a carbon source because the 2,3-BDO synthetic enzymes play a role of the oxidation of NADH arising from glucose conversion to pyruvate. Since K. oxytoca has insufficient power of NAD oxidation by the 2,3-BDO synthetic enzymes, some organic acids are produced from pyruvate. To increase 2,3-BDO yield from glucose, organic acid production should be reduced. In this study, chemical mutation and evolutionary engineering were employed to obtain mutant strains of K. oxytoca containing the reduced ability of organic acid production. K. oxytoca mutant library was cultured in a medium containing both sodium bromide and sodium bromate, of which compound, bromine can kill high acid-producing strain and hence K. oxytoca mutants with reduced acid-forming ability were screened. Batch fermentation was carried out to validate the effect of organic acid production on 2,3-BDO production.

The yeast pichia pastoris has been well known as a highly successful system for the production of various recombinant heterologous proteins. To improve scFv production in pichia pastoris, parameters such as cultivation temperature, initial pH of medium and cell density were examined. Investigation of culture conditions revealed that the maximal cell growth and functional scFv expression were achieved at pH 6.0-7.0 and at 20 ℃ which is relatively low temperature. The N-terminal sequence of expressed anti-BNP scFv was analyzed and two Ste13 sites were not cut out at all unlike our expectation. However the function or activity of anti-BNP scFv was not affected significantly. The optimized culture conditions determined through this study will be used from the efficient production of anti-BNP scFv.

Chemokinese are best known for their vital role in leukocyte chemotaxis, as part of the larger inflammatory response. These are large, diverse group of small cytokines that can be classified into several families, including the CC and CXC chemokines that are characterized by two adjacent cysteines near their amino terminus. Although a sequence for Japanese flounder Paralichthys olivaceus CC and CXC chemokines have been reported, it had no similarity to the other chemokines but had more than 32% and 72% amino acid identity with the sequences from Ictalurus furcatus. In this study, we report molecular cloning of a full-length cDNA encoding CC and CXC chemokines from Japanese flounder. The expression profiles of the chemokines genes in healthy fish were determined by semi RT-PCR. Also, cDNA fragments containing the genes were inserted into Bombyx mori baculovirus vectors under the control of the polyhedron promoter. We tried to expression of the recombinant protein from P. olivaces by using baculovirus expression system. ※ This work has been conducted with support through the research program of KORDI with contract No. PM55551 to HSR.

The catalytic activity of oxygenase-based whole-cell biocatalysts are heavily influenced by substrate and product toxicity because of cell membrane permeabilizing effects and protein denaturating effects of lipophilic substrates and products. In this presentation, we will report a novel approach to increase stability of oxygenase-based whole-cell biocatalyst, a recombinant Escherichia coli expressing chnB the gene of cyclohexanone monooxygenase of Acinetobacter calcoaceticus NCIMB 9871. Oxygenation activity of the recombinant E. coli expressing chnB alone was rapidly decreased during biotransformation of cyclohexanone into ε-caprolactone. However, the catalytic activity was rather maintained when the genes encoding chaperones GroEL-ES, DnaKJ-GrpE or ClpB, IbpAB, DnaKJ-GrpE were coexpressed. The chaperoning proteins were assumed to assist protein folding to the native conformation and/or disaggregating unfolded proteins. Thus, we conclude that stability of oxygenase-based whole-cell biocatalysts can be enhanced via coexpression of the genes encoding chaperones.

Bioethanol is a promising alternative biofuel for automobile. The current bioethanol is produced with corn and sugar cane, which are also used as foods and feeds. To replace the starch and sugar-based bioethanol, the 2nd and 3rd generation bioethanol technology have been developed using cellulosic and marine biomass, respectively. Among marine biomass, red algae is mostly composed of cellulose and galactan. Galactan is a representative polymer consisting of D-galactose and anhydro-galactose. For efficient production of bioethanol from red algae, both glucose from cellulose and D-galactose from galactan should be fermented simultaneously. In this study, food-grade alcohol yeast, Saccharomyces cerevisiae was engineered to utilize both glucose and galactose simultaneously. A laboratory strain, S. cerevisiae D452-2 was subjected to evolutionary engineering by serial batch culture with 2-deoxy-D-glucose (an analog of D-glucose) and D-galactose. After five times serial cultures, the mutant strains were selected on YP solid medium containing both 2-deoxy-D-glucose and D-galactose. The fermentative characteristics of the yeast mutants were investigated in batch fermentation at different culture conditions.

Citrobacter amalonaticus Y19 (CAY19) was isolated from anaerobic sludge digester. The facultative anaerobe has fermentative metabolism and is tolerant to about 40% CO. CAY19 has unique capability to oxidize CO with reduction of protons to produce H2gas. None of the other Citrobacter species is reported to have CO induced H2 production. To get a deeper insight of the water-gas shift reaction, genome of CAY19 was explored. From the contiguous gene analysis it was found that apart from the metabolic genes for catabolic pathway of hexose sugars, unique genes for hydrogen production are present. Carbon monoxide dehydrogenase (codh) gene cluster (3.8 kb) was found on contig CAY00120. The hydrogenase (hyd) gene cluster (8 kb) was structurally found to be in the reverse direction with respect to codh cluster on contig CAY00120. Functional analyses of genes suggest that the CODH and hydrogenase proteins in Y19 are closely related to other type IV CODH and Ni-Fe hydrogenases. In this study, we report the first genetic evidence of CO induced H2 production in Citrobacter. The high hydrogen productivity (27.1 mmol/g cell h) can have direct ramifications on H2 industry. The in-depth genetic analysis can provide clue about nature’s ability to engineer microbes by way of adaptive evolution.

We investigated the optimal strategy for ethanol production using flocculent Sacchromyces cerevisiae ATCC 96581. Considering the characteristic of flocculent yeast, a repeated fed-batch ethanol fermentation was designed, in which non-sterile glucose powder was fed every 12 hours and, after cell flocculation, new feeding medium was exchanged every 24 or 36 hours. We particularly compared this fermentation process with those when cell flocculation was not carried out. Finally, the maximal total ethanol production was 825 g-ethanol during 120 hours, in which the time interval of withdrawal-fill of feeding medium was 24 hours and cell flocculation was carried out.

The study about masking odors and reducing oxidation was performed. Encapsulating properties of iota carrageenan (IC) and sodium casein (SC) for the coating of the fish oil were evaluated. Retention of fish oil (oil red o staining) after freeze-drying was observed by measuring absorbance. Highest amount of fish oil (70 ± 0.3% of initially added amount) was retained in a matrix consisting of IC and SC, in the ratio of 0.1:1:1 (w/w). Particle size varied from 2.0 to 3.2 μm. The fishy odor of raw fish oil was considerably reduced by encapsulation of IC and SC. A mixture consisting of IC-SC is an efficient encapsulant for fish oil encapsulation.

Nanomaterials exhibit a wide spectrum of properties stemming from their nanoscale dimension and morphological features. Biomolecules have attracted increasing attention in the field of nanobiotechnology due to their exquisite specific recognition ability toward various inorganic materials including metals and semiconductors.1) Many biomolecules were shown to mediate the formation of inorganic nanocrystal structures from their precursors under ambient condition through a process called biomineralization. However, most of biomineralized inorganic nanostructure reported elsewhere showed deductive morphology (i.e. morphology is determined after trial and error based biomineralization) rather than inductive morphology (i.e. morphology is predicted prior to biomineralization). This raises a need for an alternative approach capable of giving rise to a patterned structure precisely mimicking a pre-designed biomolecular scaffold which is conducive to designing practical devices with a desired performance in a predictable manner. To explore a novel strategy for achieving inductive nanostructure, this study aims to synthesize TiO2 nanoparticles and its patterned nanostructure capable of exhibiting new physical properties at nanometer scale which would otherwise be difficult to realize by the conventional metal oxide nanofabrication. A core strategy for formation of TiO2 nanoparticles and subsequent simple patterned nanostructure is to mimic exquisite biomineralization mechanism evolved by living entities in nature. For this, we created a designer protein LacI-STB1 harboring TiO2 cognitive peptide at the C-terminus of LacI while leaving DNA binding domain (DBD) of LacI intact. By using LacI-STB1 as a linker to position TiO2 molecules on the plasmid DNA (pDNA) harbouring lacO at a specific position, it was demonstrated that TiO2 nanoparticles could be positioned at a specific position on pDNA/LacI-STB1 scaffold (i.e. a simple inductive structure). Ongoing study will further test a possibility of biomineralizing TiO2 at programmed positions with the use of DNA/Lac-STB1 scaffold as an inductive structure where LacI-STB1 acts as a mineralizing catalyst as well as an anchor to position such mineralized TiO2 particles. In parallel, various 1-, 2- and 3-D structures of DNA by harnessing DNA origami technology were synthesized and LacI-STB1 binding to these diverse DNA scaffolds was confirmed, highlighting a potential of realizing complicated inductive nanostructured metal oxide with the use of pre-designed biomolecular scaffolds.

This study was conducted to investigate the physiological activities of extracts from Suaeda japonica harvested in different season for its possibility as a functional material in food or cosmetic composition. The total mineral content of S. japonica harvested in summer was about 89.8 g/kg, and it comprised a little more content than one in winter (86.7g/kg). The Na content of S. japonica did not show a remarkable contrast on harvest season whereas the K and Ca contents of summer were decreased to half or increased to double in winter. In addition, the antioxidative activity of each extract from S. japonica changed depending on harvest season. For S. japonica harvested in summer, the ethyl acetate extract showed the highest DPPH radical scavenging activity, but in winter the butanol extract fraction had the highest value. However, FRAP values were the highest in butanol extracts from S. japonica harvested in summer and winter. Total phenolic contents in the extracts were in proportion to the antioxidative activities.

We synthesized a hydroquinone glucoside (HG) as a potential skin whitening agent using Leuconostoc mesenteroides dextransucrase with hydroquinone (HQ) as an acceptor and sucrose as a donor. The product was purified using butanol partitioning and silica-gel column chromatography. The structure of the purified HG was determined by NMR. HG was identified as 4-hydroxyphenyl-α-D-glucopyranoside. The optimum condition of HG synthesis, determined using a response surface methodology, was 450 mM HQ, 215 mM sucrose, and 0.55 U/mL dextransucrase; the final HG produced was 544 mg/L HG potently inhibited oxidation. The IC50 of diphenylpicryl-hydrazyl scavenging activity was 3.85 mM indicating a higher anti-oxidant activity compared to β-arbutin (IC50 = 6.04 mM). HG-mediated inhibition of lipid peroxidation was 3.51% that of HQ (100%) and much higher than that of β-arbutin (0.81% of HQ). In addition the IC50 value of nitrite scavenging activity was 14.76 mM showing a superior scavenging activity to that of β-arbutin (IC50 = 27.09 mM)

In this study, the effects of fucoidan on aspirin-induced ulcers in rats were evaluated: both biochemical and immunological parameters were taken into consideration. The status of stomach tissue glycogen storage and histological changes were also examined. Examination of basic biochemical parameters showed significant (p <0.01) alterations in aspartate (AST) and alanine (ALT) transaminases in ulcer-induced rats. Also, moderate alterations (p <0.05) were observed in the levels of cholesterol and blood urea nitrogen (BUN). Analysis of serum cytokines of aspirin-induced rats showed a moderate decrease in interleukin-10 (IL-10) with considerable increase of interleukin-6 (IL-6) and interferon-γ (INF-γ) when compared with control. Administration of fucoidan showed considerable (p <0.05) protection against ulceration by inhibiting the acute alterations of AST, ALT, cytokines and stomach glycogen. However, aggravated serum INF-γ was observed in the fucoidanpretreated group. These findings suggest that the anti-ulcer property of fucoidan might contribute in protecting the inflammatory cytokinemediated oxidative damage to gastric mucosa.

In this study, in vivo antioxidant and anticancer activity of mixture extracts with fishes and papurika were measured for medical application. The fishes used in this study were four: Paralichthys livaceus, Mugil cephalus, and Muraenesox cinereus. Two sea weeds and seven mushrooms were used to prepare the extract mixture. To assay anticancer activity, three different origins of cells (pancreas, liver and stomach) were used. Cell viability was measured using MTT assay. Relatively large amount of mushrooms (60 g/kg) should be administered to show rather high anticancer activity. This research was financially supported by the Ministry of Education, Science Technology (MEST) and Korea Institute for Advancement of Technology(KIAT) through the Human Resource Training Project for Regional Innovation(2010)

In this study, in vivo antioxidant and anticancer activity of hot-water extracts of Muraenesox cinereus were measured for medical application. To assay anticancer activity, three different origins of cells (pancreas, liver and stomach) were used. Cell viability was measured using MTT assay. MTT values and viability data showed that no anticancer activity was not found within the concentration range studied. This research was financially supported by the Ministry of Education, Science Technology (MEST) and Korea Institute for Advancement of Technology(KIAT) through the Human Resource Training Project for Regional Innovation (2010)

In this study, in vivo antioxidant and anticancer activity of hot-water extracts of Mugil cephalus were measured for medical application. To assay anticancer activity, three different origins of cells (pancreas, liver and stomach) were used. Cell viability was measured using MTT assay. MTT values and viability data showed that no anticancer activity was not found within the concentration range studied. This research was financially supported by the Ministry of Education, Science Technology (MEST) and Korea Institute for Advancement of Technology(KIAT) through the Human Resource Training Project for Regional Innovation(2010)

Mushrooms-derived polysaccharides are regarded as one of the well-known functional food and biopharmaceutical materials with a great deal of interest. The polysaccharides are the major component of mushrooms that displays various biological activities such as anti-cancer and immunomodulatory effects. It was assessed whether polysaccharides were able to stimulate the functional activation of macrophages and spleenocyte proliferation. The polysaccharides are able to enhance strongly upregulate the phenotypic functions of macrophages such as phagocytic uptake, release of cytotoxic molecules and cytokine secretion. Also, Polysaccharides are able to cell proliferation and cytokine expression on spleenocytes.In this study, we focus on investigating the potential immunomodulatory effects of mushroom with regard to macrophage activation and splenocyte proliferation.

Supercritical carbon dioxide (SCO2) and an organic solvent, hexane were used to extract oil from anchovy The SCO2 extraction was carried out at temperatures ranging from 35 to 60ºC and pressures ranging from 20 to 40 MPa. The flow rate of CO2 was 22 g/min and it was constant entire the extraction period. The extracted oil was analysed by Gas Chromatography (GC) for fatty acids compositions. A high percentage of polyunsaturated fatty acids especially eicosapentaenoic acid (EPA, C20:5) and docosahexaenoic acid (DHA, C22:6) were found in the anchovy oil obtained by SCO2 extraction. EPA and DHA have been shown to be important for the prevention of a range of human diseases and disorders

As a part of search for antioxidants from marine organisms, antioxidant activities of two seagrasses Zostera japonica and Z. asiatica were investigated. Each of them was extracted two times with acetone-CH2Cl2(1:1) and MeOH in turn. Crude extracts were combined for each sample. Each of the combined crude extracts was partitioned between CH2Cl2 and water. The organic layer was further partitioned between 85% aq MeOH and n-hexane, and the aqueous layer was fractionated with n-BuOH and H2O, successively. The protective effect of each fraction from Z. japonica and Z. asiatica on H2O2-induced oxidative stress was examined in Raw 264.7 cells. The levels of intracellular ROS and GSH were measured using 2',7'-dichlorofluorescin diacetate and monobromobimane as fluorescence probe, respectively. All solvent fractions including their crude extracts not only significantly decreased levels of intracellular ROS in a dose-dependent manner but also increased intracellular GSH level. Moreover, they exhibited a significant protective effect on membrane lipid peroxidation in TBARS assay system. These results suggest that potential natural antioxidants related to oxidative stress could be isolated from Z. japonica and Z. asiatica

Krill is not utilized for human consumption due to the lack of proper technology for protein recovery. The aim of this study was to isolate protein concentrate from the krill residues extracted by supercritical carbon dioxide and to determine the nutritional value. Using supercritical carbon dioxide (SCO2) extraction, with optimal conditions among the range applied of 45°C and 25 MPa ; most of the neutral lipids were extracted. Proximate analysis of protein recovered by isoelectric solubilzation/precipitation indicated that protein concentrate was composed of approximately 79% protein and 2% fat (dry weight). Result indicates it is a high quality protein that is suitable as a protein source in foods designated for consumption with the advantage of being a rich source of omega-3 polyunsaturated fatty acids. Further studies are needed to determine the health benefits associated with consuming Krill protein.

2-methoxy-ρ-benzoquinone (2-MBQ) and 2,6-dimethoxy-ρ-benzoquinone (2,6-DMBQ) have antimicrobial- and immune-stimulatory effect. These compounds are abundant in wheat germ as a form of glycone. It is well known that bioconversion of wheat germ glycosidic bond to 2-MBQ and 2,6-DMBQ requires glucosidase. This study was performed in an attempt to investigate the possible application of lactic acid and yeast producing β-glucosidase as starter cultures for production of 2-MBQ and 2,6-DMBQ from wheat germ. Lactobacillus zeae and Pichia pjperi, selected through β-glucosidase enzyme assay for 38 yeast strains and 6 lactic acid bacteria, were used for fermentation of wheat germ. It was found that Lb. zeae, compared with P. pjperi, was more efficient for production of 2-MBQ and 2,6-DMBQ from wheat germ. Concentration of 2-MBQ produced by mixed culture of Lb. zeae and P. pijperi for 48 hr was approximately 1.6-fold higher than that produced by pure culture of Lb. zeae. Contrary to expectation, concentration of 2,6-DMBQ was not enhanced significantly by mixed culture of Lb. zeae and P. pijperi.

Hydroquinone galactoside (HQ-Gal) as a potential skin whitening agent was synthesized by the reaction of lactase (β-galactosidase) from Kluyveromyces lactis, Aspergillus oryzae, Bacillus circulans and Thermus sp. with lactose as a donor and HQ as an acceptor. Among these lactases, the acceptor reaction involving HQ and lactose with K. lactis lactase showed a higher conversion ratio to HQ-Gal (60.27%). HQ-Gal was purified using butanol partitioning and silica-gel column chromatography. The structure of the purified HQ-Gal was determined by nuclear magnetic resonance and the ionic product was observed at m/z 295 (C12H16O7 Na)+ using matrix assisted laser desorption ionization time-of-flight mass spectrometry. HQ-Gal was identified as 4-hydroxyphenyl-β-D-galactopyranoside. The optimum conditions for HQ-Gal synthesis by K. lactis determined using response surface methodology were 50 mM HQ, 60 mM lactose and 20 U mL-1 lactase. These conditions produced a yield of 2.01 g L-1 HQ-Gal. The half maximal inhibitory concentration (IC50) of diphenylpicryl-hydrazyl scavenging activity was 3.31 mM indicating a similar anti-oxidant activity compared to β-arbutin (IC50 = 3.95 mM). The Ki value of HQ-Gal (0.75 mM) against tyrosinase was smaller than that of β-arbutin (Ki = 1.97 mM), indicating its superiority as an inhibitor. HQ-Gal inhibited (23%) melanin synthesis without being significantly toxic to the cells, while β-arbutin exhibited only 8% reduction of melanin synthesis in B16 melanoma cells compared with the control. These results indicate that HQ-Gal may be a suitable functional component in the cosmetics industry.

The precedent studies of Sparassis Crispa (SC) as immunostimulator were conducted mainly using Sparassis crispa glucan (SCG, β-glucan), a bioactive component, and there is a difficulty in commercializing SC as a food in economic aspects, since the extract yield of SC using extraction method applied to foods is too low. Thus, in this study, we have tried to prove the immunostimulatory effect of SC suitable for commercialization in consideration of economic value. The aim for this study was to obtain the beneficial effect on powder (contains about 30% of β-glucan) and water extracts (contains about 10% of β-glucan) of SC which were extracted at various temperatures as immunostimulator in BALB/C mice at 6 weeks of age. The powder and water extracts of SC were orally treated every day for 8 weeks and the secreted cytokines (IL-2, IL-4, IL-12, IFN- γ, and TNF-α) in the blood were measured by ELISA kit. The results show that the SC treatment has led to the increased levels (2~5 times higher) of cytokines (such as IL-2, IL-12 and IFN- γ) compared to the normal control group, as well as the increased level of IL-4 (2.7 times higher at max.), associated with Th1 and Th2 immune after 5~8 weeks. In addition, the level of TNF-α that controls immune response by inducing the creation of many kinds of cytokine has increased by 3.7 times higher with the SC treatment. Especially, it was found that SC powder was more effective than water extracts in immunostimulatory activity and this immunostimulatory effect of SC was proved similar to that of red ginseng (positive control), a typical health food. The results also suggest that SC can be considered as a useful, high value-added food material with the function of immunostimulator.