As the mechanisms of biological functions have been successively clarified, synthetic biology has become a feasible approach. One of the goals to synthetic biologists is to reproduce behaviors in the natural systems and ultimately to create artificial life . We have applied synthetic biological approaches to tissue engineering. Three-dimensional (3D) tissue construction from individual cells is an important process in regenerative medicine to enhance cell function. For transplanting tissue-engineered constructs, a limitation of oxygen/nutrient supply due to an insufficient vascular network causes cell death. Thus, it is necessary to develop a system for inducing a vascular network under hypoxic condition to 3D tissue constructs. In our previous study , we developed a hypoxia-inducible transgene expression system in which a target gene can be expressed in response to hypoxic stress using hypoxia-responsive promoter RTP801 as a trigger, tTA transactivator as an amplifier, and ODD sequence as a noise canceler. In this study, to improve hypoxia and nutritional limitation inside the genetically engineered 3D tissue constructs, a hypoxia-responsive expression system of vascular endothelial growth factor (VEGF) was introduced into cells. Then we demonstrated that genetically modified cells could control the VEGF expression autonomically in an oxygen concentration dependent manner. Using the genetically modified cells, 3D tissue constructs were fabricated. The tissue constructs were transplanted into mice, and we evaluated the feasibility of the hypoxia-responsive VEGF gene expression system in vivo. The results indicate that the hypoxia-responsive VEGF gene expression system is promising for preparing 3D tissue constructs in regenerative medicine.
 S. A. Benner and A. M. Sismour, 窶彜YNTHETIC BIOLOGY,窶・Nature, 2005, vol. 6, no. 7, pp. 533–543.
 Ono A, et al. 窶廩ypoxia-responsive transgene expression system using RTP801 promoter and synthetic transactivator fused with oxygen-dependent degradation domain.窶・J. Biosci. Bioeng., 2017;124:115-124.
The polymerization of proteins can create active and large bio-macromolecular assemblies that exhibit unique functionalities depending on the properties of protein units in the polymers. Herein, we report the polymerization of proteins and its functions in diagnostic application. We engineered proteins; Escherichia coli alkaline phosphatase (BAP), horseradish peroxidase (HRP), and chimeric antibody-binding proteins (pG2pAs) for conducting the polymerization reaction. The proteins were fused with a tyrosine-containing peptide tag (Y-tag) through a flexible linker at the N- and/or C-termini. The recombinant proteins were then expressed in E. coli or silkworm (Bombyx mori). Trametes sp. laccase (TL) was used to activate the tyrosine residues in the Y-tags with molecular O2 to form a tyrosyl free radical, which initiated the tyrosine coupling reaction between the Y-tagged enzyme units. The proteins without Y-tag were not recognized as substrates by TL even though there are several tyrosine residues in the primary structure of both enzymes. Conversely, the Y-tagged proteins showed sufficient reactivity to conduct TL-catalyzed formation of large and active proteinaceous polymers. Furthermore, the TL also enabled co-polymerization of two Y-tagged proteins; BAPs and a chimeric antibody-binding protein, Y-pG2pA-Y, to form bifunctional proteins. While, the Y-tagged HRPs were also co-polymerized with Y-HRP-pG, a fusion protein of HRP with an antibody-binding protein. In these constructs, enzymes play a role as reporter and other parts are for specific binding with IgG, making them compatible with the enzyme-linked immunosorbent assay (ELISA). The detection limit of the ELISA of ovalbumin (OVA) with anti-OVA IgG depended on the molar ratio of enzyme and pG-conjugated proteins in the TL-catalyzed cross-linking reaction. In general, the higher molar ratio of enzymes, the higher ELISA signal. The present enzymatic polymerization of functional proteins promises to provide highly active protein polymers as diagnostics probes for biomedical applications.
Immobilized metal affinity chromatography (IMAC) has been widely used for the one-step purification of recombinant proteins. In light of its superior selectivity and binding strength, we have previously reported the employment of IMAC adsorbents for the purification and immobilization of enzymes. However, the biocatalysts thus prepared exhibited low operational stability in repeated-batch operations due to the desorption of enzymes. In this study, we report the preparation and applications of loofa sponge-based bifunctional epoxy-IMAC adsorbents for the selective adsorption and covalent immobilization of His-tagged enzymes. Loofa sponge is the fibrous vascular reticulated system of the matured dried fruit of Luffa cylindrical. Its high porosity, structural stability and resistance to cellulose-mediated degradation make loofa sponge an ideal matrix for the development of supermacroporous adsorbent for the purification of recombinant proteins from unclarified cell homogenates. To increase the density of the accessible functional groups for activation, the loofa sponge was grafted with hydroxyethyl cellulose (HEC). The results of equilibrium study showed that upon HEC grafting the adsorption capacity of the loofa sponge-based IMAC adsorbent was increased by 62% from 1.51 mg/g to 2.45 mg/g. By controlling the concentration of iminodiacetic acid (IDA) and reaction time during conjugation, a series of bifunctional epoxy-IMAC adsorbents containing both metal chelating groups for selective adsorption of His-tagged proteins and epoxy groups for covalent immobilization of the adsorbed enzymes were prepared. The difference in kinetics of protein adsorption and covalent immobilization allowed the sequential adsorption and immobilization of the model enzyme, His-tagged trehalose synthase. The immobilized trehalose synthase thus prepared with the bifunctional epoxy-IMAC adsorbents exhibited superior operational stability in repeated-batch operations, retaining 96% of the initial activity after 20 cycles. Furthermore, the superporous structure of loofa sponge allowed the adsorption and immobilization of proteins from unclarified cell homogenate without observable fouling.
In this study, an immobilized metal ion affinity (IMAM) approach was adopted for coupling cellulases. The mini-scaffolding protein gene (CipA) and the endoglucanase gene (CelA) were constructed via recombinant genetic engineering approach. The immobilized nickel ion IMAM membrane was used to purify and immobilize the CipA from the fermentation broth to make an IMAM-Ni2+/CipA membrane under a 12 h adsorption. The endoglucanase (CelA) with docking domain is used as the model enzyme coupled to the above affinity membrane. The catalytic activity of the immobilized CipA-CelA was clearly higher at a wide range of pH compared to the free CelA. The optimal temperature for the immobilized CipA-CelA was also increased to 70°C Moreover the immobilized CelA could be reused 4 times with 70% of residual activity. To check the metal ion effect during the construction of IMAM, some metal ions, like iron, cobalt and copper ions would enhance the enzyme activity; however, casium and znic ions would suppress the enzyme activity. Effect of metal ions addition on the enzyme activity immobilized in IMAM is lower than that of free enzyme.
Elaeocarpus serratus L. (Ceylon-olive) leaves are the by-products in the harvest or agricultural processing of Ceylon-olive fruits, containing many high-value bio-phenols and flavonoids with anti-oxidation activity, and are valued to enhance the nutrition for humans. The purpose of this study is to investigate the effect of non-thermal ultrasound and solvent on the extraction of Ceylon-olive leaves. The fresh Ceylon-olive leaves were thoroughly cleaned, freeze-dried, defatted and ground to get raw biomass powder. The extraction efficiency was explored by using various solvents (methanol, ethanol and water) without or with ultrasound at different operation modes to extract the bioactive compounds from Ceylon-olive leaves. The main compounds of bioactive extract were determined by high performance liquid chromatography. The characteristic structure of Ceylon-olive leaves before and after extraction, including morphological structure, functional groups and crystallinity, was analyzed by using field emission scanning electron microscope, Fourier transform infrared spectroscopy, and high resolution X-ray diffractometer. Using methanol as the solvent, the total phenolic and total flavonoid contents of the Ceylon-olive leaves extract with ultrasonic extraction were 1.45 times of those without ultrasound, and myricetin and myricitrin contents with ultrasound-assisted extraction were 1.57 and 1.38 times of that without ultrasound, respectively. When using aqueous ethanol, the contents of total phenolic compounds, total flavonoids, myricetin and myricitrin were all enhanced by the increase of ethanol concentration under ultrasound. The crystallinity of Ceylon-olive leaves after various extractions showed about 21.83% to 44.13% increase, compared to that before extraction, and the morphological structures of Ceylon-olive leaves were observed to be seriously crumbled and punched by ultrasonic extraction, leading to the enhancement of bioactive compounds transported. In this study, the strategic ultrasound and appropriate solvent demonstrated the significant effect on the promotion of extraction efficiency for Ceylon-olive leaves.
Keywords: Ceylon-olive leaf, ultrasound, bioactive compounds, extraction
Phyllostachys makinoi (Makino bamboo), the endemic species of Taiwan, are natural materials with potential bioactivity beneficial to human health. In this study, the objective is to develop the green processing method for the extraction of bioactive compounds from Makino bamboo (RB) via ultrasonic pretreatment. Two different solvents (ethanol and water) were used in extraction of Makino bamboo with or without ultrasonic pretreatment (40 kHz, 60°C 3 h), and the crystallinity were examined by the high resolution X-ray diffractometer. From the observation by field emission scanning electron microscope, the microstructure of Makino bamboo was seriously crumbled and cracked via ultrasonic pretreatment before the extraction, leading to the enhancement of extraction efficiency. Using water as the solvent, the yields of the extract after ultrasonic pretreatment and without ultrasonic pretreatment were 3.98% and 3.56%, respectively, showing that ultrasonic pretreatment would give the extract yield enhanced to 1.12 times of that without ultrasound. Besides, the total phenolic content with ultrasonic pretreatment was 1.08 times of that without ultrasound. Under ultrasonic irradiation, the antioxidant capacity and total flavonoid content of the Makino bamboo extract by using water were 1.91 and 1.40 times of that using 95% ethanol, respectively. The diffraction peaks for Makino bamboo in various extraction conditions within the 2 theta range of 15-25 °were much sharper than those for RB, and the crystallinity index of RB was 47.21%; the crystallinity of Makino bamboo after extraction showed about 3.47% to 7.99% increase, compared to that before extraction (RB). The efficient method by ultrasonic pretreatment to assist the extraction of Makino bamboo has been explored, and ultrasound can be regarded as an effective, mild and non-toxic technology to extract bioactive compounds for the valorization of Makino bamboo.
Keywords: Makino bamboo, ultrasound, pretreatment, extraction
Polyacrylonitrile (PAN) nanofibrous membrane was prepared by electrospinning technique. The PAN membrane used in this work comprises a polyethylene terephthalate (PET) spunbond fabric as a supporting layer with upper and lower PAN nanofibrous membrane. After 3 M NaOH and diluted HCl treatments, the weak cationic exchange membrane (i.e., AEA-COOH) was obtained. The AEA-COOH membrane was then functionalized with ethylenediamine (EDA) and bromoacetic acid (BrA), sequentially. This modified ion exchange membrane (namely AEA-BrA) has more acidic groups as compared to the AEA-COOH membrane. In this study, lysozyme obtained from chicken egg white (CEW) was chosen as a model enzyme. The adsorption characteristics of the AEA-BrA membrane were assessed by measurements of the breakthrough curves in membrane chromatography. The influences of adsorption pH, CEW concentration, and liquid flow rate on adsorption performance of the membrane were investigated in an AKTA prime chromatographic system (GE Healthcare). On the basis of the results, the optimal conditions for the capture of lysozyme were carried out in 10% (v/v) CEW at pH 9 and flow rate of 0.1 mL/min for the ion exchange membrane process. Moreover, the two-step elution scheme (0.6 M and 1.0 M NaCl, pH 9) was chosen for eluting the adsorbed lysozyme. The lysozyme fraction eluted by second step 1.0 M NaCl was recovered with purification factor of 402 and yield of 91% in AEA-BrA ion exchange membrane chromatography.
Polyacrylonitrile nanofiber membranes (PAN) were fabricated via electrospinning technique. The PAN membrane comprises a polyethylene terephthalate (PET) spunbond fabric as a supporting layer with upper and lower PAN nanofibrous membrane. After NaOH and HCl treatment procedures, the modified membrane is a weak ion exchange membrane (namely AEA-COOH). The AEA-COOH membrane was characterized in terms of fiber diameter, porosity, specific area, pore size, ionic density, and binding capacity. In this study, lysozyme was chosen as a model protein. The adsorption efficiency of the AEA-COOH ion exchange membrane for lysozyme was assessed by the measurements of breakthrough curves in a small stirred cell system (Model Millipore 8010). The capture of lysozyme from diluted chicken egg white (CEW) was carried out with a working volume of 10 mL under the different feed concentrations, flow rates, rotating speeds, and operating pressures. The optimal adsorption conditions were found to be adsorption pH 9, 1 in 10 dilution of CEW, average flow rate of 0.1 mL/min, rotating speed of 200 rpm, and initial operating pressure of 0 psi). Furthermore, the elution of lysozyme was also investigated in stirred cell experiments. It was successful in achieving purification of lysozyme in a high yield of 98.2% with a purification factor of 62.9 in a single step.
Heat-sensitive materials (food, pharmaceutical etc.) have to be dried at low temperature. Drying method of such materials is often demanded the maintenance of shape (porous product), color and high rehydration rate. Freeze drying satisfies above demands, but it has some problems like necessity of extremely low pressure, non-uniform heating (conductive or radiant heating) and long drying time.
Some problems of the freeze drying are considered to be solved by our method. Figure 1 shows the outline of experimental apparatus in our study. The frozen materials are put in the vacuum chamber. In the vacuum chamber, air with low temperature (about 273 K) and low humidity circulates under the reduced pressure. Frozen materials are heated by the convective heating due to circulating air. The drying progresses with the sublimation in materials. The sublimation occurs at the relatively high pressure (10 kPa in our study).
The drying characteristics of frozen materials (carrot and brick plate) for our drying method were studied experimentally and theoretically. The changes in temperature and moisture content of material during drying were measured in the experiment. The properties (color, volume and rehydration characteristic) of material (carrot) after drying were examined. Equations of heat and mass transfers were used for the numerical analysis. The effects of operational conditions on the drying characteristics of frozen material (brick plate) were examined by using the numerical analysis.
Sublimation rate on the material surface is much higher than that in the inner part, i.e., the present drying method is suitable for small or thin material. The changes in volume and color during drying are smaller than those in our previous study (fluidized bed drying of frozen carrot under reduced pressure, at 333 K and 12 kPa). The humidity and temperature of circulating air largely affects the drying rate.
β-Cryptoxanthin (BCX) is contained in citrus fruits such as Wenzhou orange and is expected to have various functions such as osteoporosis prevention, diabetes improvement, cancer prevention by immune improvement, and skin care effect. As BCX cannot be synthesized in human body, it must be taken from food. However, BCX is very hydrophobic and the absorption rate in oral administration should be low. In the present study, bioaccessibility of BCX after complexation with sodium caseinate was assessed using the Caco-2 cell model. BCX was recovered from a citrus fruit kumquat (Fortunella spp.) cultivated in Miyazaki by extraction using a mixture of ethanol and pyrogallol. The extracted BCX was complexed with casein by mixing an ethanol solution of BCX and an aqueous solution of sodium caseinate, followed by evaporation of ethanol in vacuo and lyophilization. For cell permeability test, Caco-2 cell was cultured onto 6-well plates for 3 weeks to prepare the cell monolayers. After pseudo-digestion by enzymatic hydrolysis using pepsin in acidic media followed by that using pancreatin in neutral media, the complex between BCX and casein or the blank sample without containing casein was injected to apical side of the cell monolayers. After incubation for 8-24 h, the concentration of BCX in basolateral side was analyzed to determine the cell-permeability of BCX. The permeability of BCX for the blank sample was negligible. In contrast, BCX complexed with casein was efficiently permeated the Caco-2 cell monolayer. The result suggests that complexation with casein would enhance the bioaccessibility of BCX.
It is not necessary to use preservatives and colorants, since frozen storage has good shelf life and quality retention. Frozen shrimp products are usually distributed in the form of frozen ice blocks. Ice-blocked shrimp cannot be removed intact unless ice is completely melted. When thawing time is getting longer, freshness of shrimp will rapidly decrease. Shortening of thawing time is required to maintain freshness and texture. In this study, we examined a new processing method to crush and separate ice on the surface of ice-blocked shrimp.
Cylindrical pure water ice and saline water ice (20%) were used as glazed-ice samples. Cylindrical fish sausage and minced shrimp were used as food samples. Compression test and split test were performed at each test temperature (-20邃ミ-70°C by using material tester. The results showed that breaking stress of pure water ice and saline water ice is always smaller than fish sausage and minced shrimp at each test temperature. Predicted that pure water ice and saline water ice are destroyed preferentially over fish sausage and minced shrimp.
Four types of model ice glazed food were prepared by subjecting fish sausage (with and without packaging film) to ice glaze treatment of pure water or saline water. Split test was performed with constraint condition (fish sausage without packaging film) or unconstraint condition (fish sausage with packaging film) at each test temperature (-20邃ミ-70°C . The result support previous prediction and indicates crushing saline water ice glaze was easier than pure water ice glaze at same temperature. It becomes possible to maintain their original flavor and nutrition. It also have advantage for cost reduction. It is considered that the saline water ice is easy to melt and break, so it is suitable for separating the ice glaze of complex shaped foods.
The blanching is carried out with 90 to 100°C hot water and uses a large amount of water in order to suppress the color tone, texture change and bacterial growth of vegetables. The tissue is seen to be transparent and soft. By blanching whole vegetables in a short time at high temperature with microwaves, the amount of water used decreases, and physical properties, the number of bacteria, etc. can be improved.
A fiber optic thermometer is placed at the center and at the end of the cabbage, and when the center temperature reaches 100°C it is taken out, water-cooled and shredded and stored at 5°C and 15°C I checked. As a comparison, what was immersed in 100°C hot water for 0 second, and was water-cooled was used. The color was determined by removing chlorophyll from cabbage, powdering and measuring Hunter color difference 竓ｿE. Texture investigated the maximum breaking strength of the cabbage leaf vein with the creep mete.
As a result, it was found that microwave blanching is superior to hot water in terms of color tone and texture, and the number of bacteria is equal
There was no change in oxygen concentration and ethylene concentration in both microwave and hot water blanching, and it was presumed that the enzyme was inactivated although it was indirect
The lettuce changes its color from green to white to black upon heating. The launching temperature is 50°C for both microwave and hot water. It is presumed that this is because milky white lactucanium contained in cabbage is eluted at around 50°C and oxidized at a higher temperature. Lettuce blanching is very difficult.
Protein aggregates can be modified by freezing and aging process. In this research, sodium caseinate suspensions (1%) were adjusted to different pH (5.5 and 8.0) and salt concentrations (0.01 and 0.1 M NaCl), followed by being frozen at –35 °C for 1 hour, 2 days or 5 days. After thawing, samples were analyzed by a tunable resistive pulse sensing (TRPS) particle analyzer. According to the results, particle diameter decreased after being frozen for 1 hour and increased after being frozen for days. While particle concentration increased after the 1-hour freezing treatment and decreased after days of freezing. It was found that the change of particle diameter and particle concentration in the 1st hour can be strengthened by repeating the freeze-thaw cycle. The changes of particle diameter and particle concentration were also related to pH and salt concentration of the sample. Particle surface characteristics were also found being related to freezing process, pH and salt concentration. In addition, the difference in freezing temperatures (–5 °C, –20 °C and –35 °C) did not strongly influence the change of particle properties during freezing process. Based on the results, freezing processing is a potential technique to control the properties of sodium caseinate suspensions.
The main component of alcoholic beverages is ethanol, and they form the complex aroma and taste by adding the other ingredients. We have been tried to apply the infrared spectroscopy, which could grasp the information of the quantitative multiple components and the interactions between the molecules, to the evaluation the aroma and taste related information. However, the alcoholic beverages are usually drunk at different temperatures. This study then aims to experimentally understand the influence of the ethanol concentration in the aqueous solution and the temperature on the infrared spectroscopic characteristics.
Ethanol aqueous solutions (12 to 22 vol%) were used as the simplest alcoholic beverage model. A Fourier transform infrared spectrometer equipped with a high-pressure gas cell was used for spectrum measurement of the gas phase. The gas over the sample was circulated from the sample compartment to the gas cell using a pump. The absorption spectra were continuously collected at 290 to 303 K.
The absorption peak intensities corresponding to the vibration modes of ethanol molecule around 1067, 1395, 2901 and 2988 cm-1 continuously became higher until the equilibrium state. At such absorption peaks, the linearity was observed between the natural logarithm of the absorption at equilibrium state and the reciprocal of the absolute temperature of the sample, and the influence of the ethanol concentration on the slope value was negligible. In addition, for the Arrhenius plot using the rate constant obtained under the assumption that the kinetic volatile behavior of ethanol could be expressed by the first order reaction equation, the slope values could be constant among the samples used in this study. These results suggested that the influences of the ethanol concentration in the aqueous solution and the temperature on the kinetic volatile behavior of ethanol could be grasped based on the infrared spectroscopic information.
Boiled red beans (azuki beans) called as "Niazuki" is very popular in Japan. After dipping the beans in water at the room temperature, the beans are boiled. Usually, the dipping water is once disposed in order to decrease the astringent ingredients in boiled red beans, and this operation called as "Shibukiri". However, the drained water contains not only astringent ingredients but also the functional ones. The heating method without "Shibukiri" operation has been then proposed empirically, and the scientific and objective method for the quality evaluation through the "Niazuki" production process is desirable. The aim of this study is to grasp the quality change behavior of the boiled red beans during the heating process using an infrared spectroscopic method, which could be based on the information of vibrational modes between molecular compounds and be simple, rapid, non-chemical and non-destructive.
Firstly, we constructed the stable and quantitative infrared spectroscopy measurement method of the cross section of the boiled red beans using a Fourier transform infrared spectrometer equipped with an attenuated total reflection accessory. Additionally, by comparing the spectral feature changes of the cross section of the red beans during heating with those of the boiled soups, the spectral feature changes of the red beans during heating were experimentally suggested to be quantitatively evaluated based on the soup spectral information that could be easily collected during the heating operation. Moreover, the infrared spectral absorption bands relating to the astringent ingredients in the soup could be extracted by performing the sensory evaluation test. Consequently, this study could be a very important step in the developments of the infrared spectroscopic monitoring for the control of the heating process of the "Niazuki" production.
1-Methylcyclopropene (1-MCP) is known as ethylene inhibitor agent that effective to delay ripening rate in fruits. Controlled release packaging with 1-MCP as active compound is an alternative method to extend the shelf life of fruits. Currently, 1-MCP coated paper has been investigated to develop the functional packaging for fruits. Therefore, this research is aimed to investigate the effectiveness 1-MCP coated paper during preservation of apple.
The formation of 1-MCP coated paper was done by dissolving a weight of 1-MCP inclusion complexes (1-MCP ICs) powder into 0.5 mL of shellac solution. The effectiveness of 1-MCP coated paper were examined in the apple fruits with different weight of 1-MCP ICs powder: 5, 10, 30 and 50 mg for over 30 days. All EPS were closed and stored at 4 °C for initial 15 days. Thereafter, apples were transferred to 20 °C storage temperature for another 15 days. After this treatment, the apples were stored at 20 °C for another 15 days to evaluate shelf life of apple and were also measured the physicochemical properties.
The results showed that apples treated with 1-MCP coated paper inhibited the increase in ethylene production rate during storage time compared to control. During storage time at 4 °C, the release of ethylene from apples with treatment of 1-MCP coated paper were less than the control apples. Low ethylene production rate of 0.22 nL/g FW/h could be observed at 50 mg of 1-MCP ICs powder. On the other hand, for the control apples without treatment of 1-MCP coated paper, the ethylene production rate was 44.7 nL/g FW/h. The finding results indicated that 1-MCP coated paper with 50 mg of inclusion complex was effective to delay the ripening rate and maintain the quality of apple during storage time.
Sweet potato has individual differences in property such as shape, starch content, amount of enzyme. When baked using household cooking tools, the taste differs depending on the individual property even under the identical cooking condition. This study aims at predicting suitable cooking condition of sweet potato for maximizing on the maltose production by the enzymatic reaction during cooking process. The sweetness of sweet potato mainly contributed by maltose. Starch contained in sweet potato is gelatinized by heating, and maltose is generated by the hydrolysis of the gelatinized starch by β-amylase. Gelatinization is accelerated by heating, whereas β-amylase is inactivated by heat. Therefore, heating condition must be optimized in order to maximize resultant amount of maltose. First, in order to investigate the gelatinization kinetics under non-isothermal condition, the degree of gelatinization was measured under selected heating rate. A diagram of the gelatinization rate as a function of temperature and gelatinization degree was prepared to predict the gelatinization behavior of sweet potato at selected cooking condition. Second, the inactivation kinetics of β-amylase was separately measured after extracting the enzyme from a sweet potato. The measurement was carried out under isothermal condition at selected temperatures. The obtained results can predict the remaining enzymatic activity throughout cooking process. Finally, the heat transfer in sweet potato under oven cooking was calculated by the finite element method by counting heat balance between the heat received by the sweet potato from the oven and the heat used to evaporate on the sweet potato surface. Based on the simulated temperature profiles, the amount of maltose produced as a result of baking was predicted. This simulation could be useful to optimize a cooking condition that controls the amount of targeted components.
Freeze-drying is known as one of the best drying method for maintaining original product qualities. Each product has a particular operational temperature limit known as collapse temperature. When temperature of in-drying product reaches over collapse temperature, structural collapse occurs and it may lead quality loss of products. However, some extent of collapse is more or less accepted in industry if it does not lead serious quality defect. In this study, model freeze-dried food products were prepared and influence of the degree of collapse during freeze-drying on quality of products was examined. Samples with different collapse temperatures were obtained by changing the amount of sodium chloride. Collapse temperature was defined as a point at which the significant electrical conductivity change was observed in the temperature ramp test from -60°C to 10°C A linear correlation was confirmed between collapse temperature and the degree of collapse that was evaluated by alternative test (i.e. the tapping test) on a prepared sample freeze-dried at fixed drying condition. Prepared samples confirmed flavor components, that is methionol and hexanoic acid. The amount of flavor components before and after freeze-drying was measured by gas chromatography. It found that the collapse temperature was clearly correlated with the retention of methionol. On the other hand, the retention of hexanoic acid did not obviously change depending on the collapse temperature. This result indicated that the degree of collapse affects the retention of some flavor components. The present study clarifies a rational choice in better freeze-drying conditions that realize product quality at desired level.
Potato pulp is an agricultural waste containing large amount of arabinogalactan. The compound is known to behave as prebiotics which modify the gut microbiota as nutrients to support the growth of lactic acid bacteria such as the genus Lactobacillus and Bifidobacterium. Potato-pulp-arabinogalactan "PPA" have not used as probiotics commercially. The development of a cost-effective PPA extraction is promise for the industrial application. In this study, the effect of the extracting procedures were investigated on the growth of gut-microflora by in vitro experiments. Crude saccharides "CS" were prepared by 50 g dried potato pulp suspended in 1 L of pure water. The suspensions were autoclaved at 121°C for 2 h, and filtered by a filter paper. The filtrate were concentrated around 1/4 by a rotary evaporator, and aggregated by 4 × volume of ethanol. Dialyzed saccharides (DS) were prepared to dialyzed CS by a dialysis membrane (cut off MW < 10,000). Enzyme-treated saccharides (ES) were prepared to digest CS by a crude amylase, Gruku-Gin (Amano Enzyme) and dialyzed. All saccharides were lyophilized or heat-dried to store. According to size-exclusion chromatography, the molecular distribution of CS were between 400 and 200,000. A peak of oligosaccharides was observed around MW 800. The peak were disappeared in charts of DS and ES. Based on the saccharide composition profiling after acid hydrolysis, glucan were occupied 50% in the total amount of glycan in CS and DS, and decreased to approx. 5% in ES. When thirteen strains of gut microbes were cultivated in GAM broth including the saccharides instead of glucose, CP and EP selectively stimulated several non-pathogenic strains in the genus Bifidobacterium and Clostridium, but not pathogenic Enterobacteriaceae. From the results, PPA would behaved as prebiotics without dependence of the extracting procedures, and can be extract by further simple processes.
There is currently great interest in the study of compounds from food sources with biological activities, as these compounds are generally considered highly safe. In particular, polyphenols are thought to have various health benefits and are found in many health foods, as well as in vegetables and fruits. However, the structure-activity relationships (SARs) of polyphenols are not well understood, because they are typically obtained as a mixture of various analogs, which makes purification difficult.
"Tannin" is a mixture extracted from plants and contains a large amount of water-soluble polyphenols that is used in the plant 窶徼anning窶・process of converting animal skin to leather, which relies on the polyphenol–collagen protein interaction in the skin. We have previously confirmed that polyphenol compounds having a galloyl group and high radical scavenging activity interact more strongly with gelatin (the material obtained after the acid or basic hydrolysis of collagen). Therefore, it was thought that it could be possible to predict the functionality of the polyphenol compound present in the mixture by confirming whether or not it binds to gelatin.
Results and discussion
Green tea leaves and raspberries were selected as plants containing polyphenol compounds. Green tea leaves are rich in polyphenol compounds such as tea catechin, (–)-epigallocatechin-3-O-gallate (EGCG) (1). Raspberry also contains many kinds of polyphenol compounds such as condensed tannin and hydrolyzable tannin in addition to (+)-catechin (2), (–)-epicatechin (3). Green tea leaves and raspberries were each extracted with methanol to prepare a polyphenol crude extract. The compounds bound when gelatin was added to these extracts were a group of compounds that tended to have high radical scavenging activity. It was suggested that functionality such as radical scavenging activity can be predicted by confirming whether it is strongly bound to gelatin.
Ion channels are important membrane proteins that allow ions to pass through the channel pore for cell function regulation. Their protein functions include establishing the control of electric potentials accompanying with the ligand binding to the channel and the opening/closing the pore. Currently, while the expansion of ion channel measurement demands for cell function evaluation and regulation, the intact membrane fabrication on conductive substrate, especially for the cytoplasmic face analysis, is still challenging.
In this study, we investigated the design of peptide interface between cell membrane and highly oriented pyrolytic graphite (HOPG) substrate. By using the bifunctional peptides capable of substrate binding and strong cell membrane binding, immobilization of cell membrane attaching onto substrate after osmotic shock was evaluated.
In order to screen cell membrane strong binding peptides, we designed peptide array that allow multi-peptides synthesis on a cellulose membrane and binding assay with NIH-3T3 cells was conducted. Screened candidate peptide was fused with HOPG binding peptide and evaluated the bi-functionality expression using chemically synthesized peptide. After the surface modification of HOPG with peptide candidates, cultured NIH-3T3 cell was sowed and cultured. Prior to the cell disruption, cell membrane was stained by Cell mask orange and the cell culture surface was soaked in cold MilliQ for osmotic shock. The cell membrane structure was observed with fluorescent microscope.
Herein, we show that bi-functional peptides screened using peptide array are suitable interface enabling surface functionalization just by dropping peptide solution on the graphite. The peptide interface revealed some superior properties for cell culturing and for stable immobilization of cell membrane attaching onto substrate even after the cell disruption process. The developed peptide-based interfaces on HOPG substrate open the door for new biosensor developments for ion channel activity evaluation from the stimuli by intracellular ligand molecules.
n-3 Fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been associated with cardiovascular function and other diseases. However, EPA and DHA are easily prone to oxidation due to many double bonds. This study focused on the oxidation stability of EPA and DHA encapsulated with maltodextrin by spray drying. Krill oil was emulsified with saponin and sodium caseinate emulsifiers by mechanical and high-pressure methods and then the emulsion was spray dried in an Ohkawara L-8 spray dryer to produced powders. The oil droplet size after the spray drying was measured as well as the amount of surface oil, the oxidation stability and the retention of the EPA and DHA after storage at 25, 50 or 70 °C for 1, 2 and 4 weeks. The results showed that the amount of surface oil and oil-droplet size affected the oxidation stability of krill oil in spray-dried powders. Krill oil of small oil droplet size was more stable than that of large one.
DHA, EPA, Krill oil, Oxidation stability, Powder, Spray dryer
Recent advancement of genome-editing technology allows the site-specific integration of gene of interest (GOI). The information of genomic loci that support high level expression of GOI, in combination with such genome-editing technology, will be useful to rapidly establish good CHO cell lines suitable for the production of biopharmaceuticals. In this study, we aimed to screen the DNA regions that strongly express transgenes.
CHO-K1 cells were obtained from Riken Cell Bank and maintained in F12 medium supplemented with 10% fetal calf serum and antibiotics. HIV- or mouse stem cell virus-based vectors that expressed eGFP under the control of CMV promoter were produced. CHO cells were infected with the vectors in low multiplicity of infection to prevent multiple integrations. Cells with strong fluorescence intensity were sorted by flowcytometer and expanded for further analysis. Integration sites were determined by inverse-PCR. To integrate model antibody expression unit, reverse reaction of Cre recombinase on mutated lox P sequence or CRISPR/Cas9 integration was performed.
3. Results and discussion
Several integration sites were determined for the clones with high/low expression. The integration sites of lentivirus vectors are not completely random (preferences: transcribed region). Chromosome immunoprecipitation analysis of parental CHO cells suggested that some histone modification was higher in integration sites of high-expressing clones, suggesting that genomic loci that support high level expression of GOI were partly related to specific epigenetic status. The expression of model antibody was higher in higher eGFP-expressing locus.
This research is partially supported by the developing key technologies for discovering and manufacturing pharmaceuticals used for next-generation treatments and diagnoses both from the Ministry of Economy, Trade and Industry, Japan (METI) and from Japan Agency for Medical Research and Development (AMED).
Meropenem is an antibacterial drug used as a final-line against multidrug-resistant bacterial infections. In general, overdose of antibacterials damages kidney, but underdose allows creation of the resistant bacteria. Thus, therapeutic drug monitoring (TDM) of antibacterials are strongly required. Development of TDM of meropenem is urgent because there is no effective therapeutic method against infection of meropenem-resistant bacteria. Thus, we are developing a real-time meropenem sensor using molecularly imprinted polymer (MIP). A MIP is synthetic polymer whose matrix is imprinted with structure of the target molecule during the synthesis process. Copolymer of methacrylic acid (functional monomer) and N,N-methylenebisacrylamide (MBAA: crosslinking monomer) imprinted with meropenem was grafted on the surface of graphite particles. The particles ware mixed with silicone oil to prepare a carbon paste electrode for sensing meropenem. Cyclic voltammetry of 5 mM potassium ferrocyanide was performed with the electrode. Fig. (A) shows the correlation between the current and the concentration of meropenem or imipenem which is the analogue of meropenem. Both of the currents depended on meropenem and imipenem with the similar sensitivity We thought that the poor selectivity was due to the high hydrophilicity and flexibility of MBAA. Therefore, ethylene glycol dimethacrylate (EDMA), which is more hydrophobic crosslinker, and MBAA were blended (molar ratio of 1:1) to prepare a new MIP carbon paste electrode, and the same experiment was performed. As shown in Fig. (B), the current at this electrode was approximately twice as sensitive to meropenem as to imipenem. Therefore, more rigid MIP of the electrode, may detect meropenem without interaction in blood and will enable the TDM of meropenem.
Bioreactor design for Solid-State Fermentation of lignocellulosic biomass using filamentous fungi needs mathematical models of their growth on solids, but these are currently unreliable due to difficulty in measuring the mass of fungal mycelia infiltrating the media. To solve this problem, we have developed a method for determining the biomass of Aspergillus niger and the cellulase hyperproducer Trichoderma reesei RUT C30 growing on solid lignocellulosic substrate, using real-time Polymerase Chain Reaction (rt-PCR). This method relates fungal genomic DNA to biomass based on the automatic duplication of nuclei (mitosis) in filamentous fungi when the ratio of cytoplasmic volume to number of nuclei exceeds a critical value. The rt-PCR assay used primers and probes unique to the individual fungal species. Candidate primer-probe sets were designed with either Primer-BLAST (NCBI) or PrimerQuest (IDT), and their amplification of the target DNA sequences verified by conventional PCR before selection for rt-PCR. The specificity of each primer-probe combination was also challenged using other fungal species. The measured amounts of DNA were converted to fungal biomass using an experimentally-determined DNA-to-biomass ratio for the mycelia of T. reesei and A. niger. The growth curves of each fungal species were obtained by separately cultivating them on wheat straw, with sampling at set time intervals. The T. reesei and A. niger DNA in the samples were quantified with the rt-PCR assay, and the equivalent fungal biomass calculated. The resulting growth curves showed the expected lag, exponential, and decline phases. The specific growth rates of T. reesei and A. niger were calculated from their corresponding exponential phases.
In this work, sodium nitroprusside-releasing chitosan-based (CS/SNP) nanofibers were fabricated via electrospinning. Prepared CS/SNP nanofibers were capable of sustainably releasing 37 μg SNP/mg for up to 7 days. SNP is known to release nitric oxide (NO), a radical of interest in bone tissue engineering, upon reduction and photo-degradation. NO–releasing nanofibers have been prepared previously by other groups, however their applicability to bone tissue engineering has never been investigated. This work serves to fill this gap.
To improve nanofiber stability and mechanical properties, one-step photo-crosslinking of blended CS/SNP nanofibers was carried out by addition of tetraethylene glycol diacrylate (TTEGDA) and 2,2-dimethoxy-2-phenylacetophenone (DMPA), and incorporation of UV irradiation into the electrospinning process. Photo-crosslinked nanofibers were characterized via scanning electron microscopy (SEM), Fourier transform infrared sprectroscopy and swelling test. Application of photo-crosslinking was found to significantly improve nanofiber stability in aqueous environments. SEM images revealed that the porous nanofibrous structure could be maintained up to 24 hours. Biocompatibility of CS/SNP nanofibers towards mouse osteoblasts was also significantly improved.
Addition of SNP into the nanofibrous scaffolds were found to improve their biocompatibility to osteoblasts and gingival fibroblasts (GF). Cell viability of 7F2 mouse osteoblasts and human GF cells were affected by SNP content in a dose- and time-dependent manner. MTT assays revealed that 7F2 cell viability increased with increasing SNP content, whereas GF cell viability peaked in CS/20% SNP nanofibers. Fluorescence microscope images also revealed that CS/SNP nanofibers improved cell attachment, spreading and proliferation. Osteogenic differentiation and mineralization were also enhanced by the nanofibers, as evidenced by elevated expressions of osteogenic differentiation markers including alkaline phosphatase (ALP), osteopontin (OPN) and calcium. Photo-crosslinked electrospun CS/SNP nanofibers are thus shown to have excellent potential as bone tissue engineering scaffolds
In this study, electrospun polyvinylidene fluoride (PVDF) with 1,2 and 3% of zinc oxide nanoparticles with enhanced piezoelectric properties was fabricated and the optimum scaffold was identified. Then, dexamethasone-loaded chitosan nanoparticles were dispersed in polycaprolactone (PCL) solution and the final scaffold was electrospun using dual electrospinning setup and the fibers were characterized using X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM). Finally, the drug release from chitosan nanoparticles was investigated. The results showed that the PVDF fibers with 1% ZnO nanoparticles enhances the piezoelectric properties of the scaffold up to 68%. Also, the young's modulus PVDF/ZnO and PCL scaffolds reached to 38.4 MPa. Controlled drug delivery of the PCL/PVDF/ZnO fibers with 0,0.4, 0.8, 1.2% chitosan nanoparticles was carried out in a period of 14 days, and the results showed that the by incorporating the chitosan nanoparticles, burst release of dexamethasone could be diminished. Moreover, bone marrow-derived mesenchymal stem cells (MSCs) cultivation on electrospun scaffold with dexamethasone nanoparticles resulted in increased cell growth during 14 days culturing. Furthermore, the obtained results revealed that adding piezoelectric ZnO nanoparticles and released dexamethasone from the scaffold could increase the alkaline phosphatase activity in a dose dependent manner which may show the effect of the fabricated scaffold on MSCs differentiation toward bone leneage. In summary, the result of this study shows that the composite fibers of PCL/PVDF/ZnO incorporated with dexamethasone-loaded chitosan nanoparticles can be used as a potential bioactive scaffold for bone tissue engineering.
This study focuses on the synthesis and characterization of silica coated magnetic iron oxide nanoparticles (IONP-SiO2) through a hydrothermal method. These nanoparticles will act as carriers of doxorubicin (DOX) drug for the treatment of liver cancer. Core-shell of monodispersed with spherical particle sizes of about 20-50 nm in diameter were obtained using the CryoTEM. Furthermore, the XRD patterns suggested the crystallization of the prepared nanoparticles with diffraction peak of at 2θ = 35.44 ° corresponded to that of magnetite (Fe3O4) (311). The XANES spectra of the Fe atom in the different samples demonstrated an absorbance feature (Fe = 7112 eV) of a 1s to 3d transition. The characteristic bands almost at 1085, 800 and 460 cm-1 are correspond to the stretching, bending and out of plane of Si-O bonds, respectively, indicating the successful coating of SiO2 on the surface of IONPs. The small angle neutron scattering (SANS) spectra revealed SiO2' poor thermo-sensitivity; however after surface modification with temperature sensitive Pluronic P123, changes in the core-shell structure were observed. SANS studies, help us to predict the changes in the core-shell structure of the nanoparticles as results of drug loading and release. Based on using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, no significant cell toxicity was observed among the cells (293T, HepG2 and Huh7) treated with different concentrations of IONPs and IONP-SiO2. Interestingly, MTT test showed that after DOX encapsulation, the IONP-SiO2-DOX complex was able to induce more cell apoptosis. This study presents an interdisciplinary work to establish new formulation of DOX in treatment of liver cancer with consequent improvement treatment. IONP-SiO2 based drug delivery system would be a potential tool to enhance targeting delivery of conventional chemotherapeutic drugs and MRI imaging.
Keywords: Drug delivery, Magnetic iron oxide nanoparticles, Silica oxide, Pluronic P123, Doxorubicin, Liver cancer.
Liver cancer remains a leading cause of cancer-related mortality worldwide, claiming the lives of millions globally. It accounts for about 9.1% of all cancer deaths worldwide with 782,000 new cases diagnosed in 2012. Application of nano-drug delivery system (NDDS) has attracted remarkable attention in the medical field. The NDDS can not only improve targeted drug delivery to the liver or improve the bioavailability of drug, but also can reduce the side effects of chemotherapy. Therefore, the objective of the study was to develop dextran and pluronic F127 stabilized magnetic iron oxide nanoparticles (DIONPs and PIONPs) through a solvothermal method. The developed nanoparticles will be used as carriers of doxorubicin (DOX) drug for the treatment of liver cancer. Furthermore, the XRD patterns indicated the crystallization of the prepared nanoparticles with diffraction peak of at 2θ = 35.44 ° corresponded to that of magnetite (Fe3O4) (311). XANES spectra of the Fe atom in the different samples depicted an absorbance feature (Fe = 7112 eV) of a 1s to 3d transition. The SANS spectra was used to probe changes in the core-shell structure of dextran/pluronic F127 micelles as a function of increasing DOX and polymer concentrations in order to predict the drug loading and release mechanisms. Regarding the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, no significant cell toxicity was observed among the cells (293T, HepG2 and Huh7) treated with different concentrations of IONPs, DIONPs, and PIONPs. This study established a multidisciplinary work to establish new formulation of DOX in treatment of liver cancer with consequent improvement treatment. DIONPs and PIONPs based drug delivery system would be a potential tool to enhance targeting delivery of conventional chemotherapeutic drugs and MRI imaging.
Keywords: Drug delivery, Magnetic iron oxide nanoparticles, Dextran, Pluronic F127, Doxorubicin, Liver cancer.
Protease from Janthinobacterium sp. isolated from Antarctic region was purified. At the first step, gradient precipitation was carried out. and it was confirmed that protease was deposited in the 30–80% range of ammonium sulfate. Next, DEAE-Sepharose column was used for the binding of the protease under various conditions. The protease was purified with 29% recovery yield. This result can be useful for the purification of other cold-adapted protein.
Expression, purification, and characterization of lipase from the psychrophile, Janthinobacterium sp. were investigated. The gene encoding lipase from Janthinobacterium sp. was cloned into a pET28a(+) vector and heterologously expressed in Escherichia coli BL21 (DE3). Recombinant E. coli harboring the Janthinobacterium lipase gene were induced by addition of isopropyl-β-D-thiogalactopyranoside, and Ni2+-NTA affinity chromatography was used to purify the lipase. The obtained specific activity was 107.9 U/mg protein. The effect of temperature and pH on the activity of lipase was measured. The stability of the lipase at low temperatures indicated it is a cold-adapted enzyme. Analysis of the lipase activity using various p-nitrophenyl esters showed a strong preference toward short acyl chains of the esters, indicating the ability of the cold-adapted lipase to hydrolyze short-chain esters.
Polysaccharides, also known as glycans, are the most abundant form of carbohydrate materials in marine algae. A semi-continuous system, i. e., with fixed harvesting frequency, was employed to cultivate Halamphora sp. for the production of cell mass and polysaccharides. The first three cultivation sets (set1 – set3) are performed according to different harvesting volumes (2, 3 or 1 L) per 3 days and three more sets (set4 – set6) were performed with the addition of Na2CO3 and a harvesting volume of 1, 2 or 3 L per 3 days. At least, six continuous batches were run for each set of cultures. The addition of Na2CO3 increases both cell mass and polysaccharide production. A high average cell concentration (0.61 g/L) and a high average specific polysaccharide production (44.68 g/g dried cell weight) were found for the cultures in set 5, i. e., with the addition of Na2CO3 and a harvesting volume of 2 L per 3 days. The polysaccharides produced by diatom are divided into intracellular, adhesive and extracellular polysaccharides. Among them, soluble intracellular carbohydrates were applied to an anion-exchange resin for polysaccharide characterization. Following anion-exchange chromatography, three soluble intracellular polysaccharides extracted from Halamphora sp. were eluated at different NaCl concentrations (0.1, 0.5, or 1M). Based on uronic acid detection, FT-IR and monosaccharides composition analysis, the polysaccharide eluated by 1M of NaCl is mainly composed of fucose and galactose, and is a sulfated acid polysaccharide . The polysaccharide eluated by 0.1M of NaCl is mainly composed of glucose, fucose and galactose and is a neutral polysaccharide.
We believe they are many broad candidates available in nature, which can move beyond difficulties to out boundary so that the good platform for fermentation of lignocellulosic biomass could be built without too much genetic modification. However, it is not easy to achieve the candidates due to the unique adaptation possessed by many yeasts upon the environmental stress.
In this study, we have developed the simultaneous method for screening the candidate yeasts, which capable to grow and ferment lignocellulosic lysate into ethanol and fine chemicals such as lactate. By using culture collection in National Bio-resource Collection (NBRC) Japan, and Indonesian National Culture Collection (InaCC), the screening of the yeast strain, having a potential of growing and fermentation in the medium containing fermentation inhibitors, was conducted. Regarding the possibility of bio-resources in Asian for bio-refinery, we would like to discuss also in this presentation.
In view of the energy shortage, the idea of developing and utilizing renewable and clean energy solar energy is put forward. Combining with the artificial chloroplast technology which has been successfully realized, this project proposes to apply the artificial photosynthesis using solar energy to the synthesis of chemical products. Ethylene, a basic chemical product widely used in chemical industry, is selected as the target product of solar power plant. The research ideas are put forward as follows: Using the related genes of EFE pathway (ethylene-forming enzyme as the key enzyme) from Pseudomonas as the source of ethylene synthesis genes, the obtained ethylene synthesis genes will be introduced into host cells and express stably. Dunaliella salina and cyanogreen algae are selected as host cells to compare differences between eukaryotic expression system and prokaryotic expression system in ethylene synthesis. In the reaction network for ethylene synthesis, the key genes of the step will be modified to increase the yield of the ethylene synthesis reaction by combining with the analysis of metabolic network to find a key rate-control step. At the same time, by the modification of the genes, the possibility of coupling the tricarboxylic acid cycle and the ethylene synthesis in the eukaryotic expression system may be increased. Besides the ethylene-producing research in Dunaliella salina, other photosynthetic cells with different light-absorbing pigments were used to analyze the light-harvesting antenna synthesis gene. The gene will be used to broaden the absorbing spectral range of the ethylene-producing Dunaliella salina, and to optimize the photosynthetic system after increasing the light absorption rate, so as to further improve the utilization efficiency of light energy, ethylene yield and even achieve sustainable development.