A Quantitative Method for Acesulfame K Using the Taste Sensor.

We have developed a method to quantify the sweetness of negatively charged high-potency sweeteners coexisting with other taste substances. This kind of sweetness sensor uses lipid polymer membranes as the taste-sensing part. Two types of outputs have been defined in the measurement of the taste sensor: one is the relative value and the other is the CPA (the change in membrane potential caused by adsorption) value. The CPA value shows a good selectivity for high-potency sweeteners. On the other hand, the relative value is several times higher than the CPA value, but the relative value is influenced by salty substances. In order to obtain both high sensitivity and selectivity, we established a model for predicting the concentration of sweeteners with a nonlinear regression analysis method using the relative values of both the sweetness sensor and the saltiness sensor. The analysis results showed good correlations with the estimated concentration of acesulfame potassium coexisting with salty substances, as represented by R2 = 0.99. This model can correspond well to the prediction of acesulfame K in a concentration of 0.2-0.7 mM, which is commonly used in food and beverages. The results obtained in this paper suggest that this method is useful for the evaluation of acesulfame K using the taste sensors.

Development of Taste Sensor to Detect Non-Charged Bitter Substances.

A taste sensor with lipid/polymer membranes is one of the devices that can evaluate taste objectively. However, the conventional taste sensor cannot measure non-charged bitter substances, such as caffeine contained in coffee, because the taste sensor uses the potentiometric measurement based mainly on change in surface electric charge density of the membrane. In this study, we aimed at the detection of typical non-charged bitter substances such as caffeine, theophylline and theobromine included in beverages and pharmaceutical products. The developed sensor is designed to detect the change in the membrane potential by using a kind of allosteric mechanism of breaking an intramolecular hydrogen bond between the carboxy group and hydroxy group of aromatic carboxylic acid (i.e., hydroxy-, dihydroxy-, and trihydroxybenzoic acids) when non-charged bitter substances are bound to the hydroxy group. As a result of surface modification by immersing the sensor electrode in a modification solution in which 2,6-dihydroxybenzoic acid was dissolved, it was confirmed that the sensor response increased with the concentration of caffeine as well as allied substances. The threshold and increase tendency were consistent with those of human senses. The detection mechanism is discussed by taking into account intramolecular and intermolecular hydrogen bonds, which cause allostery. These findings suggest that it is possible to evaluate bitterness caused by non-charged bitter substances objectively by using the taste sensor with allosteric mechanism.

Development of a Sensor with a Lipid/Polymer Membrane Comprising Na+ Ionophores to Evaluate the Saltiness Enhancement Effect.

The saltiness enhancement effect is the effect whereby saltiness is enhanced by adding specific substances to salt (sodium chloride). Since this effect can be used in the development of salt-reduced foods, a method to objectively evaluate the saltiness with this effect is required. A taste sensor with lipid/polymer membranes has been used to quantify the taste of food and beverages in recent years. The sensor electrodes of this taste sensor have the feature of selectively responding to each of the five basic tastes, which is realized by the lipid/polymer membranes. In this study, we developed a new saltiness sensor based on the lipid/polymer membrane with the aim of quantifying the saltiness enhancement effect. In addition to the conventional components of a lipid, plasticizer, and polymer supporting reagent, the membrane we developed comprises ionophores, which selectively capture sodium ions. As a result, the response of the sensor increased logarithmically with the activity of NaCl in measured samples, similarly to the taste response of humans. In addition, all of the sensor responses increased upon adding saltiness-enhancing substances, such as citric acid, tartaric acid and branched-chain amino acids (BCAAs), to NaCl samples. These findings suggest that it is possible to quantify the saltiness enhancement effect using a taste sensor with lipid/polymer membranes.

Improved Durability and Sensitivity of Bitterness-Sensing Membrane for Medicines.

This paper reports the improvement of a bitterness sensor based on a lipid polymer membrane consisting of phosphoric acid di-n-decyl ester (PADE) as a lipid and bis(1-butylpentyl) adipate (BBPA) and tributyl o-acetylcitrate (TBAC) as plasticizers. Although the commercialized bitterness sensor (BT0) has high sensitivity and selectivity to the bitterness of medicines, the sensor response gradually decreases to almost zero after two years at room temperature and humidity in a laboratory. To reveal the reason for the deterioration of the response, we investigated sensor membranes by measuring the membrane potential, contact angle, and adsorption amount, as well as by performing gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS). We found that the change in the surface charge density caused by the hydrolysis of TBAC led to the deterioration of the response. The acidic environment generated by PADE promoted TBAC hydrolysis. Finally, we succeeded in fabricating a new membrane for sensing the bitterness of medicines with higher durability and sensitivity by adjusting the proportions of the lipid and plasticizers.

Study of the relationship between taste sensor response and the amount of epigallocatechin gallate adsorbed onto a lipid-polymer membrane.

A taste sensor using lipid-polymer membranes has been developed to evaluate the taste of foods, beverages and medicines. The response of the taste sensor, measured as a change in the membrane potential caused by adsorption (CPA), corresponds to the aftertaste felt by humans. The relationships between the CPA value and the amount of adsorbed taste substances, quinine and iso-α acid (bitterness), and tannic acid (astringency), have been studied so far. However, that of epigallocatechin gallate (EGCg) has not been clarified, although EGCg is abundantly present in green tea as one of its astringent substances. This study aimed at clarifying the response of the taste sensor to EGCg and its relationship with the amount of EGCg adsorbed onto lipid-polymer membranes. The lipid concentration dependence of the CPA value was similar to that of the amount of adsorbed EGCg, indicating a high correlation between the CPA value and the amount of adsorbed EGCg. The CPA value increased with increasing amount of adsorbed EGCg; however, the CPA value showed a tendency of leveling off when the amount of adsorbed EGCg further increased.

Analysis of a Lipid/Polymer Membrane for Bitterness Sensing with a Preconditioning Process.

It is possible to evaluate the taste of foods or medicines using a taste sensor. The taste sensor converts information on taste into an electrical signal using several lipid/polymer membranes. A lipid/polymer membrane for bitterness sensing can evaluate aftertaste after immersion in monosodium glutamate (MSG), which is called "preconditioning". However, we have not yet analyzed the change in the surface structure of the membrane as a result of preconditioning. Thus, we analyzed the change in the surface by performing contact angle and surface zeta potential measurements, Fourier transform infrared spectroscopy (FTIR), X-ray photon spectroscopy (XPS) and gas cluster ion beam time-of-flight secondary ion mass spectrometry (GCIB-TOF-SIMS). After preconditioning, the concentrations of MSG and tetradodecylammonium bromide (TDAB), contained in the lipid membrane were found to be higher in the surface region than in the bulk region. The effect of preconditioning was revealed by the above analysis methods.

Development of a sweetness sensor for aspartame, a positively charged high-potency sweetener.

Taste evaluation technology has been developed by several methods, such as sensory tests, electronic tongues and a taste sensor based on lipid/polymer membranes. In particular, the taste sensor can individually quantify five basic tastes without multivariate analysis. However, it has proven difficult to develop a sweetness sensor, because sweeteners are classified into three types according to the electric charges in an aqueous solution; that is, no charge, negative charge and positive charge. Using membrane potential measurements, the taste-sensing system needs three types of sensor membrane for each electric charge type of sweetener. Since the commercially available sweetness sensor was only intended for uncharged sweeteners, a sweetness sensor for positively charged high-potency sweeteners such as aspartame was developed in this study. Using a lipid and plasticizers, we fabricated various lipid/polymer membranes for the sweetness sensor to identify the suitable components of the sensor membranes. As a result, one of the developed sensors showed responses of more than 20 mV to 10 mM aspartame and less than 5 mV to any other taste. The responses of the sensor depended on the concentration of aspartame. These results suggested that the developed sweetness sensor had high sensitivity to and high selectivity for aspartame.

Relationship between the amount of bitter substances adsorbed onto lipid/polymer membrane and the electric response of taste sensors.

The bitterness of bitter substances can be measured by the change in the membrane electric potential caused by adsorption (CPA) using a taste sensor (electronic tongue). In this study, we examined the relationship between the CPA value due to an acidic bitter substance and the amount of the bitter substance adsorbed onto lipid/polymer membranes, which contain different lipid contents, used in the taste sensor. We used iso-α-acid which is an acidic bitter substance found in several foods and beverages. The amount of adsorbed iso-α-acid, which was determined by spectroscopy, showed a maximum at the lipid concentration 0.1 wt % of the membrane, and the same phenomenon was observed for the CPA value. At the higher lipid concentration, however, the amount adsorbed decreased and then remained constant, while the CPA value decreased monotonically to zero. This constant adsorption amount was observed when the membrane potential in the reference solution did not change with increasing lipid concentration. The decrease in CPA value in spite of the constant adsorption amount is caused by a decrease in the sensitivity of the membrane as the surface charge density increases. The reason why the peaks appeared in both the CPA value and adsorption amount is based on the contradictory adsorption properties of iso-α-acid. The increasing charged lipid concentration of the membrane causes an increasing electrostatic attractive interaction between iso-α-acid and the membrane, but simultaneously causes a decreasing hydrophobic interaction that results in decreasing adsorption of iso-α-acid, which also has hydrophobic properties, onto the membrane. Estimates of the amount of adsorption suggest that iso-α-acid molecules are adsorbed onto both the surface and interior of the membrane.

Development of a portable taste sensor with a lipid/polymer membrane.

We have developed a new portable taste sensor with a lipid/polymer membrane and conducted experiments to evaluate the sensor's performance. The fabricated sensor consists of a taste sensor chip (40 mm × 26 mm × 2.2 mm) with working and reference electrodes and a portable sensor device (80 mm × 25 mm × 20 mm). The working electrode consists of a taste-sensing site comprising a poly(hydroxyethyl)methacrylate (pHEMA) hydrogel layer with KCl as the electrolyte layer and a lipid/polymer membrane as the taste sensing element. The reference electrode comprises a polyvinyl chloride (PVC) membrane layer with a small hole and a pHEMA layer with KCl. The whole device is the size of a USB memory stick, making it suitable for portable use. The sensor's response to tannic acid as the standard astringency substance showed good accuracy and reproducibility, and was comparable with the performance of a commercially available taste sensing system. Thus, it is possible for this sensor to be used for in-field evaluations and it can make a significant contribution to the food industry, as well as in various fields of research.

Scalable spin Seebeck thermoelectric generation using Fe-oxide nanoparticle assembled film on flexible substrate.

We fabricated Fe3O4 nanoparticle (NP)-assembled films on flexible polyimide sheets with Pt or Ta cap layer using a spin coating method and DC sputtering. The films were elaborated for spin Seebeck thermoelectric generator applications, and their spin Seebeck voltages were observed. We showed that the thermoelectric power of [Pt film/Fe3O4 NP]n multilayered films increases with increasing number of stacking n. Additionally, we prepared spin Seebeck thermopile devices in which the Fe3O4 NP-assembled films capped by Pt and Ta are connected alternately in series. We demonstrated that spin Seebeck voltages of the thermopile devices are larger than those of single [Pt or Ta film/Fe3O4 NP]n piece. Our results indicate that the spin Seebeck thermoelectric power of Fe3O4 NPs can be enhanced using a simple fabrication process without lithography technique.

Development of two-dimensional qualitative visualization method for isoflavones secreted from soybean roots using sheets with immobilized bovine serum albumin.

A visualization method for the qualitative evaluation of soybean isoflavones secreted from soybean roots by transferring them onto a sheet with immobilized bovine serum albumin (BSA) was developed. BSA was chemically bonded onto a glass microfiber filter. The fluorescence quenching resulting from the interaction of BSA with soybean isoflavones such as daidzein and daidzin was utilized. Fluorescence images before and after soybean roots were placed in contact with the sheets with immobilized BSA were taken with an electron-multiplying charge-coupled device camera. The fluorescence quenching in the images was visualized and analyzed. Soybean isoflavones were extracted from the sheets for quantitative analysis, and the correlation coefficient between the quenched fluorescence intensity per sheet and the total amount of soybean isoflavones was 0.78 (p < 0.01), indicating a high correlation. The quenched fluorescence intensity was lower in pumpkin roots, which do not secrete soybean isoflavone. It was found from analyzed images that soybean isoflavone is secreted in larger amounts from the basal region of the taproot and the tips of the lateral roots of soybean.

Development and evaluation of a miniaturized taste sensor chip.

A miniaturized taste sensor chip was designed for use in a portable-type taste sensing system. The fabricated sensor chip (40 mm × 26 mm × 2.2 mm) has multiple taste-sensing sites consisting of a poly(hydroxyethyl methacrylate) hydrogel with KCl as the electrolyte layer for stability of the membrane potential and artificial lipid membranes as the taste sensing elements. The sensor responses to the standard taste substances showed high accuracy and good reproducibility, which is comparable with the performance of the sensor probe of the commercialized taste sensing system. Thus, the fabricated taste sensor chip could be used as a key element for the realization of a portable-type taste sensing system.

Bitterness compounds in coffee brew measured by analytical instruments and taste sensing system.

We investigated the bitter compounds in coffee brews using multivariate analysis of the data obtained from analytical instrument and electronic taste sensor experiments. Coffee brews were prepared from coffee beans roasted to four different degrees. Each brew was fractionated into four fractions by liquid-liquid extraction. The relative amounts of 30 compounds in each fraction were analyzed by analytical instruments, and the bitterness response value of each fraction was analyzed by a taste sensor. Candidate bitter compounds in the coffee brews were identified with reference to their variable importance in projection and by coefficient of projection to latent structure regression (PLS-R) analysis. PLS-R analysis suggested that nicotinic acid, l-lactic acid, and nicotinamide contributed to the bitterness of the coffee brews. In fact, the coffee brews with added nicotinic acid, l-lactic acid, and nicotinamide had an increased bitterness response value compared to those without.

Taste Sensor: Electronic Tongue with Lipid Membranes.

Taste is of five basic types, namely, sourness, saltiness, sweetness, bitterness and umami. In this review, we focus on a potentiometric taste sensor that we developed and fabricated using lipid polymer membranes. The taste sensor can measure the taste perceived by humans and is called an electronic tongue with global selectivity, which is the property to discriminate taste qualities and quantify them without discriminating each chemical substance. This property is similar to the gustatory system; hence, the taste sensor is a type of biomimetic device. In this paper, we first explain the sensing mechanism of the taste sensor, its application to beer evaluation and the measurement mechanism. Second, results recently obtained are introduced; i.e., the application of the senor to high-potency sweeteners and the improvement of the bitterness sensor are explained. Last, quantification of the bitterness-masking effect of high-potency sweeteners is explained using a regression analysis based on both the outputs of bitterness and sweetness sensors. The taste sensor provides a biomimetic method different from conventional analytical methods.

Identification of characteristic compounds of moderate volatility in breast cancer cell lines.

In this study, we were challenging to identify characteristic compounds in breast cancer cell lines. GC analysis of extracts from the culture media of breast cancer cell lines (MCF-7, SK-BR-3, and YMB-1) using a solid-phase Porapak Q extraction revealed that two compounds of moderate volatility, 1-hexadecanol and 5-(Z)-dodecenoic acid, were detected with markedly higher amount than those in the medium of fibroblast cell line (KMST-6). Furthermore, LC-TOF/MS analysis of the extracts clarified that in addition to the above two fatty acids, the amounts of five unsaturated fatty acids [decenoic acid (C10:1), decadienoic acid (C10:2), 5-(Z)-dodecenoic acid (C12:1), 5-(Z)-tetradecenoic acid (C14:1), and tetradecadienoic acid (C14:2)] in MCF-7 medium were higher than those in medium of KMST-6. Interestingly, H2O2-oxidation of 5-(Z)-dodecenoic acid and 5-(Z)-tetradecenoic acid produced volatile aldehydes that were reported as specific volatiles in breath from various cancer patients, such as heptanal, octanal, nonanal, decanal, 2-(E)-nonenal, and 2-(E)-octenal. Thus, we concluded that these identified compounds over-produced in breast cancer cells in this study could serve as potential precursors producing reported cancer-specific volatiles.

Metabolome Analysis Identified Okaramines in the Soybean Rhizosphere as a Legacy of Hairy Vetch.

Inter-organismal communications below ground, such as plant-microbe interactions in the rhizosphere, affect plant growth. Metabolites are shown to play important roles in biological communication, but there still remain a large number of metabolites in soil to be uncovered. Metabolomics, a technique for the comprehensive analysis of metabolites in samples, may uncover the molecules that intermediate these interactions. We conducted a multivariate analysis using liquid chromatography (LC)-mass spectrometry (MS)-based untargeted metabolomics in several soil samples and also targeted metabolome analysis for the identification of the candidate compounds in soil. We identified okaramine A, B, and C in the rhizosphere soil of hairy vetch. Okaramines are indole alkaloids first identified in soybean pulp (okara) inoculated with Penicillium simplicissimum AK-40 and are insecticidal. Okaramine B was detected in the rhizosphere from an open field growing hairy vetch. Okaramine B was also detected in both bulk and rhizosphere soils of soybean grown following hairy vetch, but not detected in soils of soybean without hairy vetch growth. These results suggested that okaramines might be involved in indirect defense of plants against insects. To our knowledge, this is the first report of okaramines in the natural environment. Untargeted and targeted metabolomics would be useful to uncover the chemistry of the rhizosphere.

Comparison of cathepsin L activity in cheek and forearm stratum corneum in young female adults.

PURPOSE: The objective is to determine the usefulness of cathepsin L (Cat L) activity derived from the stratum corneum as an index of skin conditions by comparing the cheek and the forearm, in 20 female young adults. METHODS: The sebum quantity, hydration index, melanin index and pH were measured with a sensor system consisting of a sebumeter, a corneometer, a mexameter and a pH meter. The stratum corneum was collected using tape stripping. Cat L and total protein (TP) were analyzed and the Cat L/TP ratio was calculated. RESULTS: Our results indicated that (i) chronic sun exposure causes an increase in Cat L activity; and (ii) in order to cancel out the influence of the collecting technique, it is very important to measure the Cat L/TP ratio. CONCLUSION: It is considered that the Cat L/TP ratio may be a useful index of the skin conditions in humans.

Evaluating the Reduced Hydrophobic Taste Sensor Response of Dipeptides by Theasinensin A by Using NMR and Quantum Mechanical Analyses.

The current study demonstrated that theasinensin A (TSA) had a potential to form the complex with hydrophobic Trp-containing dipeptides, and to reduce their membrane potential by artificial-lipid membrane taste sensor. At a 1:3 molar ratio of the 6 Trp-containing dipeptides together with TSA, we observed a significant chemical shift of the protons of the dipeptides (Δδ) to a high magnetic field, when analyzed using 1H-nuclear-magnetic resonance (NMR) spectroscopy. The Δδ values were correlated with the hydrophobicity (log P) of the dipeptides and significant correlations were obtained (P = 0.022, R2 = 0.77); e.g., Trp-Leu with the highest log P value of 1.623 among the tested dipeptides showed the highest Δδ value of 0.105 ppm for the H7 proton of Trp-Leu, while less chemical shifts were observed in theasinensin B and epigallocatechin-3-O-gallate. Diffusion-ordered NMR spectroscopy revealed that the diffusion coefficient of 3 mM of Trp-Leu (7.6 × 10-11 m2/s) at a pulse field gradient in the range 0.05-0.3 T/m decreased in the presence of 3 mM TSA (6.6 × 10-11 m2/s), suggesting that Trp-Leu forms a complex with TSA. Quantum mechanical calculations and rotating frame nuclear Overhauser effect-NMR spectroscopy provided configuration information on the geometry of the complex that Trp-Leu formed with TSA (1:1 complex) with a ΔG energy of -8.7 kJ/mol. A sensor analysis using artificial-lipid membranes demonstrated that the changes in membrane potential of 1 mM Trp-Leu (21.8 ± 1.3 mV) and Leu-Trp (5.3 ± 0.9 mV) were significantly (P < 0.001) reduced by 1 mM TSA (Trp-Leu, 13.1 ± 2.4 mV; Leu-Trp, 3.5 ± 0.5 mV; TSA alone, 0.2 ± 0.01 mV), indicating the effective suppression of hydrophobicity of dipeptides by TSA-formed complex.

Development of Indirect Competitive Immuno-Assay Method Using SPR Detection for Rapid and Highly Sensitive Measurement of Salivary Cortisol Levels.

The monitoring of salivary cortisol as a key biomarker of an individual's stress response has been increasingly focused on. This paper describes the development of a novel cortisol immuno-assay method based on an indirect competitive method using a commercially available surface plasmon resonance instrument. The surface of an Au chip was modified with PEG6-COOH aromatic dialkanethiol self-assembled monolayers and hydrocortisone 3-(O-carboxymethyl) oxime (hydrocortisone 3-CMO) as a cortisol analog. A detection limit of 38 ppt range with a measurement range of 10 ppt-100 ppb was accomplished without the incubation of a mixing solution consisting of standard cortisol and an anti-cortisol antibody, and the time for quantification of cortisol concentration was 8 min from the sample injection. We experimentally compared our immuno-assay with a commercialized salivary cortisol enzyme-linked immunosorbent assay (ELISA) kit using human saliva samples. It was found that the results obtained by the cortisol immuno-assay had a good correlation with those obtained by ELISA assay (R = 0.96). Our findings indicate the potential utility of the cortisol immuno-assay for measurements of human salivary cortisol levels.

Secretory and continuous expression of Aspergillus niger glucose oxidase gene in Pichia pastoris.

We proposed a yeast transformant cell incorporating the Aspergillus niger glucose oxidase gene (GOX gene), which is capable of constitutively as well as secretory expression. The GOX gene has been cloned in this study. This conclusion is based on the following: first, the ligated DNA determined by electrophoresis, was a 1489-1882bp fragment, close to the size of glucose oxidase (GOD), which is 1818bp. Secondly, the single open reading frame encoded a protein of 605 amino acids. Thirdly, secreted GOD recombinant proteins in the culture supernatants of the GOX gene transformant migrated as a single band in SDS-PAGE with an apparent molecular mass of between 75,000 and 100,000 Da, which is glycosylated GOD by the Pichia pastoris X-33 host machinery during the secretion process. Finally, the clones were cultured and secreted a protein, which possessed the GOD activity of catalyzing beta-d-glucose oxidation. With regard to the pH characteristics, the activity was more than 80% of the maximum activity in the range between pH 5 and pH 7. As for the temperature characteristics, the activity was not less than 92% of the maximum in the temperature range between 10 and 45 degrees C. The GOX gene transformant was able to maintain the GOD enzyme activity and produce recombinant GOD continuously for at least 2 weeks.