奈良井（金山） 朝子

For the purpose of clarifying the relationship between pasteurization and inactivation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in Saccharomyces pastorianus cells induced by pressurized carbon dioxide microbubbles (CO2MB) treatment, a storage test of S. pastorianus cells after CO2MB treatment was conducted to ascertain their recovery, and the treatment condition in the inactivation of GAPDH in S. pastorianus cells by CO2MB was investigated. Each population of S. pastorianus for 48, 96, and 144 h at 25°C was decreased significantly by CO2MB treatment at 35°C for 3 min (MB35-3 and MB35-5) or at 40°C and 45°C for 1 and 3 min (MB40-1, MB40-3, and MB45-1). In the storage test, recovery of treated cells was not observed after storage for 144 h at 25°C. The denaturation of GAPDH in the S. pastorianus cells caused by the same treatment as the storage test was detected by using sodium dodecyl sulphate polyacrylamide gel electrophoresis. While the activities at MB35-1, MB35-3, and MB40-1 were significantly higher than those at non-treatment, and those at MB35-5, MB40-3, and MB45-1 were lower. Therefore, GAPDH denaturation, but not the activity, was associated with the inactivation of S. pastorianus cells.

To clarify the relationship between irreversible inactivation and intracellular protein denaturation of Saccharomyces pastorianus by low-pressure carbon dioxide microbubbles (CO2MB) treatment, a storage test of S. pastorianus cells treated with CO2MB was performed, and the effect on the intracellular protein was investigated. In the storage test, the S. pastorianus population, which decreased below the detection limit by CO2MB treatment at a temperature of 45 and 50 degrees C (MB45 and MB50), and thermal treatment at a temperature of 80 degrees C (T80), remained undetectable during storage for 3 weeks at 25 degrees C. However, 4.1 and 1.3-logs of the S. pastorianus populations, which survived after CO2MB treatment at temperatures of 35 and 40 degrees C (MB35 and MB40), increased gradually during storage for 3 weeks at 25 degrees C. Insolubilization of intracellular proteins in S. pastorianus increased with increasing the temperature of CO2MB treatment. Activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) identified as one of the insolubilized proteins increased at MB35 and MB40 than non-treatment but disappeared at MB45 and MB50, and T80. Therefore, it was revealed that S. pastorianus cells inactivated below the detection level by CO2MB treatment did not regrow and that the denaturation of intracellular proteins of S. pastorianus was caused by CO2MB and thermal treatments. Furthermore, it was suggested that denaturation of intracellular vital enzymes was an important factor for achieving irreversible inactivation of S. pastorianus by CO2MB and thermal treatments.

Black tea is a highly popular beverage, and its pigments, polymerized catechins such as theaflavins (TFs), are attracting attention due to their beneficial health effects. In this study, to test the inhibitory activities of TFs on the intestinal absorption of cholesterol, we investigated their effects on phosphatidylcholine (PC) vesicles in the absence or presence of a bile salt. (-)-Epicatechin gallate, (-)-epigallocatechin gallate, and TFs formed insoluble complexes with PC vesicles. Galloylated TFs such as TF2A, TF2B, and TF3 precipitated far more than other polyphenols. The subsequent addition of taurocholate redispersed the polyphenol-PC complexes, except that a large amount of TF2A remained insoluble. After incubation with taurocholate-PC micelles, TF2A elevated the turbidity of the micelle solution, providing red sediments. The TF2A-specific effect was dependent on the PC concentration. These results suggest that TF2A interacts with PC and aggregates in a specific manner different from catechins and other TFs.

<p>The influence of soymilk on the fecal microbiota, particularly Bifidobacterium species, and metabolic activities were investigated in eight healthy adult humans. During the soymilk intake period, the number of bifidobacteria in feces was significantly higher (p<0.05) on day 14 of the soymilk intake period than before the intake period, whereas that of Enterobacteriaceae was significantly lower (p<0.05) on days 7 and 14 of the soymilk intake period than before the intake period. In an investigation of Bifidobacterium at the species or group level, the numbers of all species and groups studied slightly increased during the soymilk intake period. These results show that the intake of soymilk may contribute to improving the intestinal environment.</p>

This study kinetically characterized the mechanism of the enzymatic synthesis of theaflavins (TFs) from catechins by mushroom tyrosinase (EC 1.14.18.1). In reactions containing one of four catechins, (-)-epicatechin (EC), (-)-epigallocatechin (EGC), and their galloylated forms (ECg and EGCg), they were oxidized by tyrosinase with apparent KM values of 3.78, 5.55, 0.80, and 3.05 mM, respectively, and with different consumption rates, of which EC was more than four times higher than those of the others. In reactions with binary combinations of catechins with tyrosinase, the synthesis of TF1 from EC and EGC occurred most efficiently, while the yields of mono- and di-galloylated TFs, TF2A, TF2B, and TF3, were low. Time-dependent changes in concentrations of the reactants suggested that the enzymatic oxidation of catechins and the subsequent non-enzymatic coupling redox reaction between the quinone derived from EC or ECg and the intact pyrogallol-type catechin (EGC or EGCg) proceeded concurrently. The latter reaction induced the rapid decrease of EGC and EGCg and it was remarkable for EGCg. So the efficiency of condensation of a pair of quinones from catechol- and pyrogallol-type catechins is restricted, critically influencing the yield of TFs. Using green tea extracts as mixtures of the four substrate catechins, tyrosinase produced TF1 most abundantly. Furthermore, a remarkable enhancement of production of TF2A and TF2B as well as TF1 was observed, when the initial concentration of EGCg was low. These results suggest that the catechin composition has an impact on yields of TFs.

Background Previously, we had reported that α-chymotrypsin–catalyzed polymerization of l-cysteine ethyl ester in a frozen buffer provided poly-l-cysteine (PLCys) in good yield, of which degree of polymerization had been determined to be 6–11. Almost all of SH groups in PLCys were in free forms. Such a multi-thiol peptide may cross-link proteins through thiol/disulfide (SH/SS) exchange reactions, considering the knowledge that other synthetic multi-thiol additives changes properties of protein materials. Methods This study explored the capability of PLCys to cross-link proteins using lysozyme as a model protein which has four disulfide bonds but no free SH group. The protein was incubated with PLCys at neutral pH and at below 70 °C to avoid PLCys-independent, β-elimination-mediated cross-linkings. Protein polymerization was analyzed by SDS-PAGE and SEC. PLCys peptides involved in the protein polymer, which were released by reduction with dithiothreitol, were analyzed by RP-HPLC. Conclusions Addition of urea and thermal treatment at 60 °C caused PLCys-induced lysozyme polymerization. Compared with free cysteine, a higher level of PLCys was required for the polymerization probably due to its low water solubility. RP-HPLC analyses suggested that PLCys played a role in the protein polymerization as a cross-linker. General significance Enzymatically synthesized PLCys shows promise as a peptidic cross-linker for the production of protein polymers with novel physiochemical properties and functionalities.

Di-, tri-, and tetra-tyrosine peptides with angiotensin I-converting enzyme inhibitory activity were synthesized by papain-catalyzed polymerization of L-tyrosine ethyl ester in aqueous media at 30 degrees C. Varying the reaction pH from 6.0 to 7.5 and the initial concentration of the ester substrate from 25 to 100 mM, the highest yield of oligo-tyrosine peptides (79% on a substrate basis) was produced at pH 6.5 and 75 mM, respectively. In the reaction initiated with 100 mM of the substrate, approx. 50% yield of insoluble, highly polymerized peptides accumulated. At less than 15 mM, the reaction proceeded poorly; however, from 30 mM to 120 mM a dose-dependent increase in the consumption rate of the substrate was observed with a sigmoidal curve. Meanwhile, each of the tri- and tetra-tyrosine peptides, even at approx. 5 mM, was consumed effectively by papain but was not elongated to insoluble polymers. For deacylation of the acyl-papain intermediate through which a new peptide bond is made, L-tyrosine ethyl ester, even at 5 mM, showed higher nucleophilic activity than di- and tri-tyrosine. These results indicate that the mechanism through which papain polymerizes L-tyrosine ethyl ester is as follows: the first interaction between papain and the ester substrate is a rate-limiting step; oligo-tyrosine peptides produced early in the reaction period are preferentially used as acyl donors, while the initial ester substrate strongly contributes as a nucleophile to the elongation of the peptide product; and the balance between hydrolytic fragmentation and further elongation of oligo-tyrosine peptides is dependent on the surrounding concentration of the ester substrate. (C) 2015 Elsevier Inc. All rights reserved.

The tuberous root of yacon (Smallanthus sonchifolius) is a rich source of fructooligosaccharide (FOS), which has a prebiotic effect. However, FOS significantly decreases accompanying (or FOS significantly decreases with) an increase in fructose during cool storage of harvested tuberous roots under normal pressure. The aim of this study was to investigate whether a storage condition at low temperature under reduced pressure could maintain FOS content by suppressing carbohydrate metabolism. After storage at 4? for one month, FOS in the tuberous roots stored at 0.7 atm was effectively retained. In addition, the activity of two endogenous enzymes associated with the degradation of FOS and sucrose, fructan 1- exohydrolase and invertase, respectively, was markedly suppressed at 0.7 atm. These results suggest that low-pressure storage is an effective condition for long-term preservation of FOS-rich yacon tuberous roots.

The effects of non-fermented soybean milk (NFSM) and fermented soybean milk (FSM) intake on the faecal microbiota and metabolic activities in 10 healthy volunteers were investigated. Soybean oligosaccharides, raffinose and stachyose were utilized by bifidobacteria except for Bifidobacterium bifidum, but most strains of Escherichia coli and Clostridium perfringens could not use them. During the dietary administration of FSM, the number of bifidobacteria and lactobacilli in the faeces increased (p &lt; 0.05), and clostridia decreased (p &lt; 0.05). Moreover, the concentrations of faecal sulphide were decreased (p &lt; 0.01) in the intake of FSM. During the dietary administration of NFSM, the number of bifidobacteria tended to increase. These results indicate that the consumption of soybean milk, especially FSM, is related to improvement of the intestinal environment.

Papain-catalyzed polymerization of L-tyrosine ethyl ester in aqueous media was efficient for synthesis of oligo-tyrosine peptides having angiotensin I-converting enzyme inhibitory activity. Di-, tri-, and tetra-tyrosine accumulated in the soluble fraction of reaction mixture. On the other hand, the peptide products with degree of polymerization from 5 to 10 were insoluble, yields of which were influenced by initial concentrations of the ester substrate. The precipitated products could be used as substrates for alpha-chymotrypsin in DMSO/buffer systems producing soluble oligo-tyrosine peptides. In the reaction media containing DMSO at 40-50% (v/v), most of the precipitates were converted to soluble peptides. The two-step enzymatic reaction, that is papain-catalyzed synthesis of Tyr-polymers from L-tyrosine ethyl ester followed by their hydrolytic cleavage by alpha-chymotrypsin, is expected to be a potent procedure for synthesis of biologically active di- and tri-tyrosine peptides in good yield. (C) 2010 Elsevier B.V. All rights reserved.

Oligo-tyrosine peptides such as Tyr-Tyr having angiotensin I-converting enzyme (ACE) inhibitory activity could be synthesized by α-chymotrypsin-catalyzed reaction with l-tyrosine ethyl ester in aqueous media. However, peptide yield in the reaction was below 10%. Since l-tyrosine amide showed highly nucleophilic activity for the deacylation of enzyme through which a new peptide bond was made, its application to the enzymatic peptide synthesis was evaluated in this study. Addition of tyrosine amide into the reaction produced Tyr-Tyr-NH 2, of which yield exceeded 130% on the basis of tyrosine ethyl ester. Although purified Tyr-Tyr-NH 2 did not inhibit ACE activity, α-chymotrypsin could act on the dipeptide amide and convert about 40% of it to Tyr-Tyr. The use of both ester and amide forms of tyrosine is expected to be a potent procedure for α-chymotrypsin-catalyzed synthesis of antihypertensive peptides. © 2009 Elsevier Inc. All rights reserved.

Oligo-tyrosine peptides with degrees of polymerization ranging from 2 to 5 could be synthesized by α-chymotrypsin-catalyzed reaction with l-tyrosine ethyl ester in aqueous media, although the peptide yield was low due to a preferential hydrolysis of the substrate. It was also confirmed that α-chymotrypsin efficiently converted tyrosine tetramer to the dimer which was resistant to the digestion. Both Tyr-Tyr and Tyr-Tyr-Tyr showed high inhibitory activity for angiotensin I-converting enzyme from rabbit lung, and their IC50 values were 34 μM and 51 μM, respectively. These two peptides exhibited a mix of competitive and noncompetitive inhibitions. Tyr-Tyr-Tyr was first recognized as an ACE inhibitor, suggesting that α-chymotrypsin could be applied to synthesis of novel potential materials for antihypertensive medicines. © 2008 Elsevier Inc. All rights reserved.

Papain polymerizes L-glutamic acid diethyl ester (Glu-di-OEt) regioselectively, resulting in the formation of poly (gamma-ethyl zeta-L-glutamic acid) with various degrees of polymerization of less than 13. Reaction temperatures below 20 degrees C were appropriate for the reaction in terms of suppression of non-enzymatic degradation of Glu-di-OEt and an increase in the peptide yield, while the reaction was preceded by a pronounced induction period. Mass spectrometric analyses of the reaction conducted at 0 degrees C revealed that the accumulation of the initial climerization product, L-glutaMyl-L-glutamic acid triethyl ester (Glu-Glu-tri-OEt), was limited during the induction period, and that a sequential polymer derived from a further elongation of the dimer was the tetramer, but not the trimer. Kinetic analyses of acyl transfer reactions with Glu-di-OEt and Glu-Glu-tri-OEt as acyl acceptors and Net-benzoyl-L-arginine ethyl ester as an acyl donor affirmed that Glu-Glu-tri-OEt bound more strongly than Glu-di-OEt both to the S- and S'-subsites of papain. Therefore, what occurred during the initial stage of the polymerization was interpreted as follows: the rate of the papain-catalyzed climerization of Glu-cli-OEt was extremely slow, once Glu-Glu-tri-OEt was initially synthesized it exclusively bound to the active site of papain, and then papain utilized the dimer in polymerization effectively rather than the monomer. (c) 2008 Elsevier B.V. All rights reserved.

Tuberous roots of yacon (Smallanthus sonchifolius) accumulate about 10%, on a fresh weight basis, of inulin-type fructooligosacharides (FOSs), known as a food ingredient with various healthy beriefits. However have a great difficulty to ensure these benefits because FOSs with a lower degree of polymerization (DP) decreased remarkably, and fructose increased when the tuberous roots were stored after harvesting oven under previously recommended storage conditions of low temperature with high humidity. In the present study, to elucidate the involvement of FOS-metabolizing enzymes in FOS reduction during storage at 90% relative humidity and 8 degrees C, we extracted a crude protein from yacon tuberous roots and measured the activities of invertase (beta-fructofuranosidase, EC 3.2.1.26), sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99), fructan:fructan 1-fructosyltransferase (1-FFT, EC 2.4. 1.100), and fructan 1-exohydrolase (1-FEH, EC 3.2.1.80). The enzyme activities acting on invertase and 1-SST, were weakened after storage for a month. In addition, the activity of 1-FFT acting such as 1-kestose (GF(2)) and 1-nystose (GF(3)) was higher than that of 1 -FFT. These results suggest that the continuos decline in FOSs of low DP during storage was dependent mainly on the 1-FEH activity. On the other hand,FOSs with a DP of &gt;= 9 only slightly decreased in stored yacon tuberous roots during storage, though distinct, was observed in vitro toward a high-DP inulin-type substrate, indicating that highly polymerized FOSs content was unlikely to be closely connectedwith the 1-FEH activity.

We have previously found a transepithelial electrical resistance (TEER)-decreasing protein derived from Flammulina velutipes, which was revealed to be identical to flammutoxin (FTX) that is known as a hemolytic pore-forming protein. This protein induced a rapid decrease in TEER and parallel increase in paracellular permeability in the intestinal epithelial Caco-2 cell monolayer without any cytotoxicity. An immunoblotting analysis revealed that the FTX-induced decrease in TEER was accompanied by the formation of a highmolecular-weight complex on the surface of Caco-2 cells. Intracellular Ca2+ imaging showed that exposure to FTX caused a rapid Ca2+ influx. It was observed by electron microscopy that FTX induced swelling of microvilli and expansion of the cellular surface. Staining with fluorescent phalloidin showed a marked change to filamentous actin in the FTX-treated cells. These results suggest that TEER reduction could sensitively detect small membrane pore formation by FTX in the intestinal epithelium which causes a morphological alteration and disruption of the paracellular barrier function.

The effects of marine substances with various cytotoxic mechanisms on the integrity of the human intestinal Caco-2 cell monolayer were examined by measuring the transepithelial electrical resistance (TEER). TEER was rapidly decreased by epical exposure of the monolayers to discodermin A, a membrane pore-forming substance, The decrease in TEER occurred in an earlier stage of incubation than the release of intracellular lactate dehydrogenase (LDH) which is commonly used as a parameter of cell damage or death. Mycalolide B (an actin-depolymerizing substance), calyculin A and okadaic acid (protein phosphatase inhibitors) also rapidly decreased the TEER value, although no cell membrane damage or resultant LDH release by these toxicants were detected. The TEER decrease caused by the toxicants was associated with the increased transepithelial permeability of the cell monolayer. Treatment with these toxicants, except calyculin A, caused morphological changes in the intracellular actin filament, suggesting that these toxicants altered the cytoskeletal structure, by which the tight junction was opened. Calyculin A was Likely to loosen the cellular junctions rapidly and induce cell detachment from the monolayer, Although onnamide A a protein synthesis inhibitor, did not cause any. decrease in TEER, at least during a 90-min incubation, TEER sensitively reflects the cytotoxic effects of various types of toxicants with acute toxicity. (C) 2000 Elsevier Science Ltd. All rights reserved.

A new protein that decreases transepithelial electrical resistance (TEER in the human intestinal Caco-2 cell monolayer was found in a water-soluble fraction of the mushroom Flammulina velutipes. This protein, termed TEER-decreasing protein (TDP), is not cytotoxic and does not induce cell detachment, but rapidly increases the tight junctional permeability for water-soluble marker substances such as Lucifer Yellow CH (M-r 457) through the paracellular pathway. TDP was isolated and purified from the aqueous extract of F: velutipes by chromatographic means. Purified TDP was found to be a simple, nonglycosylated protein without intermolecular disulfide bonds, and the appal ent molecular mass as estimated by SDS/PAGE and gel filtration is 30 kDa. It was revealed that the N-terminal amino-acid sequence of purified TDP is identical to the recently reported N-terminal sequence of flammutoxin, a membrane-perturbing hemolytic protein, for which the complete primary structure has not yet been reported [Tomita, T., Ishikawa, D., Noguchi, T., Katayama, E., and Hashimoto, Y. (1998) Biochem. J. 333, 24794-24799]. The cDNA coding fur TDP was cloned by 5' and 3' rapid amplification of c DNA ends. The ORF encodes a protein with 272 amino-acid residues showing no homology to known proteins. Relevant studies using TDP cDNA will provide insight into the structure-function relationships of membrane pore-forming toxins.

The transepithelial electrical resistance (TEER) of human intestinal Caco-2 cell monolayers cultured on a permeable filter rapidly decreased on apical exposure of the monolayer cells to such cytotoxic membrane perturbents as benzalkonium chloride and saponin. This decrease in TEER occurred at lower concentrations of these toxicants and at earlier stages of incubation than those for the release of intracellular lactate dehydrogenase, which is commonly used as a parameter for assessing cell membrane damage. The TEER decrease caused by the toxicants was associated with increased transepithelial permeability of the cell monolayer, as well as with subtle changes in the cytoskeletal structure, suggesting that membrane perturbation by the toxicants induced cytoskeletal changes that resulted in disruption of the tight junction and a subsequent increase in paracellular permeability. The rapid TEER change caused by the cytotoxic membrane perturbents suggests that TEER measurement would provide a simple and sensitive method for evaluating membrane-perturbing toxicants. (C) 1997 Elsevier Science Ltd.