Enzymatic determination of carnosine in meat and fish using ß-Ala-Xaa dipeptidase and histidine ammonia-lyase derived from Pseudomonas putida NBRC100650.

Carnosine is a naturally occurring dipeptide and a functional component in foods, while also showing health-promoting effects. Generally, food-derived carnosine is quantified via high-performance liquid chromatography (HPLC). We have developed a method for quantifying carnosine in foods using microbial enzymes, ß-Ala-Xaa dipeptidase (BapA) and histidine ammonia-lyase (HAL). The carnosine concentrations in extracts of chicken, pork, beef, bonito, and tuna were determined via both HPLC and enzymatic determination. The carnosine contents measured via enzymatic determination were in agreement with those determined via conventional HPLC analysis. Relative standard-deviation values of the conventional HPLC method and the enzymatic determination of carnosine in foods were 0.728-5.76% and 0.504-4.58%, respectively. The recovery of carnosine in food extracts via enzymatic determination was 97-103%. Therefore, the developed enzymatic determination technique using BapA and HAL can be used for the determination of carnosine in meats and fishes with comparable accuracy to that of conventional HPLC analysis.

Electrochemical antioxidant capacity measurement: a downsized system and its application to agricultural crops.

An on-site electrochemical antioxidant capacity measurement system was developed using a screen print electrode (SPE) and circuit tester. The antioxidant capacities of eight antioxidants were evaluated with the handheld electrochemical antioxidant capacity measurement system to compare with those measured with spectroscopic methods, namely, oxygen radical absorbance capacity (ORAC) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assays, as well as the reported electrochemical method with three conventional electrodes (a glassy carbon electrode, Ag/AgCl electrode and platinum wire electrode) and a potentiostat. Additionally, the potential shifts were proportional to the logarithm of the antioxidant concentration, which obeyed the Nernstian equation. Moreover, the antioxidant capacities of extracts from vegetables (green pepper, ginger and eggplant) were measured with a handheld electrochemical system. Each measurement was finished in only ca. 3 min. The electrochemically obtained antioxidant data were comparable to those from DPPH free-radical scavenging assays and superoxide anion scavenging activity (SOSA) assays, as well as the total phenolic compound content.

Quantification of Proline-containing Cyclic Dipeptides by LC-MS/MS.

The cyclic dipeptides, 2,5-diketopiperazines (DKPs), draw attention as bioactive and taste compounds. DKPs, especially those containing Proline (Pro), are contained in heated and fermented foods. Herein, we developed a method for a simultaneous quantitative analysis of Pro-containing DKPs using LC-MS/MS. After optimizing the LC-MS/MS conditions, the developed method was applied to quantify Pro-containing DKPs in Goishi tea, which is a post-fermented tea produced by a two-step aerobic-anaerobic fermentation process. Consequently, 17 kinds of Pro-containing DKPs could be quantified, and the total amount of Pro-containing DKPs was 3.40 mg/L. The recovery in a spiked test was 93 - 117%, which is satisfactory. We believe that the developed method can be used to elucidate the role of Pro-containing DKPs in food.

Investigation of the formation mechanism of proline-containing cyclic dipeptide from the linear peptide.

Cyclic dipeptides, 2,5-diketopiperazines (DKPs), are well-known bioactive and taste compounds in food. DKPs have also been reported in various foods and particularly, Pro-containing DKPs (cyclo(-X-Pro)) are more predominant in heated and fermented foods than other type of DKPs. However, the mechanism underlying the preferential formation of Pro-containing DKPs in food remains uncertain. Herein, we attempted to elucidate the effects of reaction conditions and the mechanism of DKPs formation. The reaction conditions (heating time, heating temperature, and pH) and amino acid sequence of the linear peptides were important for the DKPs formation from linear peptides. In addition, Pro-containing DKPs were significantly formed from linear peptides with the second amino acid from the N-terminus being Pro. Based on these results, the underlying mechanism of the enrichment of Pro-containing DKPs in foods was proposed.

Improvement of blood lipid profiles by Goishi tea polyphenols in a randomised, double-blind, placebo-controlled clinical study.

Dyslipidaemia is a risk factor for arteriosclerosis. Recent studies have shown that dyslipidaemia is effectively prevented by various polyphenols. In this clinical study (UMIN trial: 000024028), we evaluated the beneficial effects of polyphenols contained in Goishi tea on blood lipid profiles. Seventy-seven subjects with LDL cholesterol (CHO) ≧120 mg/mL were randomly divided into two groups for 12 weeks of polyphenol intake as follows: the Goishi tea group for daily consumption of Goishi tea containing 122 mg of polyphenols and the placebo group for the corresponding consumption of a placebo drink containing 12.2 mg of polyphenols. Intake of Goishi tea polyphenols tended to increase HDL CHO and suppress the elevation of triglycerides. These effects were particularly notable among the subjects with a body mass index <25 kg/m2. These findings suggest that Goishi tea polyphenols may suppress arteriosclerosis and reduce cardiovascular event risk by improving blood lipid profiles and thereby preventing dyslipidaemia.

New Electrochemical Evaluation of the Antioxidant Capacity of Beverages with Polyoxometalates as Redox Probes.

A new method was developed to evaluate antioxidant activity based on the redox properties of polyoxometalates, which are partially reduced by antioxidants to generate a limiting potential. The polyoxometalates [PMo12O40](3-), [PVW11O40](4-) and [SV2W10O40]4- formed in situ were used as electrochemical probes for the new evaluation method, and their formation conditions were optimized to evaluate the antioxidant activities of gallic acid, ellagic acid, catechin, quercetin, morin, trans-ferulic acid, sesamol, α-tocopherol, δ-tocopherol and L-ascorbic acid. The observed difference between initial potential and limiting potential (ΔE) were compared with spectrophotometrically evaluated antioxidant activities. In addition, the antioxidant capacities of five beverages (Japanese green tea, concentrated catechin-containing green tea, grapefruit juice, red wine and Japanese sake) were evaluated.

Development of LC-MS/MS analysis of cyclic dipeptides and its application to tea extract.

2,5-Diketopiperazines (DKPs), also called cyclic dipeptides, have been known to occur in various foods. Recently, DKPs have attracted attentions as bioactive components. There were some reports on analytical methods for DKPs, but the number of analyzed DKPs was only a part of all DKPs and the quantitative performance was not studied in detail. In this study, we selected 31 kinds of DKPs and developed a quantitative and simultaneous analytical method using LC-MS/MS. This method was applied to DKPs determination in Pu-erh tea, post-fermentation tea, and 18 kinds of DKPs were determined at concentration of 0.0017-0.11 ppm. As a result of spiked test, it was concluded that the developed method using LC-MS/MS was useful for estimating DKPs concentration in tea.

Content of resveratrol and glycoside and its contribution to the antioxidative capacity of Polygonum cuspidatum (Itadori) harvested in Kochi.

We quantified trans-resveratrol in each part of fresh Polygonum cuspidatum harvested in Kochi Prefecture. A small amount of trans-resveratrol was detected in the edible stem parts, the content varying with seasonal, geographical, and environmental factors. We also examined the antioxidative activity of each part, suggesting that P. cuspidatum contained many more contributing antioxidants other than resveratrol.

Applicability of the DPPH assay for evaluating the antioxidant capacity of food additives - inter-laboratory evaluation study -.

An inter-laboratory evaluation study was conducted in order to evaluate the antioxidant capacity of food additives by using a 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. Four antioxidants used as existing food additives (i.e., tea extract, grape seed extract, enju extract, and d-α-tocopherol) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were used as analytical samples, and 14 laboratories participated in this study. The repeatability relative standard deviation (RSD(r)) of the IC50 of Trolox, four antioxidants, and the Trolox equivalent antioxidant capacity (TEAC) were 1.8-2.2%, 2.2-2.9%, and 2.1-2.5%, respectively. Thus, the proposed DPPH assay showed good performance within the same laboratory. The reproducibility relative standard deviation (RSD(R)) of IC50 of Trolox, four antioxidants, and TEAC were 4.0-7.9%, 6.0-11%, and 3.7-9.3%, respectively. The RSD(R)/RSD(r) values of TEAC were lower than, or nearly equal to, those of IC50 of the four antioxidants, suggesting that the use of TEAC was effective for reducing the variance among the laboratories. These results showed that the proposed DPPH assay could be used as a standard method to evaluate the antioxidant capacity of food additives.

An automated sequential injection spectrophotometric method for evaluation of tyramine oxidase inhibitory activity of some flavonoids.

An automated sequential injection (SI) spectrophotometric system has been developed for evaluation of tyramine oxidase (TOD) inhibitory activity. The method is based on the inhibition of TOD that catalyzes the oxidation of tyramine substrate to produce aldehyde and hydrogen peroxide (H2O2). The produced H2O2 reacts with vanillic acid and 4-aminoantipyrine (4-AA) in the presence of peroxidase (POD) to form a quinoneimine dye, the absorbance of which is measured of absorbance at wavelength of 490 nm. The decrease of the quinoneimine dye is related to an increase of TOD inhibitory activity. Under the optimum conditions: 1.0 mM tyramine, 8 U mL(-1) TOD, 1.0 mM vanillic acid, 1.0 mM 4-AA and delay time of 10 s, some flavonoid compounds were examined for the TOD inhibitory activity expressed as IC50 value. It was found that flavonols (quercetin and myricetin) and flavans (epicatechin gallate (ECG) and epigallocatechin (EGC)) showed higher TOD inhibitory activity than flavones and flavanones. The results of IC50 values obtained from the proposed method and a batch-wise method were not significantly different from each other. Moreover, the SI system enabled automation of the analysis, leading to more convenient, more sensitive and faster analysis than the batch-wise method. A precise timing of the system also improves precision and accuracy of the assay, especially when the measurement of absorbance at non-steady state condition is involved.

The influence of Goishi tea on adipocytokines in obese mice.

This study investigated the influence of Goishi-tea on visceral fat weight in induced obese mice. Mice were divided into two main groups, normal and obesity. In obesity group, mice were fed with high-fat diet. Goishi-tea including its fractions (ethyl-acetate layer and water layer) was administrated in normal and obesity three sub-groups. Results showed no influence of Goishi-tea in normal group. However, visceral fat weight, size of adipose cell and cholesterol level were significantly decreased in obesity group fed Goishi-tea compared to control group. Moreover, adiponectin levels tended to increase and adipocytokines has significant values lower in obesity group fed Goishi-tea compared to control group. Interestingly, Goishi tea involved in the high-fat diet induced-obese mice can inhibit fat accumulation and maintain adiponectins without increasing tumor necrosis factor-alpha and interleukin-6. It would be beneficial for the prevention of metabolic syndrome and obesity-related disorder.

Sequential injection spectrophotometric system for evaluation of mushroom tyrosinase-inhibitory activity.

A sequential injection (SI) spectrophotometric method with absorbance detection at 475 nm has been developed for evaluating activity of some compounds on an inhibition of the mushroom tyrosinase. The method involved a reaction of 3,4-dihydroxyphenylalanine (L-DOPA) and mushroom tyrosinase to form the o-dopaquinone. The decrease of the o-dopaquinone was related to an increase of tyrosinase-inhibitory activity. Under the optimum conditions (concentration and volume of L-DOPA and mushroom tyrosinase of 2.0 mM, 60 µL and 142 U mL(-1), 15 µL, respectively), some antioxidant compounds were examined for the tyrosinase-inhibitory activity. A batch enzymatic assay of tyrosinase-inhibitory activity was applied as the reference method for comparison. The results of IC(50) values obtained from the proposed method and the batch method were correlated well, with r(2) of 0.969. The SIA provides higher precision and degrees of automation, consumes smaller amounts of chemicals and it is simpler and faster than the batch method.

Superoxide dismutase activity of Helicobacter pylori per se from 158 clinical isolates and the characteristics.

We investigated the correlation between the SOD activity of Helicobacter pylori (H. pylori) and gastroduodenal diseases and the characteristics of strains exposed to oxidative stress. Two sequenced strains, 26695 and J99, and clinical isolates from 156 Japanese patients with gastroduodenal diseases such as gastric cancer (n= 59) and non-cancer (n= 97) were used. SOD activities of all 158 isolates were measured and were divided into three groups: high-SOD activity (>0.22, n= 2), moderate-SOD activity (0.15≦≦0.22, n= 16) and low-SOD activity (<0.15, n= 140). Expressions of H. pylori Fe-SOD were examined by western blotting with anti-H. pylori Fe-SOD antibody prepared inhouse, and the profiles of Fe-SOD activity were investigated by zymogram with activity staining in native-PAGE. The characteristics of strains from high-SOD and low-SOD groups were examined under oxidative stress by paraquat. The average of H. pylori SOD activity was significantly higher in the cancer group than in the non-cancer group (P < 0.05). However, irrespective of SOD activity level, the amount of Fe-SOD expressed was variable among individual strains. Zymogram revealed a single band in moderate-SOD and low-SOD strains, but multiple bands in high-SOD strains were observed. These bands were confirmed as H. pylori Fe-SOD. Under oxidative stress with paraquat, low-SOD strains were drastically eliminated without inducible SOD activity; however, high-SOD strains were still viable with increased SOD activity. This study is the first to exhibit the characteristics of high-SOD activity strains representing multiple bands in zymograms and the correlation between H. pylori SOD activity and gastric cancer.

In vitro antimicrobial activity of aminoreductone against the pathogenic bacteria methicillin-resistant Staphylococcus aureus (MRSA).

In this study, antimicrobial activity of aminoreductone (AR), a product formed during the initial stage of the Maillard reaction, against methicillin-resistant Staphylococcus aureus (MRSA) was evaluated. The significant growth inhibition of all 51 MRSA isolates irrespective of drug susceptibility by AR was observed. The minimum inhibitory concentration (MIC) of AR ranged from 13 to 26 mM. The bactericidal activity of AR was evaluated by a killing assay with multiples of MIC, and it was recognized to depend on its dose. The combined effects of AR and antibiotics frequently used for the treatment of infections caused by Gram-positive and Gram-negative bacteria, such as amikacin (AN), ciprofloxacin, imipenem and levofloxacin, were examined. As a result, AR did not interfere with these antibiotic activities against 12 MRSA isolates selected and showed the advanced effect of growth inhibition in combination with antibiotics. Moreover, the inhibitory effects of AR were similar to those of AN, an antibiotic with known adverse effects, some serious. These findings show that AR is a naturally formed antimicrobial agent present in thermally processed foods with potential health benefits in medical practice.

Demonstration of the presence of aminoreductone formed during the Maillard reaction in milk.

The Maillard reaction is a common chemical reaction that occurs in food and it generates multiple reaction products. Aminoreductone (AR) is one of the early-stage Maillard reaction products. At present the formation of AR has only been demonstrated in a model system consisting of a monosaccharide or disaccharide and an amino group-containing compound. There is no direct evidence to show the presence of AR in food. In this study, we demonstrated the formation and presence of AR in milk using a combination of 2,4-dinitrophenylhydrazine (DNP) and Cu(2+). A DNP derivative of AR oxidised by Cu(2+) was isolated and its detailed structure was identified by NMR analysis. We thus directly demonstrated the formation and presence of AR in milk.

Antimicrobial activity of aminoreductone against Helicobacter pylori.

Anti-Helicobacter pylori (H. pylori) effects of aminoreductone (AR), a Maillard reaction product, were evaluated in this study. AR effectively inhibited the growth of all 24 strains (19 clinical isolates and 5 isogenic mutants) irrespective of susceptibility to antibiotics and clinical manifestation. The minimum inhibitory concentration (MIC) of AR ranged from 0.5 to 5 mM. A killing assay with multiples of MIC was performed, demonstrating that the killing activity of AR was significantly higher than that of its derived melanoidin, an inhibitor of H. pylori urease-gastric mucin adherence, formed in the final stage of the Maillard reaction. These significant effects of AR on H. pylori were observed even in acidic conditions (pH 3). At most, 25 mM AR effectively exhibited bactericidal activity in all strains. These results rise up the possibility that foods containing AR, such as milk and dairy products, are valuable sources for preventing colonization of H. pylori in the stomach and its associated tissue damages.

Flow injection analysis of angiotensin I-converting enzyme inhibitory activity with enzymatic reactors.

Assay of angiotensin I-converting enzyme (ACE) inhibitory activity always draws much attention because of diverse applications in the field of antihypertension and related pathogenesis. Recently, the use of a new synthetic substrate, 3-hydroxybutyrylglycyl-glycyl-glycine (3HB-GGG), for the assay of ACE inhibitory activity has been confirmed. To construct a rapid, economical, and automatic determination system of ACE inhibitory activity using 3HB-GGG, a flow injection analysis (FIA) system with enzymatic reactors was developed in this study. Enzyme reactors were composed of aminoacylase and 3-hydroxybutyrate dehydrogenase immobilized separately on CNBr-activated Sepharose 4B. The assay condition was optimized in terms of the conversion of 3HB-G into NADH by the enzymatic reactors when the reaction solution containing 3HB-G generated from 3HB-GGG (after the incubation with ACE) was repetitively injected into the FIA system. Under the optimized conditions, 3HB-G was converted to 3HB, and then 3HB was oxidized by NAD(+) to form NADH. The developed system successfully detected practical ACE inhibitors with a great sensitivity, high sampling frequency (10 samples h(-1)) and a durable stability of the enzymatic reactors.

Assay of angiotensin I-converting enzyme-inhibiting activity based on the detection of 3-hydroxybutyrate with water-soluble tetrazolium salt.

A newly synthesized substrate, 3-hydroxybutyrylglycyl-glycyl-glycine (3HB-GGG), was applied to the assay of ACE-inhibiting activity to overcome the smaller selectivity and sensitivity of the conventional method. In this study, an ACE-inhibiting assay was improved by the use of a water-soluble tetrazolium salt, 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt (WST-1), for the detection of 3-hydroxybutyrate, derived from 3HB-GGG. The optimized conditions were as follows: 0.333 mM NAD(+), 0.333 mM WST-1, 0.1 mM EDTA, 0.633 U ml(-1) diaphorase, and 0.700 U ml(-1) 3-hydroxybutyrate dehydrogenase. The developed assay was efficiently applicable to evaluate the ACE-inhibiting activity of practical ACE inhibitors.

Determination of antioxidant activity and characterization of antioxidant phenolics in the plum vinegar extract of cherry blossom (Prunus lannesiana).

Sakura-cha (salted cherry blossom tea) is a Japanese tea that is traditionally served at celebrations such as wedding ceremonies. The production of Sakura-cha includes the immersion of cherry blossom flowers in Japanese plum vinegar, and through this process, the byproduct (plum vinegar extract of cherry blossom) is obtained. In this study, the antioxidant activity of the plum vinegar extract of cherry blossom was examined. The plum vinegar extract of cherry blossom had a greater superoxide anion scavenging activity compared with red wine, which is a well-known strong antioxidant. Liquid chromatography-mass spectrometry analysis showed that cyanidin-3-glucoside, cyanidin-3-rutinoside, and caffeic acid were the major components in the phenolic extract prepared from plum vinegar extract of cherry blossom, and they possessed superoxide anion scavenging activity. Caffeic acid is mainly responsible for the scavenging activity of phenolic extract; the contributions of cyanidin-3-glucoside and cyanidin-3-rutinoside were minor.

Electron spin resonance analysis of superoxide anion radical scavenging activity with spin trapping agent, diphenyl-PMPO.

Recently, a novel electron spin resonance (ESR) spin-trapping agent, 2-(diphenylphosphinoyl)-2-methyl-3,4-dihydro-2H-pyrrole N-oxide (Diphenyl-PMPO), was synthesized. Because it had some advantages in stability and reactivity over conventional spin-trapping agents, we applied it to the ESR analyses of superoxide anion radical scavenging activity (SOSA) of superoxide dismutase (SOD) and of well-known natural antioxidants (green tea, oolong tea, and red wine). At the same time, the results with Diphenyl-PMPO were compared with 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Our results revealed that Diphenyl-PMPO showed higher detectability than DMPO in the SOSA assays of SOD and natural antioxidants, so it could be available for the ESR analyses as an alternative to conventional spin-trapping agents.