Understanding Antidiabetic Potential of Oligosaccharides from Red Alga Dulse Devaleraea inkyuleei Xylan by Investigating α-Amylase and α-Glucosidase Inhibition.

In this study, the α-glucosidase (maltase-glucoamylase: MGAM) and α-amylase inhibitory properties elicited by xylooligosaccharides (XOSs) prepared from dulse xylan were analysed as a potential mechanism to control postprandial hyperglycaemia for type-2 diabetes prevention and treatment. Xylan was purified from red alga dulse powder and used for enzymatic hydrolysis using Sucrase X to produce XOSs. Fractionation of XOSs produced xylobiose (X2), ß-(1→3)-xylosyl xylobiose (DX3), xylotriose (X3), ß-(1→3)-xylosyl-xylotriose (DX4), and a dulse XOS mixture with n ≥ 4 xylose units (DXM). The different fractions exhibited moderate MGAM (IC50 = 11.41-23.44 mg/mL) and α-amylase (IC50 = 18.07-53.04 mg/mL) inhibitory activity, which was lower than that of acarbose. Kinetics studies revealed that XOSs bound to the active site of carbohydrate digestive enzymes, limiting access to the substrate by competitive inhibition. A molecular docking analysis of XOSs with MGAM and α-amylase clearly showed moderate strength of interactions, both hydrogen bonds and non-bonded contacts, at the active site of the enzymes. Overall, XOSs from dulse could prevent postprandial hyperglycaemia as functional food by a usual and continuous consumption.

Enhanced overexpression of secreted enzymes by discrete repeat promoters in Streptomyces lividans.

Streptomyces lividans is an efficient host for extracellular overproduction of recombinant proteins. To enhance the overexpression strength of S. lividans, we designed several kinds of expression plasmids with different positioning of repeat promoters. The effect of repeat promoters was evaluated by measuring the accumulated amounts of a stable transglutaminase or an unstable carboxypeptidase that was secreted into the medium. Successive tandem positions of repeat promoters upstream of the normal promoter did not enhance the expression of transglutaminase. Discrete positions of repeat promoters both upstream and downstream of the normal promoter enhanced the expression of transglutaminase to 2-fold, and the downstream ones also enhanced the expression of carboxypeptidase to 1.7-fold. On the other hand, there were still some constructs of plasmids with discrete repeat promoters that did not promote the expression of the target enzymes, indicating the complexity of the mechanisms of repeat promoters working on gene expression.

Construction and development of a novel dual-gene coexpression system to promote heterologous protein secretion for Streptomyces.

Streptomyces lividans is a potent host for the extracellular overproduction of heterologous proteins. To further improve the usability and productivity of S. lividans, a dual gene expression vector of "pTSKr duet" containing two strong constitutive promoters, scmpPc and kasOp*, was constructed. The success in the overproduction of two secretory enzymes simultaneously without interference with each other indicated that the "pTSKr duet" vector can realize the coexpression of two genes simultaneously and independently. Further, using the two-gene coexpression vector, we screened the effects of the overexpression of five factors that possibly promote secretion on the extracellular overproduction of heterologous secretory proteins. Interestingly, the coexpression of a quality control regulator (CssR) promoted the overproduction level to 1.3-fold for a stable heterologous protein of SMTG (transglutaminase from S. mobaraensis), while other four factors limited the overproduction of SMTG at different degrees.

Isolation of the hemeoxygenase-1 inducer from rice-derived peptide.

Bioactive peptides with various health benefits have been reported from rice protein hydrolysates. We previously showed that rice-derived peptides (RP) increased intracellular glutathione levels and induced the expression of γ-glutamylcysteine synthetase, which is regulated by nuclear transcription factor-erythroid 2-related factor 2 (Nrf2). Heme oxygenase-1 (HO-1) is an important Nrf2 downstream antioxidant enzyme that protects against oxidative stress. This study aimed to investigate the protective effects of RP on hydrogen peroxide (H2O2)-induced oxidative stress in human hepatoblastoma cell line HepG2 and identified HO-1 induced peptides from RP. Pretreatment of cells with RP reduced the cytotoxicity caused by H2O2 in a dose-dependent manner. Moreover, RP induced HO-1 expression in a concentration- and time-dependent manner. Next, we attempted to isolate the HO-1 inducer from RP by bioactivity-guided fractionation. Purification of the active peptides using a Sep-Pak C18 cartridge and reversed-phase HPLC, followed by sequence analysis by mass spectrometry, led to the identification of the three peptides. These peptides effectively reduced H2O2-induced oxidative stress. Among them, only P3 (peptide sequence: RSAVLLSH) increased HO-1 protein expression. Additionally, the knockdown of Nrf2 suppressed the induction of HO-1 expression by P3. Our results indicated that P3 identified from RP induced HO-1 by activating the Nrf2 signaling pathway.

Improvement in hyperexpression with a combination of truncated scmp promoter and Streptomyces lividans.

We have previously reported a powerful promoter from the Streptomyces cinnamoneus TH-2 strain named "scmp" and created an expression vector of pTONA5a for expression using S. lividans. The full-length scmp promoter sequence consists of 424 bp upstream of a metalloendoprotease gene in the S. cinnamoneus TH-2 genome. The promoter works in the presence of inorganic phosphate and glucose. In this study, we present the essential region of the scmp promoter (promoter C), which lacks 358 bp of the 5' region of the full-length promoter. Promoter C was very short and contained only 63 bp. Using promoter C, we succeeded in the extracellular production of the Streptomyces enzymes of leucine aminopeptidase, ferulic acid esterase, and transglutaminase, which possessed signal peptides for secretion via the type II secretion pathway, at high levels.

Molecular investigation of proanthocyanidin from Alpinia zerumbet against the influenza A virus.

Proanthocyanidins (PACs) have various bioactivities, such as being anti-bacterial, anti-cancer, and anti-oxidant. Consequently, they have been vigorously studied for the development of new natural bioactive compounds. Recently, PAC was isolated from leaves and pseudostems of the medicinal plant Alpinia zerumbet (Pers.) B.L. Burtt and R.M. Smith, and it had shown in vitro antiviral activity against influenza A H1N1 viruses (IAVs). The 50% endpoint dilution method indicated that 0.1 mg/mL A. zerumbet-derived PAC (AzPAC) reduced the titer of IAVs by >3 logs. The antiviral activity of AzPAC means that it can interact directly with viral particles since the antiviral activity test was done by coincubation of PAC with and IAVs before viral infection. However, few studies have investigated the preventive mechanism utilized by AzPAC on influenza virus replication. In this study, the composition of AzPAC and the affinity between AzPAC and IAVs was investigated in detail. We found that AzPAC was composed of an epicatechin, which was linked by inter-flavan bonds between the C4 and C8 positions (B2-type) and the C4 and C6 positions (B5-type) in the terminal units. A quenching assay indicated that AzPAC interacted with IAV membrane proteins, hemagglutinin and neuraminidase. Additionally, circular dichroism analysis indicated that AzPAC affected the change in the secondary structure rate of the viral membrane proteins. AzPAC was able to impair the infective process of IAVs via direct interaction with their viral membrane proteins. These results indicate that A. zerumbet is a bioresource for the development of preventive drugs against IAV infection.

Inactivation of plant and animal viruses by proanthocyanidins from Alpinia zerumbet extract.

Alpinia zerumbet (Pers.) B.L. Burtt and R.M. Smith belongs to the Alpinia genus in the Zingiberaceae family. In East Asia, Alpinia zerumbet has been widely used as food and traditional medicine. Previously, we identified proanthocyanidins (PACs), an anti-plant-virus molecule in A. zerumbet, using Nicotiana benthamiana and tomato mosaic virus (ToMV). Here, we found that PACs from A. zerumbet, apple, and green tea effectively inhibited ToMV infection. Additionally, the PACs from A. zerumbet exhibited greater antiviral activity than those from apple and green tea. The PACs from A. zerumbet also effectively inactivated influenza A virus and porcine epidemic diarrhea virus (PEDV), which acts as a surrogate for human coronaviruses, in a dose-dependent manner. The results from the cytopathic effect assays indicated that 0.1 mg/ml PACs from A. zerumbet decreased the titer of influenza A virus and PEDV by >3 log. These findings suggested that the direct treatment of viruses with PACs from A. zerumbet before inoculation reduced viral activity; thus, PACs might inhibit infections by an influenza virus, coronaviruses, and plant viruses.

Identification of an anti-plant-virus molecule in Alpinia zerumbet.

In plants, viral diseases are second only to fungal diseases in terms of occurrence, and cause substantial damage to agricultural crops. The aqueous extracts of shell ginger, Alpinia zerumbet exhibit inhibitory effects against virus infections in belonging to the Solanaceae family. In this study, we isolated an anti-plant-virus molecule from the extracts using a conventional method involving a combination of reversed phase column chromatography, dialysis, and lyophilization. The anti-plant-virus molecule was identified as proanthocyanidin, which mostly consisted of epicatechin and exhibited more than 40 degrees of polymerization.

Preparation of ß(1→3)/ß(1→4) xylooligosaccharides from red alga dulse by two xylanases from Streptomyces thermogriseus.

Red alga dulse contains xylan with ß(1→3)/ß(1→4) linkages. We previously prepared xylooligosaccharides (XOSs) from dulse xylan; however, the product contained many D-xylose residues and fewer XOSs with ß(1→3) linkages. To improve the efficiency of XOS production, we prepared two recombinant endoxylanases from Streptomyces thermogriseus (StXyl10 and StXyl11). Comparing the kcat/Km values for dulse xylan, this value from StXyl10 was approximately two times higher than that from StXyl11. We then determined the suitable conditions for XOS production. As a result, dulse XOS was prepared by the successive hydrolysis of 10 mg/mL dulse xylan by 0.5 µg/mL StXyl10 for 4 h at 50 °C and then 2.0 µg/mL StXyl11 for 36 h at 60 °C. Xylan was converted into 95.8% XOS, including 59.7% XOS with a ß(1→3) linkage and 0.97% D-xylose. Our study provides useful information for the production of XOSs with ß(1→3) linkages.

Hepatoprotective Activity of Yellow Chinese Chive against Acetaminophen-Induced Acute Liver Injury via Nrf2 Signaling Pathway.

Glutathione, the most abundant intracellular antioxidant, protects cells against reactive oxygen species induced oxidative stress and regulates intracellular redox status. We previously demonstrated that yellow Chinese chive (ki-nira) increased the intracellular glutathione levels. Acetaminophen (APAP) is a commonly used analgesic. However, an overdose of APAP causes severe hepatotoxicity via depletion of the hepatic glutathione. In this study, we investigated the hepatoprotective effects of yellow Chinese chive extract (YCE) against APAP-induced hepatotoxicity in mice. YCE (25 or 100 mg/kg) was administered once daily for 7 d, and then APAP (700 mg/kg) was injected at 6 h before the mice were sacrificed. APAP treatment markedly increased the serum biological markers of liver injury such as alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and alkaline phosphatase. Pretreatment with YCE significantly prevented the increases in the serum levels of these enzymes. Histopathological evaluation of the livers also revealed that YCE prevented APAP-induced centrilobular necrosis. Pretreatment with YCE dose-dependently elevated glutathione levels, but the difference was not significant. Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a critical role in APAP-induced hepatotoxicity by regulating the antioxidant defense system. Therefore, we investigated the expression of Nrf2 and its target antioxidant enzyme. YCE led to an increased expression of Nrf2 and its target antioxidant enzymes, NAD(P)H quinone oxidoreductase 1 (NQO1), glutathione peroxidase (GPx), cystine uptake transporter (xCT), especially hemeoxygenase-1 (HO-1) in mice livers. These results suggest that YCE could induce HO-1 expression via activation of the Nrf2 antioxidant pathway, and protect against APAP-induced hepatotoxicity in mice.

Protective Effects of Rice Peptide Oryza Peptide-P60 against Oxidative Injury through Activation of Nrf2 Signaling Pathway In Vitro and In Vivo.

We previously showed that commercially available rice peptide Oryza Peptide-P60 (OP60) increased the intracellular glutathione levels. This study aimed to evaluate the antioxidant potential of this peptide and assess its mechanism of action. Pretreatment of HepG2 cells with OP60 reduced the cytotoxicity caused by H2O2 or acetaminophen (APAP) (47.7 ± 1.3% or 12.2 ± 1.3% of the cytotoxicity for 5 mg/mL OP60 pretreatment compared to that in H2O2- or APAP-treated groups, respectively; p < 0.01) through the restoration of glutathione homeostasis. Moreover, OP60 elevated the mRNA level of genes encoding heavy and light subunits of γ-glutamylcysteine synthetase (γ-GCS) by 2.9 ± 0.1-fold and 2.7 ± 0.2-fold (p < 0.001), respectively, at 8 h and also increased the level of mRNA encoding other antioxidant enzymes. Besides, OP60 promoted Nrf2 nuclear translocation by 2.2 ± 0.3-fold (p < 0.05) after 8 h. Conversely, knockdown of Nrf2 inhibited the increase of the intracellular glutathione levels and suppressed the induction of antioxidant enzyme expression by OP60. In animal studies, OP60 prevented APAP-induced liver injury by suppressing glutathione depletion (from 0.19 ± 0.02 mmol/mg protein to 0.90 ± 0.02 mmol/mg protein; p < 0.01, by pretreatment with 500 mg/kg OP60) and increasing heavy subunit of γ-GCS and heme oxygenase-1 expression in the liver. Our results indicated that OP60 exhibits a cytoprotective effect via the Nrf2 signaling pathway and is one of the few peptides with excellent antioxidant properties.

Inhibitory effects of Alpinia zerumbet extract against plant virus infection in solanaceous plants.

Tomato mosaic virus (ToMV) and tobacco mosaic virus (TMV) are critical pathogens causing severe crop production losses of solanaceous plants. The present study was undertaken to evaluate the antiviral effects of extracts of Alpinia plants on ToMV and TMV infection in Nicotiana benthamiana. The aqueous extracts of Alpinia zerumbet (Pers.) B.L. Burtt and R.M. Smith and Alpinia kumatake, which grow widely in subtropical and tropical regions including East Asia, were effective in reducing ToMV infection when plants were treated prior to virus inoculation. We also found that the extracts of A. zerumbet isolated from Okinawa (Japan), locally referred to as shima-gettou, strongly inhibited ToMV and TMV infection. To obtain an active fraction, the aqueous extract of A. zerumbet isolate OG1 was separated by ethyl acetate, and the antiviral active compound was found to be present in the water layer. Based on our results, the extract of Alpinia plants has potential as an antiviral reagent for practical application in solanaceous crop production.

Molecular insights into the mechanism of substrate recognition of Streptomyces transglutaminases.

The microbial TGase from Streptomyces mobaraensis has used in various food industries. However, the detailed substrate specificities of TGases from the Streptomyces species toward the natural peptides remains to be unclear. In this study, we conducted the comparison of two different TGases from Streptomyces mobaranensis (SMTG) and Streptomyces cinnamoneus (SCTG). To clarify the region associated with the characteristics of enzymes, we constructed a chimeric enzyme of CM, of which is consisted of N-terminal half of SCTG and C-terminal half of SMTG. To reveal the differences in the substrate specificity between SCTG and SMTG toward natural peptides, we investigated the time dependence of TGase activity on the productivity of cross-linking peptide with tryptic casein and lysine by using LC-MS. We identified two peptides of "VLPVPQK" and "AVPYPQR" as substrates for both of the TGases.

Enzymatic Degradation of Allergen Peptides from Bovine Casein by a Combination of Streptomyces Aminopeptidases.

Cow's milk is one of the most common allergenic foods. Cow's milk allergy is mainly an IgE-mediated hypersensitivity reaction, and the major allergens from cow's milk have been found to be caseins, ß-lactoglobulin, and α-lactalbumin. Several peptides derived from bovine casein are known allergens in cow's milk. To reduce their allergenicity, these proteins can be degraded by food-grade peptidases. We succeeded in detection of two peptides, VLPVPQK and FFVAPFPEVFGK, from bovine casein-derived allergen peptides by using an ion trap LC-MS apparatus. This study focuses on the synergistic effects of Streptomyces aminopeptidases belonging to the M1, M24, and M28 families on the degradation of the allergen peptides. From these results, we demonstrated that the combination of M1 and M24 aminopeptidases was the most effective for degrading the abovementioned allergenic peptides.

Active site pocket of Streptomycesd-stereospecific amidohydrolase has functional roles in aminolysis activity.

d-Stereospecific amidohydrolase from Streptomyces sp. 82F2 (DAH) recognizes d-amino acyl ester derivatives as substrates and catalyzes hydrolysis and aminolysis to yield d-amino acids and d-amino acyl peptides or amide derivatives, respectively. Crystallographic analysis has revealed that DAH possesses a large cavity with a small pocket at the bottom. Because the pocket is close to the catalytic center and is thought to interact with substrates, we examined the function of the eight residues that form the pocket in terms of substrate recognition and aminolysis via mutational analysis. Formation of the acyl-enzyme intermediate and catalysis of aminolysis by DAH were changed by substitutions of selected residues with Ala. In particular, I338A DAH exhibited a significant increase in the condensation product of Ac-d-Phe methyl ester and 1,8-diaminooctane (Ac-d-Phe-1,8-diaminooctane) compared with the wild-type DAH. A similar effect was observed by the mutation of Ile338 to Gly and Ser. The pocket shapes and local flexibility of the mutants I338G, I338A, and I338S are thought to resemble each other. Thus, changes in the shape and local flexibility of the pocket of DAH by mutation presumably alter substrate recognition for aminolysis.

Loop of Streptomyces Feruloyl Esterase Plays an Important Role in the Enzyme's Catalyzing the Release of Ferulic Acid from Biomass.

Feruloyl esterases (FAEs) are key enzymes required for the production of ferulic acid from agricultural biomass. Previously, we identified and characterized R18, an FAE from Streptomyces cinnamoneus NBRC 12852, which showed no sequence similarity to the known FAEs. To determine the region involved in its catalytic activity, we constructed chimeric enzymes using R18 and its homolog (TH2-18) from S. cinnamoneus strain TH-2. Although R18 and TH2-18 showed 74% identity in their primary sequences, the recombinant proteins of these two FAEs (recombinant R18 [rR18] and rTH2-18) showed very different specific activities toward ethyl ferulate. By comparing the catalytic activities of the chimeras, a domain comprised of residues 140 to 154 was found to be crucial for the catalytic activity of R18. Furthermore, we analyzed the crystal structure of rR18 at a resolution of 1.5 Å to elucidate the relationship between its activity and its structure. rR18 possessed a typical catalytic triad, consisting of Ser-191, Asp-214, and His-268, which was characteristic of the serine esterase family. By structural analysis, the above-described domain was found to be present in a loop-like structure (the R18 loop), which possessed a disulfide bond conserved in the genus Streptomyces Moreover, compared to rTH2-18 of its parental strain, the TH2-18 mutant, in which Pro and Gly residues were inserted into the domain responsible for forming the R18 loop, showed markedly high kcat values using artificial substrates. We also showed that the FAE activity of TH2-18 toward corn bran, a natural substrate, was improved by the insertion of the Gly and Pro residues.IMPORTANCEStreptomyces species are widely distributed bacteria that are predominantly present in soil and function as decomposers in natural environments. They produce various enzymes, such as carbohydrate hydrolases, esterases, and peptidases, which decompose agricultural biomass. In this study, based on the genetic information on two Streptomyces cinnamoneus strains, we identified novel feruloyl esterases (FAEs) capable of producing ferulic acid from biomass. These two FAEs shared high similarity in their amino acid sequences but did not resemblance any known FAEs. By comparing chimeric proteins and performing crystal structure analysis, we confirmed that a flexible loop was important for the catalytic activity of Streptomyces FAEs. Furthermore, we determined that the catalytic activity of one FAE was improved drastically by inserting only 2 amino acids into its loop-forming domain. Thus, differences in the amino acid sequence of the loop resulted in different catalytic activities. In conclusion, our findings provide a foundation for the development of novel enzymes for industrial use.

Sake lees hydrolysate protects against acetaminophen-induced hepatotoxicity via activation of the Nrf2 antioxidant pathway.

Acetaminophen is a commonly used analgesic. However, an overdose of acetaminophen causes severe hepatotoxicity via depletion of hepatic glutathione. Here, we investigated the protective effects of sake lees hydrolysate against acetaminophen-induced hepatotoxicity in mice. Sake lees hydrolysate was administered orally to ICR mice for seven days. Six hours after acetaminophen treatment, the mice were sacrificed, and blood and liver samples were collected for analysis. Treatment with acetaminophen markedly increased the levels of serum alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and alkaline phosphatase. Pretreatment with sake lees hydrolysate significantly prevented the increases in the serum levels of these enzymes and inhibited acetaminophen-mediated glutathione depletion. In addition, histopathological evaluation of the livers also revealed that sake lees hydrolysate prevented acetaminophen-induced centrilobular necrosis. The expression of γ-glutamylcysteine synthetase (γ-GCS), hemeoxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) in the liver were decreased after acetaminophen treatment, whereas pretreatment with sake lees hydrolysate led to an increased expression of all three proteins. Furthermore, sake lees hydrolysate induced the expression of these proteins in HepG2. These results suggested that sake lees hydrolysate could induces HO-1 and γ-GCS expression via activation of the Nrf2 antioxidant pathway, and protects against acetaminophen-induced hepatotoxicity in mice.

Anti-oxidative Activity of Hydrolysate from Rice Bran Protein in HepG2 Cells.

Glutathione (GSH) is an ubiquitous thiol-containing tripeptide, which plays important roles in cellular protection from oxidative stress. In our search for a dietary source that can increase GSH levels, we discovered that a 24 h treatment of HepG2 cells with rice bran protein hydrolysate (RBPH), prepared by Umamizyme G-catalyzed hydrolysis, increased the GSH content in a dose-dependent manner. RBPH elevated the expression levels of γ-glutamylcysteine synthetase (γ-GCS), which constitutes the rate-limiting enzyme of GSH synthesis, and of another two enzymes, hemeoxygenase-1 (HO-1) and reduced nicotinamide adenine dinucleotide (phosphate): quinone oxidoreductase 1 (NQO1). This induction was preceded by the accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2) inside the nucleus, which is a key transcription factor for the expression of the γ-GCS, HO-1, and NQO1. Pre-treatment of cells with RBPH produced a significant protective effect against cytotoxicity caused by H2O2 or ethanol. These results indicate that RBPH exerts a protective effect against oxidative stress by modulating GSH levels and anti-oxidative enzyme expression via the Nrf2 pathway.

Hepatoprotective effects of rice-derived peptides against acetaminophen-induced damage in mice.

Glutathione, the most abundant intracellular antioxidant, protects cells against reactive oxygen species induced oxidative stress and regulates intracellular redox status. We found that rice peptides increased intracellular glutathione levels in human hepatoblastoma HepG2 cells. Acetaminophen is a commonly used analgesic. However, an overdose of acetaminophen causes severe hepatotoxicity via depletion of hepatic glutathione. Here, we investigated the protective effects of rice peptides on acetaminophen-induced hepatotoxicity in mice. ICR mice were orally administered rice peptides (0, 100 or 500 mg/kg) for seven days, followed by the induction of hepatotoxicity via intraperitoneal injection of acetaminophen (700 mg/kg). Pretreatment with rice peptides significantly prevented increases in serum alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase levels and protected against hepatic glutathione depletion. The expression of γ-glutamylcysteine synthetase, a key regulatory enzyme in the synthesis of glutathione, was decreased by treatment with acetaminophen, albeit rice peptides treatment recovered its expression compared to that achieved treatment with acetaminophen. In addition, histopathological evaluation of the livers also revealed that rice peptides prevented acetaminophen-induced centrilobular necrosis. These results suggest that rice peptides increased intracellular glutathione levels and could protect against acetaminophen-induced hepatotoxicity in mice.

Low accumulation of chlorogenic acids represses reddening during flesh browning in Japanese peach "Okayama PEH7".

In peaches, fruit flesh browns unattractively after peeling or cutting. A recently developed cultivar, Okayama PEH7, was distinct from other Japanese cultivars, including Okayama PEH8, with respect to its reduced browning potential. Homogenate prepared from Okayama PEH7 flesh had significantly less reddening during the browning reaction. Okayama PEH7 had less soluble phenolic compounds and higher polyphenol oxidase activity than Okayama PEH8. Reduced browning was observed even when phenols prepared from Okayama PEH7 were incubated with crude extract from Okayama PEH8, suggesting that phenols lower the browning potential of Okayama PEH7. In Okayama PEH7, contents of chlorogenic acid and its isomers were about one-tenth compared to Okayama PEH8. Exogenous addition of chlorogenic acid to Okayama PEH7 homogenate increased the browning potential and visibly enhanced reddening. These results indicate that the reduced browning of Okayama PEH7 flesh is due to a defect in chlorogenic acid accumulation.