Soymilk yogurt fermented using Pediococcus pentosaceus TOKAI 759 m improves mice gut microbiota and reduces pro-inflammatory cytokine production.

This study aimed to determine the anti-inflammatory activities and bioactive compounds of soymilk yogurt prepared using Lactiplantibacillus plantarum TOKAI 17 or Pediococcus pentosaceus TOKAI 759 m. Mice were divided into five groups: normal diet (ND), soymilk (SM), soymilk yogurt using L. plantarum TOKAI 17 (SY 17) or P. pentosaceus TOKAI 759 m (SY 759 m), and 0.5 × 109 cells of each starter strain (BC 17 or BC759m). In the SY 759 m group, the serum pro-inflammatory cytokine levels and the cytotoxicity of natural killer cells were attenuated compared to the ND group. In the cecum microbiota, the abundances of butyrate-producing bacteria increased in the SY 759 m and BC 17 groups. Furthermore, SY 759 m metabolites contained high levels of aglycone isoflavone, adenine and showed a significant decrease in CCL-2 and IL-6 production in LPS-induced macrophage. In conclusion, soymilk yogurt produced using P. pentosaceus TOKAI 759 m modulates the gut microbiota and can potentially prevent pro-inflammatory cytokine production.

Regulatory effects of antioxidants on indoxyl sulfate-enhanced intracellular oxidation and impaired phagocytic activity in differentiated U937 human macrophage cells.

Indoxyl sulfate (IS), a uremic toxin, is a physiologically active sulfated metabolite, specifically in kidney failure patients. Our previous studies have shown that IS downregulates phagocytic immune function in a differentiated HL-60 human macrophage cell model. However, it remains unclear whether IS exerts similar effects on macrophage function in other cell types or in lipopolysaccharide (LPS)-sensitive immune cell models. Therefore, this study aimed to investigate the effects of IS on intracellular oxidation levels and phagocytic activity in a differentiated U937 human macrophage cell model, both in the absence and presence of LPS. Our results demonstrated that IS significantly increases intracellular oxidation levels and decreases phagocytic activity, particularly in cells activated by LPS. Furthermore, we found that 2-acetylphenothiazine, an NADH oxidase inhibitor, attenuates the effects of IS in LPS-activated macrophage cells. Representative antioxidants, trolox, α-tocopherol, and ascorbic acid, significantly mitigated the effects of IS on the macrophages responding to LPS.

Lactic acid bacterial exopolysaccharides strongly bind histamine and can potentially be used to remove histamine contamination in food.

The symptoms of foodborne histamine poisoning are similar to those of IgE-mediated food allergies. In this study, we investigated the histamine-binding capacity of lactic acid bacteria (LAB) strains as potential preventive agents against histamine poisoning. Histamine biosorption capacity was determined for 16 LAB strains. Leuconostoc mesenteroides TOKAI 51 m, Lactobacillus paracasei TOKAI 65 m, Lactobacillus plantarum TOKAI 111 m and Pediococcus pentosaceus TOKAI 759 m showed especially high biosorption rates and reached saturation within 30 min. Adsorption isotherms showed better conformance to the Freundlich model than to the Langmuir model. Analyses after heat, periodic acid and guanidine hydrochloride treatments suggested that histamine was bound to the bacterial cell surface. HPLC analysis revealed that exopolysaccharides produced by Lact. paracasei TOKAI 65 m strongly bound to histamine. In the detachment test with 1 mol l-1 HCl solution, the dissociation rate of histamine for Lact. paracasei TOKAI 65 m was <10 %. This strain is presumably a suitable candidate for use against histamine poisoning.

Effect of heat-moisture treatment of germinated black rice on the physicochemical properties and its utilization by lactic acid bacteria.

The heat susceptibility of starch granule structure has been considering as significant limitation of germinated black rice (GBR) using in food processing industry. Therefore, this study aimed to improve the physicochemical and antioxidation property as well as its effect on the probiotics of GBR by heat moisture treatment (HMT). Black rice germinated at 37.5 °C for 12, 24, and 36 h were studied. Ultrastructural image of each sample was visualized through scanning electron microscope. The results illustrated 24 h-GBR retain its former shape with rough surface. Subsequently, 24 h-GBR was structurally modified by HMT with moisture levels of 20% and 25% for 1 and 2 h. The results showed that pasting properties of HMT-treated GBR were improved. This was particularly on, GBR using HMT condition of 25% moisture for 2 h decrease in breakdown viscosity was shown. Moreover, phenolic content of HMT-treated GBR was higher than those of GBR. Besides, the number of the Lactobacillus paracasei TOKAI 13 was increased in GBR and HMT-treated GBR with counts of 10.08 ± 0.83 Log CFU/ml and 9.31 ± 0.33 Log CFU/ml, respectively, with significant increases in antioxidant property. Therefore, the HMT-GBR could be utilized as an alternative functional ingredient in food processing products.

Investigation of the effects of indoxyl sulfate, a uremic toxin, on the intracellular oxidation level and phagocytic activity using an HL-60-differentiated human macrophage cell model.

Indoxyl sulfate (IS), a uremic toxin, is a sulfate-conjugated metabolite originated from tryptophan. Accumulating uremic toxins may worsen renal diseases and further complicate related disorders including impaired immune functions under oxidative stress conditions. However, it has remained unclear whether or not IS can directly cause the cellular immune dysfunction. We investigated the effects of IS on the intracellular oxidation level and phagocytic activity in a HL-60-differantiated human macrophage cell model. Incubation of the cells in the presence of IS resulted in increasing intracellular oxidation level and decreasing phagocytic activity. In addition to inhibitors for NADH oxidase (NOX), organic anion transporting polypeptide2B1 (OATP2B1), protein kinase C (PKC), and phosphoinositide 3-kinase (PI3K), a representative antioxidant Trolox, was also shown to significantly relieve the IS-induced oxidation and restore weakened phagocytosis. Collectively, IS may directly down-regulate the phagocytic immune function of macrophages through the oxidation mechanisms including OATP2B1, PKC, PI3K, and NOX pathways. Abbreviations: CKD: Chronic kidney disease; IS: Indoxyl sulfate; ROS: Reactive oxygen species; NOX: NADH oxidase; OATP2B1: Organic anion transporting polypeptide2B1; PKC: Protein kinase C; PI3K: Phosphoinositide 3-kinase; 2-APT: 2-acetylphenothiazine.

Increased phenolic content and antioxidant capacity of the heated leaves of yacon (Smallanthus sonchifolius).

We investigated the content of phenolic compounds and antioxidant capacity of two batches of non-heated and heated leaves of the yacon cultivar "Andes no yuki", grown in Japan. Lyophilized yacon leaves heated at 160°C for 20 min and 100°C for 60 min had a 1.96 to 9.69-times higher total phenolic content than that of the non-heated leaves. Heated leaves exhibited a 1.98 to 4.07-times higher antioxidant capacity than that of the non-heated leaves in three different free radical scavenging assays. Heated leaves were more efficient at attenuating the superoxide anion radical production in human granulocytic cells than the non-heated leaves. High-performance liquid chromatography analysis revealed that, in the heated leaves, the caffeic acid content was 2.13 to 3.64-times higher and the chlorogenic acid content was slightly lower than those in the non-heated leaves. Hence, heat processing may affect the active constituent contents in yacon leaves, potentiating its antioxidant capacity.Abbreviations: ABTS+: 2,2'-azinobis(2-ethylbenzothiazoline-6-sulfonic acid) cation; DPPH: 1,1-diphenyl-2-picrylhydrazyl, HPLC: high-performance liquid chromatography; NBT: nitroblue tetrazolium; O2-: superoxide anion; PMA: phorbol 12-myristate 13-acetate; PMS: phenazine methosulfate; TEAC: Trolox equivalent antioxidant capacity.

Unique antioxidant effects of herbal leaf tea and stem tea from Moringa oleifera L. especially on superoxide anion radical generation systems.

This study aimed to investigate the unique antioxidative effects of Japanese moringa products, herbal leaf tea and stem tea, using established free radical assays, focusing on superoxide anion (O2-) radical generation systems. Hot-water extracts from moringa teas resulted in different but lower scavenging activities than Trolox in four synthetic free radical models. Interestingly, these extracts further showed higher O2- radical scavenging effects than Trolox in the phenazine methosulfate-NADH-nitroblue tetrazolium and xanthine oxidase assay systems. Incubating human neutrophils in the presence of these tea extracts rather than Trolox effectively suppressed cellular O2- radical generation. Among the eight known phenolic constituents of moringa leaves, caffeic acid and chlorogenic acid may be responsible for the O2-specific radical scavenging capacity stronger than that of Trolox. These results suggest that moringa herbal teas are a good source of natural antioxidants for preventing O2- radical-mediated disorders. Abbreviations: O2-: superoxide anion; ROS: reactive oxygen species; H2O2: hydrogen peroxide; XOD: xanthine oxidase; DPPH: 1,1-diphenyl-2-picrylhydrazyl; ABTS+: 2,2'-azinobis(2-ethylbenzothiazoline-6-sulfonic acid) cation; CPZ+: chlorpromazine cation; PMS: phenazine methosulfate; NBT: nitroblue tetrazolium; PMA: phorbol 12-myristate 13-acetate.

Analysis of the sexual development-promoting region of Schizophyllum commune TRP1 gene.

This study aims to elucidate the mechanism of sexual development of basidiomycetous mushrooms from mating to fruit body formation. Sequencing analysis showed the TRP1 gene of basidiomycete Schizophyllum commune encoded an enzyme with three catalytic regions of GAT (glutamine amidotransferase), IGPS (indole-3-glycerol phosphate synthase), and PRAI (5-phosphoribosyl anthranilate isomerase); among these three regions, the trp1 mutant (Trp(-)) had a missense mutation (L→F) of a 338th amino acid residue of the TRP1 protein within the IGPS region. To investigate the function of IGPS region related to sexual development, dikaryons with high, usual, and no expression of the IGPS region of TRP1 gene were made. The dikaryotic mycelia with high expression of the IGPS formed mature fruit bodies earlier than those with usual and no expression of the IGPS. These results showed that the IGPS region in TRP1 gene promoted sexual development of S. commune.

Biosorption of Heavy Metals by Lactic Acid Bacteria for Detoxification.

The gradual accumulation of heavy metals can have detrimental effects on health. Lactic acid bacteria (LAB) are common microbes used as probiotics; various LAB strains are consumed in food products, especially in fermented foods. Many studies have suggested that LAB with high affinity to harmful heavy metals can be used as efficient detoxification tools. Accordingly, it is important to test the biosorption of various heavy metals, e.g., cadmium, lead, arsenic, and mercury, by LAB. Here, I describe protocols to quantify the binding ability of LAB and to identify their heavy metal-binding proteins.

Inhibition of Advanced Glycation End Products (AGEs) by Fermented Foods Using Lactic Acid Bacteria.

Glycation is an important nonenzymatic reaction between reducing sugars and amines. Advanced glycation end products (AGEs) accumulation in the human body is associated with secondary complications related to diabetes in hyperglycemic environments. These observations suggest that the inhibition of AGEs formation is important for preventing diabetes mellitus (DM) progression and the development of diabetes-related complications. Lactic acid bacteria (LAB) are probiotics commonly used in fermented foods and food additives. Therefore, it is necessary to identify starter strains of LAB to produce fermented food to decrease the risk of DM and its complications. This chapter introduces the protocols that are inhibition assay of fermented food using LAB on AGEs such as Nω-(carboxymethyl) arginine (CMA), Nε-(carboxymethyl) lysine (CML), and fluorescent AGEs.

Biosorption of Histamine by Lactic Acid Bacteria for Detoxification.

Histamine accumulates in fish and fish products such as tuna, mackerel, skipjack, and bonito by work microorganisms. And it causes allergy reactions like IgE-mediated ones. Lactic acid bacteria (LAB) are known as one of the probiotic bacteria that indicate various health functionalities for humans. And some previous studies report that LAB can adsorb and excrete various toxic molecules. Here, this chapter introduces the methods to quantify the histamine-binding ability of LAB.

Fluorescence of phytochrome adducts with synthetic locked chromophores.

We performed steady state fluorescence measurements with phytochromes Agp1 and Agp2 of Agrobacterium tumefaciens and three mutants in which photoconversion is inhibited. These proteins were assembled with the natural chromophore biliverdin (BV), with phycoerythrobilin (PEB), which lacks a double bond in the ring C-D-connecting methine bridge, and with synthetic bilin derivatives in which the ring C-D-connecting methine bridge is locked. All PEB and locked chromophore adducts are photoinactive. According to fluorescence quantum yields, the adducts may be divided into four different groups: wild type BV adducts exhibiting a weak fluorescence, mutant BV adducts with about 10-fold enhanced fluorescence, adducts with locked chromophores in which the fluorescence quantum yields are around 0.02, and PEB adducts with a high quantum yield of around 0.5. Thus, the strong fluorescence of the PEB adducts is not reached by the locked chromophore adducts, although the photoconversion energy dissipation pathway is blocked. We therefore suggest that ring D of the bilin chromophore, which contributes to the extended π-electron system of the locked chromophores, provides an energy dissipation pathway that is independent on photoconversion.

An adhesin-like protein, Lam29, from Lactobacillus mucosae ME-340 binds to histone H3 and blood group antigens in human colonic mucus.

A cell-surface 29-kDa protein (Lam29, cysteine-binding protein of the ABC transporter) from Lactobacillus mucosae ME-340 showed an adhesin-like property for human ABO blood group antigens expressed on the gastrointestinal mucosa. In addition, here we report that Lam29 also bound to an 18-kDa protein on human colonic mucus. By ligand blot assay and N-terminal amino acid sequence of the protein, it was identified as human histone H3. By ligand blot and microplate binding assays with recombinant histone H3, binding between Lam29 and histone H3 was confirmed. The adhesion of ME-340 cells to histone H3 was significantly inhibited by 26% after the addition of 2.5 mg/mL Lam29 as compared to the absence of Lam29 (p<0.01). By GHCl extraction and transcription attenuation of ME-340 cells, binding reduction of ME340 cells against histone H3 was detected at 12% and 13% respectively, as compared to control cells by the BIACORE assay (p<0.01). These data indicate that Lam29 shows multiple binding activities to blood group antigens and histone H3 in human colonic mucus. This is the first report to indicate that lactobacilli expressing Lam29 adhere to histone H3 on gastrointestinal mucosa.

Different effects on the tonus of colon and ileum isolated from mouse by resin glycoside (pharbitin) of Pharbitidis Semen.

BACKGROUND: Pharbitidis Semen (the seeds of Pharbitis nil), traditionally used as a purgative in Japan, China and Korea, contains a resin glycoside fraction named pharbitin, which is known as a purgative ingredient. Due to the complex nature of pharbitin, little is known about either the action on intestinal tension caused by resin glycoside itself or by its components. METHODS: In this study, we investigated the effects of pharbitin, the glycosidic acid fraction (pharbitic acid) and the aglycone fraction (phar-genin) generated from pharbitin on peristalsis of colon and ileum isolated from mice with the Magnus method. RESULTS: We demonstrated that pharbitin (3-30 µg/mL) concentration-dependently increased tonus of mice colon via acetylcholine receptors, its components phar-genin (1.27-12.7 µg/mL) and pharbitic acid (10-1000 µg/mL) also had the increment on colon tonus. On the other hand, ileum tension decreased in the presence of pharbitin. CONCLUSIONS: The effects of resin glycoside of Pharbitidis Semen on colon tonus are different with those on ileum tonus isolated from mice. In the next step it is necessary to investigate details of its pharmacological mechanism.

Investigation of radical scavenging effects of acetaminophen, p-aminophenol and their O-sulfated conjugates.

Acetaminophen (APAP) and p-aminophenol (p-AP) are the analogous simple phenolic compounds that undergo sulfate conjugation (sulfation) by cytosolic sulfotransferases. Sulfation is generally thought to lead to the inactivation and disposal of endogenous as well as xenobiotic compounds. This study aimed to investigate the antioxidative effects of O-sulfated form of APAP and p-AP, i.e., APAPS and p-APS, in comparison with their unsulfated counterparts. Using a 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay, the antioxidant capacity of APAPS was shown to be approximately 126-times lower than that of APAP. In contrast, p-APS displayed comparable activity as unsulfated p-AP. Similar trends concerning the suppressive effects of these chemicals on cellular O2- radical generation were found using an activated granulocytic neutrophil cell model. Collectively, these results indicated that, depending on the presence of an additional "active site", sulfation may not always decrease the antioxidant activities of phenolic compounds.

Structural insights into vinyl reduction regiospecificity of phycocyanobilin:ferredoxin oxidoreductase (PcyA).

Phycocyanobilin:ferredoxin oxidoreductase (PcyA) is the best characterized member of the ferredoxin-dependent bilin reductase family. Unlike other ferredoxin-dependent bilin reductases that catalyze a two-electron reduction, PcyA sequentially reduces D-ring (exo) and A-ring (endo) vinyl groups of biliverdin IXalpha (BV) to yield phycocyanobilin, a key pigment precursor of the light-harvesting antennae complexes of red algae, cyanobacteria, and cryptophytes. To address the structural basis for the reduction regiospecificity of PcyA, we report new high resolution crystal structures of bilin substrate complexes of PcyA from Synechocystis sp. PCC6803, all of which lack exo-vinyl reduction activity. These include the BV complex of the E76Q mutant as well as substrate-bound complexes of wild-type PcyA with the reaction intermediate 18(1),18(2)-dihydrobiliverdin IXalpha (18EtBV) and with biliverdin XIIIalpha (BV13), a synthetic substrate that lacks an exo-vinyl group. Although the overall folds and the binding sites of the U-shaped substrates of all three complexes were similar with wild-type PcyA-BV, the orientation of the Glu-76 side chain, which was in close contact with the exo-vinyl group in PcyA-BV, was rotated away from the bilin D-ring. The local structures around the A-rings in the three complexes, which all retain the ability to reduce the A-ring of their bound pigments, were nearly identical with that of wild-type PcyA-BV. Consistent with the proposed proton-donating role of the carboxylic acid side chain of Glu-76 for exo-vinyl reduction, these structures reveal new insight into the reduction regiospecificity of PcyA.

Protection of human intestinal epithelial cells from oxidative stress caused by mercury using lactic acid bacteria.

Heavy metals are harmful to human health. Therefore, we investigated the biosorption of heavy metals by lactic acid bacteria (LAB). Of all the tested heavy metals, biosorption by LAB was highest for mercury, followed by lead, cadmium, and finally arsenic. The viability of HCT-116 cells was reduced by half in the presence of 7.5 µg/mL mercury but recovered after the addition of selected LAB strains. HCT-116 cells showed increased superoxide dismutase and catalase activities, whereas glutathione peroxidase activities decreased significantly. Addition of Lactobacillus sakei TOKAI 57m recovered all antioxidant enzyme activities. Our results suggest that this strain can be used for cellular detoxification.

Functions of small RNAs in Lactobacillus casei-Pediococcus group of lactic acid bacteria using fragment analysis.

Small RNAs (sRNA) are non-cording RNAs composed of 50∼400 nt responsible for coordinating the adaption of Escherichia coli and other bacteria to changing environmental conditions, including pH and temperature. However, the role of sRNAs in lactic acid bacteria (LAB) has not yet been clarified. In this study, we used the Lactobacillus casei-Pediococcus group to evaluate the function of sRNAs in LAB, using RNA sequencing in the exponential growth phase and stationary phase to map and analyze sRNA fragments, which were categorized as Pediococcus pentosaceus and Lactobacillus paracasei. We evaluated the role of sRNAs in nutrient synthesis for cell growth in exponential growth phase and in protein and biofilm biosynthesis for cell body durability. During exponential growth, the sRNA fragments were found to be involved in the stress response in Pediococcus pentosaceus and in environmental adaption in Lactobacillus paracasei. The results suggest that the function of sRNA can be characterized from sRNA fragments using RNA sequencing during the exponential growth and stationary phases in Lactobacillus casei-Pediococcus group.

Effects of indole and indoxyl on the intracellular oxidation level and phagocytic activity of differentiated HL-60 human macrophage cells.

Indoxyl, a derivative of indole originating from tryptophan, may undergo phase-II sulfate-conjugation pathway, thereby forming indoxyl sulfate (IS) in vivo. We previously reported that IS, a well-known uremic toxin, can increase the intracellular oxidation level and decrease the phagocytic activity in a differentiated HL-60 human macrophage cell model. Using the same cell model, the current study aimed to investigate whether indole and indoxyl (the metabolic precursors of indoxyl and IS, respectively) may cause macrophage immune dysfunction. Results obtained indicated that intracellular oxidation level and cytotoxicity markedly increased upon treatment with indole and indoxyl, in comparison with IS. Incubation of the cells with indole and indoxyl also resulted in attenuated phagocytic activity. Human serum albumin (HSA)-binding assay confirmed that tryptophan and IS, but not indole and indoxyl, could selectively bind to the site II in HSA. Collectively, the results indicated that indole and indoxyl may strongly down-regulate the phagocytic immune function of macrophages, whereas IS, formed upon sulfate conjugation of indoxyl, may exhibit enhanced HSA-binding capability, thereby reducing the adverse effects of indoxyl.

Assembly of Agrobacterium phytochromes Agp1 and Agp2 with doubly locked bilin chromophores.

The natural chromophore of most bacterial and fungal phytochromes is biliverdin (BV), which is incorporated in a covalent manner into the protein. Upon photoconversion between the red light-absorbing form Pr and the far-red light-absorbing form Pfr, the stereochemistry of the chromophore around the C15 methine bridge changes from Z anti to E anti. Recombinant phytochromes Agp1 and Agp2 from Agrobacterium tumefaciens were assembled with a set of synthetic chromophores, including 2,18-Et-BV, 3,18-Et-BV, and the doubly locked 5Ea15Ea-BV, 5Es15Ea-BV, 5Za15Ea-BV, and 5Zs15Ea-BV. In all chromophores, covalent bond formation is restricted. As shown by spectral changes and desalting column separation, all chromophores are bound to Agp1 and Agp2. Adducts with 2,18-Et-BV and 3,18-Et-BV undergo normal photoconversion between Pr and Pfr. As opposed to typical phytochromes, the BV-Agp2 adduct converts from Pr to Pfr in darkness. However, the 2,18-Et-BV-Agp2 and 3,18-Et-BV-Agp2 adducts can undergo dark conversion from Pr to Pfr and Pfr to Pr, showing that ring A of the chromophore has a direct impact on the direction of dark conversion. The doubly locked chromophores were designed to probe for the stereochemistry of the C5 methine bridge in the Pfr form. The adducts with 5Es15Ea-BV and 5Zs15Ea-BV absorbed in the blue spectral range only. Therefore, the C5 E syn and Z syn stereochemistries are unlikely for the Pfr chromophore of Agp1 and Agp2. According to our spectra, the Agp2 chromophore most likely adopts an E anti stereochemistry at its C5 methine bridge. Thus, during Pr to Pfr conversion, the C5 methine bridge of the chromophore might undergo a Hula-twist isomerization. In Agp1, the Pfr chromophore is most likely in the C5 Z anti stereochemistry. We propose that the stereochemistry of the C5 methine bridge might differ between different phytochromes, most particularly in the Pfr form.