Recent Advances in Microbial Production of cis,cis-Muconic Acid.

cis,cis-Muconic acid (MA) is a valuable C6 dicarboxylic acid platform chemical that is used as a starting material for the production of various valuable polymers and drugs, including adipic acid and terephthalic acid. As an alternative to traditional chemical processes, bio-based MA production has progressed to the establishment of de novo MA pathways in several microorganisms, such as Escherichia coli, Corynebacterium glutamicum, Pseudomonas putida, and Saccharomyces cerevisiae. Redesign of the metabolic pathway, intermediate flux control, and culture process optimization were all pursued to maximize the microbial MA production yield. Recently, MA production from biomass, such as the aromatic polymer lignin, has also attracted attention from researchers focusing on microbes that are tolerant to aromatic compounds. This paper summarizes recent microbial MA production strategies that involve engineering the metabolic pathway genes as well as the heterologous expression of some foreign genes involved in MA biosynthesis. Microbial MA production will continue to play a vital role in the field of bio-refineries and a feasible way to complement various petrochemical-based chemical processes.

Anti­photoaging effect of fermented agricultural by­products on ultraviolet B­irradiated hairless mouse skin.

Processed products from agricultural produce generate a large number of agricultural by­products that contain a number of functional substances. These are often discarded owing to the lack of suitable processing methods. The present study investigated the anti­photoaging properties of fermented rice bran (FRB), soybean cake (FSB) and sesame seed cake (FSC) on ultraviolet B (UVB)­irradiated hairless mouse skin. Results indicated that the oral administration of FRB, FSB and FSC effectively inhibited the UVB irradiation­induced expression of matrix metalloproteinase (MMP)­2, MMP­9, MMP­3 and MMP­13. Reverse transcription­quantitative polymerase chain reaction results also demonstrated that FRB, FSB and FSC significantly inhibited the UVB­induced expression of the genes encoding tumor necrosis factor­α, inducible nitric oxide synthase, interleukin (IL)­6 and IL­1ß when compared with the UVB­vehicle group (P<0.05). Additionally, collagen degradation and mast cell infiltration were reduced in hairless mouse skin. Furthermore, UVB­induced wrinkle formation was also significantly reduced in mouse skin compared with the UVB­vehicle group (P<0.05). These results reveal that fermented agricultural by­products may serve as potential functional materials with anti­photoaging activities.

The composition of a bioprocessed shiitake (Lentinus edodes) mushroom mycelia and rice bran formulation and its antimicrobial effects against Salmonella enterica subsp. enterica serovar Typhimurium strain SL1344 in macrophage cells and in mice.

BACKGROUND: Human infection by pathogenic Salmonella bacteria can be acquired by consuming of undercooked meat products and eggs. Antimicrobial resistance against antibiotics used in medicine is also a major concern. To help overcome these harmful effects on microbial food safety and human health, we are developing novel antimicrobial food-compatible formulations, one of which is described in the present study. METHODS: The composition of a bioprocessed (fermented) rice bran extract (BPRBE) from Lentinus edodes liquid mycelia culture was evaluated using gas chromatography and mass spectrometry, and the mechanism of its antibacterial effect against Salmonella Typhimurium, strain SL1344 was investigated in macrophage cells and in mice. RESULTS: BPRBE stimulated uptake of the bacteria into RAW 264.7 murine macrophage cells. Activation of the cells was confirmed by increases in NO production resulting from the elevation of inducible nitric oxide synthase (iNOS) mRNA, and in protein expression. Salmonella infection down-regulated the expression of the following protein biomarkers of autophagy (a catabolic process for stress adaptation of cellular components): Beclin-1, Atg5, Atg12, Atg16, LC3-I and LC3-II. BPRBE promoted the upregulation of protein expressions that induced bacterial destruction in autolysosomes of RAW 264.7 cells. ELISA analysis of interferon IFN-ß showed that inflammatory cytokine secretion and bactericidal activity had similar profiles, suggesting that BPRBE enhances cell-autonomous and systemic bactericidal activities via autophagic capture of Salmonella. The treatment also elicited increased excretion of bacteria in feces and their decreased translocation to internal organs (cecum, mesenteric lymph node, spleen, and liver). CONCLUSIONS: The antibiotic mechanism of BPRBE involves the phagocytosis of extracellular bacteria, autophagic capture of intracellular bacteria, and prevention of translocation of bacteria across the intestinal epithelial cells. The new bioprocessing combination of mushroom mycelia and rice brans forms a potentially novel food formulation with in vivo antimicrobial properties that could serve as a functional antimicrobial food and medical antibiotic.

Changes in the Anti-Allergic Activities of Sesame by Bioconversion.

Sesame is an important oilseed crop, which has been used as a traditional health food to ameliorate the prevention of various diseases. We evaluated the changes in the anti-allergic activities of sesame by bioconversion. SDS-PAGE of non-fermented sesame proteins showed major allergen bands, while that of fermented sesame showed only a few protein bands. Additionally, we investigated the effectiveness of fermented sesame by bioconversion in tumor necrosis factor-α (TNF-α)- and interferon-γ (IFN-γ)-induced HaCaT cells. In HaCaT cells, fermented sesame inhibited the mRNA expression of interleukin-6 (IL-6) and interleukin-1ß (IL-1ß), thymus and macrophage-derived chemokine (MDC/CCL22), activation-regulated chemokine (TARC/CCL17), and intercellular adhesion molecule-1 (ICAM-1). Moreover, fermented sesame inhibited the activation of nuclear factor-κB (NF-κB) and signal transducer and activator of transcription 1 (STAT1). Fermented sesame exerts anti-allergic effects by suppressing the expression of chemokines and cytokines via blockade of NF-κB and STAT1 activation.

Mechanism of Antibacterial Activities of a Rice Hull Smoke Extract (RHSE) Against Multidrug-Resistant Salmonella Typhimurium In Vitro and in Mice.

The present study tested antibacterial activity of a rice hull smoke extract (RHSE) against a multidrug-resistant strain of Salmonella Typhimurium and examined its mode of suppressive action in vitro and in mice. In vitro studies showed that the minimum inhibitory concentration (MIC) value of RHSE was 1.29% (v/v). The inactivation was confirmed by complete loss of cell viability in the range of 104 to 107 colony forming units of the resistant Salmonella Typhimurium strain. Agarose and sodium dodecyl sulfate-polyacrylamide gel electrophoreses were used to evaluate the integrities of bacterial genomic DNA and total cellular protein profiles. The antibacterial action of RHSE results from a leakage of intracellular macromolecules following rupture of bacterial cells. Scanning electron microscopy of the cells shows that RHSE also induced deleterious morphological changes in the bacterial cell membrane of the pathogens. In vivo antibacterial activity of RHSE at a 1 × MIC concentration was examined in a bacterial gastroenteritis model using Balb/c mice orally infected with the Salmonella Typhimurium. The results show greatly decreased excretion of the bacteria into the feces and suppressed translocation of the bacteria to internal organs (cecum, mesenteric lymph node, spleen, and liver) compared with the infected mice not subjected to the RHSE treatment. Collectively, the present findings indicate that the mechanism of the antibacterial activities both in vitro and in the gastroenteritis environment of the animal model is the result of the direct disruption of cell structure, leading to cell death. RHSE has the potential to serve as a multifunctional food additive that might protect consumers against infections by antibiotic-resistant microorganisms. PRACTICAL APPLICATION: The rice hull derived liquid smoke has the potential to complement widely used wood-derived smoke as an antimicrobial flavor and health-promoting formulation for application in foods and feeds.

Turmeric Bioprocessed with Mycelia from the Shiitake Culinary-Medicinal Mushroom Lentinus edodes (Agaricomycetes) Protects Mice Against Salmonellosis.

This study investigated the suppressive mechanisms of an extract from bioprocessed Lentinus edodes mycelial liquid culture supplemented with turmeric (bioprocessed Curcuma longa extract [BPCLE]) against murine salmonellosis. The BPLCE extract from the bioprocessed mycelia of the Salmonella Typhimurium into murine RAW 264.7 macrophage cells, elimination of intracellular bacteria, and elevation of inducible nitric oxide synthase expression. Dietary administration of BPCLE activated leukocytes from the mice infected with Salmonella through the intraperitoneal route. The enzyme-linked immunosorbent assay of the cytokines produced by splenocytes from infected mice showed significant increases in the levels of Th1 cytokines, including interleukin (IL)-1ß, IL-2, IL-6, and IL-12. Histology showed that dietary administration of BPCLE protected against necrosis of the liver resulting from a sublethal dose of Salmonella. In addition, the treatment (1) extended the lifespan of lethally infected mice, (2) suppressed the invasion of Salmonella into human Caco-2 colorectal adenocarcinoma cells, (3) increased excretion of the bacterium in the feces, (4) suppressed the translocation of the Salmonella to internal organs, and (5) increased total immunoglobulin A in both serum and intestinal fluids. BPCLE protected the mice against salmonellosis via cooperative effects that include the upregulation of the Th1 immune reaction, prevention of translocation of bacteria across the intestinal epithelial cells, and increased immunoglobulin A production in serum and intestinal fluids.

Comparative Analysis of γ-Oryzanol, ß-Glucan, Total Phenolic Content and Antioxidant Activity in Fermented Rice Bran of Different Varieties.

Rice bran, a by-product derived from processing rice, is a rich source of bioactive compounds. Recent studies have suggested that the fermentation can improve their biological activities. This study aimed to determined the level of γ-oryzanol, ß-glucan and total phenol contents of fermented rice bran from 21 Korean varieties, as well as to evaluate their antioxidant activities. We also assessed the validation of the analytical method for determining γ-oryzanol content in fermented rice brans. Among the fermented rice brans, the Haedam rice bran contained the highest level of total phenol content (156.08 mg gallic acid equivalents/g), DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity (71.30%) and ORAC (Oxygen radical absorbance capacity) value (1101.31 µM trolox equivalents/g). Furthermore, the fermented Migwang rice bran showed the highest level of γ-oryzanol content (294.77 ± 6.74 mg/100 g).

A Bioprocessed Polysaccharide from Lentinus edodes Mycelia Cultures with Turmeric Protects Chicks from a Lethal Challenge of Salmonella Gallinarum.

Our previous studies demonstrated that a bioprocessed polysaccharide (BPP) isolated from Lentinus edodes mushroom mycelia cultures supplemented with black rice bran can protect mice against Salmonella lipopolysaccharide-induced endotoxemia and reduce the mortality from Salmonella Typhimurium infection through upregulated T-helper 1 immunity. Here, we report that a BPP from L. edodes mushroom mycelia liquid cultures supplemented with turmeric (referred to as BPP-turmeric) alters chicken macrophage responses against avian-adapted Salmonella Gallinarum and protects chicks against a lethal challenge from Salmonella Gallinarum. In vitro analyses revealed that the water extract of BPP-turmeric (i) changed the protein expression or secretion profile of Salmonella Gallinarum, although it was not bactericidal, (ii) reduced the phagocytic activity of the chicken-derived macrophage cell line HD-11 when infected with Salmonella Gallinarum, and (iii) significantly activated the transcription expression of interleukin (IL)-1ß, IL-10, tumor necrosis factor α, and inducible nitric oxide synthase in response to various Salmonella infections, whereas it repressed that of IL-4, IL-6, interferon-ß, and interferon-γ. We also found that BPP-turmeric (0.1 g/kg of feed) as a feed additive provided significant protection to 1-day-old chicks infected with a lethal dose of Salmonella Gallinarum. Collectively, these results imply that BPP-turmeric contains biologically active component(s) that protect chicks against Salmonella Gallinarum infection, possibly by regulating macrophage immune responses. Further studies are needed to evaluate the potential efficacy of BPP-turmeric as a livestock feed additive for the preharvest control of fowl typhoid or foodborne salmonellosis.

Acute and subchronic (13-week) toxicity of fermented Acanthopanax koreanum extracts in Sprague Dawley rats.

The biological fermentation of plants is usually used to improve their product properties, including their biological activity. Acanthopanax koreanum is a plant indigenous to Jeju, Korea; however, fermented A. koreanum (FAK) has not been guaranteed to be safe. Therefore, in this study, a safety evaluation of aqueous extracts of FAK was performed using Sprague Dawley rats. The acute toxicity of FAK did not influence animal mortality, body weight changes or the animals' clinical appearance at a concentration of 5000 mg/kg body weight. Using doses of 500, 1000 and 2000 mg/kg/day in a subchronic (13-week) toxicity study, the administration of FAK in male rats increased their body weight, food consumption, absolute liver weight, liver-associated enzymes and total cholesterol content. However, these effects of FAK were not considered toxic because the changes were not accompanied by any evidence of clinical signs or any change in the histopathological examination. On the other hand, the FAK-treated female rats did not exhibit significant changes in their body weight, food consumption, absolute and relative organ weights or liver enzymes. These results suggest that the acute oral administration of FAK is non-toxic to rats, and 13 weeks of repeated dosing demonstrated no FAK-related toxicity at a concentration of 2000 mg/kg. Therefore, the no-observed-adverse-effect level (NOAEL) of FAK was determined to be 2000 mg/kg/day for both male and female rats.

Elm Tree (Ulmus parvifolia) Bark Bioprocessed with Mycelia of Shiitake (Lentinus edodes) Mushrooms in Liquid Culture: Composition and Mechanism of Protection against Allergic Asthma in Mice.

Mushrooms can break down complex plant materials into smaller, more digestible and bioactive compounds. The present study investigated the antiasthma effect of an Ulmus parvifolia bark extract bioprocessed in Lentinus edodes liquid mycelium culture (BPUBE) against allergic asthma in chicken egg ovalbumin (OVA)-sensitized/challenged mice. BPUBE suppressed total IgE release from U266B1 cells in a dose-dependent manner without cytotoxicity. Inhibitory activity of BPUBE against OVA-specific IgE secretion in bronchoalveolar lavage fluid (BALF) was observed in OVA-sensitized/challenged asthmatic mice. BPUBE also inhibited OVA-specific IgG and IgG1 secretion into serum from the allergic mice, suggesting the restoration of a Th2-biased immune reaction to a Th1/Th2-balanced status, as indicated by the Th1/Th2 as well as regulatory T cell (Treg) cytokine profile changes caused by BPUBE in serum or BALF. Inflammatory cell counts in BALF and lung histology showed that leukocytosis and eosinophilia induced by OVA-sensitization/challenge were inhibited by the oral administration of BPUBE. Amelioration of eosinophil infiltration near the trachea was associated with reduced eotaxin and vascular cell adhesion molecule-1 (VCAM-1) levels. Changes in proinflammatory mediator levels in BALF suggest that BPUBE decreased OVA-sensitization-induced elevation of leukotriene C4 (LTC4) and prostaglandin D2 (PGD2). The finding that asthma-associated biomarker levels of OVA-sensitized/challenged mice were much more inhibited with BPUBE treatment than NPUBE (not-bioprocessed Ulmus parvifolia extract) treatment suggested the production of new bioactive compounds by the mushroom mycelia that may be involved in enhancing the observed antiasthmatic properties. The possible relation of the composition determined by proximate analysis and GC/MS to observed bioactivity is discussed. The results suggest that the elm tree (Ulmus parvifolia) bark bioprocessed with mycelia of shiitake (Lentinus edodes) mushrooms has the potential to prevent and/or treat allergic asthma.

The antioxidant activity and their major antioxidant compounds from Acanthopanax senticosus and A. koreanum.

The antioxidant activity and chlorogenic acid and caffeic acid contents were investigated from different parts of Acanthopanax senticosus and A. koreanum. Antioxidant activity was assessed by various in vitro assays such as DPPH, ABTS, FRAP, reducing power assays and ORAC, and the chlorogenic acid and caffeic acid were validated by HPLC chromatography. Among the various extracts, the fruit extracts of A. senticosus and A. koreanum exhibited strongest antioxidant activities including ABTS, FRAP, reducing power and ORAC, however, strongest DPPH radical scavenging activity was observed from the leaf extract of A. senticosus. In addition, the antioxidant activities of various extracts were correlated with total phenolic and proanthocyanidin contents. The major phenolic contents from various parts of these plants observed that leaf extract of A. senticosus expressed higher levels of chlorogenic acid (14.86 mg/dry weigh g) and caffeic acid (3.09 mg/dry weigh g) than other parts. Therefore, these results suggest that the leaf of A. senticosus may be an excellent natural source for functional foods and pharmaceutical agents, and the validated method was useful for the quality control of A. senticosus.

Application of new guidelines for the primary prevention of atherosclerotic cardiovascular disease in a Korean population.

AIM: We investigated the proportion of people who would qualify for statin treatment with an atherosclerotic cardiovascular disease (ASCVD) 10-year risk of ≥7.5% and who exhibit an LDL cholesterol (LDL-C) of 70 to 189 mg/dL according to the new ACC/AHA guidelines for the treatment of increased cardiovascular risk. METHODS: The study population (8,742 subjects) included individuals who underwent health examinations at Kangbuk Samsung Hospital in South Korea in 2010. We also evaluated the data obtained from the 2008-2010 Korea National Health and Nutrition Examination Survey (KNHANES) of 16,892 adults. RESULTS: Approximately 90% of men ≥60 years of age and women ≥70 years of age had an ASCVD 10-year risk of ≥7.5% and LDL-C level of ≥70 mg/dL. The proportions of subjects with a Framingham 10-year risk of ≥10%, coronary artery calcium score of >20 and >100 and fatty liver each increased in association with an increasing ASCVD 10-year risk quartile in both sexes. Furthermore, age was significantly associated with the ASCVD 10-year risk in both sexes (all p-value <0.001). The KNHANES data also showed that over 85.0% of men ≥60 years of age and 95.0% of women ≥70 years of age had an ASCVD 10-year risk of ≥7.5% and an LDL-C level of ≥70 mg/dL. CONCLUSIONS: Adopting the new ASCVD prevention guidelines would result in the treatment of almost all Korean men and women (≥60 years and ≥70 years of age, respectively) without evidence of cardiovascular disease. Therefore, Asian-specific guidelines are needed to avoid unnecessary over treatment in an aging global population.

A Polysaccharide isolated from the liquid culture of Lentinus edodes (Shiitake) mushroom mycelia containing black rice bran protects mice against salmonellosis through upregulation of the Th1 immune reaction.

The present study investigated the antibacterial effect of a bioprocessed polysaccharide (BPP) isolated from Lentinus edodes liquid mycelial culture supplemented with black rice bran against murine salmonellosis. BPP was not bactericidal in vitro, it did, however, stimulate uptake of the bacteria into RAW 264.7 murine macrophage cells, as indicated by increased colony-forming unit (CFU) counts of the contents of the lysed macrophages incubated with Salmonella Typhimurium for 30 and 60 min. Two hours postinfection, the bacterial counts drastically increased in the macrophages, but 4 and 8 h postinfection BPP extract-treated cells showed lower bacterial counts than the vehicle (saline phosphate pH 7.4 buffer, PBS)-treated control. BPP elicited altered morphology and markedly elevated inducible nitric oxide (NO) synthase (iNOS) mRNA and protein expression in the infected macrophage cells. BPP also activated leukocytes in S. Typhimurium-infected mice, as determined by spleen lymphocyte proliferation and IFN-γ levels in mice sera. ELISA analysis on cytokine production by Th1 and Th2 immune cells from splenocytes of infected mice showed significant increases in the levels of the following Th1 cytokines: IL-1ß, IL-2, IL-6, and IL-12. Histology assays of the livers of mice infected with a sublethal dose (1 × 10(4) CFU) of S. Typhimurium showed that BPP, administered daily through an intraperitoneal (ip) or oral route, protected against necrosis of the liver, a biomarker of in vivo salmonellosis. The lifespan of mice similarly infected with a lethal dose of S. Typhimurium (1 × 10(5) CFU) was significantly extended by ip injection or oral administration of the BPP without side effects. These results suggest that the activity of BPP against bacterial infection in mice occurs mainly through the activation of macrophage-mediated immune response resulting from augmented Th1 immunity. The significance of the results for microbial food safety and human health and further research needs are discussed.

A polysaccharide isolated from the liquid culture of Lentinus edodes (Shiitake) mushroom mycelia containing black rice bran protects mice against a Salmonella lipopolysaccharide-induced endotoxemia.

Endotoxemia (sepsis, septic shock) is an inflammatory, virulent disease that results mainly from bacterial infection. The present study investigates the inhibitory effect of a bioprocessed polysaccharide (BPP) isolated from the edible Lentinus edodes liquid mycelial mushroom culture supplemented with black rice bran against murine endotoxemia induced by the Salmonella lipopolysaccharide and d-galactosamine (LPS/GalN). BPP was obtained after dialysis against water using a cellulose tube with a molecular weight cutoff of 10000. BPP eluted as a single peak on an HPLC chromatogram. Acid hydrolysis of BPP showed the presence of the following sugars: fucose, galactose, galactosamine, glucose, glucosamine, mannose, rhamnose, and xylose. Treatment of BPP with ß-glucanase reduced its immunostimulating activity, suggesting that the polysaccharide has a ß-glucan structure. Pretreatment of mice with BPP via oral or intraperitoneal (ip) administration for 2 weeks resulted in the suppression of LPS/GalN-induced catalase, superoxide dismutase (SOD), and transaminase (GOT/GPT) liver enzymes, amelioration of necrotic liver lesions, and reduction of tumor necrosis factor α (TNF-α) and nitrite serum levels as well as myeloperoxidase (MPO) activity, an index of necrotic injury. Immunostimulating macrophage activity was up to 5.4-fold greater than that observed with the culture without the rice bran. BPP also extended the lifespan of the toxemic mice. These positive results with inflammation biomarkers and lifespan studies suggest that the BPP can protect mice against LPS/GalN-induced liver, lung, and kidney injuries and inflammation by blocking oxidative stress and TNF-α production, thus increasing the survival of the toxic shock-induced mice. The polysaccharide has the potential to serve as a new functional food.