Jaboticaba (Myrciaria cauliflora) Fruit Extract Suppressed Aberrant Crypt Formation in 1,2-Dimetylhydrazine-Induced Rats.

Early intervention can significantly improve the colorectal cancer survival rate. Foods rich in phenolic compounds, such as jaboticaba (Myrciaria cauliflora), may prevent tumorigenesis. We investigated the effectivity of jaboticaba whole fruit ethanolic extract (FEX) in suppressing aberrant crypt foci (ACF), the earliest lesion of colorectal cancer (CRC), in 1,2-dimethylhydrazine (DMH)-induced rats and the underlying mechanisms related to the gut microbiota composition and short chain fatty acid (SCFA). This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Providence University (Trial Registration Number 20180419A01, registration date: 22 December 2018). The FEX contains gallic acid and an especially high ellagic acid concentration of 54.41 ± 1.80 and 209.79 ± 2.49 mg/100 g FEX. The highest total ACF number (150.00 ± 43.86) was recorded in the DMH control (D) group. After 56 days of oral FEX treatment, the total ACF number in the low FEX dosage (DL) group was significantly lower compared to the D group (p < 0.05). The large-sized ACF (> 5 foci), which has a higher probability of progressing to later stage, was significantly decreased in the high FEX dosage (DH) group. The 16s rDNA metagenomic sequencing of the cecal material revealed that the CRC biomarker Lachnoclostridium was significantly suppressed in the DH group (p < 0.05), whereas some SCFA-producing taxa and the cecal butyrate concentration were significantly elevated in the DL and DH groups (p < 0.05). This study demonstrated the potential of jaboticaba whole fruit in CRC prevention, especially in the initial stage, by shifting gut microbiota composition and improving cecal butyrate level.

A New Schiff Base Chemodosimeter: Selective Colorimetric and Fluorescent Detection of Fe (III) Ion.

A new efficient chemosensor 1 was prepared, for the detection of Fe(3+) in solutions as a colorimetric and fluorescent sensor. The visual and fluorescent behaviors of the receptor toward various metal ions were also explored. The receptor shows exclusive response toward Fe(3+) ions and also distinguishes Fe(3+) from other cations by color change and fluorescence enhancement in hydroalcoholic solution (MeOH/H2O = 9/1, v/v). Thus, the receptor can be used as a colorometric and fluorescent sensor for the determination of Fe(3+) ion. The fluorescence microscopy experiments showed that the chemosensor is efficient for detection of Fe(3+) in vitro, developing a good image of the biological organelles. Graphical Abstract ᅟ.

A New Schiff Base Chemodosimeter for Fluorescent Imaging of Ferric Ions in Living Cells.

A new and efficient chemodosimeter for ferric ions has been developed. The visual and fluorescent behaviors of the compound toward various metal ions were investigated: ferric ions are distinguished from other cations by selective color change and unusual fluorescence enhancement in mixed aqueous solution. Fluorescence microscopy experiments showed that this receptor is effective for detection of Fe(3+) in vitro, developing a good image of the biological organelles. The sensing mechanism is shown to involve a hydrolysis process.

Non-competitive inhibition of acetylcholinesterase by jaboticaba (Myrciaria cauliflora) peel ethanolic extracts.

Specific anthocyanins and phenolic compounds exhibit acetylcholinesterase inhibitory (AChEi) activity. In this study, the AChEi activity of jaboticaba peel extracts were investigated based on their high phenol contents. Jaboticaba peel ethanolic extract (PEX) and aqueous extract (PAX) were prepared by extracting jaboticaba peel with 95% ethanol and boiling water, respectively. Through HPLC-MS/MS and HPLC-PDA analysis, gallic acid was identified in PAX with a concentration of 598.13 ± 42.43 mg/100 g extract, and ellagic acid in PEX with a concentration of 350.47 ± 8.53 mg/100 g extract. Both PEX and PAX showed dose-dependent inhibition against AChE activity, with IC50 values of 3.54 and 4.07 mg/mL, respectively. The mechanism of inhibition of PEX was determined to be non-competitive inhibition based on the decreasing V max and relatively constant K m with increasing PEX concentration, as determined using a Lineweaver-Burk plot.

The Association between Delayed Gut Microbiota Maturity in Pre-Term Infants and the Feeding Intolerance-A Pilot Study.

This study compared gut (fecal) microbiota profiles between pre-term and full-term infants, assuming that pre-term infants without feeding intolerance would have gut microbiota similar to those of full-term infants. A total of 13 pre-term infants (gestational age < 37 weeks, birthweight ≤ 2500 g) and 10 full-term infants were included. The pre-term infants were assigned to the feeding tolerance (FT) group (n = 7) if their daily intake exceeded 100 mL/kg/day at two weeks after birth, or the feeding intolerance (FI) group (n = 6). Microbial DNA from weekly fecal samples was analyzed. The microbiota profiles of the pre-term infants and full-term infants were significantly different (p = 0.0001), as well as the FT and FI groups (p = 0.0009). The full-term group had more diversity, with higher concentrations of facultative anaerobes such as Bifidobacteriaceae and Lactobacteriaceae. The FT group's gut microbiota matured over four weeks, with higher levels of digestion-related bacteria, while the FI group had more pathogens. In the FI group, a significant difference was observed between the first and second weeks, with no significant differences noted between the first week and the third or fourth weeks. The delay in the development of the pre-term infants' gut microbiota may be associated with the FI.

Improving antioxidant status in aged mice by 50% ethanol extract from red bean fermented by Bacillus subtilis.

BACKGROUND: The purpose of this study was to investigate the effects of 50% ethanol extracts from red bean non-fermented (RBE) and fermented by Bacillus subtilis (RBNE) on the antioxidant status of aged ICR mouse. RESULTS: Compared to 2-month-old ICR mouse, the plasma total antioxidant status (TAS) in 12-month-old ICR mouse decreased about 57%, while malondialdehyde (MDA) levels in the liver and brain of 12-month-old ICR mouse increased 56% and 30%, respectively. Orally administration of RBE or RBNE could completely recover the changes of MDA and plasma TAS levels due to the aging process. Vitamin E contents declined 88% in the liver and 74% in the brain of aged ICR mouse. At a level of 0.3 or 0.6 g kg(-1) body weight, RBNE raised vitamin E content in the liver and brain; however, RBE showed no significant influence. All antioxidant enzymes activities in the liver and brain of aged ICR mouse decreased compared to those activities in 2-month-old ICR mouse. RBNE could significantly enhance the superoxide dismutase activity in the brain of aged ICR mouse. CONCLUSION: Oral administration of RBE or RBNE could improve antioxidant status in aged ICR mouse. Fermentation by Bacillus subtilis could enhance the antioxidant properties of red bean.

Construction of Recombinant Saccharomyces cerevisiae with Ethanol and Aldehydes Tolerance via Overexpression of Aldehyde Reductase.

Furfural and hydroxy-methyl-furfural (HMF) are produced by lignocellulosic biomass during heat or acid pretreatment and are toxic to yeast. Aldehyde reductase is the main enzyme to reduce furfural and HMF. To improve the conversion efficiency of lignocellulosic biomass into ethanol, we constructed Saccharomyces cerevisiae with overexpression of aldehyde reductase (encoded by ari1). The gene of aldehyde reductase (encoded by ari1) was cloned via polymerase chain reaction (PCR) and ligated with the expression vector pGAPZαC. Western blot coupled with anti-His tag confirmed overexpression of the ari1 gene. The growth curves of the wild and ari1-overexpressed strain in the YPD medium were found to be almost identical. Compare to the ari1-overexpressed strain, the wild strain showed a longer doubling time and lag phase in the presence of 20 mM furfural and 60 mM HMF, respectively. The real-time PCR results showed that furfural was much more potent than HMF in stimulating ari1 expression, but the cell growth patterns showed that 60 mM HMF was more toxic to yeast than 20 mM furfural. S. cerevisiae with ari1 overexpression appeared to confer higher tolerance to aldehyde inhibitors, thereby increasing the growth rate and ethanol production capacity of S. cerevisiae in an aldehyde-containing environment.

Effects of fermented red bean extract on nephropathy in streptozocin-induced diabetic rats.

BACKGROUND: The antioxidant effects of Bacillus subtilis-fermented red bean (natto-red bean) extract (NRBE) in young (6 weeks old) Sprague-Dawley rats and aged (12 months old) mice had been reported previously. OBJECTIVE: To evaluate the antioxidant and anti-inflammatory effects of NRBE in the kidneys of streptozocin-induced diabetic rats. DESIGN: Normal control rats and diabetic rats were orally gavaged with saline and low-dose NRBE (100 mg/kg body weight [BW]), medium-dose NRBE (200 mg/kg BW), and high-dose NRBE (500 mg/kg BW), for 12 weeks and then sacrificed. Concentration of fasting glucose, adiponectin, renal function markers, antioxidative markers, and pro-inflammatory markers were measured. RESULTS: Oral administration of 50% ethanolic extract of NRBE with a dosage of 100 mg/kg BW, 200 mg/kg BW, or 500 mg/kg BW could improve the symptoms of kidney enlargement and renal function. Supplementation of NRBE can effectively inhibit the formation of renal reactive oxygen species and advanced-glycation end-products and increase renal glutathione content and serum adiponectin. A low dose of NRBE (100 mg/kg BW) decreased fasting blood sugar and renal interleukin (IL)-6 expression. Serum C-reactive protein, renal tumor necrosis factor-α, and monocyte chemoattractant protein-1 concentrations were decreased, and renal superoxide dismutase activity was increased in the medium-dose NRBE group. Twenty-four hour creatinine clearance and urinary albumin excretion also improved by medium-dose NRBE supplementation. In NRBE, total phenols and flavonoids were 6.3 mg gallic acid equivalent/g and 12.02 mg rutin equivalent/g, respectively, and kampherol was the major active antioxidant compound. CONCLUSION: This study demonstrated that appropriate amount of NRBE, 200 mg/kg BW in rats, could prevent diabetic nephropathy by improving antioxidant status and inhibiting inflammation in renal tissue.

Production of bioactive human alpha-defensin 5 in Pichia pastoris.

Human alpha-defensin 5 (HD5), a small cationic peptide, is expressed in Paneth cell granules of small intestinal crypts. HD5 exhibits high antimicrobial activity against a broad spectrum of pathogenic agents, including bacteria, fungi, and viruses. In this study, the constitutive expression of HD5 antimicrobial peptide was achieved using the methylotrophic yeast, Pichia pastoris (P. pastoris). HD5 cDNA was amplified by polymerase chain reaction (PCR) using human lung cell cDNA as template. The 96-bp DNA fragment encoding mature HD5 peptide (amino acid 63-94) was subcloned into the yeast expression vector and transfected into P. pastoris X-33 expression host by electroporation. The recombinant HD5 (rHD5) was detected in the supernatant of transfected yeast by western blot analysis. The recombinant HD5 crude extract from transfected P. pastoris showed antimicrobial activities against Salmonella typhimurium, Staphylococcus aureus and pathogenic E. coli. However, rHD5 did not inhibit the growth of lactic acid bacteria such as Lactobacillus bulgaricus, Bifidobacterium bifidum, or B. longum. These results indicated that the rHD5 expressed in P. pastoris selectively inhibited the growth of specific bacteria.

In vitro antioxidant and antiproliferative activity of the stem extracts from Graptopetalum paraguayense.

This study was aimed to evaluate the antioxidant abilities of water (SGWE), 50% ethanolic (SGE50) and 95% ethanolic (SGE95) extracts from the stem of Graptopetalum paraguayense, and the extract with the highest antioxidant activity was assayed for its inhibitory effect on proliferation of human hepatoma (Hep G2) cell line. Antioxidant abilities of extracts were assessed their radical-scavenging abilities and effects on Fe/ascorbate-induced lipid peroxidation in a liposome model system. The results of this study showed that antioxidant activities were increased with the increase of the extracts concentrations, and the activities correlated with both the total phenol and anthocyanin contents. A comparison of the 50% inhibition concentration (IC(50)) values of different antioxidant reactions revealed that SGWE was the more effective at scavenging superoxide anion radical and preventing lipid peroxidation than SGE50 and SGE95 (p<0.05). The flow cytometry results indicated that SGWE lowered cell viability, and induced G1 phase arrest and apoptosis in Hep G2 cells. These results demonstrated the antioxidant and anti-hepatoma potential of stem of Graptopetalum paraguayense.

Safety evaluation of water extracts of Toona sinensis Roemor leaf.

The purposes of this study were to evaluate the safety of water extracts of Toona sinensis Roemor leaf (TSL-1). The mutagenic properties of TSL-1 was investigated using the Ames test, and no mutagenicity was found toward all tester strains (Salmonella typhimurium TA98, TA100, TA102 and TA1535). In the acute oral toxicity study, a single limit dose of 5000 mgTSL-1/kg bw was given to male and female ICR mice, then observed for a 14-day period. In the subacute study, TSL-1 was administered as oral daily dose of 1000 mg/kg bw/day for 28 days. The results showed no acute lethal effect at a maximal tested dose of 5000 mg/kg bw TSL-1 in male and female mice. The subacute toxicity showed the oral administration of 1000 mg/kg bw for consecutive 28 days was safe in male mice. TSL-1 treated female mice showed decreases of food intake and kidney relative weight in acute oral toxicity test, and decreases of body weight gain, food intake and lung relative weight in subacute toxicity trial. However, no remarked toxic effects were found in the biochemical and histopathological parameters of TSL-1 treated female mice. These effects whether related to the major components or other ingredients in TSL-1 need to elucidate in the further studies.

Biochemical properties of genetic recombinant xylanase II.

The aim of this study was to overexpress the xylanase II gene of Trichoderma reesei in Escherichia coli and determine the characteristics of the recombinant enzyme. Recombinant xylanase II gene was constructed by ligating the cDNA of xylanase, obtained from reverse transcriptase-polymerase chain reaction, and fused with NusA protein of pET-431b plasmid. An Ni2+-NTA affinity column was used to further purify the recombinant xylanase II. The molecular mass of the recombinant enzyme measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approx 76 kDa (including 55 kDa of NusA and 21 kDa of xylanase II), and the isoelectric point and specific activity were 7.5 and 225 U/mg, respectively. The optimal reaction temperature and pH for the recombinant enzyme were 50 degrees C and 4.0, respectively. The recombinant enzyme was stable at a pH range of 5.0-10.0 and maintained 95% residual activity after incubating at 30-35 degrees C for 30 min. The kinetic parameters KM and Vmax of the recombinant xylanase II were 13.8 mg/mL and 336 micromol/(mg.min), respectively, using birchwood xylan as the substrate.

Metabolic engineering of Saccharomyces cerevisiae for improvement in stresses tolerance.

Lignocellulosic biomass is an attractive low-cost feedstock for bioethanol production. During bioethanol production, Saccharomyces cerevisiae, the common used starter, faces several environmental stresses such as aldehydes, glucose, ethanol, high temperature, acid, alkaline and osmotic pressure. The aim of this study was to construct a genetic recombinant S. cerevisiae starter with high tolerance against various environmental stresses. Trehalose-6-phosphate synthase gene (tps1) and aldehyde reductase gene (ari1) were co-overexpressed in nth1 (coded for neutral trehalase gene, trehalose degrading enzyme) deleted S. cerevisiae. The engineered strain exhibited ethanol tolerance up to 14% of ethanol, while the growth of wild strain was inhibited by 6% of ethanol. Compared with the wild strain, the engineered strain showed greater ethanol yield under high stress condition induced by combining 30% glucose, 30 mM furfural and 30 mM 5-hydroxymethylfurfural (HMF).

Protective effect of medicinal fungus Xylaria nigripes mycelia extracts against hydrogen peroxide-induced apoptosis in PC12 cells.

Xylaria nigripes ( XN) is a medicinal fungus that was used traditionally as a diuretic, nerve tonic, and for treating insomnia and trauma. In this study, we elucidated possible mechanisms of neuroprotective effects of XN mycelia extracts. XN mycelia were produced by fermentation. Hot water extract and 70% ethanol extract of XN mycelia were evaluated on hydrogen peroxide (H2O2)-induced apoptosis in PC12, a rat pheochromocytoma cell line. Both XN extracts effectively protected PC12 cells against H2O2-induced cell damage by inhibiting release of lactate dehydrogenase, decreasing DNA damage, restoring mitochondrial membrane potential, and arresting abnormal apoptosis through upregulation of Bcl-2 and downregulation of Bax and caspase 3. Compared to water extract, ethanol extract showed not only greater neuroprotective effects but also a higher antioxidant activity by scavenging DPPH radicals, inhibiting lipid peroxidation, and reducing power. High phenolic content and antioxidant activity may provide the neuroprotective properties of XN ethanol extract.

Engineering Saccharomyces cerevisiae for improvement in ethanol tolerance by accumulation of trehalose.

A genetic recombinant Saccharomyces cerevisiae starter with high ethanol tolerance capacities was constructed. In this study, the gene of trehalose-6-phosphate synthase (encoded by tps1), which catalyzes the first step in trehalose synthesis, was cloned and overexpressed in S. cerevisiae. Moreover, the gene of neutral trehalase (encoded by nth1, trehalose degrading enzyme) was deleted by using a disruption cassette, which contained long flanking homology regions of nth1 gene (the upstream 0.26 kb and downstream 0.4 kb). The engineered strain increased its tolerance against ethanol and glucose stress. The growth of the wild strain was inhibited when the medium contained 6 % or more ethanol, whereas growth of the engineered strain was affected when the medium contained 10 % or more ethanol. There was no significant difference in the ethanol yield between the wild strain and the engineered strain when the fermentation broth contained 10 % glucose (p > 0.05). The engineered strain showed greater ethanol yield than the wild type strain when the medium contained more than 15 % glucose (p < 0.05). Higher intracellular trehalose accumulation by overexpression of tps1 and deletion of nth1 might provide the ability for yeast to protect against environmental stress.

Characterization of a Chitosanase from Jelly Fig (Ficus awkeotsang Makino) Latex and Its Application in the Production of Water-Soluble Low Molecular Weight Chitosans.

A chitosanase was purified from jelly fig latex by ammonium sulfate fractionation (50-80% saturation) and three successive column chromatography steps. The purified enzyme was almost homogeneous, as determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and gel activity staining. The molecular mass of the enzyme was 20.5 kDa. The isoelectric point (pI) was <3.5, as estimated by isoelectric focusing electrophoresis on PhastGel IEF 3-9. Using chitosan as the substrate, the optimal pH for the enzyme reaction was 4.5; the kinetic parameters Km and Vmax were 0.089 mg mL-1 and 0.69 µmol min-1 mg-1, respectively. The enzyme showed activity toward chitosan polymers which exhibited various degrees of deacetylation (21-94%). The enzyme hydrolyzed 70-84% deacetylated chitosan polymers most effectively. Substrate specificity analysis indicated that the enzyme catalyzed the hydrolysis of chitin and chitosan polymers and their derivatives. The products of the hydrolysis of chitosan polymer derivatives, ethylene glycol (EG) chitosan, carboxymethyl (CM) chitosan and aminoethyl (AE) chitosan, were low molecular weight chitosans (LMWCs); these products were referred to as EG-LMWC, CM-LMWC and AE-LMWC, respectively. The average molecular weights of EG-LMWC, CM-LMWC and AE-LMWC were 11.2, 11.2 and 8.89 kDa, respectively. All of the LMWC products exhibited free radical scavenging activities toward ABTS•+, superoxide and peroxyl radicals.

Enzymatic saccharification and fermentation of xylose-optimized dilute acid-treated lignocellulosics.

The cellulose reactivity of two lignocellulosic feedstocks, switchgrass and poplar, was evaluated under straight saccharification (SS) and simultaneous saccharification and fermentation (SSF) conditions following dilute sulfuric acid pretreatments designed for optimum xylose yields. The optimum pretreatment conditions, within the constraints of the experimental system (Parr batch reactor), were 1.2% acid, 180 degrees C, and 0.5 min for switchgrass and 1% acid, 180 degrees C, and 0.56 min for poplar. The cellulase enzyme preparation was from Trichoderma reesei and fermentations were done with Saccharomyces cerevisiae. Time courses for SS were monitored as the sum of glucose and cellobiose; those for SSF as the sum of glucose, cellobiose, and ethanol. Percentage conversions under SS conditions were 79.1% and 91.4% for the pretreated poplar and switchgrass feedstocks, respectively. Analogous values under SSF conditions were 73.0% and 90.3% for pretreated poplar and switchgrass, respectively.

Red Bean Extract Inhibits Lipopolysaccharide-Induced Inflammation and H2O2-Induced Oxidative Stress in RAW 264.7 Macrophages.

Red bean (Phaseolus radiatus L. var. Aurea) is a leguminous seed and mainly used as one of the popular ingredients in oriental desserts. The objective of this study was to evaluate the anti-inflammatory activity of 50 g/kg ethanolic extract of red bean (RBE) by measuring lipopolysaccharide (LPS)-induced expressions of nitric oxide (NO), inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in RAW 264.7 macrophages. On the other hand, the antioxidant activity of RBE was determined by thiobarbituric acid reactive substances method and comet assay using H2O2-induced macrophages. The results showed that RBE at the concentrations of 50-200 µg/mL can significantly suppress the inflammatory responses in LPS-stimulated macrophages through the reduction of cellular NO and downregulation of the gene expressions of iNOS, COX-2, TNF-α, and IL-6 in a dose-dependent manner. Furthermore, RBE can diminish H2O2-induced oxidative damage in RAW 264.7 macrophage. Phenolic compounds and cyanidin-3-O-glucoside from BRE may have efficacy as overall in vitro anti-inflammatory and antioxidant agents. Red bean exerts an anti-inflammatory response and has potential as a health-promoting ingredient.

Antioxidative activity and safety of the 50 ethanolic extract from red bean fermented by Bacillus subtilis IMR-NK1.

This study aimed at evaluating the antioxidative activities and the safety of 50% ethanolic extract from red bean fermented by Bacillus subtillis IMR-NK1. The antioxidative activities, including alpha,alpha-diphenyl-beta-picryl-hydrazyl (DPPH) radicals scavenging effects, Fe(2+)-chelating ability, and reducing power, were studied in vitro. It was found that the antioxidative activity increased with the concentrations of the extract to a certain extent and then leveled off as the concentration further increased. As compared to the commercial antioxidants, the fermented red bean extract showed less scavenging effect on the DPPH radical and reducing power than alpha-tocopherol and BHT, but better Fe(2+)-chelating ability. No mutagenicity or toxicity effect toward all tester strains was found in the 50% ethanolic extract of fermented red bean by means of the Ames test. The results suggested that the 50% ethanolic extract was safe in genotoxicity.

Evaluation of antioxidative and mutagenic properties of 50% ethanolic extract from red beans fermented by Aspergillus oryzae.

Various bean products fermented by microorganisms are commonly consumed in Asian diets; however, the safety or functional properties of fermented beans can vary with different microbial species and with different processes being applied to different beans. The objectives of this study were to evaluate the antioxidative and mutagenic properties of 50% ethanolic extracts from red beans fermented by Aspergillus oryzae. The extracts' antioxidative activities, including alpha,alpha;-diphenyl-beta-picryl-hydrazyl (DPPH) radical-scavenging effects, Fe(2+)-chelating ability, and reducing power, were studied in vitro. The antioxidative effects provided by the extracts depended strongly on their concentrations. In general, antioxidative activity increased with extract concentration to a certain point and then leveled off as the concentration further increased. The fermented red bean extracts showed less of a scavenging effect on the DPPH radical and less reducing power than the commercial antioxidants alpha-tocopherol and butylated hydroxytoluene, but better Fe(2+)-chelating ability. No mutagenicity or toxicity effect on any of the tested strains (Salmonella Typhimurium TA97, TA98, TA100, TA102, and TA1535) was found for the 50% ethanolic extracts of fermented red beans with the Ames mutagenicity assay. These results suggest that the 50% ethanolic extracts were not mutagenic.