Effect of lysin EN4 in combination with sodium bicarbonate on reduction of Salmonella in chilled and thawed chicken meat.

Lysin EN4 is a peptidoglycan-degrading enzyme. Like other lysins against Gram-negative bacteria, EN4 requires cell-wall destabilizing agents, such as ethylenediamine tetraacetic acid (EDTA) to facilitate it to the peptidoglycan layer. This study aimed to use EN4 in reducing Salmonella in chilled and thawed raw chicken meat. However, the use of EDTA is limited to some types of foods. An alternative to EDTA was explored. Sodium bicarbonate was identified as an effective alternative to EDTA. The combination of EN4 with 0.1 % NaHCO3, pH 7.4 showed a wide lytic spectrum against Salmonella spp. The combination showed efficiency in reduction of Salmonella Enteritidis and Typhimurium in raw chicken meat during storage at 4 °C for 48 h, with the maximum reduction of 1.0-1.3log CFU/g. The efficiency of the combination against Salmonella was evaluated in frozen chicken meat during proper and improper defrosting. A significant reduction of Salmonella was observed in EN4-treated meat compared to the untreated control through 48 and 4 h of defrosting at 4 and 30 °C, respectively, with the greatest reduction of 1.2-1.6 log CFU/g. The results indicated that EN4 in combination with NaHCO3 has a potential use for controlling growth of Salmonella in chilled and thawed chicken meat.

The functional starter and its genomic insight for histamine degradation in fish sauce.

Histamine is a biogenic amine significantly formed in fish sauce leading to a major concern in consumers. This study aimed to identify a halophilic bacterium for histamine degradation in fish sauce, and understand its genomic insight to enhance histamine degradation activity. We discovered the novel halophilic bacterium, Bacillus piscicola FBU1786, degrading histamine and other biogenic amines. Its histamine breakdown was growth-associated in a wide range of NaCl concentrations, pH, and temperature from 4% to 18%, 6.0 to 9.0, and 30 to 45 °C, respectively. Genome sequencing revealed the presence of Cu2+-binding oxidase-encoding genes and their heterologous expression with Cu2+ supplementation triggered histamine degradation in E. coli. The degree of histamine breakdown in B. piscicola FBU1786 could be enhanced by Cu2+ addition. Histamine degradation of the culture was evaluated in raw fish sauce mixtures to partially mimic the condition during fish sauce fermentation. Histamine degradation was suppressed to the extent of raw fish sauce, but could be restored by Cu2+ supplementation. Together, this study disclosed B. piscicola FBU1786 with the potent histamine degradation activity, identified Cu2+-binding oxidases responsible for histamine breakdown, and enhanced histamine degradation of the culture using Cu2+ supplementation.

Suppression of inflammatory mediators and matrix metalloproteinase (MMP)-13 by Morus alba stem extract and oxyresveratrol in RAW 264.7 cells and C28/I2 human chondrocytes.

This study aimed to investigate the effects of Morus alba stem extract (MSE) and oxyresveratrol on the suppression of pro-inflammatory mediators in LPS-stimulated RAW 264.7 macrophages and IL-1ß-stimulated C28/I2 human chondrocyte cell line. The chondroprotective effect was also investigated using the chondrocyte cell line. First, MSE was prepared and analyzed for the amount of oxyresveratrol. The anti-inflammatory effects of MSE at various concentrations were evaluated through the inhibition of nitric oxide (NO), prostaglandin (PG)-E2 and cyclooxygenase (COX)-2 production. Oxyresveratrol at the equivalent amount found in the extract was investigated in the same manner. The chondroprotective effect was investigated through the suppression of MMP-13 production. The results showed that oxyresveratrol content in MSE was 15%. In RAW 264.7 cells, MSE (5-50 µg/mL) could inhibit the NO (24-30%) and PGE2 (11-82%) production. Oxyresveratrol at 0.75 and 7.5 µg/mL could suppress NO and also inhibited PGE2 but at only at high concentration. In the chondrocyte cell line, MSE at 5-100 µg/mL significantly decreased the PGE2 and COX-2 production by 44-93% and 17-65%, respectively. Again, oxyresveratrol at both concentrations could significantly inhibit PGE2 production by 50-92% but it inhibited COX-2 only at high concentration. In addition, MSE and oxyresveratrol was shown to significantly inhibit MMP-13 production by 14-57% and 16-56%, depending on their concentrations. The MSE demonstrates the potential to be used as an alternative treatment for reducing inflammation and preventing cartilage degradation. Its component, oxyresveratrol, may exert these effects to some extent.

Selection of Potential Probiotic Lactobacillus with Inhibitory Activity Against Salmonella and Fecal Coliform Bacteria.

Three hundred and sixty presumptive lactic acid bacteria (LAB) isolated from pregnant sows, newborn, suckling, and weaned piglets were preliminarily screened for anti-Salmonella activity. Fifty-eight isolates consisting of Lactobacillus reuteri (n = 32), Lactobacillus salivarius (n = 10), Lactobacillus mucosae (n = 8), Lactobacillus johnsonii (n = 5), and Lactobacillus crispatus (n = 3) were selected and further characterized for probiotic properties including production of antimicrobial substances, acid and bile tolerance, and cell adherence to Caco-2 cells. Eight isolates including Lact. johnsonii LJ202 and Lact. reuteri LR108 were identified as potential probiotics. LJ202 was selected for further use in co-culture studies of two-bacterial and multiple-bacterial species to examine its inhibitory activity against Salmonella enterica serovar Enteritidis DMST7106 (SE7106). Co-culture of LJ202 and SE7106 showed that LJ202 could completely inhibit the growth of SE7106 in 10 h of co-culture. In co-culture of multiple-bacterial species, culturable fecal bacteria from pig feces were used as representative of multiple-bacterial species. The study was performed to examine whether interactions among multiple-bacterial species would influence antagonistic activity of LJ202 against SE7106 and fecal coliform bacteria. Co-culture of SE7106 with different combinations of fecal bacteria and probiotic (LJ202 and LR108) or non-probiotic (Lact. mucosae LM303) strains revealed that the growth of SE7106 was completely inhibited either in the presence or in the absence of probiotic strains. Intriguingly, LJ202 exhibited notable inhibitory activity against fecal coliform bacteria while LR108 did not. Taken together, the results of co-culture studies suggested that LJ202 is a good probiotic candidate for further study its inhibitory effects against pathogen infections in pigs.

In Vitro Anti-Inflammatory Activity of Morus alba L. Stem Extract in LPS-Stimulated RAW 264.7 Cells.

Morus alba L., also known as white mulberry or Mhon, has long been used in traditional medicines. This study was aimed to investigate anti-inflammatory activities of mulberry stem ethanolic extract (MSE) in lipopolysaccharide- (LPS-) stimulated RAW 264.7 macrophage cell line. The MSE was first prepared and then investigated for cell viability using the MTT assay. The anti-inflammatory activities were investigated through the inhibition of inducible nitric oxide synthase (iNOS), cyclooxygenase- (COX-) 2 mRNA expression, and iNOS protein expression using reverse transcription-polymerase chain reaction (RT-PCR) assay and immunoblotting analysis, respectively. The inhibition of nitric oxide production of the MSE was also investigated using the Griess reaction assay. The MSE concentration ranging from 10 to 40 µg/ml yielded cell viability higher than 80%. The MSE at concentrations of 20 and 40 µg/ml demonstrated anti-inflammatory activity through the inhibition of nitric oxide production via suppression of both the iNOS mRNA and protein. It was also found to inhibit the expression of COX-2 mRNA in LPS-induced RAW 264.7 cells. This study is the first to report the anti-inflammatory potential of the extract prepared from the stem of mulberry.

Identification and characterization of R2R3-MYB and bHLH transcription factors regulating anthocyanin biosynthesis in gentian flowers.

Gentian plants have vivid blue-colored flowers, caused by accumulation of a polyacylated anthocyanin 'gentiodelphin'. We previously performed expression analysis of gentiodelphin biosynthetic genes, and hypothesized that the white-flowered gentian cultivar 'Polarno White' might have resulted from the mutation of certain regulatory factors responsible for anthocyanin biosynthesis in flower petals. In this study, we isolated 26 R2R3-MYB gene fragments including four full-length cDNAs (GtMYB2a, GtMYB2b, GtMYB3 and GtMYB4) and one basic helix-loop-helix (bHLH) gene (GtbHLH1) from blue-flowered gentian by degenerate PCR and rapid amplification of cDNA ends (RACE). Phylogenetic tree analysis showed that GtMYB3 was categorized into a clade involved in anthocyanin biosynthesis including petunia AN2 and Arabidopsis PAP1. On the other hand, GtbHLH1 exhibited high identity with petunia AN1 based on both phylogenetic and genomic structural analyses. Temporal profiles of GtMYB3 and GtbHLH1 transcript levels corresponded well with those of gentiodelphin accumulation and their biosynthetic genes in petals. Yeast two-hybrid analysis showed that GtbHLH1 interacted with GtMYB3. Moreover, transient expression analysis indicated that the co-expression of GtMYB3 and GtbHLH1 could enhance the promoter activities of late anthocyanin biosynthetic genes in tobacco BY2 cells. We also revealed that in cv. 'Polarno White' the GtMYB3 genes were mutated by insertions of transposable elements or uncharacterized sequences, indicating that the white coloration was caused by GtMYB3 mutation. These results strongly suggested that GtMYB3 and GtbHLH1 are involved in the regulation of gentiodelphin biosynthesis in gentian flowers.