Vitamin K2 Modulates Mitochondrial Dysfunction Induced by 6-Hydroxydopamine in SH-SY5Y Cells via Mitochondrial Quality-Control Loop.

Vitamin K2, a natural fat-soluble vitamin, is a potent neuroprotective molecule, owing to its antioxidant effect, but its mechanism has not been fully elucidated. Therefore, we stimulated SH-SY5Y cells with 6-hydroxydopamine (6-OHDA) in a proper dose-dependent manner, followed by a treatment of vitamin K2. In the presence of 6-OHDA, cell viability was reduced, the mitochondrial membrane potential was decreased, and the accumulation of reactive oxygen species (ROS) was increased. Moreover, the treatment of 6-OHDA promoted mitochondria-mediated apoptosis and abnormal mitochondrial fission and fusion. However, vitamin K2 significantly suppressed 6-OHDA-induced changes. Vitamin K2 played a significant part in apoptosis by upregulating and downregulating Bcl-2 and Bax protein expressions, respectively, which inhibited mitochondrial depolarization, and ROS accumulation to maintain mitochondrial structure and functional stabilities. Additionally, vitamin K2 significantly inhibited the 6-OHDA-induced downregulation of the MFN1/2 level and upregulation of the DRP1 level, respectively, and this enabled cells to maintain the dynamic balance of mitochondrial fusion and fission. Furthermore, vitamin K2 treatments downregulated the expression level of p62 and upregulated the expression level of LC3A in 6-OHDA-treated cells via the PINK1/Parkin signaling pathway, thereby promoting mitophagy. Moreover, it induced mitochondrial biogenesis in 6-OHDA damaged cells by promoting the expression of PGC-1α, NRF1, and TFAM. These indicated that vitamin K2 can release mitochondrial damage, and that this effect is related to the participation of vitamin K2 in the regulation of the mitochondrial quality-control loop, through the maintenance of the mitochondrial quality-control system, and repair mitochondrial dysfunction, thereby alleviating neuronal cell death mediated by mitochondrial damage.

Effect of static magnetic field on morphology and growth metabolism of Flavobacterium sp. m1-14.

Increasing evidence shows that static magnetic fields (SMFs) can affect microbial growth metabolism, but the specific mechanism is still unclear. In this study, we have investigated the effect of moderate-strength SMFs on growth and vitamin K2 biosynthesis of Flavobacterium sp. m1-14. First, we designed a series of different moderate-strength magnetic field intensities (0, 50, 100, 150, 190 mT) and exposure times (0, 24, 48, 72, 120 h). With the optimization of static magnetic field intensity and exposure time, biomass and vitamin K2 production significantly increased compared to control. The maximum vitamin K2 concentration and biomass were achieved when exposed to 100 mT SMF for 48 h; compared with the control group, they increased by 71.3% and 86.8%, respectively. Interestingly, it was found that both the cell viability and morphology changed significantly after SMF treatment. Second, the adenosine triphosphate (ATP) and glucose-6-phosphate dehydrogenase (G6PDH) metabolism is more vigorous after exposed to 100 mT SMF. This change affects the cell energy metabolism and fermentation behavior, and may partially explain the changes in bacterial biomass and vitamin K2 production. The results show that moderate-strength SMFs may be a promising method to promote bacterial growth and secondary metabolite synthesis.

Improvement of menaquinone-7 production by Bacillus subtilis natto in a novel residue-free medium by increasing the redox potential.

Bacillus subtilis natto is a GRAS bacterium. Nattokinase, with fibrinolytic and antithrombotic activities, is one of the major products of this organism. It is being gradually recognized that B. subtilis natto can also be used as a biosynthetic strain for vitamin K2, which has phenomenal benefits, such as effects in the prevention of cardiovascular diseases and osteoporosis along with antitumor effects. Knocking out of the aprN gene by homologous recombination could improve the redox potential and slightly increase the concentration of MK-7. By detecting the change in redox potential during the growth of B. subtilis natto, a good oxygen supply and state of the cell membrane were found to be beneficial to vitamin K2 synthesis. A two-step RSM was used to optimize the operation parameters and substrate concentration in the new residue-free fermentation culture. The optimal conditions for the residue-free medium and control were determined. The optimum concentrations of soybean flour, corn flour, and peptone were 78.9, 72.4, and 24.8 g/L, respectively. The optimum rotational speed and volume of the culture medium using a shaking flask were 117 rpm and 10%, respectively. The state and composition of the cell membranes were more stable when engineered bacteria were cultured in this residue-free fermentation medium. Finally, the concentration of MK-7 increased by 37% to 18.9 mg/L, and the fermentation time was shortened by 24 h.

A simple and efficient method for the extraction and separation of menaquinone homologs from wet biomass of Flavobacterium.

Menaquinone homologs (MK-n), that is, MK-4, MK-5, and MK-6, can be produced by the fermentation of Flavobacterium. In this study, we proposed a simple and efficient method for the extraction of menaquinones from wet cells without the process of drying the biomass. Meanwhile, a rapid and effective solution for the separation of menaquinone homologs was developed using a single organic solvent, which was conducive to the recovery of the solvent. The results showed that the highest yield was obtained with pretreatment using absolute ethanol at a ratio of 6:1 (v/m) for 30 min and then two extractions of 30 min each using methanol at a ratio of 6:1 (v/m). The recovery efficiency of the menaquinones reached to 102.8% compared to the positive control. The menaquinone homologs were effectively separated using methanol as eluent at a flow rate of 0.52 mL/min by a glass reverse-phase C18 silica gel column with a height-to-diameter ratio of 5.5:1. The recovery of menaquinones achieved was 99.6%. In conclusion, the methods for extraction and separation of menaquinone homologs from wet Flavobacterium cells were simple and efficient, which makes them suitable not only on a laboratory scale but also for application on a large scale.

Optimization Medium Composition for Vitamin K2 by Flavobacterium sp. using Response Surface Methodology and Addition of Arachis hypogaea

ABSTRACT The purpose of this research was to enhance the production of vitamin K2 by fermentation optimization and Arachis hypogaea supplementation in Flavobacterium sp. mutant SP-L-01. Optimized culture condition were as follows: 6-days shake-flask culture at 37oC with initial pH value 7.0 ± 0.2, shaking speed in 120 r/min and medium volume of 30 mL with 2% inoculums. After optimization of fermentation medium by response surface methodology (RSM), optimized medium were maltose 23.8 g/l, glucose 9.69 g/l, beef extract 15 g/l, K2HPO4 4.5 g/l，NaCl 3.0 g/l and MgSO4·7H2O 0.3 g/l. Production of vitamin K2 after optimization reached to 10.97 mg/l, which is 79.25% higher than that before optimization (6.12 mg/l). 3 mg/mL of arachis hypogaea was added into the medium at 72 h of shake-flake cultivation, which improved the production of menaquinone-4 (MK4) up to 371% and menaquinone-6 (MK6) up to 149% higher than those of the original medium. D-(+)-catechin, one of the components of arachis hypogaea, was added alone into the medium, which also improved the vitamin K2 synthesis.

Enhanced Production of Vitamin K2 from Bacillus subtilis (natto) by Mutation and Optimization of the Fermentation Medium

The aim of this study was to enhance the production of vitamin K2 by using N-methyl-N-nitro-N-nitroso-guanidine (NTG) and low energy ion beam implantation and optimizing the fermentation medium. Mutation resulted in 1.66-fold higher production of vitamin K2 than that of the parentl strain. The production by the mutant BN-P15-11-1was increased 55% and reached 3.593±0.107 mg/L by using the Plackett-Burman and Box-Behnken designs to optimize the fermentation medium. The optimal fermentation culture medium was composed of (g/L) glycerol 69.6, sucrose 34.5, K2HPO4 4.0, peptone 20, yeast extract 25 and fermented at 37 °C and 150 rpm for 72 h. The results showed that the NTG and low energy ion beam implantation mutations and optimizing fermentation medium were effective methods to enhance vitamin K2 production.

[Protection of polysaccharides-2b from mudan cortex of Paeonia suffruticosa andr on diabetic cataract in rats].

OBJECTIVE: To observe the the protective effection of polysaccharides-2b of mudan cortex of Paeonia suffruticosa andr (PSM2b) on diabetic cataract. METHOD: The animal model of diabetic cataract in rats was induced by streptozotocin (STZ) and freund's adjuvant complete (CFA). The initial opacity occurrence time in lens was investigated with cranny lamp, and opacity degree of lens was compared too. The activities of glutathione peroxidase (GSH-pX), superoxide dismutase (SOD), catalase (CAT), the content of malonaldehyde (MDA) in serum and lens were detected. At the same time, the activities of Na(+) -K(+) -ATPase, the content of macromolecular weight protein and infusibility protein in lens were detected too. RESULT: The results examinated by cranny lamp showed that PSM2b could significantly postpone the occurrence and alleviate opacity degree of lens. Compared with model group, every treatment group of PSM2b could lower the level of MDA, high and middle dose groups could increase the levels of SOD, GSH-pX, CAT in serum and lens in evidence, and enhance the activity of Na(+) -K(+) -ATPase. These indexes present favorable positive correlation between dose and effect. CONCLUSION: All these results demonstrated that PSM2b had apparently protective effection on diabetic cataract in rats.

[Protection of polysaccharide ATPS-2 from Armillariella tabescens on immunological liver injury in mice induced by BCG plus LPS].

OBJECTIVE: To observe the effect of polysaccharide ATPS-2 from Armillariella tabescens on the immunological liver injury in mice induced by BCG plus LPS. METHOD: BCG and LPS were adopted to establish BCG plus LPS liver injury model in mice. The content of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and NO, the activity of superoxide dismutase (SOD) and malondiadehyde (MDA) content of liver homogenate in mice were measured by colorimetric method. The content of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), on serum were measured by enzyme linked immunosorbent assay (ELISA) , and T- and B-lymphocyte proliferation were measured by MTT. Index of liver, spleen and thymus were calculated after treatment. RESULT: Polysaccharide ATPS-2 from A. tabescens (25, 50, 100 mg x kg(-1)) could obviously reduce the high level of ALT, AST, NO and TNF-alpha, IL-1 on serum, inhibit the high level of MDA, increase the low activity of SOD in liver homogenate and enhance T-and B-lymphocyte proliferation, elevate the spleen, thymic index and decrease liver index of the mice to different extent. CONCLUSION: Polysaccharide ATPS-2 from A. tabescens had apparently protective effects in the immunological liver injury mice induced by BCG plus LPS.