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Vitamin K2, also known as menaquinone, has become a significant research focus, particularly in fermented products. This study aims to investigate the content of menaquinone‐7 (MK‐7), an essential form of vitamin K, and other fat‐soluble vitamins (A, D, E) during the fermentation of various fermented milk products. The vitamin contents of six fermented milk products were analyzed: two yogurt samples (produced with commercial starter and probiotic starter), two kefir samples (produced with commercial starter and kefir grain), and milk fermented with Lactobacillus acidophilus or Bifidobacterium bifidum. Throughout the fermentation process, continuous pH monitoring was conducted, and fermentation was terminated based on pH levels. Fat‐soluble vitamins and vitamins K1, K2 were extracted from samples taken at specific fermentation periods and quantitatively determined using the reverse phase liquid chromatography (RPLC) method. The developed method was validated according to ICH guidelines. Simultaneously, the microbial content of the samples was analyzed. Among the fermented dairy products analyzed, the highest MK‐7 content (4.82 μg/100 g) was found in the kefir sample produced with kefir grain (KG). The diverse microorganisms in kefir grains necessitated detailed research to explain their role in this complex mechanism. In yogurt samples, the presence of L. acidophilus in the YB sample suggests that this bacterium may be responsible for the observed differences. This is supported by the LA sample, where production with L. acidophilus strain showed an increase from 0.97 to 1.70 at the 20th hour of fermentation. It was noted that the concentration of menaquinone‐7 increased throughout the fermentation period. Given the critical health effects of essential fat‐soluble vitamins and MKs, determining their content in commonly consumed fermented dairy products and understanding the influence of starter cultures, which are potential probiotics, on vitamin production underscores the importance of this research. It is important to highlight the potential of fermented products in nutritional recommendations. [ABSTRACT FROM AUTHOR]

Menaquinone‐7 (MK‐7), a form of vitamin K2, supports bone health and prevents arterial calcification. Microbial fermentation for MK‐7 production has attracted widespread attention because of its low cost and short production cycles. However, insufficient substrate supply, unbalanced precursor synthesis, and low catalytic efficiency of key enzymes severely limited the efficiency of MK‐7 synthesis. In this study, utilizing Bacillus subtilis BSAT01 (with an initial MK‐7 titer of 231.0 mg/L) obtained in our previous study, the glycerol metabolism pathway was first enhanced to increase the 3‐deoxy‐arabino‐heptulonate 7‐phosphate (DHAP) supply, which led to an increase in MK‐7 titer to 259.7 mg/L. Subsequently, a combination of knockout strategies predicted by the genome‐scale metabolic model etiBsu1209 was employed to optimize the central carbon metabolism pathway, and the resulting strain showed an increase in MK‐7 production from 259.7 to 318.3 mg/L. Finally, model predictions revealed the methylerythritol phosphate pathway as the major restriction pathway, and the pathway flux was increased by heterologous introduction (Introduction of Dxs derived from Escherichia coli) and fusion expression (End‐to‐end fusion of two enzymes by a linker peptide), resulting in a strain with a titer of 451.0 mg/L in a shake flask and 474.0 mg/L in a 50‐L bioreactor. This study achieved efficient MK‐7 synthesis in B. subtilis, laying the foundation for large‐scale MK‐7 bioproduction. [ABSTRACT FROM AUTHOR]

In this study, a novel colorimetric screening method for identifying menaquinone-7 (MK-7) producing strains was established using potassium permanganate. To our knowledge, this method represents the first direct screening methodology for the identification of MK-7 producing strains. Utilizing this screening method, a new MK-7 producing strain, Bacillus subtilis GSA-184, was identified from the soil of the Tibetan Plateau. Under the optimized fermentation medium (50 g/L glycerol, 30 g/L yeast extract powder, 100 g/L soybean peptone, 1 g/L KH2PO4, and 1 g/L MnSO4), the production of MK-7 was increased to 25.7 mg/L. Additionally, the maximum production of MK-7 reached 36.46 mg/L after 48 h in a 5-L fermenter. [ABSTRACT FROM AUTHOR]

A recombinant strain (BS168-Δ bdhA) that increased co-production of MK-7 and NK was constructed by knocking out the 2,3-butanediol dehydrogenase gene (bdhA) of Bacillus subtilis 168 to reduce the carbon spillover. The results showed that the 2,3- butanediol content in the recombinant strain was reduced by 64% to 2.76 g/L. The recombinant strain had an MK-7 production of 30.6 mg/L and an NK activity of 6.5 FU/mL. RNA-Seq analysis showed that: bdhA knockdown blocked the carbon flux of 2,3-butanediol and promoted glycerol uptake, and the carbon flux flowed more to the MK-7 and NK synthesis pathways. The negative regulator of NK, codY , was down-regulated by 2.19-fold. secA , which is responsible for the energy supply of transmembrane proteins, was down-regulated by 0.37-fold, the Tat secretion pathway was up-regulated by 2.8-fold and 0.5-fold for tatAD and tatC , respectively. This study revealed the mechanism of co-production of MK-7 and NK in recombinant strains for the first time and revealed potential molecular targets to promote the co-production of MK-7 and NK, which will provide a new strategy for metabolic engineering to increase the production of MK-7 and NK. [Display omitted] • First use of metabolically engineered commons nattokinase and NK. • bdhA knockdown promotes redistribution of carbon flow towards MK-7 and NK synthesis. • bdhA knockdown promotes extracellular secretion of MK-7 and NK. [ABSTRACT FROM AUTHOR]

In this study, we developed a column-switching high-performance liquid chromatography (HPLC) method with fluorescence detection for the analysis of vitamin K. Column-switching is accomplished by changing the direction of flow using a switching valve with a set time program. Using this method, three vitamin K, phylloquinone (PK), menaquinone-4 (MK-4), and menaquinone-7 (MK-7), were separated and identified with high sensitivity, and impurities were eliminated. This method was used to determine the vitamin K content in meat, fish meat, snails, bivalves, sea urchins, seaweeds, vegetables, tea, soy products, milk products, and supplements. The results showed that chicken showed the highest content of MK-4 (15.35 ± 0.35 μg/100 g), matcha showed the highest content of PK (3069.66±80.10 μg/100 g), and dried natto showed the highest content of MK-7 (3997.57±79.42 μg/100 g). This method can also be used to analyze vitamin K in supplements and pharmaceuticals. The results of this study revealed that different manufacturers add different types of vitamin K to their commercial supplements and infant formulas. The developed method provides highly reproducible and quantitative results and allows for the rapid analysis of the three vitamin K types. Thus, the method developed in this study may aid the sequential analysis of vitamin K in different samples to assess food nutrients. [ABSTRACT FROM AUTHOR]

Background: In observational studies, high levels of desphospho-uncarboxylated matrix gla protein (dp-ucMGP) that result from vitamin K deficiency were consistently associated with poor clinical outcomes during COVID-19. Vitamin K-activated matrix gla protein (MGP) is required to protect against elastic fibre degradation, and a deficiency may contribute to pathology. However, intervention trials assessing the effects of vitamin K supplementation in COVID-19 are lacking. Methods: This is a single-centre, phase 2, double-blind, randomised, placebo-controlled trial investigating the effects of vitamin K2 supplementation in 40 hospitalised COVID-19 patients requiring supplemental oxygen. Individuals were randomly assigned in a 1:1 ratio to receive 999 mcg of vitamin K2—menaquinone-7 (MK-7)—or a placebo daily until discharge or for a maximum of 14 days. Dp-ucMGP, the rate of elastic fibre degradation quantified by desmosine, and hepatic vitamin K status quantified by PIVKA-II were measured. Grade 3 and 4 adverse events were collected daily. As an exploratory objective, circulating vitamin K2 levels were measured. Results: Vitamin K2 was well tolerated and did not increase the number of adverse events. A linear mixed model analysis showed that dp-ucMGP and PIVKA-II decreased significantly in subjects that received supplementation compared to the controls (p = 0.008 and p = 0.0017, respectively), reflecting improved vitamin K status. The decrease in dp-ucMGP correlated with higher plasma MK-7 levels (p = 0.015). No significant effect on desmosine was found (p = 0.545). Conclusions: These results demonstrate that vitamin K2 supplementation during COVID-19 is safe and decreases dp-ucMGP. However, the current dose of vitamin K2 failed to show a protective effect against elastic fibre degradation. [ABSTRACT FROM AUTHOR]

Patients with chronic kidney disease (CKD) suffer disproportionately from a high burden of cardiovascular disease, which, despite recent scientific advances, remains partly understood. Vascular calcification (VC) is the result of an ongoing process of misplaced calcium in the inner and medial layers of the arteries, which has emerged as a critical contributor to cardiovascular events in CKD. Beyond its established role in blood clotting and bone health, vitamin K appears crucial in regulating VC via vitamin K-dependent proteins (VKDPs). Among these, the matrix Gla protein (MGP) serves as both a potent inhibitor of VC and a valuable biomarker (in its inactive form) for reflecting circulating vitamin K levels. CKD patients, especially in advanced stages, often present with vitamin K deficiency due to dietary restrictions, medications, and impaired intestinal absorption in the uremic environment. Epidemiological studies confirm a strong association between vitamin K levels, inactive MGP, and increased CVD risk across CKD stages. Based on the promising results of pre-clinical data, an increasing number of clinical trials have investigated the potential benefits of vitamin K supplementation to prevent, delay, or even reverse VC, but the results have remained inconsistent. [ABSTRACT FROM AUTHOR]

Menaquinone-7 (MK-7) is an important class of vitamin K2 that is essential in human health and can prevent osteoporosis and cardiovascular disease. However, due to the complex synthesis pathway, the synthesis efficiency is low. The main objective of this study was to explore the effect of enhanced supply of precursors in Bacillus natto. Three precursors of pyruvate, shikimic acid, and sodium glutamate were chosen to investigate the effect of enhanced supply of precursors on MK-7 synthesis. Then, the optimal concentrations, different combinations, and different adding times were systematically studied, respectively. Results showed that the combination of shikimic acid and sodium glutamate could boost MK-7 production by 2 times, reaching 50 mg/L of MK-7 titer and 0.52 mg/(L·h) of MK-7 productivity. Furthermore, adding shikimic acid and sodium glutamate initially and feeding pyruvate at 48 h and 72 h increased MK-7 production to 58 mg/L. At the same time, the expression of the three related genes was also significantly upregulated. Subsequently, a new fermentation strategy combining the precursors enhancement and product secretion was proposed to enhance MK-7 yield and MK-7 productivity to 63 mg/L and 0.45 mg/(L·h). This study proposed a new fermentation regulation strategy for the enhancement of vitamin K2 biosynthesis. [ABSTRACT FROM AUTHOR]

Menaquinone-7 (MK-7), a multipotent vitamin K2, possesses a wide range of biological activities, a precise curative effect and excellent safety. A simple and rapid LC-APCI-MS/MS method for the determination of MK-7 in human plasma with single liquid–liquid extraction (LLE) extraction and 4·5-min analysis time has been developed and validated. Four per cent bovine serum albumin (BSA) was used as surrogate matrix for standard curves and endogenous baseline subtraction. This method was reproducible and reliable and was used to analyse of MK-7 in human plasma. The endogenous circadian rhythm and bioavailability of MK-7 were investigated in two randomised single-dose, open, one-way clinical trials (Study I and Study II). A total of five healthy male subjects were enrolled in Study I and 12 healthy male subjects in Study II. Single-dose (1 mg) of MK-7 was given to each subject under fasting condition, and all eligible subjects were given a restricting VK2 diet for 4 d prior to drug administration and during the trial. The experiment results of Study I demonstrated that endogenous MK-7 has no circadian rhythm in individuals. Both studies showed MK-7 are absorbed with peak plasma concentrations at about 6 h after intake and has a very long half-life time. [ABSTRACT FROM AUTHOR]

Patients with chronic kidney disease (CKD) suffer disproportionately from a high burden of cardiovascular disease, which, despite recent scientific advances, remains partly understood. Vascular calcification (VC) is the result of an ongoing process of misplaced calcium in the inner and medial layers of the arteries, which has emerged as a critical contributor to cardiovascular events in CKD. Beyond its established role in blood clotting and bone health, vitamin K appears crucial in regulating VC via vitamin K-dependent proteins (VKDPs). Among these, the matrix Gla protein (MGP) serves as both a potent inhibitor of VC and a valuable biomarker (in its inactive form) for reflecting circulating vitamin K levels. CKD patients, especially in advanced stages, often present with vitamin K deficiency due to dietary restrictions, medications, and impaired intestinal absorption in the uremic environment. Epidemiological studies confirm a strong association between vitamin K levels, inactive MGP, and increased CVD risk across CKD stages. Based on the promising results of pre-clinical data, an increasing number of clinical trials have investigated the potential benefits of vitamin K supplementation to prevent, delay, or even reverse VC, but the results have remained inconsistent.

Background: Type 2 diabetes mellitus (T2DM) is a prevalent disease with metabolic consequences. Lower levels of adiponectin and higher levels of leptin were reported in T2DM. This study aimed to evaluate the effect of menaquinone-7 (MK-7) supplementation on adiponectin and leptin in overweight/obese T2DM patients. Methods: Sixty men and women with T2DM and 27 ≤ body mass index < 35 kg/m2 , participated in this double-blind placebo-controlled randomized clinical trial for 12 weeks. The subjects were allocated into intervention (200 µg/day MK-7) or placebo groups. Three-day food records, anthropometrics and physical activity were assessed and fasting blood samples were collected at the pre- and post-intervention. Serum adiponectin, leptin, fasting blood sugar (FBS) and fasting insulin (FI) were measured and adiponectin to leptin ratio (A/L ratio) was calculated. Results: MK-7 decreased fasting blood sugar (P: 0.018) and fasting insulin (P: 0.012), according to a within group analysis of the 45 patients who finished the research. Fasting blood sugar (P: 0.024) and leptin levels (P: 0.032) were lower in the MK-7 group, but there were no significant changes between the groups in terms of adiponectin or the adiponectin to leptin (A/L) ratio. Conclusion: Current study showed that MK-7 supplementation could lead to significant reduction in leptin and FBS, but it has no effect on the adiponectin and A/L ratio in overweight/ obese T2DM patients.

Vitamin Ks are expected to contribute bone and cardiovascular health. Especially, menaquinone-7 has a higher bioavailability and a longer half-life than other vitamin Ks in the human body. However, their low water-solubility limits their application. On the other hand, Bacillus subtilis natto produces a water-soluble complex, which comprises menaquinone-7 and peptides. The peptide named K-binding factor (KBF) has been reported as the main component of the complex. In the present, the structural characteristics of KBF were studied. Mass spectrometry showed significant peaks at m / z  = 1050, while the previous PAGE suggested that molecular weight of KBF was ~ 3k. Amino acid analysis revealed that the 1k peptides were the various combinations of nine amino acids, among which Asx, Glx, Val, Leu and Met were found to be the most abundant. The peptides could serve as detergent properties. The 1k peptides could be isolated by reverse-phase high performance liquid chromatography. The bundle of three 1k detergent-like peptides would participate to the micelle structure containing menqauinone-7 inside. In conclusion, a basic unit of KBF would be the ~ 1k peptides, and the three basic unit assemble to the ~ 3k bundle, then the bundle form a water-soluble micelle including menqauinone-7 inside. [ABSTRACT FROM AUTHOR]

Background Vitamin K deficiency is highly prevalent in patients on dialysis and may contribute to their low bone mineral density (BMD) and increased risk of fracture. This study investigated the effect of menaquinone-7 (MK-7) supplementation on BMD in patients on chronic dialysis. Methods In a multicentre, double-blind, placebo-controlled intervention trial, 123 patients on chronic dialysis were randomised to a daily oral supplement of either MK-7 360 µg or placebo for 2 years. BMD of the distal radius (1/3, mid, ultradistal and total), femoral neck, lumbar spine (L1–L4) and whole body was assessed by dual-energy X-ray absorptiometry. Serum levels of vitamin K1 and MK-7 and plasma levels of total osteocalcin, dephosphorylated-uncarboxylated matrix Gla protein and protein induced by vitamin K absence II were measured to assess vitamin K status. Results After 2 years, an accelerated BMD loss of the 1/3 distal radius was found with MK-7 supplementation {mean difference of changes relative to placebo −0.023 g/cm2 [95% confidence interval (CI) −0.039 to −0.008]}, whereas the decrease in lumbar spine BMD seen in the placebo group was prevented [mean difference of changes between groups 0.050 g/cm2 (95% CI 0.015–0.085)]. No significant effects were observed at the remaining skeletal sites. Vitamin K status strongly improved in MK-7-supplemented participants. Conclusion Compared with placebo, an accelerated BMD loss of the 1/3 distal radius was found after 2 years of MK-7 supplementation, whereas a decline in lumbar spine BMD was prevented. As such, MK-7 supplementation might modify BMD site-specifically in patients on dialysis. In aggregate, our findings do not support MK-7 supplementation to preserve bone in patients on dialysis. [ABSTRACT FROM AUTHOR]

Menaquinone-7 is a form of vitamin K2 that has been shown to have numerous healthy benefits. In this study, several surfactants were investigated to enhance the production of menaquinone-7 in Bacillus natto. Results showed that Brij-58 supplementation influenced the cell membrane via adsorption, and changed the interfacial tension of fermentation broth, while the changes in the state and the composition of the cell membrane enhanced the secretion and biosynthesis of menaquinone-7. The total production and secretion rate of menaquinone-7 increased by 48.0% and 56.2% respectively. During fermentation, the integrity of the cell membrane decreased by 82.9% while the permeability increased by 158% when the maximum secretory rate was reached. Furthermore, Brij-58 supplementation induced the stress response in bacteria, resulting in hyperpolarization of the membrane, and increased membrane ATPase activity. Finally, changes in fatty acid composition increased membrane fluidity by 30.1%. This study provided an effective strategy to enhance menaquinone-7 yield in Bacillus natto and revealed the mechanism of Brij-58 supplementation in menaquinone-7 production. Key points: • MK-7 yield in Bacillus natto was significantly increased by Brij-58 supplementation. • Brij-58 could be adsorbed on cell surface and change fermentation environment. • Brij-58 supplementation could affect the state and composition of the cell membrane. [ABSTRACT FROM AUTHOR]

Insufficient calcium intake during growth is a global public health concern. The aim of this study was to investigate the effects of dietary menaquinone-7 (MK-7) on bone accrual in growing Sprague–Dawley rats under calcium restriction. Following 13 weeks of treatment, various bone quality parameters, including microarchitecture, were measured. Fecal and cecal samples were subjected to microbiome (16S rRNA gene sequencing) analyses, while metabolomics analysis of the cecum and humerus samples was analyzed based on UHPLC-Q/TOF-MS. We found that calcium deficiency diminished the richness of the microbiome and disrupted microbiome composition, accompanied by an elevation in the relative abundance of Parasutterella. Furthermore, calcium insufficiency escalated the level of isovaleric acid and modified the metabolic profiles. MK-7 supplementation significantly increased the cortical thickness, cortical bone area, and the calcium content of the femur. Apart from improving bone calcium deposition and diminishing bone resorption, the mechanisms underlying the beneficial effects of MK on bone quality also involve the modulation of the host's metabolic pathways and the composition of gut microbiota. The gut–bone axis holds promise as an efficacious target for ameliorating calcium deficiency in children's bone quality, and MK-7 is a promising dietary supplement from this perspective. [ABSTRACT FROM AUTHOR]

Background: There is a very high prevalence of subclinical vitamin K deficiency in patients requiring hemodialysis (HD), and this problem is associated with vascular calcification and arterial stiffness. Vitamin K2 (MK-7) supplementation can improve vitamin K status in HD patients. However, the benefits of vitamin K supplementation on arterial stiffness have still not been established. The present study was conducted to evaluate the efficacy of menaquinone-7 (MK-7) supplementation on arterial stiffness in chronic HD patients. Methods: This open-label multicenter randomized clinical trial was conducted in 96 HD patients who had arterial stiffness, defined by high carotid femoral pulse wave velocity (cfPWV ≥ 10 m/s). The patients were randomly assigned to receive oral MK-7 (375 mcg once daily) for 24 weeks (n = 50) or standard care (control group; n = 46). The change in cfPWV was the primary outcome. Results: Baseline parameters were comparable between the two groups. There was no significant difference in the change in cPWV at 24 weeks between the MK-7 group and standard care [−6.0% (−20.2, 2.3) vs. −6.8% (−19.0, 7.3), p = 0.24]. However, we found that MK-7 significantly decreased cPWV in patients with diabetes [−10.0% (−15.9, −0.8) vs. 3.8% (−5.8, 11.6), p = 0.008]. In addition, the MK-7 group had a lower rate of arterial stiffness progression, compared to controls (30.2% vs. 39.5%, p = 0.37), especially in diabetes patients (21.4% vs. 72.7%, p = 0.01). No serious adverse events were observed during the 24 weeks. Conclusion: Vitamin K supplements provided a beneficial impact in lowering the rate of arterial stiffness progression in chronic hemodialysis patients with diabetes. Possible benefits on cardiovascular outcomes require further investigation. [ABSTRACT FROM AUTHOR]

Abstract Menaquinone-7 (MK-7) is a kind of vitamin K2 playing an important role in the treatment and prevention of cardiovascular disease, osteoporosis and arterial calcification. The purpose of this study is to establish an adaptive evolution strategy based on a chemical modulator to improve MK-7 biosynthesis in Bacillus natto. The inhibitor of 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase), glyphosate, was chosen as the chemical modulator to perform the experiments. The final strain ALE-25–40, which was obtained after 40 cycles in 25 mmol/L glyphosate, showed a maximal MK-7 titer of 62 mg/L and MK-7 productivity of 0.42 mg/(L h), representing 2.5 and 3 times the original strain, respectively. Moreover, ALE-25–40 generated fewer spores and showed a higher NADH and redox potential. Furthermore, the mechanism related to the improved performance of ALE-25–40 was investigated by comparative transcriptomics analysis. Genes related to the sporation formation were down-regulated. In addition, several genes related to NADH formation were also up-regulated. This strategy proposed here may provide a new and alternative directive for the industrial production of vitamin K2. Graphical Abstract

Abstract Background Menaquinone-7 (MK-7), which is associated with complex and tightly regulated pathways and redox imbalances, is produced at low titres in Bacillus subtilis. Synthetic biology provides a rational engineering principle for the transcriptional optimisation of key enzymes and the artificial creation of cofactor regeneration systems without regulatory interference. This holds great promise for alleviating pathway bottlenecks and improving the efficiency of carbon and energy utilisation. Results We used a bottom-up synthetic biology approach for the synthetic redesign of central carbon and to improve the adaptability between material and energy metabolism in MK-7 synthesis pathways. First, the rate-limiting enzymes, 1-deoxyxylulose-5-phosphate synthase (DXS), isopentenyl-diphosphate delta-isomerase (Fni), 1-deoxyxylulose-5-phosphate reductase (DXR), isochorismate synthase (MenF), and 3-deoxy-7-phosphoheptulonate synthase (AroA) in the MK-7 pathway were sequentially overexpressed. Promoter engineering and fusion tags were used to overexpress the key enzyme MenA, and the titre of MK-7 was 39.01 mg/L. Finally, after stoichiometric calculation and optimisation of the cofactor regeneration pathway, we constructed two NADPH regeneration systems, enhanced the endogenous cofactor regeneration pathway, and introduced a heterologous NADH kinase (Pos5P) to increase the availability of NADPH for MK-7 biosynthesis. The strain expressing pos5P was more efficient in converting NADH to NADPH and had excellent MK-7 synthesis ability. Following three Design-Build-Test-Learn cycles, the titre of MK-7 after flask fermentation reached 53.07 mg/L, which was 4.52 times that of B. subtilis 168. Additionally, the artificially constructed cofactor regeneration system reduced the amount of NADH-dependent by-product lactate in the fermentation broth by 9.15%. This resulted in decreased energy loss and improved carbon conversion. Conclusions In summary, a "high-efficiency, low-carbon, cofactor-recycling" MK-7 synthetic strain was constructed, and the strategy used in this study can be generally applied for constructing high-efficiency synthesis platforms for other terpenoids, laying the foundation for the large-scale production of high-value MK-7 as well as terpenoids.

Background: Vitamin K2 refers to a series of naphthoquinone derivatives, which have a variety of physiological and pharmacological functions for the human body. The most important type of vitamin K2 is menaquinone-7 (MK-7), an expensive raw material with no local manufacturers in Iran. Objectives: Since there was no report on the yield of MK-7 produced by the currently available Bacillus subtilis natto species in Iran, this study aims to optimize the culture condition for the production of MK-7 using this Bacillus species. Materials and Methods: The base medium (BM) for MK-7 production contained glycerol (6.3%), soybean peptone (3%), and yeast extract (0.51%). The selected factors for optimizing the MK-7 production included the incubation temperature (30, 37, and 40°C) and incubation time (72, 96, and 120hr) with/without the addition of K2HPO4 to the fermentation medium. Three sets of experiments with six modes in each set were designed based on these parameters. MK-7 content was analyzed by the HPLC method. Results: Two experiments showed the highest MK-7 production yields of 0.319 and 0.3158 mg/L. The culture condition for both of these yields was as follows:120 hours incubation time in the presence of K2HPO4. However, the incubation temperature was different in these two experiments. The incubation temperature of 30°C resulted in 0.319 mg/L MK-7 concentration, and 37°C yielded 0.3158 mg/L. Conclusion: B. subtilis natto (IBRC-M 11153) is suitable to be used as a basic platform for the mutation and production of a high-producer species. Optimizing the culture conditions using the wild-type species is not beneficial in increasing the production ability of the bacterium. It is necessary to use different methods for enhancing the production yield of MK-7 to lower the cost of microbial production and make the industrial process economic.

Menaquinone-7 (MK-7) is a kind of vitamin K2 playing an important role in the treatment and prevention of cardiovascular disease, osteoporosis and arterial calcification. The purpose of this study is to establish an adaptive evolution strategy based on a chemical modulator to improve MK-7 biosynthesis in Bacillus natto. The inhibitor of 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase), glyphosate, was chosen as the chemical modulator to perform the experiments. The final strain ALE-25–40, which was obtained after 40 cycles in 25 mmol/L glyphosate, showed a maximal MK-7 titer of 62 mg/L and MK-7 productivity of 0.42 mg/(L h), representing 2.5 and 3 times the original strain, respectively. Moreover, ALE-25–40 generated fewer spores and showed a higher NADH and redox potential. Furthermore, the mechanism related to the improved performance of ALE-25–40 was investigated by comparative transcriptomics analysis. Genes related to the sporation formation were down-regulated. In addition, several genes related to NADH formation were also up-regulated. This strategy proposed here may provide a new and alternative directive for the industrial production of vitamin K2. [ABSTRACT FROM AUTHOR]

Insufficient calcium intake during growth is a global public health concern. The aim of this study was to investigate the effects of dietary menaquinone-7 (MK-7) on bone accrual in growing Sprague–Dawley rats under calcium restriction. Following 13 weeks of treatment, various bone quality parameters, including microarchitecture, were measured. Fecal and cecal samples were subjected to microbiome (16S rRNA gene sequencing) analyses, while metabolomics analysis of the cecum and humerus samples was analyzed based on UHPLC-Q/TOF-MS. We found that calcium deficiency diminished the richness of the microbiome and disrupted microbiome composition, accompanied by an elevation in the relative abundance of Parasutterella. Furthermore, calcium insufficiency escalated the level of isovaleric acid and modified the metabolic profiles. MK-7 supplementation significantly increased the cortical thickness, cortical bone area, and the calcium content of the femur. Apart from improving bone calcium deposition and diminishing bone resorption, the mechanisms underlying the beneficial effects of MK on bone quality also involve the modulation of the host’s metabolic pathways and the composition of gut microbiota. The gut–bone axis holds promise as an efficacious target for ameliorating calcium deficiency in children’s bone quality, and MK-7 is a promising dietary supplement from this perspective.

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In this study, 10 different plant materials (seeds/beans) were fermented by Bacillus subtilis var. natto. The influence of the process on vitamin K2 MK-7 content during different fermentation periods was assessed. Fermented plant samples were analyzed by the procedure using HPLC UV/DAD. The fermented sunflower seeds, mung beans and peas appeared to be the most promising plants, reaching values of K2 of 1080.18±55.11 µg/100g, 806.45±60.95 µg/100g and 636.92±59.86 µg/100g, respectively. The experiments showed that extending of the fermentation time to 5–6 days was favorable for the menaquinone-7 yield. The results show that almost all fermented seeds/beans, apart from soybean, can be good source of vitamin K2 MK-7 and represent a new perspective, especially in terms of lower the phytoestrogen content.

Menaquinone-7 (MK-7) is a form of vitamin K 2 with health-beneficial effects. A novel fermentation strategy based on combining soy protein hydrolysates (SPHs) with biofilm-based fermentation was investigated to enhance menaquinone-7 (MK-7) biosynthesis by Bacillus subtilis natto. Results showed the SPHs increased MK-7 yield by 199.4% in two-stage aeration fermentation as compared to the SP-based medium in submerged fermentation, which was related to the formation of robust biofilm with wrinkles and the enhancement of cell viability. Moreover, there was a significant correlation between key genes related to MK-7 and biofilm synthesis, and the quorum sensing (QS) related genes, Spo0A and SinR , were downregulated by 0.64-fold and 0.39-fold respectively, which promoted biofilm matrix synthesis. Meanwhile, SPHs also enhanced the MK-7 precursor, isoprene side chain, supply, and MK-7 assembly efficiency. Improved fermentation performances of bacterial cells during fermentation were attributed to abundant oligopeptides (Mw < 1 kDa) and moderate amino acids, particularly Arg, Asp, and Phe in SPHs. All these results revealed that SPHs were a potential and superior nitrogen source for MK-7 production by Bacillus subtilis natto. [Display omitted] • Robust biofilm formation could enhance MK-7 biosynthesis in Bacillus subtilis natto. • A two-stage aeration fermentation strategy further improved fermentation performance. • Soy protein hydrolysates supply promoted biofilm formation and delayed sporulation. • Abundant oligopeptides in soy protein hydrolysates contributed to MK-7 production. [ABSTRACT FROM AUTHOR]

Abstract Background This study was aimed to examine the effects of vitamin K2 supplementation on atherogenic status, assessed by insulin resistance (IR)-related indexes, in patients with type 2 diabetes mellitus (T2DM). Methods In this double-blind, controlled trial, 68 patients with T2DM on the oral glucose-lowering medications were randomly allocated into two groups receiving daily intakes of 360 μg MK-7 or placebo for 12 weeks. Eight different IR-related indexes were calculated at the baseline and end of the trial. Results At the end of the study, atherogenic coefficient (mean ± SD: − 0.21 ± 0.45 vs. 0.02 ± 0.43; p = 0.043), triglyceride-glucose index (8.88 ± 0.55 vs. 9.23 ± 0.69; p = 0.029), and atherogenic index of plasma (0.37 ± 0.27 vs. 0.51 ± 0.24; p = 0.031) were significantly lower in the vitamin K2 group, compared to the placebo. However, after accounting for their baseline values, the differences were no more significant. No significant differences were observed in Castelli’s Ӏ and ӀӀ risk indexes, the ratio of triglycerides to high-density lipoprotein cholesterol, lipoprotein combine index, and the metabolic score for insulin resistance index between the two groups at the end of the study. Conclusions Daily intakes of 360 μg vitamin K2 in the form of MK-7 for 12 weeks could not improve the IR-related indexes of Cardiovascular Diseases risk. Trial registration The trial was registered on Iranian Registry of Clinical Trials registry (Trial ID. IRCT20190824044592N1) on 22 December 2019. The record can be found at https://en.irct.ir/trial/41728 .

Background: There is a very high prevalence of subclinical vitamin K deficiency in patients requiring hemodialysis (HD), and this problem is associated with vascular calcification and arterial stiffness. Vitamin K2 (MK-7) supplementation can improve vitamin K status in HD patients. However, the benefits of vitamin K supplementation on arterial stiffness have still not been established. The present study was conducted to evaluate the efficacy of menaquinone-7 (MK-7) supplementation on arterial stiffness in chronic HD patients. Methods: This open-label multicenter randomized clinical trial was conducted in 96 HD patients who had arterial stiffness, defined by high carotid femoral pulse wave velocity (cfPWV ≥ 10 m/s). The patients were randomly assigned to receive oral MK-7 (375 mcg once daily) for 24 weeks (n = 50) or standard care (control group; n = 46). The change in cfPWV was the primary outcome. Results: Baseline parameters were comparable between the two groups. There was no significant difference in the change in cPWV at 24 weeks between the MK-7 group and standard care [−6.0% (−20.2, 2.3) vs. −6.8% (−19.0, 7.3), p = 0.24]. However, we found that MK-7 significantly decreased cPWV in patients with diabetes [−10.0% (−15.9, −0.8) vs. 3.8% (−5.8, 11.6), p = 0.008]. In addition, the MK-7 group had a lower rate of arterial stiffness progression, compared to controls (30.2% vs. 39.5%, p = 0.37), especially in diabetes patients (21.4% vs. 72.7%, p = 0.01). No serious adverse events were observed during the 24 weeks. Conclusion: Vitamin K supplements provided a beneficial impact in lowering the rate of arterial stiffness progression in chronic hemodialysis patients with diabetes. Possible benefits on cardiovascular outcomes require further investigation.

Background: Menaquinone-7 (MK-7), which is associated with complex and tightly regulated pathways and redox imbalances, is produced at low titres in Bacillus subtilis. Synthetic biology provides a rational engineering principle for the transcriptional optimisation of key enzymes and the artificial creation of cofactor regeneration systems without regulatory interference. This holds great promise for alleviating pathway bottlenecks and improving the efficiency of carbon and energy utilisation. Results: We used a bottom-up synthetic biology approach for the synthetic redesign of central carbon and to improve the adaptability between material and energy metabolism in MK-7 synthesis pathways. First, the rate-limiting enzymes, 1-deoxyxylulose-5-phosphate synthase (DXS), isopentenyl-diphosphate delta-isomerase (Fni), 1-deoxyxylulose-5-phosphate reductase (DXR), isochorismate synthase (MenF), and 3-deoxy-7-phosphoheptulonate synthase (AroA) in the MK-7 pathway were sequentially overexpressed. Promoter engineering and fusion tags were used to overexpress the key enzyme MenA, and the titre of MK-7 was 39.01 mg/L. Finally, after stoichiometric calculation and optimisation of the cofactor regeneration pathway, we constructed two NADPH regeneration systems, enhanced the endogenous cofactor regeneration pathway, and introduced a heterologous NADH kinase (Pos5P) to increase the availability of NADPH for MK-7 biosynthesis. The strain expressing pos5P was more efficient in converting NADH to NADPH and had excellent MK-7 synthesis ability. Following three Design-Build-Test-Learn cycles, the titre of MK-7 after flask fermentation reached 53.07 mg/L, which was 4.52 times that of B. subtilis 168. Additionally, the artificially constructed cofactor regeneration system reduced the amount of NADH-dependent by-product lactate in the fermentation broth by 9.15%. This resulted in decreased energy loss and improved carbon conversion. Conclusions: In summary, a "high-efficiency, low-carbon, cofactor-recycling" MK-7 synthetic strain was constructed, and the strategy used in this study can be generally applied for constructing high-efficiency synthesis platforms for other terpenoids, laying the foundation for the large-scale production of high-value MK-7 as well as terpenoids. [ABSTRACT FROM AUTHOR]

Menaquinone-7 (MK-7) is an important vitamin K2, synthesized from the menaquinone parent ring and seven isoprene side chains. Presently, the synthesis of MK-7 stimulated by environmental stress primarily focuses on oxygen stress, while the effect of alkali stress is rarely studied. Therefore, this study researched the effects of alkali stress on the fermentation performance and gene expression of Bacillus subtilis natto. The organism's growth characteristics, biomass, sporogenesis, MK-7 biosynthesis, and gene expression were analyzed. After a pH 8.5 stress adaptation treatment for 0.5 h and subsequent fermentation at pH 8.5, which promoted the growth of the strain and inhibited the spore formation rate. In addition, biomass was significantly increased (P < 0.05). The conversion rate of glycerol to MK-7 was 1.68 times higher than that of the control group, and the yield of MK-7 increased to 2.10 times. Transcriptomic analysis showed that the MK-7 high-yielding strain had enhanced carbon source utilization, increased glycerol and pyruvate metabolism, enhanced the Embden-Meyerhof pathway (EMP), tricarboxylic acid (TCA) circulation flux, and terpenoid biosynthesis pathway, and promoted the accumulation of acetyl-CoA, the side-chain precursor of isoprene. At the same time, the up-regulation of transketolase increased the metabolic flux of the pentose phosphate (HMP) pathway, which was conducive to the accumulation of D-erythrose 4-phosphate, the precursor of the menadione parent ring. This study's results contribute to a better understanding of the effects of environmental stress on MK-7 fermentation by Bacillus subtilis natto and the molecular regulatory mechanism of MK-7 biosynthesis. [ABSTRACT FROM AUTHOR]

Menaquinone-7 (MK-7) is an important vitamin K2, synthesized from the menaquinone parent ring and seven isoprene side chains. Presently, the synthesis of MK-7 stimulated by environmental stress primarily focuses on oxygen stress, while the effect of alkali stress is rarely studied. Therefore, this study researched the effects of alkali stress on the fermentation performance and gene expression of Bacillus subtilis natto. The organism’s growth characteristics, biomass, sporogenesis, MK-7 biosynthesis, and gene expression were analyzed. After a pH 8.5 stress adaptation treatment for 0.5 h and subsequent fermentation at pH 8.5, which promoted the growth of the strain and inhibited the spore formation rate. In addition, biomass was significantly increased (P < 0.05). The conversion rate of glycerol to MK-7 was 1.68 times higher than that of the control group, and the yield of MK-7 increased to 2.10 times. Transcriptomic analysis showed that the MK-7 high-yielding strain had enhanced carbon source utilization, increased glycerol and pyruvate metabolism, enhanced the Embden-Meyerhof pathway (EMP), tricarboxylic acid (TCA) circulation flux, and terpenoid biosynthesis pathway, and promoted the accumulation of acetyl-CoA, the side-chain precursor of isoprene. At the same time, the up-regulation of transketolase increased the metabolic flux of the pentose phosphate (HMP) pathway, which was conducive to the accumulation of D-erythrose 4-phosphate, the precursor of the menadione parent ring. This study’s results contribute to a better understanding of the effects of environmental stress on MK-7 fermentation by Bacillus subtilis natto and the molecular regulatory mechanism of MK-7 biosynthesis.

Purpose: To investigate the beneficial effects of menaquinone-7 (MK-7), an isoform of vitamin K2, against beta-amyloid (Aβ) production and aggregation in Alzheimer's disease using in vitro assays. Methods: The cytotoxicity of MK-7 was determined by MTT assay. The amount of Aβ produced and secreted into the supernatant by APP-CHO cells treated with MK-7 was evaluated by ELISA. The expression of β-secretases and ADAM10, a representative α-secretase, was determined using western blot analysis. The production of sAPPβ and sAPPα fragments generated by β-secretases and αsecretase, respectively, were also determined by western blot analysis. The effect on Aβ aggregation was assessed using Thioflavin T (Th T) assay. Results: MK-7 (up to 75 nM) significantly decreased Aβ production in APP-CHO cells. This was accompanied by decreased expression of β-secretase and lower production of sAPPβ (p < 0.05). However, expression of ADAM10 and production of sAPPα were not significantly affected. In contrast, MK-7 significantly decreased Aβ aggregation in a dose-dependent manner (p < 0.05). Conclusion: MK-7 exerts anti-amyloidogenic effects via decreased production and lower aggregation of Aβ into oligomers and fibrils. Therefore, dietary supplementation with MK-7 may be beneficial for the prevention of Alzheimer’s disease. [ABSTRACT FROM AUTHOR]

Background Arterial calcification is associated with cardiovascular mortality in dialysis patients. Active matrix Gla protein (MGP) is a vitamin K-dependent inhibitor of arterial calcification. Elevated plasma concentrations of inactive MGP, i.e. dephosphorylated-uncarboxylated MGP (dp-ucMGP), are prevalent in dialysis patients. MGP inactivity might contribute to arterial calcification. We investigated whether vitamin K supplementation had an effect on arterial calcification in chronic dialysis patients. Methods In a 2-year, double-blind, placebo-controlled intervention trial, 48 dialysis patients were randomized to vitamin K [menaquinone-7 (MK-7), 360 µg daily] or placebo. MK-7 in serum and dp-ucMGP in plasma were used to assess vitamin K status. Carotid-femoral pulse wave velocity (cfPWV) and scores of coronary arterial calcification (CAC) and abdominal aortic calcification (AAC) were used to assess arterial calcification. Results Thirty-seven participants completed Year 1, and 21 completed Year 2. At Year 2, serum MK-7 was 40-fold higher, and plasma dp-ucMGP 40% lower after vitamin K supplementation compared with placebo {mean dp-ucMGP difference: −1380 pmol/L [95% confidence interval (CI) −2029 to −730]}. There was no significant effect of vitamin K supplementation on cfPWV [mean difference at Year 2: 1.2 m/s (95% CI −0.1 to 2.4)]. CAC Agatston score increased significantly in vitamin K supplemented participants, but was not significantly different from placebo [mean difference at Year 2: 664 (95% CI −554 to 1881)]. AAC scores increased in both groups, significantly so within the placebo group at Year 1, but with no significant between-group differences. Conclusions Vitamin K supplementation improved vitamin K status, but did not hinder or modify the progression of arterial calcification in dialysis patients. [ABSTRACT FROM AUTHOR]

The production of nutraceutical compounds through biosynthetic approaches has received considerable attention in recent years. For example, Menaquinone-7 (MK-7), a sub-type of Vitamin K2, biosynthesized from Bacillus subtilis (B. subtilis), proved to be more efficiently produced than the conventional chemical synthesis techniques. This is possible due to the development of B. subtilis as a chassis cell during the biosynthesis stages. Hence, it is imperative to provide insights on the B. subtilis membrane permeability modifications, biofilm reactors, and fermentation optimization as advanced techniques relevant to MK-7 production. Although the traditional gene-editing method of homologous recombination improves the biosynthetic pathway, CRISPR-Cas9 could potentially resolve the drawbacks of traditional genome editing techniques. For these reasons, future studies should explore the applications of CRISPRi (CRISPR interference) and CRISPRa (CRISPR activation) system gene-editing tools in the MK-7 anabolism pathway.

Bacillus subtilis fermented soybeans (natto) contain high vitamin K2 levels, mostly as menaquinone-7 (MK-7), and must be avoided by warfarin-dependent patients. This is the first report which demonstrates the characteristics and clinical relevance of a low MK-7 natto for such patients. We generated a novel, mutant B. subtilis strain TTCC2051 with short-term fermentation and reduced MK-7 production, yielding 19–24% of the normal MK-7 content. After functional assessments and a preclinical trial, 10 warfarin-dependent patients underwent a clinical trial with a 7-day ingestion test of the low MK-7 natto. Functional assessments were satisfactory, and the preclinical trial showed no increases in plasma MK-7 levels after 7 days of ingestion. In the clinical trial, 20 g/day of the low MK-7 natto significantly increased plasma MK-7 levels while 10 g/day did not. However, neither dose of low MK-7 natto changed international normalized ratio of prothrombin time (PT-INR) values in either group. The low MK-7 natto neither changed PT-INR values nor precipitated adverse events if ingested with a once-daily maximum of 20 g (46 μg of MK-7). Thus, this novel food product has potential for consumption by warfarin-dependent patients. [ABSTRACT FROM AUTHOR]

The objective of the present research is to develop and optimize a chewable tablet containing synergistic combination of magnesium orotate dihydrate (MOD), cholecalciferol (CHOL) and menaquinone-7 (MK-7) as per product development guidelines of ICH Q8 (R2). The effects of critical variables on quality attributes of chewable tablets were evaluated using 30 runs based design of experiment (DoE) after risk assessment. Optimized formation was found to be the one that was prepared with moderate granulation time of 7.23 min and contained 14 mg/tablet binder, 31 mg/tablet disintegrant and 11.377 mg/tablet lubricant. Prepared tablets were evaluated for prescribed pharmacopoeial and regulatory quality checks. Optimized formulation was found to have very low disintegration time of 6.06 min and 87.39% dissolution of MOD within 15 min in acidic media (0.1 N HCl), which ensure that the developed formulation behaves as a solution following oral administration. Stability studies under accelerated conditions revealed that the developed formulation can retain its quality characteristics throughout its shelf life. Pharmacokinetics study of chewable tablets in male Wistar rats shows that the time to reach maximum plasma or serum concentration (Tmax) was 3 h for MOD and 6 h for both CHOL and MK-7. Maximum plasma or serum concentration (Cmax) of MOD, CHOL and MK-7 was found to be 7.233 ± 1.159, 8.182 ± 0.783 and 8.331 ± 0.863 mg/mL [mean ± standard deviation (SD)], respectively. The area under the curve (AUC 0-t) for MOD, CHOL and MK-7 was 80.692 ± 11.197, 124.325 ± 17.101 and 126.568 ± 12.064 mg.mL.h-1 (mean ± SD), respectively. Comparison of pharmacokinetic data of chewable tablets with a mixture of pure drugs proves that the developed formulation can efficiently deliver all the three nutrients in blood and is capable to elicit desired pharmacological response. [ABSTRACT FROM AUTHOR]

Background: This study was aimed to examine the effects of vitamin K2 supplementation on atherogenic status, assessed by insulin resistance (IR)-related indexes, in patients with type 2 diabetes mellitus (T2DM). Methods: In this double-blind, controlled trial, 68 patients with T2DM on the oral glucose-lowering medications were randomly allocated into two groups receiving daily intakes of 360 μg MK-7 or placebo for 12 weeks. Eight different IR-related indexes were calculated at the baseline and end of the trial. Results: At the end of the study, atherogenic coefficient (mean ± SD: − 0.21 ± 0.45 vs. 0.02 ± 0.43; p = 0.043), triglyceride-glucose index (8.88 ± 0.55 vs. 9.23 ± 0.69; p = 0.029), and atherogenic index of plasma (0.37 ± 0.27 vs. 0.51 ± 0.24; p = 0.031) were significantly lower in the vitamin K2 group, compared to the placebo. However, after accounting for their baseline values, the differences were no more significant. No significant differences were observed in Castelli’s Ӏ and ӀӀ risk indexes, the ratio of triglycerides to high-density lipoprotein cholesterol, lipoprotein combine index, and the metabolic score for insulin resistance index between the two groups at the end of the study. Conclusions: Daily intakes of 360 μg vitamin K2 in the form of MK-7 for 12 weeks could not improve the IR-related indexes of Cardiovascular Diseases risk. [ABSTRACT FROM AUTHOR]

Menaquinone-7 is involved in bone metabolism and can be used to prevent and treat osteoporosis. However, as a fat-soluble vitamin, menaquinone-7 has poor water solubility. As a surfactant, hydrophobins can change the affinity/hydrophobicity of the covered interface. In this study, menaquinone-7 was modified by hydrophobins, and the different addition ratios were explored. Moreover, Fourier transform infrared (FTIR), X-ray photoelectron spectroscopy (XPS), and water contact angle (WCA) measurements indicated that hydrophobins effectively bind to menaquinone-7 and greatly increase the hydrophilicity of the surface of menaquinone-7. Studies on the metabolism of MC3T3-E1 cells showed that compared with native menaquinone-7, HGFI-modified menaquinone-7 can significantly promote osteoblast differentiation but inhibit osteoclast differentiation. Besides, the Mito-Tracker Green experiments show that HGFI-modified menaquinone-7 can significantly promote the activity of mitochondria in cells. These findings indicate that hydrophobins can be used as an effective biomaterial to modify menaquinone-7, promote the formation of osteoblasts, and better to bone balance. [ABSTRACT FROM AUTHOR]

BACKGROUND Starch can be used as a carbon source for producing menaquinone‐7 (MK‐7) by Bacillus sp., but the final titer and productivity are unsatisfactory because starch cannot be effectively hydrolyzed to fermentable sugars. This article focuses on developing thermo‐tolerable MK‐7 high‐producing strains by temperature‐induced mutagenesis‐based adaptive evolution. RESULTS: Here, we isolated a thermo‐tolerable MK‐7 high‐producing strain B. amyloliquefaciens MK50‐36 after multi‐rounds of acclimation at high temperature. Strain MK50‐36 showed a large growth with soluble starch as carbon source at 50 °C. Compared with the parental strain B. amyloliquefaciens H.β.D.R.‐5 cultivated at 37 °C, MK‐7 yield of strain MK50‐36 increased about twofold at 50 °C when soluble starch was used as the sole carbon resource (17.5 ± 1.5 mg L–1 versus 37.1 ± 2.1 mg L–1). In addition, strain MK50‐36 showed an improvement in starch utilization rate as compared with strain H.β.D.R.‐5 because α‐amylase activity of strain MK50‐36 was the highest at 50 °C. Moreover, MK‐7 yield of strain MK50‐36 was further increased by feeding fresh broth to control the amount of fermentation liquid in fed‐batch fermentation because fermentation broth will be easy to volatilize and thus affect the production of MK‐7 at high temperature, which reached up to 52.7 ± 4.6 mg L–1. CONCLUSION: These results indicated that soluble starch can act as an effective carbon source for producing MK‐7, and strain MK50‐36 has a superior capacity to produce MK‐7 from soluble starch. © 2020 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Development of clinical stem cell interventions are hampered by immature cell progeny under current protocols. Human mesenchymal stem cells (hMSCs) are characterized by their ability to self-renew and differentiate into multiple lineages. Generating hMSCs from pluripotent stem cells (iPSCs) is an attractive avenue for cost-efficient and scalable production of cellular material. In this study we generate mature osteoblasts from iPSCs using a stable expandable MSC intermediate, refining established protocols. We investigated the timeframe and phenotype of cells under osteogenic conditions as well as the effect of menaquinone-7 (MK-7) on differentiation. From day 2 we noted a significant increase in RUNX2 expression under osteogenic conditions with MK-7, as well as decreases in ROS species production, increased cellular migration and changes to dynamics of collagen deposition when compared to differentiated cells that were not treated with MK-7. At day 21 OsteoMK-7 increased alkaline phosphatase activity and collagen deposition, as well as downregulated RUNX2 expression, suggesting to a mature cellular phenotype. Throughout we note no changes to expression of osteocalcin suggesting a non-canonical function of MK-7 in osteoblast differentiation. Together our data provide further mechanistic insight between basic and clinical studies on extrahepatic activity of MK-7. Our findings show that MK-7 promotes osteoblast maturation thereby increasing osteogenic differentiation.

Vitamin K (VK) is a fat-soluble vitamin that is indispensable for the activation of vitamin K-dependent proteins (VKDPs). It has been shown to play an important role in the proper calcium deposit at the bone level, hindering that on the vascular walls. The deficiency of this vitamin in European populations is frequent and unknown. It is related to several factors, poor dietary intake, altered intestinal absorption or altered production by bacteria, indicating possible dysbiosis. For Vitamin K2 (VK2), there is currently no official reference daily intake (RDI). However, the effects of VK2 on the improvement of health in cardiovascular diseases, on bone metabolism, on chronic kidney diseases have been the subject of research in recent decades. The microbiota in the gastrointestinal tract plays an important role: Bacteroides are primarily capable of synthetizing very long chain forms of menaquinones and, in addition to the bacteria present in the intestinal flora, VK2 is also produced by bacteria used in food fermentation processes. This review provides an update on the current literature regarding the origin of VK2 and its implications in what is called the "calcium paradox", namely the lack of calcium in the bone and its storage in the wall of the vessel.

Epidemiological outburst of type 2 diabetes is of great global concern. T2D starts with Insulin Resistance (IR) which arises largely due to environmental factors and to a lesser extent due to genetic factor. IR gradually develops into T2D and encompasses a wide array of conditions including Impaired Glucose Tolerance (IGT), hyperinsulinemia, Impaired Fasting Glucose (IFG), and Impaired Insulin Release (IIR). Initiation of IR increases the risk of Cardiovascular Diseases (CVD). Therefore, early diagnosis and management of IR and its related outcomes (hyperinsulinemia, hyperglycemia, and dyslipidemia) should be the prime focus of intervention therapies. Present research aimed to evaluate the synergistic combination of Magnesium orotate (MOD), Menaquinone- 7 (MK-7), and Cholecalciferol (CHOL) for the management of these therapeutic targets in the Streptozotocin--Nicotinamide-induced T2D Wistar rat model. Synergistic combination was found to be superior over its individual components in management of hyperglycemia, impaired insulin secretion, Homeostatic Model Assessment of Insulin Resistance (HOMA-IR), and dyslipidemia (p < 0.01 or p < 0.05). Its effect was found to be equivalent or better than reference drugs (p < 0.01 or p < 0.05). Histopathological analysis depicted that combination treatment was able to regenerate and preserve pancreatic β-cell mass in diabetic rats. In conclusion, combination studied in present research can be evaluated further under clinical settings for management of IR and its related outcomes. [ABSTRACT FROM AUTHOR]

Abstract Following an application from NattoPharma ASA submitted for authorisation of a health claim pursuant to Article 13(5) of Regulation (EC) No 1924/2006 via the Competent Authority of Ireland, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on the scientific substantiation of a health claim related to MenaQ7® and maintenance of the elastic properties of the arteries. The scope of the application was proposed to fall under a health claim based on newly developed scientific evidence. The food proposed by the applicant as the subject of the health claim is MenaQ7®, which contains 97% menaquinone‐7 by weight. The Panel considers that MenaQ7® is sufficiently characterised. The claimed effect proposed by the applicant is ‘improves arterial stiffness’. Upon a request from EFSA, the applicant agreed that the claimed effect is maintenance of elastic properties of the arteries (by measuring the arterial stiffness). The Panel considers that maintenance of the elastic properties of the arteries is a beneficial physiological effect. No conclusions could be drawn from the three human intervention studies submitted for scientific substantiation of the claim. In the absence of evidence for an effect of MenaQ7® on maintenance of the elastic properties of the arteries in vivo in humans, the studies on the proposed mechanisms by which the food/constituent could exert the claimed effect cannot be used as a source of evidence for the scientific substantiation of the claim. The Panel concludes that a cause and effect relationship has not been established between the consumption of MenaQ7® and maintenance of the elastic properties of the arteries.

Chronic Kidney Disease (CKD) patients are at high risk of presenting with arterial calcification or stiffness, which confers increased cardiovascular mortality and morbidity. In recent years, it has become evident that VC is an active process regulated by various molecules that may act as inhibitors of vessel mineralization. Matrix Gla Protein (MGP), one the most powerful naturally occurring inhibitors of arterial calcification, requires vitamin K as a co-factor in order to undergo post-translational γ-carboxylation and phosphrorylation and become biologically active. The inactive form of MGP (dephosphorylated, uncarboxylated dp-ucMGP) reflects vitamin K deficiency and has been repeatedly associated with surrogate markers of VC, stiffness, and cardiovascular outcomes in CKD populations. As CKD is a state of progressive vitamin K depletion and VC, research has focused on clinical trials aiming to investigate the possible beneficial effects of vitamin K in CKD and dialysis patients. In this study, we aim to review the current evidence regarding vitamin K supplementation in uremic patients.

Objective: The aim of this study was to investigate the effect of oral vitamin K2 (Menaquinone‐7 [MK‐7]) on clinical and biochemical parameters in polycystic ovary syndrome (PCOS) patients. Methods: In this randomized, double‐blind, placebo‐controlled clinical trial, 84 PCOS patients were randomly assigned into the treatment (90 µg Menaquinone‐7 daily for 8 weeks) and placebo groups. Insulin resistance, lipid profile, endocrine biomarkers, and body composition of the participants were measured before and after the intervention. This study was performed in Ghadir Mother & Child Hospital affiliated to Shiraz University of Medical Sciences, Shiraz, Iran. Results: Menaquinone‐7 supplementation, when compared to placebo, significantly decreased serum fasting insulin (p =.002), homeostasis model of assessment insulin resistance (p =.002), and homeostasis model of assessment β‐cell function (p =.02) in addition to a significant increase in quantitative insulin sensitivity check index (p =.001). Also, MK‐7 administration led to significant declines in serum triglyceride (p =.003) and dihydrotestosterone (DHT; p =.03) levels, free androgen index (p <.001), waist circumference (p =.03), and body fat mass (p <.001) as well as significant increases in skeletal muscle (p <.001) and sex hormone binding globulin (SHBG, p <.001). Conclusions: This study highlights the beneficial effects of MK‐7 on insulin resistance, fat mass, skeletal muscle, and serum levels of triglyceride, DHT, and SHBG in PCOS patients. Therefore, it seems that MK‐7 supplementation might be an appropriate additive treatment for PCOS patients. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Astrocytes play a key role in hypoxic brain injury. The aim of our research was to determine the effects of menaquinone- 7 (MK-7), a subtype of vitamin K2 (VK2), on astrocytes during hypoxia and its potential mechanisms. MATERIALS AND METHODS: Astrocytes from the palliums of newborn Sprague Dawley rats were cultured. An astrocyte-hypoxia model was established using a hypoxia workstation. Cell Counting Kit-8 (CCK-8) and BrdU assays were used to determine the effects of MK-7 on hypoxic astrocytes. 2ʹ,7ʹ-Dichlorodihydrofluorescein diacetate (DCFDA) or dihydroethidium (DHE) assays were conducted to detect the levels of reactive oxygen species (ROS). An ATP assay was used to measure intracellular ATP production. The levels of proinflammatory cytokines and chemokines containing interleukin- 6 (IL-6), tumor necrosis factor-alpha (TNF-α), CC-chemokine ligand 2 (CCL2), and CXC-chemokine ligand 10 (CXCL10), as well as vitamin K-dependent protein growth arrest-specific 6 (Gas6), were determined in hypoxia-induced astrocytes, in the presence or absence of MK-7 pretreatment. Small interfering RNA (siRNA) was used to knockdown Gas6 expression to determine its role in hypoxic astrocytes pretreated with MK-7. RESULTS: Hypoxia reduced astrocyte viability and proliferation significantly; however, when pretreated with MK-7, these conditions remarkably increased. MK-7 also inhibited hypoxia-induced ROS production and enhanced ATP generation of hypoxic astrocytes. Pretreatment with MK-7 effectively reduced the expression of IL-6, TNF-α, CCL2, and CXCL10 but enhanced the expression of Gas6 in hypoxic astrocytes. Gas6 inhibition markedly attenuated the decline in MK- 7-induced ROS generation and IL-6 expression, and weakened MK-7-induced cell viability and ATP production in hypoxic astrocytes. CONCLUSIONS: Our study is the first to confirm that MK-7 can protect astrocytes from hypoxia- induced cytotoxicity, possibly by inhibiting mitochondrial dysfunction and the expression of proinflammatory cytokines. Gas6 may also participate in these protective effects. [ABSTRACT FROM AUTHOR]

Vitamin menaquinone-7 (MK-7) supplementation improves bone health and reduces the incidence of osteoporosis. Despite the recent developments in MK-7 fermentation using Bacillus subtilis natto, low fermentation yields, as well as complicated downstream processing steps, are still the main reasons for the expensive final product. To overcome these issues, developing a fermented dairy-based product rich in MK-7 by avoiding costly downstream steps and optimising the fermentation operating conditions to enhance the MK-7 concentration would be an alternative approach. The present study, therefore, aims to evaluate the role of agitation and aeration as the key operating conditions on MK-7 production by Bacillus subtilis natto using a milk media. The agitation and aeration rates of 525 RPM and 5 VVM were found to be the optimum levels leading to the production of 3.54 mg/L of MK-7. Further, the sensory evaluation was performed to compare the sensory properties of the freeze-dried fermented samples with non-fermented milk samples. The results illustrated that the fermented samples had a significant saltiness with intense aroma resulting in the less acceptability of them by the panellists. [ABSTRACT FROM AUTHOR]

Previous studies indicated a positive effect of vitamin K2 (VK2) supplementation on bone turnover biomarkers and bone mineral density (BMD), but the doses varied, and few studies have focused on the difference between VK2 supplementation alone and in combination with calcium and vitamin D3. The aim of this study was to explore a low and effective dose of VK2 for improving BMD, and to examine whether the co-supplementation of VK2, calcium and vitamin D3 would bring greater effects. In this trial, a total of 311 community-dwelling men and postmenopausal women aged 50 and 75 years were randomly assigned to four groups, receiving placebo, 50 µg/day, 90 µg/day or co-supplementation with calcium (500 mg/day) and vitamin D3 (10 µg/day) for 1 year. At the endpoint, the bone loss of femoral neck was significantly lower in postmenopausal women in the two 90 µg groups (treatment × time, p = 0.006) compared with placebo, but no effects in men. Serum biomarkers cOC/ucOC ratio increased in the intervention groups (treatment × time, p < 0.001). VK2 supplementation in dose of 90 µg/day performed a significant effect on reducing bone loss in postmenopausal women, but in combination with calcium and vitamin D3 brought no additional effects.Trial registration This trial was registered at http://www.chictr.org.cn as chiCTR1800019240. [ABSTRACT FROM AUTHOR]

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Following an application from NattoPharma ASA submitted for authorisation of a health claim pursuant to Article 13(5) of Regulation (EC) No 1924/2006 via the Competent Authority of Ireland, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on the scientific substantiation of a health claim related to MenaQ7® and maintenance of the elastic properties of the arteries. The scope of the application was proposed to fall under a health claim based on newly developed scientific evidence. The food proposed by the applicant as the subject of the health claim is MenaQ7®, which contains 97% menaquinone‐7 by weight. The Panel considers that MenaQ7® is sufficiently characterised. The claimed effect proposed by the applicant is ‘improves arterial stiffness’. Upon a request from EFSA, the applicant agreed that the claimed effect is maintenance of elastic properties of the arteries (by measuring the arterial stiffness). The Panel considers that maintenance of the elastic properties of the arteries is a beneficial physiological effect. No conclusions could be drawn from the three human intervention studies submitted for scientific substantiation of the claim. In the absence of evidence for an effect of MenaQ7® on maintenance of the elastic properties of the arteries in vivo in humans, the studies on the proposed mechanisms by which the food/constituent could exert the claimed effect cannot be used as a source of evidence for the scientific substantiation of the claim. The Panel concludes that a cause and effect relationship has not been established between the consumption of MenaQ7® and maintenance of the elastic properties of the arteries. [ABSTRACT FROM AUTHOR]