Isolation, Purification And Physicochemical Characterization Of Vitamin K₂ From Flavobacterium

Vitamin K₂ refers to a series of naphthoquinone derivatives,which has a variety of physiological and pharmacological functions for human body,and is also called menaquinone-n?MK-n,n=1?14?,where n denotes the number of isoprene units in the side chain.Currently,Word Health Organization,the US Food and Drug Administration and the European Union Food and Drug Administration and so on have recommended that people use vitamin K₂ in daily life and clinical medication.However,these valuable compounds are not readily available at affordable prices because several tedious and inefficient unit operations are required for their production and downstream purification.There is no mature separation and purification methods for vitamin K₂ in our country especially,seriously limited the wide application of vitamin K₂.Flavobacterium is a Gram-negative bacterium that can be fermented to produce multiple vitamin K₂ homologs.The production of vitamin K₂ by Favobacterium fermentation has great advantages and potentials in improving the yields,types,bioactivity and biocompatibility of vitamin K₂.Due to complex composition of fermentation broth,small product concentration,poor stability and high quality requirements,it is necessary to create novel and innovative technologies to overcome these existing challenges and obtain high purity menaquinones.In this study,the bacteria separation of Flavobacterium fermentation was achieved by the membrane separation technology.The freeze-drying method and spray-drying method were used to dry wet cells,respectively.A method of extracting vitamin K₂ from dry cells was established by optimizing several parameters including the type of organic solvents,the ratio of organic solvents to dry cells weight,processing time and extraction times.The results showed that the highest yield?95%?was obtained using methanol at a ratio of 5:1?v/m?with three extractions of 30 minutes each.In order to further reduce the dry time and energy consumption during the drying process of wet cells,we proposed a rapid and simple method of direct extraction from wet cells with organic solvents.The optimized results showed that the highest yield was obtained with pretreatment using absolute ethanol at a ratio of 6:1?v/m?for 30 minutes and then two extractions of 30 minutes 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 composition analysis for vitamin K₂ extract indicates that there are a large number of strong polar fats.Condering the characteristics of the menaquinones,the macroporous adsorption resin HZ816 was selected for further study.The optimized results showed that the recovery rates and purities of the menaquinones reached 96%and 17.3%,respectively,when methanol-dichloromethane?1:1,v/v?was selected as eluent at a flow rate of 0.75 mL/min through a glass chromatography column with a height-to-diameter ratio of 80:10?mm:mm?.The processing capacity of the menaquinones reached 4.78 mg menaquinones per gram resin support.Different purity calculation methods show that there are many similar polarity components without UV absorption present in the solution purified by macroporous adsorption resin.A prepared gel permeation chromatography column?15 mm I.D.×255 mm?containing Bio-Beads S-X3 support was performed to remove these components.The results indicate that the recovery rates and purities of the menaquinones reached 99.9%and 57.3%,respectively,when dichloromethane was selected as eluent at a flow rate of 0.25 mL/min.And the processing capacity of the menaquinones reached 7.0 mg menaquinones per gram Bio-BeadsTM S-X3 support.Vitamin K₂ homologues have structural and functional differences.HPLC chromatograms show that reverse-phase C18 silica gel chromatography has great potential in the purification of vitamin K₂ homologues.The menaquinones were loaded on the C18 column with a ratio of height to diameter of 400:30?mm:mm?.MK-4,MK-5 and MK-6 were eluted with volume ratios of 9:1,6:1 and 3:1 methanol-dichloromethane?V/V?,respectively,at a flow rate of 3.0 mL/min.The purities of MK-4,MK-5 and MK-6 reached 85.5%,94.3%and 98.6%.And their recovery rates reached no less than 99.5%.The maximum processing capacity was 0.95 mg menaquinones per gram silica gel packing.After crystallization using methanol,the purity of vitamin K₂ homologues reached from 96.3%to 99.2%and their recovery rate reached no less than 92.1%.Throughout the entire process,the only organic solvents used were methanol and dichloromethane,facilitating the recycling of these solvents and,hence,application of the method on a large scale.The vitamin K₂ homologs were identified as MK-4,MK-5,MK-6 and MK-7 by UV.IR,MS and 1H-NMR.And MK-7 was firstly discovered in Flavobacterium cells.The SEM shows that all the homologue crystals are flaky,about 10 ?m in size.DSC and TGA analysis demonstated that the melting points of MK-5,MK-6 and MK-7 were 39.44 0C,47.92 0C and 54.07 ?,respectively,and the boiling points were 223.94 ?,233.81 ? and 245.13 ?,respectively.Light stability experiments showed that the degradation rate of vitamin K₂ in brown glass bottles was significantly reduced under indoor conditions.In conclusion,this study has important theoretical and practical significance for the industrialization and appliacation of vitamin K₂.