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A method for extracting and purifying vitamin K2 from wall-broken Bacillus natto cells

A technology of bacillus natto and vitamins, applied in the field of bioengineering and bioseparation engineering, can solve the problems of complex overall process, low extraction rate, high cost, etc., and achieve the effect of simple process, high efficiency and high extraction rate

Active Publication Date: 2021-07-27
HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Preparation of Vitamin K by Fermentation of Bacillus Natto 2 Among them, Bacillus natto has more metabolites, and at the same time, due to its cell autolysis, it will produce a variety of impurities, such as miscellaneous proteins, nucleic acids, oils, etc., resulting in the acquisition of highly purified vitamin K 2 more difficult
[0005] Currently, organic solvents are widely used to extract vitamin K from Bacillus natto fermentation broth 2 , this method requires pretreatment operations such as bacterial crushing, dehydration and drying, and subsequent purification methods also require macroporous resin and reversed-phase silica gel, etc. The overall process is complex, expensive, and the extraction rate is low

Method used

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  • A method for extracting and purifying vitamin K2 from wall-broken Bacillus natto cells
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Embodiment 1

[0030] Example 1: A method for extracting and purifying vitamin K2 from Bacillus natto thallus

[0031] The high-performance liquid chromatography analyzer used in the embodiment of the present invention is purchased from Japan Shimadzu Corporation, is equipped with ultraviolet detector and reverse phase C18 column (4.6mm I.D * 250mm), mobile phase: methanol / methylene chloride=4: 1. Detection wavelength: 248nm, column temperature: 35°C, injection volume: 20ul, flow rate: 1ml / min.

[0032] All the reagents used in the examples of the present invention are analytically pure, except those used in high performance liquid chromatography are chromatographically pure.

[0033] The fermented liquid that present embodiment is processed is bacillus natto in by glycerol 69.6g / L, glucose 34.5g / L, K 2 HPO 4 4.0g / L, 20g / L peptone, 25g / L yeast extract, cultivated at 250rpm, 37°C for five days.

[0034] Add the fermentation liquid to the ultrafiltration membrane equipment, open the valves,...

Embodiment 2

[0059] Example 2: A method for extracting and purifying vitamin K2 from Bacillus natto thallus

[0060] A method for extracting and purifying vitamin K2 from the fermentation product of Bacillus natto, comprising the following steps:

[0061] Step 1: Filtrate the Bacillus natto fermentation liquid with an ultrafiltration membrane device having a ceramic membrane with a pore size of 5 nm, centrifuge the obtained concentrated solution at a speed of 5000 rpm for 20 min, discard the supernatant, and collect the precipitated substance as sodium Fresh thallus of Bacillus bean;

[0062] Step 2: Put the fresh bacteria of Bacillus natto into the ion beam equipment, bombard the bacteria with nitrogen ion beam, the energy of the ions used is 15keV, and the amount of implanted ions is 1×10 15 N + / cm 2 , inject for 10 seconds, stop injecting for 5 seconds, and repeat the operation for 60 minutes to obtain the broken Bacillus natto cells;

[0063] Step 3: including the first leaching a...

Embodiment 3

[0076] Example 3: A method for extracting and purifying vitamin K2 from Bacillus natto thallus

[0077] A method for extracting and purifying vitamin K2 from the fermentation product of Bacillus natto, comprising the following steps:

[0078] Step 1: Filter the Bacillus natto fermentation broth with a ceramic membrane with a pore size of 10um, centrifuge the obtained solution at a speed of 18,000rpm for 20min, discard the supernatant, and collect the precipitated substance that is the fresh bacteria of Bacillus natto ;

[0079]Step 2: Put the fresh bacteria of Bacillus natto into the ion beam equipment, bombard the bacteria with nitrogen ion beam, the energy of the ions used is 35keV, and the amount of implanted ions is 5×10 19 N + / cm 2 , inject for 10 seconds, stop injecting for 5 seconds, and repeat the operation for 30 minutes to obtain the broken Bacillus natto cells;

[0080] Step 3: including the first leaching and the second leaching;

[0081] The first leaching: ...

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Abstract

A method for extracting and purifying vitamin K2 from broken Bacillus natto cells, separating the fermented microorganisms from solid and liquid by ultrafiltration and centrifugation to obtain wet cells, and treating the wet cells with ion beams to break the walls , Extracted with absolute ethanol, not only will obtain a large amount of vitamin K2, but also extract less oil, rough purification is carried out during the extraction process, which provides a good solution for the subsequent one-step high-efficiency purification. The extract is extracted through a column, and organic reagents with different polarities are used for extraction and purification, and impurities of different polarities are extracted, so that the purity of the solution is greatly improved. It is then frozen and crystallized to obtain vitamin K2 crystals with a purity of 95%. The method omits the processes of drying and cell crushing in the traditional method, and improves the extraction efficiency and the extraction purity. And through one-step purification to obtain vitamin K with higher purity 2 .

Description

technical field [0001] The invention relates to a method for extracting and purifying vitamin K2 from broken Bacillus natto thallus, belonging to the fields of bioengineering and bioseparation engineering. Background technique [0002] Vitamin K 2 It is a general term for a series of terpene side chain compounds containing 2-methyl-1,4-naphthoquinone core and C3 with different numbers of isoprene structural units. According to the carbon element on the terpene side chain The number can be divided into K2(10), K2(20), K2(35), K2(40) and so on. Vitamin K 2 It is a fat-soluble vitamin, a derivative of a naphthoquinone group with biological activity of phylloquinone, and is one of the indispensable important vitamins in the human body. With research on its function, it has been proven that vitamin K 2 It has the effect of preventing osteoporosis, cardiovascular, Parkinson, liver cancer and other diseases. Therefore, functional foods, medical products, cosmetics and other fi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07C46/10C07C50/14
Inventor 郑之明方志伟王鹏王丽赵根海刘会王晗吴荷芳倪文枫孙小雯杨强唐恒芳
Owner HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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