Ion pair dual water phase method for extracting and separating pyrroloquinoline quinone

Abstract

The invention discloses an ion pair dual water phase method for extracting and separating pyrroloquinoline quinine. The method comprises the steps of preparing mixed liquid containing the pyrroloquinoline quinine, adopting an ion pair dual water phase extraction system to extract the mixed liquid containing the pyrroloquinoline quinine, separating an upper layer to obtain an organic layer rich in the pyrroloquinoline quinine, covering the organic layer rich in the pyrroloquinoline quinine with anion exchange resin, washing with double-distilled water and diluted acid in sequence, collecting the diluted acid part, freezing, drying, and purifying to obtain the pyrroloquinoline quinine. According to the characteristics of a PQQ (pyrroloquinoline quinine) structure, namely the structure of the PQQ contains three carboxyls and is acidic, and the ion pair dual water phase system is used for separating and purifying the structure. Tetrabutyl ammonium hydroxide serves as an ion pair reagent, and is combined with the PQQ, so that the PQQ is selectively distributed in an organic phase, while other water-solubility impurities are distributed into the water phase, so that the PQQ is separated and purified effectively and selectively.

Description

Technical field

[0001] The invention relates to a method for separating pyrroloquinoline quinone by ion-pair aqueous two-phase extraction and belongs to the field of biochemical industry. Background technique

[0002] Pyrroloquinolinequinine (PQQ) is a water-soluble quinone compound, a coenzyme of glucose dehydrogenase and alcohol dehydrogenase, and is considered a new B vitamin (Nature 2003; 422: 832). The molecular weight of PQQ is 330. It was first discovered in microorganisms, and subsequent studies have shown that it also exists in animals and plants. The structural formula is shown in the following formula:

[0003]

[0004] It has been proven that PQQ has important physiological functions, such as maintaining skin health, stimulating nerve growth factors, enhancing immune function, promoting cell growth, anti-oxidation, scavenging free radicals, enhancing the tolerance of bacteria to extreme environmental conditions, and participating in cell signal transduction Wait. In t...

Examples

Embodiment 1

[0039] The method of ion-pair aqueous two-phase extraction and separation of pyrroloquinoline quinone in this embodiment includes the following steps:

[0040] (1) Preparation: Gluconobacter oxydans was inoculated into the fermentation medium according to the inoculation amount of 5%, and the seed solution was obtained by shaking culture at 28°C for 3 days; then the seed solution was inoculated into the fermentation medium according to the inoculation amount of 10%. Enrich the medium, culture with shaking at 28°C for 5 days, centrifuge the culture solution at 5°C, 9000r / min for 15min, collect the supernatant, and obtain the fermentation broth containing pyrroloquinoline quinone;

[0041] (2) Extraction: The ion-pair two-phase extraction system is used to extract the fermentation broth containing pyrroloquinoline quinone. The volume ratio of the extraction system and the fermentation broth containing pyrroloquinoline quinone is 4:2. The upper layer is separated and the lower layer is...

Embodiment 2

[0045] The method of ion-pair aqueous two-phase extraction and separation of pyrroloquinoline quinone in this embodiment includes the following steps:

[0046] (1) Preparation: Gluconobacter oxydans was inoculated into the fermentation medium according to the inoculum of 5%, cultured with shaking at 28°C for 3 days to obtain the seed liquid; then the seed liquid was inoculated with the inoculum of 10% Enrich the medium, culture with shaking at 28°C for 5 days, centrifuge the culture solution at 5°C, 9000r / min for 15min, collect the supernatant, and obtain the fermentation broth containing pyrroloquinoline quinone;

[0047] (2) Extraction: The ion-pair two-phase extraction system is used to extract the fermentation broth containing pyrroloquinoline quinone. The volume ratio of the extraction system and the fermentation broth containing pyrroloquinoline quinone is 5:3. The upper layer is separated and the lower layer is separated. The extraction was repeated 3 times, and the separate...

Embodiment 3

[0051] The method of ion-pair aqueous two-phase extraction and separation of pyrroloquinoline quinone in this embodiment includes the following steps:

[0052] (1) Preparation: Gluconobacter oxydans was inoculated into the fermentation medium according to the inoculation amount of 5%, and the seed solution was obtained by shaking culture at 28°C for 3 days; then the seed solution was inoculated into the fermentation medium according to the inoculation amount of 10%. Enrich the medium, culture with shaking at 28°C for 5 days, centrifuge the culture solution at 5°C, 9000r / min for 15min, collect the supernatant, and obtain the fermentation broth containing pyrroloquinoline quinone;

[0053] (2) Extraction: The ion-pair aqueous two-phase extraction system is used to extract the fermentation broth containing pyrroloquinoline quinone. The volume ratio of the extraction system and the fermentation broth containing pyrroloquinoline quinone is 7:4. The upper layer is separated and the lower ...

Technical field

[0001] The invention relates to a method for separating pyrroloquinoline quinone by ion-pair aqueous two-phase extraction and belongs to the field of biochemical industry. Background technique

[0002] Pyrroloquinolinequinine (PQQ) is a water-soluble quinone compound, a coenzyme of glucose dehydrogenase and alcohol dehydrogenase, and is considered a new B vitamin (Nature 2003; 422: 832). The molecular weight of PQQ is 330. It was first discovered in microorganisms, and subsequent studies have shown that it also exists in animals and plants. The structural formula is shown in the following formula:

[0003]

[0004] It has been proven that PQQ has important physiological functions, such as maintaining skin health, stimulating nerve growth factors, enhancing immune function, promoting cell growth, anti-oxidation, scavenging free radicals, enhancing the tolerance of bacteria to extreme environmental conditions, and participating in cell signal transduction Wait. In t...

Examples

Embodiment 1

[0039] The method of ion-pair aqueous two-phase extraction and separation of pyrroloquinoline quinone in this embodiment includes the following steps:

[0040] (1) Preparation: Gluconobacter oxydans was inoculated into the fermentation medium according to the inoculation amount of 5%, and the seed solution was obtained by shaking culture at 28°C for 3 days; then the seed solution was inoculated into the fermentation medium according to the inoculation amount of 10%. Enrich the medium, culture with shaking at 28°C for 5 days, centrifuge the culture solution at 5°C, 9000r / min for 15min, collect the supernatant, and obtain the fermentation broth containing pyrroloquinoline quinone;

[0041] (2) Extraction: The ion-pair two-phase extraction system is used to extract the fermentation broth containing pyrroloquinoline quinone. The volume ratio of the extraction system and the fermentation broth containing pyrroloquinoline quinone is 4:2. The upper layer is separated and the lower layer is...

Embodiment 2

[0045] The method of ion-pair aqueous two-phase extraction and separation of pyrroloquinoline quinone in this embodiment includes the following steps:

[0046] (1) Preparation: Gluconobacter oxydans was inoculated into the fermentation medium according to the inoculum of 5%, cultured with shaking at 28°C for 3 days to obtain the seed liquid; then the seed liquid was inoculated with the inoculum of 10% Enrich the medium, culture with shaking at 28°C for 5 days, centrifuge the culture solution at 5°C, 9000r / min for 15min, collect the supernatant, and obtain the fermentation broth containing pyrroloquinoline quinone;

[0047] (2) Extraction: The ion-pair two-phase extraction system is used to extract the fermentation broth containing pyrroloquinoline quinone. The volume ratio of the extraction system and the fermentation broth containing pyrroloquinoline quinone is 5:3. The upper layer is separated and the lower layer is separated. The extraction was repeated 3 times, and the separate...

Embodiment 3

[0051] The method of ion-pair aqueous two-phase extraction and separation of pyrroloquinoline quinone in this embodiment includes the following steps:

[0052] (1) Preparation: Gluconobacter oxydans was inoculated into the fermentation medium according to the inoculation amount of 5%, and the seed solution was obtained by shaking culture at 28°C for 3 days; then the seed solution was inoculated into the fermentation medium according to the inoculation amount of 10%. Enrich the medium, culture with shaking at 28°C for 5 days, centrifuge the culture solution at 5°C, 9000r / min for 15min, collect the supernatant, and obtain the fermentation broth containing pyrroloquinoline quinone;

[0053] (2) Extraction: The ion-pair aqueous two-phase extraction system is used to extract the fermentation broth containing pyrroloquinoline quinone. The volume ratio of the extraction system and the fermentation broth containing pyrroloquinoline quinone is 7:4. The upper layer is separated and the lower ...

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