CN112961199B - Method for extracting NMN from fruits and vegetables - Google Patents

Method for extracting NMN from fruits and vegetables Download PDF

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CN112961199B
CN112961199B CN202110201826.4A CN202110201826A CN112961199B CN 112961199 B CN112961199 B CN 112961199B CN 202110201826 A CN202110201826 A CN 202110201826A CN 112961199 B CN112961199 B CN 112961199B
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nmn
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CN112961199A (en
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游林
贺金华
毛艳
周佳佳
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Chengdu Xiyu Leisurely Biotechnology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C07H1/08—Separation; Purification from natural products
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/048—Pyridine radicals
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids

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Abstract

The invention discloses a method for extracting NMN from fruits and vegetables. The method comprises the following steps: (1) crushing fruit and vegetable materials; (2) adding the powder obtained after crushing into alkali liquor, stirring and mixing uniformly, and adding a mixed solution of EDTA and sodium chloride for ultrasonic treatment; (3) after the ultrasound in the step (2) is finished, continuously adding a mixed solution containing CTAB, sulphobetaine and formic acid, and continuously performing ultrasound; (4) and (4) filtering the product obtained in the step (3), collecting supernatant, concentrating, crystallizing and drying the solid-phase product. The invention can extract NMN with higher yield and purity through the optimized extraction process and extraction reagent.

Description

Method for extracting NMN from fruits and vegetables
Technical Field
The invention belongs to the technical field of compound extraction, and particularly relates to a method for extracting NMN from fruits and vegetables.
Background
Nmn (nicotinamide mononuleotide): the full name of beta-nicotinamide mononucleotide is naturally-occurring bioactive nucleotide. The substances inherent in the human body are also rich in some fruits and vegetables. NMN is NAD in humans + The most direct precursor, which functions by NAD + Is shown. The decline of NAD during aging is considered to be a major cause of diseases and disabilities such as hearing and vision loss, cognitive and motor dysfunction, immune deficiency, arthritis due to autoimmune inflammatory response disorders, metabolic disorders and cardiovascular diseases. Therefore, NMN supplementation increases in vivo NAD + And the content of the extract can be used for delaying, improving and preventing various phenotypes related to aging, or age-induced metabolic disorders, senile diseases and the like. However, the existing extraction method has the problems of low extraction rate and low purity when extracting NMN.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a method for extracting NMN from fruits and vegetables, which can effectively solve the problem of low extraction rate of the existing method.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
(1) crushing fruit and vegetable materials;
(2) adding the powder obtained after crushing into alkali liquor, stirring and mixing uniformly, adding a mixed solution formed by EDTA and sodium chloride, and then carrying out ultrasonic treatment at 200-600W for 10-20 min;
the material-liquid ratio of the powder to the mixed solution is 1: 5-10; the content of EDTA in the mixed solution is 0.1-5 wt%, and the content of sodium chloride is 0.1-2 wt%;
(3) after the ultrasound in the step (2) is finished, continuously adding a mixed solution containing CTAB, sulfobetaine and formic acid, and performing ultrasound for 40-100 min at 600-800W and 60-80 ℃; the material-liquid ratio of the powder to the mixed liquid is 1: 20-30 (unit is g/mL);
the CTAB content in the mixed solution is 0.05-1.5 wt%; the sulfobetaine content is 0.01-1 wt%; the content of formic acid is 0.5-1 wt%;
(4) and (4) filtering the product obtained in the step (3), collecting supernatant, concentrating, crystallizing and drying the solid-phase product.
Further, the volume of the alkali liquor is 5-10 times of the weight of the powder.
Further, the alkali liquor is sodium hydroxide or ammonia water.
Further, the content of EDTA in the mixed solution was 1wt%, and the content of sodium chloride was 1.5 wt%.
Further, the ratio of the powder to the mixed solution was 1:22 (unit is g/mL).
Further, the CTAB content in the mixed solution is 1.5 wt%; sulfobetaine content 0.3 wt%; the formic acid content was 0.5% by weight.
Further, the sulfobetaine is lauramidopropyl hydroxysulfobetaine.
Further, the ultrasonic conditions in the step (3) are as follows: ultrasonic treatment at 800W and 60 deg.C for 80 min.
Further, the fruit and vegetable is avocado, green soy bean, cabbage, broccoli or tomato.
The beneficial effects of the invention are as follows:
1. the alkaline solution is adopted to treat the powder, so that the fruit and vegetable cells can be damaged in the ultrasonic environment, the components in the fruit and vegetable cells are dissolved out into the solution, and the alkaline environment can denature protein components in the cells, reduce the content of impurities possibly existing in a subsequent extraction product and facilitate the subsequent extraction.
2. The extraction is carried out by adopting the mixed solution of EDTA and sodium chloride, so that the extraction rate of the NMN obtained by extraction can be effectively ensured. EDTA can inhibit the activity of enzyme capable of degrading NMN in cells, and sodium chloride can separate NMN from polysaccharide components in the cells, so that the extraction rate and purity of NMN in the product are ensured.
3. The application adopts the mixed solution that CTAB, sulfobetaine and formic acid formed to be used for further extracting NMN, and CTAB can make NMN a large amount of enrichments under the condition of low ion concentration, the effectual extraction rate that has guaranteed NMN, and the amide molecule in the NMN has fine hydrophilicity, and NMN degrades easily when the temperature surpasses the room temperature. Therefore, by adding the zwitterion sulfobetaine as a carrier for bearing the NMN, the stability of the NMN at the temperature of 60-80 ℃ can be effectively ensured, the degradation amount of the NMN is reduced, and the extraction rate of the NMN is further ensured.
4. Formic acid is added into the mixed liquid in the step (3), and multiple experiments prove that after formic acid is added, the extraction rate can be ensured, the using amount of the mixed liquid in the step (3) can be effectively reduced, and the extraction cost can be greatly reduced.
5. Under the coordination of the mixed liquid in the step (3) and ultrasonic conditions of 600-800W, 60-80 ℃ and 40-100 min, the extraction rate of NMN in the product can be maximized.
Detailed Description
The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.
Example 1
A method for extracting NMN from fruits and vegetables comprises the following steps:
(1) crushing green soy beans;
(2) adding the powder obtained after crushing into ammonia water, stirring and mixing uniformly, then adding a mixed solution of EDTA and sodium chloride, and carrying out ultrasonic treatment at 200W for 10 min; wherein the material-liquid ratio of the powder to the mixed solution is 1: 5; the content of EDTA in the mixed solution is 1wt%, and the content of sodium chloride in the mixed solution is 1.5 wt%;
(3) after the ultrasound in the step (2) is finished, continuously adding a mixed solution containing CTAB, lauramide propyl hydroxy sulfobetaine and formic acid, and carrying out ultrasound for 80min at 800W and 60 ℃; wherein the material-liquid ratio of the powder to the mixed liquid is 1: 22; CTAB content 1.5 wt%; sulfobetaine content 0.3 wt%; the formic acid content is 0.5 wt%;
(4) and (4) filtering the product obtained in the step (3), collecting supernatant, concentrating, crystallizing and drying the solid-phase product.
Example 2
A method for extracting NMN from fruits and vegetables comprises the following steps:
(1) crushing green soy beans;
(2) adding the powder obtained after crushing into a sodium hydroxide solution, stirring and mixing uniformly, then adding a mixed solution of EDTA and sodium chloride, and carrying out ultrasonic treatment at 300W for 15 min; the material-liquid ratio of the powder to the mixed solution is 1: 6; the content of EDTA is 0.5wt%, and the content of sodium chloride is 2 wt%;
(3) after the ultrasound in the step (2) is finished, continuously adding a mixed solution containing CTAB, lauramide propyl hydroxy sulfobetaine and formic acid, and carrying out ultrasound for 60min at 600W and 80 ℃; wherein the material-liquid ratio of the powder to the mixed liquid is 1: 20; CTAB content 0.3 wt%; sulfobetaine content 0.8 wt%; formic acid content 1 wt%;
(4) and (4) filtering the product obtained in the step (3), collecting supernatant, concentrating, crystallizing and drying the solid-phase product.
Example 3
A method for extracting NMN from fruits and vegetables comprises the following steps:
(1) crushing green soy beans;
(2) adding the powder obtained after crushing into ammonia water, stirring and mixing uniformly, then adding a mixed solution of EDTA and sodium chloride, and carrying out ultrasonic treatment at 600W for 20 min; the material-liquid ratio of the powder to the mixed solution is 1: 10; the EDTA content is 5wt%, and the sodium chloride content is 2 wt%;
(3) after the ultrasound in the step (2) is finished, continuously adding a mixed solution containing CTAB, lauramidopropyl hydroxysulfobetaine and formic acid, and carrying out ultrasound for 60min at 600W and 75 ℃; wherein the material-liquid ratio of the powder to the mixed liquid is 1: 30; CTAB content of 1 wt%; sulfobetaine content 0.1 wt%; formic acid content 1 wt%;
(4) and (4) filtering the product obtained in the step (3), collecting supernatant, concentrating, crystallizing and drying the solid-phase product.
Example 4
Step (3) was omitted as compared with example 1, and the rest was the same as example 1.
Example 5
Compared with the example 1, the formic acid is lacked in the step (3), the feed-liquid ratio is 1:50, and the rest is the same as the example 1.
Example 6
Compared with the example 1, the ultrasonic conditions in the step (3) are 600W, 60 ℃ and 60min, and the rest is the same as the example 1.
Example 7
The procedure of example 1 was repeated except that the CTAB and lauramidopropyl hydroxysultaine in step (3) were replaced with SDS, and the procedure was as in example 1.
Weighing 10g of green soy beans, respectively treating the green soy beans by the methods described in examples 1-7, and detecting the NMN content in the product by the method described in the literature, "determination of nicotinamide mononucleotide in vegetables and fruits by liquid chromatography-mass spectrometry", wherein the results are shown in Table 1.
TABLE 1 NMN extraction yield and purity
Figure BDA0002949262400000051
Figure BDA0002949262400000061
As can be seen from the data in Table 1, the product extracted by the method in the embodiments 1-3 of the present application has high NMN yield and purity, and the extraction effect of the embodiment 1 is the best due to certain differences among the ultrasonic conditions, the feed-liquid ratio and the use amounts of EDTA and sodium chloride in the embodiments 1-3.
In example 4, because of the lack of EDTA, a great amount of NMN is degraded in the extraction process, and the yield is low; in example 5, the feed-to-liquid ratio is increased, and although the yield of NMN is also increased, the cost is also increased due to the increase of the feed-to-liquid ratio, and after the feed-to-liquid ratio is increased, more impurities may be extracted, so that the purity of NMN is greatly influenced; the ultrasonic frequency was reduced and the ultrasonic time was reduced in example 6, and it can be seen that the difference between the NMN content and the purity is not so large as in example 1, and that in example 7, CTAB and lauramidopropyl hydroxysultaine were replaced with SDS, and the NMN yield was significantly reduced as compared to example 1. Therefore, the technical scheme described in the embodiment 1 of the application can be further used for extracting and obtaining NMN with higher yield and purity.

Claims (8)

1. A method for extracting NMN from fruits and vegetables is characterized by comprising the following steps:
(1) crushing green soy beans;
(2) adding the powder obtained after crushing into alkali liquor, stirring and mixing uniformly, adding a mixed solution formed by EDTA and sodium chloride, and then carrying out ultrasonic treatment at 200-600W for 10-20 min;
the material-liquid ratio of the powder to the mixed solution is 1: 5-10; the content of EDTA in the mixed solution is 0.1-5 wt%, and the content of sodium chloride is 0.1-2 wt%;
(3) after the ultrasound in the step (2) is finished, continuously adding a mixed solution containing CTAB, sulfobetaine and formic acid, and performing ultrasound for 40-100 min at 600-800W and 60-80 ℃; the material-liquid ratio of the powder to the mixed liquid is 1: 20-30;
the CTAB content in the mixed solution is 0.05-1.5 wt%; the sulfobetaine content is 0.01-1 wt%; the content of formic acid is 0.5-1 wt%;
(4) and (4) filtering the product obtained in the step (3), collecting supernatant, concentrating, crystallizing and drying the solid-phase product.
2. The method for extracting NMN from fruits and vegetables according to claim 1, wherein the volume of said alkali solution is 5-10 times of the weight of said powder.
3. The method for extracting NMN from fruits and vegetables according to claim 1 or 2, wherein said alkali solution is sodium hydroxide or ammonia.
4. The method for extracting NMN from fruits and vegetables according to claim 1, wherein the content of EDTA in said mixed solution is 1wt% and the content of sodium chloride is 1.5 wt%.
5. The method for extracting NMN from fruits and vegetables according to claim 1, wherein the ratio of powder to mixed liquor is 1: 22.
6. The method for extracting NMN from fruits and vegetables according to claim 1, wherein CTAB content in the mixed solution is 1.5 wt%; sulfobetaine content 0.3 wt%; the formic acid content was 0.5% by weight.
7. The method for extracting NMN from fruits and vegetables according to claim 1 or 6, wherein said sulfobetaine is lauramidopropyl hydroxysulfobetaine.
8. The method for extracting NMN from fruits and vegetables according to claim 1, wherein the ultrasonic conditions in the step (3) are as follows: performing ultrasonic treatment at 800W and 60 ℃ for 80 min.
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