CN111579667A - Beta-carotene standard sample and preparation method and application thereof - Google Patents
Beta-carotene standard sample and preparation method and application thereof Download PDFInfo
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- CN111579667A CN111579667A CN202010442334.XA CN202010442334A CN111579667A CN 111579667 A CN111579667 A CN 111579667A CN 202010442334 A CN202010442334 A CN 202010442334A CN 111579667 A CN111579667 A CN 111579667A
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- G—PHYSICS
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Abstract
The invention relates to a beta-carotene standard sample and a preparation method and application thereof, comprising the following steps: step 1), preparation of a test solution: preparing a beta-carotene raw material into a test solution; step 2), liquid chromatography preparation: carrying out liquid chromatography on the test solution in the step 1) to separate pure fractions, wherein the chromatographic conditions are as follows: the chromatographic column is selected from PREP-ODS (H) -KIT or WatersXTrraRP 18; the mobile phase is a mixed solution of dichloromethane and acetonitrile; step 3), collecting: drying and crushing the pure fraction obtained in the step 2) under inert atmosphere to obtain the high-purity low-purity. The invention adopts a semi-preparative liquid chromatography system to purify the beta-carotene standard sample with high content (more than 99 percent) from the beta-carotene raw material (96 percent), has simple process, high preparation speed, stable product quality and good uniformity, and fills the blank of the current beta-carotene standard sample deletion.
Description
Technical Field
The invention relates to the field of compound extraction and purification, and particularly relates to a beta-carotene standard sample and a preparation method and application thereof.
Background
Beta-carotene is a fat-soluble carotenoid, has good anti-oxidation, anti-cancer and cancer-prevention effects, has certain effects on preventing cardiovascular diseases, enhancing the immunity of organisms, delaying aging and the like, and is a functional natural pigment with development value.
The production of beta-carotene raw material mainly includes natural extraction method, chemical synthesis method and microbial fermentation method. According to relevant regulations, the content of the beta-carotene food additive is above 96%, the content of the sample is high, but due to the existence of the positive and negative beta-carotene and some impurities with similar structures, the beta-carotene sample cannot be further separated and purified by the conventional extraction methods such as recrystallization, countercurrent chromatography and the like.
In order to meet the requirements of analysis and detection and quality control of related products and ensure the accuracy, comparability and traceability of detection results, a beta-carotene standard sample is urgently needed to be prepared.
Disclosure of Invention
Aiming at the defects and shortcomings in the prior art, the invention provides a beta-carotene standard sample and a preparation method and application thereof.
One of the purposes of the invention is to provide a preparation method of a standard beta-carotene sample, which comprises the following steps:
step 1), preparation of a test solution: preparing a beta-carotene raw material into a test solution;
step 2), liquid chromatography preparation: carrying out liquid chromatography on the test solution in the step 1) to separate pure fractions, wherein the chromatographic conditions are as follows: the chromatographic column is selected from PREP-ODS (H) -KIT or WatersXTrraRP 18; the mobile phase is a mixed solution of dichloromethane and acetonitrile;
step 3), collecting: drying and crushing the pure fraction obtained in the step 2) under inert atmosphere to obtain the high-purity low-purity.
According to some preferred embodiments of the present invention, in step 1), the beta-carotene raw material is beta-carotene crystals, beta-carotene powder, an intermediate liquid containing beta-carotene in a fermentation broth or an intermediate liquid containing beta-carotene during chemical synthesis, preferably beta-carotene crystals with a content of more than 96%.
According to some preferred embodiments of the invention, in step 1), the beta-carotene starting material is dissolved in dichloromethane and dissolved by sonication, and then filtered through a 0.22 μm or 0.45 μm filter.
According to some preferred embodiments of the invention, in step 2), the column is PREP-ODS (H) KIT (250 mm. times.20 mm,5 μm), or WatersXTrerRP 18(250 mm. times.20 mm,5 μm); mobile phase: dichloromethane: acetonitrile 50:50 solution; the flow rate is 5-15 mL/min, preferably 10mL/min, the sample injection amount is 0.1-1 mL, and fractions with the time of 18.5-20min are collected.
According to some preferred embodiments of the present invention, in step 2), the column temperature: 30 ℃; operating time: 20 min; detection wavelength: 470 nm.
According to some preferred embodiments of the present invention, in step 3), the solvent is removed by vacuum distillation, and then the mixture is lyophilized at-60 to-40 ℃ for 20 to 30 hours, and then is milled and pulverized, and then is lyophilized again for 20 to 30 hours.
According to some preferred embodiments of the present invention, in step 3), the inert atmosphere is nitrogen or helium, preferably nitrogen.
According to some preferred embodiments of the present invention, the standard sample of β -carotene prepared in step 3) is stored at-18 ℃ in a dark environment sealed under vacuum.
According to some preferred embodiments of the present invention, the isolated β -carotene standard sample is prepared using a semi-preparative liquid chromatography system.
The invention also aims to provide a beta-carotene standard sample prepared by the method.
The invention further aims to provide the beta-carotene standard sample prepared by the method or the beta-carotene standard sample for qualitative and quantitative detection of beta-carotene addition in food or feed.
The invention has the beneficial effects that: the invention provides an economic, rapid and effective preparation method of a standard beta-carotene sample, which can make up for the technical blank in the development aspect of the standard beta-carotene sample; meanwhile, the invention solves the problem that the standard sample is lacked when qualitative and quantitative detection research of the addition of the beta-carotene in the food and the feed is carried out. The invention adopts a semi-preparative liquid chromatography system to prepare the separated beta-carotene standard sample, has high content, simple process, high preparation speed, stable product quality and good uniformity, can obtain high content (more than 99 percent), is obviously superior to the product with the content of 95 to 97 percent obtained by adopting a recrystallization method in the prior art, has less pollution of related solvents, and can realize clean production.
Drawings
FIG. 1 is a standard curve for the preparation of beta-carotene standards;
FIG. 2 is a Raman spectrum of a prepared standard sample of beta-carotene;
FIG. 3 is a 1H-NMR chart of a prepared standard sample of β -carotene;
FIG. 4 is a mass spectrum of a prepared standard sample of beta-carotene;
FIG. 5 is an HPLC chromatogram of a prepared standard sample of beta-carotene.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications.
In the present invention, the instruments and the like used are conventional products which are purchased from regular vendors, not indicated by manufacturers. The raw materials used in the invention can be conveniently bought in domestic product markets. In the following examples, the preparation of standard samples of beta-carotene was carried out in a semipreparative liquid chromatography system in the form of semer femorax, using the beta-carotene crystals obtained by extraction of blakeslea trispora by the applicant, which are commercially available and have an HPLC content of 96.3%.
Example 1
A preparation method of a standard beta-carotene sample comprises the following steps:
(1) preparation of beta-carotene solution: preparing a beta-carotene standard sample by taking beta-carotene crystals as raw materials, dissolving the beta-carotene raw materials in dichloromethane, dissolving the dichloromethane by ultrasonic, and filtering the solution by a filter membrane of 0.22 mu m to obtain a beta-carotene solution;
(2) preparing liquid chromatography: injecting the beta-carotene solution into preparative chromatographic separation pure fraction, wherein the chromatographic condition is that the chromatographic column is PREP-ODS (H) -KIT (250mm multiplied by 20mm,5 mu m); mobile phase: dichloromethane: acetonitrile 50: 50; flow rate: 10 mL/min; column temperature: 30 ℃; operating time: 15 min; detection wavelength: 210nm, the sample injection amount is 0.2mL, and fractions within 18.5-20min are collected;
(3) and (3) fraction collection: the distillate was received in a brown bottle pre-filled with nitrogen, surrounded by tinfoil;
(4) and (3) drying: placing the distillate in a rotary evaporator at 55-75 ℃ by adopting a reduced pressure distillation method, then placing the distillate in a freeze dryer for freeze drying at-50 ℃ for 24 hours, grinding and crushing the freeze-dried beta-carotene solid, and freeze drying for 24 hours again to remove free water to the greatest extent to obtain a beta-carotene standard sample; the content of the standard sample is determined to be 99.4%.
(5) And (3) storage: the prepared pure beta-carotene solid is subpackaged in a relatively independent and clean glove box by adopting a 2mL brown sample bottle, sealed by a black bottle cap with a polytetrafluoroethylene lining, and independently and plastically packaged in vacuum by adopting a plastic bag, wherein the validity period is 1 year.
The peak area of β -carotene in the standard is shown in FIG. 1, the Raman spectrum of the β -carotene standard prepared in this example is shown in FIG. 2,1the H-NMR spectrum is shown in FIG. 3, the mass spectrum is shown in FIG. 4, and the HPLC detection spectrum is shown in FIG. 5. The standard of FIG. 1 was obtained from sigma and contained at 96%.
From the Raman spectrum of the β -carotene standard sample prepared in FIG. 2, it can be seen that 2923cm-1And 2853cm-1The nearby strong absorption peak is-CH3and-CH2The absorption peak of 11C-C bonds in the molecular chain is very weak due to the β -carotene has an infrared inactive symmetric structure, such as 1600-1680cm in the figure-1Shown by the weak absorption peak at (b). 1461cm-1Has a medium absorption peak of-CH3Antisymmetric bending vibration and-CH2Overlapping spectral bands of symmetric bending vibrations. 1376cm-1Has a medium-intensity absorption peak of-CH3Is symmetric bending vibration. 965cm-1The strong absorption peak is the out-of-plane bending vibration absorption peak of C-H on the carbon/carbon double bond.
From the 1H-NMR chart of the β -carotene standard sample prepared in FIG. 3, it can be seen that the chemical shift peak at 2.17 is the peak of the solvent CD3 OD. The spectrum has 7 groups of H-NMR peaks, which represent 7 hydrogen atoms with similar chemical environment in the structure of beta-carotene. The single peak at a chemical shift of 1.03 is the resonance peak of 12 hydrogen atoms. The multiple peak of the chemical shift at 1.47 is the formant of four hydrogen atoms. The multiple peak at a chemical shift of 1.62 is the formant of eight hydrogen atoms. The single peak at a chemical shift of 1.72 is the formant of six hydrogen atoms. The single peak at a chemical shift of 1.97 is the resonance peak of 12 hydrogen atoms. The multiple peaks with chemical shifts between 6.11 and 6.37 are resonance peaks of 10 hydrogen atoms. The multiple peaks with chemical shifts between 6.62 and 6.68 are the four hydrogen resonance peaks.
As can be seen from the mass spectrum of the β -carotene standard sample prepared in fig. 4, the mass spectrum shows that M/Z-536.5 is a molecular ion peak of β -carotene, and M/Z-444 is one of characteristic ion peaks of β -carotene.
According to the infrared spectrum, the nuclear magnetic spectrum and the mass spectrum, the structural information of the carotene sample prepared by the invention is consistent with the beta-carotene standard substance.
The standard sample prepared in this example was diluted to the concentration range of the standard curve in fig. 1 to obtain an HPLC profile as shown in fig. 5, and the detection method was that the mobile phase was acetonitrile: water (9:1), ethyl acetate gradient elution, flow rate of 1mL/min, detection wavelength of 455nm, from which peak area of 26.3067 was obtained, and substituting into the standard curve of fig. 1, the content of β -carotene in the carotene sample prepared by the present invention was obtained as 99.4%.
Example 2
A preparation method of a standard beta-carotene sample comprises the following steps:
(1) preparation of beta-carotene solution: the raw material for preparing the beta-carotene standard sample is a beta-carotene food additive, the beta-carotene raw material is dissolved in dichloromethane and dissolved by ultrasonic, and then the beta-carotene raw material is filtered by a filter membrane of 0.22 mu m to obtain a beta-carotene solution;
(2) preparing liquid chromatography: injecting the beta-carotene solution into preparative chromatographic separation pure fraction, wherein the chromatographic condition is that the chromatographic column is PREP-ODS (H) -KIT (250mm multiplied by 20mm,5 mu m); mobile phase: dichloromethane: acetonitrile 50: 50; flow rate: 15 mL/min; column temperature: 30 ℃; operating time: 20 min; detection wavelength: 210nm, the sample injection amount is 0.2mL, and fractions within 18.5-20min are collected;
(3) and (3) fraction collection: the distillate was received in a brown bottle pre-filled with nitrogen, surrounded by tinfoil;
(4) and (3) drying: placing the distillate in a rotary evaporator at 55-75 ℃ by adopting a reduced pressure distillation method, then placing the distillate in a freeze dryer for freeze drying at-50 ℃ for 24 hours, grinding and crushing the freeze-dried beta-carotene solid, and freeze drying for 24 hours again to remove free water to the greatest extent to obtain a beta-carotene standard sample; the standard sample was determined to have a beta-carotene content of 99.2%.
(5) And (3) storage: the prepared pure beta-carotene solid is subpackaged in a relatively independent and clean glove box by adopting a 2mL brown sample bottle, sealed by a black bottle cap with a polytetrafluoroethylene lining, and independently and plastically packaged in vacuum by adopting a plastic bag, wherein the validity period is 1 year.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A preparation method of a standard beta-carotene sample is characterized by comprising the following steps:
step 1), preparation of a test solution: preparing a beta-carotene raw material into a test solution;
step 2), liquid chromatography preparation: carrying out liquid chromatography on the test solution in the step 1) to separate pure fractions, wherein the chromatographic conditions are as follows: the chromatographic column is selected from PREP-ODS (H) -KIT or WatersXTrraRP 18; the mobile phase is a mixed solution of dichloromethane and acetonitrile;
step 3), collecting: drying and crushing the pure fraction obtained in the step 2) under inert atmosphere to obtain the high-purity low-purity.
2. The process according to claim 1, wherein the beta-carotene raw material in the step 1) is beta-carotene crystals, beta-carotene powder, an intermediate liquid containing beta-carotene in a fermentation broth or an intermediate liquid containing beta-carotene during a chemical synthesis, preferably beta-carotene crystals having a content of 96% or more.
3. The method of claim 2, wherein: in step 1), the beta-carotene raw material is dissolved in dichloromethane and dissolved by ultrasonic, and then filtered by a 0.22 μm or 0.45 μm filter membrane.
4. The method according to claim 1, wherein in step 2), the column is PREP-ODS (H) -KIT (250 mm. times.20 mm,5 μm), or WatersXTrerRP 18(250 mm. times.20 mm,5 μm); mobile phase: dichloromethane: acetonitrile 50:50 solution; the flow rate is 5-15 mL/min, preferably 10mL/min, the sample injection amount is 0.1-1 mL, and fractions with the time of 18.5-20min are collected.
5. The method according to claim 4, wherein in step 2), the column temperature: 30 ℃; operating time: 20 min; detection wavelength: 470 nm.
6. The preparation method according to any one of claims 1 to 5, wherein in the step 3), the solvent is removed by reduced pressure distillation, and then the mixture is lyophilized at-60 to-40 ℃ for 20 to 30 hours, milled and pulverized, and then lyophilized again for 20 to 30 hours.
7. The method according to any one of claims 1 to 6, wherein in step 3), the inert atmosphere is nitrogen or helium, preferably nitrogen.
8. The method according to any one of claims 1 to 7, wherein the standard sample of β -carotene prepared in step 3) is stored in a dark environment sealed under vacuum at-18 ℃.
9. A standard sample of β -carotene produced by the method of any one of claims 1-8.
10. The standard sample of beta-carotene prepared by the method according to any one of claims 1 to 8 or the standard sample of beta-carotene according to claim 9 for use in the qualitative and quantitative detection of the addition of beta-carotene to food or feed.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2001019542A1 (en) * | 1999-09-14 | 2001-03-22 | Natural Compounds, Ltd. | Naturally extracted and synthetic hypoglycemic or hypolipidemic compositions |
| CN103995079A (en) * | 2014-05-29 | 2014-08-20 | 中国计量学院 | Quantitative detection method of beta-carotene content in beta-carotene extract |
| CN105717240A (en) * | 2016-03-24 | 2016-06-29 | 中国标准化研究院 | Method for preparing Alitame standard substance |
| CN107827799A (en) * | 2017-11-21 | 2018-03-23 | 嘉必优生物技术（武汉）股份有限公司 | A kind of bata-carotene and preparation method thereof, application |
| CN110483357A (en) * | 2019-09-03 | 2019-11-22 | 西南大学 | A kind of isolation and purification method of tangerine peel major carotenoids |
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2020
- 2020-05-22 CN CN202010442334.XA patent/CN111579667A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001019542A1 (en) * | 1999-09-14 | 2001-03-22 | Natural Compounds, Ltd. | Naturally extracted and synthetic hypoglycemic or hypolipidemic compositions |
| CN103995079A (en) * | 2014-05-29 | 2014-08-20 | 中国计量学院 | Quantitative detection method of beta-carotene content in beta-carotene extract |
| CN105717240A (en) * | 2016-03-24 | 2016-06-29 | 中国标准化研究院 | Method for preparing Alitame standard substance |
| CN107827799A (en) * | 2017-11-21 | 2018-03-23 | 嘉必优生物技术（武汉）股份有限公司 | A kind of bata-carotene and preparation method thereof, application |
| CN110483357A (en) * | 2019-09-03 | 2019-11-22 | 西南大学 | A kind of isolation and purification method of tangerine peel major carotenoids |
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| Title |
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| K.N.CHIDAMBARA MURTHY: "PRODUCTION OF β-CAROTENE FROM CULTURED DUNALIELLA sp. AND EVALUATION OF BIOLOGICAL", 《DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY OF UNIVERSITY OF MYSORE》 * |
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| 米佳 等: "枸杞色泽与其类胡萝卜素含量和组成的相关性", 《食品科学》 * |
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