CN115989866A - Preparation method and application of astaxanthin encapsulated by soy protein nanofiber - Google Patents
Preparation method and application of astaxanthin encapsulated by soy protein nanofiber Download PDFInfo
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Images
Abstract
The invention discloses a preparation method and application of astaxanthin encapsulated by soybean protein nanofibers, and belongs to the field of nutrient substance delivery protection. The invention enhances the astaxanthin loading capacity by fibrillating the soybean protein isolate, and obtains the protein/astaxanthin compound by an emulsification-evaporation method, hydrophobic acting force and hydrogen bond between the protein and the astaxanthin, thereby solving the problems of poor water solubility, unstable chemical property and low bioavailability of the astaxanthin. Meanwhile, the preparation method is simple and convenient, and the material sources are wide.
Description
Technical Field
The invention relates to the field of food engineering, in particular to a preparation method and application of astaxanthin encapsulated by soybean protein nanofibers.
Background
Astaxanthin (astaxantin) is a hydrophobic ketocarotenoid that is widely found in algae such as haematococcus pluvialis and crustaceans such as shrimp and crab. The molecular structure of natural astaxanthin comprises a c=c double-chain conjugated olefin structure, the specificity of which provides it with an ability to efficiently eliminate active oxygen and scavenge free radicals. A large number of in vitro studies and in vivo experiments show that astaxanthin has various biological activities such as antioxidant activity, anti-inflammatory activity, anti-tumor activity, vision protection, central nervous system protection and the like. However, astaxanthin has a special unsaturated conjugated double bond structure, so that the astaxanthin has the problems of poor water solubility, low chemical stability, low bioavailability and the like. In actual processing and storage, astaxanthin is often degraded and discolored due to changes in factors such as illumination, temperature, oxygen content and the like. This places a great limit on its use in the food, pharmaceutical and cosmetic industries.
In recent years, astaxanthin delivery systems based on natural proteins have been developed, which have been considered as one of the effective strategies for improving the dispersibility, chemical stability and bioavailability of astaxanthin in aqueous environments. In this regard, different food proteins such as whey protein, beta-lactoglobulin, potato protein and zein have been used as carriers for enhancing the water solubility and antioxidant activity of astaxanthin. However, the natural protein has a compact structure, most of hydrophobic groups and charged amino acid residues are buried in the protein, and the loading capacity of the prawn penicillin is limited. At present, the capability of the natural protein for loading bioactive substances can be remarkably improved by changing the protein structure of the natural protein in physical, chemical, biological modification and other modes and inducing the hydrophobic groups and charged amino acid residues of the natural protein to be exposed on the surface of the protein. In addition, the natural proteins can be disassembled and reassembled in different types to expose more functional groups, so that the binding capacity with astaxanthin molecules is improved.
The invention adopts soybean protein isolate which is a natural protein capable of being industrially produced, and induces self-assembly of the soybean protein isolate under the conditions of acidity and heating to form nano-sized fiber aggregate embedded astaxanthin so as to improve the water solubility, chemical stability and bioavailability of the astaxanthin. Meanwhile, the industrial application of the astaxanthin in the industries of foods, medicines and cosmetics is improved to a certain extent.
Disclosure of Invention
The invention aims to provide a preparation method of encapsulated astaxanthin, which can better protect the bioactivity of the astaxanthin, improve the effect of embedding the astaxanthin, improve the water solubility and chemical stability of the astaxanthin and has excellent capability of resisting environmental stress and stability.
Meanwhile, the invention provides application of the nano-embedded astaxanthin in food and health care, which is used for improving the gastrointestinal digestion resistance of the astaxanthin and improving the bioavailability of the astaxanthin in human bodies.
Meanwhile, the invention provides a food with nano-encapsulated astaxanthin.
Meanwhile, the invention provides a health care product of nano-encapsulated astaxanthin.
The invention is realized by the following technical scheme:
(1) Dissolving natural Soybean Protein Isolate (SPI) in deionized water to prepare 1-3% (w/v) SPI solution, magnetically stirring for 2h, adjusting pH to 2.0 with HCl solution, and hydrating the SPI solution overnight at 4deg.C. The next day the protein solution was heated in a 85 ℃ water bath with constant stirring for 12h. After heat treatment, the protein fibrils were cooled in an ice-water bath and the resulting samples were stored in a refrigerator at 4 ℃ or lyophilized for use.
(2) And (3) regulating the soybean protein nanofiber solution obtained in the step (1) to pH3.0 by using a NaOH solution. Dissolving astaxanthin in ethanol-dichloromethane solution (5:1, v/v) to obtain astaxanthin solution, gradually adding astaxanthin solution into soybean protein nanofiber solution, and emulsifying the obtained mixture solution with high speed shearing machine at 8000-10000rpm for 2min.
(3) And (3) performing rotary evaporation on the soy protein nanofiber-astaxanthin composite solution obtained in the step (2) by using a vacuum rotary evaporator to remove the organic solvent, wherein the final concentration of astaxanthin in the composite solution is 0.1-0.2mg/mL. The resulting complex solution was stored in a refrigerator at 4 ℃ or lyophilized for use.
Further defined, the protein content of the natural isolated soy protein of step (1) should be above 90% and the HCl solution used should be at a concentration of 1-2mol/L.
Further defined, the concentration of NaOH solution used in step (2) is 0.5-1mol/L.
Further defined, the reduced pressure rotary evaporator in step (3) is used at a temperature of 38-45 ℃ for 10-15min.
The invention has the following beneficial effects:
the invention provides a protein carrier for nano-embedding astaxanthin, a preparation method and application thereof, which are used for preparing a soybean protein nanofiber-astaxanthin compound by separating natural soybean protein nanofibers and utilizing hydrophobic interaction and hydrogen bonding capability between the protein nanofibers and astaxanthin. The soybean protein nanofiber prepared by the invention has good astaxanthin nano-embedding effect, and greatly enhances the water solubility and the environmental stress resistance of astaxanthin.
The soybean protein nanofiber also has good protease resistance, and has excellent gastrointestinal digestion resistance when being used as a nano carrier, so that the bioavailability of astaxanthin is effectively improved.
Drawings
FIG. 1 is a technical roadmap of the invention;
FIG. 2 is a graph of the final water-soluble content of astaxanthin;
FIG. 3 shows the change in retention of astaxanthin during heat treatment at 90 ℃;
FIG. 4 shows the change in astaxanthin retention during 15W ultraviolet light irradiation;
fig. 5 is a graph of the bioavailability of astaxanthin after simulated digestion in vitro.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The isolated soy protein is prepared in a laboratory by alkali dissolution and acid precipitation of defatted soybean meal, and the protein content is 92.71+/-0.61% as measured by a Kjeldahl nitrogen determination method. Defatted soybean meal was purchased commercially from Shandong Yuwang group.
Astaxanthin is commercially available from Shanghai source leaf Biotechnology Inc.
Other chemical reagents were all analytically pure.
Example 1.
(1) Natural Soy Protein Isolate (SPI) is dissolved in deionized water to prepare 3% (w/v) SPI solution, the solution is magnetically stirred for 2 hours, the pH of the solution is regulated to 2.0 by using 2mol/LHCl solution, and the SPI solution is hydrated overnight at 4 ℃. The next day the protein solution was heated in a 85 ℃ water bath with constant stirring for 12h and 24h. After heat treatment, the protein fibrils were cooled in an ice-water bath and the resulting samples were stored in a refrigerator at 4 ℃ or lyophilized for use.
(2) And (3) regulating the soybean protein nanofiber solution obtained in the step (1) to pH3.0 by using a NaOH solution. Astaxanthin was dissolved in an ethanol-dichloromethane solution (5:1, v/v) to obtain an astaxanthin solution, and the astaxanthin solution was gradually added to the soy protein nanofiber solution, and the obtained mixture solution was emulsified for 2min by a high-speed shearing machine at 8000 rpm.
(3) And (3) spin-evaporating the soy protein nanofiber-astaxanthin composite solution obtained in the step (2) at 40 ℃ by using a vacuum rotary evaporator to remove the organic solvent, wherein the final concentration of astaxanthin in the composite solution is 0.2mg/mL. The resulting complex solution was stored in a refrigerator at 4 ℃ or lyophilized for use.
Comparative example 1.
(1) Natural Soy Protein Isolate (SPI) is dissolved in deionized water to prepare 3% (w/v) SPI solution, the solution is magnetically stirred for 2 hours, the pH of the solution is regulated to 2.0 by using 2mol/LHCl solution, and the SPI solution is hydrated overnight at 4 ℃. The resulting samples were stored in a refrigerator at 4 ℃ or lyophilized for use.
(2) The soy protein isolate solution obtained in step (1) was adjusted to pH3.0 using a 1mol/LNaOH solution. Astaxanthin was dissolved in an ethanol-dichloromethane solution (5:1, v/v) to obtain an astaxanthin solution, and the astaxanthin solution was gradually added to the SPI solution, and the obtained mixture solution was emulsified by a high-speed shearing machine at 8000rpm for 2min.
(3) Spin-evaporating the soy protein isolate-astaxanthin complex solution obtained in the step (2) at 40 ℃ by using a vacuum rotary evaporator to remove the organic solvent, wherein the final concentration of astaxanthin in the complex solution is 0.2mg/mL. The resulting complex solution was stored in a refrigerator at 4 ℃ or lyophilized for use.
The following experiment was used to verify the experimental effect: experimental effects were verified with example 1 and comparative example 1:
1. astaxanthin water solubility assay: the astaxanthin complex was centrifuged (2000 Xg, 10min,4 ℃) to remove free astaxanthin. The centrifuged supernatant was mixed with an absolute ethanol-dichloromethane (5:1, v/v) solution in a centrifuge tube, and centrifuged (5000 Xg, 10min,4 ℃ C.) after vortexing for 1 min. The centrifuged supernatant was again filtered through a 0.22 μm organic filter and analyzed for astaxanthin content by High Performance Liquid Chromatography (HPLC). HPLC conditions were as follows: using a photodiode detector with a detection wavelength of 470 nm, C 18 (250 mm. Times.4.6 mm,5 μm) liquid chromatography column with 95% (v/v) methanol solution as mobile phase at a flow rate of 1mL/min and a sample volume of 20. Mu.L. And a calibration curve of astaxanthin was established and the solubility of astaxanthin in water was calculated.
The results are shown in FIG. 1: the solubility of natural astaxanthin in water is very poor, and the solubility of the astaxanthin in water is only 5.44 mug/mL by HPLC analysis, but after SPI loading, the solubility is increased to 18.68 mug/mL, and the water solubility of astaxanthin is obviously increased, but still at a very low level. After fibrosis of SPI, astaxanthin water solubility was significantly increased (P < 0.05). Wherein the content of astaxanthin loaded by the isolated soy protein for 12 hours is the maximum, and is 152.14 mug/mL, and the water solubility of the isolated soy protein is improved by nearly 28 times compared with that of free astaxanthin.
2. Environmental stress stability
Thermal stability determination: astaxanthin solution in methylene chloride-ethanol solution (5:1, V/V) was added to deionized water to make the astaxanthin content consistent with that in the protein fiber sample. 10mL of each sample was taken and placed in a stoppered test tube, and then heat treated in a water bath at 90℃to sample at 15, 30, 60 and 120min, respectively, and immediately after the sampling, the samples were placed in an ice-water bath, and then the astaxanthin content was measured.
The results are shown in FIG. 2: the retention rate of astaxanthin after heating the sample for 2 hours is detected by HPLC, and the decomposition rate of free astaxanthin is found to be relatively fast under the heating condition of 90 ℃, and the astaxanthin is degraded by nearly 66% after heating for 2 hours. After SPI, the thermal stability of astaxanthin is obviously improved, but astaxanthin is still degraded by nearly half after heat treatment for 2 hours at 90 ℃. Whereas the thermal stability of the soy protein-loaded astaxanthin for 12h and 24h of fibrosis was significantly increased, 63% and 66% respectively remained in the complex system after heat treatment at 90 ℃ for 2h. This is probably due to the fact that after the astaxanthin is embedded in the protein, the macromolecular structure of the protein effectively protects the oxidative degradation thereof, and that the heat treatment at 90 ℃ and the pH3.0 are close to the conditions at which the protein fibrils are formed, the protein fibrils may continue to self-assemble for cross-linking, better protecting the astaxanthin in their fiber aggregates.
Ultraviolet light stability determination: astaxanthin solution in methylene chloride-ethanol solution (5:1, V/V) was added to deionized water to make the astaxanthin content consistent with that in the protein fiber sample. 10mL of each sample was placed in a horizontally placed petri dish (diameter: 90 mm), and then irradiated with a 15W ultraviolet lamp at a short distance (20 cm), and sampled at 30, 60, 120, 240min, respectively, to determine the astaxanthin content therein.
The results are shown in FIG. 3: the astaxanthin content after irradiation with UV light for 8 hours was detected by means of an ultraviolet spectrophotometer at 478 nm. It can be seen that free astaxanthin is highly sensitive to uv light and degrades very fast under uv light. After 8h of uv exposure, only about 37% of the astaxanthin remained, the uv stability of the astaxanthin encapsulated in SPI was significantly improved, with approximately 45% of the astaxanthin remaining in the SPI/AST complex. The astaxanthin in the encapsulated soy protein nanofiber has better protection effect on ultraviolet light-induced decomposition, wherein about 61% of astaxanthin remains in the compound. This is probably due to the fact that the macromolecular proteins absorb ultraviolet light, which can prevent the ultraviolet light from directly irradiating the astaxanthin molecules, and the ultraviolet light stability of astaxanthin is improved. Meanwhile, the complex protein fiber structure induces exposure of aromatic amino acid residues and double bonds buried in SPI molecules, and the ultraviolet light absorption capacity of the protein molecules is improved.
3. In vitro simulated bioavailability assay: 10mL of the different samples were mixed with 30mL of artificial gastric juice containing 2mg/mL NaCl and 3.2mg/mL pepsin in an Erlenmeyer flask. And incubated at 37℃and 120rpm in a shaking incubator at full temperature for 1h. After gastric digestion is completed, it is adjusted to pH7.4 to stop gastric juice digestion. To this was added artificial intestinal juice, and the final concentrations of trypsin and bovine bile salt were 4mg/mL and 5mg/mL, respectively. And incubated in a shaking incubator at room temperature for 2 hours (37 ℃ C., 120 rpm), and after the digestion was completed, a volume of the sample was mixed with 20% acetic acid in equal volume and centrifuged (12000 Xg, 10 min) to determine the astaxanthin content by HPLC.
The results are shown in FIG. 3: the in vitro simulated bioavailability of free astaxanthin is very low, only 13.7%. This is because carotenoids such as astaxanthin decompose in harsh environments such as the gastrointestinal tract, while the aqueous environment in the gastrointestinal tract also limits their absorption. After protein embedding, the bioavailability of the astaxanthin is obviously improved (P is less than 0.05), and the soybean protein nanofiber nano-encapsulated astaxanthin has the highest bioavailability (69.6%), and compared with free astaxanthin, the bioavailability of the astaxanthin is improved by 3.45 times. This is probably due to the fact that the protein fibrils obtained by heating under acidic conditions are more easily adapted to the harsh gastric environment, preventing astaxanthin from being degraded, and the modified protein fibrils are more easily replaced by bile salts, increasing the astaxanthin content entering the bile salt micelles.
Example 2.
(1) Natural Soy Protein Isolate (SPI) is dissolved in deionized water to prepare 3% (w/v) SPI solution, the solution is magnetically stirred for 2 hours, the pH of the solution is regulated to 2.0 by using 2mol/LHCl solution, and the SPI solution is hydrated overnight at 4 ℃. The next day the protein solution was heated in a 85 ℃ water bath with constant stirring for 12h and 24h. After heat treatment, the protein fibrils were cooled in an ice-water bath and the resulting samples were stored in a refrigerator at 4 ℃ or lyophilized for use.
(2) The soy protein nanofiber solution obtained in step (1) was adjusted to pH3.0 using 1mol/LNaOH solution. Astaxanthin is dissolved in ethanol-dichloromethane solution (5:1, v/v) to obtain astaxanthin solution, and the astaxanthin solution is gradually added into soybean protein nanofiber solution under the condition of magnetic stirring to promote the complexation between astaxanthin and protein fibrils. The resulting complex solution was stirred further for 3h at 37℃under light-shielding conditions.
(3) And (3) spin-evaporating the soy protein nanofiber-astaxanthin composite solution obtained in the step (2) at 40 ℃ by using a vacuum rotary evaporator to remove the organic solvent, wherein the final concentration of astaxanthin in the composite solution is 0.2mg/mL. The resulting complex solution was stored in a refrigerator at 4 ℃ or lyophilized for use.
Claims (7)
1. A preparation method and application of the soybean protein nanofiber encapsulated astaxanthin are characterized in that the soybean protein nanofiber encapsulated astaxanthin is obtained through an emulsification-evaporation method.
2. Use of the soy protein nanofiber encapsulated astaxanthin of claim 1 for the preparation of a health product for delivery of astaxanthin.
3. Use of the soy protein nanofiber encapsulated astaxanthin of claim 1 for the preparation of a food product for delivering astaxanthin.
4. The method for preparing the soy protein nanofiber encapsulated astaxanthin according to claim 1, wherein the preparation method comprises the following steps:
(1) Dissolving natural Soybean Protein Isolate (SPI) in deionized water to prepare 1-3% (w/v) SPI solution, magnetically stirring for 2h, adjusting pH to 2.0 with HCl solution, and hydrating the SPI solution overnight at 4deg.C. The next day the protein solution was heated in a 85 ℃ water bath with constant stirring for 12h. After heat treatment, the protein nanofibers were cooled in an ice water bath and the resulting samples were stored in a refrigerator at 4 ℃ or lyophilized for use.
(2) The soy protein nanofiber solution obtained in step (1) was adjusted to pH3.0 using NaOH solution. Dissolving astaxanthin in ethanol-dichloromethane solution (5:1, v/v) to obtain astaxanthin solution, gradually adding astaxanthin solution into soybean protein nanofiber solution, and emulsifying the obtained mixture solution with high speed shearing machine at 8000-10000rpm for 2min.
(3) And (3) performing rotary evaporation on the soy protein nanofiber-astaxanthin composite solution obtained in the step (2) by using a vacuum rotary evaporator to remove the organic solvent, wherein the final concentration of astaxanthin in the composite solution is 0.1-0.2mg/mL. The resulting complex solution was stored in a refrigerator at 4 ℃ or lyophilized for use.
5. The process of claim 4, wherein the isolated natural soy protein of step (1) has a protein content of 90% or more and is obtained using a HCl solution having a concentration of 1-2mol/L.
6. The process according to claim 4, wherein the concentration of NaOH solution used in the step (2) is 0.5 to 1mol/L.
7. The method according to claim 4, wherein the reduced pressure rotary evaporator in step (3) is used at a temperature of 38 to 45℃for a rotary evaporation time of 10 to 15 minutes.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110592701A (en) * | 2019-09-04 | 2019-12-20 | 东北农业大学 | Method for preparing nanofibers by self-assembly of soybean protein enzymatic hydrolysate |
| CN111328953A (en) * | 2020-03-02 | 2020-06-26 | 大连医诺生物股份有限公司 | Astaxanthin nanoemulsion with good reconstitution property and preparation method thereof |
| CN113368006A (en) * | 2021-06-10 | 2021-09-10 | 河南工业大学 | Preparation method and application of high-water-dispersion H-type or J-type aggregate astaxanthin/lactalbumin/chitosan nano compound |
| CN113812615A (en) * | 2021-08-11 | 2021-12-21 | 华南理工大学 | Water-soluble protein-based astaxanthin product and preparation method thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110592701A (en) * | 2019-09-04 | 2019-12-20 | 东北农业大学 | Method for preparing nanofibers by self-assembly of soybean protein enzymatic hydrolysate |
| CN111328953A (en) * | 2020-03-02 | 2020-06-26 | 大连医诺生物股份有限公司 | Astaxanthin nanoemulsion with good reconstitution property and preparation method thereof |
| CN113368006A (en) * | 2021-06-10 | 2021-09-10 | 河南工业大学 | Preparation method and application of high-water-dispersion H-type or J-type aggregate astaxanthin/lactalbumin/chitosan nano compound |
| CN113812615A (en) * | 2021-08-11 | 2021-12-21 | 华南理工大学 | Water-soluble protein-based astaxanthin product and preparation method thereof |
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