A polysaccharide-protein based co-assembly with high efficiency of curcumin loading and its preparation method and application

Abstract

The present invention discloses a polysaccharide-protein-based co-assembly for efficiently loading curcumin, and a preparation method and application thereof, and belongs to the technical field of functional food processing. The present invention first prepares a protein-based co-assembly loaded with curcumin by a pH cycle strategy, and then covers a pectin coating on the surface of the protein-based co-assembly by polyelectrolyte surface deposition to prepare a polysaccharide-protein-based co-assembly loaded with curcumin. The polysaccharide-protein-based co-assembly loaded with curcumin prepared by the present invention can realize efficient loading of curcumin, and can effectively improve the in vitro antioxidant activity of curcumin and the bioaccessibility after simulated gastrointestinal digestion. At the same time, the preparation process is simple and low in energy consumption, which meets the requirements of green, low-carbon and sustainable development concepts. The present invention expands the application scope of fat-soluble polyphenols such as curcumin in the field of food processing; improves the added value of tilapia protein isolate, and promotes the development of tilapia deep processing industry.

Description

Technical Field

[0001] The present invention relates to the technical field of functional food processing, and in particular to a polysaccharide-protein based co-assembly for efficiently loading curcumin, and a preparation method and application thereof. Background Art

[0002] The outbreak of the Japanese nuclear wastewater incident has attracted global attention, which has not only had a serious impact on the marine environment, but also brought severe challenges to the marine food processing industry. People have begun to pay more attention to the development and utilization of freshwater fish resources, hoping to seek alternative food sources to ensure food safety and health. Tilapia is an important freshwater fish in my country's aquatic product processing and export industry. It has the advantages of strong environmental adaptability, fast growth rate, low fat and high protein. At present, the main processed products include frozen tilapia, fish fillets and fish paste gel, but the degree of deep processing is low. This is mainly attributed to the low water solubility of tilapia protein.

[0003] As a natural compound, curcumin has a variety of biological activities that are beneficial to health. However, the bioavailability of curcumin is low, and the absorption rate after human ingestion is not high. A higher dose may be required to achieve the desired effect. At the same time, curcumin is easily affected by factors such as light, heat, and oxygen and loses its activity, reducing its efficacy. Tilapia protein isolate contains a large number of hydrophobic groups, which leads to its poor water solubility and difficulty in application. At the same time, these hydrophobic groups can also provide binding sites for fat-soluble polyphenols such as curcumin. Therefore, how to improve the stability and water solubility of tilapia protein isolate and make it a good carrier for fat-soluble polyphenols such as curcumin is a key scientific issue to improve the market application of tilapia protein isolate and curcumin.

[0004] The water solubility of poorly soluble proteins can be effectively improved by recombining hydrophilic proteins with hydrophobic proteins. The addition of hydrophilic soy protein isolate interacts with wheat alcohol-soluble proteins under pH cycle induction, thereby greatly improving the solubility of wheat alcohol-soluble proteins. At the same time, pH cycle induction can change the protein structure, providing an effective means for loading hydrophobic polyphenols. In this process, the protein can encapsulate hydrophobic molecules without relying on organic solvents and chemical denaturants, showing extremely high economy and practicality. The structure of kidney bean protein can be changed and its functional properties can be improved by pH driving, so that bioactive substances can be better embedded and their loading capacity can be improved. When the pH value of the mixed solution changes from extreme pH value to neutral pH value, the active substance molecules are buried in the hydrophobic region of the internal structure of the carrier in an amorphous form. Based on the different reactivity of different groups to different binding sites, the protein structure can meet the requirements of loading different bioactive substances through hydrogen bonding and hydrophobic attraction. However, pure protein-based carriers have the defects of low environmental stability and easy hydrolysis in the digestive tract, which causes the leakage of loaded active substances and brings challenges to the stable delivery of active substances. Using polyelectrolyte surface deposition technology to add polysaccharide coating on the surface of protein-based carriers can effectively improve the environmental stability and gastrointestinal digestion stability of the carriers, and promote the embedding and delivery of active substances. However, the research on the development of curcumin and other polyphenol loading systems using tilapia protein isolate and pectin as the substrate using a green and simple pH cycle induction strategy and polyelectrolyte surface deposition technology has not been reported. Summary of the invention

[0005] The purpose of the present invention is to provide a polysaccharide-protein based co-assembly for efficiently loading curcumin and a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art. The present invention utilizes a pH cycle induction method and a polyelectrolyte surface deposition technology to prepare a polysaccharide-protein based co-assembly for loading curcumin with both high embedding rate and loading amount.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for preparing a curcumin-loaded polysaccharide-protein based co-assembly, comprising the following steps:

[0008] Extracting tilapia protein isolate, dispersing it in water, adjusting the pH value of the solution to alkaline, stirring until the tilapia protein isolate is completely dissolved, adding curcumin and sodium caseinate, stirring to dissolve, adjusting the pH value of the solution to neutral, stirring, and obtaining a protein-based co-assembly dispersion loaded with curcumin;

[0009] The curcumin-loaded protein-based co-assembly dispersion is mixed with an equal volume of a polysaccharide solution, stirred, dialyzed, and freeze-dried to obtain a curcumin-loaded polysaccharide-protein-based co-assembly.

[0010] Preferably, the preparation method of the tilapia protein isolate is:

[0011] Crush the thawed tilapia meat, mix with distilled water, and homogenize with a high-speed disperser;

[0012] Adjust the pH value to alkaline, stir, centrifuge at low temperature, take the supernatant, and filter the supernatant through cotton gauze;

[0013] The pH value is adjusted to neutral, centrifuged at low temperature, the precipitate is mixed with water, the pH value is adjusted to neutral, dialyzed, and freeze-dried to obtain tilapia protein isolate.

[0014] Preferably, the mass ratio of the curcumin, the sodium caseinate and the tilapia protein isolate is 1:5:5.

[0015] Preferably, the polysaccharide solution is a pectin solution.

[0016] More preferably, the mass ratio of pectin to the tilapia protein isolate is (1-5):10.

[0017] The present invention also provides a polysaccharide-protein based co-assembly loaded with curcumin prepared by the above preparation method.

[0018] The present invention also provides the use of the curcumin-loaded polysaccharide-protein based co-assembly in improving the bioaccessibility of curcumin.

[0019] The present invention also provides the use of the curcumin-loaded polysaccharide-protein based co-assembly in the preparation of health food, medicine and cosmetics.

[0020] The present invention also provides an antioxidant drug, which uses the curcumin-loaded polysaccharide-protein based co-assembly as the only active ingredient.

[0021] Preferably, a pharmaceutically acceptable excipient is also included.

[0022] The present invention discloses the following technical effects:

[0023] The preparation method of the curcumin-loaded polysaccharide-protein based co-assembly proposed in the present invention has the advantages of simple process, low energy consumption, and no use of organic solvents and chemical denaturants, and meets the requirements of the green, low-carbon and sustainable development concept.

[0024] The preparation method of the polysaccharide-protein co-assembly loaded with curcumin proposed by the present invention has the advantages of high embedding rate and high loading amount, and is easy to realize industrial production. When the mass ratio of curcumin to protein is 1:10 and the amount of pectin added is 0.05g, the embedding rate and loading amount of curcumin are 97.87% and 8.90% respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 The figure is a test result diagram of the embedding efficiency and loading capacity of the protein-based co-assembly with different curcumin addition amounts; wherein the co-assembly with different curcumin to protein mass ratios is marked as C / TN=(1:x), TN represents the total mass of tilapia protein isolate and sodium caseinate, C represents curcumin, and x=10, 20, 40, 80, 100; different letters indicate that the results are significantly different;

[0027] Figure 2 The particle size, PDI and potential detection result diagrams of polysaccharide-protein based co-assemblies with different pectin addition amounts; wherein, a) is the particle size and PDI detection result diagram, and b) is the potential detection result diagram; TN represents the protein-based co-assembly loaded with curcumin, and the mass ratio of curcumin to protein is 1:10; different letters indicate that the results are significantly different;

[0028] Figure 3 The figure shows the embedding efficiency and loading capacity of curcumin in polysaccharide-protein co-assembly with different pectin addition amounts; TN represents the protein-based co-assembly loaded with curcumin, and the mass ratio of curcumin to protein is 1:10; different letters indicate significant differences in the results;

[0029] Figure 4 The DPPH free radical scavenging rate and ABTS free radical scavenging rate test results of polysaccharide-protein based co-assembly with different pectin addition amounts are shown in the figure; wherein, a) is the DPPH free radical scavenging rate test result, b) is the ABTS free radical scavenging rate test result; CUR represents free curcumin, TN represents the protein-based co-assembly loaded with curcumin, and the mass ratio of curcumin to protein is 1:10; different letters indicate that the results are significantly different;

[0030] Figure 5 The figure shows the bioaccessibility test results of curcumin after simulated gastrointestinal digestion of polysaccharide-protein based co-assemblies with different pectin addition amounts; CUR represents free curcumin, TN represents protein-based co-assemblies loaded with curcumin, and the mass ratio of curcumin to protein is 1:10; different letters indicate significant differences in the results. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] In the following examples, the method for preparing curcumin-loaded egg white protein particles by pH shifting method adopts the method used in the article pH-shifting encapsulation of curcumin in egg white protein isolate for improved dispersity, antioxidant capacity and thermal stability published in the journal Food Research International;

[0037] The invention relates to a method for preparing curcumin-loaded particles using an organic solvent and a chemical denaturant, and adopts the method used in the article Disulfide bond-breaking induced structural unfolding and assembly of soy protein acting as ananovehicle for curcumin published in the journal Innovative Food Science & Emerging Technologies.

[0038] Example 1 Preparation of a protein-based co-assembly with high efficiency of curcumin loading

[0039] 1. Tilapia protein isolate extraction:

[0040] Take a certain amount of tilapia meat and thaw it at 4°C. The thawed fish meat was minced and mixed with ice distilled water at a mass ratio of 1:9 (w / w), and homogenized by a high-speed disperser at 10000rpm for 2min. Use 1M NaOH to adjust the pH of the solution to 12, stir for 30min, and then use a centrifuge to centrifuge at 9000×g, 4°C for 20min, and take the supernatant. The supernatant was filtered through cotton gauze to remove fat, and the pH value was adjusted to 7 using 1M HCl, and centrifuged again at 9000×g, 4°C for 20min. The precipitate (tilapia protein isolate) was mixed with a small amount of ice water to disperse, and the pH value of the dispersion was adjusted to 7, and dialyzed for 48h using a 8000-14000Da dialysis bag. The dialyzed protein solution was freeze-dried into powder by a freeze dryer to obtain tilapia protein isolate.

[0041] 2. Preparation of curcumin-loaded protein-based co-assemblies induced by pH cycling:

[0042] 0.5 g of tilapia protein isolate was dissolved in 50 mL of deionized water, and the pH was adjusted to 12 using 1 M NaOH, and the mixture was stirred at 550 rpm to fully dissolve the protein; 0.01 g of curcumin and 0.5 g of sodium caseinate were added, 1.0 M NaOH was added to maintain a constant pH, and the mixture was stirred at 550 rpm for 30 min. The solution pH was adjusted to 7, and the mixture was stirred at 550 rpm for 10 min to obtain a dispersion of a protein-based co-assembly loaded with curcumin.

[0043] 3. Determine the ratio of curcumin to protein

[0044] The above experimental steps were repeated, and the addition amount of curcumin was adjusted to 0.0125 g, 0.025 g, 0.05 g, and 0.1 g, respectively, to prepare curcumin-loaded protein-based co-assemblies with different ratios of curcumin to protein.

[0045] 4. Encapsulation efficiency and loading capacity detection

[0046] The protein-based co-assemblies loaded with curcumin at different ratios of curcumin to protein were mixed with dichloromethane, vortexed for 10 seconds to extract free curcumin, and the extraction was repeated 4 times. After drying with a nitrogen evaporator, the mixture was re-dissolved in alcohol, and the free curcumin content was detected by a UV spectrophotometer to calculate the encapsulation rate and loading amount of curcumin.

[0047] The results of the embedding efficiency and loading capacity test are as follows Figure 1 As shown, considering the two indicators, the protein-based co-assembly loaded with curcumin prepared under the condition of 0.1 g of curcumin addition (the mass ratio of curcumin to protein was 1:10) was selected for subsequent experiments.

[0048] Example 2 Preparation of a polysaccharide-protein based co-assembly with high efficiency of curcumin loading

[0049] The experiment was carried out using the curcumin-loaded protein-based co-assembly prepared in Example 1 (the mass ratio of curcumin to protein was 1:10).

[0050] 1. Preparation of curcumin-loaded polysaccharide-protein based co-assemblies by polyelectrolyte surface deposition technology:

[0051] Dissolve 0.05 g of pectin in 25 mL of deionized water. Subsequently, add an equal volume of the protein-based co-assembly solution loaded with curcumin, adjust the pH to 7.10 using 0.1 M NaOH, and stir for 20 min to prepare a curcumin-loaded polysaccharide-protein-based co-assembly dispersion. The prepared dispersion was dialyzed for 24 h using a dialysis bag of 8000 to 14000 Da to remove sodium chloride. The freshly prepared polysaccharide-protein-based co-assembly dispersion can be used directly for analysis or freeze-dried into powder using a freeze dryer.

[0052] 2. Detection Method

[0053] (1) Particle size and Zeta potential detection: The sample was diluted with deionized water to a protein concentration of 1 mg / mL, and the particle size and Zeta potential were analyzed using a laser particle size analyzer.

[0054] (2) Detection of embedding efficiency and loading capacity: Take a small amount of sample and mix it with dichloromethane, vortex for 10 seconds to extract free curcumin, and repeat the extraction 4 times. After drying with a nitrogen evaporator, use alcohol to re-dissolve, and use a UV spectrophotometer to detect the free curcumin content, so as to calculate the embedding efficiency and loading capacity of curcumin.

[0055] (3) Antioxidant activity detection

[0056] ①DPPH free radical scavenging rate

[0057] The curcumin-loaded co-assembly dispersion was diluted to a suitable concentration range (20-200 μg / mL) with deionized water. 2 mL of DPPH radical ethanol solution (100 μM) was mixed with 2 mL of sample, incubated in a dark room at 25°C for 30 min, and the absorbance of the resulting solution at 517 nm was measured using an ultraviolet spectrophotometer. The DPPH radical scavenging activity of the sample was calculated by the following formula:

[0058]

[0059] Among them A 对照 and A 样品 represent the absorbance of control and sample solutions, respectively.

[0060] ②ABTS free radical scavenging rate

[0061] Mix equal volumes of ABTS solution (7mM) and potassium persulfate solution (2.45mM) into a working solution and store in the dark at 25°C for 12-16h. Dilute the ABTS working solution to an appropriate concentration with phosphate buffer (pH 7.4, 5mM). Dilute the polysaccharide-protein based co-assembly dispersion loaded with curcumin with deionized water to an appropriate concentration range (4-40μg / mL). Take 2.5mL of sample solution and mix it with 2.5mL of ABTS solution, react at room temperature for 18min, and use a UV spectrophotometer to measure the absorbance of the reaction solution at 734nm. The ABTS free radical scavenging activity of the sample is calculated by the following formula:

[0062]

[0063] Among them A 对照 and A 样品 represent the absorbance of control and sample solutions, respectively.

[0064] (4) Bioaccessibility testing

[0065] Take 5mL of sample and mix it with 45mL of HCl (0.1mol / L), shake it for 10min (37℃, 100rpm), then add 10mg of pepsin and adjust the pH to 2.0 for simulated gastric digestion. After 60min, add 250mg of bile salt, adjust the pH to 7.0, stir for 10min, add 20mg of pancreatic enzyme, and simulate intestinal digestion (SIF) for 120min. After SIF digestion, take 2mL of sample and centrifuge (2000×g, 30min), collect the supernatant, and obtain a mixed micelle phase. The digested samples before and after centrifugation were dissolved in dimethyl sulfoxide respectively, and the absorbance at 428nm was measured by ultraviolet spectrophotometer. The bioaccessibility of curcumin was calculated by the following formula:

[0066]

[0067] Among them, C 混合胶束层姜黄素浓度 and C 小肠姜黄素总浓度 are the concentration of curcumin in the mixed micelle phase and the concentration of curcumin in the small intestine, respectively.

[0068] Under this condition, the embedding efficiency of curcumin was 97.87% and the loading capacity was 8.90%, which was 22.18% and 185.26% higher than that of the egg white protein particles loaded with curcumin prepared by pH shift method.

[0069] Example 3 Preparation of a polysaccharide-protein based co-assembly with high efficiency of curcumin loading

[0070] The difference from Example 2 is that the amount of pectin used is 0.10 g.

[0071] Under this condition, the embedding efficiency of curcumin was 98.78% and the loading capacity was 8.23%, which was 23.32% and 163.78% higher than that of the egg white protein particles loaded with curcumin prepared by pH shift method.

[0072] Example 4 Preparation of a Polysaccharide-Protein Co-Assembly with High Efficiency Loading Curcumin

[0073] The difference from Example 2 is that the amount of pectin used is 0.15 g.

[0074] Under this condition, the embedding efficiency of curcumin was 98.86% and the loading capacity was 7.60%, which was 23.42% and 143.59% higher than that of the egg white protein particles loaded with curcumin prepared by pH shift method.

[0075] Example 5 Preparation of a polysaccharide-protein based co-assembly with high efficiency of curcumin loading

[0076] The difference from Example 2 is that the amount of pectin used is 0.20 g.

[0077] Under this condition, the embedding efficiency of curcumin was 99.05% and the loading amount was 7.10%, which was 23.66% and 127.56% higher than that of the egg white protein particles loaded with curcumin prepared by pH shift method.

[0078] Example 6 Preparation of a polysaccharide-protein based co-assembly with high efficiency of curcumin loading

[0079] The difference from Example 2 is that the amount of pectin used is 0.25 g.

[0080] Under this condition, the embedding efficiency of curcumin was 99.15% and the loading amount was 6.61%, which was 23.78% and 111.86% higher than that of the egg white protein particles loaded with curcumin prepared by pH shift method.

[0081] The relevant indicators of the polysaccharide-protein based co-assembly loaded with curcumin prepared in the example were tested using the detection method in Example 2. The test results are as follows: Figure 2-5 As shown. Figure 3 It can be seen that the polysaccharide-protein co-assembly loaded with curcumin prepared by the present invention has an embedding rate that increases with the increase of the amount of pectin added, and a loading amount that decreases with the increase of the amount of pectin added. Figure 4 It can be seen that the scavenging rate of the curcumin-loaded polysaccharide-protein-based co-assembly prepared by the present invention for DPPH free radicals and ABTS free radicals is significantly higher than that of free curcumin and the protein-based co-assembly loaded with curcumin, and the scavenging rate increases with the increase of the amount of pectin added. Figure 5 It can be seen that the bioaccessibility of the polysaccharide-protein-based co-assembly loaded with curcumin prepared by the present invention is significantly higher than that of free curcumin and the protein-based co-assembly loaded with curcumin, and the bioaccessibility increases with the increase of the amount of pectin added. In short, the polysaccharide-protein-based co-assembly loaded with curcumin prepared by the present invention has obvious advantages in all aspects.

[0082] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a polysaccharide-protein based co-assembly loaded with curcumin, characterized in that: The following steps are involved: Extracting tilapia protein isolate, dispersing it in water, adjusting the pH value of the solution to alkaline, stirring until the tilapia protein isolate is completely dissolved, adding curcumin and sodium caseinate, stirring to dissolve, adjusting the pH value of the solution to neutral, stirring, and obtaining a protein-based co-assembly dispersion loaded with curcumin; The protein-based co-assembly dispersion loaded with curcumin is mixed with an equal volume of a polysaccharide solution, stirred, dialyzed, and freeze-dried to obtain a polysaccharide-protein-based co-assembly loaded with curcumin; Wherein, the preparation method of the tilapia protein isolate is: Crush the thawed tilapia meat, mix with distilled water, and homogenize with a high-speed disperser; Adjust the pH value to alkaline, stir, centrifuge at low temperature, take the supernatant, and filter the supernatant through cotton gauze; Adjusting the pH value to neutral, centrifuging at low temperature, taking the precipitate and mixing it with water, adjusting the pH value to neutral, dialyzing, and freeze-drying to obtain the tilapia protein isolate; The polysaccharide solution is a pectin solution; The mass ratio of the curcumin, the sodium caseinate and the tilapia protein isolate is 1:5:5; The mass ratio of pectin to the tilapia protein isolate is (1-5):

10.

2. The curcumin-loaded polysaccharide-protein based co-assembly obtained by the preparation method according to claim 1.

3. Use of the curcumin-loaded polysaccharide-protein based co-assembly as claimed in claim 2 in improving the bioaccessibility of curcumin.

4. Use of the curcumin-loaded polysaccharide-protein based co-assembly as claimed in claim 2 in the preparation of health foods, medicines and cosmetics.

5. An antioxidant drug, characterized in that: The curcumin-loaded polysaccharide-protein based co-assembly according to claim 2 is the only active ingredient.

6. The drug according to claim 5, characterized in that Pharmaceutically acceptable excipients are also included.

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