WO2021098492A1 - 一种高荷载姜黄素的大豆多肽基纳米颗粒及其pH驱动制备方法与应用 - Google Patents

一种高荷载姜黄素的大豆多肽基纳米颗粒及其pH驱动制备方法与应用 Download PDF

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WO2021098492A1
WO2021098492A1 PCT/CN2020/125606 CN2020125606W WO2021098492A1 WO 2021098492 A1 WO2021098492 A1 WO 2021098492A1 CN 2020125606 W CN2020125606 W CN 2020125606W WO 2021098492 A1 WO2021098492 A1 WO 2021098492A1
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curcumin
soybean polypeptide
soybean
load
polypeptide
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PCT/CN2020/125606
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English (en)
French (fr)
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赵谋明
袁丹
周非白
沈鹏辉
张远红
郑淋
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华南理工大学
广州现代产业技术研究院
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Publication of WO2021098492A1 publication Critical patent/WO2021098492A1/zh

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
    • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
    • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
    • A23J3/346—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of vegetable proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
    • A23P10/00—Shaping or working of foodstuffs characterised by the products
    • A23P10/30—Encapsulation of particles, e.g. foodstuff additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5169—Proteins, e.g. albumin, gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the invention relates to a functional nano-biological product, in particular to a soybean polypeptide-based nano-particle with a high load of curcumin, and a pH-driven preparation method and application thereof.
  • Curcumin is a low-molecular-weight natural plant polyphenol compound, which has been proven to have a wide range of biological activities including anti-inflammatory, anti-oxidant, anti-tumor cell proliferation, anti-bacterial, anti-rheumatic, anti-atherosclerosis, etc.
  • curcumin has poor water solubility and unstable structure, and its low bioavailability and bioavailability in the body have severely restricted its industrial application. It is currently mainly used as a pigment in food production.
  • nanocarriers are the most widely used form of nanocarriers.
  • proteins, lipids, oligosaccharides, polysaccharides and starches can be used as wall materials for the preparation of nanoparticles for the embedding and delivery of active factors.
  • multiple hydrophobic regions formed by non-polar amino acid fragments in the polypeptide chain of proteins can be used as binding sites for hydrophobic biologically active factors, and theoretically have a higher loading capacity. Compatibility and physiological activity are more advantageous.
  • soy protein has abundant sources and low prices.
  • soy protein as a nano-carrier material can effectively improve the solubility and bioavailability of curcumin.
  • soy protein due to the compact structure of soy protein, the high content of hydrophobic amino acids, and the limited water dispersibility, its application is largely limited.
  • soybean peptides obtained by enzymatic hydrolysis of soybean protein not only have many physiological activities (anti-oxidation, anti-hypertension, anti-bacterial, enhanced mineral binding, etc.) that the protein does not have, but its water dispersibility is also significantly increased. It can be dispersed and stable in a wide pH range.
  • polypeptides are also biologically amphiphilic.
  • the hydrophobic groups existing in soybean peptides still have high reactivity, and are easy to interact with hydrophobic active factors, and have great potential as a new type of food functional factor embedding carrier.
  • the anti-solvent method is widely used.
  • the anti-solvent process consumes a large amount of organic reagents and pure water, which does not conform to the environmental protection and green production principles of the modern food industry.
  • many studies have shown that nanoparticles prepared by the anti-solvent method are prone to bridging aggregation or dissociation due to their structural properties are not stable enough, and generally require the addition of chemical reagents for secondary cross-linking.
  • the pH-driven method is to adjust the acidity and basicity of the microenvironment of the solute in the solution, so that the solute molecules interact in this process to form a new stable system.
  • the pH-driven method to load biologically active substances is a low-energy, low-cost, green, safe and efficient technology.
  • the purpose of the present invention is to provide a high-load curcumin-loaded soybean polypeptide-based nanoparticle and a pH-driven preparation method and application thereof.
  • the primary purpose of the present invention is to improve the water solubility and stability of curcumin, and to provide a method for preparing high-load curcumin-loaded soybean polypeptide-based nanoparticles by pH driving.
  • Another object of the present invention is to provide curcumin-loaded soybean polypeptide-based nanoparticles prepared by the above method.
  • Another object of the present invention is to provide the application of the curcumin-loaded soybean polypeptide-based nanoparticles.
  • the present invention provides a pH-driven method for preparing high-load curcumin-loaded soybean polypeptide-based nanoparticles, which includes the following steps:
  • Adjust step (1) The pH value of the soybean polypeptide dispersion is alkaline (preferably adjusted by NaOH solution), then add curcumin powder to the soybean polypeptide dispersion, stir evenly to obtain a mixed solution, and adjust The pH of the mixed solution is neutral (preferably adjusted by HCl solution), and the supernatant is centrifuged to obtain the high-loaded curcumin-loaded soybean polypeptide-based nanoparticles, which can be dried to obtain powdered curcumin-rich soybeans Peptide-based nano products.
  • step (1) in the process of adjusting the pH of the soy protein isolate dispersion liquid and the enzymatic hydrolysis reaction liquid, the pH of the system can be stabilized by stirring.
  • the mass-volume ratio of the soy protein isolate to water in step (1) is 1-10:100 (w/v, g/mL).
  • the mass-volume ratio of the soy protein isolate to water in step (1) is 4-10:100 (w/v, g/mL).
  • step (1) the pH of the soy protein isolate dispersion is adjusted so that the adjusted pH of the soy protein isolate dispersion is 7.0-9.0.
  • the pH of the soy protein isolate dispersion liquid is adjusted so that the adjusted pH of the soy protein isolate dispersion liquid is 7.0-8.5.
  • the stirring rate in the stirring state is 150-200 rpm
  • the hydrolase is bromelain (bro), papain (pap), alkaline protease (alc), neutral protease (neu ), one or more of flavor protease (neu), compound protease (pro) and trypsin (try)
  • the mass of the hydrolase is 0.1wt%-1wt% of the mass of the soybean protein isolate
  • the temperature is 35°C-60°C
  • the enzymolysis reaction time is 0.5-2.0h.
  • the stirring rate in the stirring state in step (1) is 160-180 rpm.
  • the mass of the hydrolase in step (1) is 0.1wt%-0.5wt% of the mass of the soybean protein isolate.
  • the temperature of the enzymatic hydrolysis reaction is 35-40°C, and further, the temperature of the enzymatic hydrolysis reaction is 35-37°C.
  • the hydrolase in step (1) is selected from one of bromelain, papain, alkaline protease, neutral protease, flavor protease and composite protease
  • the temperature of the enzymatic hydrolysis reaction is 50-60°C
  • the temperature of the enzymatic hydrolysis reaction is 50-55°C.
  • the time of the enzymatic hydrolysis reaction in step (1) is 1-2h.
  • the temperature of the enzyme inactivation treatment in step (1) is 95-100° C.
  • the time of the enzyme inactivation treatment is 10-20 min.
  • the pH of the enzymatic hydrolysis reaction solution needs to be maintained In 7.0-7.5.
  • the time for the enzyme inactivation treatment in step (1) is 10-15 min.
  • step (1) the concentration of the soybean polypeptide dispersion after dilution is 5-50 mg/L.
  • the concentration of soybean polypeptide in the soybean polypeptide dispersion in step (2) is 30-50 mg/mL. Under this polypeptide concentration condition, the loading capacity of soybean polypeptide for curcumin can be Reach a higher level.
  • step (2) in the process of adjusting the pH of the soybean polypeptide dispersion liquid and the mixed liquid, the pH of the system can be stabilized by stirring.
  • step (2) adjusting the pH value of the soybean polypeptide dispersion liquid is to make the adjusted pH of the soybean polypeptide dispersion liquid 10.0-12.0.
  • the mass ratio of the curcumin in step (2) to the soy protein isolate in step (1) is 1:100-1:5 (w/w).
  • step (2) the adjustment of the pH value of the soybean polypeptide dispersion liquid is to make the adjusted pH of the soybean polypeptide dispersion liquid 11.0-12.0.
  • the pH of the system can be stabilized by stirring for 20-40 minutes (more preferably 30-40 minutes).
  • the mass ratio of curcumin in step (2) to soy protein isolate in step (1) is 1:50-1:10 (w/w).
  • step (2) after the curcumin powder is added, it can be stirred for 30-50 min (preferably 30-40 min) to make the curcumin powder uniformly dispersed.
  • step (2) the pH of the mixed solution is adjusted to be neutral.
  • the pH of the mixed liquid is adjusted to be neutral, and the pH of the mixed liquid can be adjusted to a value of 7.0-7.5.
  • the speed of the centrifugation in step (2) is 3000-8000 g, and the time of the centrifugation is 10-20 min.
  • the centrifugation rate in step (2) is 3000-5000g.
  • the time of centrifugation in step (2) is 10-15 min.
  • the drying method in step (2) is one of freeze drying and spray drying; the temperature of the freeze drying is -40°C to -50°C, the vacuum degree of the freeze drying is less than 1 mbar, and the freeze drying time It is 20-30 hours; the inlet air temperature of the spray drying is 160-180°C, and the exhaust air temperature of the spray drying is 70-90°C.
  • the freeze-drying time is 20-25h.
  • the inlet air temperature of the spray drying is 170-180°C
  • the exhaust air temperature of the spray drying is 80-90°C.
  • the present invention provides a high-load curcumin soybean polypeptide-based nanoparticle prepared by the above-mentioned pH-driven preparation method.
  • the high-load curcumin-loaded soybean polypeptide-based nanoparticle provided by the present invention is a composite obtained by embedding curcumin with soybean polypeptide as a wall material, and its average particle size is less than 150 nm, and the curcumin loading amount is 5-90mg/g , The curcumin loading amount can reach about 90 mg/g soybean peptide.
  • the high-load curcumin-loaded soybean polypeptide-based nanoparticles provided by the present invention can be used in the preparation of anti-inflammatory and antioxidant foods or medicines.
  • Curcumin is a low-molecular-weight natural plant polyphenol compound, which has been proven to have a wide range of biological activities including anti-inflammatory, anti-oxidant, anti-tumor cell proliferation, anti-bacterial, anti-rheumatic, anti-atherosclerosis, etc. As the most widely used plant protein produced and used in the food industry, soy protein has abundant sources and low prices.
  • the high-load curcumin-loaded soybean polypeptide-based nanoparticles prepared by the present invention can realize the delivery and directional release of curcumin, and can not only be directly used in the research and development of functional foods, but also can be used as a new type of functionality with high nutritional value and physiological activity. Food ingredients are added to the food system.
  • the invention discloses a soybean polypeptide-based nanoparticle with high load of curcumin and a pH-driven preparation method and application thereof.
  • the present invention uses soybean protein isolate as raw material to obtain soybean polypeptide by enzymatic hydrolysis, uses soybean polypeptide as wall material, and utilizes the solubility characteristics of curcumin under specific pH conditions to prepare a high-load curcumin-based soybean polypeptide-based nanometer through molecular self-assembly technology Particles.
  • the preparation method provided by the present invention includes: enzymatically hydrolyze soybean protein isolate to obtain soybean polypeptide, adjust the pH to make it stable in an alkaline environment, add curcumin powder, stir for a certain period of time and then adjust the pH to neutral, and collect the supernatant after centrifugation Then, the curcumin-rich soybean polypeptide-based nano particles can be obtained, and the powdered curcumin-rich soybean polypeptide-based nano products can be obtained after drying.
  • the prepared nanoparticles have a nanometer size, with an average particle size less than 150 nm, the maximum curcumin loading can reach about 90 mg/g soybean peptides.
  • the nanoparticles prepared by the present invention have the advantages of good stability, high loading rate and loading capacity, and good biocompatibility; and the preparation process does not involve organic reagents such as alcohols, and is safe and non-toxic and side effects , The process is simple to operate and can be produced on a large scale.
  • the present invention has the following advantages and beneficial effects:
  • the pH-driven preparation method of high-load curcumin-loaded soybean peptide-based nanoparticles uses soybean polypeptide as a carrier material to embed curcumin for the first time to form high-load curcumin-loaded soybean peptide-based nanoparticles. It can improve the water solubility, stability and bioavailability of curcumin.
  • the pH-driven preparation method of high-load curcumin-loaded soybean polypeptide-based nanoparticles involves simple preparation steps, easy industrial production, and the preparation process does not involve organic reagents such as alcohols, and is green, safe, and non-toxic and side effects.
  • Example 1 is a diagram showing the particle size distribution of 7 parts of high-load curcumin-loaded soybean polypeptide-based nanoparticles prepared in Example 1 of the present invention
  • Example 2 is a diagram showing the particle size distribution of high-load curcumin-loaded soybean polypeptide-based nanoparticles prepared in Example 2 of the present invention
  • Example 3 is a diagram showing the particle size distribution of high-load curcumin-based soybean polypeptide-based nanoparticles prepared in Example 3 of the present invention
  • Example 4 is a transmission electron micrograph of high-load curcumin-based soybean polypeptide-based nanoparticles prepared in Example 3 of the present invention.
  • Fig. 5 is a diagram showing the particle size distribution of high-load curcumin-loaded soybean polypeptide-based nanoparticles prepared in Example 4 of the present invention.
  • weight unit can be grams, kilograms, etc., and can also be any other amount commonly used in the art.
  • the binding rate of curcumin and the loading amount of curcumin can be calculated by the following formula:
  • a pH-driven preparation method of high-load curcumin-loaded soybean polypeptide-based nanoparticles includes the following steps:
  • soy protein isolate dispersion (1) 7 parts of soy protein isolate were added to 7 parts of water and mixed uniformly to obtain 7 parts of soy protein isolate dispersion.
  • the mass volume percentage concentration of the soy protein isolate dispersion was 4% (w/v, g/ mL), adjust the pH of 7 parts of soy protein isolate dispersion to 8.5, and add bromelain (bro), papain (pap) and alkali to 7 parts of soy protein isolate dispersion under stirring at 180rpm.
  • the hydrolysis reaction takes 2 hours, but the enzymatic hydrolysis reaction temperature is different.
  • the enzymatic hydrolysis reaction temperature of the soybean protein dispersion solution with trypsin is 35°C, and the rest are 55°C; after the enzymolysis is completed, adjust 7 parts of the enzymatic hydrolysis reaction solution.
  • the pH value is 7.0, and the enzyme inactivation treatment is carried out at 95°C.
  • the enzyme inactivation treatment time is 10 minutes.
  • soybean polypeptide dispersion After dilution, 7 parts of soybean polypeptide dispersion are obtained, and the concentration of 7 parts of soybean polypeptide dispersion is 30 mg/ml;
  • the 7 soybean peptide dispersions were named SPI bro , SPI pap , SPI alc , SPI neu , SPI fla , SPI pro , and SPI try ;
  • the average particle size and the polymer dispersibility index (PDI) data of the 7 high-load curcumin-loaded soybean polypeptide-based nanoparticles prepared in Example 1 were measured by a Malvern Nano-ZS, and based on the curcumin content The binding rate and loading data of curcumin were calculated, and the results are shown in Table 1 below.
  • PDI polymer dispersibility index
  • Fig. 1 is a diagram showing the particle size distribution of 7 kinds of nanoparticles (highly loaded curcumin-based soy polypeptide-based nanoparticles) prepared by soybean polypeptide and curcumin in Example 1. It can be seen from Table 1 and Figure 1 that the average particle size of the 7 high-load curcumin-loaded soybean polypeptide-based nanoparticles prepared in Example 1 is 60-110 nm, and the PDI is all less than 0.3.
  • the high-load curcumin-loaded soybean peptide-based nanoparticles SPI neu and the high-load curcumin-loaded soybean peptide-based nanoparticles SPI fla have a relatively low loading amount of soy peptides to curcumin, and the rest of the high-load curcumin-loaded soybean peptides In the base nanoparticles, the amount of curcumin loaded by soybean peptides is not much different.
  • a pH-driven preparation method of high-load curcumin-loaded soybean polypeptide-based nanoparticles which specifically includes the following steps:
  • the average particle size of the prepared curcumin-loaded soybean peptide-based nanoparticles, PDI data, and the binding rate and loading data of curcumin calculated based on the curcumin content were measured by the Malvern Nano-ZS, see below Table 2.
  • curcumin: soy protein isolate represents the mass ratio of curcumin powder in step (2) to soy protein isolate in step (1), so the ratios in Table 2 are 1:50, 1:20, and 1:5 They are respectively expressed as high-load curcumin soybean polypeptide-based nanoparticles prepared by adding curcumin powders of different qualities in step (2) in Example 2.
  • FIG. 2 is a diagram of the particle size distribution of the curcumin-loaded soy polypeptide SPI neu obtained by the enzymatic hydrolysis of 3 neutral protease in Example 2 of the present invention .
  • SPI neu Expressed as soybean peptides obtained by neutral protease, Cur represents curcumin, 2% Cur, 5% Cur, and 10% Cur respectively represent high-load curcumin-based soybean peptide-based nanoparticles prepared by adding curcumin powder of different quality.
  • 2%, 5%, and 10% represent that the mass ratio of the curcumin powder to the soy protein isolate in step (1) is 1:50, 1:20, and 1:5, respectively.
  • a pH-driven preparation method of high-load curcumin-loaded soybean polypeptide-based nanoparticles which specifically includes the following steps:
  • the quality of each part of the hydrolase is 0.1% (w/w) of the quality of each part of soybean protein isolate
  • the temperature of the enzymatic hydrolysis reaction is 37°C
  • the time of the enzymatic hydrolysis reaction is 0.5h
  • the concentration of the peptide dispersion is 20mg/mL;
  • the average particle size and PDI data of the prepared curcumin-loaded soybean peptide-based nanoparticles were measured with a Malvern Nano-ZS, and the binding rate and loading data of curcumin calculated based on the curcumin content are shown below table 3.
  • Figure 3 is the particle size distribution diagrams of 3 high-load curcumin-loaded soybean polypeptide-based nanoparticles prepared in Example 3; SPI try represents the soybean polypeptide obtained by trypsin, Cur represents curcumin, 2% Cur, 5 %Cur and 10%Cur respectively represent high-load curcumin-loaded soybean peptide-based nanoparticles prepared by adding curcumin powders of different qualities, wherein 2%, 5%, and 10% represent the curcumin powder and step (1 )
  • the mass ratios of the soybean protein isolate are 1:50, 1:20, and 1:5, respectively. It can be seen from Fig.
  • a pH-driven preparation method of high-load curcumin-loaded soybean polypeptide-based nanoparticles which specifically includes the following steps:
  • the time of enzymatic inactivation treatment is 20min. Dilute them to obtain 4 parts of soybean polypeptide dispersion, 4 parts The concentrations of the soybean polypeptide dispersion are 5 mg/mL, 10 mg/mL, 20 mg/mL, and 50 mg/mL;
  • the average particle size and PDI data of the prepared high-loaded curcumin-loaded soybean peptide-based nanoparticles were measured with a Malvern Nano-ZS, and the binding rate and loading data of curcumin calculated based on the curcumin content, see Table 4 below.
  • Soy peptide dispersion (mg/ml) Average particle size (nm) Polymer Dispersibility Index Combination rate (%) Load capacity (mg/g) 5 72.05 0.28 59.16 59.16 10 68.26 0.27 62.23 62.23 20 80.69 0.25 64.35 64.35 50 90.68 0.26 89.17 89.17
  • Figure 5 is a diagram showing the particle size distribution of high-load curcumin-loaded soybean polypeptide-based nanoparticles prepared in Example 4; 5 mg/mL, 10 mg/mL, 20 mg/mL, and 30 mg/mL in Figure 5 are represented by the respective concentrations. Soy polypeptide-based nanoparticles with high loading of curcumin prepared from 5mg/mL, 10mg/mL, 20mg/mL and 50mg/Ml soybean polypeptide dispersions.
  • soybean polypeptide-curcumin nanoparticle (high-load curcumin-loaded soybean polypeptide-based nanoparticle) product prepared by the invention has the advantages of high loading, small particle size, good storage stability and the like.
  • the poor water solubility of curcumin limits its bioavailability, while soybean peptides have a small molecular weight, good water solubility, and have certain physiological activities.
  • soybean peptides are used to embed them to form nanoparticles, which not only increases curcumin The solubility and also have higher physiological activity.
  • the technical method of the present invention can be used to produce curcumin-rich functional foods;
  • the pH-driven method adopted in the present invention is green and safe, has low energy consumption, and has greater advantages than traditional anti-solvent methods.
  • Advantages and applicability; the soybean polypeptide prepared in the present invention can effectively increase the loading of hydrophobic physiologically active substances, and will have a wide range of applications in the production fields of food, medicine, cosmetics and the like.
  • the preparation method provided by the present invention the raw materials are natural and healthy, the preparation process is green and safe, the energy consumption is low, the process operation is simple, and the rapid and continuous production can be carried out. Value.

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Abstract

一种高荷载姜黄素的大豆多肽基纳米颗粒及其pH驱动制备方法与应用。该方法包括：酶解大豆分离蛋白得到大豆多肽，调节pH使其在碱性环境中稳定后，加入姜黄素粉末，搅拌一定时间再回调pH至中性，离心后收集上清液即可得到富载姜黄素的大豆多肽基纳米颗粒，经干燥处理可得粉末状富载姜黄素的大豆多肽基纳米制品。所制备出的纳米颗粒具有纳米级尺寸，平均粒径小于150nm，姜黄素荷载量最高可达到约90mg/g大豆多肽。与传统的制备方法相比，所述方法制备的纳米颗粒具有稳定性好，包载率及荷载量高、生物相容性好等优点；且制备过程未涉及醇类等有机试剂，安全无毒副作用，工艺操作简单，可规模化生产。

Description

一种高荷载姜黄素的大豆多肽基纳米颗粒及其pH驱动制备方法与应用 技术领域
本发明涉及功能性纳米生物制品，具体涉及一种高荷载姜黄素的大豆多肽基纳米颗粒及其pH驱动制备方法与应用。
 背景技术
现代食品工业越来越注重食品对人类营养及健康改善的功能，大量功能活性因子被应用于各类功能性食品的研发。但食源性功能因子如疏水性植物多酚等多存在难溶于水、稳定性差、吸收利用率低等问题，是功能性食品开发应用亟待解决的技术难题。姜黄素（Curcumin）是一种低分子量的天然植物多酚化合物，已证实其具有广泛的生物活性包括抗炎、抗氧化、抗肿瘤细胞增殖、抗菌、抗风湿、抗动脉粥样硬化等。但是姜黄素水溶性差且结构不稳定，在体内生物利用率和生物效价低，严重制约了其产业化应用，目前主要作为色素用于食品生产中。
随着纳米技术的发展，通过载体包埋可有效实现活性因子的稳态化，其中纳米颗粒是应用最广泛的纳米载体形式。许多研究表明，蛋白质、脂类、低聚糖、多糖以及淀粉质等均可作为制备纳米颗粒的壁材，用于活性因子的包埋输送。相比于其他生物大分子，蛋白质的多肽链中非极性氨基酸片段形成的多个疏水区域均可作为疏水性生物活性因子的结合位点，理论上具有更高的荷载量，同时其在生物相容性及生理活性方面更具优势。大豆蛋白作为现今食品工业生产和利用最广泛的植物蛋白，其来源丰富，价格低廉。已有研究发现利用大豆蛋白作为纳米载体材料可以有效提高姜黄素的溶解度和生物利用率。然而由于大豆蛋白结构致密，疏水性氨基酸含量高，水分散性有限，很大程度上限制了它的应用。
通过生物酶法制备具有健康益处的蛋白多肽因其来源广泛、生物相容性好、功能多样、安全性高，已逐渐成为现代功能性食品开发的重要途径。相关研究表明，大豆蛋白经酶解处理后得到的大豆多肽不仅具有蛋白所不具备的诸多生理活性（抗氧化、抗高血压、抗菌、增强矿物质结合等），其水分散性也明显增加，在较宽的pH值范围内均可分散且稳定。同时，作为介于氨基酸和蛋白质之间的一类化合物，多肽还具有生物两亲性。大豆多肽中存在的疏水基团仍具有较高的反应活性，容易和疏水性活性因子发生相互作用，具有作为新型食品功能因子包埋载体的巨大潜力。
基于姜黄素等活性因子在有机溶剂中的溶解特性，为了提高其包埋率，反溶剂法被广泛应用。但是反溶剂过程中消耗大量的有机试剂以及纯水，不符合现代食品工业环保绿色的生产原则。此外，许多研究表明经反溶剂法制得的纳米颗粒，容易发生因本身结构性质不够稳定导致的桥连聚集或解离，一般还需通过加入化学试剂来进行二次交联。pH驱动法是通过调节溶液中溶质所处微环境的酸碱性，使溶质分子在这一过程中发生相互作用形成新的稳定体系。与传统反溶剂等荷载方法相比，通过pH驱动法来荷载生物活性物质是一种低耗能、低成本、绿色安全高效的技术。
基于上述大豆多肽的优异性质和pH驱动法的绿色高效，探讨利用pH驱动制备高荷载生物活性物质的大豆多肽基纳米颗粒具有强大的开发前景和应用价值。
 技术解决方案
为了克服现有技术存在的上述不足，本发明的目的是提供一种高荷载姜黄素的大豆多肽基纳米颗粒及其pH驱动制备方法与应用。
本发明的首要目的是为了提高姜黄素的水溶解度和稳定性，提供一种通过pH驱动制备高荷载姜黄素的大豆多肽基纳米颗粒的方法。
本发明的另一目的在于提供由上述方法制得的荷载姜黄素的大豆多肽基纳米颗粒。
本发明的再一目的在于提供上述荷载姜黄素的大豆多肽基纳米颗粒的应用。
本发明的目的至少通过如下技术方案之一实现。
本发明提供的一种pH驱动制备高荷载姜黄素的大豆多肽基纳米颗粒的方法，包括以下步骤：
（1）将大豆分离蛋白加入水中，混合均匀，得到大豆分离蛋白分散液，调节大豆分离蛋白分散液的pH为中性或碱性（优先为7.0-9.0），在搅拌状态下加入水解酶进行酶解反应，酶解完成后调节酶解反应液的pH为7.0-7.5，接着进行灭酶处理，稀释得到所需浓度的大豆多肽分散液；
（2）调节步骤（1）所述大豆多肽分散液的pH值为碱性（优选NaOH溶液来调节），然后往所述大豆多肽分散液中加入姜黄素粉末，搅拌均匀，得到混合液，调节所述混合液的pH为中性（优选HCl溶液来调节），离心取上清液，得到所述高荷载姜黄素的大豆多肽基纳米颗粒，经干燥处理可得粉末状富载姜黄素的大豆多肽基纳米制品。
进一步地，步骤（1）中，在调节所述大豆分离蛋白分散液及酶解反应液的pH过程中，可以通过搅拌使体系的pH稳定。
进一步地，步骤（1）所述大豆分离蛋白与水的质量体积比为1-10:100（w/v，g/mL）。
优选地，步骤（1）所述大豆分离蛋白与水的质量体积比为4-10:100（w/v，g/mL）。
进一步地，步骤（1）中，调节大豆分离蛋白分散液的pH，使调节后的大豆分离蛋白分散液的pH为7.0-9.0。
优选地，所述调节大豆分离蛋白分散液的pH，使调节后的大豆分离蛋白分散液的pH为7.0-8.5。
进一步地，步骤（1）所述在搅拌状态下的搅拌速率为150-200rpm，所述水解酶为菠萝蛋白酶（bro）、木瓜蛋白酶（pap）、碱性蛋白酶（alc）、中性蛋白酶（neu）、风味蛋白酶（neu）、复合蛋白酶（pro）及胰酶（try）中的一种以上，所述水解酶的质量为大豆分离蛋白质量的0.1wt%-1wt%；所述酶解反应的温度为35℃-60℃，所述酶解反应的时间为0.5-2.0h。
优选地，步骤（1）所述在搅拌状态下的搅拌速率为160-180rpm。
优选地，步骤（1）所述水解酶的质量为大豆分离蛋白质量的0.1wt%-0.5wt%。
优选地，步骤（1）所述水解酶选用胰酶时，所述酶解反应的温度为35-40℃，更进一步地，所述酶解反应的温度为35-37℃。
优选地，步骤（1）所述水解酶选用菠萝蛋白酶、木瓜蛋白酶、碱性蛋白酶、中性蛋白酶、风味蛋白酶及复合蛋白酶中的一种时，所述酶解反应的温度为50-60℃，更进一步地，所述酶解反应的温度为50-55℃。
优选地，步骤（1）所述酶解反应的时间为1-2h。
进一步地，步骤（1）所述灭酶处理的温度为95-100℃，所述灭酶处理的时间为10-20min，在所述灭酶处理中，所述酶解反应液的pH需维持在7.0-7.5。
优选地，步骤（1）所述灭酶处理的时间为10-15min。
进一步地，步骤（1）中，稀释以后的大豆多肽分散液的浓度为5-50mg/L。
优选地，在步骤（1）稀释后，步骤（2）所述大豆多肽分散液中，大豆多肽的浓度为30-50mg/mL，在此多肽浓度条件下，大豆多肽对于姜黄素的荷载量能达到较高的水平。
进一步地，步骤（2）中，在调节所述大豆多肽分散液及所述混合液的pH过程中，可以通过搅拌使体系的pH稳定。
进一步地，步骤（2）中，调节所述大豆多肽分散液的pH值，是使调节后的大豆多肽分散液的pH为10.0-12.0。
进一步地，步骤（2）所述姜黄素与步骤（1）所述大豆分离蛋白的质量比为1:100-1:5（w/w）。
优选地，步骤（2）中，所述调节所述大豆多肽分散液的pH值，是使调节后的大豆多肽分散液的pH为11.0-12.0。
优选地，在步骤（2）调节述大豆多肽分散液的pH值为碱性过程中，可以通过搅拌20-40min（进一步优选为30-40min），使得体系的pH稳定。
优选地，步骤（2）姜黄素 与步骤（1）所述大豆分离蛋白的质量比为1:50-1:10（w/w）。
优选地，步骤（2）中，加入姜黄素粉末后，可以搅拌30-50min（优选为30-40min），使得姜黄素粉末分散均匀。
进一步地，步骤（2）中调节所述混合液的pH为中性。
优选地，步骤（2）所述调节所述混合液的pH为中性，可以调节混合液的pH值为7.0-7.5。
进一步地，步骤（2）所述离心的速率为3000-8000g，所述离心的时间为10-20min。
优选地，步骤（2）所述离心速率为3000-5000g。
优选地，步骤（2）所述离心的时间为10-15min。
进一步地，步骤（2）所述干燥的方式为冷冻干燥及喷雾干燥中的一种；所述冷冻干燥的温度为-40℃～-50℃，冷冻干燥的真空度小于1mbar，冷冻干燥的时间为20-30h；所述喷雾干燥的进风温度为160-180℃，喷雾干燥的排风温度为70-90℃。
优选地，步骤（2）干燥的方式选用冷冻干燥的方式时，所述冷冻干燥的时间为20-25h。
优选地，步骤（2）干燥的方式选用喷雾干燥的方式时，所述喷雾干燥的进风温度为170-180℃，所述喷雾干燥的排风温度为80-90℃。
本发明提供一种由上述的pH驱动制备方法制得的高荷载姜黄素的大豆多肽基纳米颗粒。
本发明提供的高荷载姜黄素的大豆多肽基纳米颗粒，是由大豆多肽作为壁材包埋了姜黄素得到的复合物，其平均粒径小于150 nm，姜黄素荷载量为5-90mg/g，姜黄素荷载量最高可达到约90 mg/g 大豆多肽。
本发明提供的高荷载姜黄素的大豆多肽基纳米颗粒能够应用在制备抗炎抗氧化的食品或药品中。
姜黄素（Curcumin）是一种低分子量的天然植物多酚化合物，已证实其具有广泛的生物活性包括抗炎、抗氧化、抗肿瘤细胞增殖、抗菌、抗风湿、抗动脉粥样硬化等。大豆蛋白作为现今食品工业生产和利用最广泛的植物蛋白，其来源丰富，价格低廉。
本发明制备的高荷载姜黄素的大豆多肽基纳米颗粒可实现对姜黄素的输送和定向释放，不仅可直接用于功能性食品的研发，也可作为具有高营养价值及生理活性的新型功能性食品配料添加到食品体系中。
本发明公开了一种高荷载姜黄素的大豆多肽基纳米颗粒及其pH驱动制备方法与应用。本发明以大豆分离蛋白为原料酶解得到大豆多肽，将大豆多肽作为壁材，利用姜黄素在特定pH条件下的溶解特性，通过分子自组装技术制备一种高荷载姜黄素的大豆多肽基纳米颗粒。
本发明提供的制备方法包括：酶解大豆分离蛋白得到大豆多肽，调节pH使其在碱性环境中稳定后，加入姜黄素粉末，搅拌一定时间再回调pH至中性，离心后收集上清液即可得到富载姜黄素的大豆多肽基纳米颗粒，经干燥处理可得粉末状富载姜黄素的大豆多肽基纳米制品。所制备出的纳米颗粒具有纳米级尺寸，平均粒径小于150 nm，姜黄素荷载量最高可达到约90 mg/g大豆多肽。
 有益效果
与传统的制备方法相比，本发明制备的纳米颗粒具有稳定性好，包载率及荷载量高、生物相容性好等优点；且制备过程未涉及醇类等有机试剂，安全无毒副作用，工艺操作简单，可规模化生产。
与现有技术相比，本发明具有如下优点和有益效果：
（1）本发明提供的高荷载姜黄素的大豆多肽基纳米颗粒的pH驱动制备方法，首次利用大豆多肽作为载体材料对姜黄素进行包埋，形成高荷载姜黄素的大豆多肽基纳米颗粒，可以很好的提高姜黄素的水溶性、稳定性和生物利用率。
（2）本发明提供的高荷载姜黄素的大豆多肽基纳米颗粒的pH驱动制备方法，涉及的制备步骤简单，易工业化生产，且制备过程未涉及醇类等有机试剂，绿色安全无毒副作用。
 附图说明
图1为本发明实施例1制得的中7份高荷载姜黄素的大豆多肽基纳米颗粒的粒径分布图；
图2为本发明实施例2制得的高荷载姜黄素的大豆多肽基纳米颗粒的粒径分布图；
图3为本发明实施案例3制得的高荷载姜黄素的大豆多肽基纳米颗粒的粒径分布图；
图4为本发明实施案例3制得的高荷载姜黄素的大豆多肽基纳米颗粒的透射电镜图；
图5为本发明实施例4制得的高荷载姜黄素的大豆多肽基纳米颗粒的粒径分布图。
 本发明的实施方式
以下结合实例对本发明的具体实施作进一步说明，但本发明的实施和保护不限于此。需指出的是，以下若有未特别详细说明之过程，均是本领域技术人员可参照现有技术实现或理解的。所用试剂或仪器未注明生产厂商者，视为可以通过市售购买得到的常规产品。
以下实施例及对比例所用到的重量（质量）份数，作为举例，重量单位可以为克、千克等，也可以是本领域常用的任意其他用量。
以下的实施例中，姜黄素的结合率和姜黄素的荷载量可通过下面的公式计算得到：
Figure dest_path_image001
 （1） ；
Figure 182461dest_path_image002
 （2） 。
实施例1
一种高荷载姜黄素的大豆多肽基纳米颗粒的pH驱动制备方法，包括以下步骤：
（1）分别将7份大豆分离蛋白加入7份水中，混合均匀，得到7份大豆分离蛋白分散液，所述大豆分离蛋白分散液的质量体积百分比浓度均为4%（w/v，g/mL），分别调节7份大豆分离蛋白分散液的pH为8.5，在转速为180rpm的搅拌状态下，往7份大豆分离蛋白分散液中分别加入菠萝蛋白酶（bro）、木瓜蛋白酶（pap）、碱性蛋白酶（alc）、中性蛋白酶（neu）、风味蛋白酶（fla）、复合蛋白酶（pro）、胰酶（try），然后在转速为180rpm的搅拌状态下下，进行酶解反应；其中，酶解反应均为2小时，但酶解反应温度不同，加入胰酶的大豆蛋白分散液的酶解反应温度为35℃，余者均为55℃；酶解完成后分别调节7份酶解反应液的pH值为7.0，然后分别在95℃条件下进行灭酶处理，灭酶处理的时间为10min，稀释得到7份大豆多肽分散液，7份大豆多肽分散液的浓度均为30 mg/ml；将这7份大豆多肽分散液分别命名为SPI bro、SPI pap、SPI alc、SPI neu、SPI fla、SPI pro、SPI try；
（2）分别调节步骤（1）所述7份大豆多肽分散液的pH值为10.0，在调节pH过程中，搅拌30min以使体系的pH稳定，然后往7份所述大豆多肽分散液中分别加入姜黄素粉末，所述姜黄素粉末与步骤（1）所述大豆分离蛋白的质量比均为1:100（w/w），搅拌均匀，得到7份混合液，分别调节7份所述混合液的pH为中性（即pH为7.0），离心取上清液（离心时间为15min，离心速率为8000g），得到7份上清液，采用纳米粒度仪（Malvern Nano-ZS）分别测定7份上清液中的纳米颗粒的粒径、聚合物分散性指数（PDI）及姜黄素含量；冷冻干燥（冷冻干燥的温度为-40℃，冷冻干燥的真空度小于1mbar，冷冻干燥的时间为20h）得到7份所述高荷载姜黄素的大豆多肽基纳米颗粒粉末制品；这7份高荷载姜黄素的大豆多肽基纳米颗粒分别命名为高荷载姜黄素的大豆多肽基纳米颗粒SPI bro、高荷载姜黄素的大豆多肽基纳米颗粒SPI pap、高荷载姜黄素的大豆多肽基纳米颗粒SPI alc、高荷载姜黄素的大豆多肽基纳米颗粒SPI neu、高荷载姜黄素的大豆多肽基纳米颗粒SPI fla、高荷载姜黄素的大豆多肽基纳米颗粒SPI pro及高荷载姜黄素的大豆多肽基纳米颗粒SPI try。
采用纳米粒度仪（Malvern Nano-ZS）测定实施例1制得的7份高荷载姜黄素的大豆多肽基纳米颗粒的平均粒径、聚合物分散性指数（PDI）的数据，并根据姜黄素含量计算出姜黄素的结合率及荷载量的数据，结果见下表1所示。
表1
高荷载姜黄素的大豆多肽基纳米颗粒 平均粒径（nm） 聚合物分散性指数 结合率（%） 荷载量（mg/g）
SPI bro 106.55 0.26 74.70 7.47
SPI pap 98.405 0.27 73.30 7.33
SPI alc 99.62 0.28 76.70 7.67
SPI neu 68.74 0.28 68.10 6.81
SPI fla 79.42 0.25 59.70 5.97
SPI pro 89.755 0.24 81.60 8.16
SPI try 59.14 0.29 78.20 7.82
图1是实施例1中大豆多肽与姜黄素制备得到的7种纳米颗粒（高荷载姜黄素的大豆多肽基纳米颗粒）粒径分布图。由表1和图1可知，实施例1制得的7种高荷载姜黄素的大豆多肽基纳米颗粒的平均粒径大小为60-110 nm，PDI均小于0.3。其中， 高荷载姜黄素的大豆多肽基纳米颗粒SPI neu与高荷载姜黄素的大豆多肽基纳米颗粒SPI fla中的大豆多肽对于姜黄素的荷载量相对较低，其余的高荷载姜黄素的大豆多肽基纳米颗粒中，大豆多肽荷载姜黄素的量相差不大。
实施例2
一种高荷载姜黄素的大豆多肽基纳米颗粒的pH驱动制备方法，具体包括以下步骤：
（1）将3份大豆分离蛋白加入3份水中，混合均匀，得到3份大豆分离蛋白分散液，3份所述大豆分离蛋白分散液的质量体积百分比浓度均为2.0%（w/v，g/mL）；分别调节3份大豆分离蛋白分散液的pH为7.0，在转速为150rpm的搅拌状态下分别往3份大豆分离蛋白分散液中加入3份水解酶进行酶解反应（此处选用中性蛋白酶neu），每一份所述水解酶的质量均为每一份大豆分离蛋白质量的0.1%（w/w），酶解反应的温度为50℃，酶解反应的时间为0.5h，酶解完成后调节3份酶解反应液的pH为7.5，接着在98℃的条件下进行灭酶处理，灭酶处理的时间为15min，稀释得到3份大豆多肽分散液，3份所述大豆多肽分散液的浓度均为20mg/mL；
（2）分别调节步骤（1）所述3份大豆多肽分散液的pH值为11.0，在调节pH过程中，搅拌20min以使体系的pH稳定，然后往3份所述大豆多肽分散液中分别加入3份质量不同的姜黄素粉末，所述姜黄素粉末与步骤（1）所述大豆分离蛋白的质量比分别为1：50、1：20及1：5；搅拌均匀，得到3份混合液（3份混合液中含有的姜黄素粉末质量均不同），分别调节3份所述混合液的pH为中性，分别离心取上清液（离心的速率为3000g，离心的时间为10min），得到3份上清液，采用纳米粒度仪（Malvern Nano-ZS）分别测定3份上清液中的纳米颗粒的粒径、聚合物分散性指数（PDI）及姜黄素含量；然后干燥（采用喷雾干燥，所述喷雾干燥的进风温度为160℃，排风温度为70℃）得到3份所述高荷载姜黄素的大豆多肽基纳米颗粒粉末制品。
采用纳米粒度仪（Malvern Nano-ZS）测定制备的荷载姜黄素的大豆多肽基纳米颗粒的平均粒径、PDI的数据以及根据姜黄素含量计算出的姜黄素的结合率及荷载量数据，见下表2。
表2
Figure dest_path_image004a
表2中，姜黄素：大豆分离蛋白表示步骤（2）所述姜黄素粉末与步骤（1）所述大豆分离蛋白的质量比，因此表2中的1：50、1：20及1：5分别表示为实施例2中步骤（2）加入不同质量的姜黄素粉末制得的高荷载姜黄素的大豆多肽基纳米颗粒。
图2为本发明实施案例2提供的3份中性蛋白酶酶解得到的大豆多肽SPI neu荷载姜黄素形成的纳米颗粒（高荷载姜黄素的大豆多肽基纳米颗粒）的粒径分布图， SPI neu表示为利用中性蛋白酶得到的大豆多肽，Cur表示姜黄素，2%Cur、5%Cur及10%Cur分别表示加入不同质量的姜黄素粉末制得的高荷载姜黄素的大豆多肽基纳米颗粒，其中，2%、5%及10%表示为所述姜黄素粉末与步骤（1）所述大豆分离蛋白的质量比分别为1：50、1：20及1：5。由图2以及表2可知，随着姜黄素浓度的增加，所述高荷载姜黄素的大豆多肽基纳米颗粒的粒径、PDI无明显变化，体系均呈现较好的稳定性。此外，随着姜黄素浓度的增加，姜黄素的荷载量也逐渐增加。
实施例3
一种高荷载姜黄素的大豆多肽基纳米颗粒的pH驱动制备方法，具体包括以下步骤：
（1）将3份大豆分离蛋白加入3份水中，混合均匀，得到3份大豆分离蛋白分散液，3份所述大豆分离蛋白分散液的质量体积百分比浓度均为2.0%（w/v，g/mL）；分别调节3份大豆分离蛋白分散液的pH为7.0，在转速为150rpm的搅拌状态下分别往3份大豆分离蛋白分散液中加入3份水解酶进行酶解反应（此处选用胰酶try），每一份所述水解酶的质量均为每一份大豆分离蛋白质量的0.1%（w/w），酶解反应的温度为37℃，酶解反应的时间为0.5h，酶解完成后分别调节3份酶解反应液的pH为7.5，接着在98℃的条件下进行灭酶处理，灭酶处理的时间为15min，稀释得到3份大豆多肽分散液，3份所述大豆多肽分散液的浓度均为20mg/mL；
（2）分别调节步骤（1）所述3份大豆多肽分散液的pH值为11.0，在调节pH过程中，搅拌20min以使体系的pH稳定，然后往3份所述大豆多肽分散液中分别加入3份质量不同的姜黄素粉末，所述姜黄素粉末与步骤（1）所述大豆分离蛋白的质量比分别为1：50、1：20及1：5；搅拌均匀，得到3份混合液（3份混合液中含有的姜黄素粉末质量均不同），分别调节3份所述混合液的pH为中性，分别离心取上清液（离心的速率为3000g，离心的时间为10min），得到3份上清液，采用纳米粒度仪（Malvern Nano-ZS）分别测定3份上清液中的纳米颗粒的粒径、聚合物分散性指数（PDI）及姜黄素含量；然后干燥（采用喷雾干燥，所述喷雾干燥的进风温度为160℃，排风温度为70℃）得到3份所述高荷载姜黄素的大豆多肽基纳米颗粒粉末制品。
采用纳米粒度仪（Malvern Nano-ZS）测定制备的荷载姜黄素的大豆多肽基纳米颗粒的平均粒径、PDI的数据以及根据姜黄素含量计算出的姜黄素的结合率及荷载量数据，见下表3。
表3
Figure dest_path_image006a
图3分别是实施例3制得的3份高荷载姜黄素的大豆多肽基纳米颗粒的粒径分布图；SPI try表示为利用胰酶得到的大豆多肽，Cur表示姜黄素，2%Cur、5%Cur及10%Cur分别表示加入不同质量的姜黄素粉末制得的高荷载姜黄素的大豆多肽基纳米颗粒，其中，2%、5%及10%表示为所述姜黄素粉末与步骤（1）所述大豆分离蛋白的质量比分别为1：50、1：20及1：5。由图3以及表3可知，随着姜黄素浓度的增加，所述高荷载姜黄素的大豆多肽基纳米颗粒的粒径、PDI无明显变化，体系均呈现较好的稳定性。此外，随着姜黄素浓度的增加，姜黄素的荷载量也逐渐增加。图4是实施例3制得的所述高荷载姜黄素的大豆多肽基纳米颗粒粉末（姜黄素：大豆分离蛋白（w/w）=1：50）复溶后的透射电镜图，由图4中可以看出大豆分离蛋白荷载姜黄素后形成的纳米颗粒是紧密、规则的球形。
实施例4
一种高荷载姜黄素的大豆多肽基纳米颗粒的pH驱动制备方法，具体包括以下步骤：
（1）将4份大豆分离蛋白加入4份水中，混合均匀，得到4份大豆分离蛋白分散液，4份所述大豆分离蛋白分散液的质量体积百分比浓度均为5.0%（w/v，g/mL）；分别调节4份大豆分离蛋白分散液的pH为8.0，在转速为200rpm的搅拌状态下分别往4份大豆分离蛋白分散液中加入4份水解酶进行酶解反应（此处选用碱性蛋白酶alc），每一份所述水解酶的质量均为每一份大豆分离蛋白质量的1%（w/w），酶解反应的温度为60℃，酶解反应的时间为1h，酶解完成后分别调节4份所述酶解反应液的pH为7.0，接着在100℃的条件下进行灭酶处理，灭酶处理的时间为20min，分别稀释得到4份大豆多肽分散液，4份所述大豆多肽分散液的浓度分别为5mg/mL、10mg/mL、20mg/mL及50mg/mL；
（2）分别调节步骤（1）所述4份大豆多肽分散液的pH值为12.0，在调节pH过程中，搅拌40min以使体系的pH稳定，然后往4份所述大豆多肽分散液中分别加入4份质量相同的姜黄素粉末，所述姜黄素粉末与步骤（1）所述大豆分离蛋白的质量比分别为1：10（w/w）；搅拌均匀，得到4份混合液（4份混合液中含有的姜黄素粉末质量均不同），分别调节4份所述混合液的pH为中性（pH值为7.0），分别离心取上清液（离心的速率为5000g，离心的时间为20min），得到4份上清液，采用纳米粒度仪（Malvern Nano-ZS）分别测定4份上清液中的纳米颗粒的粒径、聚合物分散性指数（PDI）及姜黄素含量；然后干燥（采用喷雾干燥，所述喷雾干燥的进风温度为180℃，排风温度为90℃）得到4份所述高荷载姜黄素的大豆多肽基纳米颗粒粉末制品。
采用纳米粒度仪（Malvern Nano-ZS）测定制备的高荷载姜黄素的大豆多肽基纳米颗粒的平均粒径、PDI的数据以及根据姜黄素含量计算出的姜黄素的结合率及荷载量数据，见下表4。
表4
大豆多肽分散液（mg/ml） 平均粒径（nm） 聚合物分散性指数 结合率（%） 荷载量（mg/g）
5 72.05 0.28 59.16 59.16
10 68.26 0.27 62.23 62.23
20 80.69 0.25 64.35 64.35
50 90.68 0.26 89.17 89.17
图5是实施例4制得的高荷载姜黄素的大豆多肽基纳米颗粒的粒径分布图；图5中的5mg/mL、10mg/mL、20mg/mL及30mg/mL分别表示为由浓度分别为5mg/mL、10mg/mL、20mg/mL及50mg/Ml大豆多肽分散液制得的高荷载姜黄素的大豆多肽基纳米颗粒。由图5以及表4可知，随着大豆多肽分散液浓度的增加，制得的高荷载姜黄素的大豆多肽基纳米颗粒的粒径逐渐增加，PDI无显著变化，体系均呈现较好的稳定性。此外，随着大豆多肽分散液的增加，姜黄素的结合率及荷载量也逐渐增加。
本发明制备的大豆多肽-姜黄素纳米颗粒（高荷载姜黄素的大豆多肽基纳米颗粒）产品具有荷载量高、粒径小，贮藏稳定性好等优点。姜黄素的水溶性差限制了其生物利用率，而大豆多肽分子量小、水溶性好且其自身具有一定的生理活性，本发明中利用大豆多肽对其进行包埋形成纳米颗粒，不仅增加了姜黄素的溶解性，也具有更高的生理活性。在工业化生产功能性食品时，可采用本发明的技术方法生产出富含姜黄素的功能性食品；本发明采用的pH驱动法绿色安全、能耗低，相比传统的反溶剂法具有更大的优势和应用性；本发明中制备的大豆多肽能够有效提高对疏水性生理活性物质的荷载量，在食品、药物、化妆品等生产领域将会有广泛的应用空间。本发明提供的制备方法中，原料天然健康，制备过程绿色安全、能耗低，工艺操作简单，能够进行快速连续化生产，制备的产品可应用于功能性食品、医药及化妆品中，具有巨大的应用价值。
以上实施例仅为本发明较优的实施方式，仅用于解释本发明，而非限制本发明，本领域技术人员在未脱离本发明精神实质下所作的改变、替换、修饰等均应属于本发明的保护范围。

Claims (10)

  1. 一种高荷载姜黄素的大豆多肽基纳米颗粒的pH驱动制备方法，其特征在于，包括以下步骤：
    （1）将大豆分离蛋白加入水中，混合均匀，得到大豆分离蛋白分散液，调节大豆分离蛋白分散液的pH为中性或碱性，在搅拌状态下加入水解酶进行酶解反应，酶解完成后调节酶解反应液的pH为7.0-7.5，接着进行灭酶处理，稀释得到大豆多肽分散液；
    （2）调节步骤（1）所述大豆多肽分散液的pH值为碱性，然后往所述大豆多肽分散液中加入姜黄素粉末，搅拌均匀，得到混合液，调节所述混合液的pH为中性，离心取上清液，即得到所述高荷载姜黄素的大豆多肽基纳米颗粒。
  2. 根据权利要求1所述的高荷载姜黄素的大豆多肽基纳米颗粒的pH驱动制备方法，其特征在于，步骤（1）所述大豆分离蛋白与水的质量体积比为1-10：100 （w/v，g/mL）。
  3. 根据权利要求1所述的高荷载姜黄素的大豆多肽基纳米颗粒的pH驱动制备方法，其特征在于，步骤（1）所述在搅拌状态下的搅拌速率为150-200rpm；所述水解酶为菠萝蛋白酶、木瓜蛋白酶、碱性蛋白酶、中性蛋白酶、风味蛋白酶、复合蛋白酶及胰酶中的一种以上；所述水解酶的质量为大豆分离蛋白质量的0.1wt%-1wt%；所述酶解反应的温度为35℃-60℃，所述酶解反应的时间为0.5-2.0h。
  4. 根据权利要求1所述的高荷载姜黄素的大豆多肽基纳米颗粒的pH驱动制备方法，其特征在于，步骤（1）中，调节大豆分离蛋白分散液的pH，使调节后的大豆分离蛋白分散液的pH为7.0-9.0。
  5. 根据权利要求1所述的高荷载姜黄素的大豆多肽基纳米颗粒的pH驱动制备方法，其特征在于，步骤（1）所述灭酶处理的温度为95-100℃，所述灭酶处理的时间为10-20min；所述大豆多肽分散液的浓度为5-50mg/mL。
  6. 根据权利要求1所述的高荷载姜黄素的大豆多肽基纳米颗粒的pH驱动制备方法，其特征在于，步骤（2）中，调节所述大大豆多肽分散液的pH值，是使调节后的大豆多肽分散液的pH为10.0-12.0。
  7. 根据权利要求1所述的高荷载姜黄素的大豆多肽基纳米颗粒的pH驱动制备方法，其特征在于，步骤（2）所述姜黄素与步骤（1）所述大豆分离蛋白的质量比为1:100-1:5；步骤（2）所述离心的速率为3000-8000g，所述离心的时间为10-20min。
  8. 根据权利要求1所述的高荷载姜黄素的大豆多肽基纳米颗粒的pH驱动制备方法，其特征在于，步骤（2）中，所述高荷载姜黄素的大豆多肽基纳米颗粒经过干燥处理后，可得到粉末状富载姜黄素的大豆多肽基纳米制品；所述干燥的方式为冷冻干燥及喷雾干燥中的一种；所述冷冻干燥的温度为-40℃～-50℃，冷冻干燥的真空度小于1mbar，冷冻干燥的时间为20-30h；所述喷雾干燥的进风温度为160-180℃，喷雾干燥的排风温度为70-90℃。
  9. 一种由权利要求1-8任一项所述的pH驱动制备方法制得的高荷载姜黄素的大豆多肽基纳米颗粒。
  10. 权利要求9所述的高荷载姜黄素的大豆多肽基纳米颗粒在制备抗炎抗氧化的食品或药品中的应用。
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