WO2021008171A1

WO2021008171A1 - Amphiphilic starch nanoparticles and preparation method therefor

Info

Publication number: WO2021008171A1
Application number: PCT/CN2020/082730
Authority: WO
Inventors: 黄强; 李松南; 孙瑞云; 张斌; 扶雄
Original assignee: 华南理工大学
Priority date: 2019-07-15
Filing date: 2020-04-01
Publication date: 2021-01-21
Also published as: CN110606961B; CN110606961A

Abstract

Disclosed are amphiphilic starch nanoparticles and a preparation method therefor. The preparation method comprises the following steps: (1) formulating a starch raw material into a starch slurry using an acetic acid-sodium acetate buffer solution, adjusting the pH to 4.5-6.5, gelatinizing same in a boiling water bath, then cooling same to a temperature suitable for enzymes, adding a debranching enzyme, continuously stirring same, then heating same in a boiling water bath, and then centrifuging and layering same to obtain a supernatant that is a short amylose dispersion, and dissolving a protein in an ethanol-water solution, and stirring until same is sufficiently dissolved to obtain a protein dispersion; and (2) dropwise adding the protein dispersion to the short amylose dispersion, intensively mixing same, then continuously stirring same, centrifuging and layering same, recrystallizing the resultant supernatant, subjecting same to vacuum concentration or centrifugation, and drying same to obtain the amphiphilic starch nanoparticles.

Description

Amphiphilic starch nano particles and preparation method thereof

Technical field

The present invention relates to protein complex induced debranched starch recrystallization, in particular to a method for preparing amphiphilic starch nanoparticles. The method involves protein complex induced debranched starch recrystallization to make it self-assemble, thereby obtaining amphiphilic The purpose of starch nanoparticles belongs to the field of food industry.

Background technique

Bio-based nanomaterials have the advantages of both "nano effect" and "green environmental protection", and are widely used in the fields of biology, materials, chemicals, food and medicine. Starch is a natural non-toxic biological macromolecule with abundant resources, good biocompatibility and biodegradability, and can be used as a raw material for bio-based nanomaterials. Starch nanoparticles have many potential applications in materials, medicine, food and other fields. For example, they can enhance the tensile strength of starch films and composite materials, improve water vapor permeability, and can be used in food-grade packaging materials; they can carry functional activities. Ingredients, such as flufenamic acid (Jain et al., 2008, European Journal of Pharmaceutics and Biopharmaceutics, 69(2), 426-435) and insulin (Santander-Ortega et al., 2010, Journal of Controlled Release, 141(1), 85-92), as a drug delivery carrier, used in the medical field; also as an emulsifier, used to prepare food-grade emulsion systems or fat-soluble active substance delivery carriers.

The current methods for preparing starch nanoparticles mainly include acid hydrolysis, precipitation, mechanical and microemulsion methods. The acid hydrolysis method is to hydrolyze the amorphous regions of starch particles under acidic conditions to obtain tightly arranged nano-scale crystals, but the preparation time is long, the yield is low, and a large amount of inorganic acid is consumed (Putaux et al., 2003, Biomacromolecules, 4(5), 1198-1202). The precipitation method is to gradually add the fully dissolved starch solution to the non-solvent system or vice versa, so that the starch molecules gradually precipitate to form starch nanoparticles, but the recrystallization process is not well controlled, and the formed starch nanoparticles agglomerate seriously (Chin et al., 2011, Carbohydrate Polymers, 86(4), 1817-1819; Qin et al., 2016, Industrial Crops and Products, 87, 182-190). The mechanical method uses physical means such as ball milling, homogenization, extrusion, irradiation, and ultrasound to gradually reduce the size of starch particles to nanometers, but requires higher equipment, large power consumption, and long processing time (Song et al., 2011, Carbohydrate Polymers, 85(1), 208-214; Bel Haaj et al., 2013, Carbohydrate Polymers, 92(2), 1625-1632; Singh et al., 2011, Carbohydrate Polymers, 83(4), 1521-1528). The microemulsion method is to form a uniform and stable water-in-oil emulsion from a fully dissolved starch solution and an incompatible organic solution through the emulsification of a surfactant, and crosslink the starch molecules in the water phase under the action of a crosslinking agent Starch nanoparticles are formed and precipitated, but the requirements for equipment are relatively high, the energy consumption is large, and a large amount of organic reagents are introduced (Shi et al., 2011, Carbohydrate Polymers, 83(4), 1604-1610). The above preparation methods require high preparation equipment, and at the same time use too many inorganic acids and chemical reagents, causing environmental problems, and toxic chemical reagents will also limit the application of these starch nanoparticles in the fields of food and medicine. .

technical problem

The starch nanoparticles prepared at present are mostly hydrophilic. The introduction of hydrophobic groups into the starch molecule makes it have both hydrophilic and hydrophobic amphiphilic characteristics. It can be used in food, medicine, biodegradable materials, cosmetics and High value-added fields such as chemical catalysis have wider applications. At present, hydrophobically modified starches are mainly concentrated in starch octenyl succinate (OS starch), a chemically modified starch formed by the reaction of octenyl succinic anhydride (OSA) and starch under alkaline conditions. The amphiphilic OS starch can carry hydrophobic active substances as a carrier for oral delivery; it can be used as a food emulsifier and thickener in the fields of microencapsulation and beverage flavors; alternative sources are limited and quality The unstable gum arabic relieves China’s dependence on imported gum arabic; it can be used as a fluid powder carrier to gradually replace the traditional use of calcium phosphate and talc in the flour industry; it has slow digestion properties and can be used as a low GI food accessories; it can be mixed with lubricating oil for light yarn sizing to improve textile performance and sizing ability. However, the US Food and Drug Administration (FDA) stipulates that the amount of OSA added in the preparation of OS starch in food should not be higher than 3%, and the degree of substitution should not exceed 0.02, which limits the application of highly substituted OS starch in the food field. Moreover, in the preparation process of OS starch, the starch and long-chain hydrophobic anhydride undergo esterification heterogeneous reaction in the aqueous dispersion system, resulting in low substitution and low reaction efficiency. The organic reagents and other physical methods or enzymes introduced in the improved method are improved. Pretreatment of the preparation undoubtedly increases its safety and production costs. Therefore, it is particularly important to invent a "green", "environmental protection" and "simple" preparation method for amphiphilic starch nanoparticles.

Technical solutions

The purpose of the present invention is to provide a method for preparing amphiphilic starch nanoparticles with simple process, rich source of raw materials, and environmental protection. The resulting product is nano-spherical and has a particle size range of 200-500. nm, the contact angle is 72-81°, it can be used as food-grade microcapsule wall material, emulsifier and drug delivery carrier, and is used in food, cosmetics and medicine.

The technical scheme of the present invention is to first fully gelatinize the starch raw materials in a boiling water bath, add debranching enzymes under suitable conditions for enzymatic hydrolysis, heat the boiling water bath again, centrifuge at a medium speed, and precipitate into long amylose and starch. The debranching enzyme is inactivated, and the supernatant is short amylose dispersion. Add the protein ethanol aqueous dispersion to the short amylose dispersion drop by drop, mix well and continue stirring, centrifuge at low speed for layering, and precipitate into uncomplexed longer chain short amylose and protein aggregates. The clear liquid is a compound solution of short amylose and protein. Finally, the short amylose and protein composite solution is self-assembled and recrystallized under certain conditions, concentrated in vacuum or centrifuged, and dried to obtain amphiphilic starch nanoparticles.

The purpose of the present invention is achieved through the following technical solutions:

A preparation method of amphiphilic starch nanoparticles includes the following steps:

(1) Preparation of short amylose and protein dispersion

The starch raw material is prepared with acetic acid-sodium acetate buffer solution into a starch slurry with a dry basis mass fraction of 5-15%, and the pH is adjusted to 4.5-6.5. After gelatinization in a boiling water bath, it is cooled to the appropriate temperature for the enzyme, and the debranching enzyme is added. Continue to stir, then heat in a boiling water bath, and then centrifuge for layering. The supernatant is the short amylose dispersion;

Dissolve the protein in the ethanol aqueous solution, continue to stir to make it fully dissolved, and prepare a protein dispersion;

(2) Add the protein solution dropwise to the short amylose dispersion, mix well, continue stirring, centrifuge for layering, recrystallize the supernatant obtained, concentrate in vacuum or centrifuge, and dry to obtain the amphiphilic Starch nanoparticles.

Preferably, in step (1), the molar concentration of the acetic acid-sodium acetate buffer solution is 0.05 to 0.2 mol/L; the starch raw material is waxy corn starch, ordinary corn starch or tapioca starch.

Preferably, in step (1), the boiling water bath gelatinization time is 0.5-2 h, and the cooling temperature is 45-65°C; the addition amount of debranching enzyme is 35-55 U/g, based on the quality of dry starch.

Preferably, in the preparation of the short amylose dispersion in step (1), the stirring method is mechanical stirring or magnetic stirring, and the stirring time is 12-24 h; the boiling water bath heating time is 1 to 2 h; The centrifugal force is 5000~10000 g , and the centrifugal time is 10~20 min.

Preferably, in step (1), the protein is zein, soy protein isolate or peanut protein; the volume fraction of the ethanol aqueous solution is 60-80%.

Preferably, in the preparation of the protein solution in step (1), the protein is 5-20 parts by mass dissolved in 100 parts by volume of ethanol aqueous solution; the stirring mode is mechanical stirring or magnetic stirring, and the stirring time is 1 to 2 h.

Preferably, in step (2), the short amylose dispersion is 1 to 4 parts by volume, and the protein solution is 1 to 2 parts by volume; the stirring method is mechanical stirring or magnetic stirring, and the stirring time is 2 to 4 parts by volume. h; The centrifugal force is 3000-5000 g , and the centrifugal time is 5-15 min.

Preferably, in step (2), the recrystallization temperature is 4-25°C, and the recrystallization time is 12-24 h; the drying method is freeze drying or blast drying, and the drying time is 24-48 h.

The amphiphilic starch nanoparticles prepared by the above method are nano-spherical, with a particle size ranging from 200 to 500 nm, the contact angle is 72~81°.

In the present invention, the starch raw material is fully gelatinized in a boiling water bath, then a debranching enzyme is added under suitable conditions for enzymatic hydrolysis, the boiling water bath is heated, and the medium speed centrifugation is separated to precipitate into long amylose and inactivated debranching. Enzyme, the supernatant is the short amylose dispersion. Add the protein ethanol dispersion to the short amylose dispersion drop by drop, mix well and continue stirring, centrifuge at low speed to layer, and precipitate into uncomplexed longer chain short amylose and protein aggregates, supernatant The liquid is a compound solution of short amylose and protein. Finally, the short amylose and protein composite solution is self-assembled and recrystallized under certain conditions, concentrated in vacuum or centrifuged, and dried to obtain amphiphilic starch nanoparticles.

Beneficial effect

Compared with the prior art, the present invention has the following advantages:

1) The method of the present invention is based on the principle of amylase debranching and protein composite induction recrystallization, by dropping the protein ethanol aqueous dispersion into the short amylose dispersion, mixing thoroughly and continuously stirring, centrifuging at low speed, and the precipitation is not The long chain short amylose and protein aggregates are compounded, and the short amylose and protein compound solution in the supernatant is self-assembled and recrystallized, so as to achieve the purpose of preparing amphiphilic starch nanoparticles. This method is different from the traditional hydrophobic modification chemical method and the preparation method of starch nanoparticles (acid hydrolysis method, precipitation method, mechanical method and microemulsion method). The modification process does not introduce organic reagents and other physical methods or enzyme preparations. Processing, the degree of modification increases with the increase of protein compounding rate and is not restricted by FDA limit. This method is simple to prepare, requires low equipment and equipment, low production cost, safety and environmental protection, and is a new type of amphiphilic starch nanoparticles The preparation method.

2) The starch nanoparticles obtained in the present invention are nano-spherical, with a particle size ranging from 200 to 500 nm and a contact angle of 72 to 81°. They are amphiphilic starch nanoparticles and can be used as food-grade microcapsule wall materials, Emulsifiers and drug delivery vehicles are used in the fields of food, cosmetics and medicine.

3) The present invention uses starch and protein from different sources as raw materials, through the method of amylase debranching and protein composite induction recrystallization. The process is simple, the source of raw materials is rich, and it is environmentally friendly. It is a kind of amphiphilic starch nanoparticles. Preparation.

Description of the drawings

Fig. 1 is a scanning electron microscope image (a), a particle size distribution diagram (b) and a contact angle diagram (c) of ordinary starch nanoparticles prepared under the conditions of a comparative example.

2 is a scanning electron microscope image (a), a particle size distribution diagram (b), and a contact angle diagram (c) of the amphiphilic starch nanoparticles prepared under the conditions of Example 1.

3 is a scanning electron microscope image (a), a particle size distribution diagram (b), and a contact angle diagram (c) of the amphiphilic starch nanoparticles prepared under the conditions of Example 2.

4 is a scanning electron microscope image (a), a particle size distribution diagram (b), and a contact angle diagram (c) of the amphiphilic starch nanoparticles prepared under the conditions of Example 3. ...

Embodiments of the invention

In order to better understand the present invention, the following further describes the present invention with reference to the drawings and embodiments, but the scope of protection claimed by the present invention is not limited to the scope of the embodiments. The related test methods in the embodiment are described as follows:

Particle microstructure analysis: Disperse the test sample evenly on the double-sided conductive adhesive of the sample stage, and then observe the microstructure of the test sample in the Carl Zeiss EVO 18 scanning electron microscope after spraying the gold with the gold spraying instrument.

Particle size distribution measurement: The test sample is prepared into a 0.01% water dispersion, ultrasonically dispersed for 1 min, and placed in the MPT-2 laser nanoparticle sizer of Malvern, UK for particle size analysis. The refractive index and absorption rate of starch granules were set to 1.52 and 0.01, respectively, and the refractive index of dispersant water was set to 1.33.

Particle amphiphilic analysis: The amphiphilic nature of solid particles is characterized by measuring its three-phase contact angle θ at the oil-water interface. When θ is less than 90°, the particles are hydrophilic; when θ is close to 90°, the solid particles are amphiphilic, the adsorption energy at the oil-water interface is the largest, and the emulsification effect is the best; when θ is greater than 90°, the particles appear Hydrophobicity.

The test procedure is as follows: the test sample is made into a sheet (2 mm thick and 13 mm diameter) with a tablet press, and then immersed in the soybean oil sample stage of the OCA 20 device, and 2 μL of water droplets are dropped on the OCA 20 device using a high-precision syringe system. The tablet surface, installed on OCA The high-speed camera on the 20 records the evolution of the droplet shape at a rate of 10 frames per second, and automatically fits the contour data of the droplet to the LaPlace-Young equation to determine the contact angle of the particle.

对比实施例Comparative example

The preparation of ordinary starch nanoparticles includes the following steps:

(1) Prepare waxy corn starch with 0.1 mol/L acetic acid-sodium acetate buffer solution into a starch slurry with a dry mass fraction of 15%, and adjust the pH to 5, gelatinize in a boiling water bath for 1 hour, and cool to 55°C According to the dry starch quality, add 40 U/g debranching enzyme, continue to stir for 24 h, heat in a boiling water bath for 1 h, centrifuge at 8000 g for 20 min, and layer, the supernatant is the short amylose dispersion;

(2) Directly add 1 part of 70% ethanol aqueous solution dropwise to 2 parts of the short amylose dispersion obtained in step (1), mix well, continue stirring for 2 h, centrifuge at 4000 g for 10 min to separate, set After recrystallization at 25°C for 24 h, vacuum concentration, and freeze-drying, ordinary starch nanoparticles can be obtained.

After testing, the ordinary starch nanoparticles obtained in the above steps are nano-spherical, but the agglomeration phenomenon between the nanoparticles is more serious (Figure 1a), which may be caused by their strong hydrophilicity and the attraction of hydrogen bonds. The particle size distribution of ordinary starch nanoparticles showed double peaks at 200-500 nm and 700-1100 nm respectively (Figure 1b), which can also explain the phenomenon of particle agglomeration; its contact angle is 46° (Figure 1c), indicating its relatively high Strong hydrophilicity.

实施例Example 11

The preparation of amphiphilic starch nanoparticles includes the following steps:

(1) Prepare waxy corn starch with 0.05 mol/L acetic acid-sodium acetate buffer solution into a starch slurry with a dry basis mass fraction of 15%, adjust the pH to 5, gelatinize in a boiling water bath for 1 hour, and cool to 55°C Add 45 U/g of debranching enzyme according to the quality of dry starch. After stirring for 24 h, heating in a boiling water bath for 1 h, centrifugation at 6000 g for 20 min, the supernatant is the short amylose dispersion;

Dissolve 5 parts of zein in 100 parts of ethanol aqueous solution with a volume fraction of 70%, continue to stir for 1 h to fully dissolve;

(2) Add 1 part by volume of the zein solution obtained in (1) dropwise to 2 parts by volume of the short amylose dispersion obtained in step (1), mix well, and continue stirring for 2 h, 3000 g After centrifugation for 15 min, the layering, recrystallization at 25°C for 24 h, vacuum concentration, and freeze-drying can obtain amphiphilic starch nanoparticles.

After testing, the obtained starch nanoparticles are nano-spherical with an average particle size of 256 nm and a contact angle of 81°. They are amphiphilic starch nanoparticles, as shown in Figure 2.

实施例Example 22

(1) Prepare ordinary corn starch with 0.2 mol/L acetic acid-sodium acetate buffer solution into a starch slurry with a dry mass fraction of 10%, adjust the pH to 5, gelatinize in a boiling water bath for 2 hours, and cool to 55°C. Add 55 U/g of debranching enzyme according to the quality of dry starch, continue to stir for 24 h, heat in a boiling water bath for 2 h, centrifuge at 10,000 g for 10 min, and separate the supernatant as the short amylose dispersion;

Dissolve 5 parts of soy protein isolate in 100 parts of ethanol aqueous solution with a volume fraction of 70%, and continue to stir for 2 h to fully dissolve;

(2) Add 1 part of the soybean protein isolate solution obtained in (1) dropwise to 2 parts of the short amylose dispersion obtained in step (1), mix well, continue stirring for 2 hours, and centrifuge at 5000 g for 5 min After layering, recrystallization at 25°C for 24 h, vacuum concentration, and air drying, amphiphilic starch nanoparticles can be obtained.

After testing, the obtained starch nanoparticles are nano-spherical with an average particle size of 295 nm and a contact angle of 77°. They are amphiphilic starch nanoparticles, as shown in Figure 3.

实施例Example 33

(1) Mix tapioca starch with 0.1 mol/L acetic acid-sodium acetate buffer solution into a starch slurry with a dry basis mass fraction of 15%, adjust the pH to 5, gelatinize in a boiling water bath for 1 h, and cool to 55°C Add 35 U/g debranching enzyme to the dry starch quality, continue stirring for 24 h, heat in a boiling water bath for 1 h, centrifuge at 8000 g for 20 min, and separate, the supernatant is the short amylose dispersion;

Dissolve 10 parts of peanut protein in 100 parts of ethanol aqueous solution with a volume fraction of 70%, and continue to stir for 2 h to fully dissolve;

(2) Add 1 part of the peanut protein solution obtained in (1) dropwise to 3 parts of the short amylose dispersion obtained in step (1), mix thoroughly, continue stirring for 4 h, and centrifuge at 4000 g for 10 min. The layer is recrystallized at 4°C for 24 h, concentrated in vacuo, and freeze-dried to obtain amphiphilic starch nanoparticles.

After testing, the obtained starch nanoparticles are nano-spherical with an average particle size of 270 nm and a contact angle of 72°. They are amphiphilic starch nanoparticles, as shown in Figure 4.

Claims

A method for preparing amphiphilic starch nanoparticles is characterized in that it comprises the following steps:

(1) Preparation of short amylose and protein dispersion

The starch raw material is prepared with acetic acid-sodium acetate buffer solution into a starch slurry with a dry basis mass fraction of 5-15%, and the pH is adjusted to 4.5-6.5. After gelatinization in a boiling water bath, it is cooled to the appropriate temperature for the enzyme, and the debranching enzyme is added. Stirring continuously, heating in a boiling water bath, and then centrifuging and layering, and the supernatant is obtained as short amylose dispersion;

Dissolve the protein in the ethanol aqueous solution, continue to stir to make it fully dissolved, and prepare a protein dispersion;

(2) Add the protein dispersion dropwise to the short amylose dispersion, mix well, continue stirring, centrifuge for layering, recrystallize the resulting supernatant, concentrate in vacuo or centrifuge, and dry it. Affinity starch nanoparticles.
The preparation method according to claim 1, wherein in step (1), the molar concentration of the acetic acid-sodium acetate buffer solution is 0.05 to 0.2 mol/L; the starch raw material is waxy corn starch, ordinary Corn starch or tapioca starch.
The preparation method according to claim 1, wherein in step (1), the boiling water bath gelatinization time is 0.5~2 h, the cooling temperature is 45~65°C; the addition amount of debranching enzyme is 35~55 U/g (based on the quality of dry starch).
The preparation method according to claim 1, characterized in that, in the preparation of the short amylose dispersion in step (1), the stirring method is mechanical stirring or magnetic stirring, and the stirring time is 12-24 h; the boiling water The heating time of the bath is 1-2 h; the centrifugal force is 5000-10000 g , and the centrifugal time is 10-20 min.
The preparation method according to claim 1, wherein in step (1), the protein is zein, soy protein isolate or peanut protein; the volume fraction of the ethanol aqueous solution is 60-80%.
The preparation method according to claim 1, characterized in that, in the preparation of the protein solution in step (1), the protein is 5-20 parts by mass dissolved in 100 parts by volume of ethanol aqueous solution; the stirring method is mechanical stirring or Magnetic stirring, stirring time is 1~2 h.
The preparation method according to claim 1, wherein in step (2), the short amylose dispersion is 1 to 4 parts by volume, and the protein solution is 1 to 2 parts by volume; and the stirring method is mechanical Stirring or magnetic stirring, the stirring time is 2~4 h; the centrifugal force is 3000~5000 g , and the centrifugation time is 5~15 min.
The preparation method according to any one of claims 1 to 7, wherein in step (2), the recrystallization temperature is 4-25°C, the recrystallization time is 12-24 h; and the drying method is Freeze drying or blast drying, the drying time is 24 to 48 h.
Amphiphilic starch nanoparticles prepared by the method of any one of claims 1-8.
The amphiphilic starch nanoparticles of claim 9, wherein the starch nanoparticles are nano-spherical, with a particle size ranging from 200 to 500 nm, and a contact angle from 72 to 81°.