阅读说明：本技术 一种环境响应型葡糖基纳米凝胶的加工方法与应用 (Processing method and application of environment-responsive glucosyl nanogel ) 是由 缪铭 刘瑶 陈一枚 杨玉琪 张涛 胡秀婷 于 2020-11-05 设计创作，主要内容包括：本发明公开了一种环境响应型葡糖基纳米凝胶的加工方法与应用,属于生物材料加工技术领域。本发明以纳米葡糖基衍生物颗粒为原料,利用酶促扩链-原位组装协同技术得到环境响应型纳米凝胶,具体的,将纳米葡糖基衍生物颗粒配成溶液,再添加供体分子和客体分子,继续添加特异性糖酶制剂,在30-45℃反应1-24h后即得目标产物环境响应型纳米凝胶。本发明技术具有反应高效温和、进程安全可控、操作简单环保等特点,实现了绿色制备环境友好型高分子材料,所得纳米凝胶不仅高效负载功能活性物质,还能在肠道实现靶向释放,可以广泛应用在食品、日化、医药等领域。(The invention discloses a processing method and application of environment-responsive glucosyl nanogel, and belongs to the technical field of biomaterial processing. The invention takes nano glucosyl derivative particles as raw materials, utilizes an enzymatic chain extension-in-situ assembly synergistic technology to obtain the environmental response type nano gel, and concretely, the nano glucosyl derivative particles are prepared into a solution, donor molecules and guest molecules are added, specific carbohydrase preparations are continuously added, and the target product environmental response type nano gel is obtained after reaction is carried out for 1-24 hours at 30-45 ℃. The technology of the invention has the characteristics of efficient and mild reaction, safe and controllable process, simple and environment-friendly operation and the like, realizes the green preparation of the environment-friendly high polymer material, and the obtained nanogel not only can efficiently load functional active substances, but also can realize targeted release in intestinal tracts, and can be widely applied to the fields of food, daily chemicals, medicines and the like.)

1. A method for processing environment-responsive glucosyl nanogel is characterized by comprising the following steps:

(1) weighing nano glucosyl derivative particles to prepare a substrate solution with the mass percentage concentration of 2-10%;

(2) then adding donor molecules 4-10 times of the mass of the nano glucosyl derivative particles and 0.1% -100% of guest molecules, and uniformly mixing;

(3) continuously adding a specific carbohydrase preparation of 5-100U/g substrate, and reacting at 30-45 ℃ for 1-24h to obtain the target product environmental response type nanogel;

wherein the specific carbohydrase preparation comprises one or more of dextran sucrase, amyloglucosidase, and phosphorylase.

2. According toThe method of claim 1, wherein the molecular weight of the nano glucosyl derivative particles is 10⁶～10⁸g/mol, particle size of 20-100 nm, surface charge of-15 to-50 mV.

3. The method as claimed in claim 2, wherein the donor molecule comprises one or more of sucrose, maltose, maltodextrin, limit dextrin, glucose-1-phosphate.

4. The processing method of the environment-responsive glucosyl nanogel according to any one of claims 1 to 3, wherein the guest molecule comprises any one or more of a drug, a nutrient and a food.

5. The processing method of the environment-responsive glucosyl nanogel according to any one of claims 1 to 4, wherein the guest molecule comprises one or more active substances selected from bovine serum albumin, lysozyme, metformin hydrochloride, acetylsalicylic acid, chloramphenicol, antibacterial agents, and phytochemicals.

6. The environmental response type glucosyl nanogel prepared by the processing method of the environmental response type glucosyl nanogel according to any one of claims 1 to 5.

7. The environmentally responsive glucosyl nanogel according to claim 6, wherein the surface of the glucosyl nanocrystallite is grafted and extended to a size of DP 20-70.

8. Food, pharmaceutical or household chemical comprising the environmentally responsive glucosyl nanogel of claim 6 or 7.

9. The application of the processing method of the environment-responsive glucosyl nanogel in the fields of food, medicine and daily chemicals in any one of claims 1 to 5.

10. Use of the environmentally responsive glucosyl nanogel of claim 6 or 7 in the fields of food, medicine, and daily chemicals.

Technical Field

The invention relates to a processing method and application of environment-responsive glucosyl nanogel, and belongs to the technical field of biomaterial processing.

Background

With the rapid development of modern society and economy, two major problems of environmental pollution and human health are increasingly highlighted. On one hand, whether renewable biomass resources and novel fuels can be widely applied before the traditional hydrocarbon fuels are exhausted is an important problem to be solved worldwide, and for years, waste plastics taking polymers (polyethylene, polypropylene and the like) as main components cause serious pollution to the environment, so that the scientific community is forced to develop and use environment-friendly biodegradable polymers in an effort, and the biological economy is developed in a sustainable manner; on the other hand, increased awareness of health management has led consumers to become increasingly concerned about the functional benefits of everyday products, and the global functional market size is expected to exceed 2750 billion dollars. Under the background, biodegradable and renewable biomass resources derived from plants in the nature are developed through biotechnology, and environment-friendly materials with specific responsiveness are constructed and are used for designing personalized functional products, so that the research hotspot is realized.

The active substance controlled release technology is one of the fields which are developed most rapidly, have the greatest contribution to human health and have wide application prospects in biological materials. The polymer intelligent hydrogel is used as a carrier material to construct a functional factor controlled release system which has unique advantages, such as: the active ingredients are released in specific tissues and organs, active targeting is achieved, meanwhile, nutrients can be slowly released, and the active ingredients are efficiently absorbed and utilized by the target organs; on the premise of ensuring the action of the medicine, the distribution of the medicine in the body is changed, so that the administration dosage is reduced to reduce or avoid toxic and side effects and the like. However, the application in the food and medicine field requires that the hydrogel has higher biocompatibility and hemocompatibility, stimulus responsiveness to pH and temperature and biodegradability. Most of the current artificially synthesized pH response type hydrogel has no biodegradability and cytotoxicity, so that the application of the hydrogel in the food biological field is limited.

In order to design a hydrogel satisfying the in vivo delivery conditions, the following four problems of the raw materials need to be considered: good biocompatibility and non-toxicity; biodegradability; the price is low and the product is easy to obtain; groups with high reactivity are present to facilitate modification. Thus, hydrogels based on natural polymer bases such as polypeptides, proteins and polysaccharides are more advantageous. The pH response type hydrogel prepared by taking polysaccharide as a raw material reported at the present stage is mainly biased to cellulose and chitosan, and the glucosyl nanogel is relatively less. In addition, most of pH-responsive hydrogels prepared from natural polymers are compounded with other synthetic organic substances, a cross-linking agent is used for gelling, and cytotoxicity experiments are required to evaluate the safety of the hydrogel applied to human bodies, and a method for preparing intelligent gel by using modified starch as a matrix and adding no cross-linking agent is rarely reported. For the reasons, in order to widen the application field of glucosyl nanogels and increase the added value, there is an urgent need to develop a processing method of environment-responsive glucosyl nanogels.

Disclosure of Invention

The invention aims to provide a processing method and application of environment-responsive glucosyl nanogel, the method has the characteristics of efficient and mild reaction, safe and controllable process, simple and environment-friendly operation and the like, environment-friendly preparation of environment-friendly high polymer materials is realized, and the prepared products can be used as matrix materials in the fields of food, biological medicine and the like.

The first purpose of the invention is to provide a method for processing environment-responsive glucosyl nanogel, which comprises the following steps:

(1) weighing nano glucosyl derivative particles to prepare a substrate solution with the mass percentage concentration of 2-10%;

(2) then adding donor molecules 4-10 times of the mass of the nano glucosyl derivative particles and 0.1% -100% of guest molecules, and uniformly mixing;

(3) continuously adding a specific carbohydrase preparation with a substrate of 5-100U/g, and reacting at 30-45 ℃ for 1-24h to obtain the target product environmental response type nanogel.

In one embodiment of the invention, the nano glucosyl derivative particles are extracted from endosperm and algal tissues of cereal grains such as corn, sorghum, rice, barley, buckwheat, arabidopsis thaliana, blue algae, red algae and the like or are obtained by biomimetic synthesis through an amylase method, and are obtained by treating with a surface charge modification reagent, wherein the charge modification reagent comprises one or more of acetic anhydride, vinyl acetate, orthophosphate, pyrophosphate, tripolyphosphate, succinic anhydride, propylene oxide, sodium chloroacetate and the like.

In one embodiment of the invention, the molecular weight of the nano glucosyl derivative particles is 10⁶～10⁸g/mol, particle size of 20-100 nm, surface charge of-15 to-50 mV.

In one embodiment of the invention, the nano glucosyl derivative particles are prepared by the following method: adding the raw materials into a phosphate buffer solution for soaking, crushing, filtering, centrifuging and collecting a supernatant, adjusting the pH of the obtained supernatant to 4.5-5.0, placing the supernatant into a heating treatment at 80-100 ℃ for 30-60min, centrifuging and collecting the supernatant, carrying out alcohol precipitation and drying treatment, dissolving the supernatant in the phosphate buffer solution with the pH of 6.0-7.0, preparing the solution with the mass fraction of 10-30%, adding a charge modification reagent with the mass percentage of 3-10%, carrying out constant temperature reaction at 40-50 ℃ for-8 h, carrying out alcohol precipitation and drying to obtain the nano glucosyl derivative particles.

In one embodiment of the invention, the phosphate buffer has a pH of 6.0 to 7.0 and a concentration of 30 to 50 mM.

In one embodiment of the present invention, the donor molecule is one or more of sucrose, maltose, maltodextrin, ultimate dextrin, glucose-1-phosphate, and the like.

In one embodiment of the invention, the guest molecule comprises any one or more of a drug, a nutrient, a food product, and the like, preferably one that requires targeted release in the intestinal tract, and the like.

In one embodiment of the invention, the guest molecule comprises one or more of bovine serum albumin, lysozyme, metformin hydrochloride, acetylsalicylic acid, chloramphenicol, antibacterial agents, plant compounds, and like active substances.

In one embodiment of the invention, the specific carbohydrase preparation comprises one or more of dextran sucrase, amyloglucosidase, phosphorylase.

The second purpose of the invention is to provide an environment-responsive glucosyl nanogel loaded with guest molecules by using the method.

In one embodiment of the invention, the environmental response type glucosyl nanogel enzyme method has the gelling time of 1-24h, and the size of the branch chain extension grafted on the surface of the nano glucosyl derivative particle is DP 20-70. The nanogel forms a large number of linear straight chains and is mutually crosslinked, so that a space three-dimensional network structure is formed, and the load rate of the environment-responsive glucosyl nanogel on guest molecules is more than 97%.

It is a third object of the present invention to provide a food, a pharmaceutical or a daily chemical comprising the above-mentioned environment-responsive glucosyl nanogel.

The fourth purpose of the invention is to apply the environment-responsive glucosyl nanogel to the field of targeted delivery of functional materials and targeted delivery.

In one embodiment of the invention, the environment-responsive glucosyl nanogel has pH sensitivity, and the release rate of the environment-responsive glucosyl nanogel to a guest substance is 5-50% at pH 3-10; it also has digestive enzyme touch sensitivity, and has release rate of 80-95% to guest substance in simulated intestinal tract.

The environment-responsive glucosyl nanogel can be widely applied to the fields of food, medicine, daily chemicals and the like.

The invention has the following advantages:

1. the nanometer glucosyl group derivative particles are prepared from the nanometer glucosyl group particles extracted from natural plants such as grains, algae and the like through simple charge modification, and are used as the skeleton of the nanogel, so that the nanogel has biodegradability and biocompatibility, and is non-toxic and harmless. The invention converts renewable and degradable biomass resources into the nano gel material with high added value, widens the application field and approach of the polysaccharide, and improves the utilization value of the polysaccharide.

2. The environment-responsive glucosyl nanogel is prepared by an enzymatic chain extension and in-situ assembly method, an initiator and a cross-linking agent are not needed, the enzymatic method is efficient and mild, the process is safe and controllable, the operation is simple and environment-friendly, and the environment-friendly preparation of the environment-friendly biomaterial is realized.

3. The environment-responsive glucosyl nanogel has good targeted delivery and positioning release effects on guest substances, and can be used for constructing a targeted delivery system of nutrients in vivo and designing functional foods or controlled-release medicines.

Drawings

Figure 1 scanning electron microscopy images of the environmentally responsive glucosyl nanogels of example 1.

Detailed Description

The present invention will be further explained with reference to examples, but the present invention is not limited to the examples.

And (3) measuring the molecular weight: weighing a sample to be detected, preparing the sample into a 0.2% solution, then injecting the solution into a high performance gel exclusion chromatography system (HPSEC), selecting a ShodOxpak SB-805HQ gel chromatographic column, taking a 0.1mol/L sodium nitrate solution as a mobile phase, setting the flow rate to be 0.7mL/min, setting the refractive index to be dn/dc to be 0.138, and calculating a single chromatographic peak in a chromatogram by using Astra software to obtain the molecular weight.

Particle size and surface potential measurements: a sample to be tested is prepared into 0.1 percent (w/v) solution, and particle size distribution and surface potential are measured by a Malvern Nano ZS tester at 25 ℃.

Chain length determination: and (3) determining the chain length distribution condition of the sample to be detected by using high-efficiency anion exchange chromatography. A CarboPacPA 200 ion exchange column was selected and gradient elution was set, with mobile phases (A): 0.25mol/L NaOH solution; (B)1.0mol/L NaAC solution; ultrapure water. The elution program is set for 0-21 min: 1.8% a mobile phase, 0% B mobile phase; 21-30 min: 1.8% of A mobile phase, and lifting the B mobile phase from 5% to 30%; 30-50 min: 80% A mobile phase, 0% B mobile phase.

And (3) measuring the load ratio: washing a sample to be detected with deionized water, measuring a light absorption value of the obtained washing liquid through a colorimetric method, and calculating the mass of an object molecule, wherein the calculation formula is as follows: the loading rate (%) - (the mass of guest molecules in the reaction system-the mass of guest molecules in the washing solution)/the mass of nanogel x 100.

Determination of pH sensitivity: 50mg of the sample to be tested is weighed and dispersed in 10mL of buffer solutions with different pH values, and stirred and released for 2h under the condition of 200 rpm. After the sample is centrifuged and the supernatant is properly diluted, the light absorption value is measured by a colorimetric method according to the measurement of the load rate to calculate the release rate of the guest molecules.

Determination of digestive enzymatic sensitivity: weighing 100mg of a sample to be detected, dispersing the sample into 10mL of simulated intestinal fluid (pH 5.2, 0.1mol/L phosphate buffer), adding porcine pancreatic alpha-amylase and glucoamylase to enable the simulated intestinal fluid to contain 290U/mL of alpha-amylase and 15U/mL of glucoamylase, hydrolyzing for 6h at the constant temperature of 37 ℃ and the rotating speed of 150rpm, and measuring the absorbance value by a colorimetric method according to the measurement of the load rate to calculate the release rate of the guest molecules.

Dextran sucrase, amyloglucosidase, phosphorylase were purchased from Sigma company; the plant materials such as corn, sorghum, rice, barley, buckwheat, arabidopsis thaliana, blue algae, red algae and the like are purchased from Chinese academy of agricultural sciences.

Example 1

Weighing 100g of corn kernels, adding 300mL of phosphate buffer (pH 7.0, 50mM) to soak for 12h, crushing, filtering, centrifuging, adjusting the pH of the obtained supernatant to 4.9, placing at 100 ℃ for heating treatment for 30min, centrifuging, collecting the supernatant, and carrying out alcohol precipitation and drying treatment; dissolving the obtained powder in phosphate buffer solution with pH of 7.0, preparing into solution with mass fraction of 10%, adding charge modification reagent chloroacetic acid with mass percentage of 3%, reacting at constant temperature of 40 ℃ for 4h, precipitating with ethanol, and drying to obtain the corn source nano glucosyl derivative particles. The molecular weight of the corn-derived derivative particles is 2.4 multiplied by 10 according to the determination analysis⁷g/mol, average particle diameter of 82nm and surface charge of-35 mV.

Weighing 100g of red algae tissue, adding 300mL of phosphate buffer (pH 6.0, 30mM) to soak for 12h, crushing, filtering, centrifuging, adjusting the pH of the obtained supernatant to 4.5, heating at 80 ℃ for 60min, centrifuging, collecting the supernatant, precipitating with ethanol, and drying; dissolving the obtained powder in phosphate buffer with pH of 6.0Washing the solution, preparing the solution into a solution with the mass fraction of 30%, adding 5% of charge modification reagent pyrophosphate by mass percent, reacting at the constant temperature of 50 ℃ for 6 hours, and precipitating with ethanol and drying to obtain the red algae source nano glucosyl derivative particles. The molecular weight of the red algae derived derivative particles is 0.4 multiplied by 10 according to the determination analysis⁷g/mol, average particle diameter of 62nm and surface charge of-28 mV.

Weighing 100g of barley seeds, adding 300mL of phosphate buffer (pH 6.0, 50mM) to soak for 12h, crushing, filtering, centrifuging, adjusting the pH of the obtained supernatant to 5.0, heating at 100 ℃ for 50min, centrifuging, collecting the supernatant, precipitating with ethanol, and drying; dissolving the obtained powder in phosphate buffer solution with pH of 6.0, preparing into solution with mass fraction of 20%, adding 3% by mass of charge modification reagent propylene oxide, reacting at constant temperature of 45 deg.C for 4h, precipitating with ethanol, and drying to obtain barley-derived nano glucosyl derivative particles. The molecular weight of the barley-derived derivative particles is 3.8 × 10 by determination and analysis⁷g/mol, average particle diameter 73nm, surface charge-35 mV.

Example 2

Weighing the corn source nano glucosyl derivative particles prepared in the embodiment 1 to prepare a uniform substrate solution with the mass percentage concentration of 6%; adding glucose-1-phosphate and 20% lysozyme molecule with the mass 6 times of that of the substrate, and uniformly mixing; continuously adding phosphorylase with 10U/g substrate, and reacting at 37 ℃ for 10h to obtain the target product environmental response type nanogel.

Analysis and determination show that the grafted chain extension size of the nano glucosyl derivative particles is DP 54.4, the load rate of guest molecules is 99.5%, the release rate of nano gel to guest substances is over 35.2% in the range of pH 3-7, and the release rate of nano gel to guest substances is 87.5% in an experiment simulating intestinal tracts. Therefore, the environmental response type nanogel prepared by the invention is released in the gastrointestinal tract, has digestive enzyme touch sensitivity, can release most of guest molecules in the small intestine, and can be used for controlling and releasing drugs or nutrients and the like.

Example 3

Red algae-derived nano glucosyl derivative particles (molecular weight 0.4X 10) prepared in example 1 were weighed⁷g/mol, average particle size of 62nm and surface charge of-28 mV) to prepare a uniform substrate solution with the mass percentage concentration of 10 percent; adding cane sugar with the mass 10 times of that of the substrate and 4% of metformin hydrochloride molecules, and uniformly mixing; continuously adding 5U/g substrate dextran sucrase, and reacting at 40 deg.C for 8 hr to obtain the target product environmental response type nanogel.

Analysis and determination show that the grafted chain extension size of the nano glucosyl derivative particles is DP 60.1, the load rate of guest molecules is 97.5%, the release rate of nano gel to guest substances is over 48.2% in the range of pH 7-10, and the release rate of nano gel to guest substances is 94.8% in an intestinal tract simulation experiment.

Example 4

Barley-derived nano glucosyl derivative particles (molecular weight 3.8X 10) prepared in example 1 were weighed⁷g/mol, the average grain diameter is 73nm, and the surface charge is-35 mV) to prepare a uniform substrate solution with the mass percentage concentration of 8 percent; adding maltodextrin 4 times of the mass of the substrate and 1% of bovine serum albumin molecules, and uniformly mixing; continuously adding 60U/g substrate of amyloglucosidase, and reacting at 38 ℃ for 12h to obtain the target product environmental response type nanogel.

Analysis and determination show that the grafted chain extension size of the nano glucosyl derivative particles is DP 50.4, the load rate of guest molecules is 99.5%, the release rate of nano gel to guest substances is over 28.6% in the range of pH 6-10, and the release rate of nano gel to guest substances is 87.9% in an experiment simulating intestinal tracts.

When the donor molecule is maltose or limit dextrin, or when the nano glucosyl derivative particles are respectively from sorghum, rice, buckwheat, arabidopsis thaliana, blue algae and the like, the environmental response type nano gel prepared according to the embodiment also has the properties, namely the nano glucosyl derivative particle surface grafting chain extension size is DP20-70, the load rate on the guest molecule is more than 97%, the release rate of the nano gel on the guest substance is 5% -50% in the range of pH (based on different charge modification conditions) of 3-10, and the release rate of the nano gel on the guest substance is 80% -95% in the experiment of simulating intestinal tracts.

Comparative example 1

When no donor molecule was added in example 2, no hydrogel could be formed.

Comparative example 2

When the nonspecific enzyme preparation of example 2 was treated, hydrogel could not be formed

Comparative example 3

Referring to example 2, the amount of phosphorylase 10U/g substrate was replaced with 1U/g substrate and 200U/g substrate, respectively, to prepare corresponding environmental responsive nanogels. The performance results of the obtained environmentally responsive nanogels are shown in table 1.

TABLE 1 environmental response type nanogels obtained with different phosphorylase dosages

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.