CN114984871B - Double-layer alginate microsphere for delivering probiotics and preparation method thereof - Google Patents

Abstract

The invention belongs to the technical field of bioengineering, and in particular relates to a double-layer alginate microsphere for delivering probiotics and a preparation method thereof. The present invention constructs a double-layer alginate microsphere system for delivering probiotics. Under centrifugal force driven fluid flow, sodium alginate and calcium chloride solution contact to form a gel, and the double-layer microsphere with controllable size is synthesized ( 200‑500 μm). Sodium alginate was used to immobilize probiotics on the inner layer of microspheres, and RS3 was added to the outer layer of sodium alginate solution to reduce the porosity of the gel, which improved the encapsulation rate and the viability of probiotics.

Description

A double-layer alginate microsphere for delivering probiotics and its preparation method

Technical field:

The invention belongs to the technical field of bioengineering, and in particular relates to a double-layer alginate microsphere for delivering probiotics and a preparation method thereof.

Background technique:

Probiotics are a type of active microorganisms that are beneficial to the host in the human body and can effectively prevent gastroenteritis, diarrhea, obesity, etc., but they are easily affected by conditions such as gastric acid, bile salts, and pH. At present, the most commonly used protective measure is microencapsulation technology, which uses carbohydrates, vegetable gums, proteins and other substances as wall materials to protect them. However, different wall materials may have problems such as low encapsulation rate, poor protection effect, high cost, and insufficient safety, and the prepared microcapsules cannot control the distribution of probiotics, and the probiotics distributed on the surface will enter the human body faster. Inactivation, and the microcapsules prepared by the extrusion method usually have the problem of large particle size (2-5mm), which affects the mouthfeel.

Sodium alginate is a naturally occurring polysaccharide, which has the advantages of non-toxicity and low cost. Its aqueous solution has a high viscosity. The hydrogel formed with calcium ions is a new type of porous material, which is often used as a packaging material. , Wall material for delivering probiotics, but its high porosity may lead to lower encapsulation rate of probiotics and earlier leakage. Therefore, it is often used in combination with other substances to increase stability and protection.

Starch is a natural high-molecular carbohydrate, and it is a food that human beings must ingest in order to maintain life and health. Starch can be divided into fast-digesting starch, slow-digesting starch and resistant starch according to its speed and degree of digestion. Resistant starch (RS) refers to starch that cannot be digested and absorbed by the small intestine and can enter the colon to provide health benefits. Among them, type III resistant starch (RS3) is an aged starch with a high degree of stability, which is effective for controlling blood sugar concentration and body weight. Metabolites are also beneficial to the human body.

However, there are still some shortcomings and deficiencies in the research on the delivery system of probiotics in the prior art. For example, the porosity of alginate gel is relatively high, resulting in low encapsulation efficiency of alginate-based microcapsules for probiotics. There are 64.4%-79%; the structure of gellan gum-xanthan gum and chitosan-coated sodium alginate microspheres is relatively soft, and the structural stability needs to be improved [LinhPhuong Taa, Erika Bujnaa, Otilia Antal, et al.Effects of various polysaccharides (alginate, carrageenan, gums, chitosan) and their combination with prebiotics carbohydrates (resistant starch, lactosucrose, lactulose) on the encapsulation of probiotic bacteria Lactobacillus casei 01 strain [J]. International Journal of Biological Macromolecules, 202 1,183:1136-1144.] The particle size of the microcapsules made by the extrusion method is usually large, about 2-5mm, and the shape is irregular, which will have a certain impact on the quality, taste and fluidity of the food [Tian Wenjing, Zhu Yingdan, Yue Linfang, et al. Research status of probiotic microencapsulation [J]. Chinese Journal of Food Science, 2016, 16(8): 186-194.]. .

Invention content:

In order to solve some shortcomings and deficiencies in the research on the delivery system of probiotics in the prior art, the present invention constructs a double-layer alginate microsphere system for delivering probiotics. Under the fluid flow driven by centrifugal force, alginate Sodium and calcium chloride solutions are contacted to form a gel, and double-layer microspheres (200-500 μm) with controllable size are synthesized. Sodium alginate was used to immobilize probiotics on the inner layer of microspheres, and RS3 was added to the outer layer of sodium alginate to reduce the pores on the surface of gel microspheres, improve the encapsulation rate and the viability of probiotics. At the same time, the RS3 on the outer layer of the microspheres acts as a prebiotic, which is conducive to the colonization of the probiotics in the inner layer of the microspheres after reaching the large intestine.

In order to achieve the above object, the present invention is achieved through the following technical solutions, a method for preparing double-layered alginate microspheres for delivering probiotics, comprising the following steps:

(1) Sodium alginate-probiotic mixed liquid is prepared; wherein, the role of sodium alginate is to fix the probiotics on the inner layer of the microparticles.

(2) configure the sodium alginate-resistant starch mixture; wherein, the role of sodium alginate is to fix the resistant starch on the outer layer of the particle; adding resistant starch can be used to fill the pores of the formed alginate microspheres, In turn, the encapsulation efficiency is improved, and the diffusion of bile salts and the like into the microspheres is limited, so as to enhance the protective effect on probiotics. Resistant starch can enter the sodium alginate matrix, making the microspheres have greater hardness and stability, and can better resist the digestion of the human gastrointestinal tract. Resistant starch has the characteristics of not being digested and absorbed in the small intestine, which can protect the gel microspheres to be transported to specific parts such as the colon and then play a role. Resistant starch can be used as a prebiotic. After entering the colon, it becomes a nutrient source for probiotics and is used as a fermentation substrate by the colonic microbial community. The metabolites are also beneficial to the human body.

(3) Calcium chloride aqueous solution is configured; wherein, the calcium ions in the calcium chloride play a coupling role to make the sodium alginate generate gel particles.

(4) Gel granulation: Centrifuge for 10 minutes under the condition of centrifugal force 1000g, drip sodium alginate-probiotic aqueous solution and sodium alginate aqueous solution into calcium chloride aqueous solution at an even speed, soak until the hydrogel pellets are completely solidified, The hydrogel pellets were isolated.

At this time, the Na ⁺ of the carboxyl functional group of sodium alginate undergoes an ion exchange reaction with the divalent cation Ca ^{2 +} . At the same time, the carboxyl functional groups of sodium alginate are cross-linked to form a network structure, thereby forming gel microspheres.

Centrifugal force mainly affects the size and shape of microspheres, the greater the centrifugal force, the smaller the particle. Under the condition of centrifugal force of 1000g, gel microspheres with smaller particles and uniform shape can be prepared. However, when the centrifugal force is greater than 1000g, the sodium alginate particles form a "tail", resulting in irregular particle shapes, which will affect the sensory perception after it is added to food.

Further, step (1) is to configure a sodium alginate solution with a mass concentration of 1-3% (w/v); take the activated probiotic bacteria liquid and centrifuge and discard the supernatant, and prepare the prepared alginate Add the sodium solution to the sludge and vortex until homogenous.

If the concentration of sodium alginate is too high, the sodium alginate-probiotic mixture and sodium alginate-resistant starch will be too viscous, and the two mixtures cannot be dripped into the calcium chloride solution during centrifugation to prevent coagulation. The formation of gel microspheres; if the concentration of sodium alginate is too low, the solution will be too dilute, and a "thread-like" gel will be formed during centrifugation, and gel microspheres will not be formed.

Further, step (2) is to configure a sodium alginate solution with a mass concentration of 1-3% (w/v); centrifuge the resistant starch aqueous solution and discard the supernatant, and add the configured sodium alginate solution Into the pellet, vortex until homogeneous; resulting in a solution with a final concentration of resistant starch of 5-30 mg/mL.

For sodium alginate, choosing this concentration range can ensure that sodium alginate and calcium chloride can form gel microspheres, and can fix probiotics in the inner layer to obtain microspheres with more regular shape. If the concentration of sodium alginate is too high, the solution will be too viscous to drop the sodium alginate-probiotic mixture and sodium alginate-resistant starch mixture into the calcium chloride solution during centrifugation, thereby preventing gelation Formation of microspheres; if the concentration of sodium alginate is too low, the solution will be too dilute, and a "thread-like" gel will form during centrifugation, preventing the formation of gel microspheres.

For resistant starch, if the concentration of resistant starch is too high, the viscosity of sodium alginate-resistant starch aqueous solution will be affected, resulting in irregular shape of gel microspheres, or even a single layer of microspheres; if the resistance If the concentration of starch is too low, it cannot fully fill the pores of the outer layer of the gel microspheres, and reduce the protective effect on the probiotics embedded in the inner layer of the gel microspheres.

Further, the centrifugation parameters of step (1) and step (2) are centrifugation at 3000-5000rpm for 5-20min.

Selecting this range of centrifugation parameters can ensure that the centrifugation is complete. After discarding the supernatant, bacteria and resistant starch are obtained, which are used for the configuration of the mixed solution with sodium alginate. If the centrifugation speed and time are too low, the separation will not be complete, and the supernatant still contains more bacteria and resistant starch.

Further, step (3) is to configure a calcium chloride solution with a mass concentration of 5-10% (w/v).

The concentration of calcium chloride mainly affects the formation of gel microspheres and the tightness of gel network structure. This concentration range is suitable for the preparation of double-layer gel microspheres with regular shape and compact structure.

Further, step (4) is completed using a centrifuge module; the centrifuge module includes a first centrifuge tube, a second centrifuge tube and a third centrifuge tube that are nested in sequence from inside to outside; the first centrifuge tube is built into the first centrifuge tube that runs through the bottom. The needle; the bottom opening of the second centrifuge tube is connected to the second needle; the cap of the third centrifuge tube is provided with an opening matching the size of the second centrifuge tube.

Further, the first centrifuge tube is a 0.5mL centrifuge tube, the second centrifuge tube is a 1.5mL centrifuge tube, and the third centrifuge tube is a 50mL centrifuge tube.

Further, the steps of using the above-mentioned device for gel granulation are as follows: load the sodium alginate-probiotic aqueous solution configured in step (1) into a 0.5mL centrifuge tube, and load the sodium alginate-probiotic aqueous solution configured in step (2) into a 1.5mL centrifuge tube. Sodium alginate-resistant starch aqueous solution, put the calcium chloride aqueous solution configured in step (3) into a 50mL centrifuge tube, so that the distance between the needle and the liquid surface of the calcium chloride solution is 0.45-1.35cm; then centrifuge.

A double-layer alginate microsphere prepared by the above method, the inner layer is a gel network structure in which probiotics are fixed, the outer layer is a gel network structure filled with resistant starch in the pores, and the size is 200- 500μm, so as to fully protect the probiotics from the damage of the external environment.

The beneficial effects of the present invention are:

(1) On the basis of the existing technology, the present invention prepares a double-layer delivery system by improving the preparation process of the probiotic delivery system, which can well fix the probiotics in a certain area inside the microsphere, and add different probiotics to the outer layer. The resistant starch digested and absorbed by the small intestine improves the encapsulation efficiency and protection ability of probiotics, allowing them to reach the colon more to exert health benefits. Moreover, the raw materials used are safe, non-toxic and low in cost, and can be added to food.

(2) The preparation process of the present invention is simple, easy to control, low in energy consumption, environmentally friendly, low in cost, can realize the protective effect on the activity of probiotics, and the generated microspheres have regular shape and small particle size, and have little influence on the mouthfeel , suitable for application in food.

Description of drawings

Fig. 1 is a schematic structural view of the centrifuge module of the present invention.

Figure 2 is a structural diagram of a double-layer alginate microsphere.

Figure 3 is the effect of sodium alginate concentration on the morphology of microspheres. Among them, left picture: 5% (left), 3% (right) sodium alginate microspheres; middle picture: microscopic picture of 5% sodium alginate microspheres; right picture: microscopic picture of 3% sodium alginate microspheres

Figure 4 is the effect of centrifugal force on the particle size of sodium alginate microspheres.

In the figure, a first centrifuge tube 1 , a second centrifuge tube 2 , a third centrifuge tube 3 , a first needle 4 , and a second needle 5 .

Detailed ways:

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all Example. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Example 1:

A preparation method for delivering probiotic double-layer alginate microspheres, comprising the following steps:

(1) Weigh the sodium alginate powder respectively, add deionized water, configure a solution with a mass concentration of 1-3% (w/v), and stir until uniform with a DC mixer equipped with a paddle-type stirring paddle;

Take the activated Enterococcus Faecium R7 bacteria solution and centrifuge at 5000rpm for 10 minutes in a centrifuge, discard the supernatant, and add the prepared 2% (w/v) sodium alginate solution to the bacteria sludge, and vortex until uniform.

(2) Centrifuge the resistant starch aqueous solution at 5000 rpm for 10 minutes in a centrifuge, discard the supernatant, and add the prepared 1% (w/v) sodium alginate solution to the precipitate, and vortex until uniform. Wherein, the final concentration of resistant starch is 5 mg/mL.

(3) Weigh the calcium chloride powder, add deionized water, and prepare a calcium chloride solution with a mass concentration of 10% (w/v).

(4) Prepare the centrifuge module as shown in Figure 1: the centrifuge module includes the first centrifuge tube 1, the second centrifuge tube 2 and the third centrifuge tube 3 which are nested in sequence from inside to outside; Needle 4; the bottom of the second centrifuge tube is open, connected to the second needle 5; the tail of the first needle 4 is connected to the needle of the second needle 5; the third centrifuge tube 3 cap is provided with the size of the second centrifuge tube mouth opening. Cut out a hole of a certain size in the cap of the 50mL third centrifuge tube; fix the second needle at the bottom of the 1.5mL second centrifuge tube, cut a small opening at the bottom of the centrifuge tube; insert the first needle into the 0.5mL first centrifuge tube Tube, and nested assembly of two centrifuge tubes, fixed on the 50mL centrifuge tube cover.

Put 2% sodium alginate-probiotic aqueous solution into a 0.5mL centrifuge tube, put 1% sodium alginate aqueous solution into a 1.5mL centrifuge tube, put 10% calcium chloride aqueous solution into a 50mL centrifuge tube, and keep the needle from The liquid level of calcium chloride solution is kept at a certain distance. Centrifuge at a centrifugal force of 1000g for 10 minutes, drop 2% sodium alginate-probiotic aqueous solution and 1% sodium alginate aqueous solution into 10% calcium chloride aqueous solution at an even speed, soak until the hydrogel beads are completely solidified, and separate the water Gel pellets were rinsed with deionized water and stored in deionized water. Its structure was observed with an optical microscope as shown in Figure 2.

The cells of the calcium chloride solution after centrifugation were counted using a hemocytometer, and no bacteria were found in the calcium chloride solution after centrifugation, which indicated that the encapsulation rate of bacteria by double-layer alginate microspheres could reach 100%.

Example 2:

Prepare the following three solutions respectively:

3% (w/v) sodium alginate + probiotic aqueous solution;

2% (w/v) sodium alginate + 20mg/mL resistant starch aqueous solution;

8% (w/v) calcium chloride in water.

All the other are identical with embodiment 1.

Example 3:

Prepare the following three solutions respectively:

1% (w/v) sodium alginate + probiotic aqueous solution;

1% (w/v) sodium alginate + 5mg/mL resistant starch aqueous solution;

5% (w/v) calcium chloride in water.

All the other are identical with embodiment 1.

The influence of the concentration of sodium alginate on the morphology of the microspheres is shown in Figure 3, and the concentration of calcium chloride and the centrifugal force are the same as in Example 1. Microspheres prepared with 5% sodium alginate have a "tail" phenomenon, and this irregular sphere structure will affect the taste after being added to food, thereby affecting the sensory quality. Reducing the concentration of sodium alginate can reduce the viscosity of the solution, which can obviously improve this phenomenon. When the concentration is 3%, the prepared microspheres are spherical in good shape.

The effect of centrifugal force on the particle size of the microspheres is shown in Figure 4. Under the same conditions of sodium alginate concentration and calcium chloride concentration as in Example 1, the particle size of the sodium alginate microspheres decreases as the centrifugal force increases.

Claims (6)

1. a preparation method for delivering the double-layer alginate microspheres of probiotics, it is characterized in that comprising the following steps: (1) configure sodium alginate-probiotic aqueous solution; (2) configuring sodium alginate-resistant starch aqueous solution; (3) configure calcium chloride aqueous solution; (4) Gel granulation: Centrifuge for 10 minutes under the condition of centrifugal force 1000g, drip sodium alginate-probiotic aqueous solution and sodium alginate aqueous solution into calcium chloride aqueous solution at an even speed, soak until the hydrogel pellets are completely solidified, Separation of hydrogel pellets; complete with a centrifugal module; the centrifugal module includes a first centrifuge tube, a second centrifuge tube, and a third centrifuge tube that are nested in sequence from the inside to the outside; the first centrifuge tube has a built-in first centrifuge tube that runs through the bottom needle; the bottom opening of the second centrifuge tube is connected to the second needle; the third centrifuge tube cover is provided with an opening matching the size of the second centrifuge tube; The first centrifuge tube is a 0.5mL centrifuge tube, the second centrifuge tube is a 1.5mL centrifuge tube, and the third centrifuge tube is a 50mL centrifuge tube; Put the sodium alginate-probiotic aqueous solution prepared in step (1) into a 0.5mL centrifuge tube, put the sodium alginate-resistant starch aqueous solution prepared in step (2) into a 1.5mL centrifuge tube, and put it into a 50mL centrifuge tube Load the calcium chloride aqueous solution configured in step (3), so that the distance between the needle and the liquid surface of the calcium chloride solution is 0.45-1.35 cm; then perform centrifugation. 2. preparation method as claimed in claim 1 is characterized in that: step (1) is the sodium alginate solution that configuration mass concentration is 1-3% (w/v); Discard the supernatant, and add the prepared sodium alginate solution to the sludge, and vortex until uniform. 3. The preparation method as claimed in claim 1, characterized in that: step (2) is to configure a sodium alginate solution with a mass concentration of 1-3% (w/v); take the resistant starch aqueous solution and centrifuge and discard the upper Clear liquid, and add the prepared sodium alginate solution to the precipitate, vortex until uniform; obtain a solution with a final concentration of resistant starch of 5-30mg/mL. 4. The preparation method according to claim 2 or 3, characterized in that: the centrifugal parameter is 3000-5000rpm for 5-20min. 5. The preparation method according to claim 1, characterized in that: step (3) is to configure a calcium chloride solution with a mass concentration of 5-10% (w/v). 6. A double-layer alginate microsphere prepared by any method of claim 1-5.

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