A probiotic-loaded aerogel-hydrogel composite and a method of making the same

Abstract

The application provides a probiotic-loaded aerogel-hydrogel composite material and a preparation method thereof. The probiotic-loaded aerogel-hydrogel composite material comprises: a sodium alginate-gelatinized high-amylose starch aerogel; an active ingredient, wherein the active ingredient comprises probiotics, and the active ingredient is at least loaded in the pores of the sodium alginate-gelatinized high-amylose starch aerogel; and a sodium alginate-calcium chloride hydrogel coating, which is coated on the surface of the sodium alginate-gelatinized high-amylose starch aerogel loaded with the active ingredient. The probiotic-loaded aerogel-hydrogel composite material provided by the application can protect the active probiotics from passing through the stomach acid environment smoothly, and stably release the probiotics in the distal intestinal region, i.e., the colon, to achieve controllable release of the probiotics.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medical materials, and particularly relates to a probiotic-loaded aerogel-hydrogel composite material and a preparation method thereof. BACKGROUND

[0002] As a dietary supplement ingredient or functional food, probiotics are one of the prevention and treatment strategies for various diseases, which can regulate the composition and number of intestinal microorganisms, inhibit harmful bacteria, produce short-chain fatty acids, and regulate the host immune system, thereby being beneficial to human health. However, the physiological environment of the gastrointestinal tract during the oral process causes low survival rate and insufficient intestinal retention of probiotics, thereby limiting the beneficial effects of probiotics. Microencapsulation of probiotics is widely used as one of the effective ways to improve the survival rate of probiotics before reaching the target area. However, the encapsulated structure carrier releases probiotics through pH value, permeation and microbial changes, which can cause the sudden swelling, dissolution or decomposition of the encapsulated structure in the gastrointestinal tract. This poor control characteristic can cause probiotics to burst and release rapidly, making it difficult to accurately control the release rate and duration, and unable to deliver probiotics to the colon site in the distal intestinal region where the probiotics can exert their beneficial effects.

[0003] Currently, the probiotic delivery system mainly focuses on specific material synthesis strategies, including complex polymer design and cumbersome synthesis steps, which may compromise the feasibility of effective loading and is not conducive to oral administration. Food-grade aerogels represented by polysaccharides have excellent biological activity, biocompatibility and biodegradability, and have potential advantages as delivery carriers. Sodium alginate is a pH-responsive and intestinal adhesive water-soluble polysaccharide, and the use of sodium alginate to load probiotics can achieve targeted release of probiotics in the intestinal site, but the hydrophilicity and porous network of sodium alginate gel cannot fully maintain the viability of probiotics in the gastrointestinal tract. The micro-porous structure of aerogel is an important bridge connecting molecular information and macroscopic properties, and plays an important role in inheriting and even amplifying the functions and characteristics at the molecular scale. However, the porous structure of aerogel can easily cause probiotic leakage and hydrogen ion entry, which is not conducive to achieving high loading and gastrointestinal tolerance of probiotics.

[0004] Therefore, it is an urgent problem to be solved to achieve efficient loading protection of probiotics and precise controlled release in the colon site. SUMMARY

[0005] To solve all or part of the above technical problems, the present application provides the following technical solutions:

[0006] The first aspect of the present application provides a probiotic-loaded aerogel-hydrogel composite material, which comprises:

[0007] sodium alginate-gelatinized high-amylose starch aerogel;

[0008] active ingredients, the active ingredients including probiotics, the active ingredients being at least loaded in the pores of the sodium alginate-gelatinized high-amylose starch aerogel;

[0009] a sodium alginate-calcium chloride hydrogel coating, which is coated on the surface of the sodium alginate-gelatinized high-amylose starch aerogel loaded with the active ingredients.

[0010] The high-amylose starch (referring to starch with the content of amylose exceeding 50 wt%) used in the present application has a high resistant starch content and the ability of resisting digestion in the stomach and small intestine, and the formed sodium alginate-gelatinized high-amylose starch aerogel can make up for the deficiency of sodium alginate being easily eroded in the stomach and small intestine. Meanwhile, considering that the granular structure of high-amylose starch is difficult to be used as a carrier of nutrients, the present application gelatinizes the high-amylose starch to reduce the crystallinity of the high-amylose starch, change the granular morphology and diffuse out the amylose wrapped by the starch to form a biocompatible network. Moreover, the gelatinization is also conducive to prolonging the activity of the probiotics loaded in the gel.

[0011] The release behavior of the probiotics of the aerogel-hydrogel composite material loaded with probiotics provided by the present application is related to the swelling, erosion of the aerogel and the properties of food ingredients, and the release mechanism is as follows:

[0012] In the composite material provided by the present application, the aerogel loaded with probiotics is embedded in the hydrogel, and the external hydrogel can avoid the leakage of probiotics during oral administration. Moreover, in the stomach environment with a larger acidity, the external hydrogel provides protection for the probiotics, thereby improving the survival rate of the probiotics under strong acid conditions. With the increase of pH value, the carboxyl groups of sodium alginate in the sodium alginate-gelatinized high-amylose starch aerogel form anionic end groups. Due to the mutual repulsion of the same charges, the aerogel swells, the gelatinized high-amylose starch forms intermolecular and intramolecular hydrogen bonds, the erosion rate of the aerogel is controlled by adjusting the crosslinking strength, and the probiotics form larger-sized probiotic clusters through the self-aggregation ability. The probiotics closely interacting with the aerogel skeleton are combined by hydrogen bonds, and the slow rupture of the hydrogen bonds promotes the release of the probiotics. That is, the aerogel-hydrogel composite material loaded with probiotics provided by the present application can protect the active probiotics to smoothly pass through the acidic environment of the stomach, and stably release the probiotics in the distal colon region of the intestinal tract, thereby realizing the controlled release of the probiotics.

[0013] In some embodiments, the sodium alginate-gelatinized high-amylose starch aerogel has a vertical pore structure, and the active ingredients are at least loaded in the vertical pore structure. The aerogel with a vertical ordered pore structure disintegrates and releases the probiotics in the form of a peeled onion, so that the release amount of the probiotics per hour is stable, which is conducive to the controlled and sustained release of the probiotics in the intestinal tract.

[0014] In some embodiments, the sodium alginate-gelatinized high-amylose starch aerogel has a porosity of 66% to 83% and contains pores with a diameter of 10 μm to 140 μm.

[0015] In some embodiments, the sodium alginate-gelatinized high-amylose starch aerogel is obtained by freeze-drying a mixed solution containing sodium alginate and gelatinized high-amylose starch.

[0016] In some embodiments, the sodium alginate-gelatinized high-amylose starch aerogel contains 1 wt% to 4 wt% of gelatinized high-amylose starch.

[0017] In some embodiments, the active ingredient has a loading amount of 2 x 10 8 ~4 x 10 8 .

[0018] The probiotic bacteria can be any known in the art. In some embodiments, the probiotic bacteria include one or a combination of Lactobacillus rhamnosus, Lactobacillus plantarum, or Bifidobacterium.

[0019] A second aspect of the present application provides a method for preparing a probiotic bacteria-loaded aerogel-hydrogel composite material, the method comprising:

[0020] providing a mixed solution containing sodium alginate and gelatinized high-amylose starch, and preparing a sodium alginate-gelatinized high-amylose starch aerogel from the mixed solution;

[0021] loading an active ingredient into the pores of the sodium alginate-gelatinized high-amylose starch aerogel, the active ingredient including probiotic bacteria, to obtain a probiotic bacteria-loaded aerogel;

[0022] immersing the probiotic bacteria-loaded aerogel sequentially in a solution containing sodium alginate and a solution containing calcium chloride to coat the surface of the probiotic bacteria-loaded aerogel with a sodium alginate-calcium chloride hydrogel coating, and obtaining a probiotic bacteria-loaded aerogel-hydrogel composite material.

[0023] In some embodiments, the mass ratio of sodium alginate to gelatinized high-amylose starch in the mixed solution is 1:2 to 2:1.

[0024] In some embodiments, the gelatinized high-amylose starch is obtained by reacting an aqueous dispersion containing high-amylose starch at 100 to 120 °C, wherein the high-amylose starch contains 50 wt% or more of amylose.

[0025] In some embodiments, the preparation method of the gelatinized high-amylose starch comprises: mixing high-amylose starch and water at a mass-volume ratio of 2g:100mL-8g:100mL to obtain a high-amylose starch solution; and reacting the high-amylose starch solution at a temperature of 100-120℃ for 30-40min to obtain the gelatinized high-amylose starch.

[0026] In some embodiments, the method for preparing the sodium alginate-gelatinized high-amylose starch aerogel comprises directional freeze-drying.

[0027] In some embodiments, the directional freeze-drying is performed at a temperature of-30℃--20℃.

[0028] In some embodiments, the directional freeze-drying is performed for 36h-48h.

[0029] In some embodiments, the preparation method specifically comprises: formulating the probiotic bacteria into a probiotic bacteria solution with a concentration of 1x10 10 ~1x10 12 CFU / mL, and allowing the probiotic bacteria solution to enter the pores of the sodium alginate-gelatinized high-amylose starch aerogel and be cross-linked, thereby obtaining the probiotic bacteria-loaded aerogel.

[0030] In some embodiments, the cross-linking is performed for 5min-10min to allow the probiotic bacteria to be loaded into the pores of the sodium alginate-gelatinized high-amylose starch aerogel.

[0031] In some embodiments, the sodium alginate-containing solution has a concentration of 10mg / mL-20mg / mL.

[0032] In some embodiments, the calcium chloride-containing solution has a concentration of 10mg / mL-20mg / mL.

[0033] In some embodiments, the preparation method specifically comprises: immersing the probiotic bacteria-loaded aerogel in the sodium alginate-containing solution for 5min-10min, and then immersing the aerogel in the calcium chloride-containing solution for 5min-10min, to obtain the probiotic bacteria-loaded aerogel-hydrogel composite material.

[0034] The probiotic bacteria can be any known probiotic bacteria in the art. In some embodiments, the probiotic bacteria comprise one or a combination of Lactobacillus rhamnosus, Lactobacillus plantarum, or Bifidobacterium.

[0035] The third aspect of the present application provides a probiotic bacteria-loaded aerogel-hydrogel composite material, which is obtained by any of the preparation methods.

[0036] The aerogel-hydrogel composite material loaded with probiotics provided by the application can be applied to efficient delivery of probiotics in the intestinal tract.

[0037] Compared with the prior art, the application has at least the following beneficial effects:

[0038] (1) On the one hand, the aerogel-hydrogel composite material loaded with probiotics provided by the application can avoid leakage of probiotics during oral administration and improve the survival rate of probiotics in a strong acid environment in the stomach due to the protection of the external hydrogel coating; on the other hand, compared with a pure sodium alginate aerogel, the sodium alginate-gelatinized high-amylose starch aerogel used in the application is not easy to be eroded in the stomach and small intestine and can stably release probiotics in the distal colon region of the intestinal tract, so that the controlled release of probiotics in the colon can be achieved.

[0039] (2) The sodium alginate-gelatinized high-amylose starch aerogel used in the application has a vertical pore structure, which is beneficial to the controlled and stable release of probiotics; in addition, the porosity of the sodium alginate-gelatinized high-amylose starch aerogel is high, and the pore size of the pores contained therein is suitable, so that a high loading amount of probiotics can be achieved.

[0040] (3) The preparation method of the aerogel-hydrogel composite material loaded with probiotics provided by the application is simple in operation, low in cost and safe and non-toxic. BRIEF DESCRIPTION OF DRAWINGS

[0041] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments described in the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0042] Figure 1 Optical photographs of the sodium alginate-gelatinized high-amylose corn starch aerogel with a vertical pore structure prepared for Example 1 in the cross section (a) and longitudinal section (b);

[0043] Figure 2 Scanning electron microscope images of the sodium alginate-gelatinized high-amylose corn starch aerogel with a vertical pore structure prepared for Example 1 in the cross section (a) and longitudinal section (b);

[0044] Figure 3 Energy dispersive spectroscopy mapping image of the sodium alginate-gelatinized high-amylose corn starch aerogel prepared for Example 1;

[0045] Figure 4 Sodium alginate aerogel prepared for Comparative Example 1 (left) and sodium alginate-gelatinized high-amylose corn starch aerogel prepared for Example 1 (right); Figure 4 left) and sodium alginate-gelatinized high-amylose corn starch aerogel prepared for Example 1 (right);Figure 4 Scanning electron microscope images of the sodium alginate aerogel of Comparative Example 1 and the sodium alginate-gelatinized high-amylose corn starch aerogels of Examples 1-3;

[0046] Figure 5 A comparison chart of the porosities of the sodium alginate aerogel of Comparative Example 1 and the sodium alginate-gelatinized high-amylose corn starch aerogels of Examples 1-3;

[0047] Figure 6 A pore size distribution chart of the cross-section of the sodium alginate-gelatinized high-amylose corn starch aerogel of Example 3;

[0048] Figure 7 A comparison chart of the XRD spectra of the sodium alginate, gelatinized high-amylose corn starch used in Example 1 and the sodium alginate-gelatinized high-amylose corn starch aerogel produced;

[0049] Figure 8 A comparison chart of the FITR spectra of the sodium alginate-gelatinized high-amylose corn starch aerogels of Examples 1-3 and the sodium alginate aerogel of Comparative Example 1;

[0050] Figure 9 A comparison chart of the resistant starch content of the gelatinized high-amylose corn starch used in Example 1 and the sodium alginate-gelatinized high-amylose corn starch aerogel produced;

[0051] Figure 10 A comparison chart of the volume shrinkage of the sodium alginate aerogel of Comparative Example 1 and the sodium alginate-gelatinized high-amylose corn starch aerogels of Examples 1-3 before and after loading probiotics;

[0052] Figure 11 A comparison chart of the volume shrinkage of the sodium alginate aerogel of Comparative Example 1 and the sodium alginate-gelatinized high-amylose corn starch aerogels of Examples 1-3 before and after loading probiotics;

[0053] Figure 12 Scanning electron microscope images of the sodium alginate-gelatinized high-amylose corn starch aerogel of Example 1 loaded with probiotics;

[0054] Figure 13 A comparison chart of the number of probiotics loaded in the sodium alginate aerogel of Comparative Example 1 and the sodium alginate-gelatinized high-amylose corn starch aerogels of Examples 1-3;

[0055] Figure 14 Fluorescent images of the fluorescently labeled probiotics and the fluorescently labeled aerogel of Example 1;

[0056] Figure 15 Aerogel-hydrogel composites (corresponding to the "coating" row in Figure 15 ) of Examples 1-3, Comparative Example 1 loaded with probiotics, and as a control, aerogel loaded with probiotics (corresponding to the "aerogel" row inFigure 15 Fluorescence images of free fluorescently labeled probiotic bacteria in simulated gastric fluid for Example 1-3, Comparative Example 1 (corresponding to

[0057] Figure 16 OD values of the probiotic bacteria-loaded aerogel-hydrogel composites (corresponding to Figure 16 “Coated” group in Example 1-3, Comparative Example 1, and probiotic bacteria-loaded aerogels (corresponding to Figure 16 “Uncoated” group in Example 1-3, Comparative Example 1) in simulated gastric fluid;

[0058] Figure 17 Live / dead bacteria staining images of the probiotic bacteria-loaded aerogel-hydrogel composites (corresponding to Figure 17 “Coated” group in Example 1-3, Comparative Example 1, and probiotic bacteria-loaded aerogels (corresponding to Figure 17 “Uncoated” group in Example 1-3, Comparative Example 1) after treatment with simulated gastric fluid;

[0059] Figure 18 Comparison plots of the number of viable probiotic bacteria in the probiotic bacteria-loaded aerogel-hydrogel composites (corresponding to Figure 18 “Coated” group in Example 1-3, Comparative Example 1, and probiotic bacteria-loaded aerogels (corresponding to Figure 18 “Uncoated” group in Example 1-3, Comparative Example 1) treated with simulated gastric fluid;

[0060] Figure 19 Microscopic disintegration images of the aerogels in Comparative Example 1, Example 1-3 in simulated colonic fluid;

[0061] Figure 20 Morphology change images of the probiotic bacteria-loaded aerogel-hydrogel composites in Comparative Example 1, Example 1-3 in simulated small intestinal fluid and simulated colonic fluid;

[0062] Figure 21 Comparison plots of the change in swelling diameter of the aerogels in Comparative Example 1, Example 1-3 in simulated colonic fluid;

[0063] Figure 22 Probiotic bacteria cumulative release amount plots of the probiotic bacteria-loaded aerogel-hydrogel composites in Comparative Example 1, Example 1-3 in simulated small intestinal fluid and simulated colonic fluid;

[0064] Figure 23 Probiotic bacteria release amount plots of the probiotic bacteria-loaded aerogel-hydrogel composites in Comparative Example 1, Example 1-3 in simulated small intestinal fluid every 2 h and in simulated colonic fluid every 1 h;

[0065] Figure 24For the probiotic release kinetics of the probiotic-loaded aerogel-hydrogel composite material of Example 3, the cumulative release amount of probiotics was fitted to the kinetic model, including zero-order model (a) in Table 1 Figure 24 , first-order model (b) in Table 1 Figure 24 , Higuchi model (c) in Table 1 Figure 24 , and Ritger-Peppas model (d) in Table 1. Figure 24

[0066] Figure 5 , Figure 11 , Figure 13 , Figure 16 , Figure 18 The letters (a, b, c, d) involved in Table 1 are the grouping marks of statistically significant differences; wherein, the groups marked with the same letter represent no statistically significant difference; the groups marked with different letters represent statistically significant difference; the groups sharing the same letter represent no significant difference, for example, one group is marked as a and b, and the other group is marked as b, because of sharing the letter b, the difference between the two is not significant. DETAILED DESCRIPTION

[0067] The technical solutions of the present application will be described in detail below with specific examples, so that the technical personnel in the art can better understand and implement the technical solutions of the present application. The specific functional details disclosed herein should not be interpreted as limiting, but only as the basis of the claims and for teaching the skilled in the art to adopt the representative basis of the present application in different ways in any appropriate detailed embodiment in fact.

[0068] In addition, unless otherwise specified, the various raw materials used in the following examples can be obtained from the market or other channels, and the various production and testing equipment used are all known equipment in the art, and the testing methods used are any known method in the art.

[0069] Example 1

[0070] Example 1 provides a probiotic-loaded aerogel-hydrogel composite material and a preparation method thereof, which specifically comprises the following steps:

[0071] (1) Preparation method of gelatinized high amylose starch: 2 g of high amylose corn starch with amylose content of 50 wt% or more was dissolved in 100 mL of water, and the solution was transferred to a high-pressure reaction kettle after being mixed thoroughly. The solution was reacted at 120 ℃ for 30 min; after the reaction was completed, a gelatinized high amylose starch solution with a concentration of 20 mg / mL was obtained.

[0072] ​​(2) Preparation of sodium alginate-paste high-amylose corn starch aerogel with vertical channel structure: After sodium alginate (2 g) was fully dissolved in sterile water (50 mL), 50 mL of the prepared 20 mg / mL paste high-amylose corn starch solution was added, and the mixture was uniformly mixed to obtain a mixed solution; the mixed solution was transferred to a directional freezing mold, and frozen at a temperature of -20 °C, and then the frozen material was transferred to a vacuum freeze dryer for vacuum freeze drying for 48 h to obtain a sodium alginate-paste high-amylose corn starch aerogel with vertical channel structure.

[0073] (3) Preparation of probiotic-loaded aerogel: Lactobacillus rhamnosus bacteria were resuspended in a calcium chloride (20 mg / mL) solution, and the concentration of Lactobacillus rhamnosus was adjusted to 1×10 10 CFU / mL; the calcium chloride solution of Lactobacillus rhamnosus was added dropwise into the channel of the prepared aerogel, and the crosslinking time was 10 min to prepare a probiotic-loaded aerogel.

[0074] (4) Preparation of probiotic-loaded aerogel-hydrogel composite material: The probiotic-loaded aerogel was soaked in a sodium alginate solution with a concentration of 20 mg / mL for 10 min, and then soaked in a calcium chloride solution with a concentration of 20 mg / mL for crosslinking for 10 min to obtain a probiotic-loaded aerogel-hydrogel composite material.

[0075] Figure 1 Optical photographs of the sodium alginate-paste high-amylose corn starch aerogel with vertical channel structure prepared in Example 1 in the cross section (a) and the longitudinal section (b), and the aerogel can be prepared into a carrier with a size suitable for human oral administration (such as a carrier with a diameter of 8 mm and a height of 8 mm) by cutting.

[0076] Figure 2 Scanning electron microscope images of the sodium alginate-paste high-amylose corn starch aerogel with vertical channel structure prepared in Example 1 in the cross section (a) and the longitudinal section (b). As can be seen from Figure 2 , the aerogel has a vertical and ordered channel structure in the longitudinal section.

[0077] Figure 3 Energy dispersive spectroscopy mapping image of the sodium alginate-paste high-amylose corn starch aerogel prepared in Example 1. As can be seen from Figure 3 , the elements are uniformly distributed in the aerogel.

[0078] Example 2

[0079] Example 2 is substantially the same as Example 1, except that in Example 2, 4 g of high-amylose corn starch is used in step (1) to prepare a gelatinized high-amylose corn starch solution with a concentration of 40 mg / mL, which is used to prepare the sodium alginate-gelatinized high-amylose corn starch aerogel of Example 2. The rest is the same as Example 1, which is not described here again. Finally, the probiotic-loaded aerogel-hydrogel composite material is obtained.

[0080] Example 3

[0081] Example 3 is substantially the same as Example 1, except that in Example 3, 8 g of high-amylose corn starch is used in step (1) to prepare a gelatinized high-amylose corn starch solution with a concentration of 80 mg / mL, which is used to prepare the sodium alginate-gelatinized high-amylose corn starch aerogel of Example 3. The rest is the same as Example 1, which is not described here again. Finally, the probiotic-loaded aerogel-hydrogel composite material is obtained.

[0082] Example 4

[0083] Example 4 is different from Example 1 only in that in step (4), the probiotic-loaded aerogel is soaked in a sodium alginate solution with a concentration of 10 mg / mL for 5 min, and then soaked in a calcium chloride solution with a concentration of 10 mg / mL for crosslinking for 5 min, to obtain the probiotic-loaded aerogel-hydrogel composite material. The rest is the same as Example 1, which is not described here again.

[0084] The probiotic-loaded aerogel-hydrogel composite material prepared in Example 4 has an effect comparable to that of Example 1.

[0085] Comparative Example 1

[0086] Comparative Example 1 is substantially the same as Example 1, except that in Comparative Example 1, no gelatinized high-amylose starch solution is used to prepare the aerogel, and only sodium alginate is used to prepare the sodium alginate aerogel, which specifically includes: 2 g of sodium alginate is fully dissolved in 100 mL of sterile water, and then the solution is transferred to a directional freezing mold for directional freezing at -20°C; then the frozen material is transferred to a vacuum freeze dryer for vacuum freeze drying for 48 h to obtain a sodium alginate aerogel. The rest is the same as Example 1, which is not described here again, and the probiotic-loaded sodium alginate aerogel-hydrogel composite material is prepared.

[0087] Figure 4 The scanning electron microscope images of the sodium alginate aerogel prepared in Comparative Example 1 ( Figure 4 left) and the sodium alginate-gelatinized high-amylose corn starch aerogel prepared in Example 1 ( Figure 4 right). From the images, it can be seen that the sodium alginate aerogel prepared in Comparative Example 1 has a larger pore size and a more irregular pore structure than the sodium alginate-gelatinized high-amylose corn starch aerogel prepared in Example 1. Figure 4As can be seen, the surface of sodium alginate aerogel in Comparative Example 1 is smooth, while the surface of aerogel after adding gelatinized high amylose is rougher, which is beneficial for probiotics to be loaded on the surface of the aerogel.

[0088] Figure 5 This is a comparison chart of the porosity of the aerogels prepared in Comparative Example 1 and Examples 1-3. Figure 5 It is known that the porosity of aerogels can reach 60%-80%, and the porosity of aerogels gradually increases with the increase of gelatinized high amylose content. Higher porosity is beneficial for probiotic loading.

[0089] Figure 6 This is a pore size distribution diagram of the cross-section of the sodium alginate-gelatinized high amylose corn starch aerogel prepared in Example 3. Figure 6 It is known that the pore size of the aerogel is in the range of 10-140 μm, which is suitable for probiotics to enter.

[0090] Figure 7 This is a comparison of the XRD spectra of sodium alginate, gelatinized high amylose corn starch, and the prepared sodium alginate-gelatinized high amylose corn starch aerogel used in Example 1. Figure 7 It is known that the gelatinization process disrupts the crystal structure of starch molecules, causing the molecular chains to expand and exhibit a disordered state, ultimately resulting in the disappearance of characteristic peaks. Compared to uncreteurized high amylose corn starch, gelatinizing it first and then preparing aerogels can reduce the crystallinity of high amylose, change the particle morphology, and diffuse out the starch-encapsulated amylose, which can form a biocompatible network with probiotics; furthermore, gelatinization also helps prolong the activity of probiotics loaded in the gel.

[0091] Figure 8 The FITR spectra of sodium alginate-gelatinized high amylose corn starch aerogels prepared in Examples 1-3 and sodium alginate aerogels prepared in Comparative Example 1 are shown. No new absorption peaks were detected in the sodium alginate-gelatinized high amylose corn starch aerogel, indicating that the interaction between sodium alginate and gelatinized high amylose corn starch is mainly dominated by hydrogen bonds.

[0092] Figure 9 This is a comparison chart of the resistant starch content in the gelatinized high amylose corn starch used in Example 1 and the sodium alginate-gelatinized high amylose corn starch aerogel prepared in Example 1. From... Figure 9 As can be seen, compared with simple gelatinized high amylose, sodium alginate-gelatinized high amylose corn starch aerogel has a higher content of resistant starch, which is more beneficial for the aerogel to resist erosion in the stomach and small intestine.

[0093] Figure 10These are photographs showing the morphological changes of the sodium alginate aerogel in Comparative Example 1 and the sodium alginate-gelatinized high amylose corn starch aerogels in Examples 1-3 before and after loading with probiotics. Figure 10 As can be seen, compared to aerogels without probiotics, aerogels shrink in volume after probiotics are loaded into the pores.

[0094] Figure 11 This is a comparison chart of the volume shrinkage rates of sodium alginate aerogel in Comparative Example 1 and the sodium alginate-gelatinized high amylose corn starch aerogels in Examples 1-3. The aerogel volume shrinkage rate is the percentage of the difference between the initial volume of the aerogel and the volume of the aerogel after probiotic loading, relative to the initial volume of the aerogel. As the content of gelatinized high amylose corn starch increases, the volume shrinkage rate of the prepared aerogel gradually decreases. A smaller volume shrinkage rate provides favorable conditions for increasing the probiotic loading.

[0095] Figure 12 The image shown is a scanning electron microscope image of the sodium alginate-gelatinized high amylose corn starch aerogel loaded with probiotics in Example 1. Figure 12 It can be seen that a large number of probiotics are loaded in the pores of the aerogel.

[0096] Figure 13 This is a comparison chart showing the number of probiotics loaded on the sodium alginate aerogel prepared in Comparative Example 1 and the sodium alginate-gelatinized high amylose corn starch aerogels prepared in Examples 1-3. Figure 13 As can be seen, with the increase of gelatinized high-amylose corn starch content, the number of probiotics that the aerogel can support gradually increases. This is because a high content of gelatinized high-amylose corn starch can reduce the volume shrinkage of the aerogel, allowing more probiotics to be loaded within the aerogel channels. Aerogels prepared within the gelatinized high-amylose corn starch content range provided in this invention can achieve a probiotic loading capacity of 4 × 10⁻⁶. 8 With CFUs or more, sodium alginate aerogel in Comparative Example 1 can achieve a probiotic loading capacity that is more than 2 times higher.

[0097] This invention also uses Rhodamine B (red) to label the sodium alginate-gelatinized high amylose corn starch aerogel prepared in Example 1, and uses fluorescein isothiocyanate (green) to label probiotics. Figure 14 The images show fluorescently labeled probiotics and fluorescently labeled aerogels from Example 1. The sodium alginate-gelatinized high amylose corn starch aerogel loaded with probiotics exhibits both red and green light, demonstrating that the probiotics were successfully loaded into the aerogel pores.

[0098] The present invention tests the relevant properties of the probiotic-loaded aerogel-hydrogel composite materials prepared in Examples 1-3 and Comparative Example 1, and uses the probiotic-loaded aerogels in Examples 1-3 and Comparative Example 1 without hydrogel coating as a control (i.e., without step (4) in the preparation method):

[0099] 1. The survival rate of probiotic-loaded aerogel-hydrogel composite materials in simulated gastric juice was tested, specifically including:

[0100] (1) Probiotics labeled with fluorescein isothiocyanate (3 mg / mL) were loaded onto the aerogel-hydrogel composite material in Examples 1-3 and Comparative Example 1, and the aerogel loaded with probiotics without hydrogel coating. They were incubated in simulated gastric juice (pH 2.0) at 37 °C with shaking at 140 rpm for 2 h. Fluorescence images were taken at 0 h and 2 h in the gastric juice.

[0101] (2) The aerogel-hydrogel composite material loaded with probiotics and the aerogel loaded with probiotics without hydrogel coating were incubated in simulated gastric juice (pH 2.0) at 37 ℃ with shaking at 140 rpm for 2 h. The optical density values ​​of the gastric juice were measured at 0 h and 2 h respectively.

[0102] (3) Dissolve the probiotic-loaded aerogel-hydrogel composite material and the probiotic-loaded aerogel without hydrogel coating in 10 mL of sodium citrate (50 mg / mL). Then stain the solution with calcein-propidium iodide dye for live / dead bacteria for 20 min. Observe the number of live and dead bacteria under a fluorescence microscope and take pictures.

[0103] (4) Dissolve the aerogel-hydrogel composite material loaded with probiotics and the aerogel loaded with probiotics in 10 mL of sodium citrate (50 mg / mL). Spread the solution on MRS agar plates and incubate at 37 °C for 24 h. Then calculate the number of probiotics by plate counting method.

[0104] Figure 15 The aerogel-hydrogel composite materials loaded with probiotics in Examples 1-3 and Comparative Example 1 (corresponding to...) Figure 15 The row labeled "coating" in the middle), and the aerogel loaded with probiotics as a control (corresponding to...). Figure 15 Fluorescence imaging of free fluorescently labeled probiotics (in the "uncoated" row) in simulated gastric fluid. From Figure 15As can be seen, without the hydrogel coating, a greater number of fluorescent probiotics enter the simulated gastric fluid, while the amount of fluorescent probiotics loaded on the aerogel-hydrogel composite material enters the simulated gastric fluid is less. This is because, in the acidic simulated gastric fluid, the outer hydrogel coating can maintain its morphology, thus preventing probiotics from leaking out of the aerogel. Without the hydrogel coating, the probiotics loaded in the aerogel can easily diffuse into the gastric fluid, reducing the probiotic load. Simultaneously, the outer hydrogel coating can prevent hydrogen ions from entering the hydrogel interior, maintaining the activity of the probiotics loaded on the aerogel.

[0105] Figure 16 The aerogel-hydrogel composite materials loaded with beneficial bacteria in Examples 1-3 and Comparative Example 1 (corresponding to...) Figure 16 The "coating" group in the text), and the aerogel loaded with probiotics as a control (corresponding to...). Figure 16 The OD values ​​of the "uncoated" group in simulated gastric juice, from Figure 16 As can be seen, the material with hydrogel coating has a lower OD value in simulated gastric juice, indicating that the hydrogel coating can keep most of the probiotics in the aerogel-hydrogel composite material.

[0106] Figure 17 Aerogel-hydrogel composites for loading beneficial bacteria (corresponding to) Figure 17 The row labeled "coating" in the middle), and the aerogel loaded with probiotics as a control (corresponding to...). Figure 17 The "uncoated" row in the middle shows a staining image of live / dead bacteria after treatment with simulated gastric juice. Figure 17 As can be seen, after treatment with simulated gastric juice, the probiotics loaded within the aerogel remained active, regardless of whether it was uncoated or coated. This is because, during the gelatinization process of high amylose, the exudated amylose interacts with the carboxyl groups of sodium alginate through hydrogen bonds. The aerogel prepared from gelatinized high amylose corn starch exhibits a more compact microstructure, which effectively delays the entry of hydrogen ions and maintains the activity of probiotics within the aerogel pores.

[0107] Figure 18 Aerogel-hydrogel composite materials loaded with probiotics after being treated with simulated gastric juice (corresponding to) Figure 18 The row labeled "coating" in the middle), and the aerogel loaded with probiotics as a control (corresponding to...). Figure 18 A comparison chart of the number of surviving probiotics in the "uncoated" row (in the middle). From Figure 18As can be seen, the survival rate of probiotics in the composite material with hydrogel coating is increased. This is because the hydrogel coating delays the entry of hydrogen ions and the diffusion of probiotics, keeping the probiotics loaded in the aerogel channels active. At the same time, as the content of gelatinized high amylose increases, the number of surviving probiotics increases. This is because the amylose exuded from the gelatinized high amylose corn starch interacts with sodium alginate through hydrogen bonds, exhibiting a more dense aerogel microstructure, further delaying the entry of hydrogen ions and significantly improving the activity of probiotics.

[0108] 2. The present invention tests the probiotic release amount of the probiotic-loaded aerogel-hydrogel composite material under simulated intestinal fluid conditions, specifically including the following steps:

[0109] (1) First, the aerogel-hydrogel composite material loaded with probiotics was incubated in 10 mL of simulated gastric fluid at 37 °C with shaking at 140 rpm for 2 h. Then, it was exposed to 10 mL of simulated small intestinal fluid (pH 6.8) and incubated at 37 °C with shaking at 140 rpm for 6 h. Finally, it was exposed to 10 mL of simulated colonic fluid (SCF, 7.8) and incubated at 37 °C with shaking at 140 rpm until the carrier was completely dissolved. 100 μL of liquid was taken out of the simulated small intestinal fluid every 2 h and 100 μL of liquid was taken out of the simulated colonic fluid every 1 h. During this process, the same volume of simulated small intestinal fluid or simulated colonic fluid was added and the material morphology was photographed. The release amount of probiotics was calculated using the plate count method.

[0110] (2) Cut the aerogel of Example 3 into 1 mm thin slices and then put them into simulated colon fluid. They were incubated at 37 °C with shaking at 140 rpm. The aerogel morphology was photographed every 20 min and the change in aerogel diameter after swelling was statistically analyzed.

[0111] (3) The probiotic release data were fitted to four mathematical models: zero-order release kinetic model (1), first-order release kinetic model (2), Higuchi release kinetic model (3) and Ritger-Peppas release kinetic model (4).

[0112]

[0113] In models (1), (2), (3), and (4), t represents time (h). Q yes t The probiotic release amount (CFUs) is given by k, which represents the rate constant, and c represents the diffusion constant.

[0114] Figure 19 This is a microscopic disintegration diagram of the aerogel in simulated colonic fluid from Example 3. Figure 19As can be seen, the aerogel first swells and then disintegrates. As the content of gelatinized amylose increases within the range provided by this invention, the aerogel disintegration rate becomes slower.

[0115] Figure 20 This is a graph showing the state changes of the probiotic-loaded aerogel-hydrogel composite materials prepared in Comparative Example 1 and Examples 1-3 in simulated small intestinal fluid and simulated colonic fluid. From... Figure 20 As can be seen, the aerogel-hydrogel composite material of Comparative Example 1 exhibits the fastest disintegration rate, completely disintegrating within 1 hour in simulated colonic fluid. With increasing gelatinized amylose content within the range provided by this invention, the disintegration rate of the aerogel-hydrogel composite material gradually decreases. Specifically, the aerogel-hydrogel composite material of Example 1 completes disintegration within 3 hours in simulated colonic fluid, while the aerogel-hydrogel composite materials of Example 2 and Example 3 show longer disintegration times, completing disintegration within 5 hours in simulated colonic fluid.

[0116] Figure 21 This is a comparison chart showing the change in swelling diameter of the aerogels prepared in Comparative Example 1 and Examples 1-3 in simulated colonic fluid. Figure 21 As shown, the aerogel of Comparative Example 1 disintegrated the fastest, completely disintegrating in simulated colonic fluid in 40 minutes; as the content of gelatinized amylose increased, the swelling diameter of the aerogel gradually increased, with the swelling diameter of the aerogel of Example 3 reaching 13 mm, and finally completely disintegrating in 120 minutes.

[0117] Figure 22 This is a graph showing the cumulative release of probiotics from the probiotic-loaded aerogel-hydrogel composite materials in simulated small intestinal and colonic fluids in Comparative Examples 1 and Examples 1-3. Figure 22 It can be seen that the aerogel-hydrogel composite material in Example 3 exhibits the highest cumulative release, ultimately reaching 4.43 × 10⁻⁶. 8 CFUs.

[0118] Figure 23 The release rates of probiotic-loaded aerogel-hydrogel composite materials in Comparative Example 1 and Examples 1-3 were measured per 2 hours in simulated small intestinal fluid and per 1 hour in simulated colonic fluid. Figure 23 As can be seen from the data, the probiotic-loaded aerogel-hydrogel composite material in Example 3 released 1.80 × 10⁻⁶ Lactobacillus rhamnosus per 2 hours in simulated small intestinal fluid. 7 CFUs - 5.50 × 10 7 The CFUs were released at a rate of 2.50 × 10⁻⁶ per hour in simulated colonic fluid. 7 CFUs - 4.80 × 10 7CFUs. This release rate is 10 times the supplemental dose recommended by the Food and Agriculture Organization of the United Nations and the World Health Organization, meeting the body's daily probiotic needs.

[0119] Figure 24 For the probiotic release kinetics of the probiotic-loaded aerogel-hydrogel composite material in Example 3, the cumulative release amount of probiotics was fitted to the kinetic model, including the zero-order model ( Figure 24 a) First-level model ( Figure 24 (b) Higuchi model ( Figure 24 c) and Ritger-Peppas model ( Figure 24 (d) to gain a deeper understanding of the release mechanism. The correlation coefficients of the release curves are summarized in Table 1. The release of *Lactobacillus rhamnosus* from the aerogel and the first-order model (Rd) are compared. 2 =0.97659) and the Ritger-Peppas model (R 2 =0.97192) is a good match; the c value for fitting the Ritger-Peppas model is 2.66, indicating that the aerogel disintegration is mainly erosion.

[0120] Table 1

[0121]

[0122] In summary, the probiotic-loaded aerogel-hydrogel composite material provided by this invention can achieve efficient delivery of probiotics, protect active probiotics to pass smoothly through the acidic environment of the stomach, and realize the long-term stable release of probiotics in the colon region of the distal intestinal tract, making it a promising candidate for application in the field of nutrient delivery.

[0123] In addition, the inventors of this case also conducted experiments with other raw materials, process operations, and process conditions described in this specification, referring to the aforementioned embodiments, and obtained relatively ideal results in all cases.

[0124] All aspects, embodiments, features, and examples of this invention should be considered illustrative and used to explain and illustrate the invention, but not to limit the invention. The scope of the invention is defined only by the claims.

[0125] Although the invention has been described with reference to illustrative embodiments, those skilled in the art will understand that various other changes, omissions, and / or additions can be made without departing from the spirit and scope of the invention, and that elements in the described embodiments can be substituted with substantially equivalents. Furthermore, many modifications can be made without departing from the scope of the invention to adapt particular situations or materials to the teachings of the invention. Therefore, this invention is not intended to be limited to the specific embodiments disclosed, but rather to encompass all embodiments falling within the scope of the appended claims. Moreover, unless specifically stated otherwise, any use of the terms first, second, etc., does not indicate any order or importance, but is used to distinguish one element from another.

Claims

1. An aerogel-hydrogel composite material loaded with probiotics, characterized in that, include: Sodium alginate-gelatinized high amylose aerogel; The active ingredient, including probiotics, is loaded at least in the pores of the sodium alginate-gelatinized high amylose aerogel; Sodium alginate-calcium chloride hydrogel coating, which coats the surface of sodium alginate-gelatinized high amylose aerogel loaded with the active ingredients; The sodium alginate-gelatinized high amylose aerogel is obtained by freeze-drying a mixed solution containing sodium alginate and gelatinized high amylose, wherein the content of gelatinized high amylose is 1wt%~4wt%, and the gelatinized high amylose is obtained by gelatinizing high amylose with an amylose content of more than 50wt%. The sodium alginate-gelatinized high amylose aerogel has a vertical pore structure with a porosity of 66%~83% and a pore diameter of 10 μm~140 μm. The active ingredient is loaded at least in the vertical pore structure.

2. The probiotic-loaded aerogel-hydrogel composite material according to claim 1, characterized in that: The loading amount of the active ingredient in the sodium alginate-gelatinized high amylose aerogel is 2 × 10⁻⁶. 8 ~4×10 8 CFUs.

3. The probiotic-loaded aerogel-hydrogel composite material according to claim 1, characterized in that: The probiotics include one or a combination of Lactobacillus rhamnosus, Lactobacillus plantarum, or Bifidobacterium.

4. The method for preparing the probiotic-loaded aerogel-hydrogel composite material according to any one of claims 1-3, characterized in that, include: A mixed solution containing sodium alginate and gelatinized high amylose is provided, and the mixed solution is freeze-dried to prepare sodium alginate-gelatinized high amylose aerogel; Active ingredients, including probiotics, are loaded into the pores of the sodium alginate-gelatinized high amylose aerogel to obtain a probiotic-loaded aerogel. The probiotic-loaded aerogel was sequentially immersed in a solution containing sodium alginate and a solution containing calcium chloride to coat the surface of the probiotic-loaded aerogel with a sodium alginate-calcium chloride hydrogel coating, thereby obtaining a probiotic-loaded aerogel-hydrogel composite material.

5. The preparation method according to claim 4, characterized in that: The mass ratio of sodium alginate to gelatinized amylose in the mixed solution is 1:2 to 2:

1.

6. The preparation method according to claim 4, characterized in that: The gelatinized high amylose starch is obtained by reacting an aqueous dispersion system containing high amylose starch at 100℃~120℃.

7. The preparation method according to claim 4, characterized in that: A method for preparing the mixed solution into sodium alginate-gelatinized high amylose aerogel includes directional freeze drying.

8. The preparation method according to claim 7, characterized in that, The directional freeze-drying temperature is -30℃ to -20℃, and / or the directional freeze-drying time is 36 to 48 hours.

9. The preparation method according to claim 4, characterized in that, Specifically, it includes: The probiotics were prepared to a concentration of 1×10⁻⁶. 10 ~1×10 12 A probiotic solution of CFU / mL is used to allow the probiotic solution to enter at least the pores of the sodium alginate-gelatinized high amylose aerogel and cross-link, thereby obtaining the probiotic-loaded aerogel.

10. The preparation method according to claim 4, characterized in that: The concentration of the solution containing sodium alginate is 10~20 mg / mL.

11. The preparation method according to claim 4, characterized in that: The concentration of the calcium chloride-containing solution is 10-20 mg / mL.

12. The preparation method according to claim 4, characterized in that: The probiotic-loaded aerogel is first soaked in the solution containing sodium alginate for 5-10 minutes, and then soaked in the solution containing calcium chloride for 5-10 minutes to obtain a probiotic-loaded aerogel-hydrogel composite material.

13. The preparation method according to claim 4, characterized in that: The probiotics include one or a combination of Lactobacillus rhamnosus, Lactobacillus plantarum, or Bifidobacterium.

14. An aerogel-hydrogel composite material loaded with probiotics, characterized in that: It is obtained by the preparation method according to any one of claims 4-13.

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