Probiotic-loaded aerogel-hydrogel composite material and preparation method thereof

Abstract

The invention provides an aerogel-hydrogel composite material loaded with probiotics and a preparation method of the aerogel-hydrogel composite material. The aerogel-hydrogel composite material loaded with the probiotics is prepared from sodium alginate-gelatinized high amylose aerogel, and the probiotics are prepared from sodium alginate-gelatinized high amylose aerogel, sodium alginate-gelatinized high amylose aerogel and sodium alginate-gelatinized high amylose aerogel. The active component comprises probiotics, and the active component is at least loaded in a pore channel of the sodium alginate-gelatinized high amylose aerogel; the surface of the sodium alginate-gelatinized high amylose aerogel loaded with the active component is coated with the sodium alginate-calcium chloride hydrogel coating. According to the aerogel-hydrogel composite material loaded with the probiotics, the active probiotics can be protected to smoothly pass through the acid environment of the stomach, the probiotics are stably released at the colon part of the far-end area of the intestinal tract, and controllable release of the probiotics is achieved.

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 strategies for preventing and treating 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 they 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: The first aspect of the present application provides a probiotic-loaded aerogel-hydrogel composite material, which comprises: sodium alginate-gelatinized high-amylose starch aerogel; active ingredients, the active ingredients including probiotics, the active ingredients being loaded at least in the pores of the sodium alginate-gelatinized high amylose starch aerogel; 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.

[0006] The high amylose starch (referring to the starch with the content of amylose exceeding 50wt%) used in the present application has a high resistant starch content and the ability of resisting the 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. And the gelatinization is also beneficial to prolong the activity of the probiotics loaded in the gel.

[0007] 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: 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. In addition, 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, and the erosion rate of the aerogel is controlled by adjusting the crosslinking strength. The probiotics form larger size probiotic clusters through the self-aggregation ability, and the probiotics closely interacting with the aerogel skeleton are combined by hydrogen bonds. 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 pass through the acidic environment of the stomach smoothly, and stably release the probiotics in the distal region of the intestinal tract, i.e. the colon, to achieve the controllable release of the probiotics.

[0008] In some embodiments, the sodium alginate-gelatinized high amylose starch aerogel has a vertical pore structure, and the active ingredients are loaded at least in the vertical pore structure. The aerogel with a vertical ordered pore structure disintegrates and releases the probiotics in the form of peeled onions, so that the release amount of the probiotics per hour is stable, which is beneficial to the controllable and sustained release of the probiotics in the intestinal tract.

[0009] In some embodiments, the porosity of the sodium alginate-gelatinized high amylose starch aerogel is 66% to 83%, and the diameter of the pores contained is 10 μm to 140 μm.

[0010] In some embodiments, the sodium alginate-gelatin aerogel is obtained by freeze-drying a mixed solution containing sodium alginate and gelatin.

[0011] In some embodiments, the content of gelatin in the sodium alginate-gelatin aerogel is 1wt%~4wt%.

[0012] In some embodiments, the loading amount of the active ingredient in the sodium alginate-gelatin aerogel is 2×10 8 ~4×10 8 .

[0013] The probiotic bacteria can be any known probiotic bacteria in the art. In some embodiments, the probiotic bacteria include one or a combination of Lactobacillus rhamnosus, Lactobacillus plantarum or Bifidobacterium.

[0014] The second aspect of the present application provides a method for preparing a probiotic bacteria-loaded aerogel-hydrogel composite material, the method comprising: providing a mixed solution containing sodium alginate and gelatin, and preparing a sodium alginate-gelatin aerogel from the mixed solution; loading an active ingredient in the pores of the sodium alginate-gelatin aerogel, the active ingredient including probiotic bacteria, to obtain a probiotic bacteria-loaded aerogel; immersing the probiotic bacteria-loaded aerogel in a solution containing sodium alginate and a solution containing calcium chloride in sequence, to coat a sodium alginate-calcium chloride hydrogel coating on the surface of the probiotic bacteria-loaded aerogel, and obtain a probiotic bacteria-loaded aerogel-hydrogel composite material.

[0015] In some embodiments, the mass ratio of sodium alginate to gelatin in the mixed solution is 1:2~2:1.

[0016] In some embodiments, the gelatinized high-amylose starch is obtained by reacting a water dispersion system containing high-amylose starch at 100~120℃, wherein the content of amylose in the high-amylose starch is 50wt% or more.

[0017] In some embodiments, the method for preparing 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 30~40 min to obtain the gelatinized high-amylose starch.

[0018] In some embodiments, the method for preparing the sodium alginate-gelatinized high-amylose starch aerogel includes directional freeze-drying.

[0019] In some embodiments, the temperature of the directional freeze-drying is -30℃ to -20℃.

[0020] In some embodiments, the time of the directional freeze-drying is 36h to 48h.

[0021] In some embodiments, the method for preparing specifically includes: formulating the probiotic bacteria into a probiotic bacteria solution with a concentration of 1×10 10 ~1×10 12 CFU / mL, allowing the probiotic bacteria solution to enter at least the pores of the sodium alginate-gelatinized high-amylose starch aerogel and crosslinking, thereby obtaining the probiotic bacteria-loaded aerogel.

[0022] In some embodiments, the time of the crosslinking is 5min to 10min, so as to load the probiotic bacteria into the pores of the sodium alginate-gelatinized high-amylose starch aerogel.

[0023] In some embodiments, the concentration of the sodium alginate-containing solution is 10mg / mL to 20mg / mL.

[0024] In some embodiments, the concentration of the calcium chloride-containing solution is 10mg / mL to 20mg / mL.

[0025] In some embodiments, the method for preparing specifically includes: allowing the probiotic bacteria-loaded aerogel to be soaked in the sodium alginate-containing solution for 5min to 10min, and then soaked in the calcium chloride-containing solution for 5min to 10min, so as to obtain the probiotic bacteria-loaded aerogel-hydrogel composite material.

[0026] The probiotic bacteria can be any known probiotic bacteria in the art. In some embodiments, the probiotic bacteria include one or a combination of Lactobacillus rhamnosus, Lactobacillus plantarum, or Bifidobacterium.

[0027] The third aspect of the present application provides a probiotic bacteria-loaded aerogel-hydrogel composite material, which is obtained by the method for preparing according to any one of the technical solutions.

[0028] The probiotic bacteria-loaded aerogel-hydrogel composite material provided by the present application can be applied in efficient delivery of probiotic bacteria in the intestinal tract.

[0029] Compared with the prior art, the present application has at least the following beneficial effects: (1) On the one hand, the aerogel-hydrogel composite material loaded with probiotics provided by the application can avoid the leakage of probiotics during oral administration and improve the survival rate of probiotics in a strong acid environment in the stomach based on 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 of the intestinal tract, so as to realize the controllable release of probiotics in the colon; (2) The sodium alginate-gelatinized high-amylose starch aerogel used in the application has a vertical pore structure, which is beneficial to the controllable and stable release of probiotics; in addition, the porosity is high, and the pore size of the contained pores is suitable, so that a high loading amount of probiotics can be realized. (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

[0030] In order to more clearly illustrate the technical solutions in the embodiments or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments described in the application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0031] Figure 1 Optical photographs of the sodium alginate-gelatinized high-amylose corn starch aerogel with a vertical pore structure prepared in Example 1 in the cross section (a) and the longitudinal section (b); Figure 2 Scanning electron microscope images of the sodium alginate-gelatinized high-amylose corn starch aerogel with a vertical pore structure prepared in Example 1 in the cross section (a) and the longitudinal section (b); Figure 3 Energy dispersive spectroscopy mapping image of the sodium alginate-gelatinized high-amylose corn starch aerogel prepared in Example 1; Figure 4 Scanning electron microscope images of the sodium alginate aerogel prepared in Comparative Example 1 (left) and the sodium alginate-gelatinized high-amylose corn starch aerogel prepared in Example 1 (right); Figure 4 Figure 4 Scanning electron microscope images of the sodium alginate aerogel prepared in Comparative Example 1 (left) and the sodium alginate-gelatinized high-amylose corn starch aerogel prepared in Example 1 (right); Figure 5 Porosity comparison chart of the sodium alginate aerogel prepared in Comparative Example 1 and the sodium alginate-gelatinized high-amylose corn starch aerogel prepared in Examples 1-3; Figure 6 Pore size distribution chart of the cross section of the sodium alginate-gelatinized high-amylose corn starch aerogel prepared in Example 3; ​Figure 7 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 8 FTIR spectra of the sodium alginate-gelatinized high-amylose corn starch aerogel prepared in Examples 1-3, and the sodium alginate aerogel prepared in Comparative Example 1; Figure 9 Comparison of the resistant starch content in the gelatinized high-amylose corn starch used in Example 1 and the prepared sodium alginate-gelatinized high-amylose corn starch aerogel; Figure 10 Photos of 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 probiotics; Figure 11 Comparison of the volume shrinkage of the sodium alginate aerogel in Comparative Example 1 and the sodium alginate-gelatinized high-amylose corn starch aerogels in Examples 1-3; Figure 12 Scanning electron microscope images of the sodium alginate-gelatinized high-amylose corn starch aerogel loaded with probiotics in Example 1; Figure 13 Comparison of the number of probiotics loaded in 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 14 Fluorescent images of the fluorescently labeled probiotics and the fluorescently labeled aerogel of Example 1; Figure 15 Fluorescent images of the free fluorescently labeled probiotics in simulated gastric fluid for the aerogel-hydrogel composites (corresponding to the “coated” row in Figure 15 ) in Examples 1-3, Comparative Example 1, and as a control, the aerogel loaded with probiotics (corresponding to the “uncoated” row in Figure 15 ); Figure 16 OD values of the aerogel-hydrogel composites (corresponding to the “coated” group in Figure 16 ) in Examples 1-3, Comparative Example 1, and as a control, the aerogel loaded with probiotics (corresponding to the “uncoated” group in Figure 16 ) in simulated gastric fluid; Figure 17 Live / dead bacteria staining of the aerogel-hydrogel composites (corresponding to the “coated” row in Figure 17 ) in Examples 1-3, Comparative Example 1, and as a control, the aerogel loaded with probiotics (corresponding to the “uncoated” row in Figure 17 ) after treatment with simulated gastric fluid; Figure 18 Survival of probiotic bacteria in simulated gastric fluid treated aerogel-hydrogel composite loaded with probiotic bacteria (corresponding to Figure 18 in the present disclosure “coated” row), and probiotic bacteria loaded aerogel as control (corresponding to Figure 18 in the present disclosure “uncoated” row); Figure 19 Microscopic disintegration of aerogel in simulated intestinal fluid for Comparative Example 1, Examples 1-3; Figure 20 Morphology change of probiotic bacteria loaded aerogel-hydrogel composite in simulated intestinal fluid and simulated colonic fluid for Comparative Example 1, Examples 1-3; Figure 21 Swelling diameter change of aerogel in simulated colonic fluid for Comparative Example 1, Examples 1-3; Figure 22 Probiotic bacteria cumulative release of probiotic bacteria loaded aerogel-hydrogel composite in simulated intestinal fluid and simulated colonic fluid for Comparative Example 1, Examples 1-3; Figure 23 Probiotic bacteria release of probiotic bacteria loaded aerogel-hydrogel composite in simulated intestinal fluid every 2 h and in simulated colonic fluid every 1 h for Comparative Example 1, Examples 1-3; Figure 24 Probiotic bacteria release kinetics of probiotic bacteria loaded aerogel-hydrogel composite of Example 3, cumulative release of probiotic bacteria was fitted to kinetic models including zero order model (a) in the present disclosure, first order model (b) in the present disclosure, Higuchi model (c) in the present disclosure, and Ritger-Peppas model (d) in the present disclosure. Figure 24 Figure 24 Figure 24 Figure 24

[0032] Wherein, Figure 5 , Figure 11 , Figure 13 , Figure 16 , Figure 18 The letters (a, b, c, d) involved in the present disclosure are the grouping markers 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 the other group is marked as b, due to sharing the letter b, the difference between the two is not significant. DETAILED DESCRIPTION

[0033] ​​​​The technical solutions of the present application are described in detail below with reference to specific embodiments, so that those skilled 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 a basis for the claims and for teaching those skilled in the art to employ the representative basis of the present application in different ways in any appropriate detailed embodiment.

[0034] In addition, unless otherwise specified, the various raw materials used in the following examples can be obtained from the market or the like, and the various production and testing equipment used is known in the art, and the testing methods used are any known methods in the art.

[0035] Example 1 Example 1 provides a probiotic-loaded aerogel-hydrogel composite material and a method for preparing the same, which specifically comprises the following steps: (1) Preparation method of gelatinized high-amylose starch: 2 g of high-amylose corn starch with a straight-chain starch 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 thoroughly mixed. The solution was reacted at 120°C for 30 min. After the reaction was completed, a gelatinized high-amylose starch solution with a concentration of 20 mg / mL was obtained.

[0036] (2) Preparation of sodium alginate-gelatinized high-amylose corn starch aerogel with vertical pore structure: 2 g of sodium alginate was dissolved in 50 mL of sterile water, and 50 mL of the above-prepared 20 mg / mL gelatinized high-amylose starch solution was added. After being thoroughly mixed, a mixed solution was obtained. The mixed solution was transferred to a directional freezing mold and frozen at a temperature of -20°C. Then, the frozen material was transferred to a vacuum freeze dryer for vacuum freeze drying for 48 h to obtain a sodium alginate-gelatinized high-amylose corn starch aerogel with vertical pore structure.

[0037] (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 pore channels of the above-prepared aerogel, and the crosslinking time was 10 min to prepare a probiotic-loaded aerogel. (4) Preparation of probiotic-loaded aerogel-hydrogel composite material: The probiotic-loaded aerogel was soaked in a 20 mg / mL sodium alginate solution for 10 min, and then soaked in a 20 mg / mL calcium chloride solution for crosslinking for 10 min to obtain a probiotic-loaded aerogel-hydrogel composite material.

[0038] Figure 1Optical photographs of the sodium alginate-paste high-amylose corn starch aerogel with vertical channel structure prepared in Example 1 in cross section (a) and longitudinal section (b), which can be prepared into carriers with size suitable for human oral administration (such as a carrier with a diameter of 8 mm and a height of 8 mm) by cutting.

[0039] 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 cross section (a) and longitudinal section (b). It can be seen from Figure 2 that the aerogel has a vertical ordered channel structure in the longitudinal section.

[0040] Figure 3 Energy dispersive spectroscopy mapping images of the sodium alginate-paste high-amylose corn starch aerogel prepared in Example 1. It can be seen from Figure 3 that the elements are uniformly distributed in the aerogel.

[0041] Example 2 Example 2 is basically the same as Example 1, except that in Example 2, 4 g of high-amylose corn starch is taken in step (1) to prepare a paste high-amylose corn starch solution with a concentration of 40 mg / mL, which is used to prepare the sodium alginate-paste high-amylose corn starch aerogel of Example 2. The rest is the same as Example 1, which is not described here again, and finally the probiotic-loaded aerogel-hydrogel composite material is obtained.

[0042] Example 3 Example 3 is basically the same as Example 1, except that in Example 3, 8 g of high-amylose corn starch is taken in step (1) to prepare a paste high-amylose corn starch solution with a concentration of 80 mg / mL, which is used to prepare the sodium alginate-paste high-amylose corn starch aerogel of Example 3. The rest is the same as Example 1, which is not described here again, and finally the probiotic-loaded aerogel-hydrogel composite material is obtained.

[0043] Example 4 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.

[0044] The probiotic-loaded aerogel-hydrogel composite material prepared in Example 4 has an effect comparable to that of Example 1.

[0045] Comparative Example 1 Comparative Example 1 is essentially the same as Example 1, except that it does not use a gelatinized high amylose solution to prepare aerogels, but instead uses sodium alginate to prepare alginate aerogels. Specifically, 2 g of sodium alginate is fully dissolved in 100 mL of sterile water, and the solution is transferred to a directional freezing mold and directionally frozen at -20 °C. The frozen material is then transferred to a vacuum freeze dryer for vacuum freeze-drying for 48 h to obtain sodium alginate aerogels. The rest of the process is the same as in Example 1 and will not be repeated here, resulting in a sodium alginate aerogel-hydrogel composite material loaded with probiotics.

[0046] Figure 4 Sodium alginate aerogel prepared for Comparative Example 1 ( Figure 4 (Left side) and sodium alginate-gelatinized high amylose corn starch aerogel prepared in Example 1 ( Figure 4 Scanning electron microscope image (right side). From Figure 4 As 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 aerogel.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] Figure 8The FTIR spectra of the sodium alginate-paste high-amylose corn starch aerogel prepared in Examples 1-3, the sodium alginate aerogel prepared in Comparative Example 1; no new absorption peaks were detected in the sodium alginate-paste high-amylose corn starch aerogel, indicating that the interaction between sodium alginate and paste high-amylose corn starch is mainly dominated by hydrogen bonds.

[0051] Figure 9 The comparison chart of the resistant starch content in the paste high-amylose corn starch used in Example 1 and the sodium alginate-paste high-amylose corn starch aerogel prepared in Example 1. It can be seen from Figure 9 that the resistant starch content in the sodium alginate-paste high-amylose corn starch aerogel is higher than that of pure paste high-amylose corn starch, which is more advantageous for the aerogel to resist erosion in the stomach and small intestine.

[0052] Figure 10 The photos of the morphological changes of the sodium alginate aerogel in Comparative Example 1, the sodium alginate-paste high-amylose corn starch aerogel in Examples 1-3 before and after loading probiotics. It can be seen from Figure 10 that compared with the aerogel without loading probiotics, the aerogel shrinks in volume after the probiotics are loaded in the pore channels of the aerogel.

[0053] Figure 11 The comparison chart of the volume shrinkage rate of the sodium alginate aerogel in Comparative Example 1, the sodium alginate-paste high-amylose corn starch aerogel in Examples 1-3. The volume shrinkage rate of the aerogel is the difference between the initial volume of the aerogel and the volume of the aerogel after loading probiotics, divided by the initial volume of the aerogel. With the increase of the content of paste high-amylose corn starch, the volume shrinkage rate of the prepared aerogel gradually decreases, and the smaller volume shrinkage rate provides favorable conditions for the increase of the loading amount of probiotics.

[0054] Figure 12 The scanning electron microscope image of the sodium alginate-paste high-amylose corn starch aerogel loaded with probiotics in Example 1. It can be seen from Figure 12 that a large amount of probiotics are loaded in the pore channels of the aerogel.

[0055] Figure 13 The comparison chart of the number of probiotics loaded in the sodium alginate aerogel prepared in Comparative Example 1, the sodium alginate-paste high-amylose corn starch aerogel prepared in Examples 1-3. It can be seen from Figure 13 that with the increase of the content of paste high-amylose corn starch, the number of probiotics that can be loaded in the aerogel gradually increases, which is because the high content of paste high-amylose corn starch can reduce the volume shrinkage of the aerogel, so that more probiotics can be loaded in the pore channels of the aerogel. The aerogel prepared in the use amount range of paste high-amylose corn starch provided in the present application can make the loading amount of probiotics reach 4 × 10 8CFUs or more, the sodium alginate aerogel in Comparative Example 1 can achieve a 2-fold or more increase in the loading amount of probiotics.

[0056] The sodium alginate-gelatinized high-amylose corn starch aerogel prepared in Example 1 was labeled with Rhodamine B (red), and the probiotics were labeled with fluorescein isothiocyanate (green), Figure 14 The fluorescence images of the fluorescently labeled probiotics and the fluorescently labeled aerogel of Example 1 show that the sodium alginate-gelatinized high-amylose corn starch aerogel loaded with probiotics exhibits both red and green light, proving that the probiotics are successfully loaded in the pore channels of the aerogel.

[0057] The relevant properties of the probiotic-loaded aerogel-hydrogel composite materials prepared in Examples 1-3 and Comparative Example 1 were tested, and the probiotic-loaded aerogels without hydrogel coating in Examples 1-3 and Comparative Example 1 were used as controls (i.e., without step (4) in the preparation method): 1. The survival rate of the probiotic-loaded aerogel-hydrogel composite material in simulated gastric juice was tested, specifically including: (1) The fluorescein isothiocyanate (3 mg / mL) labeled probiotics were loaded into the aerogel-hydrogel composite materials in Examples 1-3 and Comparative Example 1, and the probiotic-loaded aerogels without hydrogel coating, and incubated in simulated gastric juice (pH 2.0) at 37°C with 140 rpm shaking for 2 h. The fluorescence was photographed at 0 h and 2 h in the gastric juice, respectively.

[0058] (2) The probiotic-loaded aerogel-hydrogel composite materials and the probiotic-loaded aerogels without hydrogel coating were incubated in simulated gastric juice (pH 2.0) at 37°C with 140 rpm shaking for 2 h. The optical density values of the gastric juice were measured at 0 h and 2 h in the gastric juice, respectively.

[0059] (3) 10 mL of sodium citrate (50 mg / mL) was used to dissolve the probiotic-loaded aerogel-hydrogel composite materials and the probiotic-loaded aerogels treated in simulated gastric juice (pH 2.0) for 2 h. Then the dissolved solution was stained with calcein-iodine propidium dye for 20 min, and the number of live and dead bacteria was observed and photographed under a fluorescence microscope.

[0060] (4) 10 mL of sodium citrate (50 mg / mL) was used to dissolve the probiotic-loaded aerogel-hydrogel composite materials and the probiotic-loaded aerogels, respectively. The dissolved solution was spread on MRS agar plates and incubated at 37°C for 24 h. Then the number of probiotics was calculated by plate counting method.

[0061] Figure 15Fluorescence images of free fluorescently labeled probiotic bacteria in simulated gastric fluid for the aerogel-hydrogel composites loaded with probiotic bacteria in Examples 1-3, Comparative Example 1 (corresponding to Figure 15 “coated” row in Table 1), and as a control, aerogels loaded with probiotic bacteria (corresponding to Figure 15 “uncoated” row in Table 1). It can be seen from Figure 15 that more fluorescent probiotic bacteria entered the simulated gastric fluid without the hydrogel coating, while less fluorescent probiotic bacteria loaded in the aerogel-hydrogel composites entered the simulated gastric fluid. This is because the external hydrogel coating can maintain its integrity in the acidic simulated gastric fluid, thereby preventing the probiotic bacteria from leaking out of the aerogel, while without the hydrogel coating, the probiotic bacteria loaded in the aerogel can easily diffuse into the gastric fluid, reducing the loading amount of probiotic bacteria. At the same time, the external hydrogel coating can prevent hydrogen ions from entering the interior of the hydrogel, thereby maintaining the activity of the probiotic bacteria loaded in the aerogel.

[0062] Figure 16 OD values in simulated gastric fluid for the aerogel-hydrogel composites loaded with probiotic bacteria in Examples 1-3, Comparative Example 1 (corresponding to Figure 16 “coated” group in Table 2), and as a control, aerogels loaded with probiotic bacteria (corresponding to Figure 16 “uncoated” group in Table 2). It can be seen from Figure 16 that the OD values in the simulated gastric fluid for the materials with the hydrogel coating were lower, indicating that the hydrogel coating can keep most of the probiotic bacteria in the aerogel-hydrogel composites.

[0063] Figure 17 Live / dead bacteria staining images for the aerogel-hydrogel composites loaded with probiotic bacteria (corresponding to Figure 17 “coated” row in Table 3), and as a control, aerogels loaded with probiotic bacteria (corresponding to Figure 17 “uncoated” row in Table 3) after treatment with simulated gastric fluid. It can be seen from Figure 17 that after treatment with simulated gastric fluid, whether coated or uncoated, the probiotic bacteria loaded in the aerogel remained in an active state. This is because during the gelatinization of high amylose starch, the exuded amylose interacts with the carboxyl groups of sodium alginate through hydrogen bonds, and the aerogel prepared from gelatinized high amylose corn starch exhibits a more compact microstructure, which can effectively delay the entry of hydrogen ions and maintain the activity of the probiotic bacteria in the pores of the aerogel.

[0064] Figure 18 Comparison chart of the number of surviving probiotic bacteria in the aerogel-hydrogel composites loaded with probiotic bacteria (corresponding to Figure 18 “coated” row in Table 4), and as a control, aerogels loaded with probiotic bacteria (corresponding to Figure 18 “uncoated” row in Table 4) after treatment with simulated gastric fluid.Figure 18 As can be seen, the survival amount of probiotics in the composite material with the hydrogel coating is improved, because the hydrogel coating delays the entry of hydrogen ions and the diffusion of probiotics, so that the probiotics loaded in the aerogel channel remain active, and as the gelatinized high-amylose starch content increases, the number of surviving probiotics increases, because the leached amylose in the gelatinized high-amylose corn starch interacts with sodium alginate through hydrogen bonds, showing a more compact aerogel microstructure, further delaying the entry of hydrogen ions, and significantly improving the activity of probiotics.

[0065] 2. The present application tests the release amount of probiotics of the aerogel-hydrogel composite material loaded with probiotics in simulated intestinal fluid, specifically comprising the following steps: (1) First, the aerogel-hydrogel composite material loaded with probiotics is incubated in 10 mL of simulated gastric juice at 37°C with 140 rpm oscillation for 2 h, then exposed to 10 mL of simulated small intestinal fluid (pH 6.8) at 37°C with 140 rpm oscillation for 6 h, and finally exposed to 10 mL of simulated colon fluid (SCF, 7.8) at 37°C with 140 rpm oscillation until the carrier is completely dissolved. In the simulated small intestinal fluid, 100 μL of liquid is taken out every 2 h, and in the simulated colon fluid, 100 μL of liquid is taken out every 1 h. During this process, the same volume of simulated small intestinal fluid or simulated colon fluid is supplemented, and the material morphology is photographed. The release amount of probiotics is calculated by plate counting method.

[0066] (2) The aerogel of Example 3 is cut into 1 mm thin slices, then put into simulated colon fluid, incubated at 37°C with 140 rpm oscillation, and the aerogel morphology is photographed every 20 min and the change in diameter of the swollen aerogel is counted.

[0067] (3) The probiotic release data is fitted to four mathematical models: zero-order release kinetics model (1), first-order release kinetics model (2), Higuchi release kinetics model (3), and Ritger-Peppas release kinetics model (4).

[0068]

[0069] In models (1), (2), (3), and (4), t represents time (h), Q is t the release amount of probiotics (CFUs) at time t, k represents the rate constant, and c represents the diffusion constant.

[0070] Figure 19 is the microstructure disintegration diagram of the aerogel of Example 3 in simulated colon fluid, from 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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 recommended supplementation amount by the Food and Agriculture Organization of the United Nations and the World Health Organization, which meets the daily probiotic requirement of the human body.

[0075] Figure 24 For 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 the zero-order model (Equation 1) Figure 24 a), the first-order model (Equation 2) Figure 24 b), the Higuchi model (Equation 3) Figure 24 c), and the Ritger-Peppas model (Equation 4) Figure 24 d) to further understand the release mechanism. The release curve correlation coefficients are summarized in Table 1. The release of Lactobacillus rhamnosus from the aerogel was in good agreement with the first-order model (R 2 = 0.97659) and the Ritger-Peppas model (R 2 = 0.97192); the c value fitted to the Ritger-Peppas model was 2.66, indicating that the disintegration of the aerogel was mainly erosion.

[0076] Table 1

[0077] In summary, the probiotic-loaded aerogel-hydrogel composite material provided by the present application can achieve efficient delivery of probiotics, protect active probiotics from passing through the acidic environment of the stomach, and achieve long-term stable release of probiotics in the distal colon region of the intestine, making it have good application prospects in the field of nutrient delivery.

[0078] In addition, the present inventors have also carried out tests with other raw materials, process operations, and process conditions described in the present specification with reference to the foregoing examples, and have obtained relatively ideal results.

[0079] Aspects, embodiments, features, and examples of the present application should be considered illustrative, for explanation and illustration of the present application, but not for limitation of the present application, and the scope of the present application is only defined by the claims.

[0080] While the application has been described with reference to the illustrative embodiments, those skilled in the art will appreciate that various modifications, omissions, and / or additions can be made without departing from the spirit or scope of the application. Additionally, many modifications can be made to adapt a particular situation or material to the teachings of the application without departing from its scope. Therefore, the application is not intended to be limited to the particular embodiments disclosed, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein. Further, to the extent that the terms first, second, etc. are used herein to describe various elements, these are generally used only as labels, and are not intended to impose numerical requirements on their objects.

Claims

1. A probiotic-loaded aerogel-hydrogel composite material, characterized in that, The application relates to a sodium alginate-gelatinized high-amylose starch aerogel loaded with active ingredients, and a preparation method thereof. The sodium alginate-gelatinized high-amylose starch aerogel comprises: a sodium alginate-gelatinized high-amylose starch aerogel; active ingredients, which comprise probiotics, at least loaded in the pores of the sodium alginate-gelatinized high-amylose starch aerogel; 2. The probiotic-loaded aerogel-hydrogel composite material according to claim 1, characterized by: 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. 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; and / or, the porosity of the sodium alginate-gelatinized high-amylose starch aerogel is 66% to 83%, and the diameter of the pores contained is 10 microns to 140 microns; and / or, the sodium alginate-gelatinized high-amylose starch aerogel is obtained by freeze-drying a mixed solution containing sodium alginate and gelatinized high-amylose starch; 3. The probiotic-loaded aerogel-hydrogel composite material according to claim 1, characterized by: The active ingredient is loaded in the sodium alginate-gelatinized high-amylose starch aerogel in an amount of 2 x 10 8 ~4 x 10 8 CFUs; and / or, the content of the gelatinized high-amylose starch in the sodium alginate-gelatinized high-amylose starch aerogel is 1wt% to 4wt%.

4. A method for preparing a probiotic-loaded aerogel-hydrogel composite material, characterized in that, and / or, the probiotics comprise one or a combination of Lactobacillus rhamnosus, Lactobacillus plantarum or Bifidobacterium. The application relates to a sodium alginate-gelatinized high-amylose starch aerogel loaded with active ingredients, and a preparation method thereof. The application relates to a sodium alginate-gelatinized high-amylose starch aerogel loaded with active ingredients, and a preparation method thereof. The application relates to a sodium alginate-gelatinized high-amylose starch aerogel loaded with active ingredients, and a preparation method thereof.

5. The method of claim 4, wherein: The mass ratio of sodium alginate to gelatinized high-amylose starch in the mixed solution is 1:2 to 2:1; and / or, the gelatinized high-amylose starch is obtained by reacting a water dispersion system containing high-amylose starch at 100 DEG C to 120 DEG C, wherein the content of amylose in the high-amylose starch is more than 50wt%; and / or, the method for preparing the sodium alginate-gelatinized high-amylose starch aerogel from the mixed solution comprises directional freeze-drying.

6. The production method according to claim 5, wherein The temperature of the directional freeze-drying is -30 DEG C to -20 DEG C, and / or the time of the directional freeze-drying is 36 to 48 hours.

7. The production method according to claim 4, characterized by, The application relates to a sodium alginate-gelatinized high-amylose starch aerogel loaded with active ingredients, and a preparation method thereof. The probiotic bacteria are formulated into a probiotic bacteria solution at a concentration of 1 x 10 10 1 x 10 12 CFU / mL, the probiotic bacteria solution is at least introduced into the pores of the sodium alginate-gelatinized high-amylose starch aerogel and cross-linked, thereby obtaining the probiotic bacteria-loaded aerogel.

8. The method of claim 4, wherein: The concentration of the solution containing sodium alginate is 10 to 20 mg / mL; and / or, the concentration of the solution containing calcium chloride is 10 to 20 mg / mL; and / or, the sodium alginate-gelatinized high-amylose starch aerogel loaded with probiotics is first soaked in the solution containing sodium alginate for 5 to 10 minutes, and then soaked in the solution containing calcium chloride for 5 to 10 minutes, so as to obtain the sodium alginate-gelatinized high-amylose starch aerogel-hydrogel composite material loaded with probiotics.

9. The method of claim 4, wherein: The probiotics comprise one or a combination of Lactobacillus rhamnosus, Lactobacillus plantarum or Bifidobacterium.

10. A probiotic-loaded aerogel-hydrogel composite material, characterized by: The sodium alginate-gelatinized high-amylose starch aerogel loaded with active ingredients is obtained by the preparation method in any one of claims 4 to 9.

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