A chitosan quaternary ammonium salt complex delivery system, and a preparation method and application thereof

Abstract

The application discloses a chitosan quaternary ammonium salt complex delivery system and a preparation method and application thereof. The preparation method comprises the following steps: reacting a first liquid phase mixture at least comprising yeast, chitosan quaternary ammonium salt, to obtain yeast coated with a first nano coating; and reacting a second liquid phase mixture at least comprising the yeast coated with the first nano coating, 1-triphenylmethyl-4-imidazolyl grafted carboxymethyl cellulose sodium and hydroxypropyl cellulose grafted hyaluronic acid under visible light, to obtain the chitosan quaternary ammonium salt complex delivery system. The chitosan quaternary ammonium salt complex delivery system can obviously increase the oral bioavailability of probiotics, improve the tolerance to gastric acid and the like, has good air permeability, supplies oxygen for the probiotics, improves the activity of the probiotics, and in the intestinal environment, the nano coating can be rapidly disintegrated due to the action of intestinal flora to release the probiotics, so that the probiotics can quickly exert the curative effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of probiotic delivery, and particularly relates to a chitosan quaternary ammonium salt complex delivery system, a preparation method and application thereof. BACKGROUND

[0002] There are more than 100 million microorganisms in the human intestinal tract, which constitute a huge ecosystem, i.e., gut microbiota. The number of genes possessed by these microorganisms far exceeds the human gene pool, greatly enhancing the synthetic and metabolic capacity of the human body. Studies have found that gut microbiota plays a crucial role in immune regulation, metabolic balance and host health. Microbial flora imbalance has been proven to be associated with a variety of diseases, such as intestinal inflammation, hypertension, heart disease, obesity, diabetes, depression and cancer. In addition, external factors such as the use of antibiotics, dietary habits and the invasion of foreign microorganisms can interfere with the intestinal microbial ecology, thereby causing changes in the composition and function of the intestinal microbiome.

[0003] The plasticity of intestinal microorganisms and their important role in human health provide new ideas and opportunities for the development of microbial therapy. For example, fecal microbiota transplantation (FMT) and oral probiotic therapy are emerging therapeutic approaches developed on this basis.

[0004] Due to the unique advantages of probiotics and their significant effects in treating related diseases, researchers have been committed to developing various new probiotic strains and applying them in functional foods and health products. However, probiotics are extremely sensitive to environmental factors, and maintaining their activity during storage, transportation and consumption poses many challenges. Only by maintaining high activity and successfully colonizing the intestinal tract can probiotics exert their due functions. Factors such as jolting during transportation, air oxidation, insufficient nutrition, etc. can cause probiotics to lose activity. In addition, the strong acidity of the human gastrointestinal tract, bile salts, high concentrations of reactive oxygen species (ROS), gastrointestinal peristalsis and complex pathological microenvironments all pose a threat to the survival of probiotics, reduce their bioavailability, inhibit their colonization and physiological activity, and thus limit their therapeutic effect.

[0005] The intestinal epithelial surface is covered with a layer of mucous rich in mucin, which is not only the habitat of intestinal microorganisms, but also an important barrier to protect intestinal cells from external damage, and provides attachment points and nutrient sources for oral probiotics. Therefore, probiotics must have strong resistance to the digestive tract environment and strong adhesion to the intestinal mucus layer in order to achieve colonization in the digestive tract and exert their functional value. In order to improve the survival rate of probiotics in the human body, enhance the adhesion and colonization efficiency of probiotics in the intestinal tract, and improve the functional value of probiotic foods, it is an urgent problem to provide a delivery system for probiotics to achieve colonization in the digestive tract and exert their functional value, and have strong resistance to the digestive tract environment and strong adhesion to the intestinal mucus layer. SUMMARY

[0006] The main purpose of the present application is to provide a chitosan quaternary ammonium salt complex delivery system, its preparation method and application, to overcome the shortcomings of the prior art.

[0007] To achieve the foregoing purposes of the application, the technical solutions adopted by the present application include:

[0008] The present application provides a preparation method of a chitosan quaternary ammonium salt complex delivery system, which comprises:

[0009] reacting a first liquid mixture comprising at least yeast, chitosan quaternary ammonium salt at 25-40 DEG C to form a first nanocoating on the surface of the yeast, thereby preparing yeast coated with a first nanocoating; wherein the yeast is probiotic bacteria, and the probiotic bacteria are Kluyveromyces marxianus;

[0010] and reacting a second liquid mixture comprising at least yeast coated with a first nanocoating, 1-triphenylmethyl-4-imidazolyl grafted carboxymethylcellulose sodium, and hydroxypropyl cellulose grafted hyaluronic acid under visible light at 25-40 DEG C to form a second nanocoating on the surface of the first nanocoating, thereby preparing a chitosan quaternary ammonium salt complex delivery system.

[0011] The present application also provides a chitosan quaternary ammonium salt complex delivery system prepared by the foregoing preparation method, which comprises yeast and a first nanocoating and a second nanocoating successively coated on the surface of the yeast.

[0012] The present application also provides the use of the foregoing chitosan quaternary ammonium salt complex delivery system in the preparation of products with intestinal targeted delivery of probiotics.

[0013] Compared with the prior art, the present application has the following advantages:

[0014] (1) The chitosan quaternary ammonium salt complex delivery system provided by the application is used as an encapsulation technology, the encapsulation technology shows pH-sensitive characteristics, solubility is reduced in a gastric acid environment, solubility is increased in an intestinal tract, slow release of probiotics is achieved, and an intestinal tract targeting effect is achieved;

[0015] (2) The encapsulation material used in the application promotes incomplete crosslinking between 1-triphenylmethyl-4-imidazolyl grafted carboxymethyl cellulose sodium and hydroxypropyl cellulose grafted hyaluronic acid without affecting the combination of cellulose and the first nano coating, so that the encapsulation material has good air permeability, can supply oxygen to probiotics to improve the activity of the probiotics, and the second nano coating can be rapidly disintegrated due to the action of intestinal flora in an intestinal environment to release the probiotics, so that the probiotics can quickly exert their curative effect;

[0016] (3) The chitosan quaternary ammonium salt complex delivery system in the application can significantly increase the oral bioavailability of probiotics;

[0017] (4) The particle size of the chitosan quaternary ammonium salt complex delivery system in the application is not much larger than that of probiotics, and the chitosan quaternary ammonium salt complex delivery system can be better released in the intestinal tract. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only some embodiments described in the application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0019] Figure 1 is the encapsulation rate diagram of the delivery system under different yeast concentrations in test example 1 of the application;

[0020] Figure 2 is the encapsulation rate diagram of the delivery system under different temperatures in test example 1 of the application;

[0021] Figure 3 is the encapsulation rate diagram of the delivery system under different mass ratios of chitosan quaternary ammonium salt and 1-triphenylmethyl-4-imidazolyl grafted carboxymethyl cellulose sodium in test example 1 of the application;

[0022] Figure 4 is the encapsulation rate diagram of the delivery system under different reaction times in test example 1 of the application;

[0023] Figure 5 is the encapsulation rate diagram of the delivery system under different light sources in test example 1 of the application;

[0024] Figure 6 is the TEM diagram of the chitosan quaternary ammonium salt complex delivery system in example 15 of the application;

[0025] Figure 7 is a survival rate change curve graph of the delivery system prepared in the present application, Example 15, Comparative Examples 1-3 at 4 °C;

[0026] Figure 8 is a survival rate change curve graph of the delivery system prepared in the present application, Example 15, Comparative Examples 1-3 at 25 °C;

[0027] Figure 9 is a digestion survival rate change curve graph of the delivery system prepared in the present application, Example 15, Comparative Examples 1-3;

[0028] Figure 10 is an in vivo survival rate test graph of the delivery system prepared in the present application, Example 15, Comparative Examples 1-3. DETAILED DESCRIPTION

[0029] In view of the defects of the prior art, the present applicant has long-term research and a large number of practices, and has obtained the technical scheme of the present application. The technical scheme of the present application will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0030] Specifically, as one aspect of the technical scheme of the present application, a preparation method of a chitosan quaternary ammonium salt complex delivery system comprises:

[0031] reacting a first liquid phase mixture containing at least yeast, chitosan quaternary ammonium salt at 25-40 °C to form a first nano-coating on the surface of the yeast, thereby obtaining yeast coated with a first nano-coating; wherein the yeast is probiotic bacteria, and the probiotic bacteria is Kluyveromyces marxianus;

[0032] and reacting a second liquid phase mixture containing at least yeast coated with a first nano-coating, 1-triphenylmethyl-4-imidazolyl grafted carboxymethyl cellulose sodium, and hydroxypropyl cellulose grafted hyaluronic acid under visible light at 25-40 °C to form a second nano-coating on the surface of the first nano-coating, thereby obtaining a chitosan quaternary ammonium salt complex delivery system.

[0033] The encapsulating material used in the present application promotes incomplete cross-linking between 1-triphenylmethyl-4-imidazolyl grafted carboxymethyl cellulose and hydroxypropyl cellulose grafted hyaluronic acid without affecting the combination of cellulose and the first nanocoating, and the 1-triphenylmethyl-4-imidazolyl grafted carboxymethyl cellulose and hydroxypropyl cellulose-hyaluronic acid form covalent "spot welding" at the reactive sites, leaving a large number of un-crosslinked segments, which self-assemble into a through-hole / mesoporous framework; the chitosan quaternary ammonium salt builds a reversible velvet layer on the surface of the pore wall by electrostatic and hydrogen bonds, which does not block the pore and can be opened and closed instantaneously with humidity. , low resistance diffusion of water vapor, and the dynamic velvet layer prevents the leakage of liquid water, both of which together give the material the "breathable" characteristics of high air permeability and no leakage, so that it has good air permeability, can supply oxygen to probiotics to improve their activity, and the second nanocoating can be rapidly disintegrated by the action of intestinal flora in the intestinal environment to release probiotics, so that the probiotics can quickly exert their therapeutic effect.

[0034] In some preferred embodiments, the preparation method specifically comprises: resuspending the yeast bacteria to OD 600 0.1-0.4 with sterile water, and then resuspending with a chitosan quaternary ammonium salt solution to form the first liquid mixture.

[0035] The concentration of the chitosan quaternary ammonium salt solution is 1-2 mg / mL.

[0036] In some preferred embodiments, the preparation method specifically comprises: dissolving hydroxypropyl cellulose in an alkaline solvent and adjusting the pH value of the solution to 9-10, then adding a first cross-linking agent and performing a cross-linking reaction at 60-80 ℃ for 2.5-5 h, then adding sodium hyaluronate into an alkaline solvent and adjusting the pH value to 10-11, continuing to add a second cross-linking agent and reacting at room temperature for 24-28 h to obtain hydroxypropyl cellulose grafted hyaluronic acid.

[0037] Further, the alkaline solvent includes a NaOH solution, without being limited thereto.

[0038] Further, the first cross-linking agent includes ethylene glycol diglycidyl ether, without being limited thereto.

[0039] Further, the second cross-linking agent includes divinyl sulfone, without being limited thereto.

[0040] Further, the mass ratio of the hydroxypropyl cellulose, the first cross-linking agent, the sodium hyaluronate, and the second cross-linking agent is 1:(0.15-0.2):0.5:(0.2-0.4).

[0041] In some preferred embodiments, the preparation method specifically comprises: dissolving sodium carboxymethyl cellulose in water and adjusting the pH value to 6-7, then adding EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide), N-hydroxysuccinimide and stirring the reaction at room temperature for 2-4 h, after which 1-triphenylmethyl-4-imidazole dimethylformamide solution, a catalyst are added and stirring the reaction at 40-50 ℃ for 12-24 h, then post-processing to obtain 1-triphenylmethyl-4-imidazole grafted sodium carboxymethyl cellulose.

[0042] Further, the mass ratio of sodium carboxymethyl cellulose, EDC, N-hydroxysuccinimide, 1-triphenylmethyl-4-imidazole and the catalyst is 1: (0.1-0.2): (0.05-0.1): (0.2-0.5): 0.01.

[0043] Further, the degree of substitution of the sodium carboxymethyl cellulose is 0.7-1.2.

[0044] Further, the catalyst includes 4-dimethylaminopyridine, and is not limited thereto.

[0045] In some preferred embodiments, the preparation method specifically comprises: mixing yeast coated with a first nanometer coating, 1-triphenylmethyl-4-imidazole grafted sodium carboxymethyl cellulose solution, hydroxypropyl cellulose grafted hyaluronic acid solution to form a second liquid phase mixture, then performing a reaction under blue light conditions to obtain a chitosan quaternary ammonium salt complex delivery system.

[0046] In some more specific embodiments, the preparation method of the chitosan quaternary ammonium salt complex delivery system comprises:

[0047] In some more specific embodiments, the preparation method of the chitosan quaternary ammonium salt complex delivery system comprises:

[0048] reacting a first liquid phase mixture containing uniformly mixed yeast and chitosan quaternary ammonium salt at a temperature of 25-40 ℃ to coat the first nanometer coating on the surface of the probiotic bacteria, wherein the OD value of the yeast is 0.1-0.4 and the concentration of the chitosan quaternary ammonium salt is 1-2 mg / mL. 600

[0049] ​The hydroxypropyl cellulose is completely dissolved in the alkaline solvent, the pH of the solution is adjusted to 9-10, then the crosslinking agent ethylene glycol diglycidyl ether is added, and after crosslinking reaction at 60-80 ℃ for 2.5-5 h, the sodium hyaluronate is added, then the alkaline solvent is added and the pH of the solution is adjusted to 10-11, and then the crosslinking agent divinyl sulfone is continuously added, and after reaction at room temperature for 24-28 h, the product is obtained by repeatedly washing with water and centrifugal sedimentation, and the hydroxypropyl cellulose grafted with hyaluronic acid is obtained.

[0050] 1g of sodium carboxymethyl cellulose (CMC, degree of substitution 0.7-1.2) is dissolved in 50 mL of deionized water (2% w / v), the pH is adjusted to 6-7 with 1 mol / L NaOH, 0.1-0.2 g of EDC and 0.05-0.1 g of N-hydroxysuccinimide are added, the carboxyl group of CMC is activated at room temperature for 2-4 h, 0.2-0.5 g of 1-triphenylmethyl-4-imidazole is dissolved in 10 mL of dimethylformamide, and then added dropwise to the activated CMC solution, and 0.01 g of 4-dimethylaminopyridine is added as a catalyst, and the reaction is carried out at 40-50 ℃ for 12-24 h, after the reaction is completed, the solution is neutralized with 1 mol / L HCl, and the reaction mixture is poured into cold ethanol to precipitate the product, which is washed with ethanol and acetone three times each, then the precipitate is dissolved in a small amount of water, and freeze-dried to obtain the purified product, i.e. 1-triphenylmethyl-4-imidazolyl grafted sodium carboxymethyl cellulose.

[0051] The second liquid mixture containing the probiotics coated with the first nanocoating, the 1-triphenylmethyl-4-imidazolyl grafted sodium carboxymethyl cellulose, and the hydroxypropyl cellulose grafted hyaluronic acid uniformly mixed is reacted under the conditions of a temperature of 25-40 ℃ and visible light irradiation, so as to coat the probiotics with the second nanocoating, and the concentration of the 1-triphenylmethyl-4-imidazolyl grafted sodium carboxymethyl cellulose is 1-2 mg / mL, and the concentration of the hydroxypropyl cellulose grafted hyaluronic acid is 0.5-1 mg / mL.

[0052] Another aspect of the embodiment of the present application also provides a chitosan quaternary ammonium salt complex delivery system prepared by the preparation method, which comprises the yeast and the first nanocoating and the second nanocoating coated on the surface of the yeast in sequence.

[0053] In some preferred embodiments, the thickness of the first nanocoating is 0.25-0.5 nm.

[0054] In some preferred embodiments, the thickness of the second nanocoating is 0.3-0.5 nm.

[0055] In some preferred embodiments, the particle size of the chitosan quaternary ammonium salt complex delivery system is 1.8-2 μm.

[0056] Another aspect of the present application provides use of the aforementioned chitosan quaternary ammonium salt complex delivery system in the preparation of a product having targeted delivery of probiotics to the intestinal tract.

[0057] The technical solutions of the present application are further described in detail below in conjunction with several preferred embodiments and the accompanying drawings. The embodiments are implemented on the premise of the technical solutions of the present application, and detailed implementation manners and specific operation processes are given, but the protection scope of the present application is not limited to the following embodiments.

[0058] The experimental materials used in the following examples are commercially available from conventional biochemical reagent companies, unless otherwise specified.

[0059] The preparation methods of hydroxypropyl cellulose grafted hyaluronic acid and 1-triphenylmethyl-4-imidazolyl grafted sodium carboxymethyl cellulose in the following test examples are prepared by the following method:

[0060] Hydroxypropyl cellulose is completely dissolved in an alkaline solvent, the pH of the solution is adjusted to 9-10, a crosslinking agent ethylene glycol diglycidyl ether is added, and crosslinking reaction is carried out at 60-80 ℃ for 2.5-5 h, then sodium hyaluronate is added, an alkaline solvent is added again, and the pH of the solution is adjusted to 10-11, a crosslinking agent divinyl sulfone is continuously added, and reaction is carried out at room temperature for 24-28 h, then the product is repeatedly washed with water and centrifuged to obtain hydroxypropyl cellulose grafted hyaluronic acid.

[0061] 1 g of carboxymethyl cellulose sodium CMC (degree of substitution 0.7-1.2) is dissolved in 50 mL of deionized water (2% w / v), the pH is adjusted to 6-7 with 1 mol / L NaOH, 0.1-0.2 g of EDC and 0.05-0.1 g of N-hydroxysuccinimide are added, the carboxyl group of CMC is activated by stirring at room temperature for 2-4 h, 0.2-0.5 g of 1-triphenylmethyl-4-imidazole is dissolved in 10 mL of dimethylformamide, and then added dropwise to the activated CMC solution, 0.01 g of 4-dimethylaminopyridine is added as a catalyst, and the reaction is carried out by stirring at 40-50 ℃ for 12-24 h. After the reaction is completed, the solution is neutralized with 1 mol / L HCl, and the reaction mixture is poured into cold ethanol to precipitate the product. After washing with ethanol and acetone three times each, the precipitate is dissolved in a small amount of water, and freeze-dried to obtain the purified product, i.e. 1-triphenylmethyl-4-imidazolyl grafted sodium carboxymethyl cellulose.

[0062] Test Example 1 Preparation of chitosan quaternary ammonium salt complex delivery system

[0063] 1. Test method (including Example 1-Example 17)

[0064] 1.1, Preparation of delivery system under different yeast concentrations

[0065] The yeast (Maxx Kluyveromyces) was resuspended in sterile water to OD 600 = 0.1, OD 600 = 0.2, OD 600 = 0.3, OD 600 = 0.4, OD 600 = 0.5, the yeast was centrifuged and the supernatant was discarded, and then resuspended with chitosan quaternary ammonium salt (2 mg / mL) solution, and magnetically stirred in a water bath at 30 °C for 1.5 h, with a stirring speed of 600 rpm. After stirring, centrifugation was performed to discard the supernatant, and the second liquid mixture containing uniformly mixed probiotics coated with a first nanometer coating on the surface, 1-triphenylmethyl-4-imidazolyl grafted carboxymethyl cellulose sodium, and hydroxypropyl cellulose grafted hyaluronic acid was magnetically stirred in a water bath at 30 °C for 1.5 h under the irradiation of visible light blue, with a stirring speed of 600 rpm. The mass ratio of chitosan quaternary ammonium salt to 1-triphenylmethyl-4-imidazolyl grafted carboxymethyl cellulose sodium was 2:1, and the molar ratio of the 1-triphenylmethyl-4-imidazolyl grafted carboxymethyl cellulose sodium to the hydroxypropyl cellulose grafted hyaluronic acid was 1:1. After the end of stirring, centrifugation was performed to discard the supernatant, and resuspension was performed with sterile water to obtain a chitosan quaternary ammonium salt complex delivery system (S-G-M.K).

[0066] 1.2, Preparation of delivery system under different reaction temperatures

[0067] The yeast was resuspended in sterile water to OD 600 = 0.2, the yeast was centrifuged and the supernatant was discarded, and then resuspended with chitosan quaternary ammonium salt (2 mg / mL) solution, and magnetically stirred in a water bath at 25 °C, 30 °C, 35 °C, 40 °C, and 45 °C for 1.5 h, with a stirring speed of 600 rpm. After stirring, centrifugation was performed to discard the supernatant, and the second liquid mixture containing uniformly mixed probiotics coated with a first nanometer coating on the surface, 1-triphenylmethyl-4-imidazolyl grafted carboxymethyl cellulose sodium, and hydroxypropyl cellulose grafted hyaluronic acid was magnetically stirred in a water bath at 25 °C, 30 °C, 35 °C, 40 °C, and 45 °C for 1.5 h under the irradiation of visible light blue, with a stirring speed of 600 rpm. The mass ratio of chitosan quaternary ammonium salt to 1-triphenylmethyl-4-imidazolyl grafted carboxymethyl cellulose sodium was 2:1, and the molar ratio of the 1-triphenylmethyl-4-imidazolyl grafted carboxymethyl cellulose sodium to the hydroxypropyl cellulose grafted hyaluronic acid was 1:1. After the end of stirring, centrifugation was performed to discard the supernatant, and resuspension was performed with sterile water to obtain a chitosan quaternary ammonium salt complex delivery system (S-G-M.K).

[0068] 1.3. Preparation of delivery system at different mass ratios

[0069] The yeast bacteria were resuspended in sterile water to OD 600 = 0.2, the yeast bacteria were centrifuged and the supernatant was discarded, and then resuspended in a chitosan quaternary ammonium salt (2 mg / mL) solution, and then magnetically stirred in a water bath at 30 °C for 1.5 h, with a stirring speed of 600 rpm. After stirring, the supernatant was discarded by centrifugation, and the second liquid mixture containing the uniformly mixed probiotics coated with a first nanometer coating, 1-triphenylmethyl-4-imidazolyl grafted carboxymethyl cellulose sodium, and hydroxypropyl cellulose grafted hyaluronic acid was magnetically stirred in a water bath at 30 °C for 1.5 h under irradiation of visible blue light, with a stirring speed of 600 rpm. The mass ratio of chitosan quaternary ammonium salt to 1-triphenylmethyl-4-imidazolyl grafted carboxymethyl cellulose sodium was 0.5:1, 1:1, 1.5:1, 2:1, and 2.5:1, and the molar ratio of 1-triphenylmethyl-4-imidazolyl grafted carboxymethyl cellulose sodium to hydroxypropyl cellulose grafted hyaluronic acid was 1:1. After the end of stirring, the supernatant was discarded by centrifugation, and the delivery system of chitosan quaternary ammonium salt complex (S-G-M.K) was obtained by resuspension in sterile water.

[0070] 1.4. Preparation of delivery system at different reaction times (same temperature and time in two-step reaction)

[0071] The yeast bacteria were resuspended in sterile water to OD 600 = 0.2, the yeast bacteria were centrifuged and the supernatant was discarded, and then resuspended in a chitosan quaternary ammonium salt (2 mg / mL) solution, and then magnetically stirred in a water bath at 30 °C for 1.5 h, with a stirring speed of 600 rpm. After stirring, the supernatant was discarded by centrifugation, and the second liquid mixture containing the uniformly mixed probiotics coated with a first nanometer coating, 1-triphenylmethyl-4-imidazolyl grafted carboxymethyl cellulose sodium, and hydroxypropyl cellulose grafted hyaluronic acid was magnetically stirred in a water bath at 30 °C for 1.5 h under irradiation of visible blue light, with a stirring speed of 600 rpm. The mass ratio of chitosan quaternary ammonium salt to 1-triphenylmethyl-4-imidazolyl grafted carboxymethyl cellulose sodium was 0.5:1, 1:1, 1.5:1, 2:1, and 2.5:1, and the molar ratio of 1-triphenylmethyl-4-imidazolyl grafted carboxymethyl cellulose sodium to hydroxypropyl cellulose grafted hyaluronic acid was 1:1. After the end of stirring, the supernatant was discarded by centrifugation, and the delivery system of chitosan quaternary ammonium salt complex (S-G-M.K) was obtained by resuspension in sterile water.

[0072] 1.5. Preparation of delivery system under different light irradiation

[0073] The yeast bacteria were resuspended in sterile water to OD 600= 0.2, the yeast was centrifuged to discard the supernatant, and then resuspended with a chitosan quaternary ammonium salt (2 mg / mL) solution, and then stirred in a water bath at 30 °C for 1.5 h at a stirring speed of 600 rpm. After stirring, the mixture was centrifuged to discard the supernatant, and then resuspended with sterile water to obtain a chitosan quaternary ammonium salt complex delivery system (S-G-M.K).

[0074] The encapsulation efficiency (%) of the chitosan quaternary ammonium salt complex delivery system was calculated according to the following formula:

[0075] Encapsulation efficiency (%) = number of present bacteria / number of original bacteria × 100%

[0076] 2. Test results

[0077] The encapsulation efficiencies of the various chitosan quaternary ammonium salt complex delivery systems obtained in this test example are shown in Table 1 and Figures 1-4 Figure 1 is the encapsulation efficiency of the delivery system at different yeast concentrations; Figure 2 is the encapsulation efficiency of the delivery system at different temperatures; Figure 3 is the encapsulation efficiency of the delivery system at different mass ratios of chitosan quaternary ammonium salt and 1-triphenylmethyl-4-imidazolyl grafted carboxymethyl cellulose sodium; Figure 4 is the encapsulation efficiency of the delivery system at different reaction times.

[0078] Table 1. Encapsulation efficiency of the nano-coating prepared under different conditions of the present application

[0079]

[0080] The test results show that, taking the encapsulation efficiency as an evaluation index, the effects of different bacterial concentrations, reaction temperatures, mass ratios (mass ratio of chitosan quaternary ammonium salt and 1-triphenylmethyl-4-imidazolyl grafted carboxymethyl cellulose sodium), reaction times, and light sources on the encapsulation efficiency were investigated. The optimal preparation process of the chitosan quaternary ammonium salt complex delivery system was determined, i.e., the bacterial concentration was OD 600 Preferably, OD 600 ​0.1-0.4, the optimal value is 0.2, the mass ratio of the chitosan quaternary ammonium salt and the 1-triphenylmethyl-4-imidazolyl grafted sodium carboxymethyl cellulose is 2:1, the temperature is preferably 25-40 °C, the reaction time is preferably 0.5-2 h, the optimal temperature and time are: 30 °C water bath under magnetic stirring for 1 h; the light source is preferably blue light.

[0081] Comparative Example 1: The method is the same as that of Example 15, except that the hydroxypropyl cellulose grafted hyaluronic acid is replaced by hyaluronic acid.

[0082] Comparative Example 2: The method is the same as that of Example 15, except that the 1-triphenylmethyl-4-imidazolyl grafted sodium carboxymethyl cellulose is replaced by sodium carboxymethyl cellulose.

[0083] Comparative Example 3: The method is the same as that of Example 15, except that the visible blue light is replaced by white light of the same intensity.

[0084] Comparative Example 4: The method is the same as that of Example 15, except that the hydroxypropyl cellulose grafted hyaluronic acid is absent.

[0085] Comparative Example 5: The method is the same as that of Example 15, except that the 1-triphenylmethyl-4-imidazolyl grafted sodium carboxymethyl cellulose is absent.

[0086] Comparative Example 6: The method is the same as that of Example 15, except that the first nano-coating is absent, and the second nano-coating is directly coated on the surface of the yeast, and the product is recorded as: G-M.K.

[0087] Comparative Example 7: The method is the same as that of Example 15, except that the second nano-coating is absent, and the product is recorded as: S-M.K.

[0088] Comparative Example 8: The method is the same as that of Example 15, except that the chitosan quaternary ammonium salt is replaced by chitosan.

[0089] Test Example 2 Transmission Electron Microscopy Measurement of the Chitosan Quaternary Ammonium Salt Complex Delivery System Layer

[0090] First, the chitosan quaternary ammonium salt complex delivery system suspension prepared in Example 15 is subjected to slight ultrasonic dispersion. A copper mesh is clamped with tweezers and placed on a wetted filter paper, and about 50 μL of the sample is gently dropped onto the copper mesh. After staining for about 2 min, the morphology of the nanoparticles is observed and photographed using a transmission electron microscope. As shown in FIG. 1, the morphology of the chitosan quaternary ammonium salt complex delivery system shows that two layers of protective films are formed on the outer layer of the bacteria. In addition, the particle size length shown by the scale of the TEM is relatively consistent with the measured particle size. Figure 6

[0091] Test Example 3 Storage Capacity Measurement of the Chitosan Quaternary Ammonium Salt Complex Delivery System

[0092] ​1. Experimental method

[0093] Free yeast and delivery system encapsulating probiotics in Example 15 and Comparative Examples 1-8 were prepared respectively, and 50 mL of each group of probiotic bacterial solution was stored at 4 °C and 25 °C respectively. 1 mL of each group of bacterial solution was taken at 1, 2, 3 weeks respectively, gradient dilution and plate counting were carried out, the survival rate was calculated, and the survival rate change curve was drawn.

[0094] 2. Experimental results

[0095] As shown in Figure 7 and Figure 8 : Within the 21-day storage period, the survival rate of probiotics in the M.K group at 4 °C and 25 °C decreased from 8.12 log CFU / mL to 4.86 log CFU / mL and 1.94 log CFU / mL at three weeks, the survival rate of probiotics in Comparative Example 1 at 4 °C and 25 °C was 3.80 log CFU / mL and 6.03 log CFU / mL at three weeks, the survival rate of probiotics in Comparative Examples 2-3 at 4 °C and 25 °C was lower than 3.35 log CFU / mL and 5.65 log CFU / mL at three weeks, and the survival rate of Example 15 bacteria decreased from 8.12 log CFU / mL to 6.09 log CFU / mL and 3.94 log CFU / mL respectively, greatly improving the survival rate. In addition, the survival rate of bacteria in Comparative Examples 4-8 was lower than that in Example 15, and the survival rate of probiotics at 4 °C and 25 °C was lower than 2.85 log CFU / mL and 4.90 log CFU / mL at three weeks. Obviously, the chitosan quaternary ammonium salt complex delivery system in the present application can significantly enhance the storage capacity of probiotics and improve the stability of yeast during storage. Therefore, the chitosan quaternary ammonium salt complex delivery system is a good storage strategy for probiotics.

[0096] Test Example 4 Determination of the anti-digestion capacity of the chitosan quaternary ammonium salt complex delivery system

[0097] 1. Experimental method

[0098] The simulated oral, gastric and intestinal digestive fluids were prepared according to the following formulations respectively:

[0099] 1 L of oral stock solution: sodium chloride (1.594 g), ammonium chloride (0.219 g), sodium nitrate (0.345 g), potassium phosphate (0.636 g), potassium citrate (0.308 g), sodium urate (0.021 g), urea (0.198 g), sodium lactate (0.146 g). Oral digestive fluid: 10 mL of oral stock solution was added with 0.3 g of porcine gastric mucosa protein.

[0100] 1 L Gastric juice stock solution: sodium chloride (2 g), hydrochloric acid (2 mL). Gastric juice: 10 mL of the gastric juice stock solution + 0.032 g pepsin, pH adjusted to 2.5 with 1 M hydrochloric acid.

[0101] 1 L Intestinal juice: pancreatin (10 g), sodium chloride (8.5 g), bile salts (3 g), trypsin (10 g), pH adjusted to 6.5 with 0.01 M hydrochloric acid.

[0102] Free yeast and the delivery system encapsulating probiotic bacteria in Example 15 and Comparative Examples 1-8 were prepared respectively. 1 mL of each group of yeast was digested in 9 mL of oral juice, gastric juice and intestinal juice for 1 h respectively. In addition, 1 mL of each group of yeast was sequentially digested in 9 mL of oral juice, gastric juice and intestinal juice for 5 min, 1 h, 2 h and 3 h respectively. After digestion, 1 mL of each group of yeast was taken at each digestion stage, gradiently diluted and then plate counted to calculate the survival rate and draw the survival rate change curve.

[0103] 2. Experimental results

[0104] As shown in Table 1 (simulating the process of continuous digestion in vivo, first 5 min in oral juice, 1 h in gastric juice, then in intestinal juice): Figure 9 Oral juice had little effect on the survival rate of probiotic bacteria. After 1 h of gastric juice digestion, the number of viable bacteria in the M.K group decreased to 4.333 log CFU / mL, and the survival rates of Comparative Examples 1-3 were all lower than 7.30 log CFU / mL, and the survival rate of Example 15 was 7.39 log CFU / mL. After 2 h of intestinal juice digestion, the number of viable bacteria in the M.K group decreased to 3.590 log CFU / mL, and the survival rate of Example 15 was 5.36 log CFU / mL. In addition, the survival rates of Comparative Examples 1-3 were all lower than 5.25 log CFU / mL compared with Example 15, and the survival rates of Comparative Examples 4-8 were all lower than 4.90 log CFU / mL compared with Example 15. The results showed that the chitosan quaternary ammonium salt complex delivery system could effectively enhance the anti-digestion ability of probiotic bacteria and improve the survival rate of yeast in the simulated digestion process, thereby improving the oral bioavailability of probiotic bacteria.

[0105] Test Example 5 In vivo survival rate determination of chitosan quaternary ammonium salt complex delivery system

[0106] 1. Experimental method

[0107] The activated bacteria and the prepared delivery systems for encapsulating probiotics as described in Examples 15 and Comparative Examples 1-8 were administered 200 μL via gavage to 2-month-old BALB / c mice. The probiotics were observed using an in vivo fluorescence imaging system, and their survival rate was determined at 8 and 24 h based on fluorescence intensity. After 24 h, the entire intestinal tract of the mice was harvested and observed using an in vivo fluorescence imaging system.

[0108] 2. Experimental Results

[0109] like Figure 10 As shown (where the minimum value of the color scale is 7.53e7 and the maximum value is 1.03e8), the in vivo fluorescence imaging results show that after 24 hours, almost no blue fluorescence was observed in the MK group in mouse in vivo imaging. The fluorescence of Comparative Examples 1-3 was significantly lower than that of Example 15. In Example 15, the chitosan quaternary ammonium salt complex delivery system group showed extremely strong fluorescence, indicating that the chitosan quaternary ammonium salt complex delivery system enhanced the yeast's resistance to gastrointestinal digestive fluids and ROS. By increasing the adhesion of yeast to the intestine and prolonging the bacterial residence time in vivo, it significantly improved the survival rate of yeast in mice. Compared with Example 15, the colonization efficiency of bacteria in the intestine of Comparative Examples 4-8 was also significantly lower than that of Example 15. This allows for rapid disintegration and release of probiotics, which are then targeted to the intestine, enabling the probiotics to quickly exert their therapeutic effects.

[0110] In addition, the applicant 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.

[0111] It should be understood that the technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made to the technical solutions of the present invention without departing from the spirit and scope of the claims are within the scope of protection of the present invention.

Claims

1. A method of preparing a chitosan quaternary ammonium salt complex delivery system, characterized by, Comprising: reacting a first liquid mixture containing at least yeast, chitosan quaternary ammonium salt at 25-40 DEG C to form a first nanometer coating on the surface of the yeast, thereby obtaining yeast coated with a first nanometer coating; wherein the yeast is probiotic bacteria, and the probiotic bacteria is Kluyveromyces marxianus; and, reacting a second liquid mixture containing at least yeast coated with a first nanometer coating, 1-triphenylmethyl-4-imidazole grafted carboxymethyl cellulose sodium, and hydroxypropyl cellulose grafted hyaluronic acid under visible light at 25-40 DEG C to form a second nanometer coating on the surface of the first nanometer coating, thereby obtaining a chitosan quaternary ammonium salt complex delivery system.

2. The production method according to claim 1, characterized by, Specifically comprising: The yeast cells were resuspended in sterile water to an OD 600 of 0.1-0.4, after which they were resuspended in a chitosan quaternary ammonium salt solution to form the first liquid phase mixture; wherein the concentration of the chitosan quaternary ammonium salt solution is 1-2 mg / mL.

3. The production method according to claim 1, characterized by, Specifically comprising: dissolving hydroxypropyl cellulose in an alkaline solvent and adjusting the pH of the solution to 9-10, then adding a first crosslinking agent and crosslinking at 60-80 DEG C for 2.5-5 h, then adding sodium hyaluronate, adding an alkaline solvent and adjusting the pH to 10-11, continuing to add a second crosslinking agent and reacting at room temperature for 24-28 h to obtain hydroxypropyl cellulose grafted hyaluronic acid.

4. The method of claim 3, wherein: The alkaline solvent is NaOH solution; and / or, the first crosslinking agent is ethylene glycol diglycidyl ether; and / or, the second crosslinking agent is divinyl sulfone; and / or, the mass ratio of hydroxypropyl cellulose, first crosslinking agent, sodium hyaluronate and second crosslinking agent is 1:(0.15-0.2):0.5:(0.2-0.4).

5. The production method according to claim 1, characterized by, Specifically comprising: dissolving carboxymethyl cellulose sodium in water and adjusting the pH to 6-7, then adding EDC and N-hydroxysuccinimide and stirring at room temperature for 2-4 h, then adding a dimethylformamide solution of 1-triphenylmethyl-4-imidazole and a catalyst and stirring at 40-50 DEG C for 12-24 h, and then post-treating to obtain 1-triphenylmethyl-4-imidazole grafted carboxymethyl cellulose sodium.

6. The method of claim 5, wherein: The mass ratio of carboxymethyl cellulose sodium, EDC, N-hydroxysuccinimide, 1-triphenylmethyl-4-imidazole and catalyst is 1:(0.1-0.2):(0.05-0.1):(0.2-0.5):0.01; and / or, the degree of substitution of carboxymethyl cellulose sodium is 0.7-1.2; and / or, the catalyst is 4-dimethylaminopyridine.

7. The preparation method according to claim 1, characterized in that, Specifically comprising: mixing yeast coated with a first nanometer coating, a solution of 1-triphenylmethyl-4-imidazole grafted carboxymethyl cellulose sodium, and a solution of hydroxypropyl cellulose grafted hyaluronic acid to form a second liquid mixture, and then reacting under blue light to obtain a chitosan quaternary ammonium salt complex delivery system; The molar ratio of the 1-triphenylmethyl-4-imidazolyl grafted sodium carboxymethyl cellulose to the hydroxypropyl cellulose grafted hyaluronic acid is 1:0.8-1:1.2; the concentration of the 1-triphenylmethyl-4-imidazolyl grafted sodium carboxymethyl cellulose solution is 1-2 mg / mL, and the concentration of the hydroxypropyl cellulose grafted hyaluronic acid solution is 0.5-1 mg / mL.

8. The chitosan quaternary ammonium salt complex delivery system produced by the method of any one of claims 1-7, characterized in that, The yeast is coated with a first nano coating and a second nano coating in sequence.

9. The chitosan quaternary ammonium salt complex delivery system according to claim 8, wherein: The thickness of the first nano coating is 0.25-0.5 nm; The thickness of the second nano coating is 0.3-0.5 nm. The particle size of the chitosan quaternary ammonium salt complex delivery system is 1.8-2 μm.

10. Use of the chitosan quaternary ammonium salt complex delivery system according to claim 8 or 9 in the preparation of a product for the targeted delivery of probiotics to the intestinal tract.

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