Intestinal targeted dual-bacteria multi-core gel particles and preparation method thereof

Abstract

The present invention relates to the technical field of probiotic application, and specifically to an intestinal-targeted dual-bacteria multi-core gel particle and a preparation method thereof. The gel particles are modified by adding thiolated sodium carboxymethyl cellulose to modify the wall material, which can enhance the adhesion of probiotics in the intestinal tract, and the effect is remarkable. The present invention prepares probiotic gel particles containing two strains of Bifidobacterium adolescentis and Bacillus subtilis by endogenous emulsification and extrusion. The Bacillus subtilis on the outer layer of the gel particles consumes free oxygen, providing a good anaerobic environment for the survival and colonization of Bifidobacterium adolescentis in the intestinal tract. Not only can it well protect the probiotics from the digestive effects of the gastrointestinal tract, avoid the destructive effects of gastric acid on bifidobacteria, and increase the proportion of bifidobacteria reaching the intestinal tract and surviving, but it can also be delivered to the colon in a targeted manner, and all release is achieved in the colon, significantly prolonging the residence time of the probiotics in the intestinal tract, so that it can give full play to its probiotic function.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotic application, and particularly relates to multi-core gel particles loaded with two probiotics capable of targeted release in the intestinal tract and a preparation method thereof. Background Art

[0002] Probiotics are called "live microorganisms" and have benefits such as maintaining intestinal ecological balance, regulating the immune system, improving inflammation and inhibiting pathogens. Among them, Bifidobacterium is a strictly anaerobic probiotic with a series of health-promoting properties, including preventing intestinal infections, maintaining mucosal barrier integrity and resisting pathogen colonization. Creating a strict anaerobic environment for Bifidobacterium to ensure a certain number of live bacteria and exert its probiotic function is the key. At the same time, it was found that Bacillus, as a facultative anaerobic bacterium, can consume free oxygen in the human intestine to create an anaerobic environment. It can also inhibit the proliferation of aerobic pathogens and is conducive to the growth of anaerobic probiotics. In order to achieve the desired effect in the host, the number of live probiotics must be 10 6 CFU / g or more. However, probiotics are very sensitive to the gastrointestinal fluid environment, and the number of bacteria that can enter the intestine alive after oral administration is relatively small.

[0003] At present, gel encapsulation is widely used to protect probiotics from the adverse conditions of the external environment and gastrointestinal tract, enhance the stability and vitality of probiotics, and thus enable probiotics to fully exert their probiotic effects. Researchers have found that CN115039886 involves a bifidobacterium microcapsule product, the preparation method uses porous starch as a load and protective agent, and then uses multi-layer coating to maintain the activity of probiotics after passing through the stomach, but only shows a certain tolerance to gastric acid, fails to provide a strict anaerobic environment after the release of bifidobacteria, and does not involve the targeted release of live bacteria.

[0004] Existing reports on probiotic-encapsulated gel particles are mainly based on a shell-core structure with a single chamber storage area, and the research on the targeted release of anaerobic bacteria in the intestine is not in-depth enough. The vague probiotic release site is not conducive to the effective colonization of probiotics in the posterior intestinal part. How to achieve better targeted release of anaerobic bacteria in the intestine to exert probiotic functions has been a research hotspot and difficulty in this field in recent years. Summary of the invention

[0005] In order to solve the problems of the prior art, the present invention provides a dual-bacteria multi-core gel particle that is conducive to the targeted release of anaerobic bacteria in the intestine and a preparation method thereof. The gel particles containing two probiotics, Bifidobacterium adolescentis and Bacillus subtilis, prepared by the endogenous emulsification method and the extrusion method of the present invention can resist digestion by gastrointestinal fluid, increase the adhesion of probiotics, deliver probiotics to the intestine, and create a strict anaerobic environment for anaerobic bacteria before release in the intestine, which greatly increases the number of viable bacteria of anaerobic bacteria and can better enhance its probiotic function. This intestinal targeted gel system with controlled release characteristics has both effectiveness and efficient antibacterial ability, and has broad prospects for clinical application.

[0006] One aspect of the present invention relates to a sodium mercaptocarboxymethyl cellulose, which is prepared by the following method:

[0007] Dissolve 100-200 mg sodium carboxymethyl cellulose in 20-40 mL ultrapure water, add 220-440 mg 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 130-260 mg N-hydroxysuccinimide, adjust the pH and stir for 2 h, then add 475-950 mg cysteine ​​hydrochloride and dialyze for 24 h; after dialysis, add 295-590 mg dithiothreitol, adjust the pH, and react at room temperature for 24 h; adjust the pH with 0.1 mol.L hydrochloric acid. -1 The mixture was dialyzed with a sodium chloride solution to a pH of 3.5; after 24 hours of dialysis, it was dialyzed with a hydrochloric acid solution at pH 3.5 for 48 hours, and freeze-dried to obtain thiolated sodium carboxymethyl cellulose.

[0008] One aspect of the present invention relates to the application of the thiolated sodium carboxymethyl cellulose in the production of food, health products or medicines.

[0009] The present invention also relates to a probiotic product, comprising probiotics and the above-mentioned thiolated sodium carboxymethyl cellulose.

[0010] The probiotics can be selected from any one or a combination of two or more of the genera Bacillus, Bifidobacterium, Lactobacillus, Streptococcus, Lactococcus, Leuconostoc, Propionibacterium, Saccharomyces, Pediococcus, and Staphylococcus.

[0011] The probiotics may be selected from any one or more combinations of Bacillus subtilis, Bacillus coagulans, Bacillus licheniformis, Clostridium, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium animalis, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus fermentum, Lactobacillus bulgaricus, Streptococcus thermophilus, Kluyveromyces marxianus, Pediococcus acidilactici, Pediococcus pentosaceus, Staphylococcus calvatus, Staphylococcus xylosus, Staphylococcus carnosus, Leuconostoc mesenteroides subsp. mesenteroides, Lactococcus lactis lactis subsp. lactis, Lactococcus lactis cremoris subsp., and Lactococcus lactis diacetyl subsp.

[0012] The present invention also relates to a dual-bacteria multi-core gel particle, the preparation method of which comprises the following steps:

[0013] (1) Preparation of core microspheres

[0014] Take 1-2 mL of Bifidobacterium adolescentis suspension in a centrifuge tube, and add thiolated sodium carboxymethyl cellulose at a ratio of 5-15 mg / mL, and mix well; then add 20-40 mL of sodium alginate solution with a concentration of 20-40 mg / mL and 0.1-0.2 g CaCO3, mix well, and make up to 60-120 mL with sterile water to obtain a mixed solution; add 180 mL of oil phase containing 2-3 mg / mL Span80 to the mixed solution, place it on a 400 rpm magnetic stirrer for emulsification for 5 min; add 200-400 μL CH3COOH and continue emulsification for 10 min; after emulsification, add sterile distilled water, mix well, let stand for stratification, discard the upper oil phase, take the microspheres of the lower aqueous phase, centrifuge them at 4°C and 5000 r / min for 5 min, discard the supernatant, and wash three times with sterile water to obtain inner core microspheres;

[0015] (2) Preparation of shell microspheres

[0016] 0.5-1 ml of Bacillus subtilis and 0.5-1 ml of the inner core microspheres prepared in step (1) are mixed evenly to obtain a bacterial ball mixture; then the bacterial ball mixture is fully mixed with a sodium alginate solution with a concentration of 30-50 mg / mL at a volume ratio of 1:1-3 to obtain a bacterial gel mixture; the bacterial gel mixture is squeezed dropwise into a calcium chloride solution with a concentration of 20-40 mg / mL at a flow rate of 10-15 ml / min using a sterile syringe, and the distance between the needle tip and the surface of the calcium chloride solution is controlled to be 45-55 cm during the dripping, and then the mixture is allowed to stand and harden for 30 minutes to form gel beads, which are then filtered with filter paper and washed three times with sterile water to obtain double-bacteria multi-core gel particles.

[0017] The present invention also relates to a double bacteria multi-core gel granule powder, which is prepared by freeze-drying the double bacteria multi-core gel granules.

[0018] The Bifidobacterium adolescentis is Bifidobacterium adolescentis FS2-3, and it was deposited in the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection Administration on June 10, 2022. Its address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No. 25046.

[0019] The Bacillus subtilis is Bacillus subtilis SNBS-3, which was deposited in the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection Administration on June 9, 2022. Its address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No. 25040.

[0020] The invention also relates to the application of the double-bacteria multi-core gel particles in the preparation of food, health products or medicines.

[0021] Beneficial effects of the present invention:

[0022] The thiol content of the thiolated sodium carboxymethyl cellulose prepared by the present invention is 139.02±3.14μmol / g. Compared with sodium carboxymethyl cellulose, the thiolated sodium carboxymethyl cellulose prepared by the present invention is better in enhancing the adhesion of probiotics in the intestine through in vitro mucus adhesion test and in vitro intestinal adhesion test results, which are 43.67±0.42% and 45.0±1.04% respectively, which is beneficial to the colonization of probiotics in the intestine. At the same time, the results of rheological tests and tensile tests show that the interaction between thiolated sodium carboxymethyl cellulose and intestinal mucus is more obvious.

[0023] The particle size distribution of the dual-bacteria multi-core gel particles prepared by the present invention is relatively uniform, wherein the particle size of the inner core microspheres is 85.45±10.92 μm, and the particle size of the dual-bacteria multi-core gel particles is 304.29±15.24 μm; the gel particles present a concave and wrinkled structure, and the surface is a dense reticular structure without pores, which limits the influence of acid and enzyme on probiotics, and has a better effect of encapsulating probiotics.

[0024] The dual-bacteria multi-core gel particles can effectively resist digestion by gastric juice and smoothly deliver probiotics to the intestine. In the intestine, by first releasing the Bacillus subtilis on the outer layer of the gel particles to consume free oxygen, a good anaerobic environment is provided for the survival and colonization of Bifidobacterium adolescentis in the intestine, which can significantly increase the survival rate of Bifidobacterium. After being treated in artificial intestinal fluid for 30 minutes, the number of live bacteria of Bacillus subtilis SNBS-3 located in the outer shell of the dual-bacteria multi-core gel particles was significantly higher than that of Bifidobacterium adolescentis FS2-3 located in the inner core; after 60 minutes of treatment, the number of live bacteria of Bacillus subtilis SNBS-3 reached the maximum; and as the treatment time increased, the number of live bacteria of Bifidobacterium adolescentis FS2-3 gradually increased. After 120 minutes of treatment, the number of live bacteria of Bifidobacterium adolescentis FS2-3 reached the maximum and remained stable at 6.67lg (CFU / mL).

[0025] The viable counts of Bacillus subtilis and Bifidobacterium adolescentis in the double-bacteria multi-core gel granule powder obtained after freeze-drying were 7.60×10 7 The sum of cfu / g is 8.09×10 7 cfu / g, and the storage stability of the two probiotics is high. After being stored at 4°C for 90 days, the survival rates of Bacillus subtilis SNBS-3 in the shell and Bifidobacterium adolescentis FS2-3 in the core are as high as 81.63% and 84.10%, respectively. This shows that the gel particles provided by the present invention can effectively reduce the influence of oxygen and other adverse external environments on the internal Bifidobacterium adolescentis, stabilize its effective live bacteria count, fully retain the functionality of the bacteria, and achieve unexpected technical effects.

[0026] The double-bacteria multi-core gel particles provided by the present invention can be widely used in the production of food, health products or medicines, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the potential value of sodium carboxymethyl cellulose and thiolated sodium carboxymethyl cellulose;

[0028] Figure 2 is the adhesion rate of intestinal mucus in vitro;

[0029] Figure 3 is the in vitro intestinal adhesion rate;

[0030] Figure 4 The dynamic rheological properties of the mixed solution (a: mucus and sodium carboxymethyl cellulose; b: mucus and thiolated sodium carboxymethyl cellulose);

[0031] Figure 5 The maximum separation force and total adhesion work of sodium carboxymethylcellulose and thiolated sodium carboxymethylcellulose on the intestinal surface;

[0032] Figure 6This is the particle size diagram of the inner core microsphere and the double bacteria microsphere;

[0033] Figure 7 The morphology of the double-bacteria multi-core gel particles (a: scanning electron microscopy; b: optical microscopy);

[0034] Figure 8 To characterize the encapsulation status of Bacillus subtilis and Bifidobacterium adolescentis in dual-bacteria multi-core gel particles;

[0035] Fig. 9 This is a graph showing the changes in the number of live bacteria of Bacillus subtilis and Bifidobacterium adolescentis in the dual-bacteria multi-core gel particles in artificial intestinal fluid.

[0036] Fig.10 This is a storage stability analysis chart of Bacillus subtilis and Bifidobacterium adolescentis in dual-bacteria multi-core gel particles. DETAILED DESCRIPTION

[0037] The following is a more detailed description of the specific embodiments of the present invention in conjunction with the accompanying drawings and examples, so as to better understand the scheme of the present invention and its advantages in various aspects. However, the specific embodiments and examples described below are only for the purpose of illustration, rather than for limiting the present invention.

[0038] 1. The Bifidobacterium adolescentis described in the embodiments of the present invention was screened from fresh feces of infants and young children aged 0-3 years who had not used probiotic preparations within half a year, and was named Bifidobacterium adolescentis FS2-3. It was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration (CGMCC) on June 10, 2022, with the deposit number CGMCC No.25046.

[0039] Bifidobacterium adolescentis FS2-3 has good biosafety and has obvious inhibitory effects on Salmonella, Escherichia coli and Staphylococcus aureus. Among them, it has the strongest inhibitory ability against Escherichia coli, with the diameter of the inhibition zone exceeding 14mm; it has strong tolerance to artificial gastrointestinal fluid. After 2h of treatment in artificial gastric fluid, the survival rate of FS2-3 strain is as high as 88.6%. After 24h of treatment in artificial intestinal fluid environment, it can still maintain a survival rate of 22.5%, which is significantly higher than the control bacteria Bifidobacterium adolescentis CICC6070. The strain has strong hydrophobicity, which is as high as 75%, significantly higher than the control strain; its self-agglutination rate gradually increases with time, and is significantly higher than the control strain after 6 hours; it has a certain mucin binding ability, can effectively adhere to the intestinal mucosa, and form a probiotic barrier on the surface of the intestinal mucosa to inhibit the colonization and invasion of harmful bacteria; it has strong antioxidant properties, and its complete cells and cell-free extracts have a DPPH clearance rate of more than 65%, and have good reducing power, with a range of reducing power of 72.01-57.50 (μM). Animal experimental results show that the strain can effectively alleviate bacterial colitis in mice. By gavage with Bifidobacterium adolescentis FS2-3, the symptoms of reduced food intake and weight loss caused by bacterial colitis can be significantly alleviated, the expression of inflammatory factors TNF-α, IL-1β, and IL-6 in serum can be downregulated, the level of C-reactive protein CRP can be reduced, the content of short-chain fatty acids such as acetic acid, propionic acid and butyric acid in the intestine of mice with bacterial colitis can be increased, the infiltration of inflammatory cells can be reduced, and the colon tissue of mice can be repaired well. This strain can be widely used in the production of food, health products and medicines, and has broad market prospects.

[0040] The preparation method of the Bifidobacterium adolescentis FS2-3 bacterial suspension described in the embodiment of the present invention is as follows: thaw the Bifidobacterium adolescentis FS2-3 strain preserved in glycerol in a -80°C ultra-low temperature refrigerator, streak on a Bifidobacterium solid culture medium, and culture it in an anaerobic incubator containing 5% CO2, 5% H2 and 90% N2 for 48 hours. After recovery, pick a single colony for staining, and after identification, inoculate it in a Bifidobacterium liquid culture medium and culture it for 48 hours. Inoculate it into a Bifidobacterium liquid culture medium at an inoculum rate of 1-2% for passage. Centrifuge at 4000r / min, 4°C for 20min, discard the supernatant and collect the bacteria, wash it twice with 0.9% saline, and then centrifuge it. The strain is prepared into a bacterial suspension in phosphate buffer (PBS, pH 7.4) so ​​that the effective viable count is 1×10 8 CFU / mL, and store the bacterial suspension in a 4°C refrigerator for later use.

[0041] 2. The Bacillus subtilis described in the embodiment of the present invention was screened from traditional fermented soybean paste collected in Shenyang City, Liaoning Province, and was named Bacillus subtilis SNBS-3. It was deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration on June 9, 2022, with the deposit number CGMCC No.25040.

[0042] Bacillus subtilis SNBS-3 has a significant inhibitory effect on black spot pathogens, with an inhibition rate of 99.93%, close to 100%. The strain has a significant control effect on black spot and red spot of Nanguo pear, and can significantly improve the quality and yield of fruit; compared with the control group, the incidence of black spot and red spot of Nanguo pear in the treatment group treated with Bacillus subtilis SNBS-3 powder was significantly reduced, and the control efficiency was as high as 88.28% and 71.36%, respectively. In addition, the fruits of Nanguo pear in the treatment group were larger and fuller, and the yield of healthy and disease-free fruits was 32.6% higher than that of the control group. The strain can produce high levels of chitinase and protease; after 24 hours of fermentation, the chitinase and protease activities in the fermentation supernatant were as high as 3.96U / mL and 135.25U / mL, respectively; it has a strong acid production ability; after 4 hours of fermentation, it began to produce a lot of acid, and the pH value of the fermentation liquid dropped rapidly; after 14 hours, it entered the stable period, and the pH value of the fermentation liquid was stable at around 3.6. Bacillus subtilis SNBS-3 can be used as bio-fertilizer, biocontrol agent, etc., and is widely used in agricultural production with significant effects and broad application prospects.

[0043] The preparation method of the Bacillus subtilis SNBS-3 bacterial suspension described in the embodiment of the present invention is as follows: thaw the Bacillus subtilis SNBS-3 strain preserved in glycerol in a -80°C ultra-low temperature refrigerator, take the bacterial solution and streak it on an LB solid culture medium plate, and culture it at 37°C overnight. Pick the colony with an inoculation loop, inoculate it into LB liquid culture medium and culture it at 37°C for 24 hours, and inoculate it into LB liquid culture medium for passage at an inoculation rate of 1-2%. Centrifuge at 80,000 r / min, 4°C for 15 minutes, discard the supernatant and collect the bacteria, wash it twice with 0.9% saline, and then centrifuge it. The strain is prepared into a bacterial suspension in phosphate buffer (PBS, pH7.4) so ​​that the effective viable count is 1×10 8 CFU / mL, and store the bacterial suspension in a 4°C refrigerator for later use.

[0044] The present invention is further described below in conjunction with specific implementation methods.

[0045] Example 1 Preparation method of thiolated sodium carboxymethyl cellulose

[0046] Dissolve 100-200 mg sodium carboxymethyl cellulose in 20-40 mL ultrapure water, add 220-440 mg 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 130-260 mg N-hydroxysuccinimide, adjust the pH and stir for 2 h, then add 475-950 mg cysteine ​​hydrochloride and dialyze for 24 h. After dialysis, add 295-590 mg dithiothreitol, adjust the pH, and react at room temperature for 24 h. Adjust the pH with 0.1 mol.L hydrochloric acid. -1 The mixture was dialyzed with a sodium chloride solution to a pH of 3.5, and then dialyzed with a pH 3.5 hydrochloric acid solution for 48 hours, and freeze-dried to obtain thiolated carboxymethyl cellulose sodium.

[0047] Example 2 Structural Characterization of Mercaptolated Sodium Carboxymethyl Cellulose

[0048] 1. Determination of sulfhydryl content

[0049] The thiol content of thiolated sodium carboxymethyl cellulose was measured by Ellman's method. The method for quantitatively measuring the thiol content in thiolated sodium carboxymethyl cellulose is as follows: 2.2-4.4 mg of dry sample was weighed and dissolved in 9.5-19 mL PBS (pH=7.4) buffer solution, and then 0.5-1.0 mL DTNB (6 mmol / L, containing 1 mmol / LEDTA) was added. After incubation at 37°C for 15 minutes, after DTNB and thiolated sample were fully reacted, the thiol content of the sample was quantitatively determined by a pre-determined standard curve. The cysteamine hydrochloride concentrations in the standard curve were 0.1, 0.15, 0.20, 0.25 and 0.30 mmol / L, respectively. Each sample was measured three times and the average value was taken.

[0050] Take 50-100 mg of thiolated sodium carboxymethyl cellulose and dissolve it in 100-200 ml of deionized water. Take the sample solution and test it according to the above method, and use the standard curve to calculate the thiol content.

[0051] The results showed that the thiol content in the thiolated sodium carboxymethyl cellulose prepared above was 139.02±3.14 μmol / g.

[0052] 2. Potential change analysis

[0053] The Zeta potential of sodium carboxymethyl cellulose (CMC) and thiolated sodium carboxymethyl cellulose (CMC-SH) was measured using Zetasizer Nano (ZEN1500, Malvern, UK). Figure 1 shown.

[0054] from Figure 1It can be seen that the zeta potential values ​​of unmodified and modified sodium carboxymethyl cellulose are both negative, indicating that the negatively charged groups on the surface of the substance are more than the positively charged groups. The potential of unmodified sodium carboxymethyl cellulose was measured to be -53.5mV, and that of the modified thiolated sodium carboxymethyl cellulose was -44.2mV. This shows that sodium carboxymethyl cellulose has successfully carried a thiol group.

[0055] Example 3 Effect of thiolated sodium carboxymethyl cellulose on the adhesion of probiotics

[0056] The present invention takes Bifidobacterium adolescentis FS2-3 as an example, and observes and analyzes the adhesion effect of intestinal mucus on the bacteria in the case of adding thiolated carboxymethyl cellulose sodium through in vitro adhesion test. The specific implementation method is as follows:

[0057] 1. In vitro adhesion test

[0058] (1) Preparation of intestinal mucus: Place the small intestine of the mouse on an ice box and rinse it with sterile PBS buffer. Then cut the small intestine longitudinally and slowly scrape the mucus on the inner surface of the small intestine with a scraper. Place it in a sterile centrifuge tube and add 2 to 4 times the volume of PBS buffer to mix thoroughly. Centrifuge it at 4°C, 12,000 r / min for 10 min and divide it into portions for later use.

[0059] (2) Preparation of a mixed solution of probiotics and thiolated sodium carboxymethyl cellulose

[0060] Bifidobacterium adolescentis FS2-3 was inoculated into 5-10 mL of bifidobacterium liquid culture medium (tryptone 10 g, anhydrous sodium acetate 3 g, dipotassium hydrogen phosphate 2 g, magnesium sulfate heptahydrate 0.575 g, manganese sulfate monohydrate 0.25 g, glucose 20 g, trisodium citrate 2.42 g, yeast extract 4 g, beef extract 8 g, Tween 1 ml, L-cysteine ​​hydrochloride 0.5 g, mupirocin 1 ml (0.05 g / mL anhydrous ethanol), distilled water 1 L), cultured at 37°C for 24 h, and centrifuged at 12000 r / min for 1 min to collect the cells. The cells were washed 2-3 times with PBS, then resuspended in PBS and stored at 4°C for later use.

[0061] (3) In vitro mucus adhesion test

[0062] Preparation of sodium carboxymethylcellulose / probiotic suspension: Dissolve 10-20 mg sodium carboxymethylcellulose in 2-4 mL ultrapure water to prepare sodium carboxymethylcellulose solution. Thoroughly mix 100-200 μL sodium carboxymethylcellulose solution with 1-2 mL Bifidobacterium adolescentis FS2-3 suspension;

[0063] Thiolated carboxymethyl cellulose sodium / probiotic suspension: 100-200 mg of the thiolated carboxymethyl cellulose sodium prepared in Example 1 was weighed and thoroughly mixed with 1-2 mL of Bifidobacterium adolescentis FS2-3 suspension;

[0064] Physiological saline / probiotic suspension: Mix 100-200 μL of physiological saline with 1-2 mL of Bifidobacterium adolescentis FS2-3 suspension.

[0065] Adjust the concentration of the intestinal mucus prepared in (1) and add it to a 96-well plate and place it at 4°C to fix overnight. Add sodium carboxymethyl cellulose / probiotic suspension and thiolated sodium carboxymethyl cellulose / probiotic suspension respectively, with normal saline / probiotic suspension as a control, and culture for 2 hours. Wash with PBS 2 to 3 times to remove non-adherent probiotics. Then add lysis buffer containing 1% SDS-0.1mol / L NaOH to release the adhered bacteria. Then count the live bacteria on the plate and calculate the adhesion rate according to the following formula. The results are as follows: Figure 2 shown.

[0066]

[0067] Where: A is the total bacterial count (CFU / mL);

[0068] B is the number of adhered bacteria (CFU / mL).

[0069] from Figure 2 It can be seen that the adhesion rate of Bifidobacterium adolescentis FS2-3 to intestinal mucus is 7.69%, while the adhesion rate of the bacteria in the group with thiolated sodium carboxymethylcellulose is as high as 43.67±0.42%, which is significantly higher than the adhesion rate of 24.34±0.72% in the sodium carboxymethylcellulose group. This shows that thiol-modified sodium carboxymethylcellulose can significantly increase the adhesion of probiotics to intestinal mucus.

[0070] 2. In vitro intestinal tissue adhesion test

[0071] The mouse intestine was spread on a sterile glass slide, and sodium carboxymethyl cellulose / probiotics and thiolated sodium carboxymethyl cellulose / probiotic suspension were added respectively, and saline / probiotic suspension was used as a control. Three slides were placed in a sterilized centrifuge tube and placed on a shaker at 80r / min. After 2 hours, they were taken out and rinsed with 0.9% NaCl 2 to 3 times, and the number of viable bacteria was calculated. The adhesion rate was calculated according to the following formula, and the results were obtained from Figure 3 shown.

[0072]

[0073] Where: A is the total bacterial count (CFU / mL);

[0074] B is the number of non-adherent bacteria (CFU / mL).

[0075] from Figure 3 It can be seen that compared with sodium carboxymethylcellulose, the adhesion rate of Bifidobacterium adolescentis FS2-3 to intestinal tissue in the group with thiolated sodium carboxymethylcellulose was as high as 45.0±1.04%. This also shows again that the adhesion effect of probiotics added with thiolated sodium carboxymethylcellulose on the intestine is significantly better than that of a single probiotic, and thiolated sodium carboxymethylcellulose can enhance the adhesion of probiotics to the intestine.

[0076] In summary, the thiolated sodium carboxymethyl cellulose prepared by the present invention can significantly enhance the adhesion effect of intestinal mucus on probiotics, improve the intestinal adhesion of probiotics, and thus facilitate the colonization of probiotics in the intestine. Example 4 Interaction between thiolated sodium carboxymethyl cellulose and intestinal mucus

[0077] The present invention uses a rheometer and a classical tensile test to analyze the interaction between the thiolated carboxymethyl cellulose sodium prepared in Example 1 and intestinal mucus. The specific implementation is as follows:

[0078] 1. Rheological measurement of the interaction between thiolated sodium carboxymethyl cellulose and intestinal mucus

[0079] The viscoelastic changes of intestinal mucus with sodium carboxymethyl cellulose and sodium thiolated carboxymethyl cellulose were analyzed by rheological oscillation time scanning to characterize the interaction between intestinal mucus and sodium carboxymethyl cellulose and sodium thiolated carboxymethyl cellulose.

[0080] Sodium carboxymethyl cellulose and sodium thiolated carboxymethyl cellulose were added to an equal volume of intestinal mucus at 50-100 mg / mL. The AR-1500 rheometer was used with a 60 mm diameter parallel plate titanium alloy rotor to perform linear viscoelastic region scanning: the frequency was set to 1-2 Hz, the strain was set to 0.1%-1000%, and the storage modulus (G′) and loss modulus (G″) of sodium carboxymethyl cellulose and sodium thiolated carboxymethyl cellulose were measured and recorded. The results are shown in Figure 4 shown.

[0081] from Figure 4 It can be seen that the dynamic rheological properties test results of sodium carboxymethyl cellulose show that ( Figure 4 a), as time goes by, the loss of the mixture of sodium carboxymethyl cellulose and intestinal mucus G″ is always higher than the storage modulus G′, which indicates that sodium carboxymethyl cellulose has a viscous property. The results of the mixture of thiolated sodium carboxymethyl cellulose and intestinal mucus show ( Figure 4b), the initial loss film amount G″ is higher than the storage modulus G′, and after a period of time, the loss film amount G″ and the storage modulus G′ intersect, the storage modulus G′ is higher than the loss film amount G″, and the mixture of thiolated sodium carboxymethyl cellulose and intestinal mucus shows an elastic advantage. This shows that the modification of sodium carboxymethyl cellulose by thiol groups gives it viscoelasticity, which can enhance the interaction between thiolated sodium carboxymethyl cellulose and intestinal mucus.

[0082] 2. Measuring the interaction between thiolated sodium carboxymethylcellulose and intestinal mucus by tensile test

[0083] The tensile test was set up to measure the maximum separation force and adhesion work of sodium carboxymethylcellulose and thiolated sodium carboxymethylcellulose with intestinal mucus. Mouse intestinal tissue was cut into approximately 3 cm 2 The tissue was fixed in a beaker containing an adhesive and covered with 0.1M pH 6.8 phosphate buffer, and the beaker was placed on a balance. A rope was taken, one end of which was connected to a syringe pump (V2, Guanjie, China), and the other end was used to fix a test disc (covered with a certain concentration of sodium carboxymethyl cellulose and sodium thiolated carboxymethyl cellulose) and attach the test disc to the intestinal tissue. After 20 minutes at room temperature, the test disc was peeled off from the intestinal tissue at a speed of 0.1 mm / s. The balance change data was recorded every second, and the maximum separation force (MDF) and total adhesion force (TWA) representing the area under the force / distance curve were calculated. The results are shown in Figure 5 shown.

[0084] from Figure 5 Comparing the two groups of data, the maximum separation force of thiolated carboxymethyl cellulose sodium reached 82.35±1.25mN, and the total adhesion work was 71.99±3.23μJ, which was higher than that of the carboxymethyl cellulose sodium group. This also shows that thiolated carboxymethyl cellulose sodium can adhere to the intestinal surface better.

[0085] In summary, the thiolated carboxymethyl cellulose sodium prepared by the present invention has a stronger interaction with intestinal mucus and a better bonding effect.

[0086] Example 5 Preparation method of dual-bacteria multi-core gel particles

[0087] 1. Preparation of inner core microspheres

[0088] Take 1-2 mL of Bifidobacterium adolescentis FS2-3 suspension (1×10 8CFU / mL) in a centrifuge tube, and add thiolated carboxymethyl cellulose sodium at a ratio of 5-15 mg / mL, and mix; then add 20-40 mL of a sodium alginate solution with a concentration of 20-40 mg / mL and 0.1-0.2 g CaCO3, mix, and make up to 60-120 mL with sterile water to obtain a mixed solution; add 180 mL of an oil phase containing 2-3 mg / mL Span80 to the mixed solution, place it on a 400 rpm magnetic stirrer for emulsification for 5 min; add 200-400 μL CH3COOH and continue emulsification for 10 min; after emulsification, add sterile distilled water, mix thoroughly, let stand for stratification, discard the upper oil phase, take the microspheres of the lower aqueous phase, centrifuge them at 4°C and 5000 r / min for 5 min, discard the supernatant, and wash three times with sterile water to obtain inner core microspheres;

[0089] 2. Preparation of Shell Microspheres

[0090] 0.5-1 ml of Bacillus subtilis SNBS-3 bacterial solution (1×10 8 CFU / mL) is mixed evenly with 0.5-1 mL of the inner core microspheres prepared above to obtain a bacterial ball mixture; then the bacterial ball mixture is fully mixed with a sodium alginate solution with a concentration of 30-50 mg / mL at a volume ratio of 1:1-3 to obtain a bacterial gel mixture; the bacterial gel mixture is squeezed dropwise into a calcium chloride solution with a concentration of 20-40 mg / mL at a flow rate of 10-15 ml / min using a sterile syringe, and the distance between the needle tip and the surface of the calcium chloride solution is controlled to be 45-55 cm during the dripping, and then the mixture is left to harden for 30 minutes to form gel beads, which are then filtered with filter paper and washed three times with sterile water to obtain double-bacteria multi-core gel particles.

[0091] Example 6 Characterization and Analysis of Double-bacteria Multi-core Gel Particles

[0092] 1. Particle size analysis

[0093] The particle size of the dual bacteria multi-core gel particles prepared in Example 5 was tested using a micron particle size analyzer. The results are as follows: Figure 6 shown.

[0094] from Figure 6 It can be seen that the particle size distribution of the gel particles prepared by the present invention is relatively uniform. Among them, the particle size of the inner core microsphere is 85.45±10.92μm, and the particle size of the double bacteria multi-core gel particles is 304.29±15.24μm. The particle size data shows that the outer shell microsphere is sufficient to embed the inner core microsphere.

[0095] 2. Microscopic observation of the morphology of dual-bacteria multi-core gel particles

[0096] (1) The surface of the double-bacteria multi-core gel particles fixed with double-sided tape was observed by SEM. Afterwards, the sample was gold-plated by sputtering copper tube for 2.5 min. The results are shown in Figure 7 As shown in a.

[0097] from Figure 7 a It can be seen that the dual-bacteria multi-core gel particles present a concave and wrinkled structure, and the surface is a dense reticular structure without pores, which limits the effects of acid and enzyme on probiotics and has a better effect of encapsulating probiotics.

[0098] (2) The morphology of the double-bacteria multi-core gel particles was observed using an optical microscope at 10×100×. Figure 7 As shown in b.

[0099] from Figure 7 b It can be seen that the outer shell microspheres successfully encapsulated the inner core microspheres and successfully produced dual-bacteria multi-core gel particles.

[0100] 3. Laser confocal microscopy observation

[0101] Before preparing the gel particles described in Example 5, BacLight TM Bacterial Activity Kit Instructions, using Syto 9 dye to stain two probiotics, Bifidobacterium adolescentis FS2-3 and Bacillus subtilis SNBS-3. Laser confocal microscopy was used to observe the encapsulation state of the prepared dual-bacteria multi-core gel particles. The results are as follows Figure 8 shown.

[0102] from Figure 8 It can be seen that green fluorescence is visible in the double-bacteria multi-core gel particles, proving that the probiotics are successfully embedded in the double-bacteria multi-core gel particles and are evenly distributed.

[0103] Example 7 Analysis of the release of probiotics from dual-bacteria multi-core gel particles in a simulated gastrointestinal environment

[0104] Weigh 1-2g of the double bacteria multi-core gel particles and wash them thoroughly with sterile water. Then add the double bacteria multi-core gel particles to 9-18mL preheated artificial gastric juice (the mass concentration ratio of 0.85% NaCl solution is adjusted to pH 1.5 with 0.1mol / L HCl, and then 1% pepsin is added after high pressure sterilization at 121℃ for 20min) for 2h, and then transfer to preheated artificial intestinal juice (the mass concentration ratio of 0.85% NaCl solution is adjusted to pH 8.0 with 0.01mol / L NaOH, and then 1% trypsin is added after high pressure sterilization at 121℃ for 20min), shake in a water bath shaker at 37℃ and 100rpm for 1-2h to release the bacteria, and take 1-2mL of the sample solution every 30min, perform gradient dilution, and determine the number of viable bacteria by plate counting method. The bacterial suspension is divided into two parts, one of which is spread on a bifidobacterium solid culture medium and cultured in an anaerobic box for 24h. The other portion was spread on LB solid medium and placed in a 37°C incubator for 24 hours, after which the colonies were counted. Fig. 9 shown.

[0105] from Fig. 9 It can be seen that after being treated in artificial gastric juice for 2 hours, the release amount of probiotics in the dual-bacteria multi-core gel particles in artificial intestinal juice gradually increased with the extension of treatment time, and then tended to be stable. After being treated in artificial intestinal juice for 30 minutes, the number of live bacteria of Bacillus subtilis SNBS-3 in the outer shell of the dual-bacteria multi-core gel particles was significantly higher than that of Bifidobacterium adolescentis FS2-3 in the inner core, indicating that Bacillus subtilis SNBS-3 in the gel particles was released first; after 60 minutes of treatment, the number of live bacteria of Bacillus subtilis SNBS-3 reached the maximum; and as the treatment time extended, the number of live bacteria of Bifidobacterium adolescentis FS2-3 gradually increased. After 120 minutes of treatment, the number of live bacteria of Bifidobacterium adolescentis FS2-3 reached the maximum and remained stable at 6.67lg (CFU / mL), at which time the dual-bacteria multi-core gel particles were basically completely disintegrated.

[0106] The above results show that the dual-bacteria multi-core gel particles prepared by the present invention can effectively resist digestion by gastric juice and smoothly deliver probiotics to the intestine. In the intestine, by first releasing the Bacillus subtilis on the outer layer of the gel particles to consume free oxygen, a good anaerobic environment is provided for the survival and colonization of Bifidobacterium adolescentis in the intestine, which can significantly increase the survival rate of Bifidobacterium adolescentis, and can deliver it to the colon in a targeted manner, achieve full release in the colon, significantly prolong its residence time in the intestine, and fully exert its probiotic function.

[0107] Example 8 Storage stability analysis of dual-bacteria multi-core gel particles

[0108] After freeze-drying the dual-bacteria multi-core gel particles prepared in Example 5, a powder was obtained, which was divided into 1 mL cryopreservation tubes, 1 g per tube, and then stored in a desiccator for 90 days. One tube was taken at 0, 15, 30, 45, 60, 75, and 90 days, and then lysed with cyst dissection fluid (concentration of 20-40 mg / mL trisodium citrate), and the number of viable bacteria of Bacillus subtilis and Bifidobacterium adolescentis was determined by plate colony counting method, and their survival rates were calculated. The storage stability was evaluated by recording the time.

[0109] The results showed that the viable counts of Bacillus subtilis and Bifidobacterium adolescentis in the dual-bacteria multi-core gel granule powder of the present invention were 7.60×10 7 The sum of cfu / g is 8.09×10 7 cfu / g.

[0110] from Fig.10 It can be seen that with the extension of storage time, the survival rates of the two probiotics in the shell and the inner core showed a downward trend. The survival rate of Bacillus subtilis SNBS-3 in the shell dropped to 81.63% after storage at 4°C for 90 days; while the survival rate of Bifidobacterium adolescentis FS2-3 in the inner core was as high as 84.10% after storage at 4°C for 90 days, and the number of viable bacteria at this time was 6.80×10 7 cfu / g. This shows that the gel particles provided by the present invention can effectively reduce the influence of oxygen and other adverse external environments on the internal Bifidobacterium adolescentis, stabilize the number of effective live bacteria, fully retain the functionality of the bacteria, and achieve unexpected technical effects.

Claims

1. A dual-bacteria multi-core gel particle, characterized in that: The preparation method of the dual-bacteria multi-core gel particles comprises the following steps: (1) Preparation of core microspheres Take 1-2 mL of Bifidobacterium adolescentis suspension in a centrifuge tube, add thiolated sodium carboxymethyl cellulose at a ratio of 5-15 mg / mL, and mix well; then add 20-40 mL of sodium alginate solution with a concentration of 20-40 mg / mL and 0.1-0.2 g CaCO3, mix well, and make up to 60-120 mL with sterile water to obtain a mixed solution; add 180 mL of oil phase containing 2-3 mg / mL Span80 to the mixed solution, place it on a 400 rpm magnetic stirrer for emulsification for 5 min; add 200-400 µL CH3COOH and continue emulsification for 10 min; after emulsification, add sterile distilled water, mix well, let stand for stratification, discard the upper oil phase, take the microspheres of the lower aqueous phase, centrifuge them at 4°C and 5000 r / min for 5 min, discard the supernatant, and wash three times with sterile water to obtain the inner core microspheres; (2) Preparation of shell microspheres 0.5-1 ml of Bacillus subtilis suspension and 0.5-1 ml of the inner core microspheres prepared in step (1) are mixed evenly to obtain a bacterial ball mixture; then the bacterial ball mixture is mixed evenly with a sodium alginate solution with a concentration of 30-50 mg / mL at a volume ratio of 1:1-3 to obtain a bacterial gel mixture; the bacterial gel mixture is squeezed dropwise into a calcium chloride solution with a concentration of 20-40 mg / mL at a flow rate of 10-15 ml / min using a sterile syringe, and the distance between the needle tip and the surface of the calcium chloride solution is controlled to be 45-55 cm during the dripping, and then the mixture is allowed to stand and harden for 30 minutes to form gel beads, which are then filtered with filter paper and washed three times with sterile water to obtain dual-bacteria multi-core gel particles; The sodium mercaptocarboxymethyl cellulose is prepared by the following method: Dissolve 100-200 mg sodium carboxymethyl cellulose in 20-40 mL ultrapure water, add 220-440 mg 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 130-260 mg N-hydroxysuccinimide, adjust the pH and stir for 2 h, then add 475-950 mg cysteine ​​hydrochloride and dialyze for 24 h; after dialysis, add 295-590 mg dithiothreitol, adjust the pH, and react at room temperature for 24 h; adjust the pH with 0.1 mol.L hydrochloric acid. -1 Sodium chloride solution to pH 3.5 was used to dialyze the mixture; after 24 hours of dialysis, the mixture was dialyzed with a pH 3.5 hydrochloric acid solution for 48 hours, and freeze-dried to obtain thiolated carboxymethyl cellulose sodium; The deposit number of the Bifidobacterium adolescentis is CGMCC No. 25046; The deposit number of the Bacillus subtilis is CGMCC No.25040.

2. Use of the dual-bacteria multi-core gel particles according to claim 1 in the preparation of medicines.

3. A dual-bacteria multi-core gel granule powder, characterized in that: The powder is prepared by freeze-drying the dual-bacteria multi-core gel particles described in claim 1.

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