Preparation method and application of Nisin oral delivery system based on yeast microcapsules

Abstract

The invention belongs to the technical field of drug carriers, and discloses preparation and application of a yeast microcapsule oral delivery system of Nisin. Saccharomycetes are treated by a hypertonic sodium chloride solution to become an active delivery carrier, and Nisin is packaged into yeast microcapsules with negative electricity through electrostatic deposition. The prepared Nisin yeast microcapsule delivery system has the advantages of high load rate, low release rate and pH responsiveness, and successfully realizes the protection effect on Nisin.

Description

Technical Field

[0001] The invention relates to the technical field of drug carriers, and in particular to the preparation and application of a yeast microcapsule oral delivery system of Nisin. Background Art

[0002] The colon is an important part of the intestine and the main site of inflammatory bowel diseases such as ulcerative colitis and Crohn's disease. There is a rich microbial community in the colon, and maintaining a balance of intestinal flora is essential for health. Several research reports have pointed out that ulcerative colitis is associated with changes in the intestinal microbiome, reduced biodiversity, and changes in the relative abundance of dominant flora.

[0003] Nisin is a natural peptide with strong antimicrobial activity. It has a strong inhibitory effect on many Gram-positive bacteria, including food spoilage bacteria and pathogenic bacteria. It is the only bacteriocin in the world that is allowed to be used as a food additive. Antimicrobial peptides have the effects of regulating the immune system and anti-inflammatory. By delivering antimicrobial peptides, the intestinal barrier function can be improved, the immune response can be regulated, and the chronic inflammatory response of the intestine can be alleviated. At the same time, antimicrobial peptides selectively inhibit the growth of harmful pathogens, reduce the invasion of harmful bacteria to the intestine, and help maintain the balance of intestinal microecology.

[0004] Oral administration is considered to be the most ideal route of administration because of its convenience, relatively good safety, high patient acceptance and compliance, and easy dosage adjustment. However, there are a series of challenges in the oral delivery of antimicrobial peptides, such as: antimicrobial peptides are easily degraded by digestive enzymes in the gastrointestinal tract, resulting in reduced effectiveness before entering the blood circulation. Even if antimicrobial peptides are able to pass through the gastrointestinal tract, they may face the problem of low absorption efficiency. Usually the bioavailability is low because they are degraded by digestive enzymes in the upper gastrointestinal tract. In order to reach the colon and be able to specifically deliver peptide or protein drugs, the obstacles of the upper gastrointestinal tract and the advantages of the colon environment must be taken into account.

[0005] Therefore, there is an urgent need to develop a safe and effective Nisin yeast microcapsule oral delivery system to improve the stability, release control and oral absorption efficiency of Nisin in the body, thereby alleviating and improving colitis. Summary of the invention

[0006] In order to solve the problems in the background technology, the present invention provides a Nisin oral delivery system based on yeast microcapsules. By utilizing the biological safety and natural membrane structure of yeast cells, an effective carrier can be provided to protect Nisin, promote its stable passage through the gastrointestinal tract, and control release and promote absorption.

[0007] The technical solution of the present invention is: a Nisin oral delivery system based on yeast microcapsules, wherein the yeast microcapsules are treated with a hypertonic sodium chloride solution to allow yeast cells to release water and even some water-soluble components to provide more space for core loading, and the Nisin is loaded into the yeast microcapsules through the pores of the yeast cell wall through electrostatic deposition.

[0008] Preferably, in the above-mentioned yeast microcapsule-based Nisin oral delivery system, the yeast microcapsules are obtained from edible yeast.

[0009] Preferably, in the above-mentioned Nisin oral delivery system based on yeast microcapsules, the particle size of the yeast microcapsules is 2-5 μm, and the potential is between -30 and -5 mV.

[0010] A method for preparing a Nisin oral delivery system based on yeast microcapsules comprises the following steps: (1) Take yeast in the logarithmic growth phase, wash it with excess ethanol aqueous solution, then wash it with excess deionized water, centrifuge it, add a hypertonic sodium chloride solution for treatment, then wash it with excess deionized water, then centrifuge it to freeze-dry the precipitate, and the dried substance obtained is the yeast microcapsule; (2) Prepare a solution containing Nisin and adjust the pH of the solution using hydrochloric acid or sodium hydroxide solution; (3) The yeast microcapsules obtained in step (1) and the solution containing Nisin in step (2) are mixed in a certain mass ratio, stirred evenly, and the precipitate is freeze-dried after centrifugation to obtain a dried substance, which is the Nisin yeast microcapsule oral delivery system.

[0011] Based on the above technical solution, further, the volume percentage of the ethanol aqueous solution in step (1) is 1% to 30%, and washing is performed 1 to 5 times. The most preferred condition is that the volume percentage of the ethanol aqueous solution is 10%, and the number of washing times is 2 times.

[0012] Based on the above technical solution, further, the concentration of the hypertonic sodium chloride solution in step (1) is 5%-30%, and the most preferred condition is 10% hypertonic sodium chloride solution.

[0013] Based on the above technical solution, further, in step (1), the mass volume ratio of the yeast to the hypertonic sodium chloride solution is 1:(5-30), and the most preferred condition is a mass volume ratio of 1:10.

[0014] Based on the above technical solution, further, in step (1), the reaction temperature of the yeast and the hypertonic sodium chloride solution is 20-80°C, and the most preferred condition is a reaction temperature of 55°C.

[0015] Based on the above technical solution, further, in step (1), the reaction time of the yeast and the hypertonic sodium chloride solution is 1 to 36 hours, and the most preferred condition is a reaction time of 48 hours.

[0016] Based on the above technical solution, further, the pH of the Nisin aqueous solution in step (2) is adjusted to 1-7 using hydrochloric acid and sodium hydroxide, and the most preferred condition is pH 1.7.

[0017] Based on the above technical solution, further, in step (3), the mass ratio of Nisin to yeast microcapsules is 1:(0.1-3), and the most preferred condition is a mass ratio of 1:1.

[0018] Based on the above technical solution, further, in step (3), the reaction temperature of Nisin and yeast microcapsules is 20-40°C, and the most preferred condition is a reaction temperature of 37°C.

[0019] Based on the above technical solution, further, in step (3), the reaction time of the yeast and the hypertonic sodium chloride solution is 2-24 h, and the most preferred condition is a reaction time of 12 h.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) The yeast microcapsules used in the present invention are obtained from edible yeast and have good biosafety.

[0021] (2) Nisin used in the present invention is a natural antimicrobial peptide that has been widely used in the food field.

[0022] (3) The preparation method of the present invention is simple, and the particles of the oral delivery system are small in size, with high encapsulation efficiency and high drug loading.

[0023] (4) The present invention adjusts the pH of the peptide solution to make Nisin positively charged, so that it can combine with the negatively charged yeast microcapsules treated with hypertonic sodium chloride, and load Nisin through electrostatic deposition.

[0024] (5) This oral delivery system can effectively improve the stability of Nisin in gastrointestinal fluid and has a certain sustained-release effect on Nisin. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram of the preparation of the Nisin oral delivery system based on yeast microcapsules.

[0026] Figure 2 Zeta potential diagram of Nisin oral delivery system based on yeast microcapsules.

[0027] Figure 3Particle size distribution of Nisin oral delivery system based on yeast microcapsules.

[0028] Figure 4 This is a scanning electron micrograph of the Nisin oral delivery system based on yeast microcapsules.

[0029] Figure 5 This is the infrared spectrum of the Nisin oral delivery system based on yeast microcapsules.

[0030] Figure 6 Schematic diagram of simulated digestion release of Nisin oral delivery system based on yeast microcapsules. DETAILED DESCRIPTION

[0031] In order to make the purpose, process scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments. It should be noted that these descriptions are only exemplary and are not intended to limit the scope of the present invention.

[0032] Example 1 Encapsulation efficiency and drug loading of Nisin in the Nisin oral delivery system based on yeast microcapsules

[0033] (1) Preparation of yeast microcapsules Take 5 g of commercially available active dry yeast (native yeast), wash it twice with excess 10% (v / v) ethanol solution, wash it once with excess ultrapure water, and wash it repeatedly to remove the polyol or polyether used as a cryoprotectant (provided by the supplier's documents: sorbitan monostearate). Between each wash, the cells were precipitated at 8000 rpm / min for 10 minutes, and the excess water was removed again. 50 ml of 10% NaCl solution was added to the precipitate and mixed, stirred at 180 rpm at 55°C for 48 h, washed 5 times with deionized water to remove cytoplasmic material and NaCl, and the cells were precipitated at 8000 rpm / min for 5 minutes between each wash, and the excess water was removed again. Finally, the yeast microcapsule carrier was obtained by freeze drying.

[0034] (2) Preparation of Nisin yeast microcapsule oral delivery system Nisin was dissolved in deionized water with a pH of 1.7, and an appropriate amount of freeze-dried yeast microcapsules was added to the Nisin solution, with a mass ratio of yeast microcapsules to Nisin of 1:1. After incubation at 37°C for 24 h, the precipitate was centrifuged at 8000 rpm / min for 10 min, and the resulting precipitate was the Nisin yeast microcapsule oral delivery system. The solid was rinsed with 0.5 ml of water and then freeze-dried.

[0035] (3) Determination of Nisin encapsulation efficiency and drug loading The encapsulation efficiency and drug loading of Nisin were determined by HPLC under the following conditions: The mobile phase was A (water + 0.1% trifluoroacetic acid), B (acetonitrile); the elution program was 0~0.5 min, 20%B; 0.5~6 min, 20-90%B; 6~8 min, 90%B; 8~8.1 min, 90%-20%B; 8.1~12.1 min, 20%B; injection volume, 10 µL; flow rate, 1 ml / min; column temperature, 30°C; wavelength, 200 nm.

[0036] Nisin standard was prepared into a series of standard gradient solutions with concentrations of 1, 2, 4, 6, 8, and 10 mg / ml using deionized water at pH 1.7. The standard curve measured by HPLC was: y=192.30367x-46.58557, R 2 =0.99928.

[0037] The Nisin content in the supernatant was determined by HPLC, and the encapsulation efficiency and drug loading rate of Nisin in the Nisin yeast microcapsule oral delivery system were calculated using the following formula.

[0038] Encapsulation efficiency (EE,%) = (total amount of drug added - amount of drug not loaded) / total amount of drug added × 100 Drug loading efficiency (LE,%) = drug amount loaded in yeast microcapsules / (yeast microcapsules + drug amount loaded in yeast microcapsules) × 100 The Nisin yeast microcapsule oral delivery system had an encapsulation efficiency of 95.21±0.69% and a drug loading of 47.61±0.35%, indicating that the yeast microcapsule product treated with hypertonic sodium chloride had better performance and high loading rate and drug loading rate.

[0039] Example 2 Characterization of Nisin oral delivery system based on yeast microcapsules

[0040] Appropriate amounts of yeast raw materials, yeast microcapsules, Nisin and the Nisin yeast microcapsule oral delivery system obtained in Example 1 were taken to measure their Zeta potential, particle size, Fourier transform infrared spectroscopy (FT-IR), and scanning electron microscopy (SEM).

[0041] (1) Zeta potential measurement A small amount of the above freeze-dried sample was evenly dispersed in deionized water, and 1 ml of the suspension was taken for determination using a nanoparticle size and zeta potential instrument, wherein the temperature was set at 25°C.

[0042] (2) Particle size determination A small amount of the above freeze-dried sample was taken and evenly dispersed in deionized water. 1 ml of the suspension was taken for each determination using a nanoparticle size and zeta potential instrument, wherein the temperature was set at 25°C and the measurement angle was 90 degrees.

[0043] (3) Fourier transform infrared spectroscopy Take an appropriate amount of freeze-dried sample to be tested, mix it with potassium bromide at a ratio of 1:100, grind it thoroughly and press it into tablets. Use Fourier transform infrared spectrometer (IRTracer-100) to perform FITR scanning analysis on yeast microcapsules before and after drug loading. The scanning range is set at 4000~400 cm -1 , and obtain the infrared absorption spectrum.

[0044] (4) SEM The freeze-dried sample is fixed on a metal sample stage dedicated to a scanning electron microscope with electrical tape or other conductive materials, and gold is sprayed to make the sample surface conductive. The sample is then sent to a cryo-scanning electron microscope for observation, and the fields of view at different magnifications are selected for comparison.

[0045] Depend on Figure 2 and Figure 3 It can be seen that the Zeta potential of the yeast raw material used is -17.35 mV and the particle size is 3779.80 nm, while the Zeta potential of the yeast microcapsules treated with hypertonic sodium chloride is -24.57 mV and the particle size is 1801.91 nm. The results of the particle size change can also be seen in Figure 4 This can be seen in the SEM images of yeast before and after treatment. This is because the treatment with hypertonic sodium chloride solution causes yeast cells to release water and even some water-soluble components, providing more space for core loading, while the relative increase in lipids caused by the disintegration of the plasma membrane leads to a decrease in the potential value. Figure 5 The yeast microvesicles were shown at 2854 cm -1 A new peak appears at , corresponding to the symmetric stretching vibration of CH2 lipids, also indicating a relative increase in lipids, while the changes in intensity and wavenumber of proteins and amide groups may indicate protein degradation during the decomposition process.

[0046] Depend on Figure 2-Figure 5 It can also be seen that the Zeta potential of the yeast microcapsule delivery carrier after loading Nisin is -19.69 mV, and the particle size is 2567.68 nm, which successfully achieves the loading of Nisin on the yeast microcapsule. This shows that the oral delivery system can successfully load Nisin into the yeast microcapsule and achieve carrier delivery.

[0047] Example 3 Simulated digestion release of Nisin oral delivery system based on yeast microcapsules

[0048] Take an appropriate amount of freeze-dried Nisin yeast microcapsule delivery system, add the prepared simulated gastric fluid (SGF, pH 1.2), simulated small intestinal fluid (SIF, pH 6.8), and simulated gastric fluid (SCF, pH 7.4), and incubate the mixture in a constant temperature water bath shaker at 37°C. After sampling every 1 hour, centrifuge to obtain the supernatant, and determine the release rate of Nisin in the oral delivery system by HPLC according to the method in Example 1.

[0049] Depend on Figure 6 It can be seen that Nisin is released slowly in simulated gastric juice, small intestinal juice and colonic juice, indicating that the yeast microcapsules prevent the leakage of Nisin in the gastrointestinal tract to a certain extent. The oral delivery system can protect and slow-release Nisin, and the drug release rate is different at different pH values, indicating that the oral delivery system has a certain pH responsiveness.

[0050] In summary, the present invention illustrates in detail the preparation and application of the yeast microcapsule oral delivery system of Nisin through the above-mentioned implementation cases. The method is simple to operate, can achieve high loading amount and drug loading amount of Nisin, and can also protect the stability of Nisin in the gastrointestinal tract.

[0051] In summary, the technical solution of the present invention improves the stability and efficiency of Nisin delivery through innovative design. Although the present invention is described in detail through specific embodiments, for those skilled in the art, based on the ideas provided by the present invention, several changes and modifications can still be made, and these changes and modifications all belong to the protection scope of the present invention.

Claims

1. A Nisin oral delivery system based on yeast microcapsules, characterized in that: The yeast microcapsule carrier is treated with a sodium chloride solution to obtain an active delivery carrier; the Nisin is encapsulated into the yeast microcapsule with negative charge through a spontaneous deposition effect mediated by electrostatic force to obtain the Nisin delivery system.

2. A Nisin oral delivery system based on yeast microcapsules according to claim 1, wherein the yeast microcapsules are obtained from edible yeast.

3. A Nisin oral delivery system based on yeast microcapsules according to claims 1-2, characterized in that: The preparation method comprises the following steps: (1) The edible yeast is washed with an excess of ethanol aqueous solution for multiple times, then washed with excess deionized water, and treated with a hypertonic sodium chloride solution to obtain a plasmolyzed yeast microcapsule carrier; (2) Dissolve Nisin in water or buffer and adjust the pH of the solution to acidic; (3) The yeast microcapsules and Nisin are mixed, stirred and incubated to obtain the Nisin yeast carrier.

4. The method for preparing a Nisin oral delivery system based on yeast microcapsules according to claim 3, characterized in that: The volume percentage of the ethanol aqueous solution in step (1) is 1% to 30%, and the washing is performed 1 to 5 times.

5. The method for preparing a Nisin oral delivery system based on yeast microcapsules according to claim 3, characterized in that: In step (1), the concentration of the sodium chloride solution used for the edible yeast is 5%-30%.

6. The method for preparing a Nisin oral delivery system based on yeast microcapsules according to claim 3, characterized in that: In step (2), the pH of the Nisin aqueous solution is adjusted to 1-7 using hydrochloric acid and sodium hydroxide.

7. The method for preparing a Nisin oral delivery system based on yeast microcapsules according to claim 5, characterized in that: In step (3), the ratio of the yeast microcapsules to Nisin is 1:(0.1-3).

8. The method for preparing a Nisin oral delivery system based on yeast microcapsules according to claim 5, characterized in that: In step (3), the reaction temperature of the yeast microcapsules and Nisin is 20-40°C.

9. The method for preparing a Nisin oral delivery system based on yeast microcapsules according to claim 5, characterized in that: In step (3), the reaction time of the yeast microcapsules and Nisin is 2-24 h.

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