Thiolated polysaccharide combined probiotic microcapsule as well as preparation method and application thereof

Abstract

The invention discloses a thiolated polysaccharide combined probiotic microcapsule as well as a preparation method and application thereof, and belongs to the technical field of probiotic microcapsules. Comprising the following steps: (1) mixing and incubating a probiotic suspension and a 2-imino thioalkane solution to obtain thiolated probiotics; and (2) adding the thiolated probiotic suspension into the thiolated polysaccharide solution, carrying out oscillation reaction, and centrifuging to remove unattached probiotics. Polysaccharide is firstly subjected to thiolation modification, and primary amino groups on the surfaces of bacteria are converted into free mercaptan under a cell compatibility condition through a simple one-step imide ester reaction, so that the free mercaptan can spontaneously form disulfide bonds to perform chemical bonding under a catalyst-free condition, and the primary amino groups on the surfaces of the bacteria are converted into the free mercaptan. The microcapsule prepared from the modified polysaccharide and the probiotics can also be combined with mucoprotein rich in the mucous layer of the intestinal tract to generate a catalyst-free thiol-disulfide exchange reaction, and colonization is carried out at the mucous layer in the intestinal tract, so that better intestinal disease treatment and probiotic benefits are generated.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of probiotic microcapsules, in particular to a thiolated polysaccharide combined probiotic microcapsule and a preparation method and application thereof. BACKGROUND

[0002] Probiotics are microorganisms that are beneficial to the host, which have the functions of regulating intestinal flora, enhancing immunity, reducing cholesterol, etc., and are widely used in the fields of biological engineering, agriculture, food safety and life health. The definition given by the Joint Expert Committee of Food and Agriculture Organization (FAO) / World Health Organization (WHO) in 2002 has been widely accepted by the public: probiotics refer to live microorganisms, and sufficient intake of microorganisms is beneficial to the health of the host.

[0003] In order to provide health benefits, probiotics must maintain a certain number of live bacteria, and it is generally believed that there are at least 10 7 CFU per gram or per milliliter of product. However, due to the influence factors of probiotic activity, such as temperature, pH, gas composition and mechanical force, etc., the probiotic preparation is prone to inactivation, resulting in reduced efficacy. In order to overcome the influence of the harsh environment in the gastrointestinal tract on the activity of probiotics, microencapsulation has become an effective method to protect probiotics.

[0004] Traditional microencapsulation is a process of burying core materials (solids, liquids and gases) in small closed microcapsules using materials that can form films. Microcapsules can protect core materials from various adverse factors in the environment, and provide a special and convenient microenvironment for probiotics to improve their survival rate in the gastrointestinal tract and control the release of cells in the intestine. Many microcapsulation technologies have been used in the food industry, such as spray drying, coacervation, extrusion, spray chilling / cooling, fluidized bed coating, freeze drying, molecular encapsulation, etc. However, most of the existing microcapsulation technologies are multi-microencapsulation, and the single-microencapsulation technology is also mostly formed by electrostatic interaction. However, the structure of the product obtained by the above wrapping method is relatively loose, and there are risks such as easy peeling of the protective film and easy penetration of gastrointestinal digestive juice, which reduces the survival rate of probiotics and thus reduces their health care efficacy in the host. Therefore, how to improve the binding stability of the protective film and probiotics has become a technical problem to be solved in the field. SUMMARY

[0005] The purpose of the present application is to provide a thiolated polysaccharide combined probiotic microcapsule and a preparation method and application thereof to solve the problems existing in the prior art.

[0006] To achieve the above purpose, the present application provides the following solutions:

[0007] One of the technical solutions of the present application is a preparation method of a thiolated polysaccharide combined probiotic microcapsule, comprising the following steps:

[0008] (1) the probiotic bacteria suspension is mixed with a 2-iminothiolane solution to obtain thiolated probiotic bacteria;

[0009] (2) the thiolated probiotic bacteria suspension is added to a thiolated polysaccharide solution, and the reaction is shaken and centrifuged to remove unattached probiotic bacteria.

[0010] The second technical solution of the present application is the thiolated polysaccharide combined probiotic bacteria microcapsule prepared by the preparation method.

[0011] The third technical solution of the present application is the use of the thiolated polysaccharide combined probiotic bacteria microcapsule in the preparation of a drug for treating intestinal diseases.

[0012] The fourth technical solution of the present application is a drug for treating intestinal diseases, comprising the thiolated polysaccharide combined probiotic bacteria microcapsule.

[0013] Based on the above technical solutions, the present application has the following technical effects:

[0014] The present application uses a new idea for preparing probiotic bacteria microcapsules. The polysaccharide is first modified by thiolation, and the primary amino group on the surface of the bacteria is converted to free thiol under cell-compatible conditions through a simple one-step imidoester reaction. This allows the formation of a disulfide bond for chemical combination under catalyst-free conditions. Compared with electrostatic force, covalent bonding is a strong force that can make the combination of polysaccharide and probiotic bacteria more secure. In addition, the amount of introduced thiol can be adjusted by different feed ratios. The microcapsules prepared from these modified polysaccharides and probiotic bacteria can also bind to the mucin-rich mucus layer in the intestinal tract, undergo a catalyst-free thiol-disulfide exchange reaction, and colonize the mucus layer in the intestinal tract, resulting in better treatment of intestinal diseases and probiotic benefits. DETAILED DESCRIPTION

[0015] Various exemplary embodiments of the present application will now be described in detail, and the methods used in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercially available reagents or reagents prepared by conventional methods unless otherwise specified. This detailed description should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0016] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, for example, a parameter, an individual value from the range can be expressly disclosed herein and / or clearly implied, and in such instances, include any and all expressed, implied and / or deduced values and / or sub-ranges underlying the same. All methods described herein can be performed by similar, equivalent or by other methods which achieve the same or similar results.

[0017] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference in their entirety for the disclosure and

[0018] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof. Additional implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The examples and embodiments described herein are exemplary and not intended to be limiting of the scope of the application.

[0019] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0020] The technical solutions described in the present application are conventional solutions in the art unless specifically stated otherwise. The reagents or raw materials used are commercially available or have been disclosed unless specifically stated otherwise.

[0021] The present application provides a preparation method of a thiolated polysaccharide combined probiotic microcapsule, comprising the following steps:

[0022] (1) mixing and incubating a probiotic suspension with a 2-iminothiolane solution to obtain thiolated probiotics;

[0023] (2) adding the thiolated probiotic suspension into a thiolated polysaccharide solution, oscillating the reaction, and removing the unattached probiotics by centrifugation.

[0024] In some specific embodiments, the probiotics comprise E. coli Nissle 1917.

[0025] In some specific embodiments, the density of the probiotic suspension is 10 9Log CFU / g; the density of the 2-iminothiolane solution is 2 mg / mL; the volume ratio of the probiotic suspension to the 2-iminothiolane solution is 79:1.

[0026] In some specific embodiments, the method of mixing incubation is incubation at room temperature for 90 minutes.

[0027] In some specific embodiments, the thiolated polysaccharide comprises jambolan gum; the concentration of the thiolated polysaccharide solution is 3-10 mg / mL; the addition amount of the thiolated probiotic suspension added into the thiolated polysaccharide solution is 0.03 mg / mL.

[0028] In some specific embodiments, the centrifugation condition is 6000 x g, 5 min.

[0029] The present application also provides a thiolated polysaccharide-bound probiotic microcapsule prepared by the preparation method.

[0030] The present application also provides the use of the thiolated polysaccharide-bound probiotic microcapsule in the preparation of a medicament for treating intestinal diseases.

[0031] The present application also provides a medicament for treating intestinal diseases, comprising the thiolated polysaccharide-bound probiotic microcapsule.

[0032] The thiol groups of the thiolated polysaccharide and the thiolated probiotic can chemically react to form a more stable disulfide bond covalent bond, and the disulfide bond is formed to form a stable probiotic microcapsule. The microcapsule prepared by the thiolated polysaccharide can also bind to the mucin rich in the intestinal mucus layer, undergo a catalyst-free thiol-disulfide exchange reaction, colonize in the intestine, improve jejunal mucositis, and achieve targeted positioning release of probiotics.

[0033] Example 1

[0034] 1This embodiment is completed by esterifying jambolan gum with mercaptopropionic acid in the presence of hydrochloric acid:

[0035] (1) 6 g of jambolan gum was dissolved in 50 mL of distilled water, and 3.6 mL of mercaptopropionic acid and 2 mL of 7 mol / L HCl were added.

[0036] (2) After the components were reacted at 80°C for 180 min, the thiolated jambolan gum was precipitated by pouring the above reaction mixture into 500 mL of 99% volume concentration methanol.

[0037] (3) The obtained milky white precipitate was washed with methanol three times, and frozen at -80°C for 4 hours, and then freeze-dried in a laboratory freeze dryer.

[0038] (4) The number of thiol groups / g of the polymer was determined using a calibration curve prepared by reacting a standard solution of L-cysteine with Ellman reagent.

[0039] (5) E. coli Nissle 1917 colonies were taken and cultured overnight (200 rpm, 37°C) in LB liquid medium, then the probiotic bacteria were washed twice with deionized water and resuspended in 987.5 μL of ice-cold phosphate buffer (PBS, pH 7.4) at a density of 10 9 Log CFU / g.

[0040] (6) To the bacterial suspension in step (5), 12.5 μL of 2-iminothiolane solution (2 mg / mL) was added, and then incubated at room temperature for 90 minutes to effectively thiolate the surface of the bacteria.

[0041] (7) 0.3 g of freeze-dried thiolated tamarind gum in step (3) was added to 100 mL of distilled water to prepare a 3 mg / mL thiolated tamarind gum sugar solution.

[0042] (8) 0.1 mL of thiolated probiotic bacterial suspension in step (6) was dissolved in the 3 mg / mL thiolated polysaccharide solution in step (7) to make the final concentration of thiolated probiotic bacterial suspension 0.03 mg / mL.

[0043] (9) The solution in step (8) was shaken at room temperature at 750 rpm / min for 1 hour.

[0044] (10) After the reaction in step (9) was completed, centrifugation was performed in a centrifuge (6000 x g, 5 min) and washed twice with PBS to completely remove unattached probiotic bacteria.

[0045] 2 Probiotic Bacteria Viability Test

[0046] (1) Detection of Modified Polysaccharides and Modified Probiotic Bacteria

[0047] An aqueous solution (0.5%, w / v) of thiolated TSP or TSP (control) in phosphate buffer (pH 0.1, 8M) was incubated with Ellman reagent (0.03%, w / v) in phosphate buffer (pH 8.0, 5M) at 2°C for 25 hours, and then the absorbance of the reaction mixture at 450 nm was measured. The number of thiol groups / g of the polymer was determined using a calibration curve prepared as described above using a standard solution of L-cysteine reacted with Ellman reagent, and the results showed that the modified tamarind gum contained 120 mM / g of thiol groups. The same method was used to determine the number of groups of E. coli Nissle 1917 probiotic bacteria after chemical modification, and the results showed that the thiol groups after modification were 2.5 times those of the unmodified.

[0048] (2) Viability assay

[0049] The probiotic microcapsules, due to the encapsulation of polyelectrolyte wall material, need to be released from the probiotic bacteria by using a disintegration solution before the viability of the probiotic bacteria is determined. The disintegration solution is generally phosphate buffer.

[0050] (3) Calculation of encapsulation yield and survival rate

[0051]

[0052] wherein the number of viable bacteria encapsulated in the microcapsules: after the microcapsule sample was completely dissolved in the disintegration solution, the number of viable bacteria in the disintegration solution was determined; the original number of viable bacteria of the probiotic bacteria: the number of viable bacteria in the initial bacterial slurry was determined.

[0053] The results of the encapsulation rate are shown in Table 1.

[0054] Table 1

[0055]

[0056] Note: The letters in the table represent the significance level (p < 0.05).

[0057] As can be seen from Table 1, the encapsulation rate between the thiol-modified polysaccharide and the probiotic bacteria was significantly improved after modification, which may be because the thiol groups of the polysaccharide and the thiol groups on the surface of the probiotic bacteria reacted chemically to form covalent bonds, successfully encapsulating the probiotic bacteria.

[0058] (4) Gastrointestinal release assay of microcapsules

[0059] 0.1 g of probiotic microcapsules was added to 9.9 mL of simulated gastric fluid, and mixed constantly, 1 mL of solution was taken out at 30, 60, 90, 120 min of incubation time, immediately added to 9 mL of PBS buffer solution, and stirred at 37°C for 1 h, so that the probiotic microcapsules were fully dissolved, then inoculated into LB culture plates by gradient dilution for counting. After 120 min of simulated gastric fluid incubation, the pH was adjusted to 7.0 using 1M sodium hydroxide, then 10 mL of simulated intestinal fluid was added, mixed well, and 1 mL of solution was taken out at 1, 2, 3, 4 h of incubation time to determine the survival rate of probiotics. The results are shown in Tables 2-3.

[0060] Table 2

[0061]

[0062] Note: The letters in the table represent the significance level (p<0.05).

[0063] Table 3

[0064]

[0065] Note: The letters in the table represent the significance level (p<0.05).

[0066] The results show that the probiotic microcapsules can survive well in simulated gastrointestinal fluid, the survival rate of probiotics is good, and the microcapsules of probiotics and polysaccharides after thiolation treatment significantly improve the survival rate of probiotics in the gastrointestinal tract, and the protection effect of probiotics is more significant.

[0067] (5) Determination of microcapsule bile salt resistance

[0068] 2% mass concentration of bile salt was added to PBS buffer solution at pH 8, and the survival rate of probiotics in the microcapsules in this environment was determined every 1 h at 37°C to investigate the tolerance of microcapsules to bile salt.

[0069] Table 4

[0070]

[0071] Note: The letters in the table represent the significance level (p<0.05).

[0072] The results show that the thiolated modified probiotic microcapsules have significantly improved bile salt resistance compared with the unmodified probiotic microcapsules, and have better bile salt resistance.

[0073] (6) Determination of microcapsule storage stability

[0074] Take 2 g of microcapsules in a sterile glass bottle, seal and store in an aluminum bag at 4°C in a constant temperature box, and measure the survival rate of probiotics at 90 days to investigate the long-term stability. The results are shown in Table 5.

[0075] Table 5

[0076]

[0077] Note: The letters in the table represent the significance level (p<0.05).

[0078] The results show that the survival rate of thiol-modified probiotic capsules is higher than that of unmodified probiotic microcapsules, which proves that microencapsulation can effectively improve the long-term stability of the strain.

[0079] (7) Thermal stability determination of microcapsules

[0080] Take 0.2 g of microcapsules in a sterile EP tube, add 4.8 mL of phosphate buffer, and place in 55, 65, and 75°C for 10 min. After heat treatment, add 15 mL of phosphate buffer to the sterile tube for rapid cooling, and measure the survival rate of probiotics.

[0081] Table 6

[0082]

[0083] Note: The letters in the table represent the significance level (p<0.05).

[0084] The results show that the survival rate of thiol-modified probiotic capsules is higher than that of unmodified probiotic microcapsules, which proves that microencapsulation can effectively resist heat environment.

[0085] (8) Ethanol tolerance determination of microcapsules

[0086] Take 0.2 g of microcapsules in a sterile EP tube, add 4.8 mL of phosphate buffer, and add 75% ethanol for 1 h, and measure the survival rate of probiotics.

[0087] Table 7

[0088]

[0089] Note: The letters in the table represent the significance level (p<0.05).

[0090] The results show that the survival rate of thiol-modified probiotic capsules is higher than that of unmodified probiotic microcapsules, which proves that microencapsulation can effectively improve the ethanol tolerance of the strain.

[0091] (9) 100 μL mucin solution (1 mg / mL) was dissolved in sterile phosphate buffered saline and immobilized on a 96-well plate for 1 h, then incubated at 4°C overnight. The microcapsules were suspended in PBS at a fixed ratio of 1:4 (v / v), then vortexed until all the microcapsules were dissolved, releasing the embedded probiotics. After washing with 200 μL sterile PBS for 3 times, 100 μL free probiotic suspension (OD 600 = 0.04) and the released probiotic suspension obtained in the last step were incubated at 37°C for 2 h, and all the wells were washed with 200 μL sterile citrate buffer to remove the unbound probiotics, while 200 μL 0.5% Tween 80 (v / v) was added to collect the adherent bacteria, and the viable count was determined by the dilution plating method to obtain the adhesion rate.

[0092] The results show that the adhesion ability of the intestinal mucus layer is a key factor for the colonization of probiotics in the host intestine, and this adhesion can prolong the residence time of probiotics in the intestine and avoid their rapid removal by gastrointestinal peristalsis. To achieve the health benefits of probiotics, in addition to tolerating the harsh environment of the gastrointestinal tract, they must be able to adhere and proliferate in specific parts of the intestine. The experimental results show that the microcapsules significantly improve the adhesion rate of probiotics to the simulated intestinal mucus. The adhesion rate of the control group of free probiotics is only 6.53 ± 0.23%, while that of the microcapsule group is 72.5 ± 0.75% respectively. This enhancement is mainly due to the formation of stable disulfide bonds between the cysteine residues on the surface of the Mucin coating and the SH groups in the mucus layer. These results show that microencapsulation can significantly enhance the intestinal adhesion of probiotics through intermolecular interactions, providing favorable conditions for their colonization and functional expression in the intestine.

[0093] Obviously, the above embodiments of the present application are only examples for the purpose of clear illustration, and are not intended to limit the embodiments of the present application. For ordinary skilled users in the art, other different forms of changes or variations can be made on the basis of the above description. Here, it is not necessary and impossible to exhaust all the embodiments. Any modification, equivalent replacement and improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. A process for the preparation of thiolated polysaccharide conjugated probiotic microcapsules, characterized in that, The method comprises the following steps: (1) mixing and incubating probiotic bacteria suspension with 2-iminothiolane solution to obtain thiolated probiotic bacteria; (2) adding the thiolated probiotic bacteria suspension into thiolated polysaccharide solution, oscillating the reaction, and removing unattached probiotic bacteria by centrifugation.

2. The production method according to claim 1, characterized by, The probiotic bacteria comprise E. coli Nissle 1917.

3. The preparation method according to claim 1, characterized in that The density of the probiotic suspension is 10 9 Log CFU / g; the density of the 2-iminothiolane solution is 2 mg / mL; the volume ratio of the probiotic suspension to 2-iminothiolane solution is 79:

1.

4. The method of claim 1, wherein, The method of mixing and incubating is incubating at room temperature for 90 minutes.

5. The preparation method according to claim 1, characterized in that The thiolated polysaccharide comprises tamarind gum; the concentration of the thiolated polysaccharide solution is 3-10 mg / mL; and the addition amount of the thiolated probiotic bacteria suspension added into the thiolated polysaccharide solution is 0.03 mg / mL.

6. The method of claim 1, wherein, The centrifugation condition is 6000xg, 5 min.

7. Thiolated polysaccharide-bound probiotic bacteria microcapsules prepared by the preparation method of any one of claims 1-6.

8. Use of the thiolated polysaccharide-bound probiotic bacteria microcapsules of claim 7 in the preparation of a drug for treating intestinal diseases.

9. A medicament for treating intestinal diseases, characterized by, The drug comprises the thiolated polysaccharide-bound probiotic bacteria microcapsules of claim 7.

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