Preparation method and application of novel probiotic nanocapsule

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a probiotic nanocapsule and a preparation method thereof. The probiotic nanocapsule is constructed by natural protein and polyphenol through layer-by-layer self-assembly on the basis of the characteristic that the surface of bacteria carries negative charges, and is formed in situ on the probiotic cell wall, so that single cell packaging can be realized, and the packaging efficiency is high; the nanocapsule disclosed by the invention is composed of natural-source protein and polyphenol, and is safe, non-toxic, low in cost and wide in application range; the environment tolerance of the encapsulated probiotics is improved, the intestinal adhesion performance is greatly improved, and the probiotics have a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a probiotic nanocapsule and a preparation method thereof. Background Art

[0002] Probiotics are active microorganisms that can have beneficial effects on the host when ingested in sufficient quantities. They have multiple biological activities and show great application prospects in regulating intestinal flora and improving the health of the host. However, probiotics are extremely sensitive to the complex external and human environment, and it is difficult for them to adhere to the intestines to achieve the purpose of colonization. Encapsulation technology provides an effective solution for improving the environmental tolerance and intestinal adhesion rate of probiotics.

[0003] Existing probiotic microcapsule technologies, such as a bile-resistant and gastric acid-resistant probiotic preparation and its preparation method and application proposed in CN111317140A; CN110801021A proposes a method of embedding intestinal composite probiotics using modified pectin; CN111436614A proposes a probiotic delivery microcapsule based on soluble dietary fiber of shiitake mushrooms and a preparation method. These probiotic microcapsule technologies not only have the problem of complex preparation process, but also may have problems such as insensitive response and premature leakage of probiotics in complex gastrointestinal environments, and have not yet been studied in terms of improving the intestinal adhesion performance of probiotics. Therefore, it is necessary to develop more effective and more widely applicable encapsulation technology to achieve more efficient protection and delivery of probiotics. In recent years, the use of new technologies to achieve probiotic nanoencapsulation has attracted much attention. Nanocoating is an emerging probiotic nanoencapsulation strategy in recent years. It can achieve encapsulation at the single cell level, improve the environmental resistance of probiotics, and improve their intestinal adhesion performance. It has become one of the most popular hotspots in the field of probiotic nanocapsules.

[0004] Existing probiotic nanocapsule technologies include a biomass-based encapsulation material and an encapsulation method for protecting the activity of probiotics proposed in CN113499325A; and a bacterium whose surface is modified with a bioactive drug and its application proposed in CN114181847A. Although these probiotic nanocapsule technologies provide active protection for probiotics to a certain extent, their preparation methods are slightly complicated and some of the raw materials used are of non-natural origin, which to a certain extent limits the application scope of this type of probiotic preparations. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, the present invention provides a probiotic nanocapsule with a natural source of preparation materials, high safety and wide application range. The probiotic capsule has a simple preparation process, is easy to operate, has low cost, can be encapsulated in a single cell, and is conducive to industrial production. It can be widely used in the preparation of food additives, health care product additives, feed additives and drug additives.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a probiotic nanocapsule, which is composed of probiotics and a nanolayer encapsulating the probiotics, wherein the nanolayer is composed of positively charged proteins and polyphenols.

[0008] Furthermore, the protein is a protein of natural origin; and the polyphenol is a combination of one or more plant polyphenols of natural origin in any proportion.

[0009] Furthermore, the protein is lactoferrin; and the polyphenols are selected from one or more of EGCG, tannic acid, tea polyphenols, and chlorogenic acid.

[0010] The probiotic surface and protein are in situ bound via electrostatic interaction, and the polyphenols and probiotic surface-bound proteins are prepared via non-covalent interactions to achieve nanolayer preparation.

[0011] Furthermore, the probiotics are selected from one or more strains of Clostridium tenella, Pediococcus, Bifidobacterium, Gram-positive cocci, Escherichia coli, Bacteroides, Faecalibacterium, Saccharomyces, Lactobacillus, and Bacillus.

[0012] In a second aspect, the present invention provides a method for preparing the above-mentioned probiotic nanocapsules, comprising the following steps:

[0013] S1. Preparation of probiotic suspension:

[0014] The probiotics solution was centrifuged and washed 2-3 times with the sterile first PBS buffer. After centrifugation, it was resuspended in the sterile first PBS buffer to make the concentration of the solution 1×10 5 ~1×10 9 CFU / mL;

[0015] S2, using a second PBS buffer to prepare a protein solution and a polyphenol solution and filter and sterilize;

[0016] S3, mixing the probiotic suspension in S1 with the protein solution in S2, allowing them to react fully, washing 2-3 times, centrifuging, and resuspending in a second PBS buffer to obtain a protein-coated probiotic solution;

[0017] S4. The protein-coated probiotics obtained in S3 are mixed with the polyphenol solution in S2, and the mixture is fully reacted. The mixture is washed 2-3 times and centrifuged to obtain the probiotics.

[0018] Specifically, based on the negatively charged property of the probiotic surface, the present invention uses natural proteins and polyphenols as raw materials to realize the construction of probiotic nanocapsules through layer-by-layer self-assembly. First, positively charged proteins are bound to the surface of probiotics through electrostatic interactions under acidic or weakly alkaline conditions, and polyphenols are added on this basis. Through non-covalent interactions such as hydrophobic interactions, polyphenols are combined with probiotic surface proteins to realize the construction of a nanolayer on the probiotic surface, and finally probiotic nanocapsules are successfully prepared.

[0019] Furthermore, in step S1, the first PBS buffer is a 0.01M phosphate buffer with a pH of 7.2-7.4 and containing 0.9% sodium chloride; the second PBS buffer is a 0.01M phosphate buffer with a pH of 3.0-8.0 and containing 0.9% sodium chloride.

[0020] Specifically, the fresh probiotic bacterial liquid is centrifuged, washed 2-3 times with a sterile first PBS buffer, and resuspended in a sterile second PBS buffer after centrifugation.

[0021] Furthermore, in step S2, the protein solution has a concentration of 1 to 10 mg / mL, the polyphenol solution has a concentration of 1 to 10 mg / mL, and both are sterilized by filtration through a 0.22 μm filter membrane.

[0022] Furthermore, in step S3, the concentration of the protein-coated probiotic solution is 1×10 5 ~1×10 9 CFU / mL.

[0023] Furthermore, in step S3, the volume ratio of the probiotic suspension to the protein solution is 1:(1-10), and in step S4, the concentration of the protein-coated probiotic solution is 1×10 5 ~1×10 9 CFU / mL, and the volume ratio of the polyphenol solution is 1:(1-10).

[0024] Furthermore, the full reaction in S3 and S4 is carried out by stirring or vortexing at 100 to 1000 rpm for 10 to 180 seconds.

[0025] Furthermore, the cleaning process in steps S1 to S4 is as follows: add the corresponding first PBS buffer or the second PBS buffer to the bacterial solution, mix thoroughly and then centrifuge, remove the supernatant, repeat the operation 2 to 3 times, centrifuge at 4000 to 8000 rpm, and centrifuge for 3 to 5 minutes.

[0026] In a third aspect, the present invention provides use of the above-mentioned probiotic capsule in the preparation of food additives and / or health product additives.

[0027] In a fourth aspect, the present invention provides use of the above-mentioned probiotic capsule in the preparation of pharmaceutical additives and / or feed additives.

[0028] The beneficial effects of the present invention are:

[0029] (1) The present invention is a nanolayer formed in situ on the surface of probiotics, which is used for nano-encapsulation and protection of probiotics; single-cell encapsulation of probiotics can be achieved, and the encapsulation efficiency can reach 100%;

[0030] (2) The present invention constructs nanocapsules based on the negatively charged property of bacterial surfaces, and can form a nanolayer in situ on the surface of any bacteria to achieve single-cell encapsulation of bacteria;

[0031] (3) The probiotic nanocapsules prepared by the present invention can improve their environmental tolerance and intestinal adhesion performance;

[0032] (4) The nanolayer preparation materials used in the present invention are all of natural origin and are prepared through non-covalent bond interactions between substances, which are low-cost and have no toxic side effects;

[0033] (5) The nanolayer preparation material provided by the present invention, the protein and polyphenols can be hydrolyzed or metabolized in the intestine as prebiotics to be converted into small molecules that are more easily utilized by probiotics or the human body, thereby promoting the colonization of probiotics in the intestine;

[0034] (6) The preparation method of the probiotic nanocapsules constructed by the present invention is simple, with low time and equipment costs, which is conducive to industrial production. The materials used are all of natural origin and have high safety. They can be used as food additives, health care product additives, feed additives and drug additives. The finished products have a wide range of applications and are suitable for industrial promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention.

[0036] Figure 1 This is a laser confocal image of the nano-encapsulated probiotic capsule prepared in Example 1;

[0037] Figure 2 This is a SEM image of the nano-encapsulated probiotic capsule prepared in Example 1;

[0038] Figure 3 TEM image of the nanoencapsulated probiotic nanocapsules prepared in Example 1;

[0039] Figure 4 This is the growth curve of the nano probiotic capsule prepared in Example 1;

[0040] Figure 5 The antioxidant properties of the nano-encapsulated probiotics prepared in Example 1. Different letters in the figure indicate significant differences between the two groups (p<0.05);

[0041] Figure 6 The adhesion performance of the nano-encapsulated probiotics prepared in Example 1. Different letters in the figure indicate significant differences between the two groups (p<0.05). DETAILED DESCRIPTION

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0044] In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof. In the following specific embodiments, if the experimental methods of specific conditions are not indicated, they are usually in accordance with the conventional methods and conditions of molecular biology within the art, and such techniques and conditions are fully explained in the literature. See, for example, Sambrook et al., the techniques and conditions described in "Molecular Cloning: A Laboratory Manual", or in accordance with the conditions recommended by the manufacturer.

[0045] The present invention is further described in conjunction with specific examples. The following examples are only for explaining the present invention and are not intended to limit the content thereof. If the specific experimental conditions are not specified in the examples, they are usually carried out under conventional conditions or under conditions recommended by the sales company; the materials and reagents used in the examples, unless otherwise specified, can be purchased through commercial channels.

[0046] The probiotic information involved in the embodiment is as follows:

[0047] Faecalibacterium prausnitzii (ATCC 27766) was purchased from Ningbo Mingzhou Biotechnology Co., Ltd. (ATCC China Agent);

[0048] Pediococcus pentosaceus (BNCC135034) and Lactobacillus salivarius (BNCC138618) were purchased from Beina Chuanglian Biotechnology Co., Ltd.

[0049] Materials and reagents used in the embodiments of the present invention:

[0050] Lactoferrin was purchased from Shaanxi Baichuan Biotechnology Co., Ltd., epigallocatechin gallate (EGCG) and tea polyphenols were purchased from Huagao Biotechnology Co., Ltd., and tannic acid (Cat. No. 10021918) was purchased from Sinopharm Chemical Reagent Co., Ltd. ATCC 1703 medium was purchased from Shandong Top Bioengineering Co., Ltd., and MRS medium was purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0051] The formula of ATCC 1703 culture medium is: 850ml of purified water, 50g of beef extract powder, 30g of tryptic peptone, 5g of yeast extract powder, 4g of glucose, 150mL of rumen fluid, 1g of cellobiose, 1g of maltose, 1g of starch, 5g of dipotassium hydrogen phosphate, 1mg of resazurin, 0.5g of L-cysteine ​​hydrochloride, 5mg of hematoxylin, 1mg of vitamin K, and 15g of agar powder (solid culture medium).

[0052] The formula of MRS culture medium is: 10g peptone, 5g beef extract powder, 4g yeast extract powder, 20g glucose, 2g triammonium citrate, 5g sodium acetate, 2g dipotassium hydrogen phosphate, 0.2g magnesium sulfate, 0.05g manganese sulfate, 1mL Tween-80, and 15g agar powder (solid culture medium) per liter of water.

[0053] The buffers used in the examples are: the first PBS buffer is a 0.01M phosphate buffer with a pH of 7.2-7.4 and containing 0.9% sodium chloride; the second PBS buffer is a 0.01M phosphate buffer with a pH of 3.0-8.0 and containing 0.9% sodium chloride.

[0054] Example 1

[0055] A probiotic nanocapsule, the preparation method is as follows:

[0056] (1) Preparation of bacterial suspension

[0057] After Faecalibacterium prausnitzii was activated and restored to vitality by passage on ATCC 1703 solid slant medium, it was inoculated into 50 mL of sterilized ATCC 1703 liquid medium and cultured in a 37°C constant temperature incubator for 18 h to serve as the seed solution of Faecalibacterium prausnitzii. 2% (v / v) was inoculated into 50 mL of sterilized ATCC 1703 liquid medium again and cultured anaerobically in a 37°C constant temperature incubator to make Faecalibacterium prausnitzii in the late logarithmic growth phase. 15 mL of 2×10 7 CFU / mL of fresh Clostridium prausnitzii was centrifuged at 5000 rpm for 10 min, the supernatant was removed, the cells were washed twice with the first PBS buffer, the supernatant was removed, 0.6 mL of the second PBS buffer with a pH of 6.0 was added, and the mixture was mixed to prepare a Clostridium prausnitzii suspension (5×10 8 CFU / mL).

[0058] (2) Preparation of Nanocapsules

[0059] The lactoferrin and EGGC solutions were prepared using the second PBS buffer solution with a pH of 6.0, with concentrations of 8 mg / mL and 8 mg / mL, respectively, and filtered through a sterile 0.22 micron filter. 1 mL of lactoferrin solution was added to the pre-treated probiotic solution in step (1), vortexed for 1 min to fully react, centrifuged again and cleaned twice using the second PBS buffer solution (pH 6.0) to remove unbound lactoferrin. The probiotics obtained by lactoferrin treatment were resuspended in the second PBS buffer solution (pH 6.0) of 0.6 mL and 0.6 mL of EGCG solution was added, vortexed for 1 min to fully react, and after centrifugation to remove the supernatant, the second PBS buffer solution (pH 6.0) was used to clean twice to remove unbound EGCG. The obtained suspension was centrifuged and collected to obtain EGCG / lactoferrin probiotic nanocapsules.

[0060] Example 2

[0061] A probiotic nanocapsule, the preparation method is as follows:

[0062] (1) Preparation of bacterial suspension

[0063] After the probiotic Pediococcus pentosaceus was activated and restored to vitality by passage on MRS solid slant medium, it was inoculated into 50 mL of sterilized MRS liquid medium and placed in a 37°C constant temperature incubator for 10 h to serve as the seed solution of Pediococcus pentosaceus. 2% (v / v) was inoculated into 50 mL of sterilized MRS liquid medium again and cultured anaerobically in a 37°C constant temperature incubator to make Pediococcus pentosaceus in the late logarithmic growth phase. 5 mL of 1×10 8 CFU / mL of fresh Pediococcus pentosaceus culture was centrifuged at 4000 rpm for 10 min, the supernatant was removed, the first PBS buffer was added for washing twice, the supernatant was removed, 0.5 mL of the second PBS buffer with a pH of 5.0 was added, and the mixture was mixed evenly to prepare a bacterial suspension (1×10 9 CFU / mL).

[0064] (2) Preparation of Nanocapsules

[0065] The lactoferrin and tannic acid solutions were prepared using a second PBS buffer solution with a pH of 5.0, with concentrations of 10 mg / mL and 6 mg / mL, respectively, and filtered through a sterile 0.22 micron filter. 1 mL of lactoferrin solution was added to the pretreated probiotic solution in step (1), vortexed for 1 min to fully react, centrifuged again and washed twice with a second PBS buffer solution (pH 5.0) to remove unbound lactoferrin. The probiotics obtained by lactoferrin treatment were resuspended in 0.5 mL of a second PBS buffer solution (pH 5.0) and 0.5 mL of tannic acid solution was added, vortexed for 1 min to fully react, and after centrifugation to remove the supernatant, washed twice with a second PBS buffer solution (pH 5.0) to remove unbound tannic acid. The obtained suspension was centrifuged and collected to obtain nano-encapsulated probiotics.

[0066] Example 3

[0067] A probiotic nanocapsule, the preparation method is as follows:

[0068] (1) Preparation of bacterial suspension

[0069] After the probiotic Pediococcus pentosaceus was activated and restored to vitality by passage on MRS solid slant medium, it was inoculated into 50 mL of sterilized MRS liquid medium and placed in a 37°C constant temperature incubator for 10 h to serve as the seed solution of Pediococcus pentosaceus. 2% (v / v) was inoculated into 50 mL of sterilized MRS liquid medium again and cultured anaerobically in a 37°C constant temperature incubator to make Pediococcus pentosaceus in the late logarithmic growth phase. 5 mL of 1×10 8CFU / mL of fresh Pediococcus pentosaceus bacterial suspension was centrifuged at 4000 rpm for 8 min, the supernatant was removed, the first PBS buffer was added for washing twice, the supernatant was removed, 1 mL of the second PBS buffer with a pH of 5.5 was added, and the mixture was mixed evenly to prepare a bacterial suspension of Pediococcus pentosaceus (5×10 8 CFU / mL).

[0070] (2) Preparation of Nanocapsules

[0071] The lactoferrin and EGCG solutions were prepared using the second PBS buffer solution with a pH of 5.5, with concentrations of 8 mg / mL and 8 mg / mL, respectively, and filtered through a sterile 0.22 micron filter. 1 mL of lactoferrin solution was added to the pre-treated probiotic solution in step (1), vortexed for 1 min to fully react, centrifuged again and cleaned twice using the second PBS buffer solution (pH 5.5) to remove unbound lactoferrin. The probiotics obtained by lactoferrin treatment were resuspended in the second PBS buffer solution (pH 5.5) of 0.5 mL and 0.5 mL of EGCG solution was added, vortexed for 1 min to fully react, and after centrifugation to remove the supernatant, the second PBS buffer solution (pH 5.5) was used to clean twice to remove unbound EGCG. The suspension was centrifuged and collected to obtain nano-encapsulated probiotics.

[0072] Example 4

[0073] A probiotic nanocapsule, the preparation method is as follows:

[0074] (1) Preparation of bacterial suspension

[0075] After Lactobacillus salivarius was activated and restored to vitality by passage through MRS solid slant medium, it was inoculated into 50 mL of sterilized MRS liquid medium and placed in a 37°C constant temperature incubator for 10 h to serve as the seed solution of Lactobacillus salivarius. 2% (v / v) was inoculated again into 50 mL of sterilized MRS liquid medium and cultured in a 37°C constant temperature incubator until Lactobacillus salivarius was in the late logarithmic growth phase. 5 mL of 1×10 8 CFU / mL of fresh Lactobacillus salivarius culture was centrifuged at 4000 rpm for 10 min, the supernatant was removed, the first PBS buffer was added for washing twice, the supernatant was removed, 0.5 mL of the second PBS buffer with a pH of 6.5 was added, and the mixture was mixed to prepare a bacterial suspension of Lactobacillus salivarius (1×10 9 CFU / mL).

[0076] (2) Preparation of Nanocapsules

[0077] The lactoferrin and tannic acid solution were prepared using a second PBS buffer solution with a pH of 6.5, with concentrations of 10 mg / mL and 6 mg / mL, respectively, and filtered through a sterile 0.22 micron filter. 1 mL of lactoferrin solution was added to the pre-treated probiotic solution in step (1), vortexed for 60 s to fully react, centrifuged again and washed twice with a second PBS buffer solution (pH 6.5) to remove unbound lactoferrin. The probiotics obtained by lactoferrin treatment were resuspended in 0.5 mL of a second PBS buffer solution (pH 6.5) and 0.5 mL of tannic acid solution was added, vortexed for 1 min to fully react, and after centrifugation to remove the supernatant, washed twice with a second PBS buffer solution (pH 6.5) to remove unbound tannic acid. The suspension was centrifuged and collected to obtain nano-encapsulated probiotics.

[0078] Example 5

[0079] A probiotic nanocapsule, the preparation method is as follows:

[0080] (1) Preparation of bacterial suspension

[0081] After Lactobacillus salivarius was activated and restored to vitality by passage through MRS solid slant medium, it was inoculated into 50 mL of sterilized MRS liquid medium and placed in a 37°C constant temperature incubator for 10 h to serve as the seed solution of Lactobacillus salivarius. 2% (v / v) was inoculated again into 50 mL of sterilized MRS liquid medium and cultured in a 37°C constant temperature incubator until Lactobacillus salivarius was in the late logarithmic growth phase. 10 mL of 1×10 8 CFU / mL of fresh Lactobacillus salivarius culture was centrifuged at 3500 rpm for 10 min, the supernatant was removed, the first PBS buffer was added for washing twice, the supernatant was removed, 1 mL of the second PBS buffer with a pH of 6.0 was added, and the mixture was mixed to prepare a bacterial suspension of Lactobacillus salivarius (1×10 9 CFU / mL).

[0082] (2) Preparation of Nanocapsules

[0083] Use the second PBS buffer solution of pH 6.0 to prepare lactoferrin and tea polyphenol solution, the concentration is 10mg / mL and 7mg / mL respectively, and filter through aseptic 0.22 micron filter. 1mL of lactoferrin solution is added to the pre-treated probiotic solution in step 1, stir (100rpm) 2min with agitator at low speed to make it fully react, centrifuge again and use the second PBS buffer solution (pH 6.0) to clean 2 times, remove unbound lactoferrin. The probiotics obtained by lactoferrin treatment are resuspended in the second PBS buffer solution of 1mL and add 1mL of tea polyphenol solution, stir 2min with agitator at low speed to make it fully react, centrifuge to remove supernatant, use the second PBS buffer solution (pH 6.0) to clean 2 times, remove unbound tea polyphenol. Suspension is centrifuged and collected to obtain nano-encapsulated probiotics.

[0084] Comparative Example 1

[0085] The same as Example 1, except that step (2) is not performed.

[0086] Comparative Example 2

[0087] The same as Example 1, except that in step (2), only lactoferrin is used for treatment.

[0088] Effect verification

[0089] 1. Test the encapsulation effect of the nano-encapsulation technology on probiotics in this embodiment:

[0090] FITC was used to fluorescently label the lactoferrin in Example 1, and then the FITC-lactoferrin was used to nano-encapsulate the probiotics. The encapsulation process was the same as in Example 1. The obtained probiotic nanocapsules were imaged using a laser confocal microscope. The results are as follows: Figure 1 As shown in the figure, it can be clearly observed that all the bacteria in the field of view are fluorescent green, indicating that the probiotics are successfully encapsulated and the encapsulation rate is 100%. The nano-encapsulated probiotics were tested by scanning electron microscopy and transmission electron microscopy. The results are as follows Figure 2 and Figure 3 As shown, it can be clearly observed that the surface of the probiotics is covered with a coating composed of nanoparticles.

[0091] 2. Test the effect of protein / polyphenol probiotic nanocapsules on the activity of probiotics:

[0092] The probiotic capsules prepared in Example 1 and the unencapsulated probiotics prepared in Comparative Example 1 were diluted to 5×10 7CFU / mL, added to the liquid culture medium at a rate of 2% (v / v), and its growth curve was monitored. Samples were taken every 3 hours for 36 hours, and the OD value was measured at 600nm and the growth curve was drawn. The effect of nanoencapsulation on the activity of probiotics was evaluated based on the growth curve. The test results are shown in Figure 4 As shown, it can be seen that the growth curve trends of nano-encapsulated probiotics and unencapsulated probiotics are consistent, with no significant differences, indicating that the EGCG / lactoferrin nano-coating has no effect on the biological activity of probiotics.

[0093] 3. Test the antioxidant capacity of the nano-encapsulated probiotics in this example;

[0094] DPPH free radical scavenging experiment. The probiotic capsule prepared in Example 1 was used as the sample to be tested, 1 mL of the sample to be tested and 1 mL of 0.1 mM DPPH ethanol solution were uniformly mixed, and allowed to stand at room temperature in the dark for 30 min, and then the absorbance value A1 was determined at 517 nm using anhydrous ethanol as a reference solution. At the same time, the absorbance value A0 of the mixed solution of 1 mL of anhydrous ethanol and 1 mL of 0.1 mM DPPH solution was used as the blank value, and the absorbance value A2 of the mixed solution of 1 mL of the sample to be tested and 1 mL of anhydrous ethanol was used as the background value. The calculation formula for the DPPH free radical scavenging rate is as follows:

[0095] DPPH free radical scavenging rate (%) = (A0-(A1-A2)) / A0×100

[0096] Where: A0 is the absorbance of 1 mL of 0.1 mM DPPH solution mixed with 1 mL of anhydrous ethanol;

[0097] A1 is the absorbance value of 1 mL of 0.1 mM DPPH ethanol solution mixed with 1 mL of the sample solution to be tested;

[0098] A2 is the absorbance value of 1 mL of ethanol solution mixed with 1 mL of the sample solution to be tested.

[0099] The results are as follows Figure 5 As shown, it can be seen that compared with the nano-encapsulated probiotics, the DPPH radical scavenging rate of the probiotics in Comparative Examples 1 and 2 is significantly lower than that of the nano-encapsulated probiotics prepared in Example 1.

[0100] 4. Testing the intestinal epithelial cell adhesion performance of the nano-encapsulated probiotics in this example:

[0101] Caco-2 and HT-29MTX cells were co-cultured for 17 days to establish an in vitro simulated intestinal adhesion model. After washing twice with PBS, 1 mL (3×10 7CFU) were placed in the cell model established by Caco-2 and HT-29MTX cells respectively, and cultured for 1 hour under 37°C and 5% CO2. The non-adherent bacteria were washed off with PBS, and the probiotics adhered to the cell model were collected and diluted 1000 times with PBS. 100 μL of the sample was taken for plate counting, and the effect of nanoencapsulated probiotics on the adhesion performance of intestinal epithelial cells was evaluated based on the counting results. The test results are shown in Figure 6 As shown, it can be seen that compared with nano-encapsulated probiotics, the probiotics in Comparative Examples 1 and 2 have poor intestinal epithelial cell adhesion ability, while the probiotic nanocapsules prepared by the present invention can improve the intestinal adhesion ability of probiotics to more than 7%.

[0102] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A probiotic nanocapsule, characterized in that: The method is composed of probiotics and a nanolayer encapsulating the probiotics. The nanolayer is composed of positively charged proteins and polyphenols. The surface of the probiotics is in situ bound to the proteins through electrostatic interactions, and the polyphenols and the binding proteins on the surface of the probiotics are prepared by non-covalent interactions.

2. A probiotic nanocapsule according to claim 1, characterized in that: The protein is a protein of natural origin, and preferably, the protein is lactoferrin.

3. A probiotic nanocapsule according to claim 1, characterized in that: The polyphenols are one or more natural plant polyphenols in combination in any proportion.

4. The probiotic nanocapsule according to claim 1, characterized in that: The probiotics are selected from one or more strains of Clostridium tenella, Pediococcus, Bifidobacterium, Gram-positive cocci, Escherichia coli, Bacteroides, Faecalibacterium, Saccharomyces, Lactobacillus, and Bacillus.

5. A method for preparing a probiotic nanocapsule according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1. Preparation of probiotic suspension: The probiotic bacterial solution was centrifuged, washed with a first PBS buffer, centrifuged, and resuspended in a second PBS buffer; S2, using a second PBS buffer to prepare a protein solution and a polyphenol solution and filter and sterilize; S3, mixing the probiotic suspension in S1 with the protein solution in S2, washing, and centrifuging to obtain protein-coated probiotics; S4. Mix the protein-coated probiotics obtained in S3 with the polyphenol solution in S2, wash, and centrifuge to obtain.

6. The preparation method according to claim 5, characterized in that: In step S2, the concentration of the protein solution is 1 to 10 mg / mL, and the concentration of the polyphenol solution is 1 to 10 mg / mL.

7. The preparation method according to claim 5, characterized in that: In step S3, the concentration of the protein-coated probiotic solution is 1×10 5 ~1×10 9 CFU / mL.

8. The preparation method according to claim 5, characterized in that: In step S3, the volume ratio of the probiotic suspension to the protein solution is 1:(1-10), and in step S4, the volume ratio of the protein-coated probiotic solution to the polyphenol solution is 1:(1-10).

9. Use of the probiotic nanocapsules prepared by the probiotic nanocapsules according to claims 1 to 4 or / and the probiotic nanocapsules prepared by the preparation method of claims 5 to 8 in the preparation of food additives and / or health care product additives.

10. Use of the probiotic nanocapsules prepared by the preparation method of the probiotic nanocapsules according to claims 1 to 4 or / and the probiotic nanocapsules according to claims 5 to 8 in the preparation of pharmaceutical additives and / or feed additives.

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