Preparation and application of probiotic biomimetic nanoparticles loaded with food functional factors

Abstract

The application discloses a kind of functional factor of loading food probiotic biomimetic nanoparticle preparation and its application, belong to the cross technical field of food science and biomedical application.It comprises: S1, dissolving probiotic in NaCl solution, stirring to obtain probiotic suspension solution, then adding alkaline solution, stirring to obtain mixed system;S2, the mixed system in S1 is centrifuged, and the precipitate obtained by centrifugation is washed, and the washed precipitate is resuspended with NaCl solution to obtain a bacterial shell suspension solution;S3, add nanoparticles to S2 bacterial shell suspension solution, incubate, and obtain food functional factor carrying probiotic biomimetic nanoparticles by centrifugation.The astaxanthin biomimetic nanoparticles prepared by the method have good biocompatibility and are stable to gastrointestinal digestion, can significantly improve non-alcoholic fatty liver, and have very good application prospect in the pharmaceutical and health product industries.

Description

TECHNICAL FIELD

[0001] The present application relates to the cross field of food science and biomedical applications, and relates to a kind of preparation and application of food functional factor loaded probiotic biomimetic nanoparticles. BACKGROUND

[0002] With the improvement of living standards, people's requirements for food have also greatly improved, from the requirement of "eating enough" to eating nutrition and health. In addition to the main function of providing nutritional energy for the human body, the bioactive ingredients contained in food are increasingly favored. They are functional factors in food, mainly including proteins, functional lipids, polysaccharides, terpenes, polyphenols, carotenoids, flavonoids, probiotics, minerals, vitamins, etc. These functional factors play an important role in improving food quality, regulating physiological functions, preventing diseases and regulating human health. However, food functional factors are often characterized by poor solubility, poor stability and low bioavailability, and are easily affected by factors such as temperature, oxygen, pH, light, humidity, enzymes and metal ions, making it difficult to be widely applied in the processing industry.

[0003] In order to overcome the low solubility, stability and bioavailability of food functional factors, nano-particle-based functional factor delivery systems have been widely used in functional foods. Nanoparticles are internalized into cells by endocytosis, thereby improving the utilization rate of food functional factors. However, nanoparticles still have safety problems, poor biocompatibility, low bioavailability and other limitations. Research has found that even if food functional factors are protected by nanoparticles, their retention rate is only 20-30% after digestion by gastric acid, and further improvement is needed. Therefore, a new type of carrier system is needed to improve the bioavailability of food functional factor-loaded nanoparticles. SUMMARY

[0004] TECHNICAL PROBLEM

[0005] Food functional factor-loaded nanoparticles have safety problems, poor biocompatibility, low bioavailability and other limitations. Generally, only 20-30% of nanoparticles can pass through the gastric acid to reach the intestine, so a more effective carrier system is needed.

[0006] TECHNICAL CONTENT

[0007] In view of the above technical problems, the purpose of the present application is to select natural probiotics as the main wall material, embed nanoparticles by punching the bacterial shell and vacuum solute replacement method, and prepare a new type of shell-shell structure carrier system to promote the bioavailability of food functional factors.

[0008] The first object of the present application is to provide a preparation method of probiotic biomimetic nanoparticles loaded with food functional factors, which comprises the following steps:

[0009] S1, adding an alkaline solution to a probiotic suspension solution to obtain a mixed system;

[0010] S2, centrifuging the mixed system in S1, washing the obtained precipitate, and resuspending the washed precipitate with a NaCl solution to obtain a bacterial shell suspension solution;

[0011] S3, adding nanoparticles to the bacterial shell suspension solution in S2, incubating, and centrifuging to obtain probiotic biomimetic nanoparticles loaded with food functional factors.

[0012] Further, the probiotics in step S1 include one or more of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus bulgaricus, and Bifidobacterium.

[0013] Further, the concentration of the probiotics in the probiotic suspension solution in step S1 is OD 600 0.5-1.5.

[0014] Further, the solvent in the probiotic suspension solution in step S1 is a NaCl solution with a concentration of 0.3-0.6 mg / mL.

[0015] Further, the alkaline solution in step S1 is an alkaline solution prepared with a NaCl solution; the concentration of the NaCl solution is 0.3-0.6 mg / mL; and the alkali used in the alkaline solution includes sodium hydroxide and / or potassium hydroxide.

[0016] Further, the final concentration of sodium chloride in the mixed system is 0.3-0.6 mg / mL.

[0017] Further, the final concentration of hydroxide in the mixed system is 0.8-1.2 mg / mL.

[0018] Further, the centrifugation in step S2 is performed at 6000-8000 rpm for 6-10 minutes.

[0019] Further, the concentration of the NaCl solution in step S2 is 0.3-0.6 mg / mL.

[0020] Further, the concentration of the bacterial shell in the bacterial shell suspension solution in step S2 is 0.1-2 mg / mL.

[0021] Further, the nanoparticles in step S3 include sugar-based protein nanoparticles or liposome nanoparticles embedded with food functional factors.

[0022] The food functional factor includes lutein, astaxanthin, curcumin or other fat-soluble functional substances.

[0023] Further, the mass ratio of the nanoparticles and the chitin in the chitin suspension solution in step S3 is 1:0.01-0.001.

[0024] Further, the incubation in step S3 is performed under vacuum.

[0025] The vacuum degree is 0.1-0.5 MPa.

[0026] The temperature of the incubation is 35-40 DEG C, and the time is 20-24 hours.

[0027] Further, the centrifugation in step S3 is performed at 6000-10000 rpm for 5-10 minutes.

[0028] The application provides a food functional factor-loaded probiotic biomimetic nanoparticle obtained by the above method.

[0029] The application provides an application of the food functional factor-loaded probiotic biomimetic nanoparticle in the preparation of food, health products and medicines.

[0030] The application also provides an embedding method, which comprises the following steps: perforating a chitin shell, and embedding a substance to be embedded into the chitin shell by using a solute replacement method.

[0031] Further, the bacteria of the chitin shell can be probiotic bacteria.

[0032] Further, the probiotic bacteria include one or more of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus bulgaricus and Bifidobacterium.

[0033] Further, the perforation is performed by stirring the bacteria in an alkaline solution.

[0034] Further, the concentration of hydroxide in the alkaline solution is 0.8-1.2 mg / mL.

[0035] Further, the alkaline solution also contains 0.3-0.6 mg / mL of NaCl.

[0036] Further, the substance to be embedded includes a food functional factor, a glycosylated protein nanoparticle embedding a food functional factor or a liposome nanoparticle.

[0037] Further, the food functional factor includes lutein, astaxanthin, curcumin or other fat-soluble functional substances.

[0038] Further, the solute replacement is a vacuum solute replacement method, specifically, under a vacuum degree of 0.1-0.5 MPa, a temperature of 35-40 DEG C, and stirring incubation for 20-24 hours.

[0039] Advantages

[0040] (1) The present application keeps the activity of the active substance under mild conditions, has simple preparation process, low energy consumption, green environmental protection, controllable and relatively uniform size of the prepared biomimetic nanoparticles;

[0041] (2) The present application uses the nanoparticles of the probiotic bacteria shadow loaded and wrapped with the fat-soluble functional factor, significantly improves the problems of poor water solubility and poor stability of the fat-soluble functional factor, improves the bioavailability, and has good biocompatibility;

[0042] (3) The prepared biomimetic nanoparticle carrier system of the food functional factor has high loading rate and good biocompatibility, can ensure that a large amount of functional factors reach the intestinal tract, and the prepared probiotic biomimetic nanoparticles can effectively regulate the pathological level of intestinal inflammation and fatty liver mice, and significantly improve the effect of inflammation and fatty liver tissue degeneration, as proved by the examples. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 It is a SEM scanning electron microscope graph of the probiotic bacteria shadow prepared by the NaOH concentration of 2.5 mg / mL in the embodiment 1 of the present application.

[0044] Figure 2 It is a SEM scanning electron microscope graph of the probiotic bacteria shadow prepared by the NaOH concentration of 4 mg / mL in the embodiment 1 of the present application.

[0045] Figure 3 It is a SEM scanning electron microscope graph of the probiotic bacteria shadow prepared by the NaOH concentration of 1 mg / mL in the embodiment 1 of the present application.

[0046] Figure 4 It is a TEM graph of the ultrathin section of the probiotic biomimetic nanoparticles in the embodiment 1 of the present application.

[0047] Figure 5 It is a metabolic kinetics graph of the probiotic biomimetic nanoparticles in the embodiment 1 of the present application in blood.

[0048] Figure 6 It is a metabolic kinetics graph of the probiotic biomimetic nanoparticles in the embodiment 1 of the present application in liver.

[0049] Figure 7The mouse liver HE staining of the blank control group, the model group, the astaxanthin group, the astaxanthin nanoparticle group and the probiotic biomimetic nanoparticle group in Example 1 of the present application, wherein Control represents the blank control group, Model represents the model group, AXT represents the astaxanthin group, AWG represents the astaxanthin nanoparticle group, and AWG@LBGs represents the probiotic biomimetic nanoparticle group.

[0050] Figure 8 The mouse liver oil red O staining of the blank control group, the model group, the astaxanthin group, the astaxanthin nanoparticle group and the probiotic biomimetic nanoparticle group in Example 1 of the present application.

[0051] Figure 9 The mouse liver triglyceride content diagram of the blank control group, the model group, the astaxanthin group, the astaxanthin nanoparticle group and the probiotic biomimetic nanoparticle group in Example 1 of the present application.

[0052] Figure 10 The mouse liver total cholesterol content diagram of the blank control group, the model group, the astaxanthin group, the astaxanthin nanoparticle group and the probiotic biomimetic nanoparticle group in Example 1 of the present application; (a) is the total cholesterol content, and (b) is the free fatty acid content. DETAILED DESCRIPTION

[0053] Raw material source

[0054] The Lactobacillus plantarum is purchased from the China Industrial Microbial Culture Collection Center, and the preservation number is CICC 24936;

[0055] The preparation of the sugar-based whey protein isolate nanoparticle embedding astaxanthin is disclosed in the invention patent with the publication number CN115844837B.

[0056] Example 1

[0057] The preparation method of the probiotic biomimetic nanoparticle loaded with a food functional factor includes the following steps:

[0058] S1, dissolve 1 gram of Lactobacillus plantarum in a NaCl solution, and obtain a probiotic suspension solution by magnetic stirring at room temperature; the OD value of the natural probiotic final concentration is 1, the NaCl solution concentration is 0.5 mg / mL, and the magnetic stirring is performed at room temperature for 60 min to obtain the bacterial suspension. 600 The OD value of the natural probiotic final concentration is 1, the NaCl solution concentration is 0.5 mg / mL, and the magnetic stirring is performed at room temperature for 60 min to obtain the bacterial suspension.

[0059] S2, adding a basic solution prepared with NaCl solution (NaCl concentration of 0.5 mg / mL) to the S1 bacterial suspension, stirring at 37°C for 24 hours to obtain a mixed system; the final concentration of NaCl in the mixed system is 0.5 mg / mL, the base is NaOH, and the final concentration of NaOH is 2.5 mg / mL; the bacterial cell wall suspension solution is subjected to SEM scanning electron microscopy imaging, and the results are shown in Figure 1 .

[0060] S3, centrifuging the mixed system in S2, washing the precipitate obtained by centrifugation, and then resuspending the precipitate with NaCl solution to obtain a bacterial cell wall suspension solution. The centrifugal force is 8000 rpm, and the centrifugation time is 6 minutes; the washing condition is 0.5 mg / mL NaCl solution, and the washing time is 3 times; the resuspension uses NaCl solution with a concentration of 0.5 mg / mL.

[0061] S4, adding 1 g of astaxanthin-embedded glycosylated whey protein isolate nanoparticles to 10 mL of the bacterial cell wall suspension solution (bacterial cell wall concentration of 1 mg / mL) prepared in S3, incubating at 37°C under vacuum conditions for 24 hours, and centrifuging to obtain probiotic biomimetic nanoparticles carrying food functional factors, with an embedding rate of more than 60%. The vacuum condition is 0.1 MPa, the centrifugal force is 8000 rpm / min, and the centrifugation time is 6 minutes.

[0062] Example 2

[0063] Referring to Example 1, only the final concentration of NaOH in the mixed system in step S2 is adjusted to 4 mg / mL and 1 mg / mL to prepare the bacterial cell wall suspension solution.

[0064] The bacterial cell wall suspension solution is subjected to SEM scanning electron microscopy imaging, and the results are shown in Figure 2 and Figure 3 .

[0065] It can be found by comparison that for Lactobacillus plantarum, the final concentration of NaOH determines the size of the bacterial cell wall opening. Obviously, too low an alkali concentration will result in too small an opening, and too high an alkali concentration will result in too large an opening. Therefore, the final concentration of NaOH should be 2.5 mg / mL.

[0066] Example 3

[0067] Referring to Example 1, only the concentration of the bacterial cell wall suspension solution in step S4 is adjusted to 0.1 mg / mL to prepare the probiotic biomimetic nanoparticles.

[0068] The loading rate of the probiotic biomimetic nanoparticles is detected, and the loading rate is 88.28%.

[0069] Example 4

[0070] Reference is made to Example 1, wherein only the embedding astaxanthin sugar-based whey protein isolate nanoparticles in step S4 is replaced by embedding astaxanthin phospholipid liposome nanoparticles, the preparation method of which is as follows:

[0071] 1 g of phospholipid and 50 mg of astaxanthin are dissolved in 10 mL of ethanol, 2 g of sugar alcohol is added, and after being mixed well, a dry powder is obtained by rotary evaporation at 60°C and 1000 rpm. A small amount of powder is hydrated to obtain astaxanthin-embedded liposomes.

[0072] The preparation method of the probiotic biomimetic nanoparticles is as follows:

[0073] S4, 1 g of astaxanthin-embedded phospholipid liposome nanoparticles is added to 10 mL of the cell wall suspension solution prepared in S3 (cell wall concentration is 1 mg / mL), and incubated at 37°C for 24 hours under vacuum conditions. The probiotic biomimetic nanoparticles carrying food functional factors are obtained by centrifugation. The vacuum condition is 0.1 MPa, the centrifugal force is 8000 rpm / min, and the centrifugation time is 6 minutes.

[0074] The embedding rate of the probiotic on the phospholipid liposome nanoparticles is 80±4.52%, and the astaxanthin retention rate in the simulated gastrointestinal digestion is more than 90%, indicating that the method described in Example 1 has a certain universality.

[0075] Example 5

[0076] I. The probiotic cell shadow carrier obtained in Example 1 is subjected to SEM scanning electron microscopy imaging. The SEM scanning electron microscopy imaging results show that the probiotic cell shadow morphology is intact, the cell shadow outer wall has pores, and there is no probiotic sub-organ inside, indicating that the probiotic cell shadow carrier is successfully prepared.

[0077] II. The probiotic biomimetic nanoparticles obtained in Example 1 are subjected to ultrathin sectioning and transmission electron microscopy imaging. Compared with the single probiotic cell shadow, the probiotic nanoparticles have astaxanthin-embedded sugar-based whey protein isolate nanoparticles, as shown in Figure 4 , indicating that the probiotic cell shadow successfully embeds sugar-based whey protein isolate nanoparticles.

[0078] III. Metabolic kinetics analysis is performed on the probiotic biomimetic nanoparticles obtained in Example 1, as shown in Figure 5 , the results show that the astaxanthin content in the blood of the probiotic biomimetic nanoparticle group is higher than that of the astaxanthin nanoparticle group and the free astaxanthin group. At the same time, as shown in Figure 6 , it is also found that the astaxanthin content in the mouse liver of the probiotic biomimetic nanoparticle group is higher than that of the astaxanthin nanoparticle group and the free astaxanthin group.

[0079] Four, take the probiotic biomimetic nanoparticles obtained in Example 1 to intervene in non-alcoholic fatty liver.

[0080] The non-alcoholic fatty liver experiment was carried out by using 6-week-old C57BL / 6 male mice, and the experiment was divided into 5 groups, namely a blank group, a model group, an astaxanthin group (AXT), an astaxanthin nanoparticle group (AWG) and a probiotic biomimetic nanoparticle group (AWG@LBGs). From the 0th day, except that the blank group was fed with a control feed, the rest groups were fed with a high-fat feed lacking in choline for 8 weeks, and then nutritional intervention was carried out. The astaxanthin group, the astaxanthin nanoparticle group and the probiotic biomimetic nanoparticle group were respectively given free astaxanthin, astaxanthin nanoparticles and probiotic biomimetic nanoparticles samples containing an equal amount of astaxanthin (300 mg / each / day) 0.2 mL for intervention for 6 weeks. The blank group and the model group were given normal saline. After six weeks, the mice were sacrificed, and the tissues and organs of the mice were taken out.

[0081] Figure 1 is a comparison chart of the liver histology of mice. Figure 7 Figure 1 is a comparison chart of the liver histology of mice.

[0082] Figure 2 is an oil red O staining of the liver of mice. Figure 8 Figure 2 is an oil red O staining of the liver of mice. Figure 9 Figure 3 is a comparison chart of the triglyceride content of the liver of mice. Figure 10 Figure 4 is a comparison chart of the total cholesterol content of the liver of mice.

[0083] Although the present application has been disclosed with reference to the preferred embodiments above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. A method for preparing probiotic biomimetic nanoparticles loaded with food functional factors, characterized in that: Here are the steps: S1. Add alkaline solution to the probiotic suspension solution and stir to obtain a mixed system; the probiotic is Lactobacillus plantarum; the OD of the probiotic at the concentration of the probiotic suspension solution is 600 The value is 1; the final concentration of NaOH in the mixed system is 2.5 mg / mL, and the final concentration of sodium chloride is 0.5 mg / mL; S2, centrifuging the mixed system in S1, washing the precipitate obtained by centrifugation, and resuspending the washed precipitate in a NaCl solution to obtain a shell suspension solution; the concentration of the NaCl solution is 0.5 mg / mL; the shell concentration in the shell suspension solution is 1 mg / mL; S3, adding nanoparticles to the shell suspension solution of S2, incubating, and centrifuging to obtain probiotic biomimetic nanoparticles carrying food functional factors; the nanoparticles are liposome nanoparticles encapsulating astaxanthin; the preparation method of the phospholipid liposome nanoparticles is as follows: dissolving 1 g of phospholipid and 50 mg of astaxanthin in 10 mL of ethanol, adding 2 g of sugar alcohol, mixing, and rotary evaporating at 60°C and 1000 rpm to obtain a dry powder, and hydrating the powder to obtain liposomes encapsulating astaxanthin; The mass ratio of the nanoparticles to the shells in the shell suspension solution is 1:0.01; the incubation is performed under vacuum; the incubation temperature is 37° C., and the time is 24 hours.

2. A probiotic biomimetic nanoparticle loaded with food functional factors, characterized in that: The probiotic biomimetic nanoparticles are prepared by the preparation method according to claim 1.

3. Use of the probiotic biomimetic nanoparticles loaded with food functional factors as claimed in claim 2 in the preparation of food or medicine.

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