Active component-loaded food-borne biological vesicle as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a food-borne biological vesicle loaded with active ingredients as well as a preparation method and application of the food-borne biological vesicle. The preparation method of the food-borne biological vesicles loaded with the active ingredients comprises the following steps: obtaining supernate of a plant-derived raw material or a milk-derived raw material; homogenizing a mixed solution obtained by mixing the active ingredients and the supernate to obtain a homogenized solution; extruding the homogenized liquid for 4-6 times through a first filter membrane, a second filter membrane and a third filter membrane in sequence to obtain an extruded solution; and carrying out centrifugal separation treatment on the extruded solution to obtain the food-borne biological vesicles loaded with the active components. The food-borne biological vesicle loaded with the active ingredients has the characteristics of complete vesicle structure, gastrointestinal digestion resistance and high encapsulation efficiency, and can obviously improve the stability of the active ingredients in gastrointestinal tracts, thereby being beneficial to improving the bioavailability of the active ingredients.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a food-borne biological vesicle loaded with active ingredients, and a preparation method and application thereof. Background Art

[0002] Oral active ingredients need to cross the triple barrier of the gastrointestinal tract before they can be absorbed by the small intestinal epithelial cells and enter the human blood circulation to exert their relevant effects. This triple barrier includes the biochemical barrier (Biochemical barrier) composed of the acidic environment inside the stomach (lower pH) and various metabolic enzymes, the mucosal barrier (Mucus barrier) composed of mucus secreted by gastrointestinal mucous cells and tight junctions between cells, and the cellular barrier (Cellular barrier) of transcellular or paracellular transport of small intestinal epithelial cells. Studies have shown that various metabolic enzymes such as pepsin, trypsin, chymotrypsin and the acidic environment (lower pH) in the gastrointestinal tract will degrade the structure of the active ingredients and make them inactive.

[0003] Polydeoxyribonudeotide (PDRN) is an active ingredient with negative charge, hydrophilicity and large molecular weight. It has poor stability in the gastrointestinal tract and is easily affected by the acidic environment of the gastrointestinal tract after oral administration, causing hydrolysis and breakage of the glycosidic bonds and phosphodiester bonds of PDRN, depurination or degradation, resulting in reduced bioavailability.

[0004] In order to improve the bioavailability of active ingredients (such as PDRN), the prior art generally uses carrier systems such as cyclodextrin, liposome, zein to protect and deliver active ingredients. For example, the Chinese patent technology with publication number CN 113368054A discloses a method for preparing a flexible nanoliposome of polydeoxyribonucleotides, by dissolving soybean lecithin, cholesterol and polyoxyethylene sorbitan monooleate in ether solvent to prepare blank liposomes, then adding PDRN and sodium cholate to the blank liposomes, successively through ultrasound, pH adjustment, rotary evaporation hydration and filtering steps, finally obtaining PDRN flexible nanoliposomes, but the method uses an organic solvent (ether) in the process of preparing PDRN flexible nanoliposomes, and this organic solvent is relatively volatile, not only will it have potential harm to the health of the operator, but the residue of the organic solvent may affect the safety of the product, and it is difficult to achieve large-scale production.

[0005] Therefore, how to develop a safe and efficient oral active ingredient delivery system without using organic solvents has become a technical problem that needs to be solved urgently. Summary of the invention

[0006] The present invention provides a method for preparing food-borne biological vesicles loaded with active ingredients. The method can be used to prepare food-borne biological vesicles loaded with active ingredients. The food-borne biological vesicles loaded with active ingredients have the characteristics of complete vesicle structure, ability to resist gastrointestinal digestion and high encapsulation rate, and can significantly improve the stability of the active ingredients in the gastrointestinal tract, thereby facilitating the improvement of the bioavailability of the active ingredients. The preparation method does not require the use of organic solvents, is simple to operate, is conducive to large-scale production, and is suitable for wide promotion and application.

[0007] The present invention also provides a food-borne biological vesicle loaded with an active ingredient, which is prepared by the above-mentioned preparation method. Therefore, the food-borne biological vesicle loaded with the active ingredient has the characteristics of a complete vesicle structure, the ability to resist gastrointestinal digestion and a high encapsulation rate. The use of the food-borne biological vesicle loaded with the active ingredient can improve the stability of the active ingredient in the gastrointestinal tract, thereby facilitating the improvement of the bioavailability of the active ingredient.

[0008] The present invention also provides an oral preparation, which includes food-borne biological vesicles loaded with active ingredients prepared by the above-mentioned preparation method. Therefore, the oral preparation has the characteristics of complete vesicle structure, ability to resist gastrointestinal digestion and high encapsulation rate. The use of the oral preparation can improve the stability of the active ingredient in the gastrointestinal tract, thereby facilitating the improvement of the bioavailability of the active ingredient.

[0009] The first aspect of the present invention provides a method for preparing a food-borne biological vesicle loaded with an active ingredient, comprising the following steps:

[0010] obtaining a supernatant of a plant-derived raw material or a milk-derived raw material;

[0011] homogenizing the mixed solution obtained by mixing the active ingredient with the supernatant to obtain a homogenous solution;

[0012] The homogenized liquid is sequentially passed through a first filter membrane, a second filter membrane, and a third filter membrane for extrusion treatment 4-6 times to obtain an extruded solution, wherein the pore size of the first filter membrane is larger than the pore size of the second filter membrane, and the pore size of the second filter membrane is larger than the pore size of the third filter membrane;

[0013] The extruded solution is subjected to centrifugal separation treatment to obtain the food-borne biological vesicles loaded with active ingredients.

[0014] In the method for preparing food-borne biological vesicles loaded with active ingredients as described above, the pore size of the first filter membrane is 0.7-0.9 μm, the pore size of the second filter membrane is 0.4-0.5 μm, and the pore size of the third filter membrane is 0.15-0.25 μm.

[0015] In the method for preparing food-borne biological vesicles loaded with active ingredients as described above, in the homogenous solution, the mass volume ratio of the active ingredient to the supernatant is 1 g: (80-120 mL).

[0016] In the method for preparing food-borne biological vesicles loaded with active ingredients as described above, the speed of the homogenization treatment is 8000-10000 rpm and the time is 2-5 min.

[0017] The method for preparing the food-borne biological vesicles loaded with active ingredients as described above, wherein the extruded solution is subjected to a centrifugal separation treatment, comprises:

[0018] The extruded solution is centrifuged through an ultrafiltration tube with a molecular weight cutoff of 100 kDa, and the collected filtrate is the food-borne biological vesicles loaded with active ingredients.

[0019] The method for preparing food-borne biological vesicles loaded with active ingredients as described above, wherein the active ingredient is any one of polydeoxyribonucleotides, pyrroloquinoline quinone, collagen peptide, and nicotinamide.

[0020] The method for preparing the food-derived biological vesicles loaded with active ingredients as described above, wherein the plant-derived raw material is selected from fresh summer black grapes, fresh blood oranges, fresh pomegranates or fresh American ginseng;

[0021] The milk source raw material is selected from fresh milk or pasteurized milk.

[0022] The method for preparing the food-derived biological vesicles loaded with active ingredients as described above, wherein the supernatant of the plant-derived raw material or the milk-derived raw material is obtained, comprises:

[0023] The plant-derived raw material is pretreated and then subjected to a first centrifugation treatment at 2-8°C and 2000-4000g for 20-40 minutes, and after removing plant residues, a second centrifugation treatment is performed at 3-5°C and 8000-12000g for 25-40 minutes to obtain a supernatant;

[0024] Alternatively, the milk-derived raw material is subjected to a third centrifugation treatment at 2-8°C and 2000-4000g for 20-40min, the whey is collected and the pH value is adjusted to 4.58-4.62, and then a fourth centrifugation treatment is performed at 2-8°C and 8000-12000g for 40-60min to obtain a supernatant.

[0025] The second aspect of the present invention provides a food-borne biological vesicle loaded with an active ingredient, which is prepared by the method for preparing the food-borne biological vesicle loaded with an active ingredient.

[0026] The third aspect of the present invention provides an oral preparation, which comprises the food-borne biological vesicles loaded with active ingredients.

[0027] The solution of the present invention has at least the following effects:

[0028] The method for preparing food-borne biological vesicles loaded with active ingredients provided by the present invention comprises the following steps: homogenizing, extruding and centrifuging a mixed solution obtained by mixing the active ingredient with the supernatant of a plant-derived raw material or a milk-derived raw material, so as to prepare food-borne biological vesicles loaded with active ingredients having a complete vesicle structure, the ability to resist gastrointestinal digestion and a high encapsulation rate, which can significantly improve the stability of the active ingredient in the gastrointestinal tract, thereby facilitating the improvement of the bioavailability of the active ingredient; the preparation method does not require the use of an organic solvent, is simple to operate, is conducive to large-scale production, and is suitable for wide promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] 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 or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 TEM structural diagrams of test sample A, test sample B and test sample C of the present invention;

[0031] Figure 2 Schematic diagram of the particle size distribution of food-borne biological vesicles loaded with pyrroloquinoline quinone in test sample C of the present invention;

[0032] Figure 3 Schematic diagram of the particle size distribution of food-borne biological vesicles loaded with pyrroloquinoline quinone in test sample A of the present invention;

[0033] Figure 4 Schematic diagram of the particle size distribution of foodborne biological vesicles loaded with pyrroloquinoline quinone in test sample B of the present invention;

[0034] Figure 5 A schematic diagram of the ingestion of the food-borne biological vesicles loaded with pyrroloquinoline quinone by Caco-2 cells;

[0035] Figure 6 It is a statistical diagram of the relative expression of CAT gene in human skin fibroblasts after treatment in each group of the present invention;

[0036] Figure 7 It is a statistical diagram of the NQO1 gene expression in human skin fibroblasts after treatment in each group of the present invention;

[0037] Figure 8 is the pyrroloquinoline quinone (PQQ) standard curve of the present invention;

[0038] Fig. 9 is the peak area of ​​PQQ at a concentration of 0.25 mg / mL in the present invention;

[0039] Fig.10 is the peak area of ​​the food-borne biological vesicles loaded with pyrroloquinoline quinone in Example 1 of the present invention;

[0040] Fig.11 is the peak area of ​​the food-borne biological vesicles loaded with pyrroloquinoline quinone in Comparative Example 6 of the present invention;

[0041] Fig.12 is the peak area of ​​the food-borne biological vesicles loaded with pyrroloquinoline quinone in Comparative Example 7 of the present invention;

[0042] Fig.13 TEM structural diagrams of test samples D, E and F of the present invention;

[0043] Fig.14 Schematic diagram of particle size distribution of food-borne biological vesicles loaded with polydeoxyribonucleotides in test sample F of the present invention;

[0044] Fig.15 Schematic diagram of particle size distribution of food-borne biological vesicles loaded with polydeoxyribonucleotides in test sample D of the present invention;

[0045] Fig.16 Schematic diagram of particle size distribution of food-borne biological vesicles loaded with polydeoxyribonucleotides in test sample E of the present invention;

[0046] Fig.17 It is a statistical graph of the PDRN content in each test sample of the present invention;

[0047] Fig.18 Schematic diagram of the transmission electron microscopy structure of the food-borne biological vesicles loaded with polydeoxyribonucleotides in Example 2 and Comparative Examples 1 to 5 of the present invention;

[0048] Fig.19 This is the polydeoxyribonucleotide (PDRN) standard curve of the present invention. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] Unless otherwise specified, the raw materials and reagents used in the following examples can be obtained from commercial sources; the processes used, unless otherwise specified, are conventional processes in the art.

[0051] It should be noted that the descriptions involving “first”, “second”, “third”, “fourth”, etc. in the present invention are used to distinguish similar objects, but are not used to describe a specific order or sequence, and therefore cannot be understood as a limitation of the present invention.

[0052] The first aspect of the present invention provides a method for preparing a food-borne biological vesicle loaded with an active ingredient, comprising the following steps:

[0053] obtaining a supernatant of a plant-derived raw material or a milk-derived raw material;

[0054] homogenizing the mixture of the active ingredient and the supernatant to obtain a homogenous solution;

[0055] The homogenized liquid is sequentially passed through a first filter membrane, a second filter membrane, and a third filter membrane for extrusion treatment 4-6 times to obtain an extruded solution, wherein the pore size of the first filter membrane is larger than the pore size of the second filter membrane, and the pore size of the second filter membrane is larger than the pore size of the third filter membrane;

[0056] The extruded solution is subjected to centrifugal separation treatment to obtain food-borne biological vesicles loaded with active ingredients.

[0057] The present invention does not particularly limit the specific source of the plant-derived raw material or the milk-derived raw material, and the raw material can be purchased through commercial channels.

[0058] The present invention does not specifically limit the specific equipment for homogenization, and equipment well known in the art can be used for homogenization. In some embodiments, a high-speed disperser can be used to homogenize the mixed solution after the active ingredient and the supernatant are mixed.

[0059] The present invention does not specifically limit the specific equipment for extrusion treatment, and equipment well known in the art can be used for extrusion treatment. In some embodiments, an extruder can be used to sequentially pass the homogenized liquid through the first filter membrane, the second filter membrane, and the third filter membrane for 4-6 times.

[0060] In the present invention, the specific process of passing the homogeneous liquid through the first filter membrane, the second filter membrane and the third filter membrane in sequence for 4-6 times of extrusion treatment is as follows: step 1, passing the homogeneous liquid as the starting liquid through the first filter membrane to obtain the first liquid; step 2, passing the first liquid through the second filter membrane to obtain the second liquid; step 3, passing the second liquid through the third filter membrane to obtain the third liquid; the above steps 1 to 3 are repeated 4-6 times, each time using the third liquid obtained by the previous filtration as the starting liquid, and passing it through the first filter membrane, the second filter membrane and the third filter membrane in sequence again.

[0061] In the present invention, the supernatant obtained from the plant-derived raw material or the milk-derived raw material is rich in various biomembrane components such as extracellular vesicles, cell membranes, and organelle membranes.

[0062] The object of the present invention is to prepare food-borne biological vesicles loaded with active ingredients. Specifically, the supernatant of a plant-derived raw material or a milk-derived raw material is first obtained, and then the obtained plant or milk supernatant is mixed with the active ingredient so that the active ingredient is dissolved in the supernatant to obtain a mixed solution; then the mixed solution is homogenized to further disperse the components such as extracellular vesicles, cell membranes and organelle membrane fragments in the mixed solution, increase the contact area with the active ingredient, and obtain a homogenous solution; then the homogenous liquid is sequentially passed through a first filter membrane, a second filter membrane and a third filter membrane for extrusion treatment 4-6 times to make the active ingredient The active ingredients are recombined with extracellular vesicles, cell membranes and organelle membrane fragments to form a food-borne biological vesicle solution loaded with active ingredients, namely, an extruded solution, wherein the pore size of the first filter membrane is larger than the pore size of the second filter membrane, and the pore size of the second filter membrane is larger than the pore size of the third filter membrane. By using filter membranes with pore sizes from large to small for step-by-step filtration, impurities in the homogeneous liquid can be gradually removed; finally, the extruded solution is centrifuged to remove unbound active ingredients and free impurities, so as to obtain food-borne biological vesicles loaded with active ingredients with a complete vesicle structure, the ability to resist gastrointestinal digestion and a high encapsulation rate.

[0063] In a specific embodiment, the pore size of the first filter membrane is 0.7-0.9 μm, the pore size of the second filter membrane is 0.4-0.5 μm, and the pore size of the third filter membrane is 0.15-0.25 μm. Further, the pore size of the first filter membrane may be preferably 0.8 μm, the pore size of the second filter membrane may be preferably 0.45 μm, and the pore size of the third filter membrane may be preferably 0.2 μm.

[0064] When the parameters of the pore size of the first filter membrane, the pore size of the second filter membrane and the pore size of the third filter membrane are each within the above range, the above-mentioned homogeneous liquid can be filtered step by step and the vesicles can be reorganized step by step, and impurities in the homogeneous liquid can be gradually removed, which is conducive to the subsequent preparation of food-borne biological vesicles loaded with active ingredients with a complete vesicle structure.

[0065] In a specific embodiment, in the above homogeneous solution, the mass volume ratio of the active ingredient to the supernatant is 1g:(80-120mL).

[0066] When the mass volume ratio of the active ingredient to the supernatant in the homogeneous solution is within the above range, the active ingredient can be effectively dispersed and dissolved in the supernatant, avoiding precipitation or aggregation due to excessive active ingredient.

[0067] In a specific embodiment, the rotation speed of the homogenization treatment is 8000-10000 rpm and the time is 2-5 min.

[0068] When the parameters of the rotation speed and time of the homogenization treatment are respectively within the above ranges, the components such as extracellular vesicles, cell membranes and organelle membrane fragments in the mixed solution are further dispersed, increasing the contact area with the active ingredients, and the appropriate time (2-5 minutes) can prevent the degradation of the active ingredients due to excessive homogenization.

[0069] In a specific embodiment, the centrifugal separation of the extruded solution comprises:

[0070] The extruded solution is centrifuged through an ultrafiltration tube with a molecular weight cutoff of 100 kDa, and the collected filtrate is the food-borne biological vesicles loaded with active ingredients.

[0071] The present invention uses the ultrafiltration tube with a molecular weight cutoff of 100 kDa for centrifugal separation, thereby removing free unencapsulated small molecule active ingredients and other impurities, and collecting foodborne biological vesicles loaded with active ingredients with a molecular weight cutoff of 100 kDa.

[0072] In a specific embodiment, the above active ingredient is any one of polydeoxyribonucleotide, pyrroloquinoline quinone, collagen peptide, and nicotinamide.

[0073] When the above substances are used as active ingredients, food-borne biological vesicles loaded with active ingredients can be successfully prepared, thereby improving the bioavailability of the active ingredients.

[0074] The present invention does not particularly limit the specific sources of the plant-derived raw materials or milk-derived raw materials, and can be selected according to actual needs. In some embodiments, the plant-derived raw materials are selected from fresh summer black grapes, fresh blood oranges, fresh pomegranates, or fresh American ginseng; the milk-derived raw materials are selected from fresh milk or pasteurized milk.

[0075] When the above-mentioned substances are used as plant-derived raw materials or milk-derived raw materials, food-derived raw materials are provided for preparing food-derived biological vesicles loaded with active ingredients, thereby obtaining the supernatant of the plant-derived raw materials or milk-derived raw materials. Using the supernatant of the plant-derived raw materials or milk-derived raw materials, food-derived biological vesicles loaded with active ingredients can be successfully prepared.

[0076] In a specific embodiment, obtaining the supernatant of the plant comprises:

[0077] The plant-derived raw material is pretreated and then subjected to a first centrifugation treatment at 2-8°C and 2000-4000g for 20-40 minutes. After removing plant residues, the raw material is subjected to a second centrifugation treatment at 3-5°C and 8000-12000g for 25-40 minutes to obtain a supernatant.

[0078] The present invention does not particularly limit the specific method of the above-mentioned pretreatment, and the operation is performed according to actual conditions. In some embodiments, the pretreatment of the plant-derived raw material includes: the plant-derived raw material can be washed and crushed, and then subjected to a first centrifugal treatment at 2-8°C and 2000-4000g for 20-40min, and the plant-derived raw material can be washed, peeled, and crushed, and then subjected to a first centrifugal treatment at 2-8°C and 2000-4000g for 20-40min.

[0079] After pre-treating the plant-derived raw materials, the present invention can discard larger plant residues to obtain a supernatant. At this time, the supernatant is rich in extracellular vesicles naturally secreted by plants (including exosomes, microvesicles and apoptotic bodies, etc.) and various biomembrane components such as plant cell membranes and organelle membranes after cell wall breaking.

[0080] In another specific embodiment, obtaining the supernatant of milk includes: subjecting the milk-derived raw material to a third centrifugation treatment at 2-8°C and 2000-4000g for 20-40min, collecting the whey and adjusting the pH value to 4.58-4.62, and then subjecting the raw material to a fourth centrifugation treatment at 2-8°C and 8000-12000g for 40-60min to obtain the supernatant.

[0081] The present invention can remove lipid and protein components in milk after pretreatment of milk-derived raw materials to obtain a supernatant. At this time, the supernatant is rich in extracellular vesicles (including exosomes, microvesicles and apoptotic bodies, etc.) naturally secreted by mammary epithelial cells, as well as various biomembrane components such as cell membranes and organelle membranes retained in the emulsion.

[0082] The second aspect of the present invention provides a food-borne biological vesicle loaded with an active ingredient, which is prepared by the above-mentioned method for preparing a food-borne biological vesicle loaded with an active ingredient. Therefore, the food-borne biological vesicle loaded with an active ingredient has a complete vesicle structure, the ability to resist gastrointestinal digestion and a high encapsulation rate. The use of the food-borne biological vesicle loaded with an active ingredient can improve the stability of the active ingredient in the gastrointestinal tract, thereby facilitating the improvement of the bioavailability of the active ingredient.

[0083] The third aspect of the present invention provides an oral preparation, which includes the above-mentioned food-borne biological vesicles loaded with active ingredients. The oral preparation includes the food-borne biological vesicles loaded with active ingredients prepared by the above-mentioned preparation method, so the oral preparation has the characteristics of complete vesicle structure, ability to resist gastrointestinal digestion and high encapsulation rate. The use of the oral preparation can improve the stability of the active ingredient in the gastrointestinal tract, thereby facilitating the improvement of the bioavailability of the active ingredient.

[0084] The present invention is further described below through specific embodiments.

[0085] Example 1

[0086] This embodiment provides a method for preparing food-borne biological vesicles loaded with pyrroloquinoline quinone, comprising the following steps:

[0087] Step (1): fresh summer black grapes are cleaned and put into a wall breaking machine for crushing, and then centrifuged at 4°C and 4000g for 20 minutes, and after removing plant residues, centrifuged at 4°C and 10000g for 30 minutes to obtain a supernatant;

[0088] Step (2): Pyrroloquinoline quinone (PQQ) and the supernatant in step (1) are fully mixed at a mass volume ratio of 1 g:100 mL to obtain a mixed solution;

[0089] Step (3): homogenizing the mixed solution in step (2) using a high-speed disperser at a speed of 10,000 rpm for 3 min to obtain a homogenous solution;

[0090] Step (4): extruding the homogenized solution in step (3) through a first filter membrane, a second filter membrane, and a third filter membrane for five times in sequence to obtain an extruded solution, wherein the pore size of the first filter membrane is 0.8 μm, the pore size of the second filter membrane is 0.45 μm, and the pore size of the third filter membrane is 0.2 μm;

[0091] Step (5): The extruded solution in step (4) is centrifuged through an ultrafiltration tube with a molecular weight cutoff of 100 kDa to remove unbound PQQ and free impurities, and the filtrate is collected, i.e., the foodborne biological vesicles loaded with pyrroloquinoline quinone.

[0092] Example 2

[0093] This embodiment provides a method for preparing food-borne biological vesicles loaded with polydeoxyribonucleotides, comprising the following steps:

[0094] Step (1): clean and peel the fresh blood orange, put it into a wall breaking machine for crushing, and then centrifuge it at 4°C and 4000g for 20 minutes, remove plant residues, add 0.02g / L pectinase, perform enzymolysis at 40°C for 60 minutes, collect the enzymolysis solution, and centrifuge it at 4°C and 10000g for 30 minutes to obtain a supernatant;

[0095] Step (2): fully mixing polydeoxyribonucleotide (PDRN) with the supernatant in step (1) at a mass volume ratio of 1 g:100 mL to obtain a mixed solution;

[0096] Step (3): homogenizing the mixed solution in step (2) using a high-speed disperser at a speed of 10,000 rpm for 3 min to obtain a homogenous solution;

[0097] Step (4): extruding the homogenized solution in step (3) through a first filter membrane, a second filter membrane, and a third filter membrane for five times in sequence to obtain an extruded solution, wherein the pore size of the first filter membrane is 0.8 μm, the pore size of the second filter membrane is 0.45 μm, and the pore size of the third filter membrane is 0.2 μm;

[0098] Step (5): The extruded solution in step (4) is centrifuged through an ultrafiltration tube with a molecular weight cutoff of 100 kDa to remove unbound PDRN and free impurities, and the filtrate is collected, i.e., the foodborne biological vesicles loaded with polydeoxyribonucleotides.

[0099] Example 3

[0100] The embodiment provides a method for preparing food-derived biological vesicles loaded with collagen peptides, comprising the following steps:

[0101] Step (1): clean and peel the fresh blood orange, put it into a wall breaking machine for crushing, and then centrifuge it at 4°C and 4000g for 20 minutes, remove plant residues, add 0.02g / L pectinase, perform enzymolysis at 40°C for 60 minutes, collect the enzymolysis solution, and centrifuge it at 4°C and 10000g for 30 minutes to obtain a supernatant;

[0102] Step (2): the collagen peptide is fully mixed with the supernatant in step (1) at a mass volume ratio of 1 g:100 mL to obtain a mixed solution;

[0103] Step (3): homogenizing the mixed solution in step (2) using a high-speed disperser at a speed of 10,000 rpm for 3 min to obtain a homogenous solution;

[0104] Step (4): extruding the homogenized solution in step (3) through a first filter membrane, a second filter membrane, and a third filter membrane for five times in sequence to obtain an extruded solution, wherein the pore size of the first filter membrane is 0.8 μm, the pore size of the second filter membrane is 0.45 μm, and the pore size of the third filter membrane is 0.2 μm;

[0105] Step (5): The extruded solution in step (4) is centrifuged through an ultrafiltration tube with a molecular weight cutoff of 100 kDa to remove unbound collagen peptides and free impurities, and the filtrate is collected, i.e., the foodborne biological vesicles loaded with polydeoxyribonucleotides.

[0106] Example 4

[0107] The embodiment provides a method for preparing food-borne biological vesicles loaded with nicotinamide, comprising the following steps:

[0108] Step (1): centrifuging fresh milk at 4°C and 2000g for 20 minutes to separate the fat layer from the whey, collecting the whey and adjusting the pH to 4.60, and then centrifuging at 4°C and 10000g for 50 minutes to obtain a supernatant;

[0109] Step (2): nicotinamide and the supernatant in step (1) are fully mixed at a mass volume ratio of 1 g:100 mL to obtain a mixed solution;

[0110] Step (3): homogenizing the mixed solution in step (2) using a high-speed disperser at a speed of 10,000 rpm for 3 min to obtain a homogenous solution;

[0111] Step (4): extruding the homogenized solution in step (3) through a first filter membrane, a second filter membrane, and a third filter membrane for five times in sequence to obtain an extruded solution, wherein the pore size of the first filter membrane is 0.8 μm, the pore size of the second filter membrane is 0.45 μm, and the pore size of the third filter membrane is 0.2 μm;

[0112] Step (5): The extruded solution in step (4) is centrifuged through an ultrafiltration tube with a molecular weight cutoff of 100 kDa to remove unbound nicotinamide and free impurities, and the filtrate is collected, i.e., the foodborne biological vesicles loaded with polydeoxyribonucleotides.

[0113] Comparative Example 1

[0114] The preparation method of the food-borne biological vesicles loaded with polydeoxyribonucleotides provided in this comparative example is basically the same as that in Example 2, except that:

[0115] Step (4): The homogenized solution in step (3) is sequentially extruded through a first filter membrane, a second filter membrane, and a third filter membrane to obtain an extruded solution, wherein the pore size of the first filter membrane is 0.8 μm, the pore size of the second filter membrane is 0.45 μm, and the pore size of the third filter membrane is 0.2 μm.

[0116] Comparative Example 2

[0117] The preparation method of the food-borne biological vesicles loaded with polydeoxyribonucleotides provided in this comparative example is basically the same as that in Example 2, except that:

[0118] Step (4): The homogenized solution in step (3) is sequentially extruded through a first filter membrane, a second filter membrane, and a third filter membrane for 10 times to obtain an extruded solution, wherein the pore size of the first filter membrane is 0.8 μm, the pore size of the second filter membrane is 0.45 μm, and the pore size of the third filter membrane is 0.2 μm.

[0119] Comparative Example 3

[0120] The preparation method of the food-borne biological vesicles loaded with polydeoxyribonucleotides provided in this comparative example is basically the same as that in Example 2, except that:

[0121] Step (4): The homogenized solution in step (3) is sequentially extruded through a first filter membrane, a second filter membrane, and a third filter membrane for 20 times to obtain an extruded solution, wherein the pore size of the first filter membrane is 0.8 μm, the pore size of the second filter membrane is 0.45 μm, and the pore size of the third filter membrane is 0.2 μm.

[0122] Comparative Example 4

[0123] The preparation method of the food-borne biological vesicles loaded with polydeoxyribonucleotides provided in this comparative example is basically the same as that in Example 2, except that:

[0124] Step (4): The homogenized solution in step (3) is extruded through a filter membrane with a pore size of 0.8 μm for 5 times to obtain an extruded solution.

[0125] Comparative Example 5

[0126] The preparation method of the food-borne biological vesicles loaded with polydeoxyribonucleotides provided in this comparative example is basically the same as that in Example 2, except that:

[0127] Step (4): The homogenized solution in step (3) is extruded through the first filter membrane and the second filter membrane for 5 times in sequence to obtain an extruded solution, wherein the pore size of the first filter membrane is 0.8 μm and the pore size of the second filter membrane is 0.45 μm.

[0128] Comparative Example 6

[0129] The preparation method of the food-borne biological vesicles loaded with pyrroloquinoline quinone provided in this comparative example is basically the same as that in Example 1, except that:

[0130] Step (4): The homogenized solution in step (3) is sequentially extruded through a first filter membrane, a second filter membrane, and a third filter membrane for 10 times to obtain an extruded solution, wherein the pore size of the first filter membrane is 0.8 μm, the pore size of the second filter membrane is 0.45 μm, and the pore size of the third filter membrane is 0.2 μm.

[0131] Comparative Example 7

[0132] The preparation method of the food-borne biological vesicles loaded with pyrroloquinoline quinone provided in this comparative example is basically the same as that in Example 1, except that:

[0133] Step (4): The homogenized solution in step (3) is sequentially extruded through a first filter membrane, a second filter membrane, and a third filter membrane for 20 times to obtain an extruded solution, wherein the pore size of the first filter membrane is 0.8 μm, the pore size of the second filter membrane is 0.45 μm, and the pore size of the third filter membrane is 0.2 μm.

[0134] Performance Testing

[0135] 1. Performance test of food-borne biological vesicles loaded with pyrroloquinoline quinone

[0136] (1) Gastrointestinal digestibility of food-borne biovesicles loaded with pyrroloquinoline quinone

[0137] The gastrointestinal digestibility test of the food-borne biological vesicles loaded with pyrroloquinoline quinone in Example 1 of the present invention is carried out, and the specific method is as follows:

[0138] Experimental group: ① 500 μL of the foodborne biological vesicles loaded with pyrroloquinoline quinone in Example 1 and 500 μL of artificial saliva (artificial saliva warmed in a 37° C. water bath) were fully mixed and placed in a 37° C. water bath for 5 min to obtain a solution digested by saliva;

[0139] ② The solution digested by saliva in ① was thoroughly mixed with 500 μL artificial gastric juice (artificial gastric juice warmed in a 37°C water bath) and placed in a 37°C water bath for 2 h. The solution digested by saliva and gastric juice was recorded as test sample A;

[0140] ③ Thoroughly mix the solution digested by saliva and gastric juice in ② with 1000 μL artificial intestinal juice (artificial intestinal juice warmed in a 37°C water bath) and place in a 37°C water bath for 1 hour. The solution digested by saliva, gastric juice and intestinal juice is recorded as test sample B.

[0141] Control group: Normal saline was used as a control, and an equal volume of normal saline was used instead of artificial saliva, artificial gastric juice and artificial intestinal juice:

[0142] ① Take 500 μL of the food-borne biological vesicles loaded with pyrroloquinoline quinone in Example 1 and 500 μL of normal saline (normal saline after warm bath in a 37° C. water bath), mix thoroughly, place in a 37° C. water bath for 5 min, and collect the first solution;

[0143] ② Thoroughly mix the first solution collected in ① with 500 μL of normal saline (normal saline warmed in a 37°C water bath), place in a 37°C water bath for 2 h, and collect the second solution;

[0144] ③ The second solution collected in ② was mixed with 1000 μL of normal saline (normal saline warmed in a 37°C water bath) and placed in a 37°C water bath for 1 hour to obtain a third solution, which was recorded as test sample C;

[0145] The control group was set up mainly to eliminate the effect of long-term 37°C water bath treatment on the vesicle structure.

[0146] Take test sample A, test sample B and test sample C and drop them on the front surface of the copper mesh respectively, use phosphotungstic acid negative staining solution (2%) to stain and then wash, place under infrared light to dry, and then take photos under transmission electron microscope. The results are as follows: Figure 1 As shown; the particle size and concentration of test sample A, test sample B and test sample C were tested respectively, and the data were analyzed based on the average particle size and concentration, as shown in FIG. Figure 2-Figure 4 As shown in Table 1; the particle size and concentration can be directly tested by nanoparticle tracking analyzer. The equipment brand used in this test is PARTICLE METRIX, model PMX120.

[0147] Table 1 Average particle size and concentration of test sample A, test sample B and test sample C

[0148] project Average particle size (Diameter / nm) Concentration (Particles / mL) Test sample C 147.7 2.4E+10 Test sample B 140.6 1.6E+10 Test sample A 140.6 4.9E+10

[0149] Depend on Figure 1 It can be seen that the food-borne biological vesicles loaded with pyrroloquinoline quinone in Example 1 of the present invention can maintain the integrity of the vesicle structure after being digested by saliva, gastric juice and intestinal juice.

[0150] Depend on Figure 2-Figure 4As shown in Table 1, the average particle size of the food-borne biological vesicles loaded with pyrroloquinoline quinone in Example 1 of the present invention after digestion by saliva, gastric juice and intestinal juice is not much different from the average particle size of the food-borne biological vesicles loaded with pyrroloquinoline quinone in the normal saline control group, and the concentration has no obvious change.

[0151] (2) Uptake of food-derived biovesicles loaded with pyrroloquinoline quinone by human colorectal cancer cells (Caco-2 cells)

[0152] The uptake of food-derived biovesicles loaded with pyrroloquinoline quinone by human colorectal cancer cells (Caco-2 cells) was tested as follows:

[0153] ① Take out human colorectal cancer cells (Caco-2 cells) of the 6th generation from the cell bank, resuscitate and inoculate into culture bottles, and culture them in a 37°C, 5% CO2 incubator until the cells grow to 80% confluence to obtain cultured Caco-2 cells;

[0154] ②5×10 4 / well: Inoculate the Caco-2 cells cultured in ① into the confocal dish, add 1 mL to each well, and then place in a 37°C, 5% CO2 incubator for 24 hours to obtain the inoculated Caco-2 cells;

[0155] ③ Use PHK 26 fluorescent labeling dye (Umibio, PKH26) to fluorescently label the food-borne biological vesicles loaded with pyrroloquinoline quinone in Example 1. The specific operation is as follows: take 1 mL of the food-borne biological vesicles loaded with pyrroloquinoline quinone in Example 1, adjust its concentration to 1 mg / mL using PBS buffer, and obtain vesicles with a concentration of 1 mg / mL; subsequent fluorescent labeling light-proof operation, dilute the PHK 26 fluorescent labeling dye 10 times to prepare a dye working solution with a concentration of 100 μM; then add 50 μL of the dye working solution to 1 mL of the vesicles with a concentration of 1 mg / mL for fluorescent labeling, use a 100 kDa ultrafiltration tube for ultrafiltration and centrifugation to remove free fluorescence, and obtain fluorescently labeled food-borne biological vesicles loaded with pyrroloquinoline quinone (referred to as food-borne biological vesicles loaded with PQQ-PHK26);

[0156] ④ The fluorescently labeled food-borne biological vesicles loaded with pyrroloquinoline quinone in ③ were co-incubated with the Caco-2 cells inoculated in ② for 7 hours to obtain treated Caco-2 cells.

[0157] ⑤ The Caco-2 cells treated in ④ were fixed and stained, and the uptake of food-borne biological vesicles loaded with pyrroloquinoline quinone by Caco-2 cells was observed under a fluorescence microscope.

[0158] Depend on Figure 5It can be seen that after the fluorescently labeled food-borne biological vesicles loaded with pyrroloquinoline quinone were co-incubated with the inoculated Caco-2 cells for 7 hours, more red fluorescence appeared in the cytoplasm region of the Caco-2 cells, indicating that the food-borne biological vesicles loaded with pyrroloquinoline quinone can be taken up by Caco-2 cells.

[0159] (3) Antioxidant capacity of food-borne biological vesicles loaded with pyrroloquinoline quinone

[0160] The antioxidant capacity of the food-borne biological vesicles loaded with pyrroloquinoline quinone in Example 1 of the present invention was tested, and the specific method was as follows:

[0161] ① Take out human skin fibroblasts (HFB cells) of the sixth generation from the cell bank, resuscitate them into a culture bottle, and culture them in a 37°C, 5% CO2 incubator until the cells are 80% confluent, thereby obtaining cultured human skin fibroblasts;

[0162] ②6×10 4 / well: Inoculate the human skin fibroblasts cultured in ① into a 6-well plate, add 2 mL to each well, and then place in a 37°C, 5% CO2 incubator for further culturing for 24 hours to obtain the inoculated human skin fibroblasts;

[0163] ③ A control group, a model group, a PQQ group and an experimental group were set up, the dosage of each well was 2 mL, and 3 replicates were set up in each well. The cells were co-incubated with the human skin fibroblasts inoculated in ② for 24 h in a 37°C, 5% CO2 incubator to obtain treated human skin fibroblasts. The specific treatment conditions of each group are shown in Table 2:

[0164] Table 2 Treatment conditions of each group

[0165]

[0166] ④ The total RNA of human skin fibroblasts after treatment in each group was collected, and the expression levels of marker genes catalase gene (CAT gene) and NAD(P)H quinone oxidoreductase 01 gene (NQO1 gene) were detected by fluorescence quantitative PCR (qRT-PCR) technology.

[0167] Depend on Figure 6 It can be seen that compared with the control group, the relative expression of CAT gene in the model group decreased significantly, indicating that the UVB-induced model was successfully established. Compared with the model group, the relative expression of CAT gene in the PQQ group and the experimental group increased, but the relative expression of CAT gene in the experimental group was higher than that in the PQQ group. Figure 7It can be seen that compared with the control group, the relative expression of NQO1 gene in the model group decreased significantly, indicating that the UVB-induced model was successfully established. Compared with the model group, the relative expression of NQO1 gene in the PQQ group and the experimental group increased, but the relative expression of NQO1 gene in the experimental group was higher than that in the PQQ group. The above results show that the antioxidant capacity of food-borne biological vesicles loaded with pyrroloquinoline quinone is stronger than that of pyrroloquinoline quinone.

[0168] (4) Effect of different extrusion methods on the PQQ encapsulation efficiency of food-borne biological vesicles loaded with pyrroloquinoline quinone

[0169] The PQQ encapsulation efficiency of the foodborne biological vesicles loaded with pyrroloquinoline quinone in Example 1 of the present invention and Comparative Examples 6-7 was respectively detected. PQQ was used as a standard product, and a standard curve was drawn according to the concentration gradient. The PQQ encapsulation efficiency was obtained by peak area and concentration. The specific method is as follows: PQQ (McLean, M864625) was used as a standard product to prepare a concentration gradient, and high performance liquid chromatography (HPLC) was used for detection. The peak area was used as the x-axis and the concentration was used as the y-axis to draw a standard curve. The standard curve equation is: y=4E-05x+0.0105, R 2 =0.9995, as shown in Table 3 and Figure 8 As shown;

[0170] 100 μL of the food-borne biological vesicles loaded with pyrroloquinoline quinone in Example 1 of the present invention and Comparative Examples 6-7 were respectively diluted 20 times to obtain a dilution solution, and another 2 mL of the food-borne biological vesicles loaded with pyrroloquinoline quinone in Example 1 of the present invention and Comparative Examples 6-7 were simultaneously detected by high performance liquid chromatography (HPLC) for peak area (such as Figure 10-12 As shown in the figure, the peak area data of the test were substituted into the standard curve to obtain the corresponding concentration, and the data whose test results were within the linear range of the standard curve were selected for calculation. If there was dilution, the loaded drug (PQQ) concentration was obtained by multiplying it by the dilution multiple. The PQQ encapsulation efficiency data were obtained according to the drug concentration. The specific results are shown in Table 4.

[0171] Table 3 Peak area and concentration corresponding to PQQ standard curve

[0172] Serial number Peak area Concentration (mg / mL) 1 69.86 0.0078 2 201.91 0.0156 3 511.07 0.031 4 1244.31 0.0625 5 2890.97 0.125 6 6406.14 0.25 7 12927.93 0.5

[0173] Table 4 PQQ encapsulation efficiency

[0174]

[0175]

[0176] As can be seen from Table 4, the PQQ encapsulation rate of the food-borne biological vesicles loaded with pyrroloquinoline quinone in Example 1 of the present invention is 35.0%, the PQQ encapsulation rate of the food-borne biological vesicles loaded with pyrroloquinoline quinone in Comparative Example 6 is 0.7%, and the PQQ encapsulation rate of the food-borne biological vesicles loaded with pyrroloquinoline quinone in Comparative Example 7 is 0.5%, indicating that the method for preparing food-borne biological vesicles loaded with pyrroloquinoline quinone provided in Example 1 of the present invention has an excellent PQQ encapsulation rate.

[0177] 2. Performance testing of food-borne biological vesicles loaded with polydeoxyribonucleotides

[0178] (1) Gastrointestinal digestibility of food-borne biological vesicles loaded with polydeoxyribonucleotides

[0179] The gastrointestinal digestibility test of the food-borne biological vesicles loaded with polydeoxyribonucleotides in Example 2 of the present invention was performed, and the specific method was as follows:

[0180] ① Take 500 μL of the foodborne biological vesicles loaded with polydeoxyribonucleotides in Example 2, mix thoroughly with 500 μL of artificial saliva (artificial saliva warmed in a 37° C. water bath), and place in a 37° C. water bath for 5 min to obtain a solution digested by saliva;

[0181] ② The solution digested by saliva in ① was thoroughly mixed with 500 μL artificial gastric juice (artificial gastric juice warmed in a 37°C water bath) and placed in a 37°C water bath for 2 h. The solution digested by saliva and gastric juice was recorded as test sample D;

[0182] ③ Thoroughly mix the solution digested by saliva and gastric juice in ② with 1000 μL artificial intestinal juice (artificial intestinal juice warmed in a 37°C water bath) and place in a 37°C water bath for 1 hour. The solution digested by saliva, gastric juice and intestinal juice is recorded as test sample E.

[0183] The food-borne biological vesicles loaded with polydeoxyribonucleotides were treated with physiological saline as a control, and the specific method was as follows:

[0184] ① Take 500 μL of the food-borne biological vesicles loaded with polydeoxyribonucleotides in Example 2, mix thoroughly with 500 μL of normal saline (normal saline after warm bath in a 37°C water bath), place in a 37°C water bath for 5 minutes, and collect solution A;

[0185] ② Mix the solution A collected in ① with 500 μL of normal saline (normal saline warmed in a 37°C water bath) and place in a 37°C water bath for 2 h to collect solution B;

[0186] ③ The solution B collected in ② was thoroughly mixed with 1000 μL of physiological saline (physiological saline warmed in a 37°C water bath) and placed in a 37°C water bath for 1 hour. The solution treated with physiological saline was recorded as test sample F.

[0187] Take test samples D, E and F and drop them on the front surface of the copper mesh respectively, use phosphotungstic acid negative staining solution (2%) to stain and then wash, place under infrared light to dry, and then take photos under transmission electron microscope. The results are as follows: Fig.13 As shown; the particle size and concentration of test sample D, test sample E and test sample F were tested respectively, and the data were analyzed based on the average particle size and concentration. The results are as follows Fig.14 , Fig.15 , Fig.16 And as shown in Table 5.

[0188] Table 5 Average particle size and concentration of test samples D, E and F

[0189] project Average particle size (Diameter / nm) Concentration (Particles / mL) Test sample D 126.6 8.4E+11 Test Sample E 126.2 6.6E+11 Test sample F 153.9 6E+11

[0190] Depend on Fig.13 It can be seen that the food-borne biological vesicles loaded with polydeoxyribonucleotides in Example 2 of the present invention can maintain the integrity of the vesicle structure after being digested by saliva, gastric juice and intestinal juice.

[0191] Depend on Fig.14 , Fig.15 , Fig.16 As shown in Table 5, the average particle size of the food-borne biological vesicles loaded with polydeoxyribonucleotides in Example 2 of the present invention after digestion by saliva, gastric juice and intestinal juice is not much different from the average particle size of the food-borne biological vesicles loaded with pyrroloquinoline quinone in the normal saline control group, and the concentration has no obvious change.

[0192] The gastrointestinal digestibility test of polydeoxyribonucleotide (PDRN) is as follows:

[0193] ① Take 500 μL of polydeoxyribonucleotide aqueous solution (3 mg / mL, m / v) and 500 μL of artificial saliva (artificial saliva warmed in a 37°C water bath), mix thoroughly and place in a 37°C water bath for 5 minutes to obtain a solution after saliva digestion;

[0194] ② Thoroughly mix the solution digested by saliva in ① with 500 μL artificial gastric juice (artificial gastric juice warmed in a 37°C water bath) and place in a 37°C water bath for 2 h. The solution digested by saliva and gastric juice is recorded as test sample G;

[0195] ③ Thoroughly mix the solution digested by saliva and gastric juice in ② with 1000 μL artificial intestinal juice (artificial intestinal juice warmed in a 37°C water bath) and place in a 37°C water bath for 1 hour. The solution digested by saliva, gastric juice and intestinal juice is recorded as test sample H.

[0196] Polydeoxyribonucleotide (PDRN) was treated with physiological saline as a control, and the specific method was as follows:

[0197] ① Take 500 μL of polydeoxyribonucleotide aqueous solution (3 mg / mL, m / v) and 500 μL of normal saline (normal saline warmed in a 37°C water bath), mix thoroughly, place in a 37°C water bath for 5 minutes, and collect solution C;

[0198] ② Mix the solution C collected in ① with 500 μL of normal saline (normal saline warmed in a 37°C water bath) and place in a 37°C water bath for 2 h to collect solution D;

[0199] ③ The solution D collected in ② was thoroughly mixed with 1000 μL of normal saline (normal saline warmed in a 37°C water bath) and placed in a 37°C water bath for 1 hour. The solution treated with normal saline was recorded as test sample I.

[0200] The absorbance of polydeoxyribonucleotide (PDRN) in test sample D, test sample E, test sample F, test sample G, test sample H and test sample I at 595 nm was detected by diphenylamine method to obtain the PDRN content in each test sample. The PDRN content in test sample I was used as a control to perform statistical analysis on the PDRN content in each test sample. The results are as follows: Fig.17 shown.

[0201] Depend on Fig.17 It can be seen that the absorbance of PDRN in test samples G and H is significantly lower than that in test sample I, and PDRN is degraded by 81.87% after digestion with saliva and gastric juice, and by 58.34% after digestion with saliva, gastric juice and intestinal juice; although the absorbance of the food-derived biological vesicles loaded with polydeoxyribonucleotides in test samples D and E is lower than that of the food-derived biological vesicles loaded with polydeoxyribonucleotides in test sample F, the food-derived biological vesicles loaded with polydeoxyribonucleotides are only degraded by 6.98% after digestion with saliva and gastric juice, and by 15.05% after digestion with saliva, gastric juice and intestinal juice. These data show that the strategy provided by the present invention (food-derived biological vesicles loaded with polydeoxyribonucleotides) has a protective effect on PDRN, can improve the stability of PDRN in the gastrointestinal tract, and improve the bioavailability of PDRN.

[0202] (2) Effects of different extrusion methods on the structure of food-borne biological vesicles loaded with polydeoxyribonucleotides

[0203] The foodborne biological vesicles loaded with polydeoxyribonucleotides in Example 2 of the present invention and Comparative Examples 1 to 5 were respectively dropped onto the front surface of the copper mesh, stained with a phosphotungstic acid negative staining solution (2%), and then washed and dried under an infrared lamp. After drying, transmission electron microscopy was performed to photograph the vesicles. The results are shown in FIG. Fig.18 shown.

[0204] Depend on Fig.18 It can be seen that in Comparative Example 1, the food-borne biological vesicles loaded with polydeoxyribonucleotides had no complete vesicle structure observed under transmission electron microscopy, and more biofilm residues were visible; in Example 2, the food-borne biological vesicles loaded with polydeoxyribonucleotides had a large number of vesicle structures with a diameter of 100-200 nm observed under transmission electron microscopy; in Comparative Example 2, the food-borne biological vesicles loaded with polydeoxyribonucleotides had no complete vesicle structure under transmission electron microscopy; in Comparative Example 3, the food-borne biological vesicles loaded with polydeoxyribonucleotides had no complete vesicle structure under transmission electron microscopy. The food-borne biological vesicles loaded with polydeoxyribonucleotides in comparative example 4 were prepared by extruding only through a 0.8 μm filter membrane for 5 times, and a large cluster structure was visible under a transmission electron microscope, without a typical vesicle structure; the food-borne biological vesicles loaded with polydeoxyribonucleotides in comparative example 5 were prepared by extruding only through 0.8 μm and 0.45 μm filter membranes for 5 times, and a partial double-layer membrane structure and a large number of incomplete cluster structures were visible under a transmission electron microscope, without a typical vesicle structure.

[0205] (3) Effects of different extrusion methods on the PDRN encapsulation efficiency of food-derived biovesicles loaded with polydeoxyribonucleotides

[0206] The PDRN encapsulation efficiency of the food-borne biological vesicles loaded with polydeoxyribonucleotides in Example 2 of the present invention and Comparative Examples 1 to 5 was respectively detected, and PDRN was used as a standard, a standard curve was drawn according to a concentration gradient, and the PDRN encapsulation efficiency was detected by the diphenylamine method, and the specific method was as follows:

[0207] A concentration gradient was prepared with PDRN as the standard, and the absorbance value was the x-axis and the concentration was the y-axis to draw a standard curve. The equation of the standard curve was: y = 0.6714x + 0.0825, R 2 =0.99, as shown in Table 6 and Fig.19 As shown;

[0208] The foodborne biological vesicles loaded with pyrroloquinoline quinone in Example 2 of the present invention and Comparative Examples 1-5 were respectively diluted 10 times, and the absorbance value (OD595nm) was detected by the diphenylamine method simultaneously. The test data was substituted into the standard curve to obtain the corresponding concentration, and the data whose test results were within the linear range of the standard curve were selected for calculation. If there was a dilution, the loaded drug (PDRN) concentration was obtained by multiplying the dilution multiple, and the PDRN blocking rate data was obtained according to the drug concentration. The specific results are shown in Table 7.

[0209] Table 6 Absorbance values ​​and concentrations corresponding to the PDRN standard curve

[0210] Serial number Absorbance value Concentration (mg / mL) 1 0.089 0 2 0.164 0.1 3 0.250 0.3 4 0.419 0.4 5 0.564 0.5

[0211] Table 7 PDRN encapsulation efficiency

[0212]

[0213]

[0214] In Table 7, “ / ” means no data.

[0215] As shown in Table 7, the PDRN encapsulation rate of the food-derived biological vesicles loaded with polydeoxyribonucleotides in Example 2 of the present invention is 36.4%, the PDRN encapsulation rate of the food-derived biological vesicles loaded with polydeoxyribonucleotides in Comparative Example 4 is 18%, and the PDRN encapsulation rate of the food-derived biological vesicles loaded with polydeoxyribonucleotides in Comparative Example 5 is 21.3%, indicating that different extrusion methods have a great influence on the PDRN encapsulation rate. In addition, since the absorbance values ​​of the food-derived biological vesicles loaded with polydeoxyribonucleotides in Comparative Examples 1-3 are lower than the detection limit, data processing is not performed.

[0216] In summary, the preparation method provided by the present invention can not only prepare food-borne biological vesicles loaded with active ingredients (such as PQQ, PDRN) with a complete vesicle structure, the ability to resist gastrointestinal digestion and a high encapsulation rate, but also improve the stability of the active ingredients (such as PQQ, PDRN) in the gastrointestinal tract, which is beneficial to improve the bioavailability of the active ingredients (such as PQQ, PDRN).

[0217] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing food-borne biological vesicles loaded with active ingredients, characterized in that: The following steps are involved: obtaining a supernatant of a plant-derived raw material or a milk-derived raw material; homogenizing the mixed solution obtained by mixing the active ingredient with the supernatant to obtain a homogenous solution; The homogenized liquid is sequentially passed through a first filter membrane, a second filter membrane, and a third filter membrane for extrusion treatment 4-6 times to obtain an extruded solution, wherein the pore size of the first filter membrane is larger than the pore size of the second filter membrane, and the pore size of the second filter membrane is larger than the pore size of the third filter membrane; The extruded solution is subjected to centrifugal separation treatment to obtain the food-borne biological vesicles loaded with active ingredients.

2. The method for preparing food-borne biological vesicles loaded with active ingredients according to claim 1, characterized in that: The pore size of the first filter membrane is 0.7-0.9 μm, the pore size of the second filter membrane is 0.4-0.5 μm, and the pore size of the third filter membrane is 0.15-0.25 μm.

3. The method for preparing food-borne biological vesicles loaded with active ingredients according to claim 1, characterized in that: In the homogeneous solution, the mass volume ratio of the active ingredient to the supernatant is 1 g: (80-120 mL).

4. The method for preparing food-borne biological vesicles loaded with active ingredients according to claim 1, characterized in that: The rotation speed of the homogenization treatment is 8000-10000 rpm and the time is 2-5 min.

5. The method for preparing food-borne biological vesicles loaded with active ingredients according to claim 1, characterized in that: The extruded solution is subjected to a centrifugal separation treatment, comprising: The extruded solution is centrifuged through an ultrafiltration tube with a molecular weight cutoff of 100 kDa, and the collected filtrate is the food-borne biological vesicles loaded with active ingredients.

6. The method for preparing food-borne biological vesicles loaded with active ingredients according to claim 1, characterized in that: The active ingredient is any one of polydeoxyribonucleotide, pyrroloquinoline quinone, collagen peptide and nicotinamide.

7. The method for preparing food-borne biological vesicles loaded with active ingredients according to claim 1, characterized in that: The plant-derived raw material is selected from fresh summer black grapes, fresh blood oranges, fresh pomegranates or fresh American ginseng; The milk source raw material is selected from fresh milk or pasteurized milk.

8. The method for preparing food-borne biological vesicles loaded with active ingredients according to claim 1, characterized in that: The method of obtaining the supernatant of the plant-derived raw material or the milk-derived raw material comprises: The plant-derived raw material is pretreated and then subjected to a first centrifugation treatment at 2-8°C and 2000-4000g for 20-40 minutes, and after removing plant residues, a second centrifugation treatment is performed at 3-5°C and 8000-12000g for 25-40 minutes to obtain a supernatant; Alternatively, the milk-derived raw material is subjected to a third centrifugation treatment at 2-8°C and 2000-4000g for 20-40min, the whey is collected and the pH value is adjusted to 4.58-4.62, and then a fourth centrifugation treatment is performed at 2-8°C and 8000-12000g for 40-60min to obtain a supernatant.

9. A food-borne biological vesicle loaded with active ingredients, characterized in that: The food-borne biological vesicle is prepared by the method for preparing the food-borne biological vesicle loaded with active ingredients according to any one of claims 1 to 8.

10. An oral preparation, characterized in that The invention comprises the food-borne biological vesicle loaded with active ingredients as claimed in claim 9.

Images