Preparation method and application of lemon vesicle embedded active probiotics

Abstract

The invention belongs to the technical field of health care products, and discloses a preparation method and application of lemon vesicle embedded active probiotics. The invention discloses lemon vesicle embedded active probiotics which comprise probiotics and lemon vesicles coating the surfaces of the probiotics, and the lemon vesicles are extracellular vesicles from lemons. The lemon vesicles are used for performing single-cell embedding on the active probiotics, so that the lemon vesicles can resist degradation of gastric acid and bile, the survival rate and colonization rate of the active probiotics in the intestinal tract through the stomach are increased, the balance of the active probiotics in regulating the intestinal microecology is promoted, various functions of the active probiotics are efficiently realized, and the effects of improving the intestinal tract microecology and improving the intestinal tract microecology are achieved. Meanwhile, the natural active ingredients of the lemon vesicles can synergistically reduce inflammatory factors of intestinal tracts. The lemon vesicles provide a brand new probiotic delivery technology for active probiotics, and a new development strategy is brought to the oral probiotic industry in the future.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of health products, and particularly relates to a preparation method of lemon vesicle-embedded active probiotics and application thereof. BACKGROUND

[0002] Probiotics are increasingly widely used in the management of intestinal health, but their oral administration is still challenged by gastric acid, bile and digestive enzymes. In order to solve this problem, researchers around the world have developed various probiotic surface coating strategies. Previous studies have mainly focused on the surface functionalization of bacteria with materials such as alginate and its derivatives, microbial polysaccharide network (MPN) coating, mineral coating and enteric coating to resist the physiological microenvironment, thereby providing defense and protection for orally administered bacteria. For example, Geng et al. modified the bacterial surface with polyphenols, enabling it to resist the physiological microenvironment and provide protection for oral bacteria. Cao et al. used a biological interface supramolecular self-assembly technology to prepare lipid-coated bacteria (LCB), which significantly improved the survival ability of LCB under extreme conditions such as antibiotics, extreme pH, ethanol and in vivo gastrointestinal environment. Zhou et al. used programmable polysaccharide coating to achieve targeted colonization of probiotics in inflamed colon sites. These coating materials not only protect probiotics from harsh gastrointestinal environments, but also enhance the adhesion of probiotics to intestinal mucosa, thereby improving the survival rate and colonization efficiency of probiotics. In recent years, significant progress has been made in probiotic engineering technology, such as using gene editing technology to enhance the resistance of probiotics to acid and bile, or displaying intestinal adhesion molecules on their surface to prolong intestinal retention time. However, current probiotic coating technologies still face problems such as high material cost, complex preparation process and the possibility of immune reactions caused by some synthetic materials, which limit their clinical translation.

[0003] Plant-derived extracellular vesicles (PDEVs) as natural exosome-like nanovesicles have attracted widespread attention due to their inherent biocompatibility and multifunctionality. Increasing evidence suggests that PDEVs participate in intestinal homeostasis by inducing intestinal stem cell proliferation, remodeling the intestinal microbiota and regulating intestinal immunity through cross-species communication with animal intestinal host cells. Notably, PDEVs have shown significant efficacy in alleviating IBD symptoms, making them a very promising therapeutic drug. For example, in a dextran sulfate sodium (DSS)-induced colitis model, grape-derived extracellular vesicles rich in polyphenols alleviate oxidative stress by activating the Nrf2 pathway. Orange-derived extracellular vesicles upregulate interleukin-22 expression by activating the aryl hydrocarbon receptor signal, enhance intestinal barrier function and inhibit inflammation. Ginger-derived extracellular vesicles containing miR159a-3p inhibit bacterial phospholipase C activity, promote docosahexaenoic acid production and downregulate the expression of programmed cell death ligand 1, thereby enhancing anti-inflammatory effects.

[0004] Lemon as a common fruit rich in bioactive components such as vitamin C, flavonoids, and polyphenols, has a wide source and relatively low cost, and has good development potential. Lemon-derived vesicles are composed of bioactive substances such as pectin, polysaccharides, lipids, and phytochemicals, and are increasingly recognized as multifunctional therapeutic agents. However, there is no reported application of lemon-derived extracellular vesicles in the field of probiotic embedding.

[0005] In view of this, the present application is proposed. SUMMARY

[0006] The present application aims to at least solve one of the technical problems existing in the prior art. The present application uses lemon vesicles to construct a probiotic embedding system. The lemon vesicles used have good biocompatibility and biological activity, and have natural acid resistance and stability under low pH conditions, which can effectively encapsulate probiotics and be administered orally to improve the survival rate of probiotics. This technology is universal and can be applied to the development and utilization of various probiotic oral drugs.

[0007] The first aspect of the present application aims to provide a lemon vesicle-embedded active probiotic.

[0008] The second aspect of the present application aims to provide a method for preparing the lemon vesicle-embedded active probiotic of the first aspect of the present application.

[0009] The third aspect of the present application aims to provide the use of the lemon vesicle-embedded active probiotic of the first aspect of the present application in the preparation of a probiotic product.

[0010] The fourth aspect of the present application aims to provide a probiotic product.

[0011] The present application aims to develop a new probiotic delivery platform by taking advantage of the dual prebiotic and therapeutic properties of lemon vesicles. This platform aims to achieve an efficient and stable passage of probiotics and other oral formulations through the gastric barrier and long-term retention in intestinal target sites, thereby significantly improving the therapeutic effect of bioactive substances on intestinal inflammation or other intestinal diseases and the intestinal health care effect.

[0012] To achieve the above-mentioned purpose, the technical solution adopted by the present application is: Lemon vesicles (LVs) are multifunctional therapeutic agents composed of bioactive substances such as pectin, polysaccharides, lipids, and phytochemicals. According to the literature, LV-derived pectin promotes the production of short-chain fatty acids (SCFAs), particularly butyrate, which plays a crucial role in strengthening intestinal barrier integrity. LVs also exhibit antioxidant and anti-inflammatory properties by modulating the ERK / NF-κB and AhR / Nrf2 pathways, thereby contributing to intestinal homeostasis and thus having a good therapeutic and health-promoting effect in the intestine. Second, the abundant pectin in lemon vesicles has natural acid resistance and stability under low pH conditions, can resist bile acids and gastric acid, and can effectively encapsulate probiotics to promote the safe arrival of probiotics to the intestinal site and oral administration. Although other plant vesicles also have great potential in biological activity, they also face the same severe situation as probiotics, and they may also be destroyed in the harsh gastrointestinal tract. Therefore, whether other vesicles can produce the same effect or have the ability to resist bile acids, gastric acid, and the like needs to be further studied. The protective effect of lemon vesicles on probiotics is a very key technology in the present application. The present application first combines naturally derived lemon vesicles with probiotics to construct a "natural protective layer- active therapeutic unit" complex system, which is innovative in terms of technology and application. The purpose is to develop a new synergistic drug delivery platform by taking advantage of the dual prebiotic and therapeutic and probiotic protective properties of LVs.

[0013] In a first aspect of the present application, a lemon vesicle-embedded active probiotic is provided, comprising a probiotic, and a lemon vesicle coated on the surface of the probiotic, wherein the lemon vesicle is an extracellular vesicle derived from lemon.

[0014] In some embodiments of the present application, the probiotic is selected from at least one of the microorganisms of Escherichia coli Nissle 1917 (EcN), Escherichia coli Lactiplantibacillus ( Lactiplantibacillus ), Lactobacillus ( Lactobacillus ), Lactococcus ( Lacticaseibacillus ), Streptococcus ( Limosilactobacillus ), and Bifidobacterium ( Streptococcus ).

[0015] In some embodiments of the present application, the probiotic of Lactiplantibacillus includes Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ).

[0016] In some embodiments of the present application, the probiotic of Lactobacillus is selected from Lactobacillus helveticus ( Lactobacillus helveticus ), Lactobacillus acidophilus ( Lactobacillus acidophilus ), Lactobacillus delbrueckii subsp. bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus ), and Lactobacillus delbrueckii subsp. lactis ( Lactobacillus delbrueckii subsp. Lactisat least one of Lactobacillus gasseri (Lactobacillus gasseri), Lactobacillus gasseri Lactobacillus crispatus (Lactobacillus crispatus), Lactobacillus crispatus Lactobacillus salivarius (Lactobacillus salivarius), Lactobacillus salivarius Lactobacillus johnsonii (Lactobacillus johnsonii), Lactobacillus johnsonii Lactobacillus kefiranofaciens subsp. kefiranofaciens (Lactobacillus kefiranofaciens subsp. kefiranofaciens), Lactobacillus kefiranofaciens subsp. kefiranofaciens

[0017] In some embodiments of the present application, the probiotic bacteria of the genus Lactobacillus are selected from at least one of Lactobacillus casei (Lactobacillus casei), Lactobacillus casei Lactobacillus paracasei (Lactobacillus paracasei) (i.e. Lactobacillus paracasei), Lacticaseibacillus paracasei Lactobacillus rhamnosus (Lactobacillus rhamnosus) (i.e. Lactobacillus rhamnosus). Lacticaseibacillus rhamnosus

[0018] In some embodiments of the present application, the probiotic bacteria of the genus Mucor are selected from at least one of Mucor ferax (Mucor ferax) (i.e. Mucor lactis) and Mucor rileyi (Mucor rileyi) (i.e. Mucor lactis). Limosilactobacillus fermentum Limosilactobacillus reuteri

[0019] In some embodiments of the present application, the probiotic bacteria of the genus Streptococcus is Streptococcus salivarius subsp. thermophilus (Streptococcus salivarius subsp. thermophilus), i.e. Streptococcus thermophilus (Streptococcus thermophilus). Streptococcus salivarius subsp. thermophilus Streptococcus thermophilus

[0020] In some embodiments of the present application, the particle size of the lemon vesicles is 150-1500 nm.

[0021] In some embodiments of the present application, the lemon vesicles are prepared by a preparation method comprising: juicing a peeled and cored lemon, gradient centrifugation, i.e. obtaining lemon vesicles.

[0022] In some preferred embodiments of the present application, the lemon is a fresh lemon.

[0023] The natural lemon-derived exovesicles contain abundant citric acid, which has good antioxidant and anti-inflammatory biological activity on the one hand, and can resist bile acids and remain stable in an acidic environment on the other hand. Therefore, the natural lemon-derived exovesicles can protect the active probiotic bacteria from degradation by gastric acid and bile, improve the survival rate and colonization rate of the active probiotic bacteria in the intestinal tract after passing through the stomach, promote the active probiotic bacteria to regulate the balance of the intestinal microecology, and the natural active ingredients of the lemon vesicles can synergistically reduce the inflammatory factors in the intestinal tract.

[0024] In some preferred embodiments of the present application, the gradient centrifugation comprises high-speed centrifugation 2-4 times and ultrahigh-speed centrifugation 1-3 times.

[0025] ​​​​​​In some preferred embodiments of the present invention, the conditions for high-speed centrifugation are: 1000 × g - 6000 × g for 10-90 minutes; further, 1000 × g - 5000 × g for 10-60 minutes; and even further, 1000 × g - 4000 × g for 10-30 minutes.

[0026] In some preferred embodiments of the present invention, the ultra-high speed centrifugation conditions are: 10000 × g - 20000 × g for 40-90 minutes; further, 10000 × g - 15000 × g for 40-70 minutes; and even further, 10000 × g - 13000 × g for 50-70 minutes.

[0027] Lemon vesicles are composed of bioactive substances such as pectin, polysaccharides, lipids, and phytochemicals, possessing bioactivities such as antioxidant, anti-inflammatory, metabolic and gut health regulation, and potential immunomodulatory effects. The pectin in lemon vesicles exhibits natural acid resistance and stability under low pH conditions, effectively encapsulating probiotics for oral administration. This encapsulation system effectively encapsulates probiotics, protecting them from the adverse effects of the gastrointestinal environment, enhancing their stability, and ensuring their safe arrival at the intestinal tract. Simultaneously, it synergistically promotes gut health. Compared to other encapsulation technologies and materials, lemon vesicles possess significant application potential and prospects in terms of intrinsic function, natural source, and economic cost.

[0028] In some embodiments of the present invention, the particle size of the lemon vesicle-encapsulated active probiotics is 1000-5000 nm.

[0029] In a second aspect, the present invention provides a method for preparing lemon vesicle-encapsulated active probiotics, comprising the following steps: ultrasonically treating lemon vesicles and mixing them with probiotics, followed by multiple physical compressions to obtain lemon vesicle-encapsulated active probiotics.

[0030] In some embodiments of the present invention, the conditions for ultrasonic treatment are: ultrasonic treatment at a power of 10-40W for 2-15 minutes; further, ultrasonic treatment at a power of 10-30W for 2-10 minutes; and even further, ultrasonic treatment at a power of 10-20W for 2-5 minutes.

[0031] In some embodiments of the present invention, based on the protein concentration in the citric acid (quantitatively detected by a BCA protein kit), each 1 mg / mL protein mixture contains (0.1-10) × 10 10 CFU probiotics; further mix (1-10) × 10 per 1 mg / mL protein. 9 CFU probiotics; further, mix 10 (2-6) times per 1 mg / mL protein. 9 CFU probiotics.

[0032] In some embodiments of the present invention, the multiple physical compressions are 10-50 physical compressions; further, 20-40 physical compressions; and even further, 20-30 physical compressions.

[0033] In some embodiments of the invention, the physical extrusion includes extrusion using a micro-extruder, such as extrusion through a polycarbonate porous membrane (pore size 1-5 μm).

[0034] In some embodiments of the invention, the method further includes a purification step to remove unbound lemon vesicles. This includes removing unbound lemon vesicles by centrifugation. The centrifugation conditions are 4000 × g to 6000 × g for 3-6 minutes.

[0035] In some embodiments of the present invention, the probiotics are cultured before mixing, including culturing the probiotics in a culture medium.

[0036] In some preferred embodiments of the present invention, the culture medium and culture conditions are adjusted according to the growth characteristics of the probiotic species. For example, when Escherichia coli Nissle 1917 is selected as the probiotic, it is cultured in LB medium at 35-39°C for 6-12 hours; When using Lactobacillus plantarum as the probiotic, culture it in MRS medium at 30-37℃ for 12-24 hours. When using Streptococcus thermophilus as the probiotic, use M17 medium and incubate at 40-42℃ for 8-24 hours. When Lactobacillus paracasei is selected as the probiotic, it should be cultured in MRS medium at 30-37℃ for 16-24 hours. When Lactobacillus rhamnosus is selected as the probiotic, it is cultured on MRS medium at 37°C for 16-30 hours. When using Lactobacillus helveticus as the probiotic, culture it in MRS medium at 37-40℃ for 18-36 hours. When using Lactobacillus fermentum as the probiotic, culture it in MRS medium at 35-37℃ for 12-24 hours. When using Lactobacillus reuteri as a probiotic, culture it in MRS medium at 37°C for 24-48 hours. When using Lactobacillus acidophilus as the probiotic, culture it in MRS medium at 35-37℃ for 18-36 hours.

[0037] This invention utilizes lemon vesicles as a protective shell for probiotics. These vesicles are mixed with active probiotics, and the mixture is then extruded using a micro-extruder. The extrusion method uses mechanical force to coat the surface of the active probiotics with the vesicle membrane, thus obtaining a technique for preparing lemon vesicle-encapsulated active probiotics. Specifically, lemon vesicles are mixed with active probiotics and repeatedly extruded through a micron-sized porous membrane, ultimately coating the surface of the active probiotics with the vesicle membrane. This protects the active probiotics in the acidic and bile environment of the stomach, allowing them to function properly in the intestines.

[0038] A third aspect of the present invention provides the application of the lemon vesicle encapsulation method of the first aspect of the present invention or the preparation method of the second aspect of the present invention in the preparation of probiotic products.

[0039] In some embodiments of the present invention, the probiotic products include pharmaceuticals, food, and health food.

[0040] In some embodiments of the present invention, the probiotic product may have any of the following functions: helping to enhance immunity, helping to resist oxidation, helping to improve memory, helping to improve sleep, weight loss, improving growth and development, regulating intestinal flora, promoting digestion, relieving constipation, having an auxiliary protective function against gastric mucosal damage, maintaining healthy blood lipid (cholesterol / triglycerides) levels, maintaining healthy blood sugar levels, anti-inflammatory, and anti-cancer.

[0041] A fourth aspect of the present invention provides a probiotic product comprising lemon vesicle-encapsulated active probiotics according to the first aspect of the present invention.

[0042] In some embodiments of the present invention, the probiotic product further includes one or more pharmaceutically or food-acceptable excipients.

[0043] In some embodiments of the invention, the pharmaceutically or food-acceptable excipient is selected from pharmaceutically or food-acceptable excipients, diluents, or carriers. Pharmaceutically or food-acceptable excipients, diluents, or carriers are well known in the pharmaceutical or food industries. Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, or sorbitol. Examples of suitable diluents include ethanol, glycerol, and water. The choice of drug carrier, excipient, or diluent can be based on the intended route of administration and standard pharmaceutical practice. The composition may contain any suitable binder, lubricant, suspending agent, coating agent, or solubilizer, or other than a carrier, excipient, or diluent. Examples of suitable binders include starch, gelatin, natural sugars, and natural or synthetic gums. Natural sugars include, for example, glucose, anhydrous lactose, free-flowing lactose, β-lactose, or corn sweeteners. Natural or synthetic gums include, for example, gum arabic, astragalus gum, sodium alginate, carboxymethyl cellulose, or polyethylene glycol. Suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, or sodium chloride. Preservatives, stabilizers, dyes, and even flavorings may be provided in the composition. Examples of preservatives include sodium benzoate, sorbic acid, or parabens. Antioxidants and suspending agents may also be used.

[0044] In some embodiments of the present invention, the probiotic product further includes prebiotics selected from at least one of inulin, mulberry leaf extract, berberine, Ganoderma lucidum, green coffee bean extract, oats, pectin, potato or its extract, citrus polyphenols, Ceylon cinnamon, chromium, ergothioneine, astaxanthin, quercetin, curcumin, proanthocyanidins, resistant dextrin, yeast β-glucan, ginseng or its extract, nutrient compounds, biotin, polydextrose, fructooligosaccharides, galactooligosaccharides, starch, cellulose, β-glucan, hemicellulose, lactulose, mannan oligosaccharides, mannan oligosaccharides, inulin rich in fructooligosaccharides, oligodextrose, tagatose, trans-galactooligosaccharides, pectin, resistant starch, or xylooligosaccharides.

[0045] The beneficial effects of this invention are: This invention utilizes lemon vesicles to encapsulate active probiotics into single cells, enabling them to resist degradation by gastric acid and bile. This improves the survival and colonization rate of active probiotics after passing through the stomach into the intestines, promoting the efficient functioning of active probiotics in regulating the balance of the intestinal microecology. Simultaneously, the natural active ingredients in lemon vesicles can synergistically reduce inflammatory factors in the intestinal tract. This lemon vesicle provides a novel probiotic delivery technology, with a synergistic mechanism protecting the delivery of probiotics and active ingredients. It offers new solutions for intestinal health and the treatment of related diseases, and brings new development strategies to the future oral probiotic industry.

[0046] The lemon vesicles used in this invention are derived from ordinary fresh lemons, which are readily available, economical, and easy to industrialize due to their simple processing. They serve as a protectant for active probiotics, while the natural active substances further enhance the health or therapeutic effects in the intestinal tract. This technology is innovative, practical, and economical, and therefore has great application prospects and can be applied to the development of various probiotic products. Attached Figure Description

[0047] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This image shows the morphological structure and particle size distribution of lemon vesicles under an electron microscope.

[0048] Figure 2 Morphological structures of active probiotics embedded in lemon vesicles under TEM and AFM electron microscopy.

[0049] Figure 3 Particle size distribution (1) and potential diagram (2) of lemon vesicles encapsulating active probiotics.

[0050] Figure 4 The stability of lemon vesicles encapsulated with active probiotics on the surface of active probiotics in 4 ℃ PBS solution (1), 4 ℃ LB medium (2), 37 ℃ LB medium (3), and 37 ℃ PBS solution (4).

[0051] Figure 5 OD values ​​of probiotics after encapsulation of active probiotics in lemon vesicles in LB medium (1), viable colony count in simulated gastric juice solution (2), viable colony count in bile solution (3), OD values ​​in simulated intestinal juice solution (4), OD values ​​in 400 μM hydrogen peroxide solution (simulated inflammation) (5), viable colony count in 1 mM hydrogen peroxide solution (simulated severe inflammation) (6).

[0052] Figure 6 The anti-inflammatory effect of lemon vesicles on macrophages after treatment with lipopolysaccharide; where (1) is the ROS level, (2) is the nitric oxide level, and (3) is the macrophage polarization level.

[0053] Figure 7 To improve the retention capacity of probiotics in the intestinal tract of mice by encapsulating active probiotics in lemon vesicles; among them, (1) is an intestinal imaging image 4 hours after oral administration, (2) is a fluorescence quantitative image of the small intestine 4 hours after oral administration, (3) is a slice image of the intestine, cecum and colon 4 hours after oral administration, and (4) is a quantitative image of the number of colonies in the intestine, cecum and colon 4 hours after oral administration. Detailed Implementation

[0054] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0055] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0056] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0057] Example 1 This embodiment prepares a lemon vesicle-encapsulated live probiotic, and the preparation method specifically includes the following steps: (1) Preparation of lemon vesicles: Take three fresh lemons, peel and remove the seeds, cut the lemon pulp into pieces and juice them. Centrifuge the lemon suspension at 1000×g for 10 minutes at 4℃ and collect the supernatant. Centrifuge at 2000×g for 20 minutes, collect the supernatant and centrifuge at 4000×g for 30 minutes. Centrifuge at 10000×g for 60 minutes for the fourth time and collect the precipitate, which is the lemon vesicle (LV). Transfer the precipitate to frozen PBS solution for resuspending and sonicate it at 17W for 5 minutes before use.

[0058] (2) Preparation of live probiotics: Escherichia coli Nissle 1917 (ECN) was cultured in LB medium at 37°C for 12 hours, centrifuged at 5000×g for 5 minutes, and then resuspended in ice-cold PBS.

[0059] (3) Preparation of live probiotics encapsulated in lemon vesicles: Take 1 mL of 1×10 9 CFU / mL E. coli Nissle 1917 was mixed with 0.5 mL of lemon vesicles (protein concentration of 0.4 mg / mL, determined by a BCA protein kit (Shanghai Beyotime Biotechnology Co., Ltd., catalog number P0012S)). After mixing, the mixture was squeezed 40 times through a 1 μm polycarbonate porous membrane using a micro-squeegee. Unbound excess lemon vesicles were centrifuged at 5000×g for 5 minutes to remove the supernatant. The precipitate was the lemon vesicle-encapsulated live probiotic (lemon vesicle-encapsulated E. coli Nissle 1917 system, denoted as E@LV).

[0060] Example 2 This embodiment prepares a lemon vesicle-encapsulated live probiotic, and the preparation method specifically includes the following steps: (1) Preparation of lemon vesicles: Take three fresh lemons, peel and remove the seeds, cut the lemon pulp into pieces and juice them. Centrifuge the lemon suspension at 1000×g for 10 minutes at 4℃ and collect the supernatant. Centrifuge at 2000×g for 20 minutes, collect the supernatant and centrifuge at 4000×g for 30 minutes. Centrifuge at 10000×g for 60 minutes for the fourth time and collect the precipitate, which is the lemon vesicle (LV). Transfer the precipitate to frozen PBS solution for resuspending and sonicate it at 17W for 5 minutes before use.

[0061] (2) Preparation of live probiotics: Escherichia coli Nissle 1917 (ECN) was cultured in LB medium at 37°C for 12 hours, centrifuged at 5000×g for 5 minutes, and then resuspended in ice-cold PBS.

[0062] (3) Preparation of live probiotics encapsulated in lemon vesicles: Take 1 mL of 1×10 9 CFU / mL E. coli Nissle 1917 was mixed with 1 mL (protein concentration 0.4 mg / mL) of lemon vesicles. After mixing, the mixture was squeezed 40 times through a 1 μm polycarbonate porous membrane using a micro-squeegee. The unbound excess lemon vesicles were centrifuged at 5000×g for 5 minutes to remove the supernatant. The precipitate was the lemon vesicle-encapsulated live probiotic (the lemon vesicle-encapsulated E. coli Nissle 1917 system, denoted as E@LV2).

[0063] Example 3 This embodiment prepares a lemon vesicle-encapsulated live probiotic, and the preparation method specifically includes the following steps: (1) Preparation of lemon vesicles: Take three fresh lemons, peel and remove the seeds, cut the lemon pulp into pieces and juice them. Centrifuge the lemon suspension at 1000×g for 10 minutes at 4℃ and collect the supernatant. Centrifuge at 2000×g for 10 minutes, collect the supernatant and centrifuge at 3000×g for 10 minutes. Centrifuge at 5000×g for 20 minutes for the fourth time and collect the supernatant. Finally, centrifuge at 15000×g for 40 minutes to collect the precipitate, which is the lemon vesicle (LV). Transfer the precipitate to frozen PBS solution for resuspension and sonicate it at 20W for 3 minutes before use.

[0064] (2) Preparation of live probiotics: Escherichia coli Nissle 1917 (ECN) was cultured in LB medium at 37°C for 12 hours, centrifuged at 5000×g for 5 minutes, and then resuspended in ice-cold PBS.

[0065] (3) Preparation of live probiotics encapsulated in lemon vesicles: Take 1 mL of 1×10 9 CFU / mL E. coli Nissle 1917 was mixed with 0.5 mL of lemon vesicles (protein concentration 0.4 mg / mL). The mixture was then squeezed 20 times through a 1 μm polycarbonate porous membrane using a micro-squeegee. Unbound excess lemon vesicles were centrifuged at 5000×g for 5 minutes to remove the supernatant. The precipitate was the lemon vesicle-encapsulated live probiotic (the lemon vesicle-encapsulated E. coli Nissle 1917 system, denoted as E@LV3).

[0066] Example 4: Identification of lemon vesicles The morphology and particle size distribution of lemon vesicles extracted in Example 1 were detected using a transmission scanning electron microscope and a particle size analyzer.

[0067] The morphological examination procedure for transmission scanning electron microscopy is as follows: Place the sealing film flat on a culture dish, and then place a copper mesh or ordinary carbon support film grid on top of the sealing film. Use a pipette to add 8-10 µL of lemon vesicle solution onto the grid, and time for 10 minutes. Gently blot the liquid dry with filter paper. Next, use a pipette to add 10 µL of phosphotungstic acid onto the grid, and time for approximately 2 minutes. Blot away excess staining with filter paper, and wait 10-20 minutes for the grid to dry before observation under the microscope.

[0068] The particle size distribution detection procedure is as follows: Take 1 mL of 0.1 mg / mL lemon vesicle solution into a particle size distribution dish for particle size analysis.

[0069] The results are as follows Figure 1 As shown, natural lemon vesicles are spherical with a distinct phospholipid bilayer structure, a diameter of approximately 150-1500 nm, and an average particle size of 500 nm.

[0070] Example 5: Morphological identification of probiotics embedded in lemon vesicles The morphology of the lemon vesicle-embedded probiotics prepared in Example 1 was examined using a transmission scanning electron microscope and an atomic force microscope.

[0071] The morphological examination procedure for transmission scanning electron microscopy is as follows: Cells are collected by centrifugation, fixed with 2.5% glutaraldehyde for 10 minutes, and then dehydrated using gradient concentrations of ethanol (25%, 50%, 75%, 90%, and anhydrous ethanol), with each concentration reacting for 10 minutes. A sealing film is laid flat on a culture dish, and a copper mesh or ordinary carbon support membrane is placed on top of the sealing film. 8-10 µL of cell solution is pipetted onto the support membrane, and the reaction time is 10 minutes. The liquid is gently blotted dry with filter paper, and the support membrane is ready for microscopic observation after drying.

[0072] The steps for morphological detection using atomic force microscopy are as follows: collect cells by centrifugation, fix with 2.5% glutaraldehyde for 10 minutes, drop the solution onto a silicon wafer, let it stand for 10 minutes, dry it with nitrogen gas, and then perform the detection.

[0073] The results are as follows Figure 2 As shown, transmission scanning electron microscopy revealed a distinct outer membrane around EcN in the EcN@LV group, while atomic force microscopy showed an increase in surface roughness of EcN after LV coating, indicating that the lemon vesicles successfully encapsulated probiotics.

[0074] Example 6: Identification of the size and potential of probiotics encapsulated in lemon vesicles The particle size and potential distribution of the lemon vesicle-embedded probiotics prepared in Examples 1 and 2 were detected using a particle size and potential analyzer.

[0075] The particle size distribution detection procedure is as follows: Take 1 mL of 0.1 mg / mL lemon vesicle-embedded probiotic solution into a particle size distribution dish for particle size analysis.

[0076] The potential distribution detection procedure is as follows: Take 1 mL of 0.1 mg / mL lemon vesicle-embedded probiotic solution into a potentiometric dish for potential detection.

[0077] The results are as follows Figure 3 As shown, the sizes of E@LV and E@LV2 increase with the increase of LV encapsulation concentration, and the potentials of E@LV and E@LV2 also increase relative to EcN.

[0078] Example 7: Study on the encapsulation stability of lemon vesicles on the surface of probiotics The E@LV and E@LV2 prepared in Examples 1 and 2 were stored under different conditions, including 4°C PBS solution, 4°C LB medium, 37°C LB medium, and 37°C PBS solution. During storage, the content of DiD dye-labeled lemon vesicles on the surface of the probiotics was detected by flow cytometry over time under different conditions.

[0079] The detection method involved incubating lemon vesicles with DiD cell membrane dye at 37°C in the dark for 30 minutes, washing with PBS, and then re-separating and washing away excess DiD dye by ultracentrifugation at 3000 Da. EcN expressing green fluorescent protein (GFP) was washed twice with ice-cold PBS, resuspended in PBS, and the GFP-expressing probiotics and DiD-stained lemon vesicles were mixed according to the ratios in Examples 1 and 2, and extruded 40 times using a micro-extruder. Subsequently, the unbound lemon vesicles were centrifuged at 5000 × g for 5 minutes.

[0080] E@LV and E@LV2 labeled with dye and fluorescence were stored under the different conditions described above, and the fluorescence intensity of DiD-labeled lemon vesicles on the APC fluorescence channel was then detected by flow cytometry.

[0081] The results are as follows Figure 4 As shown, APC fluorescence confirmed that E@LV remained stable for up to 72 hours when stored in PBS at 4°C. Figure 4 (1)); Conversely, in LB at 4°C, the LV (DiD staining, APC fluorescence) on the EcN surface gradually decreased over time, reflecting the slow growth of probiotics in LB at 4°C ( Figure 4 (2) When the temperature of LB rises to 37°C (the optimal growth condition for bacteria), LV rapidly detaches within 8 hours, indicating that EcN proliferation is unaffected and that the LV coating can detach in the intestinal environment after localization. Figure 4 (3)); In PBS at 37°C, the LV coating gradually detached over time, possibly due to slight growth of probiotics in the LV in the PBS medium ( Figure 4 (4)

[0082] Example 8: Protective effect of lemon vesicle-encapsulated active probiotic system on probiotics under different conditions The growth curves of E@LV and E@LV2 prepared in Examples 1 and 2 were detected under different conditions (including LB medium, simulated intestinal fluid, and 400 μM hydrogen peroxide solution (simulating a mild inflammatory environment)) and the number of viable colonies were detected in simulated gastric juice solution, bile solution and 1 mM hydrogen peroxide solution (simulating a severe inflammatory environment).

[0083] Simulated intestinal fluid, namely artificial small intestinal fluid (enzyme-free and sterile), was purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number R24022-500mL. Its main components include phosphate, trypsin and bile salts, pH=6.8. The simulated gastric juice, also known as artificial gastric juice (USP, sterile), was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with the product number R22155-500mL. Its main components are dilute hydrochloric acid, sodium chloride, and pepsin, with a pH of 2.5. The bile solution, also known as artificial bile, was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with product number R28028-500mL. Its main components include sodium chloride, sodium bicarbonate, potassium chloride, urea, calcium chloride, BSA, and bile salts, with a pH of 8.1.

[0084] The steps of the detection method are as follows: (1) 10 6CFU / mL EcN, E@LV and E@LV2 were cultured in LB medium for 0.5, 1, 2, 4 and 8 hours, respectively. Then, the absorbance of the EcN, E@LV and E@LV2 groups was detected at 600 nm using a microplate reader and growth curves were plotted.

[0085] (2) Place 10 7 CFU / mL EcN, E@LV, and E@LV2 were placed in simulated gastric fluid (SGF, pH=2.5) and incubated at 37 ℃ for 0.5, 1, 2, and 4 hours, respectively. After incubation, the test samples were serially diluted, usually 10-fold, to ensure that the final colony count was between 30 and 300. 100 μL of each dilution was inoculated onto solid culture medium using the spread plate method and then placed in an incubator for 24 hours. After obvious colony growth on the culture medium, the colonies were counted.

[0086] (3) Place 10 7 CFU / mL EcN, E@LV, and E@LV2 were incubated in bile solution at 37 °C for 0, 0.5, 1, 2, and 4 hours, respectively. After incubation, the test samples were serially diluted, usually 10-fold, to ensure that the final colony count was between 30 and 300. 100 μL of each dilution was inoculated onto solid culture medium using the spread plate method and then incubated in an incubator for 24 hours. After obvious colony growth on the culture medium, the colonies were counted.

[0087] (4) Place 10 7 CFU / mL EcN, E@LV, and E@LV2 were placed in simulated intestinal fluid (pH=6.8) and cultured at 37 ℃ for 0, 1, 2, 4, 8, and 12 hours, respectively. The absorbance of the EcN, E@LV, and E@LV2 groups was detected at 600 nm using a microplate reader and growth curves were plotted.

[0088] (5) Place 10 7 CFU / mL EcN, E@LV, and E@LV2 were placed in 400 μM hydrogen peroxide solution (simulating a mild inflammatory environment) and cultured at 37 °C for 0, 1, 2, 4, 8, 12, and 18 hours. The absorbance of the EcN, E@LV, and E@LV2 groups was measured at 600 nm using a microplate reader and growth curves were plotted.

[0089] (6) Place 10 7CFU / mL EcN, E@LV, and E@LV2 were incubated in 1mM hydrogen peroxide solution (simulating a severe inflammatory environment) at 37°C for 0, 1, 2, 4, and 8 hours, respectively. After incubation, the test samples were serially diluted, usually 10-fold, to ensure that the final colony count was between 30 and 300. 100 μL of each dilution was inoculated onto solid culture medium using the spread plate method and then incubated in an incubator for 24 hours. After obvious colony growth on the culture medium, the colonies were counted.

[0090] The results are as follows Figure 5 As shown, through OD 600 The monitored growth curves demonstrated that the encapsulation of EcN by LV did not affect the growth kinetics of EcN. Figure 5 (1) LV-encapsulated EcN showed a survival rate more than 10 times higher than that of the original EcN in simulated gastric fluid, attributed to pectin-mediated acid resistance ( Figure 5 (2)); Subsequent bile acid reactions showed that the activity of LV-embedded EcN was significantly improved compared to EcN, indicating that LV has a strong protective function against acid and bile acid EcN ( Figure 5 (3)); As the number of EcNs surviving under harsh conditions slowly increased, engineered probiotics showed comparable proliferation to EcNs in simulated intestinal fluid ( Figure 5 (4) In a simulated inflammatory environment (400 μM H2O2), LV-encapsulated EcN showed significantly enhanced proliferation, while unmodified EcN showed growth arrest (Figure (5)); In a simulated severe inflammatory environment (1 mM H2O2), LV-encapsulated probiotics improved probiotic survival. The above results indicate that the LV coating has an acid / bile acid shielding effect during gastric transport and protects probiotics from continuous proliferation in the inflamed gut.

[0091] Example 9: Anti-inflammatory effect of lemon vesicles on LPS-induced macrophages Following intestinal localization and subsequent LV embedding coating removal, this invention investigated the residual bioactivity of the removed LV components in the colitis microenvironment, such as anti-inflammatory effects at sites of inflammation, including regulation of reactive oxygen species levels, nitric oxide (NO) levels, and polarization regulation of macrophages in the intestinal microenvironment.

[0092] The detection method is as follows: macrophages are prepared at a concentration of 5 × 10⁻⁶. 5The culture medium was seeded into 24-well plates and incubated overnight at 37°C. After adhesion, the culture medium in each well was replaced with DMEM, DMEM with 1 μg / mL LPS, DMEM with 1 μg / mL LPS + 10 μg / mL LV (calculated based on protein concentration), and DMEM with 1 μg / mL LPS + 20 μg / mL LV, respectively. The cells were then incubated at 37°C for another 24 hours. After incubation, the cells were washed with PBS and stained with a 10 μM ROS detection fluorescent probe (DCFHDA) to detect ROS levels. The ROS content was then measured by flow cytometry.

[0093] Macrophages were 5 × 10 5 The culture medium was seeded into 24-well plates and incubated overnight at 37°C. After adhesion, the culture medium in each well was replaced with DMEM, DMEM with 1 μg / mL LPS, DMEM with 1 μg / mL LPS + 10 μg / mL LV (calculated based on protein concentration), and DMEM with 1 μg / mL LPS + 20 μg / mL LV, respectively. The plates were then incubated at 37°C for another 24 hours. After incubation, the culture medium from each well was collected for NO detection. Nitrate concentration was measured using a total nitric oxide assay kit at 540 nm using a microplate reader.

[0094] Macrophages were 5 × 10 5 The cells were seeded into 24-well plates at a concentration of [missing information - likely a specific concentration], and the plates were incubated overnight at 37°C. After adhesion, the culture medium in the wells was replaced with DMEM, DMEM with 1 μg / mL LPS, DMEM with 1 μg / mL LPS + 10 μg / mL LV, and DMEM with 1 μg / mL LPS + 20 μg / mL LV, respectively. The cells were then cultured for another 24 hours at 37°C. Cells were collected for macrophage polarization analysis. Macrophage polarization induced by inflammation was assessed by detecting markers of M1 macrophages. CD86, a marker of M1 macrophages (pro-inflammatory macrophages), was used. Macrophages were stained with CD86 antibody, and inflammatory M1 macrophages were quantified using flow cytometry.

[0095] The results are as follows Figure 6 As shown, LPS stimulation induced high levels of ROS, while the addition of LV significantly reduced ROS levels by 50%. Figure 6(1)); Consistent with the trend of ROS levels, NO levels were also induced to release at high levels under LPS stimulation, while the addition of LV significantly inhibited NO content, further confirming the anti-inflammatory performance of LV ( Figure 6 (2)); The polarization of macrophages caused by inflammation was detected by detecting CD86, a marker of M1 macrophages. The data showed that when cells were co-cultured with LV, the level of CD86 markers decreased compared with LPS stimulation alone. Figure 6 (3)

[0096] Example 10: The retention capacity of lemon vesicle-encapsulated active probiotic system in the intestine. This embodiment verifies the retention capacity of EcN, E@LV and E@LV2 prepared in Examples 1 and 2 in the mouse intestine, and observes the retention and probiotic colonization in the mouse intestine using an in vivo imaging instrument.

[0097] The detection method was as follows: Three groups of BALB / c male mice (n=3 per group) were used, and they were fasted for 24 hours before the experiment. EcN transfected with mCherry (red fluorescent protein) was administered orally on the day of the experiment, along with E@LV and E@LV2 (EcN content was 2 × 10⁻⁶). 8 The mice were euthanized 4 hours later, and their complete digestive tract was dissected and fluorescently imaged using a live imaging instrument.

[0098] In addition, the small intestine, cecum, and colon were separated and washed with PBS to remove internal components. The jejunum was embedded in Tissue-Tek®_OCT gel (Sakura, catalog number 4583) and fixed overnight at -80°C for use in cryopreservation slides. The slides were stained with DAPI (anti-fading solution), covered with coverslips, and then imaged with DMI8.

[0099] Meanwhile, portions of the small intestine, cecum, colon, and jejunum separated from each group were ground. The grinding solution was filtered through a 40μm filter and then subjected to a series of serial dilutions, usually 10-fold serial dilutions, to ensure that the final colony count was between 30 and 300. 100μL of each dilution was taken and inoculated onto a solid culture medium using the spread plate method. The medium was then placed in an incubator and incubated for 24 hours. After obvious colonies grew on the culture medium, the colonies were counted.

[0100] The results are as follows Figure 7 As shown, E@LV and E@LV2 exhibited stronger fluorescence values ​​in the intestine than the EcN group, indicating that LV has a good protective ability against EcN, allowing EcN to reach the intestinal site effectively. Figure 7In (1) and (2)); E@LV and E@LV2 in frozen sections of the small intestine, cecum and colon showed significant probiotic colonization effects. The red fluorescence was EcN transfected with mCherry (red fluorescent protein), and the blue fluorescence was DAPI-stained tissue cells. Figure 7 (3)); Plate count further quantified the survival of EcNs. Data showed that the number of LV-encapsulated EcNs delivered to the small intestine increased by 3-4 times compared to untreated EcNs. Similarly, the cecum and colon regions showed enhanced colonization of LV-protected EcNs (E@LV and E@LV2). The colonic colonization level of EcNs in E@LV2 was 2.5 times higher than that in the unmodified EcN group, indicating improved therapeutic potential for colitis.

[0101] The above experimental examples demonstrate that the lemon vesicles prepared in this invention, which encapsulate probiotics, do not affect the survival rate and proliferation of the probiotics. In simulated gastric juice, intestinal juice, bile, and simulated inflammatory environments, their stability is significantly superior to that of unencapsulated probiotics. This allows the probiotics to tolerate the harsh gastrointestinal environment, improving their survival rate upon reaching the intestines and promoting colonization. The detached LVs after reaching the intestinal site further promote intestinal repair by alleviating inflammation and promoting epithelial regeneration. From a therapeutic perspective, this cross-species symbiotic system utilizes the inherent biocompatibility of natural biomaterials and microorganisms to enhance targeted anti-inflammatory efficacy and restore microbial homeostasis. By simultaneously alleviating colitis symptoms and addressing its root cause, this strategy achieves synergistic therapeutic effects. This system surpasses traditional probiotic drug delivery methods, bringing enormous potential to the probiotic formulation and intestinal health or treatment market.

[0102] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. A lemon vesicle-encapsulated active probiotic, comprising probiotics and lemon vesicles coated on the surface of the probiotics, wherein the lemon vesicles are extracellular vesicles derived from lemons.

2. The lemon vesicle-encapsulated active probiotics according to claim 1, characterized in that, The probiotics were selected from Escherichia coli Nissle 1917 ( Escherichia coli Nissle 1917), Lactobacillus ( Lactiplantibacillus Lactobacillus ( ) Lactobacillus Lactobacillus ( ) Lacticaseibacillus ), Lactobacillus spp. Limosilactobacillus Streptococcus ( Streptococcus At least one of the microorganisms.

3. The lemon vesicle-encapsulated active probiotics according to claim 1 or 2, characterized in that, The lemon vesicles have a particle size of 150-1500 nm.

4. The lemon vesicle-encapsulated active probiotics according to claim 3, characterized in that, The lemon vesicles are prepared by the following method: juicing a seedless lemon and then centrifuging it using a gradient method to obtain lemon vesicles.

5. A method for preparing lemon vesicle-encapsulated active probiotics according to any one of claims 1-4, comprising the following steps: After ultrasonic treatment of lemon vesicles, they are mixed with probiotics and subjected to multiple physical compressions to obtain lemon vesicles encapsulated with active probiotics.

6. The preparation method according to claim 5, characterized in that, The conditions for ultrasonic treatment are 2-15 minutes of ultrasound at a power of 10-40W.

7. The preparation method according to claim 5, characterized in that, Based on the protein concentration in the citric acid, each 1 mg / mL protein mixture contains (1-10) × 10 9 CFU probiotics.

8. The preparation method according to any one of claims 5-7, characterized in that, The multiple physical compressions refer to 10-50 physical compressions.

9. The application of the lemon vesicle encapsulated active probiotics according to any one of claims 1-4 or the preparation method according to any one of claims 5-8 in the preparation of probiotic products.

10. A probiotic product comprising lemon vesicle-encapsulated active probiotics as described in any one of claims 1-4.

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