Nutritional supplement containing human milk oligosaccharide

Abstract

The invention relates to the technical field of functional food, and discloses a nutritional supplement containing human milk oligosaccharide, which comprises a core content and a coating layer, and the core content comprises human milk oligosaccharide functionalized probiotics and free human milk oligosaccharide. The human milk oligosaccharide functionalized probiotics are formed by anchoring human milk oligosaccharide on the surfaces of probiotics cells through chemical bonds. And the free human milk oligosaccharide and the human milk oligosaccharide functionalized probiotics have a synergistic effect, so that the growth and metabolism of intestinal beneficial bacteria are further promoted. The core content is wrapped in a capsule containing at least one isolating layer and multiple layers of pH response type enteric-coated materials, so that effective protection of active ingredients in the stomach and programmed targeted release from the middle rear section of the small intestine to the colon are realized. According to the invention, the survival rate and colonization ability of the probiotics are obviously improved, the synergistic interaction effect of the probiotics and the human milk oligosaccharide is enhanced, and accurate delivery and efficient utilization of active ingredients are ensured, so that the intestinal microecology is effectively regulated, and the health promotion effect is exerted.

Description

Technical Field

[0001] The present invention relates to the technical field of functional foods, and specifically to a nutritional supplement containing human milk oligosaccharides. Background Art

[0002] The balance of the gut microecosystem is crucial for maintaining human health. Probiotics, as a type of live microorganisms, can provide health benefits to the host when ingested in sufficient amounts, such as improving the gut microbiota structure, enhancing immune function, promoting nutrient absorption, etc. Human milk oligosaccharides (HMOs) are the third largest solid component in breast milk after lactose and fat, with unique structures and important biological functions, including promoting the growth of specific beneficial bacteria (such as Bifidobacterium) as selective prebiotics, directly regulating the host immune system, and inhibiting pathogen adhesion. Therefore, combining probiotics with HMOs to develop new synbiotics or functional foods has become a research hotspot in the fields of nutrition and health.

[0003] Currently, common probiotic and prebiotic combination products on the market are mostly simple physical mixtures of the two. In this form, when probiotics and HMOs enter the human digestive tract, there are many challenges in the exertion of their interaction and synergistic effects. Before reaching the intestinal site of action, probiotics need to withstand the harsh environments of gastric acid, bile salts, etc., and their survival rate is often significantly affected. At the same time, although HMOs can be utilized by certain beneficial bacteria in the intestine, in the complex competition environment of the gut microbiota, the added specific probiotics may not be able to preferentially and efficiently obtain and utilize these HMOs, thus limiting their rapid colonization and function exertion. In addition, simple physical mixing is difficult to ensure the synchronous and targeted release of probiotics and HMOs in specific regions of the intestine, which may lead to premature absorption of HMOs by the upper digestive tract or consumption by non-target microbiota, and probiotics may also not release a sufficient number of live bacteria at the optimal site of action.

[0004] To overcome these shortcomings, researchers have begun exploring more advanced probiotic-HMO combination and delivery strategies. For example, microencapsulation technology can be used to encapsulate probiotics to improve their survival in the digestive tract, or prebiotics can be combined with probiotics to enhance their targeting. However, existing technologies still leave room for improvement in achieving stable and efficient binding of HMOs and probiotics while ensuring both probiotic activity and HMO biological functions, as well as the controlled release and synergistic effects of the final product in vivo. Key challenges in this field are how to enable probiotics to more intelligently carry and utilize HMOs, while also employing efficient delivery systems to ensure precise release and maximum efficacy in specific areas of the intestine. Therefore, developing a composition and preparation method that can functionally bind HMOs to the surface of probiotics and combine them with advanced targeted release technologies is crucial for fully realizing the health potential of probiotics and HMOs. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the present invention provides a nutritional supplement containing human milk oligosaccharides, which solves the problems that when existing probiotics are used in combination with human milk oligosaccharides, the survival rate of probiotics in the digestive tract is low, the synergistic effect with human milk oligosaccharides is not fully exerted, and the active ingredients are difficult to target and effectively release in specific parts of the intestine.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions: A nutritional supplement containing human milk oligosaccharides, comprising:

[0007] (a) a core composition comprising:

[0008] Human milk oligosaccharide-functionalized probiotics, wherein one or more human milk oligosaccharides are bound to the surface of the probiotic cells by covalent linkage or specific non-covalent anchoring; the human milk oligosaccharide-functionalized probiotics account for 30-70% (w / w) of the total mass of the core composition;

[0009] Free human milk oligosaccharides, accounting for 20-60% (w / w) of the total mass of the core composition;

[0010] Optionally, pharmaceutically acceptable excipients, accounting for 0-20% (w / w) of the total mass of the core composition;

[0011] (b) a multi-layer enteric pH-responsive targeted release capsule comprising at least one inner coating layer and at least one outer pH-sensitive polymer coating layer, wherein the core composition is encapsulated in the capsule.

[0012] Preferably, the probiotic is Bifidobacterium longum subsp. infantis.

[0013] Preferably, the human milk oligosaccharide bound to the surface of the probiotic cells and / or the free human milk oligosaccharide is selected from at least one of 2'-fucosyllactose, lacto-N-neotetraose, 3'-sialyllactose, 6'-sialyllactose or a mixture thereof in any proportion.

[0014] Preferably, the human milk oligosaccharide is covalently bonded to the surface of the probiotic cells through a covalent bond selected from an amide bond or a secondary amine bond.

[0015] Preferably, the multi-layer enteric pH-responsive targeted release capsule is adapted to target the release of the contents in the mid-lower part of the small intestine or the colon, and the at least one outer layer pH-sensitive polymer coating is formed of a pH-sensitive polymer selected from acrylic copolymers.

[0016] A method for preparing a nutritional supplement containing human milk oligosaccharide, comprising the following steps:

[0017] (a) Preparing human milk oligosaccharide-functionalized probiotics (HAPs), including:

[0018] Culturing probiotic cells;

[0019] Under the condition of optimizing at least one process parameter including reactant concentration, reaction time, pH value and temperature, one or more human milk oligosaccharides are bound to the surface of the probiotic cells by covalent connection or specific non-covalent anchoring, and the optimization conditions are designed to control both the binding density and spatial conformation of the human milk oligosaccharide, or at least one of them, while maintaining the physiological activity of the probiotic and the structural integrity of the human milk oligosaccharide;

[0020] Purifying the human milk oligosaccharide-functionalized probiotics;

[0021] Mixing the human milk oligosaccharide-functionalized probiotics with one or more cryoprotectants and then freeze-drying;

[0022] (b) Preparing a core composition by mixing the freeze-dried human milk oligosaccharide-functionalized probiotics prepared in step (a) with free human milk oligosaccharide and optionally pharmaceutically acceptable excipients;

[0023] (c) Filling the core composition into a capsule;

[0024] (d) Performing a multi-layer enteric pH-responsive targeted release coating on the capsule filled with the core composition, and the coating includes applying at least one inner layer coating and at least one outer layer pH-sensitive polymer coating

[0025] Preferably, the selection of the one or more human milk oligosaccharides in step (a) is based on the specific metabolic preference of the probiotic strain used or the specific human milk oligosaccharide binding site on its cell surface, so as to achieve the specific matching and binding of the strain and the human milk oligosaccharide.

[0026] Preferably, in step (a), the human milk oligosaccharide is bound to the surface of the probiotic cells, aiming to form a multifunctional integrated layer, and the multifunctional integrated layer is used to provide nutrition, enhance the competitive inhibition ability against pathogens, and / or directly mediate immunomodulatory signals.

[0027] Preferably, the covalent connection in step (a) forms an amide bond through the EDC / NHS chemical method, or forms a secondary amine bond through reduction after the formation of a Schiff base.

[0028] Preferably, the multi-layer enteric pH-responsive targeted release coating in step (d) includes applying at least one outer coating formed by a pH-sensitive polymer selected from acrylic copolymers using a fluidized bed coating process, wherein the parameters of the coating process include an inlet air temperature of 40 - 60 °C and a product bed temperature of 30 - 45 °C.

[0029] The present invention provides a nutritional supplement containing human milk oligosaccharide. It has the following beneficial effects:

[0030] 1. By functionalizing and anchoring human milk oligosaccharide on the surface of probiotics, the present invention significantly improves the survival, colonization, and activity exertion ability of probiotics in the complex intestinal environment. This innovative "self - contained nutrient" design enables probiotics to more quickly adapt to the intestinal environment and preferentially utilize the anchored specific human milk oligosaccharide for proliferation. At the same time, the functionalized surface properties may enhance their adhesion to the intestinal mucosa, thereby more effectively exerting their probiotic effects in promoting host health and providing more direct support for the improvement of the intestinal microecology.

[0031] 2. The present invention scientifically formulates the functionalized probiotics with human milk oligosaccharide and specific types of free human milk oligosaccharide, achieving a multi - level and synergistic regulation effect on the intestinal microecology. The functionalized probiotics can play a role in a precise targeted manner, while the free human milk oligosaccharide, as a broad - spectrum prebiotic, can be utilized by more types of beneficial bacteria in the intestine, jointly promoting the balance of the intestinal flora and amplifying the overall health benefits that are difficult to achieve by a single component, especially being more significant in improving the intestinal microenvironment and supporting the growth of beneficial flora.

[0032] 3. The multi - layer enteric pH - responsive targeted release capsule technology adopted by the present invention ensures that the core active ingredients can safely and completely pass through the strong acidic environment of the stomach and the enzymatic hydrolysis in the upper digestive tract, and are programmed to release at a specific preset intestinal site. This precise delivery method not only maximally protects the activity of probiotics and the structural integrity of human milk oligosaccharide, but also significantly improves their bioavailability and targeting of action, thereby enhancing the overall efficacy of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is a flow chart of the present invention. Detailed implementation manners

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0035] Example 1:

[0036] Please refer to the attached Figure 1 , the embodiment of the present invention provides a nutritional supplement containing human milk oligosaccharides, including:

[0037] This embodiment describes the preparation of the nutritional supplement, in which human milk oligosaccharide-functionalized probiotics (HAPs) account for 50% in the core composition, free HMOs account for 40%, and excipients account for 10%. The coating process parameters adopt the intermediate values within the scope of the claims.

[0038] A. Preparation of human milk oligosaccharide-functionalized probiotics (HAPs)

[0039] A.1 Probiotic culture and pretreatment

[0040] Strain: Bifidobacterium longum subsp. infantis.

[0041] Culture: Inoculate the strain into MRS broth containing 0.05% L-cysteine hydrochloride and culture it at 37 °C under anaerobic conditions (85% N2, 10% CO2, 5% H2) for 30 hours until the end of the logarithmic growth phase.

[0042] Collection and washing: Centrifuge the culture solution at 4 °C and 5000×g for 15 minutes to collect the bacterial cells. Wash the bacterial cell precipitate three times with pre-cooled sterile 0.1M phosphate buffer saline (PBS, pH 7.2).

[0043] Bacterial suspension preparation: Resuspend the bacterial cells with 0.1M MES buffer (pH 6.0) and adjust the bacterial cell concentration in the bacterial suspension to OD 600 to approximately 2.0, which is equivalent to approximately 2×10 11 CFU / mL.

[0044] A.2 Preparation and activation of HMOs solution

[0045] HMOs for anchoring: 2'-fucosyllactose (2'-FL) and lacto-N-neotetraose (LNnT), mixed in a mass ratio of 1:1.

[0046] HMOs solution: Dissolve the above-mentioned mixed HMOs in 0.1 M MES buffer (pH 6.0) with a total concentration of 12.5 mg / mL.

[0047] Activation: Add EDC·HCl and NHS to the HMOs solution so that the molar ratio of HMOs (calculated based on an average molecular weight of about 500 Da), EDC, and NHS is 1:4:4. Stir and activate in the dark at 25 °C for 45 minutes.

[0048] A.3 Covalent linkage of HMOs to probiotics

[0049] Mixing and reaction: Slowly add the activated HMOs solution to the bacterial suspension so that the ratio of the dry weight of probiotic cells to the total mass of added HMOs is approximately 1:0.15. React with gentle stirring at 25 °C and pH 6.2 for 16 hours.

[0050] A.4 Purification and washing of HAPs

[0051] Collection and washing: After the reaction, collect HAPs by centrifugation at 4 °C and 5000 × g for 20 minutes. Wash three times with PBS (pH 7.2) containing 5% (w / v) trehalose and 2% (w / v) sucrose.

[0052] A.5 Freeze-drying of HAPs

[0053] Resuspension with cryoprotectant: Resuspend the purified HAPs with a sterile aqueous solution containing 10% (w / v) trehalose and 5% (w / v) mannitol, and adjust the concentration so that the expected viable cell count after lyophilization is about 1×10 11 CFU / g.

[0054] Pre-freezing: Pre-freeze at -70 °C for 6 hours.

[0055] Freeze-drying: Maintain the shelf temperature at -25 °C for 36 hours (primary drying, vacuum degree 0.1 mbar), and then increase the temperature programatically to 20 °C and maintain for 18 hours (secondary drying, vacuum degree 0.05 mbar).

[0056] B. Preparation of the core content

[0057] Component ratio (w / w):

[0058] Lyophilized powder of HAPs (from step A.5): 50%

[0059] Free HMOs mixture powder: 40% (this mixture contains 2'-FL, LNnT, 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL) mixed in a mass ratio of 1:1:1:1)

[0060] Microcrystalline cellulose (MCC PH101): 9%

[0061] Silica (fumed): 1%

[0062] Mixing: In a clean and low-humidity environment, mix each component in a three-dimensional motion mixer at 25 rpm for 25 minutes until homogeneous.

[0063] C. Capsule filling and multi-layer enteric coating

[0064] C.1 Capsule filling

[0065] Select size 0 HPMC hard capsules and fill 400 mg of the core content in each capsule.

[0066] C.2 Sealing layer coating

[0067] Coating solution: HPMC E5 (7% w / v) dissolved in ethanol: water (85:15, v / v), plasticizer TEC (12% of the dry weight of HPMC).

[0068] Process parameters: Bottom spray in fluidized bed, inlet air temperature 50 °C, product bed temperature 39 °C, atomization air pressure 2.0 bar.

[0069] Weight gain: 2.5% (w / w) of the capsule core weight.

[0070] C.3 First layer of enteric coating (released in the middle and posterior part of the small intestine)

[0071] Coating solution: L100-55 (10% w / v), plasticizer TEC (15% of the dry weight of the polymer), anti-adhesive talc powder (45% of the dry weight of the polymer). The solvent is isopropanol: water (93:7, v / v).

[0072] Process parameters: Inlet air temperature 50 °C, product bed temperature 38 °C, atomization air pressure 2.2 bar.

[0073] Weight gain: 9% (w / w) of the capsule core (including the sealing layer) weight.

[0074] C.4 Second layer of enteric coating (colon-targeted release)

[0075] Coating solution: S100 (12% w / v), plasticizer TEC (15% of the dry weight of the polymer), anti-adhesive talc powder (45% of the dry weight of the polymer)

[0076] ℃, product bed temperature 38 °C, atomization air pressure 2.2 bar.

[0077] Weight gain: 11% (w / w) of the capsule core (including the sealing layer and the first enteric layer) weight.

[0078] C.5 Drying and curing

[0079] After film coating, fluidized drying is carried out at 40 °C for 30 minutes, and then curing is carried out in an oven at 40 °C for 8 hours.

[0080] Example 2: Preparation of a nutritional supplement containing human milk oligosaccharides using the lower limit parameter and component ratio

[0081] This example describes the preparation of a nutritional supplement, in which HAPs account for 30% in the core composition, free HMOs account for 50%, and excipients account for 20%. The film coating process parameters use the lower limit value within the scope of the claims.

[0082] A. Preparation of human milk oligosaccharide-functionalized probiotics (HAPs)

[0083] A.1 Probiotic culture and pretreatment

[0084] The strain and culture conditions are the same as in Example 1, and the culture time is adjusted to 24 hours.

[0085] Collection, washing, and preparation of the bacterial suspension are the same as in Example 1, and the concentration of the bacterial suspension is adjusted to OD 600 about 1.5, equivalent to about 1.5×10 11 CFU / mL.

[0086] A.2 Preparation and activation of HMOs solution

[0087] HMOs for anchoring: Only 2'-FL is used.

[0088] HMOs solution: Dissolve 2'-FL in 0.1M MES buffer (pH 5.5) at a concentration of 10 mg / mL.

[0089] Activation: Add EDC·HCl and NHS to the HMOs solution so that the molar ratio of HMOs, EDC, and NHS is 1:3:3. Stir and activate in the dark at 20 °C for 30 minutes.

[0090] A.3 Covalent connection of HMOs and probiotics

[0091] Mixing and reaction: Add the activated HMOs solution to the bacterial suspension so that the ratio of the dry weight of probiotic cells to the total mass of added HMOs is about 1:0.1. Under the conditions of 20 °C and pH 6.0, gently stir and react for 12 hours.

[0092] A.4 Purification and washing of HAPs

[0093] Collection and washing: The same as in Example 1, but the washing solution is PBS (pH 7.2) containing only 5% (w / v) trehalose.

[0094] A. Lyophilization of 5HAPs

[0095] Resuspension with protective agent: Resuspend with a sterile aqueous solution containing 8% (w / v) trehalose and 4% (w / v) sucrose, and adjust the concentration so that the expected viable cell count after lyophilization is about 8×10 10 CFU / g.

[0096] Pre-freezing: Pre-freeze at -40°C for 10 hours.

[0097] Lyophilization: Maintain the shelf temperature at -30°C for 48 hours (primary drying, vacuum degree 0.15 mbar), and then increase the temperature programmatically to 15°C and maintain for 24 hours (secondary drying, vacuum degree 0.08 mbar).

[0098] B. Preparation of the core content

[0099] Component ratio (w / w):

[0100] Lyophilized powder of HAPs (from step A.5): 30%

[0101] Free HMOs mixture powder: 50% (this mixture contains 2'-FL and LNnT mixed in a mass ratio of 1:1)

[0102] Lactose (filler): 19%;

[0103] Magnesium stearate (lubricant): 1%;

[0104] Mixing: The same as in Example 1, mixing time 20 minutes.

[0105] C. Capsule filling and multi-layer enteric coating

[0106] C.1 Capsule filling

[0107] Select No. 1 HPMC hard capsule, and fill 300 mg of the core content into each capsule.

[0108] C.2 Barrier layer coating

[0109] Coating solution: HPMC E5 (5% w / v) dissolved in ethanol:water (80:20, v / v), plasticizer TEC (10% of the dry weight of HPMC).

[0110] Process parameters: Bottom spray in fluidized bed, inlet air temperature 40°C, product bed temperature 30°C, atomization air pressure 1.8 bar.

[0111] Weight gain: 2% (w / w) of the capsule core weight.

[0112] C.3 First layer of enteric coating (released in the middle and posterior part of the small intestine)

[0113] Coating solution: L100 - 55 (8% w / v), plasticizer TEC (10% of the dry weight of the polymer), anti - sticking agent talc powder (30% of the dry weight of the polymer). The solvent is isopropanol: water (95:5, v / v).

[0114] Process parameters: inlet air temperature 40 °C, product bed temperature 30 °C, atomization air pressure 2.0 bar.

[0115] Weight gain: 6% (w / w) of the weight of the capsule core (including the isolation layer).

[0116] C.4 Second - layer enteric coating (colon - targeted release)

[0117] Coating solution: S100 (10% w / v), plasticizer TEC (10% of the dry weight of the polymer), anti - sticking agent talc powder (30% of the dry weight of the polymer). The solvent is isopropanol: water (93:7, v / v).

[0118] Process parameters: inlet air temperature 40 °C, product bed temperature 30 °C, atomization air pressure 2.0 bar.

[0119] Weight gain: 8% (w / w) of the weight of the capsule core (including the isolation layer and the first enteric layer).

[0120] C.5 Drying and curing

[0121] After coating, fluidized drying is carried out at 35 °C for 20 minutes, and then curing is carried out in an oven at 35 °C for 4 hours.

[0122] Example 3: Preparation of a nutritional supplement containing human milk oligosaccharides using upper - limit parameter and component ratio

[0123] This example describes the preparation of a nutritional supplement, in which HAPs account for 70% in the core composition, free HMOs account for 30%, and excipients account for 0%. The coating process parameters adopt the upper - limit values within the scope of the claims.

[0124] A. Preparation of human milk oligosaccharide - functionalized probiotics (HAPs)

[0125] A.1 Probiotic culture and pretreatment

[0126] The strain and culture conditions are the same as in Example 1, and the culture time is adjusted to 36 hours.

[0127] Collection, washing, and preparation of the bacterial suspension are the same as in Example 1, and the concentration of the bacterial suspension is adjusted to OD 600 about 2.5, equivalent to about 2.5×10 11 CFU / mL.

[0128] A.2 Preparation and activation of HMOs solution

[0129] HMOs for anchoring: 2'-FL, LNnT, 3'-SL, and 6'-SL are mixed at a mass ratio of 1:1:1:1.

[0130] HMOs solution: Dissolve the above mixed HMOs in 0.1 M MES buffer (pH 6.5) with a total concentration of 15 mg / mL.

[0131] Activation: Add EDC·HCl and NHS to the HMOs solution so that the molar ratio of HMOs, EDC, and NHS is 1:5:5. Stir and activate in the dark at 30 °C for 60 minutes.

[0132] A.3 Covalent linkage of HMOs to probiotics

[0133] Mixing and reaction: Add the activated HMOs solution to the bacterial suspension so that the dry weight ratio of probiotic cells to the total mass of added HMOs is approximately 1:0.2. Gently stir and react at 30 °C and pH 6.5 for 24 hours.

[0134] A.4 Purification and washing of HAPs

[0135] Collection and washing: The same as in Example 1, but the washing solution is PBS (pH 7.4) containing 7% (w / v) trehalose and 3% (w / v) sucrose.

[0136] A.5 Freeze-drying of HAPs

[0137] Resuspension with cryoprotectant: Resuspend with a sterile aqueous solution containing 12% (w / v) trehalose and 6% (w / v) mannitol, and adjust the concentration so that the expected viable cell count after freeze-drying is approximately 1.5×10 11 CFU / g.

[0138] Pre-freezing: Pre-freeze at -80 °C for 4 hours.

[0139] Freeze-drying: Maintain the shelf temperature at -20 °C for 24 hours (primary drying, vacuum degree 0.08 mbar), and then increase the temperature programmatically to 25 °C and maintain for 12 hours (secondary drying, vacuum degree 0.04 mbar).

[0140] B. Preparation of the core content

[0141] Component ratio (w / w):

[0142] Freeze-dried powder of HAPs (from step A.5): 70%

[0143] Free HMOs mixture powder: 30% (this mixture contains 2'-FL, LNnT, 3'-SL, and 6'-SL mixed at a mass ratio of 1:1:1:1)

[0144] Excipients: 0%

[0145] Mixing: The same as in Example 1, with a mixing time of 30 minutes.

[0146] C. Capsule filling and multi-layer enteric coating

[0147] C.1 Capsule filling

[0148] Select size 00 HPMC hard capsules, and fill 500 mg of the core content into each capsule.

[0149] C.2 Sealing layer coating

[0150] Coating solution: HPMC E5 (10% w / v) dissolved in ethanol: water (90:10, v / v), and plasticizer TEC (15% of the dry weight of HPMC).

[0151] Process parameters: Bottom spray in a fluidized bed, inlet air temperature 60 °C, product bed temperature 45 °C, atomization air pressure 2.5 bar.

[0152] Weight gain: 3% (w / w) of the weight of the capsule core.

[0153] C.3 First layer of enteric coating (released in the middle and posterior segments of the small intestine)

[0154] Coating solution: L100-55 (12% w / v), plasticizer TEC (20% of the dry weight of the polymer), anti-adhesive talc powder (50% of the dry weight of the polymer). The solvent is isopropanol: water (90:10, v / v).

[0155] Process parameters: Inlet air temperature 60 °C, product bed temperature 45 °C, atomization air pressure 2.5 bar.

[0156] Weight gain: 10% (w / w) of the weight of the capsule core (including the sealing layer).

[0157] C.4 Second layer of enteric coating (colon-targeted release)

[0158] Coating solution: S100 (15% w / v), plasticizer TEC (20% of the dry weight of the polymer), anti-adhesive talc powder (50% of the dry weight of the polymer). The solvent is isopropanol: water (85:15, v / v).

[0159] Process parameters: Inlet air temperature 60 °C, product bed temperature 45 °C, atomization air pressure 2.5 bar.

[0160] Weight gain: 12% (w / w) of the weight of the capsule core (including the sealing layer and the first enteric layer).

[0161] C.5 Drying and curing

[0162] After film coating, fluidized drying was carried out at 45°C for 40 minutes, and then curing was carried out in an oven at 45°C for 12 hours.

[0163] Comparative Example 1: Compared with Example 1, the difference lies in the composition of Core Composition B: This composition does not contain the freeze-dried powder of HAPs, but consists of 43.5% (w / w) of the freeze-dried powder of the same subspecies of Bifidobacterium longum infantis that has not been functionalized with HMOs (this amount is calculated based on the amount of probiotics input in the HAPs anchoring reaction in Example 1 to ensure that the total amount of probiotic cells is equivalent), 46.5% (w / w) of the total free HMOs mixture powder (this mixture contains the HMOs originally planned for anchoring in Example 1 and the original free HMOs), and 10% (w / w) of excipients. That is, the core composition is a simple physical mixture of probiotics and free HMOs, and the probiotics are not functionally anchored with HMOs. The rest are the same.

[0164] Comparative Example 2: Compared with Example 1, the difference lies in the preparation of HAPs (Step A): In the step of preparing and activating the A.2 HMOs solution, EDC·HCl and NHS are not added to activate the HMOs, and the HMOs solution is directly used for the subsequent "connection" step A.3 with probiotics (that is, the chemical anchoring step is omitted, and it is only a simple mixing and incubation), and the rest are the same.

[0165] Comparative Example 3: Compared with Example 1, the difference lies in the composition of Core Composition B: It does not contain 40% (w / w) of the free HMOs mixture powder, and this part is replaced by an equal amount of microcrystalline cellulose, so that the total amount of excipients increases to 50% (w / w) (that is, the free HMOs part in the synergistic combination of HAPs and free HMOs is missing). The rest are the same.

[0166] Comparative Example 4: Compared with Example 1, the difference lies in the capsule preparation step C: Omit C.2 the coating of the isolating layer, C.3 the first layer of enteric coating ( L100-55) and C.4 the second layer of enteric coating ( S100), that is, no enteric coating treatment is carried out after capsule filling (that is, the multi-layer pH-responsive targeted release capsule technology is missing). The rest are the same.

[0167] Test Example 1:

[0168] 1. Experimental purpose

[0169] This experiment aims to verify and compare, through in vitro experimental methods, the differences in the HMOs anchoring efficiency, the selective utilization ability of specific HMOs, and the adhesion ability to intestinal epithelial cells among human milk oligosaccharide-functionalized probiotics (HAPs) under different preparation conditions (Examples 1 - 3) and probiotics that are not functionalized or are improperly treated (Comparative Examples 1 and 2), so as to highlight the advantages of the HMOs functionalized anchoring technology in the present invention.

[0170] 2. Test objects

[0171] HAPs prepared in Example 1 (Bifidobacterium longum subsp. infantis anchored with 2'-FL / LNnT, median parameter)

[0172] HAPs prepared in Example 2 (Bifidobacterium longum subsp. infantis anchored with 2'-FL, lower limit parameter)

[0173] HAPs prepared in Example 3 (Bifidobacterium longum subsp. infantis anchored with 2'-FL / LNnT / 3'-SL / 6'-SL, upper limit parameter)

[0174] Probiotic component in the core content prepared in Comparative Example 1 (Bifidobacterium longum subsp. infantis without HMOs anchoring, physically mixed with HMOs)

[0175] "HAPs" prepared in Comparative Example 2 (HMOs not effectively activated, theoretically with extremely low or no anchoring efficiency)

[0176] 3. Experimental procedures

[0177] 3.1 Determination of HMOs anchoring efficiency

[0178] 3.1.1 Sample preparation:

[0179] Accurately weigh about 50 mg of the freeze-dried powder of each test object, and record the actual weighing amount.

[0180] For Comparative Example 1, take its core content, and preliminarily separate the probiotics (precipitate) and most of the free HMOs (supernatant) by resuspending in sterile water and centrifuging at low speed (e.g., 1000×g, 5 minutes), and then wash the probiotic precipitate for subsequent steps to remove physically adsorbed HMOs, simulating the washing steps in the preparation of HAPs

[0181] 3.1.2 Washing to remove unbound HMOs:

[0182] Resuspend the weighed sample (or the probiotics treated in Comparative Example 1) thoroughly with 10 mL of pre-cooled sterile PBS (pH 7.2).

[0183] Centrifuge at 8000×g for 10 minutes at 4°C, and discard the supernatant.

[0184] Repeat the above washing steps three times to ensure that free or non-specifically adsorbed HMOs are removed sufficiently.

[0185] 3.1.3 Hydrolysis and derivatization of HMOs (taking 2'-FL as an example, assuming it is one of the main anchored HMOs):

[0186] Precisely add the washed bacterial cell precipitate to 1 mL of 2 M trifluoroacetic acid (TFA) solution.

[0187] Heat and hydrolyze at 110 °C in a water bath or metal bath for 4 hours to hydrolyze the bound 2'-FL into fucose, galactose, and glucose.

[0188] After hydrolysis, cool the sample to room temperature and blow it dry with nitrogen to remove TFA.

[0189] Add 1 mL of ultrapure water to redissolve and vortex.

[0190] Take an appropriate amount of the hydrolyzate (e.g., 100 μL) and perform derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP) for subsequent HPLC detection of fucose. Derivatization steps: Add an equal volume of 0.6 M NaOH solution and 0.5 M PMP methanol solution to the supernatant, react at 70 °C for 60 minutes. After the reaction, add an equal volume of 0.3 M HCl for neutralization, then add chloroform to extract the unreacted PMP, and take the upper aqueous phase to pass through a 0.22 μm filter membrane for testing.

[0191] 3.1.4 Determination of the content of characteristic monosaccharide (fucose) by HPLC:

[0192] Chromatographic conditions (reference): C18 reverse-phase chromatographic column (e.g., 4.6 mm × 250 mm, 5 μm); mobile phase A is acetonitrile, mobile phase B is phosphate buffer solution (e.g., 0.1 M, pH 6.7); gradient elution; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 245 nm.

[0193] Quantify the fucose content in the sample by comparing with a fucose standard of known concentration.

[0194] 3.1.5 Result calculation:

[0195] Calculate the amount of 2'-FL anchored on the surface of probiotics according to the mass ratio of fucose in 2'-FL.

[0196] The result is expressed as: mg anchored HMOs / g probiotic dry weight.

[0197] Simultaneously determine the viable count of the probiotic sample before treatment, and the result can also be expressed as: mg anchored HMOs / 10 10 CFU.

[0198] 3.2 Evaluation of the selective utilization ability of HMOs

[0199] 3.2.1 Preparation of culture media:

[0200] Basal medium: Modified MRS medium without glucose or other carbohydrates.

[0201] Test medium: Add 0.5% (w / v) of 2'-FL or LNnT (selected according to the types of main anchored HMOs in the examples) as the sole carbon source to the basal medium.

[0202] 3.2.2 Preparation and inoculation of bacterial suspensions:

[0203] Resuspend the lyophilized powder of each test object (for Comparative Example 1, take its core content) with sterile PBS and adjust the bacterial suspension concentration to OD 600 to approximately 1.0.

[0204] Inoculate into the above test medium at an inoculation amount of 1% (v / v). Set the medium without bacteria as the blank control.

[0205] 3.2.3 Anaerobic culture and growth monitoring:

[0206] Place the inoculated culture tubes or microplates in a 37°C anaerobic incubator (or use an anaerobic bag / anaerobic workstation).

[0207] At time points such as 0, 4, 8, 12, 16, 24 hours of culture, take samples and measure OD 600 values to monitor the bacterial growth.

[0208] 3.2.4 Data analysis:

[0209] Plot the growth curve.

[0210] Calculate the maximum specific growth rate (μmax) and the final OD 600 value after 24 hours of culture.

[0211] At the end of the culture, collect the fermentation broth, centrifuge to obtain the supernatant, and use HPLC to measure the concentration of the remaining HMOs in the medium to evaluate the consumption of HMOs.

[0212] 3.3 Evaluation of the adhesion ability to intestinal epithelial cells in vitro

[0213] 3.3.1 Culture of Caco-2 cells:

[0214] The human colon adenocarcinoma cell line Caco-2 cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum, 1% non-essential amino acids, and 1% penicillin-streptomycin, and placed in an incubator at 37°C with 5% CO2.

[0215] When the cells grew to 80-90% confluence, they were passaged. The cells were seeded into 24-well cell culture plates at a density of 1×10 5 cells / mL and cultured for 18-21 days until they differentiated into a monolayer of tightly connected cells. One day before the experiment, the medium was replaced with antibiotic-free DMEM medium.

[0216] 3.3.2 Preparation of bacterial suspension:

[0217] The lyophilized powder of each test subject (for Comparative Example 1, its core content was taken) was resuspended with sterile and antibiotic-free DMEM medium, and the bacterial suspension concentration was adjusted to 1×10 8 CFU / mL.

[0218] 3.3.3 Adhesion experiment:

[0219] The original medium of Caco-2 cells in the 24-well plate was aspirated, and the cell monolayer was washed twice with pre-warmed sterile PBS.

[0220] 1 mL of the above-prepared bacterial suspension (the ratio of bacteria to cells was approximately 100:1) was added to each well.

[0221] The culture plate was placed in an incubator at 37°C with 5% CO2 and incubated for 1.5 hours.

[0222] 3.3.4 Washing and cell lysis:

[0223] After incubation, the medium was carefully aspirated, and the cell monolayer was gently washed 3 times with pre-warmed PBS to remove non-adherent bacteria.

[0224] 1 mL of 0.5% Triton X-100 (dissolved in PBS) was added to each well and allowed to act at room temperature for 10 minutes to lyse Caco-2 cells and release adherent bacteria.

[0225] 3.3.5 Viable bacteria counting:

[0226] The cell lysate was serially diluted 10-fold, and an appropriate dilution of the bacterial suspension was spread on MRS agar plates.

[0227] After anaerobic culture at 37°C for 48 hours, viable bacteria counting was performed.

[0228] At the same time, the initial 1×10 8 CFU / mL bacterial suspension was also subjected to viable bacteria counting as the total number of initial bacteria.

[0229] 3.3.6 Result calculation:

[0230] Adhesion rate (%) = (Number of viable bacteria adhering to cells / Total number of initial viable bacteria added) × 100%.

[0231] Table 1: Results of the evaluation of the functional anchoring effect of HMOs and the in vitro functions of HAPs

[0232]

[0233] For the human milk oligosaccharide-functionalized probiotics prepared by the method of the present invention (as shown in Examples 1, 2, and 3), human milk oligosaccharides significantly higher than those of the control group (Comparative Examples 1 and 2) were successfully anchored on their cell surfaces. As shown in Table 1, the contents of the main HMOs anchored in Examples 1, 2, and 3 reached 18.3, 9.7, and 26.1 mg / g of cell dry weight, respectively, while the corresponding values of Comparative Example 1 (physical mixing) and Comparative Example 2 (ineffective anchoring) were only 0.8 and 1.5 mg / g. This efficient surface anchoring directly confers a metabolic advantage to the functionalized probiotics when using the corresponding HMOs as the sole carbon source. Experimental data show that when using 2'-FL as the carbon source, the probiotics in Examples 1, 2, and 3 exhibited higher maximum specific growth rates (μmax were 0.28, 0.21, 0.33 h -1 ), respectively) and cell concentrations after 24 hours of cultivation (OD 600 were 1.15, 0.88, 1.32, respectively), which were significantly better than those of Comparative Example 1 (μmax 0.14 h -1 , OD 600 0.65) and Comparative Example 2 (μmax 0.15 h -1 , OD 600 0.69). This indicates that by directly presenting HMOs on the surface of probiotic cells, it can promote the preferential and efficient utilization of specific prebiotics by them, thus obtaining a growth advantage in a nutrient-competitive environment.

[0234] The human milk oligosaccharide-functionalized probiotics of the present invention also exhibit improved characteristics in terms of their interaction with host cells. From the results of the Caco-2 cell adhesion experiment, it can be seen that the functionalized probiotics in Examples 1, 2, and 3 showed significantly enhanced in vitro intestinal epithelial cell adhesion ability, and their adhesion rates were 12.7%, 9.1%, and 15.5%, respectively. In contrast, the probiotics in Comparative Example 1 and Comparative Example 2 had lower adhesion rates of 5.3% and 5.8% respectively due to the lack of effective HMO surface modification. The surface anchoring of HMOs may enhance the binding of probiotics to intestinal epithelial cells by changing the physicochemical properties of the probiotic cell surface or through specific recognition mediated by HMO molecules, which is of great significance for the effective colonization of probiotics in the intestine and the exertion of their long-term biological effects.

[0235] In summary, the experimental results strongly demonstrate the effectiveness of the HMO-functionalized probiotic preparation method provided by the present invention. Through a specific surface anchoring technology, not only is the HMO stablely attached to the probiotic surface, but the probiotic's efficiency in utilizing specific HMOs and its ability to adhere to intestinal epithelial cells are also significantly enhanced. These in vitro functional advantages are attributed to the present invention's core component, the HMO-functionalized probiotic, which achieves tight binding between the probiotic and its preferred nutrient substrate, potentially optimizing the probiotic's surface biological properties. These properties give the present composition the potential to outperform simple mixtures of traditional probiotics and prebiotics in regulating the intestinal microbiome.

[0236] Test Example 2:

[0237] 1. Purpose of the experiment

[0238] This study aimed to evaluate the synergistic effects of combining the human milk oligosaccharide-functionalized probiotics (HAPs) described in this invention with specific free HMOs on the metabolic activities (focusing on the production of short-chain fatty acids (SCFAs)) and key microbial structures (such as the proliferation of beneficial bacteria) of the simulated intestinal flora using an in vitro simulated intestinal fermentation model, thereby validating the synergistic effect of the combination of HAPs and free HMOs.

[0239] 2. Test subjects

[0240] Core contents prepared in Example 1 (HAPs + free HMOs, intermediate value)

[0241] Core contents prepared in Example 2 (HAPs + free HMOs, lower limit)

[0242] Core contents prepared in Example 3 (HAPs + free HMOs, upper limit)

[0243] Core contents prepared in Comparative Example 1 (unanchored probiotics + total free HMOs)

[0244] Core content prepared in Comparative Example 3 (only HAPs, lacking the synergy of free HMOs)

[0245] Negative control group (containing only fecal bacterial suspension and basal culture medium, without adding any test samples)

[0246] Positive control group (optional, e.g., adding commercial prebiotic inulin)

[0247] 3. Experimental Procedure

[0248] 3.1 Preparation of human fecal bacterial suspension

[0249] Fecal sample collection: At least 3 healthy adult volunteers (who have not taken antibiotics, probiotics, prebiotics, etc. in the past 3 months) were recruited, and fresh fecal samples were collected. Immediately after sample collection, the samples were placed in an anaerobic bag and processed within 2 hours.

[0250] Preparation of bacterial suspension: Inside the anaerobic workbench, fecal samples from different volunteers were mixed evenly in equal amounts. Take 10 g of the mixed feces and add 90 mL of pre-reduced sterile phosphate buffer (PBS, 0.1 M, pH 7.0, containing 0.05% L-cysteine hydrochloride). Vigorously shake and mix well, and then filter through sterile gauze to remove large food residues to obtain a 10% (w / v) fecal bacterial suspension.

[0251] 3.2 Preparation of in vitro fermentation medium

[0252] Basic fermentation medium: For example, it contains peptone (2 g / L), yeast extract (2 g / L), NaCl (0.1 g / L), K2HPO4 (0.04 g / L), K2HPO4 (0.04 g / L), MgSO4·7H2O (0.01 g / L), CaCl2·2H2O (0.01 g / L), NaHCO3 (2 g / L), Tween 80 (2 mL / L), cysteine hydrochloride (0.5 g / L) and resazurin (0.001 g / L, as an anaerobic indicator). After autoclaving, it was maintained at 37 °C and under anaerobic conditions before adding the fecal bacterial suspension.

[0253] 3.3 In vitro fermentation experiment

[0254] Sample addition: Inside the anaerobic workbench, add 45 mL of the basic fermentation medium to a sterile fermentation bottle. Then, add the samples of each test subject respectively to make the initial concentration of HAPs (or probiotics in the control group) in the final sample reach, for example, 10 7 -10 8 CFU / mL, or the concentration of HMOs (total HMOs or free HMOs) reach, for example, 5 - 10 mg / mL. Record the actual addition amount of each group of samples.

[0255] Inoculation of fecal bacterial suspension: Add 5 mL (10% v / v) of the prepared fecal bacterial suspension to each fermentation bottle.

[0256] Anaerobic culture: Immediately seal the fermentation bottle and place it in a 37 °C constant temperature shaking incubator for anaerobic fermentation culture at a rotation speed of 100 rpm.

[0257] 3.4 Sample collection and processing

[0258] At 0 hours, 12 hours, 24 hours, and 48 hours after the start of fermentation, aseptically sample from each fermentation bottle.

[0259] pH measurement: Immediately measure the pH value of the fermentation broth.

[0260] SCFAs analysis sample: Take 1 mL of the fermentation broth, add 25 μL of 20% (v / v) metaphosphoric acid solution and an appropriate amount of internal standard (such as 2-ethylbutyric acid), mix well, and centrifuge at 4°C and 12,000×g for 10 minutes. Take the supernatant and filter it through a 0.22 μm filter membrane, and store it at -20°C for future measurement.

[0261] Microbial community DNA extraction sample: Take 1 mL of the fermentation broth, centrifuge at 4°C and 10,000×g for 10 minutes to collect the microbial cell pellet, and discard the supernatant. Store the microbial cell pellet at -80°C for subsequent DNA extraction and microbial community analysis.

[0262] 3.5 Short-chain fatty acids (SCFAs) analysis

[0263] Analyze the concentrations of SCFAs such as acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid by gas chromatography (GC).

[0264] Chromatographic conditions (for reference): Capillary column (such as HP-FFAP, 30 m × 0.25 mm × 0.25 μm); injection port temperature 250°C; detector (FID) temperature 280°C; programmed temperature rise; carrier gas is nitrogen. Quantitative analysis is performed by comparing with the standard curve.

[0265] 3.6 Analysis of the relative abundance of key microbial communities (qPCR)

[0266] DNA extraction: Use a commercial fecal DNA extraction kit to extract the total DNA from the stored microbial cell pellet.

[0267] qPCR amplification: Using the extracted DNA as a template, select specific primers for the 16S rRNA genes of total bacteria, Bifidobacterium genus, Lactobacillus genus, etc. for real-time fluorescence quantitative PCR (qPCR).

[0268] Data analysis: Calculate the change in the relative abundance of each target microbial community relative to total bacteria

[0269] Table 2: Effects of combinations of HAPs and free HMOs on in vitro simulated intestinal fermentation for 24 hours

[0270]

[0271] According to the results of the above-mentioned in vitro simulated intestinal fermentation experiment, it can be clearly observed that the core content of the present invention, comprising a combination of human milk oligosaccharide functionalized probiotics (HAPs) and specific free HMOs, has a significant positive regulatory effect on the metabolic activity and structure of the simulated intestinal flora. As shown in Table 2, compared with the negative control group, Comparative Example 1 (physical mixing of unanchored probiotics with HMOs) and Comparative Example 3 (only HAPs, no free HMOs), Examples 1, 2, and 3 all resulted in lower pH values (5.68, 5.85, and 5.53, respectively) after 24 hours of fermentation, which directly reflects a stronger acid production capacity. At the same time, the concentrations of major short-chain fatty acids (SCFAs) such as acetic acid, propionic acid, and butyric acid in the example group were also significantly higher than those in the control group. For example, the acetic acid, propionic acid, and butyric acid produced in Example 1 were 38.5, 15.1, and 20.3 μmol / mL, respectively, while those in Comparative Example 1 were 29.7, 10.5, and 14.8 μmol / mL. This suggests that the combination of HAPs and free HMOs can be more effectively utilized by the intestinal flora, thereby producing more health-promoting metabolites.

[0272] This enhanced metabolic activity is closely related to the promoting effect of the composition on specific beneficial bacteria. Experimental data show that after 24 hours of fermentation, the relative abundance of Bifidobacterium spp. in Examples 1, 2, and 3 increased by 6.8 times, 4.5 times, and 8.9 times compared to 0 hours, respectively. This increase is significantly higher than that in Comparative Example 1 (3.1 times) and Comparative Example 3 (2.7 times). Bifidobacterium is a recognized beneficial intestinal bacterium, and its proliferation is beneficial to maintaining the balance of intestinal microecology. Because HAPs themselves have HMOs anchored on their surfaces, they can preferentially utilize these "self-contained" nutrients for rapid activation and proliferation; at the same time, the additional free HMOs added to the system can not only be continuously utilized by HAPs, but can also be used as prebiotics by other native beneficial bacteria in the intestine that can utilize HMOs (including different types of bifidobacteria), thereby forming a synergistic effect and jointly promoting the growth of beneficial bacteria.

[0273] In summary, the in vitro fermentation results strongly support the rationality and superiority of the core composition design of the present invention. By scientifically combining probiotics functionalized with HMOs with specific types of free HMOs, not only is the vitality and functionality of the anchored probiotics ensured, but the supplementation of free HMOs also further expands the overall beneficial effects on the intestinal flora. This synergistic effect of HAPs and free HMOs enables the composition of the present invention to demonstrate superior potential to HAPs alone or simple mixtures of traditional probiotics and prebiotics in promoting the production of beneficial SCFAs and optimizing intestinal flora structure (particularly increasing Bifidobacterium abundance), demonstrating its unique advantages in improving the intestinal microbiome.

[0274] Test Example 3: Performance Evaluation of Multilayer Enteric-coated pH-responsive Targeted Release Capsules

[0275] 1. Experimental Purpose

[0276] The purpose of this experiment is to evaluate the programmed release characteristics of the multilayer enteric-coated pH-responsive coated capsules described in the present invention under different pH environments of the simulated gastrointestinal tract through in vitro dissolution experiments, as well as the protective effect of the coating on the core active ingredients (HAPs live bacteria and HMOs), so as to verify the effectiveness of this targeted release system.

[0277] 2. Test Objects

[0278] The capsules prepared in Example 1 (double-layer enteric coating, isolation layer + [[ID=1VI6]]L100-55 + S100, median parameter)

[0279] The capsules prepared in Example 2 (double-layer enteric coating, isolation layer + L100-55 + S100, lower limit parameter)

[0280] The capsules prepared in Example 3 (double-layer enteric coating, isolation layer + L100-55 + S100, upper limit parameter)

[0281] Control Example 4 (filling the core content of Example 1 into an ordinary HPMC capsule without any enteric coating)

[0282] (To calculate the release percentage, it is necessary to measure the initial number of live HAPs bacteria and the content of HMOs in the capsule contents of each batch)

[0283] 3. Experimental Procedures

[0284] 3.1 Determination of Initial Active Substance Content

[0285] Randomly take 3-5 capsules of each test object.

[0286] Carefully open the capsules and completely transfer the core content into an appropriate amount (e.g., 10 mL) of sterile PBS buffer (pH 7.0), and mix well to dissolve or disperse the content.

[0287] Counting of live HAPs bacteria: serially dilute the above suspension, coat it on MRS agar plates, count after anaerobic culture at 37 °C for 48 hours, and calculate the average initial number of live bacteria per capsule (CFU / capsule).

[0288] Determination of HMOs content: Take an appropriate amount of the suspension, and after appropriate treatment (such as centrifuging to obtain the supernatant, enzymatic hydrolysis or derivatization if necessary), use HPLC or other suitable methods to determine the content of one or more representative HMOs (for example, 2'-FL), and calculate the average initial HMOs content per capsule (mg / capsule).

[0289] 3.2 In vitro programmed release experiment

[0290] Dissolution apparatus: Use a dissolution tester specified in the Chinese Pharmacopoeia or USP (for example, paddle method, rotation speed 50 - 75 rpm).

[0291] Dissolution medium:

[0292] Stage 1 (simulated gastric fluid, SGF): 0.1 M HCl solution, pH 1.2 (pepsin can be added as needed). Dissolution volume 900 mL, temperature 37 ± 0.5 °C.

[0293] Stage 2 (simulated intestinal fluid, SIF): Phosphate buffer solution, pH 6.8 (pancreatin can be added as needed). Dissolution volume 900 mL, temperature 37 ± 0.5 °C.

[0294] Stage 3 (simulated colonic initial fluid, SCF): Phosphate buffer solution, pH 7.2 (or higher, such as pH 7.4, according to the dissolution characteristics of the second-layer coating material S100). Dissolution volume 900 mL, temperature 37 ± 0.5 °C.

[0295] Experimental procedure:

[0296] Place single capsules (at least n = 3 parallel experiments for each test subject) into the dissolution cups containing SGF respectively, and start the experiment.

[0297] Run in SGF for 2 hours. At the end of 2 hours, precisely sample from each dissolution cup (for example, 10 mL, and supplement an equal volume of fresh SGF to maintain the volume, or do not supplement but correct for volume changes). Immediately process the samples for viable bacteria and HMOs determination.

[0298] Carefully transfer the capsules still under the dissolution basket or paddle (if not completely disintegrated) to the preheated dissolution cups containing SIF (or replace the SGF in the dissolution cups with SIF).

[0299] Continue to run in SIF. For example, sample at 1 hour, 2 hours, 3 hours (or more time points, depending on the dissolution of the first-layer coating) after transfer to SIF. Process the samples as above.

[0300] After completing the SIF stage, carefully transfer the capsules to the preheated dissolution cups containing SCF (or replace the medium).

[0301] Continue to run in the SCF. For example, samples are taken 1 hour, 2 hours, 3 hours (or more time points, depending on the dissolution of the second-layer coating) after transferring to the SCF. The samples are processed as described above.

[0302] 3.3 Sample analysis

[0303] Viable count of HAPs: For the dissolution medium samples collected at each time point, immediately perform serial gradient dilutions, spread them on MRS agar plates, and count after anaerobic incubation at 37 °C for 48 hours. The results are expressed as the cumulative number of viable bacteria released (CFU).

[0304] Determination of HMOs content: For the dissolution medium samples collected at each time point, after appropriate treatment (such as centrifugation, filtration), the concentration of representative HMOs is determined using the same method as in 3.1. The results are expressed as the cumulative amount of HMOs released (mg).

[0305] 3.4 Data calculation and expression

[0306] Calculate the cumulative release percentage of viable HAPs and HMOs at each time point:

[0307] Cumulative release percentage (%) = (total amount detected in the dissolution medium at the current time point / initial average content of the capsule) × 100%.

[0308] Plot the release curve: with time as the abscissa and cumulative release percentage as the ordinate.

[0309] Table 3: In vitro cumulative release characteristics of different capsule formulations in simulated gastrointestinal fluids

[0310]

[0311]

[0312] Based on the above in vitro programmed release experimental results, the multi-layer enteric-coated pH-responsive coated capsules used in the present invention exhibit excellent active ingredient protection and targeted release characteristics. As shown in Table 3, under the condition of simulated gastric fluid (SGF, pH 1.2) for 2 hours, the capsules of Examples 1, 2, and 3 all maintained good integrity, and the cumulative release rates of viable HAPs bacteria in their core contents were extremely low (1.8%, 3.1%, and 0.9% respectively), and the cumulative release rates of representative HMOs (calculated as 2'-FL) were also correspondingly very low (2.5%, 4.0%, and 1.3% respectively). This indicates that the enteric coating on the outer layer of the capsule (and the possible intermediate layer) can effectively resist the erosion of the strong acid environment and prevent the premature release and inactivation of the active ingredients in the stomach. In contrast, the capsule of Comparative Example 4 without any enteric coating treatment rapidly disintegrated and released most of its contents within 0.5 hours in SGF (75.2% release of viable HAPs bacteria and 80.1% release of HMOs), demonstrating the key role of the coating technology of the present invention in protecting the core components through the stomach.

[0313] When the experimental environment was changed to simulated intestinal fluid (SIF, pH 6.8), the capsules of Examples 1, 2, and 3 began to exhibit programmed release behavior. For example, after 1 hour (total 3 hours) in SIF for Example 1, the cumulative release rates of viable bacteria and HMOs reached 15.7% and 18.3% respectively, and further increased to 58.2% and 61.5% after 3 hours (total 5 hours) in SIF. This is consistent with the mechanism of gradual dissolution of the first-layer enteric coating (such as L100-55 designed to dissolve at around pH 5.5 - 6.8) under this pH condition. Subsequently, when the capsule entered the simulated colon initial fluid (SCF, pH 7.2) environment, the release further accelerated and tended to be complete. After 1 hour (total 6 hours) in SCF for Example 1, the cumulative release rates of viable bacteria and HMOs had reached 85.9% and 88.1% respectively, and approached complete release after 2 hours (total 7 hours). This corresponds to the disintegration characteristics of the second-layer enteric coating (such as S100 designed to dissolve above pH 7.0) under this pH condition, achieving the release of the contents in the more distal intestine. L100-55) gradually dissolving under this pH condition. S100) under this pH condition, realizing the release of the contents in the more distal intestine.

[0314] In summary, the results of the in vitro release experiments strongly confirm the effectiveness of the multi-layer enteric-coated pH-responsive capsule design of the present invention. This system can not only successfully protect core active ingredients such as HAPs and free HMOs from gastric acid damage, but also achieve programmed and targeted release of the contents in the simulated mid-small intestine and colon environments through the combined application of different pH-sensitive polymers. Such controllable release characteristics are crucial for ensuring the viability of live bacteria, improving the bioavailability of HMOs, and delivering active ingredients to the intended site of action, fully demonstrating the innovation of the present invention in formulation technology, and thus providing a key guarantee for the core composition to exert its expected biological functions.

[0315] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A nutritional supplement containing human milk oligosaccharides, characterized in that, Comprising: (a) A core composition, the core composition comprising: Human milk oligosaccharide-functionalized probiotics, wherein one or more human milk oligosaccharides are bound to the surface of the probiotic cells by covalent linkage or specific non-covalent anchoring; the human milk oligosaccharide-functionalized probiotics account for 30-70% (w / w) of the total mass of the core composition; Free human milk oligosaccharides, accounting for 20-60% (w / w) of the total mass of the core composition; Optionally, pharmaceutically acceptable excipients, accounting for 0-20% (w / w) of the total mass of the core composition; (b) A multi-layer enteric pH-responsive targeted release capsule, which comprises at least one inner coating and at least one outer pH-sensitive polymer coating, and the core composition is encapsulated within the capsule.

2. A nutritional supplement containing human milk oligosaccharide according to claim 1, characterized in that, The probiotic is Bifidobacterium longum subsp. infantis.

3. A nutritional supplement containing human milk oligosaccharides according to claim 1, characterized in that, The human milk oligosaccharides bound to the surface of the probiotic cells and / or the free human milk oligosaccharides are selected from at least one of 2'-fucosyllactose, lacto-N-neotetraose, 3'-sialyllactose, 6'-sialyllactose or a mixture of any proportion thereof.

4. A nutritional supplement containing human milk oligosaccharide according to claim 1, characterized in that, The human milk oligosaccharides are linked to the surface of the probiotic cells by a covalent bond selected from an amide bond or a secondary amine bond.

5. A nutritional supplement containing human milk oligosaccharides according to claim 1, characterized in that, The multi-layer enteric pH-responsive targeted release capsule is adapted to target the release of the content in the middle and posterior segments of the small intestine or the colon, and the at least one outer pH-sensitive polymer coating is formed of a pH-sensitive polymer selected from acrylic copolymers.

6. A method for preparing a nutritional supplement containing human milk oligosaccharides, according to any one of claims 1-5, a nutritional supplement containing human milk oligosaccharides, characterized in that, Comprising the following steps: (a) Preparing human milk oligosaccharide-functionalized probiotics, including: Culturing probiotic cells; Under conditions of optimizing at least one process parameter including reactant concentration, reaction time, pH value and temperature, binding one or more human milk oligosaccharides to the surface of the probiotic cells by covalent linkage or specific non-covalent anchoring, the optimization conditions are aimed at controlling both the binding density and spatial conformation of the human milk oligosaccharides, or at least one of them, while maintaining the physiological activity of the probiotics and the structural integrity of the human milk oligosaccharides; Purifying the human milk oligosaccharide-functionalized probiotics; Mixing the human milk oligosaccharide-functionalized probiotics with one or more cryoprotectants and then performing freeze-drying; (b) Preparing a core composition by mixing the freeze-dried human milk oligosaccharide-functionalized probiotics prepared in step (a) with free human milk oligosaccharides and optionally pharmaceutically acceptable excipients; (c) Filling the core composition into a capsule; (d) Performing a multi-layer enteric pH-responsive targeted release coating on the capsule filled with the core composition, the coating including applying at least one inner coating and at least one outer pH-sensitive polymer coating.

7. A method for preparing a nutritional supplement containing human milk oligosaccharide according to claim 6, characterized in that, In step (a), the selection of the one or more human milk oligosaccharides is based on the specific metabolic preferences of the probiotic strain used or the specific human milk oligosaccharide binding sites on its cell surface, so as to achieve specific matching binding between the strain and the human milk oligosaccharides.

8. A method for preparing a nutritional supplement containing human milk oligosaccharide according to claim 6, characterized in that, In step (a), binding the human milk oligosaccharides to the surface of the probiotic cells is aimed at forming a multi-functional integrated layer, and the multi-functional integrated layer is used to provide nutrition, enhance the competitive inhibitory ability against pathogens and / or directly mediate immunomodulatory signals.

9. A method for preparing a nutritional supplement containing human milk oligosaccharide according to claim 6, characterized in that, The covalent connection in step (a) forms an amide bond through EDC / NHS chemistry or forms a secondary amine bond through reduction after the formation of a Schiff base.

10. The preparation method of a nutritional supplement containing human milk oligosaccharide according to claim 6, characterized in that, The multi-layer enteric pH-responsive targeted release coating in step (d) includes applying at least one outer coating formed of a pH-sensitive polymer selected from acrylic copolymers using a fluidized bed coating process, wherein the parameters of the coating process include an inlet air temperature of 40 - 60 °C and a product bed temperature of 30 - 45 °C.

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