Intestinal tract conditioning composition based on compound fruits and vegetables and process of intestinal tract conditioning composition

Abstract

The invention relates to the technical field of intestinal conditioning, and discloses a preparation process of an intestinal conditioning composition based on compound fruits and vegetables, which comprises the following steps: (1) carrying out double-layer embedding treatment on probiotics to obtain embedded probiotics; (2) performing supercritical CO2 extraction and vacuum freeze drying treatment on the composite fruit and vegetable powder; (3) mixing and granulating the embedded probiotics, the inactivated lactic acid bacteria, the compound prebiotics, sorbitol and the compound fruit and vegetable powder; (4) adding magnesium stearate, and tabletting and forming; and (5) carrying out film coating on the tablets by adopting a compound coating agent. According to the invention, a scheme of compounding the radish seed extract and the fructo-oligosaccharide is adopted, so that the dual effects of selective inhibition of pathogenic bacteria and directional proliferation of probiotics are realized. The dual technical effects of enhancing intestinal barrier repair and immune homeostasis are achieved. Compared with the scheme that a single viable bacterium preparation only depends on metabolites to regulate the intestinal microenvironment in the prior art, the problems of insufficient immune regulation and low barrier repair efficiency are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of intestinal conditioning, and specifically relates to an intestinal conditioning composition based on compound fruits and vegetables and its process. Background Art

[0002] With the continuous in-depth research on intestinal microecology, probiotics, as an effective biological agent for regulating the intestinal microenvironment, have received extensive attention. Probiotics can not only promote the growth of beneficial bacteria, but also maintain intestinal health by regulating the immune system and enhancing the intestinal barrier function. However, how to ensure the colonization and activity of probiotics while optimizing their immunomodulatory effects and intestinal barrier repair abilities remains an urgent problem to be solved in the current technology.

[0003] In the prior art, single probiotic preparations usually rely on metabolites to regulate the intestinal microenvironment, but ignore the enhancement of immunomodulatory effects and the effectiveness of barrier repair. In addition, traditional extraction and processing technologies have the problem of high-temperature damage to biological activity, resulting in low yields of functional components in the final product and poor bioavailability, and the multiple benefits of probiotics cannot be fully exerted. Therefore, how to ensure the activity of probiotics while solving the problem of loss of active ingredients during the processing process and improving their comprehensive effects remains an urgent technical problem to be solved. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the present invention provides an intestinal conditioning composition based on compound fruits and vegetables and its process, solving the problems of insufficient immune regulation, degradation of bioactive components, single flora regulation, and low survival rate of probiotics existing in the prior art.

[0005] To achieve the above objectives, the present invention is realized through the following technical solutions: An intestinal conditioning composition based on compound fruits and vegetables, including components in parts by mass:

[0006] Probiotics: 0.005 - 0.03%;

[0007] Inactivated lactic acid bacteria: 0.005 - 0.03%;

[0008] Compound prebiotics: 20 - 30%;

[0009] Sorbitol: 50 - 60%;

[0010] Compound fruit and vegetable powder: 3 - 8%;

[0011] Magnesium stearate: 0.5 - 1.5%;

[0012] Compound coating agent: 2 - 5%.

[0013] Preferably, the probiotics are Lactobacillus mucosae GMNL-263.

[0014] Preferably, the inactivated lactic acid bacteria include at least one of Lactiplantibacillus plantarum Lp18, Bifidobacterium animalis subsp. lactis WKB148, and Lactobacillus casei LC89.

[0015] Preferably, the compound prebiotic includes at least two of inulin, radish seed extract, and fructooligosaccharide.

[0016] Preferably, the raw materials of the compound fruit and vegetable powder include at least three of celery, pumpkin, spinach, carrot, beet, broccoli, banana, tomato, watermelon, lemon, plum, plum, white radish, towel gourd, soybean, litchi, apple, kiwifruit, and balsam pear.

[0017] Preferably, the compound coating agent includes at least two of polyvinyl alcohol, polyglycerol ricinoleate, and polyethylene glycol.

[0018] A preparation process of an intestinal conditioning composition based on compound fruits and vegetables includes the following steps:

[0019] (1) Double-layer embedding treatment of probiotics to obtain embedded probiotics;

[0020] (2) Supercritical CO2 extraction and vacuum freeze-drying treatment of the compound fruit and vegetable powder;

[0021] (3) Mixing and granulating the embedded probiotics, inactivated lactic acid bacteria, compound prebiotic, sorbitol, and compound fruit and vegetable powder;

[0022] (4) Adding magnesium stearate and then tabletting;

[0023] (5) Film coating the tablets with the compound coating agent.

[0024] Preferably, the double-layer embedding treatment in step (1) includes:

[0025] Inner layer embedding: Mixing probiotics with sodium alginate solution and dropping it into CaCl2 solution for cross-linking to form gel microspheres;

[0026] Outer layer embedding: Immersing the gel microspheres in lecithin-cholesterol solution and removing the solvent to form a liposome coating layer.

[0027] Preferably, the pressure of supercritical CO2 extraction in step (2) is 20-35 MPa, and the temperature is 35-50 °C.

[0028] Preferably, the pressure of tabletting in step (4) is 3-8 kN, and the tabletting temperature ≤ 30 °C.

[0029] Beneficial effects:

[0030] 1. The present invention uses 20-30% of compound prebiotics, specifically including inulin, radish seed extract, and fructooligosaccharide, which synergistically act as a carbon source with sorbitol (50-60%). Inulin and fructooligosaccharide selectively proliferate Bifidobacterium, and glucosinolate in radish seeds is metabolized by intestinal flora to generate isothiocyanate, inhibiting the growth of pathogenic bacteria.

[0031] 2. By using the Peltier mold temperature control technology, the tableting temperature is stabilized at 27±1°C, solving the problem of a sharp drop in the viable cell survival rate caused by local high temperature (>45°C) in the traditional process. Compared with the conventional tableting scheme, the present invention increases the survival rate of Streptococcus thermophilus from 30.4% to 87.5%, maintains the tablet hardness above 108N, and reduces the friability to 0.08%.

[0032] 3. Based on the logP value of lycopene, the extraction conditions of 28MPa / 48°C are set, breaking through the bottleneck of polyphenol degradation caused by blindly increasing pressure (≥35MPa) in the traditional scheme. In the prior art, the retention rate of apigenin is often lower than 50%, while the present invention increases it to 94.3%, increases the ORAC value by 61.2%, and reduces the energy consumption by 22%. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a schematic flow chart of the method of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0034] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with 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 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] Please refer to the attached Figure 1 , the embodiments of the present invention provide an intestinal conditioning composition based on compound fruits and vegetables and its process, including:

[0036] Probiotics (0.005-0.03%): Lactobacillus mucosae GMNL-263 is used, and the bacterial cells are protected by double-layer embedding (inner calcium alginate gel and outer lecithin-cholesterol liposome). Calcium alginate protonates in gastric acid to form a dense network (pH-responsive), blocking the penetration of gastric acid; the outer lipid layer isolates oxygen and moisture through hydrophobic interaction, delaying the oxidative inactivation during storage.

[0037] Inactivated lactic acid bacteria (0.005-0.03%): including inactivated bacterial cells such as Lactiplantibacillus plantarum Lp18 and Bifidobacterium animalis subsp. lactis WKB148. The cell wall components of inactivated bacterial cells (such as peptidoglycan) activate the intestinal TLR-2 receptor, promote the secretion of immunoglobulin A (IgA), and enhance the intestinal barrier function.

[0038] Compound prebiotics (20 - 30%): Containing inulin, radish seed extract, and fructooligosaccharide, they synergistically act as a carbon source with sorbitol (50 - 60%). Inulin and fructooligosaccharide selectively proliferate Bifidobacterium. Glucosinolates in radish seeds are metabolized by intestinal flora to generate isothiocyanates, which inhibit the growth of pathogenic bacteria (such as Escherichia coli).

[0039] Compound fruit and vegetable powder (3 - 8%): Containing 18 raw materials such as celery, pumpkin, and bitter gourd, it retains polyphenols and dietary fiber through supercritical CO2 extraction (20 - 35 MPa, 35 - 50 °C). The low-polarity property of supercritical CO2 preferentially extracts fat-soluble antioxidant components (such as lycopene). Dietary fiber increases the fecal volume by adsorbing water and stimulates intestinal peristalsis.

[0040] Compound film-forming agent (2 - 5%): Polyvinyl alcohol and polyethylene glycol are blended to form a flexible film. The hydroxyl groups of polyvinyl alcohol and the ether bonds of polyethylene glycol form a hydrogen bond network to regulate the hydrophilicity of the film, enabling the tablet to disintegrate rapidly (5 - 15 minutes) at intestinal pH (6.8).

[0041] Step 1: Probiotic double-layer embedding process

[0042] Inner layer embedding (calcium alginate gel microspheres): Mix the sodium alginate solution (1.5 - 3.0%) with probiotics and drop it into the CaCl2 solution (1.0 - 3.0%) for crosslinking for 10 - 30 minutes to form microspheres with a diameter of 150 - 300 μm. Ca 2+ Undergoes ionic crosslinking with the carboxyl groups of sodium alginate to form a three-dimensional network structure to encapsulate the bacterial cells. The crosslinking density is controlled by the CaCl2 concentration, which determines the gel pore size (5 - 20 nm) and blocks the penetration of gastric acid (HCl) molecules.

[0043] Outer layer liposome encapsulation: Immerse the microspheres in the lecithin-cholesterol solution (lecithin:cholesterol = 7:3), and remove ethanol by rotary evaporation to form a lipid layer with a thickness of 10 - 30 μm. The hydrophobic tails of lecithin and the rigid ring structure of cholesterol form a dense arrangement, reducing the permeability of the liposome membrane and inhibiting oxygen diffusion (antioxidant); cholesterol regulates the membrane fluidity to avoid lipid crystallization and rupture at low-temperature storage.

[0044] Step 2: Stabilization treatment of compound fruit and vegetable powder

[0045] Supercritical CO2 extraction (20 - 35 MPa, 35 - 50 °C): The crushed fruit and vegetable raw materials are extracted in supercritical CO2 for 2 - 4 hours to separate fat-soluble functional components. CO2 in the supercritical state has both gas diffusivity and liquid solubility, selectively dissolving non-polar components such as lycopene (logP = 8.5) and avoiding the destruction of thermosensitive substances at high temperatures.

[0046] Vacuum freeze-drying (pre-freezing at -45 to -55°C, sublimation drying at 0.01 - 0.1 Pa): The extract is mixed with inulin and then freeze-dried to form a porous powder. Rapid pre-freezing forms tiny ice crystals, reducing mechanical damage to cell walls; vacuum sublimation avoids the presence of liquid water and prevents catalytic degradation by polyphenol oxidase (such as the quinonization of apigenin).

[0047] Step 3: Low-temperature tabletting (pressure 3 - 8 kN, temperature ≤ 30°C)

[0048] The encapsulated probiotics, composite prebiotics, sorbitol, etc. are mixed and granulated (particle size D50 = 100 - 200 μm), and magnesium stearate is added for lubrication and then tabletted. Sorbitol and maltodextrin form a glassy matrix (Tg = 120°C), inhibiting molecular migration; magnesium stearate forms a lubricating film on the surface of the particles, reducing the tabletting friction and avoiding heat inactivation of probiotics caused by local heating.

[0049] Step 4: Film coating (coating solution viscosity 100 - 200 mPa·s)

[0050] Using a fluidized bed coater, a polyvinyl alcohol - polyethylene glycol solution is sprayed onto the surface of the tablet core to form a coating layer with a thickness of 30 - 60 μm. The hydroxyl groups of polyvinyl alcohol and the ether bonds of polyethylene glycol form dynamic hydrogen bonds, regulating the swelling rate of the film; the hydrogen bonds break under the alkaline environment in the intestine, accelerating disintegration.

[0051] Example 1:

[0052] Lactobacillus mucosae GMNL-263: 0.01%

[0053] Inactivated Lactiplantibacillus plantarum Lp18: 0.01%

[0054] Composite prebiotic (inulin + radish seed): 25%

[0055] Sorbitol: 54.2%

[0056] Composite fruit and vegetable powder (celery + pumpkin + bitter gourd): 8%

[0057] Magnesium stearate: 0.79%

[0058] Compound coating agent (polyvinyl alcohol + polyethylene glycol): 3%

[0059] Steps:

[0060] Encapsulated probiotics: The concentration of sodium alginate solution is 2.5%. After mixing with the probiotic suspension, it is dropped into 2% CaCl2 for cross-linking for 20 minutes, and the microsphere diameter is 200 μm.

[0061] The microspheres are soaked in a lecithin - cholesterol solution (7:3), and ethanol is removed by rotary evaporation at 35°C.

[0062] Extracted fruit and vegetable powder: Supercritical CO2 extraction with a pressure of 28 MPa, a temperature of 42 °C, and extraction for 3 hours.

[0063] Freeze-drying parameters: Pre-freezing at -50 °C for 3 hours, and sublimation at a vacuum degree of 0.05 Pa for 24 hours.

[0064] Tabletting: Sorbitol and maltodextrin are dry-blended for 5 minutes, and then embedded bacteria, inactivated bacteria, and wet granulation of fruit and vegetable powder are added (HPMC 4%, addition amount 12%). The tabletting pressure is 5 kN, and the cooling water circulation of the tabletting machine controls the temperature at 28 °C.

[0065] Coating: The viscosity of the coating solution is 150 mPa·s, the spraying rate is 8 mL / min, the inlet air temperature is 45 °C, and the coating thickness is 50 μm.

[0066] Example 2:

[0067] Bifidobacterium animalis subsp. lactis WKB148: 0.02%

[0068] Inactivated Lactobacillus casei LC89: 0.02%

[0069] Compound prebiotics (fructooligosaccharide + inulin): 23%

[0070] Sorbitol: 56%

[0071] Compound fruit and vegetable powder (carrot + tomato + kiwifruit): 7%

[0072] Magnesium stearate: 1.2%

[0073] Compound film-forming agent (polyglycerol ricinoleate): 2.76%

[0074] Steps:

[0075] Double embedding: The concentration of sodium alginate is 1.8%, the cross-linking solution is 1.5% CaCl2, the time is 15 minutes, and the microsphere particle size is 180 μm.

[0076] Liposome solution (lecithin:cholesterol = 6:4) is sprayed on the microspheres and dried at 40 °C.

[0077] Fruit and vegetable treatment: Supercritical CO2 pressure is 32 MPa, temperature is 48 °C, flow rate is 15 L / min, and extraction is for 2.5 hours.

[0078] The final moisture content of freeze-drying is 1.8%.

[0079] Granulation and tabletting: The dry-blending time is 8 minutes, the binder is HPMC 5%, and the granulation particle size D50 = 130 μm.

[0080] The tabletting pressure is 4 kN, the tablet weight is 600 mg, and the tabletting temperature is 25 °C.

[0081] Film coating: The coating solution contains 40% PEG6000, the spraying rate is 6 mL / min, and the outlet air temperature is 38°C.

[0082] Example 3:

[0083] Lactobacillus reuteri GMNL-263: 0.03%

[0084] Inactivated Bifidobacterium WKB148: 0.01%

[0085] Compound prebiotics (radish seeds + fructooligosaccharide): 28%

[0086] Sorbitol: 50%

[0087] Compound fruit and vegetable powder (spinach + broccoli + apple): 5%

[0088] Magnesium stearate: 1.5%

[0089] Compound film-forming agent (polyvinyl alcohol + maltodextrin): 5.46%

[0090] Bacterial cell protection: The concentration of sodium alginate is 3%, the concentration of CaCl2 is 2.5%, the cross-linking time is 25 minutes, and the microsphere diameter is 250 μm.

[0091] The thickness of the liposome coating is 25 μm, and the purity of lecithin is 90%.

[0092] Antioxidant treatment: The pressure of supercritical CO2 is 25 MPa, the temperature is 38°C, and the extraction time is 4 hours. The vacuum degree in the freeze-drying analysis stage is 0.01 Pa, and the temperature is 28°C.

[0093] Low-temperature forming: 0.5% sodium citrate is added during granulation, and the mixing speed is 200 rpm. The tableting pressure is 7 kN, and the surface of the tableting machine die is titanium-plated to prevent sticking.

[0094] Coating process: The coating solution contains 0.1% titanium dioxide (light-shielding agent), the spraying thickness is 60 μm, and the disintegration time is 12 minutes.

[0095] Example 4:

[0096] Lactobacillus casei LC89: 0.005%

[0097] Inactivated Lactobacillus plantarum Lp18: 0.025%

[0098] Compound prebiotics (inulin + galactooligosaccharide): 22%

[0099] Sorbitol: 58%

[0100] Compound fruit and vegetable powder (beet + lemon + soybean): 4%

[0101] Magnesium stearate: 1.0%

[0102] Compound coating agent (polyethylene glycol): 4.97%

[0103] Steps:

[0104] Embedding operation: The concentration of sodium alginate solution is 2%, the crosslinking solution is 1.8% CaCl2, and the microsphere diameter is 150 μm. The proportion of lecithin in the liposome solution is 75%, and the rotary evaporation temperature is 38°C.

[0105] Polyphenol retention: The pressure of supercritical CO2 is 30 MPa, the temperature is 45°C, and the CO2 flow rate is 18 L / min. The pre-freezing rate of freeze-drying is -2°C / min, and the final moisture content is 1.5%.

[0106] Tablet pressing control: The moisture content of the granules is 2.8%, the tablet pressing pressure is 6 kN, and the die temperature is 27°C.

[0107] Tablet hardness is 110 N, and friability is <0.1%.

[0108] Functional coating: 0.05% nano-silica (moisture-proof) is added to the coating solution, and the spraying uniformity deviation is <5%

[0109] Example 5:

[0110] Mixed probiotics (GMNL-263 + LC89): 0.015%

[0111] Inactivated bacteria (Lp18 + WKB148): 0.015%

[0112] Compound prebiotics (radish seed + inulin): 24%

[0113] Sorbitol: 55%

[0114] Compound fruit and vegetable powder (watermelon + plum + litchi): 6%

[0115] Magnesium stearate: 0.8%

[0116] Compound coating agent (polyvinyl alcohol + polyglycerol ricinoleate): 4.17%

[0117] Steps:

[0118] Embedding enhancement: The concentration of sodium alginate is 2.2%, the concentration of CaCl2 is 2%, and the crosslinking time is 18 minutes.

[0119] The proportion of cholesterol in the liposome solution is 35%, and the drying time is 40 minutes.

[0120] Extraction optimization: The pressure of the supercritical CO2 separation kettle is 8 MPa, and the purity of the collected lycopene is 95%.

[0121] The freeze-drying stage is heated in three stages: -40°C → -20°C → 25°C, and the total time is 30 hours.

[0122] Tablet pressing parameters: The Carr Index of the granule fluidity index is 18%, and the tablet pressing pressure is 5.5 kN.

[0123] The surface roughness Ra of the tablet is 1.2 μm, which is beneficial for coating adhesion.

[0124] Coating details: The solid content of the coating solution is 12%, the spraying atomization pressure is 0.3 MPa, and the coating weight gain is 3%.

[0125] Comparative Example 1 (corresponding to Example 1):

[0126] Differences: Double-layer embedding is cancelled, and only single-layer calcium alginate embedding is used.

[0127] Preparation process:

[0128] Single-layer embedding: The concentration of the sodium alginate solution is 2.5%. After mixing with the probiotic suspension, it is directly dropped into 2% CaCl2 for cross-linking for 20 minutes, without outer liposome encapsulation.

[0129] The remaining steps are the same as those in Example 1 (including tablet pressing, coating, etc.).

[0130] Comparative Example 2 (corresponding to Example 2):

[0131] Differences: The supercritical CO2 extraction parameters are out of range (high temperature destroys polyphenols).

[0132] Preparation process:

[0133] Supercritical extraction: The pressure is 38 MPa (32 MPa in the original Example 2), and the temperature is 55 °C (48 °C in the original Example 2), and other parameters are the same.

[0134] Subsequent lyophilization and granulation are the same as those in Example 2.

[0135] Comparative Example 3 (corresponding to Example 3):

[0136] Differences: Use a single prebiotic (only inulin), and cancel the radish seed extract.

[0137] Preparation process:

[0138] Formula adjustment: The composite prebiotic is replaced with pure inulin, accounting for 28% (radish seed + fructooligosaccharide in the original Example 3).

[0139] The embedding and tablet pressing processes are exactly the same as those in Example 3.

[0140] Comparative Example 4 (corresponding to Example 4):

[0141] Differences: The tablet pressing temperature exceeds the standard (high temperature causes probiotic inactivation).

[0142] Tablet pressing parameters: The cooling system of the tablet press was turned off, and the tablet pressing temperature was raised to 45 °C (27 °C in the original Example 4).

[0143] The remaining steps (embedding, coating) were the same as in Example 4.

[0144] Comparative Example 5 (corresponding to Example 5):

[0145] Difference: The compound film-forming agent was cancelled and a single-component coating was used instead.

[0146] Preparation process:

[0147] Coating adjustment: The compound film-forming agent was replaced with a single polyvinyl alcohol (accounting for 4.17%), and there was no polyglycerol ricinoleate.

[0148] The embedding and tablet pressing parameters were the same as those in the original Example 5.

[0149] Comparative Example 6 (corresponding to Example 1):

[0150] Difference: The freeze-drying process was not segmented and was directly analyzed at high temperature.

[0151] Freeze-drying adjustment:

[0152] The segmented sublimation was cancelled, and it was dried at 25 °C / 0.01 Pa for 30 hours throughout the process (the original Example 1 was segmented drying).

[0153] The other steps were the same as in Example 1.

[0154] Comparative Example 7 (corresponding to Example 2):

[0155] Difference: Traditional spray drying was used instead of freeze-drying.

[0156] Drying process: The extraction solution was directly spray-dried (inlet air temperature 180 °C, outlet air temperature 90 °C), and vacuum freeze-drying was cancelled.

[0157] The embedding and tablet pressing were the same as those in the original Example 2.

[0158] Comparative Example 8 (corresponding to Example 3):

[0159] Formulation adjustment: Sodium citrate was removed (0.5% was added in the original Example 3), and the proportion of sorbitol was increased to 55.96% accordingly.

[0160] Other process parameters were exactly the same as those in Example 3.

[0161] Experiment 1: Verification of the double-layer embedding effect of probiotics

[0162] Experiment description:

[0163] Accelerated stability test

[0164] Samples: Example 1 (double-layer embedding), Comparative Example 1 (single-layer embedding), Comparative Example 6 (non-segmented freeze-drying).

[0165] Conditions: 40°C / 75% RH, stored for 6 months.

[0166] Method: Samples were taken monthly, and the viable cell count (CFU / g) was determined by the plate counting method. The dilution gradient was 10 6 -10 8 , and anaerobically cultured at 37°C for 48 hours.

[0167] In vitro digestion simulation

[0168] Gastric juice stage: The tablets were ground and added to simulated gastric juice (0.1 M HCl, pH 1.2, containing 1% pepsin), and shaken at 37°C for 2 hours.

[0169] Intestinal juice stage: The precipitate was taken by centrifugation, added to simulated intestinal juice (0.05 M KH2PO4, pH 6.8, containing 1% trypsin), and continued to shake for 6 hours.

[0170] Detection: Survival rate in gastric juice stage (%) = (viable cell count after gastric treatment / initial viable cell count) × 100%; Intestinal juice release rate (%) = (viable cell count in intestinal juice / viable cell count after gastric treatment) × 100%.

[0171] Table 1 Accelerated stability and in vitro digestion test data

[0172]

[0173]

[0174] The double-layer embedding structure significantly improves the stability of viable bacteria. The pore size of the calcium alginate gel network is precisely controlled at 5 - 20 nm under Ca 2+ crosslinking, making it difficult for gastric acid molecules to penetrate. The hydrophobic chains of lecithin in the outer liposome are closely stacked, reducing the oxygen permeability during storage. The single-layer structure of Comparative Example 1 rapidly erodes in gastric juice, and the viable bacteria are exposed and inactivated. In Comparative Example 6 with non-segmented freeze-drying, due to the large ice crystals, the pores of the embedding layer are damaged, accelerating the infiltration of oxidation.

[0175] The segmented temperature rise in the freeze-drying process allows the water to evaporate in a gradient. In the pre-freezing stage, rapid cooling at -50°C forms micron-sized ice crystals, avoiding the extrusion of bacteria. The residual water escapes in a gaseous state under the vacuum of 0.01 Pa during the analytical drying. The continuous high-temperature drying in Comparative Example 6 destroys the fluidity of the lipid layer, and the membrane structure cracks.

[0176] The difference in intestinal fluid release rate indicates the embedding synergy. The chitosan in Example 1 is deprotonated at pH 6.8, the gel network relaxes, and live bacteria are released explosively. In Comparative Example 6, due to freeze-drying damage, some probiotics are encapsulated in the collapsed structure and the release is delayed. The spatio-temporal control characteristics of the bilayer design increase the live bacteria targeting colonization efficiency by more than 2 times.

[0177] Experiment 2: Optimization and verification of supercritical CO2 extraction parameters

[0178] Experiment description:

[0179] Detection of polyphenol retention rate

[0180] Samples: Example 2 (32 MPa / 48 °C), Comparative Example 2 (38 MPa / 55 °C), Comparative Example 7 (spray drying).

[0181] Method: Determine the contents of lycopene and apigenin by HPLC. Weigh 1 g of the sample, extract it ultrasonically with methanol for 30 minutes, and centrifuge to take the supernatant. Chromatographic conditions: C18 column, mobile phase acetonitrile - water (85:15), flow rate 1 mL / min, detection wavelength 450 nm (lycopene), 340 nm (apigenin).

[0182] Antioxidant activity test

[0183] ORAC method: Mix the sample with fluorescein, add AAPH to initiate the oxidation reaction, and detect the fluorescence decay curve. Calculate the oxygen radical absorbance capacity (μmol TE / g).

[0184] Table 2 Data of polyphenol retention rate and antioxidant activity

[0185]

[0186] The excessive increase in supercritical CO2 extraction parameters leads to irreversible degradation of polyphenol molecules. Under the conditions of 28 MPa / 48 °C in Example 2, the CO2 fluid density is maintained at 650 kg / m 3 , close to the solubility threshold (~1.2 wt%) of lycopene (logP = 18.3), achieving selective extraction. When the pressure in Comparative Example 2 is increased to 38 MPa, the system temperature rises to 55 °C, triggering the acidic hydrolysis (pH 3.2) of the C7-O-β-glucoside bond of apigenin, resulting in a glycoside aglycone loss rate exceeding 47%. In the spray drying group, due to the inlet air temperature reaching 180 °C, exceeding the glass transition temperature of polyphenols (Tg = 145 °C), the Maillard reaction is triggered, and the total phenol retention rate is only 38.6 ± 2.1%.

[0187] The attenuation of antioxidant activity is due to the change in molecular conformation. The all-trans configuration of lycopene (λmax = 472 nm) undergoes cis-isomerization at high temperature (the proportion of cis-isomers ≥ 32%), and the cleavage of the conjugated double bond system leads to a 61.2% decrease in the ORAC value. The 4',5-catechol structure of apigenin undergoes quinone oxidation in a high-pressure CO2 environment (the HPLC peak area of quinones increases by 4.8 times), resulting in the loss of free radical scavenging ability. In Example 2, by controlling the dielectric constant (ε = 1.2), the planar configuration of polyphenol molecules is maintained, and the π-electron delocalization effect makes the ORAC value reach 2850 ± 120 μmol TE / g.

[0188] The process economy needs to comprehensively evaluate efficiency and yield. Although Comparative Example 2 shortens the extraction time by 0.7 hours (ΔT = 22%), the yield of active ingredients decreases by 53%, and the unit energy consumption cost increases by 1.8 times. The specific productivity of the supercritical process under optimized parameters (28 MPa / 48 °C) reaches 4.2 g / (L·h), which is 3.5 times higher than that of spray drying. The latter causes activity loss due to thermal degradation and requires additional purification steps, resulting in a 42% increase in the comprehensive cost.

[0189] Experiment 3: Verification of the synergistic effect of composite prebiotics

[0190] Experiment description:

[0191] In vitro fermentation model

[0192] Samples: Example 3 (radish seed extract + fructooligosaccharide), Comparative Example 3 (single inulin).

[0193] Method: Take fecal samples from healthy people and prepare a 10% bacterial suspension. After mixing with the samples, inject them into anaerobic culture tubes and ferment at 37 °C for 24 hours. Monitor the pH change every 4 hours, and centrifuge to collect the supernatant at the end.

[0194] Detection:

[0195] Bacterial community abundance: The copy numbers of Bifidobacterium (primers Bif-F / R) and Lactobacillus (primers Lac-F / R) are determined by qPCR.

[0196] Short-chain fatty acids (SCFAs): The concentrations of acetic acid, propionic acid, and butyric acid (μg / mL) are determined by GC-MS.

[0197] Table 3 Data on the proliferation of bacterial communities and the generation of SCFAs

[0198]

[0199] Glucosinolates in radish seeds are hydrolyzed by intestinal microbial β-thioglucosidase to generate isothiocyanates. This compound selectively reduces the abundance of Enterobacteriaceae (Δ=-47%) by inhibiting Escherichia coli DNA gyrase (IC50=8.3 μM), creating a niche for the colonization of Bifidobacterium. In Comparative Example 3, although the single inulin group increased the proliferation of Bifidobacterium by 2.8 times, the inhibitory rate of pathogenic bacteria was less than 19%, resulting in the total production of short-chain fatty acids (SCFAs) reaching only 54% of that in Example 3. The difference in butyric acid concentration was the most significant (3.5 vs 1.2 μg / mL). As a histone deacetylase inhibitor (Ki=12 nM), it directly upregulated the expression of the occludin gene in the colonic epithelium by 2.1 times.

[0200] The synergistic effect of the composite prebiotic stems from substrate complementarity. Inulin (DP=23) specifically promotes the production of acetic acid by Bifidobacterium metabolism (r=0.93, p<0.01), while radish seed fiber (containing 35% lignin) mainly generates butyric acid after degradation by Roseburia. The dual-path metabolism reduces the colonic pH from the initial 6.9±0.2 to 5.8±0.3, exceeding the optimal survival range of most putrefactive bacteria (pH 6.0-7.5). The pH in Comparative Example 3 only decreased to 6.3±0.4, resulting in the relative abundance of Enterococcus still maintaining 12.7%.

[0201] The difference in the SCFA profile reflects functional differentiation. In the Example 3 group, the proportion of butyric acid reached 28.5%, which inhibited the NF-κB pathway by activating the GPR109A receptor (IL-8 decreased by 62%), while propionic acid (proportion 19%) entered the liver through the portal vein to regulate the expression of PGC-1α (+37%). In the single inulin group, the proportion of acetic acid exceeded 76% and the butyric acid was less than 8%, and the barrier repair efficiency decreased to 63.2±5.1%. The recovery rate of the TEER value in the composite group (89.4%) was significantly higher than that in the single group (p<0.001), confirming that the synergistic effect is not a simple superposition.

[0202] Experiment 4: Verification of the low-temperature tabletting process

[0203] Experiment description:

[0204] Viable cell heat damage test

[0205] Samples: Example 4 (tabletting temperature 27°C), Comparative Example 4 (tabletting temperature 45°C).

[0206] Method: Immediately after tabletting, take 10 tablets and grind them, dilute them in physiological saline in gradients, coat them on MRS agar plates, and count the viable cell numbers after anaerobic culture at 37°C for 48 hours.

[0207] Calculation: viable bacteria loss rate (%) = (1 - number of viable bacteria after tabletting / number of viable bacteria before tabletting) × 100%.

[0208] Tablet physical property testing

[0209] Hardness: Using a tablet hardness tester (PharmaTest PTB311E), apply vertical pressure until rupture, and record the peak force (N).

[0210] Friability: Take 20 tablets and place them in a friabilator (Erweka TA3R), rotate at 25 rpm for 4 minutes, and calculate the percentage of mass loss.

[0211] Table 4 Effects of tabletting process on viable bacteria and tablet properties

[0212]

[0213] The tabletting process temperature is significantly negatively correlated with the survival rate of probiotics. When the mold temperature exceeds 45 °C, the lipid bilayer of the cell membrane of probiotics such as Streptococcus thermophilus undergoes a phase change (Tm = 41 - 43 °C), resulting in an abnormal increase in membrane permeability (positive rate of PI staining +62%). Part of the sorbitol matrix is transformed into the β-crystal form (XRD 2θ = 18.7°) at a local high temperature (68 °C), forming a dense structure that restricts the nutrient exchange of the bacteria. The viable bacteria survival rate of Comparative Example 4 is only 30.4%, and the tablet hardness drops to 95 N (YB-III type hardness tester), and the friability exceeds 2.3 times the USP standard limit.

[0214] The low-temperature pressing process achieves the balance of structure - function through thermodynamic regulation. Example 4 uses a Peltier mold temperature control system to accurately control the tabletting interface temperature at 27 ± 1 °C (ISO7866:2012). Under this condition, sorbitol maintains a metastable amorphous structure (DSC enthalpy change ΔH = 2.3 J / g), and the porosity is stable at 18.5% to ensure the oxygen exchange of the bacteria. The viable bacteria survival rate is increased to 87.5% (p < 0.001), and the tensile strength of the tablet reaches 1.8 MPa (meeting the Eur.Ph.10.0 standard). The process validation data shows that if Comparative Example 4 is put into mass production, the unqualified rate of the product will reach 12.7%, resulting in a 41.5% increase in the unit cost.

[0215] Experiment 5: Function verification of compound coating agent

[0216] Experiment description:

[0217] Disintegration time test

[0218] Samples: Example 5 (polyvinyl alcohol + polyglycerol ricinoleate), Comparative Example 5 (single polyvinyl alcohol).

[0219] Method: Refer to the Disintegration Test Method 0931 in Chinese Pharmacopoeia. The medium is pH 6.8 phosphate buffer solution, the temperature is 37 ± 1°C, and record the time (minutes) required for the tablets to completely disintegrate.

[0220] Moisture-proof performance test

[0221] Condition: Store in a constant temperature and humidity chamber at 40°C / 75% RH for 30 days.

[0222] Detection: Use a Karl Fischer moisture analyzer to measure the moisture content (%) of the tablets before and after storage, and calculate the increment.

[0223] Table 5 Data of film coating performance and moisture-proof effect

[0224]

[0225] The hydrophobic group of polyglyceryl ricinoleate reverses the hydrophilicity of the film. In the film coating of Example 5, at intestinal pH, the dissociation of the hydroxyl groups of polyvinyl alcohol is enhanced, and the hydrogen bond network loosens. The long-chain alkyl groups of polyglycerol bind to bile salt micelles, accelerating the disintegration of the film layer. The single polyvinyl alcohol film in Comparative Example 5 swells slowly when absorbing water, and the disintegration is extended to 15 minutes, and the lag of viable bacteria release affects the colonization efficiency.

[0226] The difference in moisture-proof performance reveals the strength of intermolecular forces. The ester bond of polyglycerol in the compounded film former forms van der Waals forces with the hydroxyl groups of polyvinyl alcohol, and the penetration path of water molecules is tortuous. There are straight-through pores in the film structure of Comparative Example 5, and moisture invades directly. After 30 days, the moisture content soars to 4.8%, sorbitol absorbs moisture and cakes, and viable bacteria accelerate their death in the local high-moisture environment.

[0227] The film thicknesses are similar but the performances are very different, indicating that material compatibility determines the function. The 52-μm film layer of Example 5 forms a dense amorphous structure due to the good compatibility of the compounded components. Phase separation occurs in the 49-μm film of Comparative Example 5, and island-like crystal regions can be seen under the electron microscope, becoming the breakthrough for water intrusion. The moisture content of the uncoated tablets directly exceeds 9%, and the viable bacteria survival rate during storage will surely collapse.

[0228] Experiment 6: Verification of staged control of freeze-drying process

[0229] Experiment description:

[0230] Powder structure analysis:

[0231] Samples: Example 1 (staged freeze-drying), Comparative Example 6 (non-staged freeze-drying).

[0232] Method: Observe the cross-section of the freeze-dried powder by scanning electron microscope (SEM), with an accelerating voltage of 5 kV and a magnification of 5000×. Measure the average pore diameter (μm) and distribution uniformity (CV%).

[0233] Rehydration test:

[0234] Condition: Weigh 100 mg of the lyophilized powder, add 10 mL of deionized water, stir magnetically (500 rpm), and record the complete dissolution time (seconds).

[0235] Table 6 Data on the Structure and Rehydration Performance of the Lyophilized Powder

[0236]

[0237] In the pre-freezing stage of segmented freeze-drying, rapid cooling at -50 °C forms micron-sized ice crystals. The growth of ice crystals is restricted, the coefficient of variation (CV) of the pore distribution uniformity is only 18.7%, and the average pore diameter is 12.3 μm. This structure is conducive to the stable embedding of the encapsulated microspheres, and water molecules can penetrate rapidly during rehydration. The non-segmented process in Comparative Example 6 results in coarse ice crystals, the pore diameter soars to 28.6 μm and the distribution is disordered (CV 42.5%), and the live bacteria microspheres are damaged during pore collapse.

[0238] The difference in rehydration time directly affects the user experience. The 34-second dissolution of Example 1 is attributed to the capillary effect of uniform pores, and water diffuses rapidly along the honeycomb network. Comparative Example 6 requires 89 seconds, and part of the lyophilized powder forms lumps and can only be dispersed by stirring. The difference in the live bacteria release rate after dissolution is even greater. The release rate of Example 1 reaches 97.5%, while that of Comparative Example 6 is only 72.8%.

[0239] The pore diameter uniformity also affects the storage stability. The dense structure of Example 1 reduces the oxygen penetration path, and the integrity of the lipid coating layer is maintained for a longer time. The large pore diameter in Comparative Example 6 allows oxygen and water to freely enter and exit, and the survival rate of live bacteria decreases rapidly.

[0240] Experiment 7: Verification of the Synergistic Effect between Inactivated Bacteria and Live Bacteria

[0241] Experiment description:

[0242] Immunomodulatory function test

[0243] Samples: Example 5 (live bacteria + inactivated bacteria), Comparative Example 9 (only live bacteria).

[0244] Method: RAW264.7 macrophages were seeded in 96-well plates, and the sample extract (containing 1×10 6 CFU / mL live bacteria or equivalent inactivated bacteria) was added. After culturing for 24 hours, the concentration of TNF-α (pg / mL) in the supernatant was detected by ELISA.

[0245] Intestinal barrier repair test

[0246] Model: A monolayer of Caco-2 cells (TEER ≥ 600 Ω·cm 2 ) was damaged by TNF-α (10 ng / mL) for 6 hours.

[0247] Treatment: Add samples and co-culture for 48 hours, and monitor the recovery rate (%) of transepithelial electrical resistance (TEER) in real time.

[0248] Table 7 Data on Immunomodulation and Intestinal Barrier Repair

[0249]

[0250]

[0251] The peptidoglycan in the cell wall of heat-killed bacteria activates the Toll-like receptor 2 pathway. After macrophages are stimulated, TNF-α is released, and its concentration rises sharply to 356 pg / mL. In Comparative Example 9, there are only live bacteria, and the metabolites mainly induce anti-inflammatory factors such as IL-10, and TNF-α is only 182 pg / mL. Appropriate inflammatory signals are indispensable for initiating intestinal immune defense, but excessive inflammation will damage the barrier. The synergistic effect of Example 5 achieves a balance, activating the immune system without losing control.

[0252] The difference in TEER recovery rate reveals the mechanism of barrier repair. The DNA fragment of heat-killed bacteria promotes the secretion of mucin by goblet cells through the TLR9 pathway. Butyric acid produced by the metabolism of live bacteria directly nourishes intestinal epithelial cells. With the superposition of the two, the expression of ZO-1 protein increases by 2.7 times, and the reconstruction of tight junctions between cells is accelerated. Although the single live bacteria in Comparative Example 9 can produce acid, they lack the guidance of immune signals, and the repair efficiency is only maintained at 63%.

[0253] Heat shock treatment of heat-killed bacteria retains surface antigens. The heat-killing process of Example 5 uses 65°C / 30 minutes, and the flagellin structure is not damaged.

[0254] 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. An intestinal conditioning composition based on composite fruits and vegetables, characterized in that, Components including mass fraction ratios: Probiotics: 0.005 - 0.03%; Inactivated lactic acid bacteria: 0.005 - 0.03%; Compound prebiotics: 20 - 30%; Sorbitol: 50 - 60%; Compound fruit and vegetable powder: 3 - 8%; Magnesium stearate: 0.5 - 1.5%; Compound film-forming agent: 2 - 5%.

2. The intestinal conditioning composition based on compound fruits and vegetables according to claim 1, characterized in that The probiotics are Limosilactobacillus reuteri GMNL-263.

3. The intestinal conditioning composition based on compound fruits and vegetables according to claim 1, characterized in that, The inactivated lactic acid bacteria include at least one of Lactiplantibacillus plantarum Lp18, Bifidobacterium animalis subsp. lactis WKB148, and Lactobacillus casei LC89.

4. The intestinal conditioning composition based on composite fruits and vegetables according to claim 1, characterized in that, The compound prebiotics include at least two of inulin, radish seed extract, and fructooligosaccharide.

5. The intestinal conditioning composition based on compound fruits and vegetables according to claim 1, characterized in that, The raw materials of the compound fruit and vegetable powder include at least three of celery, pumpkin, spinach, carrot, beet, broccoli, banana, tomato, watermelon, lemon, plum, plum, white radish, towel gourd, soybean, litchi, apple, kiwifruit, and balsam pear.

6. The intestinal conditioning composition based on composite fruits and vegetables according to claim 1, characterized in that, The compound film-forming agent includes at least two of polyvinyl alcohol, polyglycerol ricinoleate, and polyethylene glycol.

7. A preparation process of an intestinal conditioning composition based on composite fruits and vegetables, according to any one of claims 1-6, an intestinal conditioning composition based on composite fruits and vegetables, characterized in that, It includes the following steps: (1) Perform double-layer embedding treatment on the probiotics to obtain embedded probiotics; (2) Subject the compound fruit and vegetable powder to supercritical CO2 extraction and vacuum freeze-drying treatment; (3) Mix and granulate the embedded probiotics, inactivated lactic acid bacteria, compound prebiotics, sorbitol, and compound fruit and vegetable powder; (4) Add magnesium stearate and then press into tablets; (5) Perform film coating on the tablets using the compound film-forming agent.

8. The preparation process of an intestinal conditioning composition based on compound fruits and vegetables according to claim 7, characterized in that, The double-layer embedding treatment in step (1) includes: Inner layer embedding: Mix the probiotics with sodium alginate solution and drop it into CaCl2 solution for cross-linking to form gel microspheres; Outer layer embedding: Immerse the gel microspheres in lecithin-cholesterol solution and remove the solvent to form a liposome coating layer.

9. The preparation process of an intestinal conditioning composition based on compound fruits and vegetables according to claim 7, characterized in that, The pressure of supercritical CO2 extraction in step (2) is 20 - 35 MPa, and the temperature is 35 - 50 °C.

10. The preparation process of an intestinal conditioning composition based on composite fruits and vegetables according to claim 7, characterized in that, The pressure of tablet pressing in step (4) is 3 - 8 kN, and the tablet pressing temperature ≤ 30 °C.

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