Preparation method and application of inulin submicrospheres capable of reducing gastrointestinal intolerance

Abstract

The invention belongs to the technical field of polysaccharide food processing, and particularly relates to inulin submicrospheres capable of reducing gastrointestinal intolerance as well as a preparation method and application of the inulin submicrospheres. According to the method, polysaccharide inulin with the molecular weight of 5-30 kDa serves as a raw material, ethyl alcohol serves as an anti-solvent, and the inulin is subjected to micro-nanocrystallization through an N2 microbubble assisted anti-solvent precipitation method; and carrying out vacuum concentration and vacuum drying through a three-stage countercurrent elution process to obtain the inulin submicrospheres capable of reducing gastrointestinal intolerance. Compared with the original inulin, the inulin submicrosphere obtained by the preparation method has the advantages that the particle size is greatly reduced, and the specific surface area is obviously increased; experiments such as in-vitro fermentation simulation and 16S rDNA sequencing prove that the abundance of microbial communities in the in-vitro fermentation bacterial liquid of the inulin submicrospheres is the highest, the diversity is the richer, histamine secretion can be effectively reduced, and the intestinal flatulence phenomenon can be relieved.

Description

Technical Field

[0001] This invention belongs to the field of inulin preparation technology, and specifically relates to an inulin microsphere that can reduce gastrointestinal intolerance, its preparation method, and its application. Background Technology

[0002] Inulin is a water-soluble dietary fiber, a mixture of natural fructans composed of D-fructofuranose linked by β-2,1 glycosidic bonds. The human body lacks the digestive enzymes to break down inulin, so it is not absorbed by the stomach and small intestine and enters the large intestine directly. In the colon, inulin is fermented by beneficial intestinal bacteria such as Bifidobacteria and Lactobacillus, breaking it down into short-chain fatty acids (such as acetic acid, propionic acid, and butyric acid) and gases (such as carbon dioxide and methane). Short-term, high-dose intake of inulin can produce more gas than the intestines can absorb or expel, leading to discomfort such as bloating, abdominal distension, and abdominal pain.

[0003] For inulin, which is a mixture of natural fructans, ensuring the uniformity and stability of the resulting submicrosphere structure is a key challenge. Although patent literature discloses a process for preparing fucoidan using antisolvent precipitation, this technology is not applicable to inulin. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes an inulin submicrosphere that can reduce gastrointestinal intolerance. The inulin submicrospheres provided by this invention are prepared using inulin with a molecular weight of 5-30 kDa as raw material via microbubble-assisted antisolvent crystallization technology. Multi-scale particle size control is achieved through precise regulation of dissolution-crystallization kinetic parameters and the hydrodynamic environment. Furthermore, the spatial conformation of the inulin submicrospheres is modified to increase their specific surface area, thereby reducing the rate of intestinal microbial fermentation of polysaccharides, alleviating intestinal bloating, and significantly reducing the risk of gastrointestinal intolerance.

[0005] The core of the antisolvent precipitation method for preparing inulin subspheres lies in utilizing the difference in solubility of inulin in good solvents and antisolvents. Inulin has good solubility in good solvents, forming a homogeneous solution. When this solution is added to a large amount of antisolvent, the concentration of inulin in the system rapidly exceeds its solubility in the poor solvent due to the extremely low solubility of inulin in the antisolvent, resulting in a supersaturated state and disrupting the thermodynamic equilibrium of the solution system. In the supersaturated state, inulin molecules begin to aggregate, first forming tiny crystal nuclei (nucleation process). Subsequently, more inulin molecules deposit on the surface of the crystal nuclei, causing the nuclei to grow and eventually form subspheres.

[0006] The technical solution of this invention is as follows: A method for preparing inulin submicrospheres that can reduce gastrointestinal intolerance includes the following steps: (1) Using inulin with a molecular weight of 5-30 kDa as raw material, prepare an aqueous solution of 6-8 mg / mL inulin as the aqueous phase; slowly pump the aqueous phase into the antisolvent, and simultaneously add 0.2% of nano-sized lactose seed crystals to the antisolvent, and place it in a 0℃ ice-water bath environment to accelerate the induction of crystallization; at the same time, simultaneously inject micron-sized N2 microbubbles, stir, and crystallize. (2) The reaction system in (1) was subjected to rotary evaporation. When the system volume was reduced to 2 / 3, isotonic replenishment was performed. This process was repeated three times to obtain a monodispersity index (PDI) < 0.30 and a D< 0.30. 50 It is an inulin submicrosphere product with a size of 10-25 μm.

[0007] Preferably, in (1), the volume ratio of the aqueous phase to the antisolvent is 1:18-25; the aqueous phase is slowly pumped into the antisolvent at a flow rate of 15-25 μL / s, wherein the antisolvent is anhydrous ethanol, to ensure that the supersaturation of the solution is in the metastable region; An antisolvent precipitation method is employed, using ethanol as the non-solvent phase to induce phase separation. The difference in miscibility between the solvent (aqueous phase) and the non-solvent (ethanol) enables the directional crystallization of inulin. Compared to traditional antisolvent methods, this process innovatively introduces microbubble-assisted technology: through the localized microturbulence and cavitation effects generated by bubble collapse, millisecond-level mixing of the polysaccharide solution and the antisolvent is achieved, effectively controlling the nucleation kinetics and significantly reducing particle aggregation.

[0008] Micron-sized N2 microbubbles are N2 microbubbles dispersed in a 0.22 μm PTFE membrane with diameters ranging from 10 to 100 μm. By utilizing the transient cavitation effect (peak pressure > 5 MPa) and microscale turbulence (Reynolds number Re ≈ 350) generated by microbubble collapse, molecular-level mixing of the solution-antisolvent system is achieved on a millisecond timescale (mixing efficiency improved by 62%).

[0009] A twin-shaft planetary stirring system is used for stirring at 800-1000 rpm and a shear rate γ = 100-200 s. -1 A mild crystallization field was constructed, and the Ostwald ripening phenomenon was suppressed by regulating the nucleation energy barrier of the β crystal form.

[0010] The volume ratio of aqueous phase to antisolvent is 1:18-25, which balances solvent replacement efficiency and product dispersibility to achieve a solubility-precipitation equilibrium system. The ordered self-assembly of solute molecules is achieved by controlling the gradient surface tension (Δσ=28 mN / m).

[0011] Preferably, in (2), the rotary evaporation conditions are 25-75 mbar and 30-60°C. Isotonic replenishment was performed using 18.2 MΩ·cm ultrapure water.

[0012] (2) A three-stage countercurrent dissolution process was designed: a rotary evaporation system was used for dynamic vacuum concentration (pressure 50 mbar, temperature 45℃). When the system volume was reduced to 2 / 3, isotonic replenishment (ultrapure water, 18.2 MΩ·cm) was implemented. The solvation layer was effectively dissociated through continuous phase microfluidic field reconstruction (solvent removal efficiency > 98%). After three cycles, inulin submicrospheres with a monodispersity index PDI < 0.30 were finally obtained (D 50 =10~25μm).

[0013] In a further preferred embodiment, in (1), the aqueous phase is slowly pumped into the antisolvent at a flow rate of 15 μL / s.

[0014] The application of the above-mentioned inulin submicrospheres in the preparation of products that alleviate gastrointestinal intolerance is also a key protected aspect of this invention.

[0015] Preferably, the above application is selected from at least one of the following: ①Prepare products to reduce the abundance of CO2-producing bacteria; ②Prepare products for reducing histamine metabolism; ③Prepare products to alleviate intestinal bloating and irritable bowel syndrome caused by consuming inulin.

[0016] The present invention has the following advantages and effects compared with the prior art: a. This invention reduces the particle size of modified inulin by anti-solvent precipitation to obtain inulin submicrospheres, thereby changing the particle size of low, medium, and high molecular weight inulin from the original 23.79±3.60μm, 24.05±4.12μm, and 34.70±3.81μm to 13.73±3.49μm, 20.95±4.13μm, and 24.87±1.87μm, while increasing the specific surface area of ​​the inulin submicrospheres; b. Furthermore, the present invention uses low, medium and high molecular weight inulin submicrospheres for in vitro fermentation to slow down the rate of polysaccharide fermentation by intestinal microorganisms and reduce its gas production effect. c. The in vitro fermentation broths obtained from the low, medium, and high molecular weight inulin submicrospheres of this invention, through in vitro fermentation, showed more changes in gut microbiota through 16S rDNA sequencing. The results showed that the high molecular weight inulin submicrosphere group could reduce Firmicutes (… Firmicutes Lactobacillus genus ( ) Lactobacillus The relative abundance of these substances prevents the rapid production of large amounts of CO2, which can significantly improve intestinal bloating. The content of histamine produced by its metabolism is also significantly reduced, while the content of butyric acid, which has intestinal protective function, is significantly increased, which can prevent abdominal pain and other irritable bowel phenomena. Therefore, high molecular weight inulin submicrospheres have the best probiotic effect and the best effect in reducing intestinal intolerance. Attached Figure Description

[0017] Figure 1 Figures showing the particle size analysis of inulin of different molecular weights and their submicrospheres; Figure 2 Scanning electron microscope (SEM) images of inulin and its submicrospheres with different molecular weights are shown. In the images, L represents low molecular weight protoinulin, M represents medium molecular weight protoinulin, H represents high molecular weight protoinulin, L-NP represents low molecular weight inulin submicrospheres, M-NP represents medium molecular weight inulin submicrospheres, and H-NP represents high molecular weight inulin submicrospheres. Figure 3 Electron micrograph of ultra-high molecular weight inulin (UL) and its submicrospheres (UL-NP); Figure 4 The graphs show the total sugar and reducing sugar content of inulin of different molecular weights and their inulin submicrospheres after 24 hours of fermentation. In the graphs, 'a' represents the total sugar content and 'b' represents the reducing sugar content. The abbreviations for different groups are the same. Figure 2 As shown; Figure 5 The graphs show the content analysis of acetic acid, propionic acid, and butyric acid in inulin of different molecular weights and their inulin microspheres after 24 hours of fermentation. In the graphs, a represents the acetic acid content analysis, b represents the propionic acid content analysis, and c represents the butyric acid content analysis. The abbreviations for different groups are the same. Figure 2 As shown; Figure 6 This is a graph showing the gas production content of protoin and inulin subspheres after 24 hours of fermentation. The abbreviations for different groups are the same. Figure 2 As shown; Figure 7 Simpson index diagrams for inulin of different molecular weights and inulin subspheres, where the abbreviations for different groups are the same. Figure 2 As shown; Figure 8 Sankey diagrams for inulin with different molecular weights and inulin submicrosphere intervention groups, where a is the abundance of bacterial communities, b is the phylum level, c is the family level, d is the class level, and e is the genus level. Figure 9 Bubble diagram analysis and histamine content analysis of fermentation metabolites of high molecular weight inulin and submicrospheres are presented. In the figure, a is the bubble diagram of fermentation metabolites, b is the histamine content analysis diagram, H represents high molecular weight inulin, and H-NP represents high molecular weight inulin submicrospheres. Detailed Implementation

[0018] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0019] Example 1: Optimization of the preparation process of inulin submicrospheres The preparation steps of inulin submicrospheres are as follows: (1) Take 0.6g of low (5-7 kDa, L group), medium (8-15 kDa, M group), and high molecular weight (15-30 kDa, H group) inulin respectively and dissolve them in 100 mL of ultrapure water. Add a rotor to the beaker and stir until completely dissolved to prepare an inulin aqueous solution as the aqueous phase. High molecular weight is difficult to dissolve in water, so it can be heated to 50℃ and stirred to dissolve it. (2) Using a peristaltic pump coupled with a PTFE microtube array device, the aqueous solution was infused into the antisolvent high-purity ethanol (anhydrous ethanol, purity 99.9%) at a constant rate of 15 μL / s (±0.5 μL).

[0020] Simultaneously, 0.2% of nano-sized lactose seed crystals (50-200 nm in diameter and 90% purity) were added to the antisolvent and placed in a 0°C ice-water bath environment to accelerate induced crystallization.

[0021] Nitrogen microbubbles (10-100 μm in diameter) dispersed in a 0.22 μm PTFE membrane are injected simultaneously. By utilizing the transient cavitation effect and microscale turbulence generated by the collapse of the microbubbles, molecular-level mixing of the solution-antisolvent system is achieved on a millisecond timescale. A dual-shaft planetary stirring system (900 rpm, shear rate γ = 150 s) is used. -1 A mild crystallization field was constructed, and the Ostwald ripening phenomenon was suppressed by regulating the nucleation energy barrier of the β crystal form. After the inulin aqueous solution was added dropwise, the volume ratio of the aqueous phase to the ethanol phase reached a dissolution equilibrium system of 1:18-25. The ordered self-assembly of solute molecules was achieved by regulating the gradient surface tension. After 180 min, rotary evaporation was carried out to concentrate the solute under reduced pressure. (3) Dynamic vacuum concentration was performed using a rotary evaporation system (pressure 50 mbar, temperature 45℃). When the system volume was reduced to 2 / 3, isotonic replenishment (ultrapure water, 18.2 MΩ·cm) was implemented. The solvation layer was effectively dissociated through continuous phase microfluidic field reconstruction (solvent removal efficiency >98%). After three cycles, submicrospheres with a monodispersity index PDI <0.30 (D) were finally obtained. 50 =10~25μm), respectively, to obtain three groups of inulin submicrospheres (L-NP, M-NP, H-NP) of low, medium and high density.

[0022] Example 2: Identification of Inulin Submicrosphere Structure 2.1 Inulin submicrosphere particle size analysis The particle size of the inulin submicrospheres obtained in Example 1 (low, medium, and high groups) was tested using scanning electron microscopy. The particle size was measured using the scale bar of ImageJ software. The test results are as follows: Figure 1 , Figure 2 And as shown in Table 1, Figure 2In this invention, L, M, and H represent low, medium, and high molecular weight protoin, respectively; L-NP, M-NP, and H-NP represent low, medium, and high molecular weight inulin submicrospheres, respectively. Unless otherwise specified, L, M, H, L-NP, M-NP, and H-NP in each embodiment of this invention have the same meaning.

[0023] Table 1. Results of submicron diameter analysis of proto-inulin and inulin. Grouping Low group / particle size Medium group / particle size High group / particle size Inulin 23.79±3.60μm 24.05±4.12μm 34.70±3.81μm Inulin submicrospheres 13.73±3.49μm 20.95±4.13μm 24.87±1.87μm

[0024] The results showed that the particle size of inulin subspheres in the low, medium, and high groups was significantly smaller than that of inulin. P <0.01 indicates that the inulin submicrospheres obtained by the antisolvent precipitation method in Example 1 have a particle size of 1-100 μm, which meets the requirements of submicrosphere particles. They change from spherical and flake-like to polymeric, with the molecular chains curling up, reducing the specific surface area.

[0025] 2.2 Molecular weight analysis of inulin submicrospheres After preparing the inulin submicrospheres using the optimized scheme in Example 1, the molecular weight (Mw) of low, medium, and high molecular weight inulin and inulin submicrospheres was determined by gel permeation chromatography using tandem PL aquagel-OH (7.5 × 50 mm, 8 μm) and PL aquagel-OH (7.5 × 300 mm, 8 μm) columns and a refractive index detector (RID). The solvent was 0.1 M NaNO3, the flow rate was set to 1 mL / min, and the injection volume was 20 μL. The detection results are shown in Table 2. Table 2. Molecular weight analysis results of protoin and inulin submicrospheres Grouping Low group / molecular weight Middle group / molecular weight High group / molecular weight Inulin 6.994 kDa 8.245 kDa 23.452 kDa Inulin submicrospheres 6.862 kDa 8.041 kDa 22.933 kDa

[0026] Results analysis: The molecular weight of inulin in the low, medium and high groups did not decrease significantly before and after preparation, indicating that the modification process only involved the aggregation state of the inulin molecular chain conformation and did not affect the change in molecular weight.

[0027] Comparative Example 1: Preparation and Analysis of Inulin Subspheres with Other Molecular Weights In Example 1, inulin with a molecular weight of 5-30 kDa was selected for the preparation of inulin subspheres. In this comparative example, inulin with a molecular weight >30 kDa (denoted as UL group) was selected, and inulin subspheres (denoted as UL-NP group) were prepared according to the methods of Examples 1 and 2 and characterized by scanning electron microscopy. Inulin with a molecular weight <5 kDa was not discussed here because its small molecular weight resulted in a too-fast fermentation rate during microbial utilization.

[0028] The results are as follows Figure 3As shown, the particle size of the UL group was 65.50±2.52μm, and the particle size of the UL-NP group was 62.33±3.79μm. The difference between the groups was not significant, indicating that inulin with a particle size >30 kDa is difficult to prepare submicrosphere particles with reduced particle size by antisolvent precipitation.

[0029] Comparative Example 2: Effect of different seed crystals on the crystallization effect of inulin submicrospheres The effects of different seed crystal additions on the crystallization rate and particle size of inulin submicrospheres were investigated during the injection of inulin aqueous solution into the antisolvent ethanol. High-grade inulin was used as the research object, and the effects of two types of seed crystals (homogeneous and heterogeneous) were examined. The homogeneous seed crystals used were inulin seed crystals (particle size 100-300 nm, purity >90%), and the heterogeneous seed crystals used were lactose seed crystals (particle size 50-200 nm, purity >90%) and fructooligosaccharide seed crystals (particle size 10-50 nm, purity >90%). The remaining procedures were the same as in Example 1.

[0030] The results are shown in Table 3 below: Lactose seed crystals crystallized faster than inulin and fructooligosaccharide seed crystals, producing inulin submicrospheres with smaller particle sizes. Inulin seed crystals, with their larger particle size, provide more surface sites for solute molecules to attach at the same supersaturated solute concentration, resulting in relatively larger inulin submicrosphere sizes. In contrast, fructooligosaccharide seed crystals at the same concentration exhibit greater competition within the solute due to their larger quantity, leading to crystal aggregation and thus a relatively larger average particle size.

[0031] Table 3. Effects of different seed crystals on the particle size and crystallization time of inulin submicrospheres index Inulin seeds Lactose seed Fructooligosaccharide seed crystals Submicrosphere average particle size 28.65±7.61μm 24.87±1.87μm 29.34±4.52μm Crystallization time 5.75 h 3.00 h 4.50 h

[0032] Comparative Example 3: Effect of different gas microbubbles on the preparation of inulin submicrospheres The effects of different gas introductions on the preparation of inulin subspheres were investigated during the injection of ethanol as an antisolvent into an inulin aqueous solution. Three common gases—nitrogen, compressed air, and carbon dioxide—were selected. The results (Table 4) showed that with the introduction of gases, the carbon dioxide group lowered the solution pH to approximately 5.9, while the introduction of compressed air and nitrogen had little effect on the solution pH, maintaining it at around 7.0 (6.8–7.1). Lower pH values ​​lead to inulin molecule degradation. Furthermore, the introduction of all gases promoted ethanol evaporation to some extent. Comparing the volume reduction, the compressed air group > carbon dioxide group > nitrogen group. From the perspective of gas diffusion, air, being a mixture of various molecules, exhibits a more complex diffusion pattern at the solution surface, effectively breaking the static layer and promoting the diffusion of ethanol molecules into the gas phase. In contrast, nitrogen molecules have a simpler diffusion pattern at the solution surface, resulting in a weaker promoting effect on ethanol molecule diffusion. Therefore, the nitrogen group showed the least volume reduction. In conclusion, nitrogen is the most effective dispersing gas.

[0033] Table 4. Effects of different gases on the preparation of inulin microspheres index Compressed air carbon dioxide Nitrogen solution pH 6.8 5.9 7.1 Decrease in solution volume most Second least

[0034] Comparative Example 4: Screening of Different Gas Dispersion Membranes 4.1 Screening of different types of gas dispersion membranes The effects of different membrane media on gas dispersion and inulin subsphere crystallization were investigated during the injection of inulin aqueous solution into the antisolvent ethanol. Four gas dispersion membranes were compared, and the results are shown in Table 5. Based on the applicable nitrogen gas requirements of Comparative Example 3, and to meet the requirements of organic phase operating environment, acid and alkali resistance, and low cost, PTFE membrane was selected as the gas dispersion membrane in this invention.

[0035] Table 5 Screening of different gas dispersion membranes Classification Applicable pressure pH tolerance Applicable solvent phase Applicable gases cost Polytetrafluoroethylene (PTFE) membrane <0.5MPa Resistant to strong acids and alkalis Resistant to organic solvents such as ethanol <![CDATA[N2、CO2、Ar]]> Low Polyvinylidene fluoride (PVDF) membrane 0.1~0.5 MPa Acid and alkali resistant Aqueous phase <![CDATA[O2, air]]> high Ceramic membrane 0.5~10 MPa Acid and alkali resistant Tolerance to organic phases <![CDATA[CO2, supercritical fluid]]> high Hollow fiber membrane 0.05~0.2 MPa Not resistant to acids and alkalis Aqueous phase <![CDATA[N2 / CO2]]> Low

[0036] 4.2 Screening of Gas Dispersion Membranes of Different Specifications Based on Comparative Example 4.1 above, different specifications of PTFE membranes were selected to investigate the effect of the formed microbubbles on the mixing effect. Moving bubbles were photographed and their size characterized using a combination of a high-speed camera and a microfocus lens. The results are shown in Table 6. A PTFE membrane with a specification of 0.22 μm can produce relatively small bubbles, which can increase the contact area between the gas and the solution, thus benefiting gas dispersion and mass transfer. A membrane with a specification of 0.1 μm produces relatively smaller bubbles and better dispersion, but has higher gas permeation resistance, and strong pressure can easily cause mechanical damage to the membrane. A PTFE membrane with a specification of 0.5 μm has low gas permeation resistance and high flux, but produces larger bubbles, which is not conducive to uniform gas dispersion and reduces the uniformity of inulin submicrosphere crystallization.

[0037] Table 6 Screening of PTFE membranes of different specifications Membrane pore size specifications bubble size 0.1μm 50~200μm 0.22μm 100~300μm 0.5μm 200~500μm

[0038] Example 3: In vitro fermentation of inulin submicrospheres Microorganisms from the feces of healthy young mice were used as inoculum donors to conduct in vitro simulated fermentation of inulin submicrospheres.

[0039] 3.1 Fermentation process Collect 5g of mouse feces and homogenize it with 50mL of pre-autoclaved PBS phosphate buffer (pH=7.0). Then filter the mixture through four layers of gauze to obtain fecal microbial culture (IGF). Prepare the culture medium using the following ingredients: 2 g / L peptone, 2 g / L yeast extract, 0.05 g / L heme chloride, 0.5 g / L bile salts, 0.1 g / L sodium chloride, 2 g / L sodium bicarbonate, 2 mL Tween, 0.04 g / L potassium dihydrogen phosphate, 0.04 g / L dipotassium hydrogen phosphate, 0.01 g / L calcium chloride, and 0.01 g / L magnesium sulfate. Autoclave the prepared culture medium at 121℃ for 20 min. Add 10 μL of V... K1 Dissolve 0.5 g of L-cysteine ​​hydrochloride in 3 mL of ultrapure water, filter through a 0.22 μm oil-based filter membrane, and then add to the culture medium.

[0040] The low, medium, and high molecular weight inulin submicrospheres and low, medium, and high molecular weight protoin obtained in Example 1 were used to prepare polysaccharide solutions of 150 mg / mL. These solutions were then mixed evenly with 1 mL IGF and 9 mL culture medium in fermentation tubes. Nitrogen gas was introduced to achieve an anaerobic environment, and fermentation was carried out at 37°C for 24 h to obtain in vitro fermentation broths of each inulin and submicrosphere.

[0041] 3.2 Analysis of total sugar and reducing sugar content The total sugar and reducing sugar contents of protoin and inulin submicrospheres after 24 hours of fermentation were analyzed. The methods for determining the total sugar and reducing sugar contents were as follows: (1) Determination of total sugar: The phenol-sulfuric acid method was used (with glucose as standard). 25.00 mg of glucose was weighed and dissolved in an appropriate amount of distilled water, then diluted to 100 mL. The solution was diluted to 2 mL to obtain final glucose concentrations of 0, 12.5, 25, 50, 75, 100, and 125 μg / mL. After mixing, 1 mL of 5% phenol solution and 5 mL of concentrated sulfuric acid were added sequentially, one tube at a time. After standing for 10 min, the solution was placed in a 30℃ water bath for 20 min. After cooling to room temperature, the absorbance at 490 nm was measured, and a standard curve was plotted. 150 mg / mL of each inulin and microsphere in vitro fermentation broth was diluted 100 times, and 2 mL of the diluted solution was taken and determined according to the above method. The total sugar content in the sample was calculated based on the standard curve.

[0042] (2) Method for determining reducing sugars: The reducing sugar assay kit (DNS microplate method) was used. Weigh 1 mL of each inulin and microsphere in vitro fermentation broth, add 14 mL of distilled water, incubate at 50 °C for 30 min, stirring occasionally to ensure complete leaching of reducing sugars. Transfer the precipitate and leachate to a 50 mL centrifuge tube, centrifuge at 4000 g for 5 min, and collect the supernatant. Add 20 mL of distilled water to the precipitate, mix well, and centrifuge again at 4000 g for 5 min, and collect the supernatant. Combine the two supernatants, dilute to 100 mL with distilled water, mix well, and use as the reducing sugar test solution.

[0043] Dilute glucose (Glu) standard: The concentration of glucose (Glu) standard is 1 mg / mL. Take a clean test tube and follow the procedure in Table 1 to obtain a series of Glu standards with different concentrations, as shown in Table 7.

[0044] Table 7. Dilution concentrations of glucose standards Glu 1 Glu 2 Glu 3 Glu 4 Glu 5 Glu standard / mL 0.01 0.02 0.03 0.04 0.05 Distilled water / mL 0.04 0.03 0.02 0.01 0.00 Standard glucose concentration (mg / mL) 0.2 0.4 0.6 0.8 1.0

[0045] Sample addition (Table 8): For each inulin and submicron reducing sugar test solution, set up blank, standard and test wells according to Table 2. Add the solution to 1mL centrifuge tubes in sequence, taking care to avoid generating air bubbles, and mix carefully.

[0046] Table 8. Sampling Table for Each Solution blank hole Standard Hole Measurement well Distilled water / μL 50 - - Series of Glu standards / μL - 50 - Each inulin reducing sugar test solution / μL - - 50 DNS detection solution / μL 100 100 100

[0047] Boil the prepared solution in a 1mL centrifuge tube in a boiling water bath for 5 minutes, remove it, cool it to room temperature with tap water, add 250μL of distilled water, and mix well.

[0048] Reducing sugar determination: 300 μL was sequentially transferred to the corresponding 96-well plate, and the absorbance of the standard well and the test well at 540 nm was measured using the blank well as the zeroing method.

[0049] like Figure 4 As shown in Table 9: Table 9. Analysis results of total sugar content of protoin and inulin submicrospheres after 24 hours of fermentation. Grouping Low group / total sugar content Middle group / total sugar content High group / total sugar content Inulin 6.29±0.38 6.26±0.40 7.09±0.43 Inulin submicrospheres 4.08±0.37 6.89±0.45 6.73±0.55

[0050] Table 10. Analysis results of reducing sugar content in protoin and inulin submicrospheres after 24 hours of fermentation. Grouping Low group / reducing sugar content Medium group / reducing sugar content High group / reducing sugar content Inulin 1.58±0.01 1.77±0.21 1.72±0.05 Inulin submicrospheres 1.26±0.17 1.23±0.03 1.55±0.19

[0051] Results analysis: After 24 hours of fermentation, the total sugar content of different inulin submicrosphere groups was as follows: Figure 4 The levels of a in the middle were significantly lower than before fermentation ( P<0.01), among which the low molecular weight inulin submicrosphere group showed the most significant decrease after fermentation compared to other inulin groups ( P <0.01), and there were no significant differences among the groups after 24 hours of fermentation in other groups. P >0.05), which is related to the fact that low molecular weight inulin is more easily utilized by microorganisms. Changes in reducing sugar content are as follows: Figure 4 Analysis of the b-values ​​showed that after preparation, the amount of reducing sugar produced in each group decreased significantly compared to before preparation. P <0.01) indicates that the fermentation rate has slowed down, which helps to alleviate the negative effects of excessive gas production caused by excessively rapid inulin fermentation.

[0052] 3.3 Analysis of short-chain fatty acid content in protoinulin and inulin submicrospheres after 24 h of fermentation The detection methods for acetic acid, propionic acid, and butyric acid in short-chain fatty acids are as follows: Preparation of standard solutions: Take 200 µL of acetic acid, propionic acid, and butyric acid standards into 10 mL volumetric flasks, and dilute to volume to prepare single standard stock solutions; take 1 mL of each single standard stock solution into the same 10 mL volumetric flask to prepare mixed standard stock solutions; take the mixed standard stock solutions and dilute them 2-fold to prepare 6 concentrations, namely 0, 2, 4, 8, and 16, to prepare standard test solutions; take 1 mL of each solution and filter it through a 0.22 μm filter membrane into a sample vial for testing.

[0053] Sample solution preparation: Take 1 mL of fermentation broth and centrifuge at 12000 rpm for 10 min at 4℃. Take 900 μL of the supernatant into a centrifuge tube, add 180 μL of 25% metaphosphoric acid, mix well, acidify at 4℃ for 1 h, centrifuge at 12000 rpm for 10 min, and take 1 mL of the supernatant through a 0.22 µm filter into a sample vial for testing.

[0054] The composition and content of short-chain fatty acids were determined by gas chromatography (GC): Chromatographic conditions: flame ionization (FID) detector, detector temperature 250℃; carrier gas nitrogen, flow rate 30 mL / min, split ratio 1:10; column temperature 100℃, retention time 1 min, temperature ramped to 250℃ at a rate of 4℃ / min; injection port temperature 250℃; hydrogen flow rate 60 mL / min, air flow rate 300 mL / min.

[0055] like Figure 5 As shown in Table 11-13: Table 11. Relative concentrations of protoin and inulin microspheres of acetic acid after 24 hours of fermentation. Figure 5 a) Content analysis results Grouping Low group / acetic acid content Middle group / acetic acid content High group / acetic acid content Inulin 0.58±0.06μg / mL 0.57±0.04μg / mL 0.45±0.07μg / mL Inulin submicrospheres 0.61±0.05μg / mL 0.56±0.07μg / mL 0.44±0.06μg / mL

[0056] Table 12. Protoin and inulin submicrosphere propionic acid after 24 hours of fermentation ( Figure 5 b) Content analysis results Grouping Low group / propionic acid content Medium group / propionic acid content High group / propionic acid content Inulin 0.072±0.008μg / mL 0.081±0.009μg / mL 0.076±0.011μg / mL Inulin submicrospheres 0.076±0.013μg / mL 0.077±0.012μg / mL 0.081±0.011μg / mL

[0057] Table 13. Protoinulin and inulin submicrosphere butyric acid after 24 hours of fermentation ( Figure 5 c) Content analysis results Grouping Low group / butyric acid content butyric acid content High butyric acid content Inulin 0.14±0.03μg / mL 0.17±0.03μg / mL 0.21±0.01μg / mL Inulin submicrospheres 0.177±0.01μg / mL 0.20±0.02μg / mL 0.23±0.01μg / mL

[0058] Results analysis: The amount of acetic acid produced by inulin subspheres obtained via antisolvent precipitation showed no significant change among the groups; the amount of propionic acid produced decreased in the low molecular weight inulin subsphere group compared to the low molecular weight inulin group; while the amount of butyric acid produced increased significantly, with the most significant increase observed in the high molecular weight inulin subsphere group. P <0.01).

[0059] 3.4 Analysis of gas production content of protoin and inulin submicrospheres after 24 hours of fermentation After 24 hours of fermentation, the gas production of low, medium, and high molecular weight inulin and submicrospheres was measured using a differential pressure gauge. Figure 6 As shown in Table 14: Table 14. Analysis results of gas production content of protoin and inulin subspheres after 24 hours of fermentation. Grouping Low group / gas production content Middle group / gas production content High group / gas production content Inulin 2.81±0.2mL 2.01±0.19 mL 2.18±0.15 mL Inulin submicrospheres 2.81±0.4 mL 2.6 ± 0.3 mL 0.8±0.24 mL

[0060] Results analysis: The gas production of the low molecular weight inulin submicrosphere group showed no significant change compared to the low molecular weight inulin group, which may be related to the fact that the particle size did not change significantly before and after nano-sizing; the gas production of the medium molecular weight inulin submicrosphere group showed an increasing trend compared to before preparation. P <0.01), while the gas production of the high molecular weight inulin submicrosphere group decreased significantly ( P <0.001).

[0061] Based on the comprehensive analysis of short-chain fatty acid and gas production content after fermentation, the high molecular weight inulin submicrosphere group showed better probiotic effects and lower gas production compared to other groups, making it a more ideal dietary fiber substrate that can be used for subsequent research.

[0062] Example 4 Microbial Analysis of Inulin Submicrospheres The inulin submicrospheres prepared using the optimized scheme in Example 1 were used to determine the bacterial community structure of the inulin fermentation broth (low, medium, and high concentrations) obtained in Example 3 after 24 hours of fermentation with the submicrospheres. Gut microbiota analysis was performed based on 16S rDNA sequencing. All bacterial samples were sent to a testing company for gut microbiota classification analysis. PCR amplification was performed on the V3-V4 region of the 16S rDNA, followed by high-throughput sequencing and bioinformatics analysis using a sequencing platform. All analytical results are based on sequencing reads and operational taxonomic units (OTUs).

[0063] 4.1 Microbial diversity analysis Results analysis: such as Figure 7 The Simpson index, which represents the diversity of the microbial community, is negatively correlated with the numerical value. Comparing different inulin groups, the high molecular weight inulin submicrosphere group showed the most significant Simpson index, indicating that this group had the highest microbial community abundance and diversity.

[0064] 4.2 Analysis of differences among microbiomes Results Analysis: Based on the Sankey diagram (Sankey) Figure 8 The results showed that the high molecular weight inulin submicrospheres were predominantly Proteobacteria (POPs). Proteobacteria Especially Escherichia coli and Shigella genus ( Escherichia-Shigella The main phylum is acetic acid, and the differences in the metabolism of acetic acid and lactic acid among the groups are not significant; the second largest phylum is Firmicutes (…). Firmicutes Lactobacillus genus ( Lactobacillus (This can produce CO2.) In summary, the high molecular weight inulin submicrosphere group significantly reduced gas production, which can reduce intestinal flatulence.

[0065] Example 5 Analysis of metabolites from inulin submicrosphere fermentation The fermentation products from Example 3 were subjected to untargeted metabolomics sequencing. The specific steps were as follows: Transfer 50 μL of sample to an EP tube, add 200 μL of extraction buffer (methanol:acetonitrile = 1:1 (V / V)), the extraction buffer contains an isotope-labeled internal standard; vortex for 30 s, sonicate for 10 min (ice-water bath); incubate at -40 ℃ for 1 h; centrifuge the sample at 4 ℃ and 13800 × g for 15 min; collect the supernatant in a sample vial for analysis; a separate QC sample is prepared by mixing equal volumes of supernatant from all samples for analysis. For polar metabolites, this project uses a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph, employing a Waters ACQUITY UPLC BEH Amide (2.1 mm × 50 mm, 1.7 μm) column for chromatographic separation of target compounds. Phase A of the liquid chromatography was aqueous, containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia; Phase B was acetonitrile. Sample tray temperature: 4 ℃; injection volume: 2 μL.

[0066] The Orbitrap Exploris 120 mass spectrometer was used for primary and secondary mass spectrometry data acquisition under the control of software (Xcalibur, version 4.4, Thermo). Detailed parameters are as follows: sheath gas flow rate: 50 Arb, auxiliary gas flow rate: 15 Arb, capillary temperature: 320 ℃, full scan (primary mass spectrometry) resolution: 60000, MS / MS resolution: 15000, collision energy: SNCE 20 / 30 / 40, spray voltage: 3.8 kV (positive ion) or -3.4 kV (negative ion). Raw data were converted to mzXML format using ProteoWizard software, and metabolite identification was performed using a collaboratively developed R package. The database used was BiotreeDB (V3.0), followed by visualization analysis using a self-developed R package.

[0067] Results analysis: Based on non-targeted omics analysis of fermentation broth, bubble diagrams of differentially metabolites were analyzed. Figure 9 The results showed significant changes in histidine metabolism. Further detailed analysis of histidine metabolism-related metabolites revealed that the high molecular weight inulin submicrosphere group had reduced histamine secretion from microbial sources compared to the original inulin group. Histamine is one of the causes of gastrointestinal intolerance, and this change can effectively reduce the risk of gastrointestinal intolerance.

[0068] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. All equivalent changes and modifications made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. A sub-microsphere of inulin which can reduce gastrointestinal intolerance, characterized in that, The inulin sub-micro-spheres are prepared from inulin with molecular weight of 5-30 kDa as raw material, and have monodispersity index PDI < 0.30, D 50 10~25 μm.

2. The method of producing inulin submicrospheres capable of reducing gastrointestinal intolerance according to claim 1, characterized in that, The steps include the following: (1) Using inulin with a molecular weight of 5-30 kDa as raw material, prepare an inulin aqueous solution with a concentration of 6-8 mg / mL as the water phase; slowly pump the water phase into the anti-solvent, simultaneously add 0.2% nano-sized lactose seed crystals into the anti-solvent, and induce crystallization; At the same time, inject micron-sized N2 micro-bubbles to disperse the seed crystals, continuously stir for 180 min, and the anti-solvent is an organic solvent; (2) The reaction system in (1) is spin-evaporated, and when the system volume is reduced to 2 / 3, isotonic liquid is added, and the process is repeated three times to obtain inulin sub-microspheres with a monodispersity index PDI < 0.30, D 50 The inulin sub-microspheres have a diameter of 10-25 μm and can reduce gastrointestinal intolerance.

3. The production method according to claim 2, characterized by, In (1), the volume ratio of the water phase to the anti-solvent is 1:18-25; slowly pump the water phase into the anti-solvent at a flow rate of 15-25 μL / s, wherein the anti-solvent is anhydrous ethanol; The nano-sized lactose seed crystals have a seed crystal particle size of 50-200 nm and a purity of >90%; The micron-sized N2 micro-bubbles are N2 micro-bubbles dispersed through a 0.22 μm polytetrafluoroethylene membrane, and the diameter is 10-100 μm; The double shaft planetary stirring system was used during stirring, at 800-1000 rpm, shear rate γ = 100-200 s -1 The mild crystallization field is constructed below.

4. The production method according to claim 2, characterized by, In (2), the rotary evaporation is performed at 25-75 mbar and 30-60°C; Use ultrapure water for isotonic replacement.

5. Use of the inulin sub-microspheres as described in claim 1 in the preparation of a product for relieving gastrointestinal intolerance.

6. Use as claimed in claim 5, characterised in that, The product for relieving gastrointestinal intolerance at least includes any one of the following products: ① A product for reducing the abundance of CO2 generating bacteria; ② A product for reducing the metabolic production of histamine; ③ A product for relieving intestinal distension and intestinal irritability caused by eating inulin.

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