Limonin-pectin emulsion gel with anti-inflammatory and intestinal probiotic functions as well as preparation and application of limonin-pectin emulsion gel

Abstract

The invention belongs to the technical field of food processing of pectin, and discloses limonin-pectin emulsion gel with anti-inflammatory and intestinal probiotic functions as well as preparation and application of the limonin-pectin emulsion gel. The method comprises the following steps: 1) dissolving pectin with the esterification degree of 10-40% and the RGI proportion of 20-40% in water to obtain a pectin solution; adjusting the pH value to 3-6, adding a calcium ion solution, and shearing to obtain pectin sol; 2) dissolving limonin in the oil phase to obtain an oil phase containing limonin; and adding the oil phase containing the limonin into the pectin sol, carrying out shearing treatment, and standing to obtain the pectin emulsion gel loaded with the limonin. The emulsion gel provided by the invention can be independently used as an emulsifier, can stably load limonin, and realizes relatively high embedding efficiency. The emulsion gel disclosed by the invention can be used for remarkably regulating intestinal flora and increasing the content of short-chain fatty acid, and has remarkable anti-inflammatory and prebiotic activity. The emulsion gel provided by the invention is used for anti-inflammatory and intestinal probiotic functional products.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing, and in particular relates to a limonin-pectin emulsion gel with anti-inflammatory and intestinal probiotic functions, and a preparation method and application thereof. Background Art

[0002] Pectin is a natural polysaccharide that is widely found in plant cell walls, especially in the peel of citrus fruits and apples. It is composed of galacturonic acid units and forms different structures through partial methylation. Pectin is divided into high-ester pectin (HMP) and low-ester pectin (LMP) according to the degree of esterification. HMP requires low pH and high sugar content to gel, while LMP can form gel in the presence of calcium ions and is suitable for low-sugar or sugar-free foods. Pectin has thickening, stabilizing, gelling and emulsifying properties and is widely used in the food industry, such as jams, jellies, yogurt and other products. In addition, pectin also has health benefits, such as lowering cholesterol and improving intestinal function.

[0003] Limonin is a tetracyclic triterpenoid compound that is abundant in citrus plants. It has been shown to have biological activities such as antioxidant, anti-inflammatory, anti-cancer, and regulation of blood sugar and blood lipids. Limonin is used as a food additive and functional ingredient in the food industry, and also has application prospects as a therapeutic agent in the medical field. However, since the bioavailability of limonin is limited by its instability and rapid metabolism in the gastrointestinal tract, its bitter taste may affect the acceptance of food. Therefore, it is often improved by encapsulation or compounding with other ingredients in the food industry.

[0004] Pectin emulsion gel is a three-dimensional network structure formed by physical or chemical crosslinking in an emulsion system using the polymer properties of pectin. It has excellent stability and controllable release characteristics. The emulsion gel system can evenly disperse the active ingredients, and the release rate of the active ingredients can be precisely controlled by adjusting the concentration, pH value and ionic strength of pectin. Due to the natural origin and biocompatibility of pectin, emulsion gel has significant advantages as a carrier for nutritional protection and sensory experience improvement in the food industry, and also shows potential for application as a drug delivery system in the medical field.

[0005] The structure of pectin is not only highly related to its gel properties, but also significantly affects its functional activity. Existing pectin emulsion gel systems only focus on the effect of esterification degree on system stability, and require the addition of multiple components such as sugar alcohols and proteins to work together to have effective loading capacity. There are defects such as unclear structure-activity relationship, single efficacy and complex operation. Different modification and extraction treatment methods will directly affect the structure of pectin, and then affect the performance of emulsion gel.

[0006] The present invention obtains pectins with different esterification degrees and RGI domain proportions by adjusting different extraction conditions; and improves the emulsification and gelling ability of pectin without the help of other biomacromolecules by combining the optimal esterification degree and RGI domain proportion, and at the same time, provides a stable high-load embedding system for limonin. The system reduces the degradation of limonin during processing and storage, masks the bitter taste, optimizes the taste of food, and significantly improves its bioavailability. The embedding of the pectin emulsion gel of the present invention helps limonin reach the colon, and cooperates with pectin to exert anti-inflammatory and prebiotic activities. Summary of the invention

[0007] In view of the shortcomings and single efficacy of the above prior arts, the purpose of the present invention is to provide a limonin-pectin emulsion gel with anti-inflammatory and intestinal prebiotic functions, and a preparation method and application thereof. The method of the present invention is simple, and the optimal combination of specific esterification degree and RGI domain ratio can significantly improve the emulsification ability of pectin, so that it can independently form an emulsion gel and stably load limonin without adding other biological macromolecules, thereby playing an active role in regulating intestinal flora. The specific esterification degree and RGI domain ratio of the present invention not only have excellent ability to regulate intestinal flora, but also can promote hydrogen bonding and enhance the stability and loading performance of the gel network, improve the efficient encapsulation of active substances and stable targeted delivery to the colon. Therefore, the present invention only needs to use Ca 2+ , it can efficiently induce RGI-rich pectin to form an emulsion gel.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] A method for preparing a limonin-pectin emulsion gel with anti-inflammatory and intestinal probiotic functions comprises the following steps:

[0010] 1) dissolving pectin with an esterification degree of 10-50% and an RGI ratio of 20-40% in water to obtain a pectin solution; adjusting the pH of the pectin solution to 3-6, adding a calcium ion solution, and subjecting the solution to shearing to obtain a pectin sol;

[0011] 2) dissolving limonin in the oil phase to obtain an oil phase containing limonin; then adding the oil phase containing limonin to the pectin sol, subjecting it to shearing treatment, and allowing it to stand to obtain a pectin emulsion gel loaded with limonin.

[0012] In step 1), the degree of esterification is preferably 20-35%, and the proportion of RGI type I is preferably 23-40%. More preferably, the degree of esterification is 24-26%, and the proportion of RGI is 37-39%.

[0013] The pectin solution in step 1) is prepared by uniformly mixing pectin and water and hydrating for 8 to 24 hours.

[0014] The mass volume percentage concentration of pectin and water in the pectin solution in step 1) is 0.5-2%, g / mL, and more preferably, the concentration is 1.5%.

[0015] The acid used to adjust the pH of the pectin solution in step 1) is at least one of nitric acid, oxalic acid, citric acid and hydrochloric acid solution, preferably citric acid.

[0016] The pH in step 1) is 4.5 to 5.5, preferably, the pH is 5.

[0017] The source of calcium ions in step 1) is at least one of calcium chloride, calcium carbonate, calcium citrate, and calcium oxalate, and more preferably, calcium chloride.

[0018] The concentration of calcium ions in the pectin sol in step 1) is 0.1-1 mmol / L, and more preferably, the concentration is 0.2-0.6 mmol / L.

[0019] The rotation speed of the shearing treatment in step 1) is 10000-15000 rpm, and the time is 1-2 min.

[0020] The oil phase in step 2) is vegetable oil, such as soybean oil, sunflower oil, corn oil, etc.; more preferably soybean oil.

[0021] In step 2), the concentration of limonin in the oil phase is 200-300 mg / kg.

[0022] The mass fraction of the oil phase in the limonin-loaded pectin emulsion gel in step 2) is 10-40%, and more preferably 15-25%.

[0023] The rotation speed of the shearing treatment in step 2) is 10000-20000 rpm, and the time is 2-5 min.

[0024] The standing temperature in step 2) is 0 to 10° C. and the standing time is 12 to 24 hours.

[0025] The pectin in step 1) is prepared by the following method:

[0026] S1. Mix citrus peel residue with water, adjust the pH to 1.5-2.5, extract at 65-75° C. for 1-2 hours, centrifuge, take the supernatant, and precipitate with alcohol to obtain primary pectin; the alcohol precipitation is carried out with ethanol; after the alcohol precipitation, the precipitate is filtered, dried, and crushed;

[0027] S2, re-dissolve the primary pectin, add pectin methylesterase, adjust the pH to 3-5, react at 40-60°C for 1-2.5 hours, centrifuge, take the supernatant, precipitate with alcohol, and obtain pectin. After alcohol precipitation, the precipitate is dried and crushed.

[0028] The citrus peel residue described in step S1 is citrus peel, lemon peel, apple peel and grapefruit peel, preferably citrus peel.

[0029] The pH in step S1 is 2, and the extraction is carried out at 70° C. for 2 hours.

[0030] After the primary pectin is redissolved in step S2, the mass volume ratio of the primary pectin to water is 2-3 g:100 mL.

[0031] The amount of pectin methylesterase added in step S2 is 150-205 U / mL. In step S2, the pH is adjusted to 3-4, and the reaction is carried out at 45-55° C. for 1.5-2 hours.

[0032] The application of the limonin-loaded pectin emulsion gel in the preparation of anti-inflammatory and intestinal probiotic functional products.

[0033] The limonin-pectin emulsion gel is used in a preparation for promoting microbial fermentation to produce short-chain fatty acids.

[0034] The invention sterilizes an anaerobic culture medium at high temperature and high pressure and cools it, then inoculates a bacterial suspension of healthy people and a limonin-pectin emulsion gel in an anaerobic workstation for fermentation to obtain a fermented sample.

[0035] The added amount of the emulsion gel is 0.5-1.5%.

[0036] The pectin emulsion gel prepared by the invention does not flow when inverted, has a high limonin loading rate, has stable gel properties and emulsion stability, and has significant anti-inflammatory and prebiotic effects of regulating intestinal flora after fermentation for 24 hours.

[0037] Compared with the prior art, the present invention has the following advantages:

[0038] (1) Due to the limitation of water solubility, the bioavailability of limonin during oral administration is not ideal. The traditional pectin emulsion gel is not stable enough and it is difficult to completely encapsulate limonin. The present invention adopts the optimal combination of specific esterification degree and RGI domain ratio and combines calcium ions to successfully prepare a stable and high-loaded pectin emulsion gel. This gel not only effectively solves the stability problem of the emulsion, but also overcomes the limitations of traditional hydrogels in delivering hydrophilic substances. The emulsion gel prepared by the method of the present invention can be used alone as an emulsifier to stably load limonin and achieve a higher encapsulation efficiency.

[0039] (2) The limonin-pectin emulsion gel of the present invention can significantly regulate intestinal flora, increase the content of short-chain fatty acids, and has significant anti-inflammatory and prebiotic activities.

[0040] (3) The method of the present invention is simple, easy to operate, economical, and practical.

[0041] The invention effectively expands the application field of pectin, and enables it to show broad application potential and market prospects in the preparation of food, health products and medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a graph showing the emulsification stability test results of the limonin-pectin emulsion gels with anti-inflammatory and intestinal probiotic functions prepared in Examples 1 to 3 and Comparative Examples 1 to 3;

[0043] Figure 2 This is a graph showing the results of measuring the embedding rate of the limonin-pectin emulsion gels with anti-inflammatory and intestinal probiotic functions prepared in Examples 1 to 3 and Comparative Examples 1 to 3;

[0044] Figure 3 The results of the determination of the rheological properties of the limonin-pectin emulsion gels with anti-inflammatory and intestinal probiotic functions prepared in Examples 1 to 3 and Comparative Examples 1 to 3;

[0045] Figure 4 A columnar accumulation diagram of the abundance of intestinal flora at the genus level of the limonin-pectin emulsion gel with anti-inflammatory and intestinal probiotic functions prepared in Examples 1 to 3 and Comparative Examples 1 to 3 after 24 hours of in vitro fermentation;

[0046] Figure 5 Changes in the total short-chain fatty acid content of the limonin-pectin emulsion gels with anti-inflammatory and intestinal probiotic functions prepared in Examples 1 to 3 and Comparative Examples 1 to 3 after in vitro fermentation for 24 hours. DETAILED DESCRIPTION

[0047] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0048] The determination method involved in the embodiment:

[0049] 1. The method for determining the degree of esterification is based on the chemical titration method in the national food safety standard GB25533-2010.

[0050] 2. Determination of monosaccharide composition: 10 mg of citrus pectin was added with 5 mL of trifluoroacetic acid and acid hydrolyzed at 121°C for 1.5 h. The trifluoroacetic acid was blown dry with a nitrogen blower, and 99.99% methanol was added for washing. The solution was then blown dry and the volume was fixed to 10 mL. The monosaccharide composition was determined using a Thermo ICS 5000+ ion chromatography system.

[0051] 3. Emulsion stability determination method: Accurately weigh a certain amount of emulsion gel and place it in a centrifuge tube and centrifuge it at 3000 revolutions per minute (rpm) for 10 minutes. After the centrifugation, observe and record the stratification phenomenon of the emulsion gel in detail to evaluate its emulsion stability.

[0052] 4. Determination of the embedding rate of limonin in the emulsion gel: Take 0.1 mg of the emulsion gel and dilute it 5 times with dimethyl sulfoxide (DMSO), then centrifuge and take the supernatant. Use the Ehrlich reagent method to measure the concentration of limonin in the solution, and finally calculate the embedding rate based on the measured concentration and the initial addition amount.

[0053]

[0054] 5. 16S rDNA sequencing: After the fermentation liquid was centrifuged for 24 hours, the precipitate was taken for 16S rDNA sequencing.

[0055] 6. Determination of short-chain fatty acid content: Ultra-high performance liquid chromatography-tandem mass spectrometry was used to determine the content of short-chain fatty acids in the fermentation broth.

[0056] 7. Determination of the inhibition rate of inflammatory factors: Use lipopolysaccharide (LPS) to intervene in macrophages to establish an inflammatory cell model and evaluate the anti-inflammatory effect of pectin emulsion gel loaded with limonin.

[0057] Macrophages (RAW264.7) with good growth status were taken and 1×10 6 RAW264.7 cells were seeded in 6-well plates at a density of 10 cells / well, 2 mL of culture medium was added to each well, and the plates were incubated at 37°C for 24 h.

[0058] The experiment was divided into a blank group, a model group, and an intervention group (each embodiment and a comparative example). The blank group: cells, no LPS, no emulsion gel; the model group: cells, LPS (2 μg / mL), no emulsion gel; the intervention group: pectin emulsion gel loaded with limonin (final concentration of 1%) of each embodiment and comparative example was added respectively, and LPS with a final concentration of 2 μg / mL was added after 2 hours of action, and the cell culture medium was collected after the culture was continued for 2 to 4 hours;

[0059] The ELISA detection kit was used to quantitatively detect the content of inflammatory factors (TNF-α and IL-6) in the cell culture fluid of each group according to the operating instructions.

[0060]

[0061] C 模型 is the content of inflammatory factors in the cell supernatant of the model group, pg / mL;

[0062] C 干预 is the content of inflammatory factors in the cell supernatant of the intervention group, pg / mL;

[0063] C 空白 is the content of inflammatory factors in the cell supernatant of the blank group, pg / mL.

[0064] Example 1

[0065] (1) Mixing citrus peel residue with water (the mass ratio of citrus peel residue to water is 1:20), adjusting the pH to 2, extracting at 70°C for 2 hours, centrifuging, taking the supernatant, precipitating with ethanol, washing the precipitate, filtering, drying and crushing to obtain pectin powder; re-dissolving the pectin powder (the mass volume ratio of pectin powder to water is 2g:100mL), adding 150U / mL of pectin methylesterase, adjusting the pH to 3.5, reacting at 50°C for 1.5 hours, precipitating with alcohol, washing, and drying to obtain pectin with an esterification degree of 32.37% and an RGI ratio of 23.75%;

[0066] (2) The pectin obtained in the above step was fully dissolved in water to obtain a 1.5% (g / mL) pectin solution, which was hydrated overnight; the pH was adjusted to 3, 4.8 mL of the pectin solution was added to a container, and calcium chloride solution was added to a calcium ion concentration of 0.25 mmol / L in the system, and sheared at 15000 rpm for 1 min to obtain a pectin sol;

[0067] (3) Soybean oil dissolved with limonin (the mass ratio of limonin to soybean oil is 260 mg / kg) is slowly added to the above pectin sol until the oil phase accounts for 20% (the mass ratio of soybean oil in the total mixed system is 20%), and high-speed shearing is performed at 15,000 rpm for 2 min. The mixture is allowed to stand at 4°C for 20 hours to obtain a limonin-pectin emulsion gel.

[0068] Example 2

[0069] (1) Mixing citrus peel residue with water (the mass ratio of citrus peel residue to water is 1:20), adjusting the pH to 2, extracting at 70°C for 2 hours, centrifuging, taking the supernatant, washing the precipitate with alcohol, filtering, drying and crushing to obtain primary pectin powder; re-dissolving the primary pectin powder, adding 200 U / mL of pectin methylesterase, adjusting the pH to 3.5, reacting at 50°C for 2 hours, washing with alcohol, and drying to obtain pectin with an esterification degree of 25.67% and an RGI ratio of 38.78%;

[0070] (2) The pectin obtained in the above step was fully dissolved in water to obtain a 1.5% (w / v) pectin solution, which was hydrated overnight; the pH was adjusted to 5, 4.8 mL of the pectin solution was added to a container, a certain volume of calcium chloride solution was added until the calcium ion concentration of the system was 0.5 mmol / L, and shearing was performed at 15000 rpm for 1 min to obtain a pectin sol;

[0071] (3) Soybean oil dissolved with limonin (the mass ratio of limonin to soybean oil is 260 mg / kg) is slowly added to the above pectin sol until the oil phase accounts for 20% (mass ratio), and high-speed shearing is performed at 15,000 rpm for 2 min. The mixture is allowed to stand at 4°C for 20 hours to obtain a limonin-pectin emulsion gel.

[0072] Example 3

[0073] (1) Mix citrus peel residue with water (the mass ratio of citrus peel residue to water is 1:20), adjust the pH to 2, extract at 70°C for 2 hours, centrifuge, take the supernatant, wash the precipitate with alcohol, filter, dry and crush to obtain pectin powder, redissolve the pectin powder, add 150U / mL of pectin methylesterase, adjust the pH to 3.5, react at 50°C for 1.5 hours, wash with alcohol, and dry to obtain pectin with an esterification degree of 32.37% and an RGI ratio of 23.75%;

[0074] (2) The pectin obtained in the above step was fully dissolved in water to obtain a 1.5% (w / v) pectin solution, which was hydrated overnight; the pH was adjusted to 3; 4.8 mL of the pectin solution was added to a 10 mL vial, a certain volume of calcium chloride solution was added until the calcium ion concentration of the system was 0.5 mmol / L, and the solution was sheared at 15000 rpm for 1 min to obtain a pectin sol;

[0075] (3) Slowly adding soybean oil dissolved with limonin into the above pectin sol until the oil phase accounts for 20%, high-speed shearing is performed at 15,000 rpm for 2 min, and standing at 4° C. for 12 hours to obtain a limonin-pectin emulsion gel.

[0076] Comparative Example 1

[0077] (1) Mix citrus peel residue with water (the mass ratio of citrus peel residue to water is 1:20), adjust the pH to 2, extract at 70°C for 2 hours, centrifuge, take the supernatant, wash the precipitate with alcohol, filter, dry and crush to obtain primary pectin powder, re-dissolve the primary pectin powder, add 150U / mL of pectin methylesterase, adjust the pH to 3.5, react at 50°C for 1.5 hours, wash with alcohol, and dry to obtain pectin with an esterification degree of 32.37% and an RGI ratio of 23.75%;

[0078] (2) The pectin obtained in the above step was fully dissolved in water to obtain a 1.5% (w / v) pectin solution, which was hydrated overnight; the pH was adjusted to 5, 4.8 mL of the pectin solution was added to a 10 mL vial, a certain volume of calcium chloride solution was added until the calcium ion concentration of the system was 0 mmol / L, and shearing was performed at 15000 rpm for 1 min to obtain a pectin sol;

[0079] (3) Slowly adding the soybean oil in which limonin is dissolved into the above pectin sol until the oil phase accounts for 20%, high-speed shearing is performed at 15,000 rpm for 2 minutes, and the mixture is allowed to stand at 4° C. for 12 hours to obtain a limonin-pectin emulsion gel.

[0080] Comparative Example 2

[0081] (1) Mix citrus peel residue with water (the mass ratio of citrus peel residue to water is 1:20), adjust the pH to 2, extract at 70°C for 2 hours, centrifuge, take the supernatant, wash the precipitate with alcohol, filter, dry and crush to obtain pectin powder, redissolve the pectin powder, add 50U / mL of pectin methylesterase, adjust the pH to 3.5, react at 50°C for 1.5 hours, wash with alcohol, and dry to obtain pectin with an esterification degree of 52.13% and an RGI ratio of 24.19%;

[0082] (2) The pectin obtained in the above step was fully dissolved in water to obtain a 1.5% (w / v) pectin solution, which was hydrated overnight; the pH was adjusted to 5, 4.8 mL of the pectin solution was added to a 10 mL vial, a certain volume of calcium chloride solution was added until the calcium ion concentration of the system was 0.5 mmol / L, and shearing was performed at 15000 rpm for 1 min to obtain a pectin sol;

[0083] (3) Slowly adding soybean oil dissolved with limonin into the above pectin sol until the oil phase accounts for 20%, high-speed shearing is performed at 15000 rpm for 2 min, and standing at 4° C. for 12 hours to obtain a limonin-pectin emulsion gel.

[0084] Comparative Example 3

[0085] (1) Mix citrus peel residue with water (the mass ratio of citrus peel residue to water is 1:20), adjust the pH to 2, extract at 55°C for 2 hours, centrifuge, take the supernatant, wash the precipitate with alcohol, filter, dry and crush to obtain primary pectin powder, redissolve the primary pectin powder, add 150U / mL of pectin methylesterase, adjust the pH to 3.5, react at 50°C for 1.5 hours, wash with alcohol, and dry to obtain pectin with an esterification degree of 32.37% and an RGI ratio of 23.75%;

[0086] (2) The pectin obtained in the above step was fully dissolved in water to obtain a 1.5% (w / v) pectin solution, which was hydrated overnight; the pH was adjusted to 6.5, 4.8 mL of the pectin solution was added to a 10 mL vial, a certain volume of calcium chloride solution was added until the calcium ion concentration of the system was 0.5 mmol / L, and shearing was performed at 15000 rpm for 1 min to obtain a pectin sol;

[0087] (3) Slowly adding the soybean oil in which limonin is dissolved into the above pectin sol until the oil phase accounts for 20%, high-speed shearing is performed at 15,000 rpm for 2 minutes, and the mixture is allowed to stand at 4° C. for 12 hours to obtain a limonin-pectin emulsion gel.

[0088] Performance Test:

[0089] (1) The emulsion gels prepared in Examples 1 to 3 and Comparative Examples 1 to 3 were subjected to emulsification stability test, embedding rate test, and in vitro fermentation experiment.

[0090] (2) The emulsion gels prepared in Examples 1 to 3 and Comparative Examples 1 to 3 were evaluated for their in vitro probiotic properties. The formula of the culture medium is as follows: 3 g / L yeast extract powder, 3 g / L tryptone, 2.0 g / L Tween 80, 4.5 g / L NaCl, 0.5 g / L bile salts, 0.5 g / L cysteine ​​HCl, 0.45 g / L KH2PO4, 0.45 g / L K2HPO4, 0.2 g / L NaHCO3, 0.07 g / L CaCl2, 0.09 g / L MgSO4-7H2O, 0.05 g / L MnSO4-H2O, 0.01 g / L resazurin sodium salt, 0.005 g / L ferrous chloride hemoglobin, and 0.005 g / L vitamin K3. After sterilization and cooling, the bacterial suspension of healthy people and 1% limonin-pectin emulsion gel (the amount of limonin-pectin emulsion gel added is 1% of the mass of the bacterial suspension) were inoculated in an anaerobic incubator. After the fermentation was completed, centrifugation was performed, the supernatant was taken to determine the content of short-chain fatty acids, and the precipitate was used for 16SrDNA sequencing.

[0091] (3) The limonin-pectin emulsion gel prepared in the above Examples 1 to 3 and Comparative Examples 1 to 3 was used to test the inhibition rate of inflammatory factors in vitro. The final concentration of the limonin-pectin emulsion gel in the cell culture medium was 1%.

[0092] Figure 1 This is a graph showing the emulsification stability test results of the limonin-pectin emulsion gels with anti-inflammatory and intestinal probiotic functions prepared in Examples 1 to 3 and Comparative Examples 1 to 3; Figure 2 This is a graph showing the results of measuring the embedding rate of the limonin-pectin emulsion gels with anti-inflammatory and intestinal probiotic functions prepared in Examples 1 to 3 and Comparative Examples 1 to 3.

[0093] Figure 3 This is a graph showing the results of measuring the rheological properties of the limonin-pectin emulsion gels with anti-inflammatory and intestinal probiotic functions prepared in Examples 1 to 3 and Comparative Examples 1 to 3.

[0094] Figure 4 This is a bar graph showing the abundance of intestinal flora at the genus level of the limonin-pectin emulsion gels with anti-inflammatory and intestinal probiotic functions prepared in Examples 1 to 3 and Comparative Examples 1 to 3 after 24 hours of in vitro fermentation. Figure 5 Changes in the total short-chain fatty acid content of the limonin-pectin emulsion gels with anti-inflammatory and intestinal probiotic functions prepared in Examples 1 to 3 and Comparative Examples 1 to 3 after in vitro fermentation for 24 hours.

[0095] The results of emulsion stability determination and embedding efficiency of the emulsion gels prepared in Examples 1-3 and Comparative Examples 1-3 are as follows: Figure 1 and Figure 2 As shown. Figure 1 It can be seen that the pectin emulsion gel without calcium ion addition (Comparative Example 1), the pectin emulsion gel with high esterification degree (Comparative Example 2) and the pectin emulsion gel with high pH (Comparative Example 3) all showed different degrees of stratification. The pectin emulsion gels of Examples 1-3 did not show demulsification or stratification after centrifugation, indicating that the emulsion gels formed had stable emulsification properties. Figure 2 It can be seen that Example 1 has a similar and highest embedding rate as Example 2. Figure 3 It can be seen that the elastic modulus (G') of the pectin emulsion gel without added calcium ions (Comparative Example 1), the pectin emulsion gel with high esterification degree (Comparative Example 2) and the pectin emulsion gel with high pH (Comparative Example 3) is less than the viscous modulus (G"), and the rheological properties show viscosity, strong fluidity and poor centrifugal stability. The pectin emulsion gels of Examples 1-3 have G'>G", showing more elastic behavior. Calcium ions, as cross-linking agents, form ion bridges with the carboxyl groups in the pectin molecules, enhance the interaction between molecules, and promote the formation of the gel network. An appropriate calcium ion concentration can optimize this cross-linking effect and improve the mechanical strength and stability of the gel. When the pH value is close to the isoelectric point of pectin, the intermolecular force is enhanced, which is conducive to gel formation. The degree of pectin esterification affects the charge state between molecules. A lower degree of esterification is conducive to the formation of more free carboxyl groups, which form cross-links with calcium ions and promote gel formation. Therefore, the appropriate calcium ion concentration, pH value and degree of pectin esterification improve the mechanical strength and stability of the pectin latex gel, which is conducive to the formation of a pectin emulsion gel with a stable load of limonin. Depend on Figure 4 and Figure 5It can be seen that compared with comparative examples 1-3, examples 1-3 with higher embedding rate and stability showed the effect of promoting the proliferation of probiotics such as Collinsella, Bifidobacterium and Prevotella_9 and inhibiting the growth of harmful bacteria such as Escherichia-Shigella, and significantly promoted the production of short-chain fatty acids. The limonin-pectin emulsion gel with a content of 1.0% has the ability to efficiently regulate intestinal flora.

[0096] The test results of the in vitro inflammatory factor inhibition rate of the emulsion gels prepared in Examples 1 to 3 and Comparative Examples 1 to 3 are shown in Table 1.

[0097] Table 1 Effect of the Inhibition Rate of TNF-α and IL-6 in Each Example and Comparative Example

[0098] serial number TNF-α inhibition rate IL-6 inhibition rate Example 1 63.56％ 69.28％ Example 2 72.28％ 79.97％ Example 3 68.87％ 77.21％ Comparative Example 1 35.18％ 33.76％ Comparative Example 2 42.97％ 29.32％ Comparative Example 3 36.64％ 32.17％

[0099] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made under the principles that do not deviate from the spirit and essence of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a limonin-pectin emulsion gel with anti-inflammatory and intestinal probiotic functions, characterized in that: The following steps are involved: 1) dissolving pectin with an esterification degree of 10-40% and an RGI ratio of 20-40% in water to obtain a pectin solution; adjusting the pH of the pectin solution to 3-6, adding a calcium ion solution, and subjecting the solution to shearing to obtain a pectin sol; 2) dissolving limonin in the oil phase to obtain an oil phase containing limonin; Subsequently, the oil phase containing limonin is added into the pectin sol, sheared and allowed to stand to obtain a pectin emulsion gel loaded with limonin.

2. The method for preparing the limonin-pectin emulsion gel with anti-inflammatory and intestinal probiotic functions according to claim 1, characterized in that: In step 1), the degree of esterification is 20-35%, and the proportion of RGI rhamnogalacturonan type I is 23-40%. The pH in step 1) is 4.5 to 5.

5.

3. The method for preparing the limonin-pectin emulsion gel with anti-inflammatory and intestinal probiotic functions according to claim 2, characterized in that: The degree of esterification in step 1) is 24-26%, and the RGI ratio is 37-39%; the pH in step 1) is 5.

4. The method for preparing the limonin-pectin emulsion gel with anti-inflammatory and intestinal probiotic functions according to claim 1, characterized in that: The concentration of calcium ions in the pectin sol in step 1) is 0.1-1 mmol / L; The source of calcium ions in step 1) is at least one of calcium chloride, calcium carbonate, calcium citrate and calcium oxalate; The shearing treatment in step 1) is performed at a speed of 10000 to 15000 rpm for 1 to 2 minutes; The shearing treatment in step 2) is performed at a speed of 10000 to 20000 rpm for 2 to 5 minutes; The oil phase in step 2) is vegetable oil; The concentration of limonin in the oil phase in step 2) is 200-300 mg / kg; The mass fraction of the oil phase in the limonin-loaded pectin emulsion gel in step 2) is 10-40%.

5. The method for preparing the limonin-pectin emulsion gel with anti-inflammatory and intestinal probiotic functions according to claim 4, characterized in that: The concentration of calcium ions in the pectin sol in step 1) is 0.2-0.6 mmol / L; The source of calcium ions in step 1) is calcium chloride; The oil phase in step 2) is soybean oil; The mass fraction of the oil phase in the limonin-loaded pectin emulsion gel in step 2) is 15-25%.

6. The method for preparing the limonin-pectin emulsion gel with anti-inflammatory and intestinal probiotic functions according to claim 1, characterized in that: The mass volume percentage concentration of pectin and water in the pectin solution in step 1) is 0.5-2%, g / mL; The pectin solution in step 1) is prepared by mixing pectin and water uniformly and hydrating for 8 to 24 hours; The acid used to adjust the pH of the pectin solution in step 1) is at least one of nitric acid, oxalic acid, citric acid, and hydrochloric acid solution; The temperature of the standing in step 2) is 0 to 10° C. and the time is 12 to 24 hours; The pectin in step 1) is prepared by the following method: S1. Mix citrus peel residue with water, adjust the pH to 1.5-2.5, extract at 65-75°C for 1-2 hours, centrifuge, take the supernatant, and precipitate with alcohol to obtain primary pectin; S2. Redissolve the primary pectin, add pectin methylesterase, adjust the pH to 3-5, react at 40-60°C for 1-2.5 hours, centrifuge, take the supernatant, and precipitate with alcohol to obtain pectin.

7. The method for preparing the limonin-pectin emulsion gel with anti-inflammatory and intestinal probiotic functions according to claim 6, characterized in that: The citrus peel residue in step S1 is citrus peel, lemon peel, apple peel and grapefruit peel; The pH in step S1 is 2, and the extraction is carried out at 70° C. for 2 hours; The mass volume ratio of the primary pectin to water after the primary pectin is re-dissolved in step S2 is 2-3 g: 100 mL; The amount of pectin methylesterase added in step S2: 150-205 U / mL; In step S2, the pH is adjusted to 3-4, and the reaction is carried out at 45-55° C. for 1.5-2 hours; The alcohol precipitation is carried out by using ethanol; after the alcohol precipitation, the precipitate is filtered, dried and crushed.

8. A limonin-pectin emulsion gel having anti-inflammatory and intestinal probiotic functions obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the limonin-pectin emulsion gel with anti-inflammatory and intestinal probiotic functions according to claim 8 in the preparation of anti-inflammatory and intestinal probiotic functional products.

10. Use of the limonin-pectin emulsion gel with anti-inflammatory and intestinal probiotic functions according to claim 8 in a preparation for promoting microbial fermentation to produce short-chain fatty acids.

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