Quinolinate phosphoribosyltransferase intestinal function improvement products and their preparation methods

Abstract

This invention discloses a quinolinate phosphoribosyltransferase (quinolinate phosphoribosyltransferase) intestinal function improvement product and its preparation method. The quinolinate phosphoribosyltransferase intestinal function improvement product includes quinolinate phosphoribosyltransferase. Based on the data from the test cases, it can be seen that oral administration of the quinolinate phosphoribosyltransferase intestinal function improvement product can intervene in the intestinal quinolinate phosphoribosyltransferase level, enhance the intestinal mucosal barrier function, maintain normal intestinal permeability, and alleviate intestinal immune dysfunction and intestinal inflammation.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a quinolinic acid phosphoribosyl transferase intestinal function improving product and a preparation method thereof. BACKGROUND

[0002] The intestine is an important digestive organ of the human body. The intestine refers to the digestive tract from the pylorus of the stomach to the anus, which is the longest section of the digestive tract and also the most important section in terms of function. As the main organ for the body to digest and absorb external nutrients and excrete harmful substances, the intestine is also one of the largest immune organs of the human body, and therefore, the health of the intestinal function is an important prerequisite for the health of the body. Due to the influence of factors such as spirit, diet, medication and environment in modern society, the number of people with abnormal intestinal function has increased rapidly and is gradually becoming younger.

[0003] Intestinal dysfunction can be manifested in many ways. Common intestinal dysfunction mainly includes abnormal changes in intestinal flora abundance, disruption of intestinal mucosal barrier, increased intestinal permeability, low intestinal immune function and occurrence of intestinal inflammation. The intestinal barrier plays an important role in preventing harmful substances in the intestinal lumen from entering the systemic circulation. The destruction of the intestinal barrier and the increase of intestinal permeability will lead to the entry of lipopolysaccharide (LPS), bacteria and related metabolites into the circulatory system, triggering a series of inflammatory reactions. The above changes not only involve intestinal diseases such as irritable bowel syndrome, but also involve various systemic diseases such as diabetes, metabolic diseases and non-alcoholic fatty liver disease. Therefore, intestinal dysfunction is a key factor in triggering various metabolic diseases.

[0004] Currently, the main treatment methods for intestinal dysfunction include the use of antibiotics or antibacterial drugs to inhibit the overgrowth of bacteria, the supplementation of probiotics and ecological preparations such as probiotic agents to increase the number of beneficial bacteria, and the supplementation of glutamine to maintain the normal permeability of the intestinal tract and protect the intestinal mucosal barrier. Usually, the above methods need to be used in combination to improve or treat intestinal dysfunction. It is of great practical significance and market value to seek new substances or methods for improving intestinal function.

[0005] Quinolinic acid phosphoribosyl transferase (QPRT) has been found to be highly expressed in the kidney, liver, placenta and spleen, and lowly expressed in the large intestine, colon, small intestine and stomach. Previous studies have found that increased expression of quinolinic acid phosphoribosyl transferase can improve lung damage caused by influenza A virus infection, and some studies have shown that quinolinic acid phosphoribosyl transferase is related to the occurrence and development of tumors. So far, there has been no report in the prior art on the improvement of intestinal function of the body by quinolinic acid phosphoribosyl transferase. SUMMARY

[0006] Therefore, it is necessary to provide a product for improving intestinal function containing quinolinate phosphoribosyltransferase.

[0007] A product for improving intestinal function, comprising quinolinate phosphoribosyltransferase.

[0008] In one embodiment, the quinolinate phosphoribosyltransferase intestinal function improvement product is immobilized quinolinate phosphoribosyltransferase microcapsule powder.

[0009] In one embodiment, the immobilized quinolinate phosphoribosyltransferase microcapsule powder includes a core material and a capsule material covering the core material;

[0010] The core material is the quinolinate phosphoribosyltransferase, and the capsule material includes liquid oil, cyclodextrin, alcohol-soluble protein, and fixative.

[0011] In one embodiment, the mass ratio of the quinolinate phosphoribosyltransferase, the liquid oil, the cyclodextrin, the prolysin, and the fixative is 0.3–10:1–10:0.05–2:1–10:0.1–1.

[0012] In one embodiment, the liquid oil is selected from at least one of corn oil, soybean oil, peanut oil, rapeseed oil, sunflower oil, and palm oil;

[0013] The prolysin is selected from at least one of zein, wheat prolysin, and progluten;

[0014] The fixative is selected from at least one of calcium alginate, carboxymethyl chitosan oligosaccharide, carboxymethyl starch, and carboxymethyl cellulose.

[0015] In one embodiment, the cyclodextrin is selected from at least one of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, its derivatives, its oligomers, its polymers, and its modifications.

[0016] A method for preparing the above-mentioned quinolinate phosphoribosyltransferase intestinal function improvement product includes the following steps:

[0017] Prepare an ethanol-water solution of alcohol-soluble protein;

[0018] Prepare a mixed aqueous solution of quinolinate phosphoribosyltransferase and cyclodextrin;

[0019] The quinolinate phosphoribosyltransferase and cyclodextrin mixed aqueous solution and the alcohol-soluble protein ethanol aqueous solution were mixed evenly, and then the ethanol was evaporated under vacuum to obtain a nano solution.

[0020] Liquid oil was added to the nano solution under stirring. After stirring evenly, quinolinate phosphoribosyltransferase nanoemulsion was obtained. After separation, quinolinate phosphoribosyltransferase microcapsule powder was obtained.

[0021] Prepare an aqueous solution of the fixative;

[0022] Under stirring, the quinolinate phosphoribosyltransferase microcapsule powder is added to the aqueous solution of the fixative. After mixing evenly, the immobilized quinolinate phosphoribosyltransferase microcapsule powder is separated. The immobilized quinolinate phosphoribosyltransferase microcapsule powder is the desired quinolinate phosphoribosyltransferase intestinal function improvement product.

[0023] In one embodiment, the alcohol-soluble protein is in an ethanol-water solution with a pH of 4.0 to 8.0 and a concentration of 60 wt% to 80 wt%, and the alcohol-soluble protein has a concentration of 10 wt% to 20 wt%.

[0024] In the mixed aqueous solution of quinolinate phosphoribosyltransferase and cyclodextrin, the concentration of quinolinate phosphoribosyltransferase is 0.3wt% to 10wt%, and the concentration of cyclodextrin is 0.05wt% to 2wt%.

[0025] The concentration of the fixative in the aqueous solution is 0.1 wt% to 5 wt%.

[0026] In one embodiment, during the process of uniformly mixing the aqueous solution of quinolinate phosphoribosyltransferase and cyclodextrin with the aqueous ethanol solution of the alcohol-soluble protein, the mass ratio of the aqueous solution of quinolinate phosphoribosyltransferase and cyclodextrin to the aqueous ethanol solution of the alcohol-soluble protein is 50-100:5-50.

[0027] In the process of adding liquid oil to the nano solution, the mass ratio of the nano solution to the liquid oil is 40-100:1-10;

[0028] In the process of adding the quinolinate phosphoribosyltransferase microcapsule powder to the aqueous solution of the fixative, the mass ratio of the aqueous solution of the fixative to the quinolinate phosphoribosyltransferase microcapsule powder is 10-30:0.5-25.

[0029] In one embodiment, during the subsequent evaporation of ethanol under vacuum, the vacuum level is 0.08 to -0.1 MPa.

[0030] The procedure for obtaining quinolinate phosphoribosyltransferase microcapsule powder after separation is as follows: the quinolinate phosphoribosyltransferase nanoemulsion is freeze-dried to obtain the quinolinate phosphoribosyltransferase microcapsule powder.

[0031] The procedure for separating the immobilized quinolinate phosphoribosyltransferase microcapsule powder after uniform mixing is as follows: centrifuge and retain the solid product, clean the solid product and freeze dry it to obtain the immobilized quinolinate phosphoribosyltransferase microcapsule powder.

[0032] The quinolinate phosphoribosyltransferase intestinal function improvement product of the present invention includes quinolinate phosphoribosyltransferase. Based on the data in the test case section, it can be seen that oral administration of the quinolinate phosphoribosyltransferase intestinal function improvement product can achieve intervention of intestinal quinolinate phosphoribosyltransferase level, which can enhance the intestinal mucosal barrier function, maintain normal intestinal permeability, and alleviate intestinal immune dysfunction and intestinal inflammation.

[0033] Preferably, the quinolinate phosphoribosyltransferase intestinal function improvement product of the present invention is immobilized quinolinate phosphoribosyltransferase microcapsule powder, thereby overcoming the destruction of quinolinate phosphoribosyltransferase by multiple barriers such as acid (gastric acid) barrier, enzyme (protease) barrier, and membrane barrier (intestinal mucosa) barrier, which promote its absorption rate in the intestine after oral administration and greatly improve the bioavailability of quinolinate phosphoribosyltransferase.

[0034] In addition, the immobilized quinolinate phosphoribosyltransferase microcapsule powder has the characteristics of small particle size, high encapsulation rate and high absorption and utilization, which allows quinolinate phosphoribosyltransferase to be utilized by intestinal cells to the greatest extent and can effectively enhance the intestinal mucosal barrier function, maintain normal intestinal permeability and alleviate intestinal inflammation.

[0035] Preferably, the immobilized quinolinate phosphoribosyltransferase microcapsule powder includes a core material and a capsule material covering the core material; the core material is the quinolinate phosphoribosyltransferase, and the capsule material includes liquid oil, cyclodextrin, prolyl protein, and a fixative.

[0036] Cyclodextrins are primarily characterized by their ability to fully utilize their hydrophobic cavity structure to effectively encapsulate various compounds, including small molecules and proteins.

[0037] Gliadin has strong water resistance and can be further covalently bound to cyclodextrin to form a stable sustained-release carrier.

[0038] The specific function of liquid oils is to allow small particles to be adsorbed at the oil-water interface to form a spatial barrier, thereby stabilizing the emulsion in the oil-in-water (O / W) system.

[0039] The specific function of the fixative is to form a film, thereby encapsulating both embedded quinolinate phosphoribosyltransferase and free quinolinate phosphoribosyltransferase in a membrane, thus improving enzyme stability. Attached Figure Description

[0040] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0041] in:

[0042] Figure 1 This is a flowchart illustrating a method for preparing a quinolinate phosphoribosyltransferase intestinal function improvement product according to one embodiment.

[0043] Figure 2 This is a comparison chart of serum FD4 levels in mice from different groups obtained in Test Example 1.

[0044] Figure 3 The image shows the Western Blot results of E-cadherin, claudin1, and ZO-1 expression in the colon tissues of mice obtained from Test Example 2.

[0045] Figure 4 This is a comparison of the grayscale values ​​of E-cadherin, claudin1, and ZO-1 bands in the colon tissues of mice in each group obtained from Test Example 2.

[0046] Figure 5 This is a comparison of the ELISA results of sIgA concentration in the intestinal fluid of mice in each group obtained from Test Example 3.

[0047] Figure 6a This is a comparison of the relative expression levels of the pro-inflammatory factor IL-1 in the colon tissues of mice in each group obtained from test example 4.

[0048] Figure 6b This is a comparison of the relative expression levels of the pro-inflammatory factor IL-6 in the colon tissues of mice in each group obtained from test example 4.

[0049] Figure 6c This is a comparison of the relative expression levels of the pro-inflammatory factor IL-12 in the colon tissues of mice in each group obtained from test example 4.

[0050] Figure 6d This is a comparison of the relative expression levels of the pro-inflammatory factor TNF-α mRNA in the colon tissues of mice in each group obtained from test example 4.

[0051] Figure 7a This is a comparison of the relative expression levels of the anti-inflammatory factor TGF-β in the colon tissues of mice in each group obtained from test example 4.

[0052] Figure 7bThis is a comparison of the relative expression levels of the anti-inflammatory factor IL-10 in the colon tissues of mice in each group obtained from test example 4. Detailed Implementation

[0053] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0054] It should be noted that all directional indications (such as up, down, left, right, front, back, etc.) in the embodiments of the present invention are only used to explain the relative positional relationship and movement of each component in a specific posture. If the specific posture changes, the directional indications will also change accordingly.

[0055] Furthermore, the use of terms such as "first" and "second" in this invention is for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, features defined with "first" and "second" may explicitly or implicitly include at least one of the stated features. Additionally, the technical solutions of the various embodiments can be combined with each other, but only on the basis of being achievable by those skilled in the art. When the combination of technical solutions is contradictory or impossible to implement, such a combination of technical solutions should be considered non-existent and not within the scope of protection claimed by this invention.

[0056] This invention discloses a quinolinate phosphoribosyltransferase intestinal function improvement product according to one embodiment, comprising quinolinate phosphoribosyltransferase.

[0057] The quinolinate phosphoribosyltransferase intestinal function improvement product of the present invention includes quinolinate phosphoribosyltransferase. Based on the data in the test case section, it can be seen that oral administration of the quinolinate phosphoribosyltransferase intestinal function improvement product can achieve intervention of intestinal quinolinate phosphoribosyltransferase level, which can enhance the intestinal mucosal barrier function, maintain normal intestinal permeability, and alleviate intestinal immune dysfunction and intestinal inflammation.

[0058] Preferably, the quinolinate phosphoribosyltransferase intestinal function improvement product of the present invention is immobilized quinolinate phosphoribosyltransferase microcapsule powder. The immobilized quinolinate phosphoribosyltransferase microcapsule powder can overcome the damage to quinolinate phosphoribosyltransferase caused by various barriers in the body, such as the acid (gastric acid) barrier, enzyme (protease) barrier, and membrane barrier (intestinal mucosa) barrier, promoting its absorption rate in the intestine after oral administration and greatly improving the bioavailability of quinolinate phosphoribosyltransferase.

[0059] In addition, the immobilized quinolinate phosphoribosyltransferase microcapsule powder has the characteristics of small particle size, high encapsulation rate and high absorption and utilization, which allows quinolinate phosphoribosyltransferase to be utilized by intestinal cells to the greatest extent and can effectively enhance the intestinal mucosal barrier function, maintain normal intestinal permeability and alleviate intestinal inflammation.

[0060] Preferably, the immobilized quinolinate phosphoribosyltransferase microcapsule powder includes a core material and a capsule material covering the core material. The core material is quinolinate phosphoribosyltransferase, and the capsule material includes liquid oil, cyclodextrin, prolyl protein, and a fixative.

[0061] Cyclodextrins are primarily characterized by their ability to fully utilize their hydrophobic cavity structure to effectively encapsulate various compounds, including small molecules and proteins.

[0062] Gliadin has strong water resistance and can be further covalently bound to cyclodextrin to form a stable sustained-release carrier.

[0063] The specific function of liquid oils is to allow small particles to be adsorbed at the oil-water interface to form a spatial barrier, thereby stabilizing the emulsion in the oil-in-water (O / W) system.

[0064] The specific function of the fixative is to form a film, thereby encapsulating both embedded quinolinate phosphoribosyltransferase and free quinolinate phosphoribosyltransferase in a membrane, thus improving enzyme stability.

[0065] Preferably, the mass ratio of quinolinate phosphoribosyltransferase, liquid oil, cyclodextrin, prolyl protein and fixative is 0.3-10:1-10:0.05-2:1-10:0.1-1.

[0066] Specifically, the liquid oil is selected from at least one of corn oil, soybean oil, peanut oil, rapeseed oil, sunflower oil, and palm oil.

[0067] Specifically, the prolysin is selected from at least one of zein, wheat prolysin, and progluten.

[0068] Specifically, the fixative is selected from at least one of calcium alginate, carboxymethyl chitosan oligosaccharide, carboxymethyl starch, and carboxymethyl cellulose.

[0069] Specifically, the cyclodextrin is selected from at least one of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, their derivatives, their oligomers, their polymers and their modifications.

[0070] Combination Figure 1 The present invention also discloses a method for preparing the above-mentioned quinolinate phosphoribosyltransferase intestinal function improvement product according to one embodiment, comprising the following steps:

[0071] S10. Prepare an ethanol-water solution of alcohol-soluble protein.

[0072] Preferably, in the ethanol-water solution of the alcohol-soluble protein, the solvent is an ethanol solution with a pH of 4.0 to 8.0 and a concentration of 60 wt% to 80 wt%, and the concentration of the alcohol-soluble protein is 10 wt% to 20 wt%.

[0073] Specifically, S10 can be: dissolving alcohol-soluble protein in an ethanol solution with a pH of 4.0 to 8.0 and a concentration of 60 wt% to 80 wt%, and magnetically stirring the solution at a temperature of 20℃ to 30℃ and a rotation speed of 800 r / min to 1500 r / min to obtain an ethanol aqueous solution of alcohol-soluble protein.

[0074] Ethanol solutions can dissolve alcohol-soluble proteins and completely evaporate during subsequent operations.

[0075] S20, prepare a mixed aqueous solution of quinolinate phosphoribosyltransferase and cyclodextrin.

[0076] In a mixed aqueous solution of quinolinate phosphoribosyltransferase and cyclodextrin, the concentration of quinolinate phosphoribosyltransferase is 0.3 wt% to 10 wt%, and the concentration of cyclodextrin is 0.05 wt% to 2 wt%.

[0077] Specifically, S20 can be: adding quinolinate phosphoribosyltransferase and cyclodextrin to water, stirring and mixing thoroughly, reacting for 5 min to 20 min to obtain an ethanol-water solution of alcohol-soluble protein.

[0078] S30. Mix the aqueous solution of quinoline phosphoribosyltransferase and cyclodextrin obtained in S20 with the aqueous solution of alcohol-soluble protein obtained in S10, and then evaporate the ethanol under vacuum to obtain a nano solution.

[0079] Preferably, in S30, the mass ratio of the mixed aqueous solution of quinolinate phosphoribosyltransferase and cyclodextrin to the ethanol aqueous solution of alcohol-soluble protein is 50-100:5-50.

[0080] Preferably, in the subsequent ethanol evaporation operation under vacuum, the vacuum degree is 0.08 to -0.1 MPa.

[0081] S40. Under stirring, liquid oil is added to the nano solution obtained in S30. After stirring evenly, quinolinate phosphoribosyltransferase nanoemulsion is obtained. After separation, quinolinate phosphoribosyltransferase microcapsule powder is obtained.

[0082] In the process of adding liquid oil to nano solution, the mass ratio of nano solution to liquid oil is 40-100:1-10.

[0083] The procedure for obtaining quinolinate phosphoribosyltransferase microcapsule powder after separation is as follows: Quinolinate phosphoribosyltransferase nanoemulsion is freeze-dried to obtain quinolinate phosphoribosyltransferase microcapsule powder.

[0084] In S40, mixing can be achieved using a high-speed homogenizer.

[0085] Specifically, S40 can be as follows: placing the nano solution in a high-speed homogenizer, setting the rotation speed to 10000 r / min to 20000 r / min, using a 5 mL syringe needle to draw up liquid oil and then drop it into the nano solution, homogenizing at high speed for 60 s to 120 s to obtain quinolinate phosphoribosyltransferase nanoemulsion, and then freeze-drying it to obtain quinolinate phosphoribosyltransferase microcapsule powder.

[0086] S50, Prepare an aqueous solution of the fixative.

[0087] The concentration of the fixative in the aqueous solution is 0.1 wt% to 5 wt%.

[0088] Specifically, S50 can be: adding the fixative to water, stirring and mixing thoroughly, and dissolving for 5 to 20 minutes (if necessary, heating to 50°C to 65°C can be used to dissolve), to obtain an aqueous solution of the fixative.

[0089] S60. Under stirring, add the quinolinate phosphoribosyltransferase microcapsule powder obtained in S40 to the aqueous solution of the fixative obtained in S50. After mixing evenly, separate to obtain the immobilized quinolinate phosphoribosyltransferase microcapsule powder. The immobilized quinolinate phosphoribosyltransferase microcapsule powder is the desired quinolinate phosphoribosyltransferase intestinal function improvement product.

[0090] In the process of adding quinolinate phosphoribosyltransferase microcapsule powder to the aqueous solution of the fixative, the mass ratio of the aqueous solution of the fixative to the quinolinate phosphoribosyltransferase microcapsule powder is 10-30:0.5-25.

[0091] The procedure for separating the immobilized quinolinate phosphoribosyltransferase microcapsule powder after uniform mixing is as follows: centrifuge and retain the solid product, clean the solid product and freeze dry to obtain quinolinate phosphoribosyltransferase microcapsule powder.

[0092] Specifically, S60 can be performed as follows: place an aqueous solution of the fixative in a magnetic stirrer, add quinolinate phosphoribosyltransferase microcapsule powder, and magnetically stir for 15 minutes at a temperature of 20℃~40℃ and a speed of 800~1500r / min. After mixing evenly, centrifuge at a speed of 2500rpm~3500rpm for 20min~40min, retain the solid product, wash the solid product with water 1~3 times, and freeze-dry to obtain immobilized quinolinate phosphoribosyltransferase microcapsule powder.

[0093] The following are specific embodiments. In the specific embodiments, wheat gliadin was purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number S24514; gliadin was purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number S25995; α-cyclodextrin was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., catalog number C6003; β-cyclodextrin was purchased from Shanghai Yien Chemical Technology Co., Ltd., catalog number R008089; quinolinate phosphoribosyltransferase was purchased from Beijing Bio-Lab Technology Co., Ltd., catalog number JN2027; corn oil was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., catalog number C805618; peanut oil was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., catalog number P914350; calcium alginate was purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number S26286; and carboxymethyl chitosan oligosaccharide was purchased from Sigma-Aldrich, USA, catalog number 926167.

[0094] Example 1

[0095] 1g of wheat gliadin was dissolved in 5g of 75% ethanol solution with pH 6.0 and magnetically stirred at 25℃ and 1000r / min to obtain an ethanol aqueous solution of wheat gliadin.

[0096] 0.05 g of β-cyclodextrin and 0.3 g of quinolinate phosphoribosyltransferase were added to 81.30 g of water, stirred thoroughly and mixed evenly, and reacted for 15 min to obtain a mixed aqueous solution of quinolinate phosphoribosyltransferase and cyclodextrin.

[0097] A mixed aqueous solution of quinolinate phosphoribosyltransferase and cyclodextrin was added to an ethanol aqueous solution of wheat gliadin, stirred for 20 min, and then the ethanol was evaporated under vacuum to obtain a nano solution.

[0098] The nano solution was placed in a high-speed homogenizer and the rotation speed was set to 15000 r / min. 1 g of corn oil was drawn up with a 5 mL syringe needle and added dropwise to the nano solution. The mixture was homogenized at high speed for 120 s to obtain quinolinate phosphoribosyltransferase nanoemulsion.

[0099] Quinolinate phosphoribosyltransferase nanoemulsion was freeze-dried to obtain quinolinate phosphoribosyltransferase microcapsule powder.

[0100] Add 0.1g of calcium alginate to 15g of water, stir and mix thoroughly, and react for 15 minutes to obtain an aqueous solution of calcium alginate.

[0101] A calcium alginate aqueous solution was placed in a magnetic stirrer, and quinolinate phosphoribosyltransferase microcapsule powder was added. The mixture was magnetically stirred for 15 min at 25°C and 1000 rpm. Then, it was centrifuged at 3000 rpm for 30 min, and the solid product was retained. After washing the solid product three times with water, it was lyophilized to obtain immobilized quinolinate phosphoribosyltransferase microcapsule powder 1.

[0102] Example 2

[0103] 5g of alcohol-soluble gluten was dissolved in 30g of 75% ethanol solution with pH 6.0 and magnetically stirred at 25℃ and 1000r / min to obtain an ethanol aqueous solution of alcohol-soluble gluten.

[0104] 1g of α-cyclodextrin and 5g of quinolinate phosphoribosyltransferase were added to 52.5g of water, stirred thoroughly and mixed evenly, and reacted for 15min to obtain a mixed aqueous solution of quinolinate phosphoribosyltransferase and cyclodextrin.

[0105] A mixed aqueous solution of quinolinate phosphoribosyltransferase and cyclodextrin was added to an ethanol aqueous solution of alcohol-soluble gluten, stirred for 20 min, and then the ethanol was evaporated under vacuum to obtain a nano solution.

[0106] The nano solution was placed in a high-speed homogenizer and the rotation speed was set to 15000 r / min. 8 g of peanut oil was drawn up with a 10 mL injection needle and added dropwise to the nano solution. The mixture was homogenized at high speed for 120 s to obtain quinolinate phosphoribosyltransferase nanoemulsion.

[0107] Quinolinate phosphoribosyltransferase nanoemulsion was freeze-dried to obtain quinolinate phosphoribosyltransferase microcapsule powder.

[0108] Add 0.4g of carboxymethyl chitosan oligosaccharide to 20g of water, stir and mix thoroughly, and react for 15 minutes to obtain an aqueous solution of carboxymethyl chitosan oligosaccharide.

[0109] A carboxymethyl chitosan oligosaccharide aqueous solution was placed in a magnetic stirrer, and quinolinate phosphoribosyltransferase microcapsule powder was added. The mixture was magnetically stirred for 15 min at 25°C and 1000 r / min. Then, it was centrifuged at 3000 rpm for 30 min, and the solid product was retained. After washing the solid product three times with water, it was lyophilized to obtain immobilized quinolinate phosphoribosyltransferase microcapsule powder 2.

[0110] Test Example 1: Immobilized quinolinate phosphoribosyltransferase microcapsule powder significantly improved intestinal inflammation in mice.

[0111] Fifty male C57BL / 6 mice (7-8 weeks old, weighing 20-22g, purchased from Shenzhen Lingfu Top Biotechnology Co., Ltd.) were randomly divided into 5 groups of 10 mice each: blank group, model group (control group), treatment group 1 (quinolinate phosphoribosyltransferase), treatment group 2 (immobilized quinolinate phosphoribosyltransferase microcapsule powder 1), and treatment group 3 (immobilized quinolinate phosphoribosyltransferase microcapsule powder 2).

[0112] The control group mice were not treated in any way (routine feeding). The model group and the treatment group mice were induced to develop enteritis mouse model by drinking water containing 2% DSS (sodium dextran sulfate, MP Biomedical, 9011-18-1) for 7 consecutive days.

[0113] Seven days after DDS induction, mice in the model group and treatment group were fed normally without any treatment. Then, mice in treatment group 1 were administered quinolinate phosphoribosyltransferase 10 mg / kg by gavage for 7 consecutive days (10 mg / kg: 10 mg quinolinate phosphoribosyltransferase per kilogram of mouse body weight by gavage); mice in treatment group 2 were administered immobilized quinolinate phosphoribosyltransferase microcapsule powder 1 containing 10 mg / kg quinolinate phosphoribosyltransferase by gavage for 7 consecutive days; and mice in treatment group 3 were administered immobilized quinolinate phosphoribosyltransferase microcapsule powder 2 containing 10 mg / kg quinolinate phosphoribosyltransferase by gavage for 7 consecutive days. During the treatment period, mice in all groups had normal diet and water.

[0114] After 14 days, all five groups of mice were deprived of water for 12 hours. Intestinal permeability of the mice was measured using FD4 (FITC-dextran, Sigma-Aldrich, 60842-46-8). FD4 was dissolved in sterile saline to a concentration of 25 mg / mL. The mice in the five groups were administered FD4 solution by gavage at a dose of 20 μL / g. Three hours later, blood was collected from the inner canthus of the eye and centrifuged to obtain serum. The serum was diluted with saline at a ratio of 1:5. The remaining FD-4 used for gavage was diluted with saline starting at an initial concentration of 0.1 mg / mL. A standard curve was plotted by down-dilution by 3 times at 7 points.

[0115] The fluorescence levels at excitation wavelength of 480 nm and emission wavelength of 520 nm were detected using a multi-functional microplate reader (Perkin Elmer, Victor Nivo 3F). The FD4 concentration was calculated based on the standard curve. Figure 2 .

[0116] The basic principle of using FD4 to determine intestinal permeability in mice is to evaluate the overall permeability of the intestine by detecting the intestinal absorption of fluorescently labeled macromolecular compounds into the bloodstream.

[0117] Depend on Figure 2It can be seen that the serum FD4 level in the model group mice was significantly higher than that in the blank group (t = 8.858, P < 0.01), indicating that the intestinal permeability of the model group mice was significantly higher than that of the blank group. The serum FD4 level in the treatment group mice was significantly lower than that in the blank group and the model group, and the order was: treatment group 3 < treatment group 2 < treatment group 1 < model group (P < 0.01), indicating that the intestinal permeability of the treatment group mice was lower than that of the model group. Furthermore, the intestinal permeability of the mice in treatment group 3 was comparable to that in treatment group 2 and lower than that in treatment group 1 (t = 4.162, P < 0.01). The results indicate that intestinal permeability in the model group mice improved after treatment, and the improvement in intestinal permeability in treatment groups 3 and 2 was better than that in treatment group 1. The intestinal permeability of mice in treatment groups 3 and 2 was close to that of mice in the blank group, indicating that the intestinal permeability of mice in treatment groups 3 and 2 recovered towards the direction of normal intestinal permeability. That is, immobilized quinolinate phosphoribosyltransferase microcapsule powder 1 and immobilized quinolinate phosphoribosyltransferase microcapsule powder 2 have a significant effect on improving intestinal permeability in mice with enteritis, and the effects of the two are comparable.

[0118] Test Example 2: Immobilized quinolinate phosphoribosyltransferase microcapsule powder significantly increased the level of intestinal barrier proteins in mice with intestinal inflammation.

[0119] In Test Example 1, after blood was collected from the inner canthus of each group of mice, all mice in the five groups were euthanized by dislocation of the neck and the abdominal cavity was quickly dissected. After collecting colonic fluid from the middle section of the colon, the colonic tissue was cleaned with physiological saline, divided into 3 tubes, and stored in a -80℃ refrigerator.

[0120] 0.2g of colon tissue from each group of mice was weighed, ground with liquid nitrogen, and proteins were extracted and quantified. Western blot was used to detect the expression of barrier proteins in the colon tissue, including E-cadherin, claudin1, and ZO-1. After standardization using mouse colon tissue β-actin protein as an internal control, each protein sample was loaded onto a PVDF membrane and separated by 10% SDS-PAGE gel electrophoresis. The membrane was then blocked with 1% BSA (ThermoFisher, 37525) for 2 hours, incubated overnight at 4°C with primary antibody (E Cadherin antibody, 1:1000 dose, 4069, CellSigning; Claudin1 antibody, 1:1000 dose, AF0127, Affinity; ZO-1 antibody, 1:1000 dose, MA5-46951, Invitrogen; β-actin antibody, 1:2000 dose, ab179467, Abcam) on a shaker, and incubated at room temperature for 2 hours with secondary antibody (goat anti-rabbit secondary antibody, 1:10000 dose, 31460, Invitrogen). The membrane was then subjected to ECL luminescence and imaged using a gel imaging system. Western blot analysis was performed on the expression of E-cadherin, claudin1, and ZO-1 in the colon tissue of mice in each group to obtain... Figure 3 The grayscale values ​​of the Western blotting bands were analyzed and measured using ImageJ software, and the ratio of intrinsic to grayscale values ​​(i.e., absorbance) was calculated to obtain... Figure 4 .

[0121] The intestinal barrier function refers to the relatively complete functional isolation zone of the normal intestine, which separates the intestinal lumen from the internal environment of the body and prevents the invasion of pathogenic antigens. The structural basis of the intestinal mucosal barrier function includes intestinal mucosal epithelial cells and intercellular tight junctions. Tight junctions are mainly composed of tight junction proteins, including claudins, the zonula occludens (ZO) family, and E-cadherin, which play an important role in maintaining intestinal homeostasis and barrier function. The expression level of tight junction proteins directly reflects the intestinal barrier function.

[0122] Combination Figure 3 and Figure 4As can be seen, the levels of intestinal barrier proteins E-cadherin, claudin1, and ZO-1 in the model group mice were significantly lower than those in the blank group, indicating that the intestinal barrier function of the model group mice was lower than that of the blank group. The levels of intestinal barrier proteins claudin1 and ZO-1 in the treatment group were significantly higher than those in the model group, and the expression trend of the above intestinal barrier proteins was: treatment group 3 > treatment group 2 > treatment group 1 > model group, indicating that the intestinal barrier function of the mice in the treatment group was significantly improved compared with that of the model group, and the effects of treatment groups 2 and 3 were better than those of treatment group 1. The levels of intestinal barrier proteins in treatment groups 2 and 3 showed a trend of recovery to the levels of intestinal barrier proteins in the blank group mice, indicating that the intestinal barrier function of mice in treatment groups 2 and 3 recovered well after treatment. That is, immobilized quinolinate phosphoribosyltransferase microcapsule powder 1 and immobilized quinolinate phosphoribosyltransferase microcapsule powder 2 have good effects on restoring the intestinal barrier function of mice, and the effects of the two are comparable.

[0123] Test Example 3: Immobilized quinolinate phosphoribosyltransferase microcapsule powder enhances the expression of secretory immunoglobulin A (sIgA) in the intestinal fluid of mice with intestinal inflammation.

[0124] Colonic fluid samples from the mid-colon of mice collected in Test Example 2 were used to detect the concentration of sIgA in the intestinal fluid of each group of mice using a mouse sIgA-ELISA kit (Elabscience, E-EL-M1040c).

[0125] Following the ELISA kit instructions, add 100 μL of standard working solution or sample to the corresponding well and incubate at 37°C for 90 minutes. Discard the liquid in the plate and immediately add 100 μL of biotinylated antibody working solution, incubate at 37°C for 60 minutes. Discard the liquid in the plate and wash the plate 3 times. Add 100 μL of HRP enzyme conjugate working solution to each well and incubate at 37°C for 30 minutes. Discard the liquid in the plate and wash the plate 5 times. Add 90 μL of substrate solution to each well and incubate at 37°C for approximately 15 minutes. Add 50 μL of stop solution to each well. Immediately use a multi-functional microplate reader (Perkin Elmer, Victor Nivo 3F) to read the data at 450 nm. Process the data and use ELISA to detect the sIgA concentration in the intestinal fluid of mice in each group. Figure 5 .

[0126] sIgA is the most abundant immunoglobulin secreted by intestinal mucosal cells and plays an important role in the immune function of the mouse intestinal mucosa. Changes in sIgA levels in mouse intestinal fluid are positively correlated to some extent with the immune function of the mouse intestinal mucosa.

[0127] Depend on Figure 5It can be seen that the sIgA level in the intestinal fluid of the model group mice was significantly lower than that of the blank group (P<0.01), indicating that the intestinal mucosal immune function of the model group mice was lower than that of the blank group. The sIgA level in the intestinal fluid of the treatment group was significantly higher than that of the model group, and the order of treatment group 3 ≈ treatment group 2 > treatment group 1 > model group (P<0.01), indicating that the intestinal mucosal immune function of the treatment group mice was significantly improved compared with that of the model group. The effects of treatment group 2 and treatment group 3 were comparable and better than those of treatment group 1. The sIgA level in the intestinal fluid of treatment group 2 and treatment group 3 was not significantly different from that of the blank group (P>0.05), indicating that after treatment, the intestinal mucosal immune function of mice in treatment group 2 and treatment group 3 was almost restored to the level of normal mouse intestinal mucosal immune function. That is, immobilized quinolinate phosphoribosyltransferase microcapsule powder 1 and immobilized quinolinate phosphoribosyltransferase microcapsule powder 2 have good effects on restoring the intestinal mucosal immune level of mice, and the effects of the two are comparable.

[0128] Test Example 4: Immobilized quinolinate phosphoribosyltransferase microcapsule powder reduced the levels of pro-inflammatory factors and increased the levels of anti-inflammatory factors in the mouse intestine.

[0129] 0.1g of colon tissue from each group of mice collected in Example 2 of the second group was weighed, ground with liquid nitrogen, and RNA was extracted and reverse transcribed into cDNA. RT-PCR was then used to detect the mRNA levels of key pro-inflammatory factors IL-1, IL-6, IL-12, and TNF-α, and the mRNA levels of anti-inflammatory factors TGF-β and IL-10. Figure 6a , Figure 6b , Figure 6c , Figure 6d , Figure 7a and Figure 7b .

[0130] Combination Figure 6a , Figure 6b , Figure 6c and Figure 6d It can be seen that, compared with the blank group mice, the expression levels of pro-inflammatory factors IL-1, IL-6, IL-12, and TNF-α genes in the colon tissue of the model group mice were significantly increased (P<0.01); compared with the model group mice, the expression levels of pro-inflammatory factors IL-1, IL-6, IL-12, and TNF-α genes in the colon tissue of the treatment group mice were significantly decreased, and the levels of pro-inflammatory factors in the colon tissue of treatment group 2 and treatment group 3 mice were comparable (P>0.05) and significantly lower than those in treatment group 1; the levels of pro-inflammatory factors in the colon tissue of treatment group 2 and treatment group 3 mice showed a trend of recovery to those in the blank group mice, that is, immobilized quinolinate phosphoribosyltransferase microcapsule powder 1 and immobilized quinolinate phosphoribosyltransferase microcapsule powder 2 have a relieving effect on intestinal inflammation, and there is no significant difference in their effects.

[0131] Combination Figure 7a and Figure 7bIt can be seen that, compared with the blank group mice, the expression levels of anti-inflammatory factors TGF-β and IL-10 mRNA in the colon tissue of the model group mice were significantly reduced (P<0.01); compared with the model group mice, the expression levels of anti-inflammatory factors TGF-β and IL-10 mRNA in the colon tissue of the treatment group mice were significantly increased, and the levels of anti-inflammatory factors in the colon tissue of treatment group 2 and treatment group 3 mice were comparable (P>0.05) and significantly higher than those in treatment group 1; the levels of pro-inflammatory factors in the colon tissue of treatment group 2 and treatment group 3 mice showed a trend of recovery to those in the blank group mice, that is, immobilized quinolinate phosphoribosyltransferase microcapsule powder 1 and immobilized quinolinate phosphoribosyltransferase microcapsule powder 2 have anti-intestinal inflammation effects, and there is no significant difference in their effects.

[0132] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A quinolinic acid phosphoribosyltransferase enterofunction-improving product, characterized by comprising a quinolinic acid phosphoribosyltransferase. The quinolinic acid phosphoribosyltransferase intestinal function improving product is a microcapsule powder of immobilized quinolinic acid phosphoribosyltransferase; the microcapsule powder of immobilized quinolinic acid phosphoribosyltransferase comprises a core material and a capsule material coated outside the core material; the core material is the quinolinic acid phosphoribosyltransferase, and the capsule material comprises liquid grease, cyclodextrin, prolamine and a fixing agent.

2. The enterotype function-improving product of quinolinic acid phosphoribosyltransferase according to claim 1, characterized by, The mass ratio of the quinolinic acid phosphoribosyltransferase, the liquid grease, the cyclodextrin, the prolamine and the fixing agent is 0.3-10:1-10:0.05-2:1-10:0.1-1.

3. The enterotype function-improving product of quinolinic acid phosphoribosyltransferase according to claim 2, characterized by, The liquid grease is at least one selected from corn oil, soybean oil, peanut oil, rapeseed oil, sunflower oil and palm oil; The prolamine is at least one selected from corn prolamine, wheat prolamine and prolamine glutelin; The fixing agent is at least one selected from calcium alginate, carboxymethyl chitosan, carboxymethyl starch and carboxymethyl cellulose.

4. The enteric function-improving product of quinolinic acid phosphoribosyltransferase according to claim 2, characterized by, The cyclodextrin is at least one selected from α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, oligomers thereof and polymers thereof.

5. A method for producing the enterotype-improving product of quinolinic acid phosphoribosyltransferase according to any one of claims 1 to 4, characterized by, The method comprises the following steps: An ethanol aqueous solution of prolamine is prepared; A mixed aqueous solution of quinolinic acid phosphoribosyltransferase and cyclodextrin is prepared; The mixed aqueous solution of quinolinic acid phosphoribosyltransferase and cyclodextrin and the ethanol aqueous solution of prolamine are uniformly mixed, and then ethanol is evaporated under vacuum to obtain a nano solution; Under stirring, liquid grease is added to the nano solution, and after uniform stirring, quinolinic acid phosphoribosyltransferase nanoemulsion is obtained, and after separation, quinolinic acid phosphoribosyltransferase microcapsule powder is obtained; An aqueous solution of a fixing agent is prepared; Under stirring, the quinolinic acid phosphoribosyltransferase microcapsule powder is added to the aqueous solution of the fixing agent, and after uniform mixing, immobilized quinolinic acid phosphoribosyltransferase microcapsule powder is obtained by separation, which is the required quinolinic acid phosphoribosyltransferase intestinal function improving product.

6. The method for producing an enterotype function-improving product of quinolinic acid phosphoribosyltransferase according to claim 5, characterized by, In the ethanol aqueous solution of prolamine, the solvent is an ethanol solution with a pH of 4.0-8.0 and a concentration of 60wt%-80wt%, and the concentration of the prolamine is 10wt%-20wt%; In the mixed aqueous solution of quinolinic acid phosphoribosyltransferase and cyclodextrin, the concentration of the quinolinic acid phosphoribosyltransferase is 0.3wt%-10wt%, and the concentration of the cyclodextrin is 0.05wt%-2wt%; In the aqueous solution of the fixing agent, the concentration of the fixing agent is 0.1wt%-5wt%.

7. The method for producing an enterotype function-improving product of quinolinic acid phosphoribosyltransferase according to claim 5, characterized by, In the operation of uniformly mixing the mixed aqueous solution of quinolinic acid phosphoribosyltransferase and cyclodextrin and the ethanol aqueous solution of prolamine, the mass ratio of the mixed aqueous solution of quinolinic acid phosphoribosyltransferase and cyclodextrin to the ethanol aqueous solution of prolamine is 50-100:5-50; In the operation of adding liquid grease to the nano solution, the mass ratio of the nano solution to the liquid grease is 40-100:1-10; In the operation of adding the quinolinic acid phosphoribosyltransferase microcapsule powder into the water solution of the fixing agent, the mass ratio of the water solution of the fixing agent to the quinolinic acid phosphoribosyltransferase microcapsule powder is 10-30:0.5-25.

8. The method for producing an enterotype function-improving product of quinolinic acid phosphoribosyltransferase according to claim 5, characterized by, In the operation of evaporating ethanol under vacuum, the vacuum degree is 0.08-0.1 MPa; The operation of obtaining the quinolinic acid phosphoribosyltransferase microcapsule powder after separation is that the quinolinic acid phosphoribosyltransferase nanometer emulsion is freeze-dried to obtain the quinolinic acid phosphoribosyltransferase microcapsule powder; The operation of obtaining the immobilized quinolinic acid phosphoribosyltransferase microcapsule powder after mixing uniformly is that the solid product is centrifuged and reserved, and the solid product is cleaned and freeze-dried to obtain the immobilized quinolinic acid phosphoribosyltransferase microcapsule powder.

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