A composition for alleviating intestinal oxidative stress damage and its application

Abstract

This invention provides a composition for alleviating intestinal oxidative stress damage and its application, the composition comprising 4'-galactosyllactose (4'-GL) and glycomacropeptide (GMP). This invention found that, compared with the individual concentrations of 4'-GL and GMP, the composition of 4'-GL and GMP more significantly promotes the release of superoxide dismutase and catalase, while also more significantly reducing the release of malondialdehyde, a marker of oxidative stress. The synergistic effect between the two allows 4'-GL and GMP to better alleviate intestinal damage in Drosophila. Simultaneously, the composition can improve the survival rate and climbing index of H2O2-induced Drosophila, prolong the intestinal length of diseased Drosophila, improve the integrity of the intestinal barrier, and induce a recovery trend in the Drosophila. This demonstrates that the composition has a mitigating effect on intestinal oxidative stress damage.

Description

Technical Field

[0001] This invention relates to the technical field of human milk oligosaccharides, and more specifically to a composition for alleviating intestinal oxidative stress damage and its application. Background Technology

[0002] Oxidative stress is an unbalanced biological response that leads to an increase in the number of free radicals in the body, which in turn damages biological macromolecules such as DNA, lipids, and proteins, playing an important role in the pathogenesis of many chronic degenerative diseases. Some habits in modern lifestyles inevitably lead to oxidative stress damage, such as processed foods, exposure to various toxic substances, and lack of exercise. The gut is a significant source of reactive oxygen species (ROS) because it is inevitably exposed to foreign substances and microbial pathogens. When harmful substances stimulate the gut and cause oxidative stress, the intestinal epithelial cells act as the first line of defense, generating a direct immune response. This process produces large amounts of ROS. At low or moderate levels, ROS are beneficial to cellular function, but excessive ROS production can disrupt important cellular processes. Gut oxidative stress can negatively impact the intestinal mucosa and gut microbiota, leading to a range of diseases and health problems. Some diseases that may be caused by gut oxidative stress include intestinal infections and inflammatory bowel disease.

[0003] To mitigate intestinal damage caused by oxidative stress, the dynamic balance of ROS production is crucial for maintaining gut homeostasis. Therefore, attention is increasingly focused on safe antioxidants obtained through diet. However, even safe antioxidants are not entirely natural foods, necessitating the exploration of more natural food sources to alleviate intestinal oxidative stress damage. Summary of the Invention

[0004] In view of this, the present invention provides a composition for alleviating intestinal oxidative stress damage and its application, the composition comprising 4'-galactosyllactose and glycomacropeptide, and the composition is verified by a Drosophila intestinal oxidative stress damage model induced by H2O2 to demonstrate that it has an alleviating effect on intestinal oxidative stress damage.

[0005] To address the technical problems mentioned in the background section, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a composition of human milk oligosaccharides, the composition comprising:

[0007] 4'-galactosyllactose and glycomacropeptide; the mass ratio of the 4'-galactosyllactose and the glycomacropeptide is (1:30) to (8:112).

[0008] Secondly, the present invention provides the use of the composition described above in the preparation of food having the following effects, including alleviating intestinal oxidative stress damage.

[0009] Furthermore, the application concentration of the composition in the food is as follows: the concentration of 4'-galactosyllactose is 0.025-0.75 mg / mL, and the concentration of glycomacropeptide is 0.2-2.8 mg / mL.

[0010] Thirdly, the present invention provides the use of the composition described above in the preparation of health foods having the following effects, including alleviating intestinal oxidative stress damage.

[0011] Furthermore, the application concentration of the composition in the health food is as follows: the concentration of 4'-galactosyllactose is 0.025-0.75 mg / mL, and the concentration of glycomacropeptide is 0.2-2.8 mg / mL.

[0012] Fourthly, the present invention provides the use of the composition described above in the preparation of a pharmaceutical product having the following effects, including the relief of intestinal oxidative stress damage.

[0013] Further, the application concentration of the composition in the pharmaceutical product is as follows: the concentration of 4'-galactosyllactose is 0.025-0.75 mg / mL, and the concentration of glycomacropeptide is 0.2-2.8 mg / mL.

[0014] Furthermore, the relief of intestinal oxidative stress damage includes at least one of the following manifestations:

[0015] Increase the release of superoxide dismutase;

[0016] Increase the release of catalase;

[0017] Reduce malondialdehyde release;

[0018] To improve the survival rate of fruit flies with H2O2-induced intestinal oxidative stress damage;

[0019] To enhance the climbing ability of fruit flies with H2O2-induced intestinal oxidative stress damage;

[0020] Extending the intestinal length of fruit flies with H2O2-induced intestinal oxidative stress damage;

[0021] Reduce intestinal permeability in fruit flies with H2O2-induced intestinal oxidative stress damage.

[0022] Furthermore, the food includes at least one of the following: infant formula, children's milk powder, adult milk powder, food for special medical purposes, nutritional products, liquid milk, complementary food, and non-alcoholic beverages.

[0023] Fifthly, the present invention provides the use of the composition described above in the preparation of foods for adjunctive treatment and / or adjunctive prevention of intestinal infections and inflammatory bowel disease caused by intestinal oxidative stress.

[0024] In a sixth aspect, the present invention provides the use of the composition described above in the preparation of health foods for adjuvant treatment and / or adjuvant prevention of intestinal infections and inflammatory bowel disease caused by intestinal oxidative stress.

[0025] In a seventh aspect, the present invention provides the use of the composition described above in the preparation of pharmaceuticals for treating and / or preventing intestinal infections and inflammatory bowel disease caused by intestinal oxidative stress.

[0026] The beneficial effects of the above-described technical solution of the present invention are as follows:

[0027] This invention provides a composition for alleviating intestinal oxidative stress damage and its application. The composition comprises 4'-galactosyllactose (4'-GL) and glycomacropeptide (GMP); the mass ratio of 4'-galactosyllactose to glycomacropeptide is (1:30) to (8:112). This invention found that, under the same concentration conditions, compared with the individual concentrations of 4'-GL and GMP, the composition of 4'-GL and GMP can more significantly promote the release of superoxide dismutase (SOD) and catalase (CAT), while also more significantly reducing the release of the oxidative stress marker malondialdehyde (MDA). The synergistic effect between the two allows 4'-GL and GMP to better alleviate intestinal damage in Drosophila. Simultaneously, the composition can improve the survival rate and climbing index of H2O2-induced Drosophila, prolong the intestinal length of diseased Drosophila, improve the integrity of the intestinal barrier, and cause the Drosophila to show a recovery trend, thus demonstrating that the composition has an alleviating effect on intestinal oxidative stress damage. Attached Figure Description

[0028] Figure 1 The figure shows the results of superoxide dismutase (SOD) release in a Drosophila intestinal oxidative stress injury model induced by H2O2 by different concentrations of 4'-galactosyllactose (4'-GL).

[0029] Figure 2 The figure shows the results of catalase (CAT) release in a Drosophila intestinal oxidative stress injury model induced by H2O2 by different concentrations of 4'-galactosyllactose (4'-GL).

[0030] Figure 3 The figure shows the results of malondialdehyde (MDA) release in a Drosophila intestinal oxidative stress injury model induced by H2O2, with different concentrations of 4'-galactosyllactose (4'-GL).

[0031] Figure 4The figure shows the results of superoxide dismutase (SOD) release in a Drosophila intestinal oxidative stress injury model induced by H2O2 using different concentrations of glycomacropeptide (GMP).

[0032] Figure 5 The figure shows the results of catalase (CAT) release in a Drosophila intestinal oxidative stress injury model induced by H2O2 with different concentrations of glycomacropeptide (GMP).

[0033] Figure 6 The figure shows the results of malondialdehyde (MDA) release in a Drosophila intestinal oxidative stress injury model induced by H2O2, with different concentrations of glycomacropeptide (GMP).

[0034] Figure 7 Figure showing the results of superoxide dismutase (SOD) release in a H2O2-induced Drosophila intestinal oxidative stress injury model using 4'-GL, GMP, and their combinations.

[0035] Figure 8 Figure showing the results of catalase (CAT) release in a Drosophila gut oxidative stress injury model induced by H2O2, using 4'-GL, GMP and their combinations.

[0036] Figure 9 Figure showing the results of malondialdehyde (MDA) release in a Drosophila gut oxidative stress injury model induced by H2O2, using 4'-GL, GMP and their combinations.

[0037] Figure 10 Figure showing the effect of 4'-GL, GMP, and their combinations on the survival rate of Drosophila;

[0038] Figure 11 Figure showing the climbing ability of Drosophila with 4'-GL, GMP and their combinations;

[0039] Figure 12 Figure showing the results of extending the intestinal length in a Drosophila intestinal oxidative stress model induced by H2O2 using 4'-GL, GMP, and their combinations;

[0040] Figure 13 The figure shows the results of the effects of 4'-GL and GMP and their combinations on intestinal barrier function in a Drosophila intestinal oxidative stress injury model induced by H2O2. Detailed Implementation

[0041] To further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the present invention.

[0042] The compositions and their effects provided in this invention are further explained below with reference to the accompanying drawings.

[0043] In a first aspect, the present invention provides a composition of human milk oligosaccharides, the composition comprising:

[0044] 4'-galactosyllactose and glycomacropeptide; the mass ratio of the 4'-galactosyllactose and the glycomacropeptide is (1:30) to (8:112).

[0045] Secondly, the present invention provides the use of the composition described above in the preparation of food having the following effects, including alleviating intestinal oxidative stress damage.

[0046] According to some embodiments of the present invention, the application concentration of the composition in the food is as follows: the concentration of 4'-galactosyllactose is 0.025-0.75 mg / mL, and the concentration of glycomacropeptide is 0.2-2.8 mg / mL.

[0047] Thirdly, the present invention provides the use of the composition described above in the preparation of health foods having the following effects, including alleviating intestinal oxidative stress damage.

[0048] According to some embodiments of the present invention, the application concentration of the composition in the health food is as follows: the concentration of 4'-galactosyllactose is 0.025-0.75 mg / mL, and the concentration of glycomacropeptide is 0.2-2.8 mg / mL.

[0049] Fourthly, the present invention provides the use of the composition described above in the preparation of a pharmaceutical product having the following effects, including the relief of intestinal oxidative stress damage.

[0050] According to some embodiments of the present invention, the application concentration of the composition in the pharmaceutical product is as follows: the concentration of 4'-galactosyllactose is 0.025-0.75 mg / mL, and the concentration of glycomacropeptide is 0.2-2.8 mg / mL.

[0051] Human milk oligosaccharides (HMOs), as the first natural prebiotics humans encounter, are specific substrates for beneficial gut bacteria. They act as anti-adhesion agents or decoy receptors, preventing viruses, bacteria, or other pathogens from binding to intestinal epithelial cells. Furthermore, they promote the maturation of the infant's immune system, acting as immunomodulators and regulating the responses of host epithelial cells and immune cells. Galactosyllactoses (GLs) are human milk oligosaccharides formed by a core of lactose residues linked by galactosidic bonds. They exist in several structurally different isomers, with 4'-galactosyllactose (4'-GL, 4'-Galactosyllactoses) being one such isomer. Galactosyllactose is abundant in breast milk, especially in colostrum secreted in the days before and after childbirth. It accounts for up to 9.5% of human milk oligosaccharides and up to 1.9 g / L in breast milk. It is the most abundant type of human milk oligosaccharide except for 2'-fucosyllactose (2'-FL) and lactose-N-neotetrasaccharide (LNnT).

[0052] Glycomaphatide (GMP) is a glycopeptide containing sialic acid, produced during cheese making by rennet cleaving casein between phenylalanine 105 and methionine 106. GMP has been shown to regulate various inflammatory pathways in different cell and animal models, and most literature indicates its anti-inflammatory role in the innate immune response. GMP can also bind directly to bacteria, preventing them from adhering to epithelial cells and reducing inflammation produced by the body.

[0053] This invention has found that single components such as 4'-GL can alleviate intestinal oxidative stress to some extent, but the effect is not ideal. The purpose of this invention is to study a composition that can effectively alleviate intestinal oxidative stress damage. This invention creatively uses increasing the levels of superoxide dismutase (SOD) and catalase (CAT) and downregulating the level of malondialdehyde (MDA), a marker of intestinal oxidative stress, as indicators for evaluating the alleviation of intestinal oxidative stress damage. This invention has found that the combination of 4'-GL and GMP can significantly enhance the alleviating effect on oxidative stress damage. Compared with single-component groups, this composition has a better alleviating effect on intestinal oxidative stress damage than single-component groups, exhibiting a synergistic effect.

[0054] According to some embodiments of the present invention, the relief of intestinal oxidative stress damage includes at least one of the following:

[0055] Increase the release of superoxide dismutase;

[0056] Increase the release of catalase;

[0057] Reduce malondialdehyde release;

[0058] To improve the survival rate of fruit flies with H2O2-induced intestinal oxidative stress damage;

[0059] To enhance the climbing ability of fruit flies with H2O2-induced intestinal oxidative stress damage;

[0060] Extending the intestinal length of fruit flies with H2O2-induced intestinal oxidative stress damage;

[0061] Reduce intestinal permeability in fruit flies with H2O2-induced intestinal oxidative stress damage.

[0062] It's important to note that superoxide dismutase (SOD) is an active substance derived from living organisms that eliminates harmful substances produced during metabolism. It is widely distributed throughout the biological world, found in almost everything from animals to plants, and even from humans to single-celled organisms. SOD is considered one of the most magical enzymes in life sciences, a scavenger of waste within the body. SOD is a natural enemy of oxygen free radicals, the number one killer of oxygen free radicals in the body, and is fundamental to health. Meanwhile, catalase (CAT) is an enzyme scavenger, a conjugated enzyme with iron porphyrin as a cofactor. It promotes the decomposition of H2O2 into molecular oxygen and water, thereby removing hydrogen peroxide from the body and protecting cells from the toxicity of H2O2. It is one of the key enzymes in the biological defense system. Furthermore, in living organisms, free radicals act on lipids, causing peroxidation reactions. The final oxidation product is malondialdehyde (MDA), which can cause cross-linking and polymerization of biomolecules such as proteins and nucleic acids, and has cytotoxic effects. MDA, an end product of lipid oxidation, affects the mitochondrial respiratory chain complex and the activity of key enzymes in mitochondria in vitro. Its production can also exacerbate membrane damage. Therefore, measuring the amount of malondialdehyde (MDA) can indirectly reflect the degree of cell damage. Thus, increasing the release of superoxide dismutase (SOD) and catalase, and decreasing the release of MDA, can reflect the elimination of reactive oxygen species and serve as a measure of the relief of intestinal oxidative stress damage.

[0063] According to some embodiments of the present invention, the food includes at least one of infant formula, children's milk powder, adult milk powder, food for special medical purposes, nutritional products, liquid milk, complementary food, and non-alcoholic beverages.

[0064] Fifthly, the present invention provides the use of the composition described above in the preparation of foods for adjunctive treatment and / or adjunctive prevention of intestinal infections and inflammatory bowel disease caused by intestinal oxidative stress.

[0065] In a sixth aspect, the present invention provides the use of the composition described above in the preparation of health foods for adjuvant treatment and / or adjuvant prevention of intestinal infections and inflammatory bowel disease caused by intestinal oxidative stress.

[0066] In a seventh aspect, the present invention provides the use of the composition described above in the preparation of pharmaceuticals for treating and / or preventing intestinal infections and inflammatory bowel disease caused by intestinal oxidative stress.

[0067] It is known that intestinal oxidative stress can negatively impact the intestinal mucosa and gut microbiota, thereby triggering diseases caused by intestinal oxidative stress, such as intestinal infections and inflammatory bowel disease. Given that the compositions provided by this invention have been proven to effectively alleviate intestinal oxidative stress, these compositions are expected to be used in the preparation of foods and health foods for the adjunctive treatment and / or prevention of intestinal infections and inflammatory bowel disease caused by intestinal oxidative stress, as well as in the preparation of pharmaceuticals for the treatment and / or prevention of intestinal infections and inflammatory bowel disease caused by intestinal oxidative stress.

[0068] The present invention will be further described below through some specific embodiments.

[0069] This invention explores the effects of 4'-GL and GMP on alleviating H2O2-induced intestinal oxidative stress damage in a model using three indicators: superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA), and screens the effective concentration ranges of 4'-GL and GMP. Based on the experimental results, effective concentration ranges are further selected for compounding 4'-GL and GMP, and the optimal compound concentration is chosen. The effects of the compound on the survival rate, climbing index, SOD, CAT, MDA content, intestinal length, and permeability of H2O2-induced fruit flies are used to verify the alleviating effect of the compound on intestinal oxidative stress damage, and the results are compared with the corresponding concentrations of the single substances.

[0070] The present invention will be further described below through some specific embodiments.

[0071] 1.1 The raw materials used in the following examples are as follows:

[0072] Fruit fly: Drosophila melanogaster W 1118 The strain was provided by Hunan Normal University.

[0073] Preparation method of glycomacropeptide (GMP):

[0074] Prepare an 8% (w / v) protein solution by dissolving an appropriate amount of whey protein concentrate. After thorough dissolution, boil in a water bath for 10 minutes and rapidly cool to room temperature. Then, add cold anhydrous ethanol at a concentration of 70% (v / v) and stir until homogeneous, allowing impurities to precipitate. Centrifuge at 4000g for 20 minutes and collect the supernatant. Adjust the pH of the supernatant to 4.5 with hydrochloric acid, allow to stand, and allow for secondary precipitation of impurities. Centrifuge at 4000g for 20 minutes and collect the supernatant. Finally, remove the ethanol from the supernatant by rotary evaporation. Adjust the pH of the remaining solution to neutral, and ultrafilter using a hollow fiber ultrafiltration membrane with a molecular weight cutoff of 5kDa. Collect the concentrate, freeze-dry, and obtain the GMP powder.

[0075] Preparation method of 4'-galactosyllactose (4'-GL):

[0076] Step (A): The oligogalactosides containing 4'-GL are fed into an activated carbon column for chromatography and eluted with a gradient of methanol solution. In this gradient elution, each time the number of elutions is increased, an aqueous solution of organic solvent with a higher concentration than that in the previous elution is used.

[0077] Step (B): Add methanol solution to the fraction obtained by final elution in step (A) to crystallize 4'-GL and obtain 4'-GL powder.

[0078] Superoxide dismutase (SOD) kit: purchased from Nanjing Jiancheng Bioengineering Institute;

[0079] Catalase (CAT) kit: purchased from Nanjing Jiancheng Bioengineering Institute;

[0080] Malondialdehyde (MDA) reagent kit: purchased from Nanjing Jiancheng Bioengineering Institute.

[0081] 1.2 Fruit fly culture

[0082] Preparation of basic culture medium: Take 130g corn flour, 77g sucrose, 30g soybean flour, 13g agar powder and 900mL distilled water and put them into a pot, boil for 30 minutes.

[0083] 4'-GL medium (experimental group medium): The preparation method is the same as that of ordinary medium, except that 4'-GL powder is added in proportion to adjust the final concentration of 4'-GL in the medium.

[0084] GMP medium (experimental group medium): The preparation method is the same as that of ordinary medium, except that GMP powder is added in proportion to adjust the final concentration of GMP in the medium.

[0085] Composite culture medium (experimental group culture medium): The preparation method is the same as that of ordinary culture medium, except that 4'-GL and GMP powder are added according to the mass ratio to adjust the final concentration of 4'-GL and GMP in the culture medium.

[0086] 1.3 Establishment of a Drosophila intestinal oxidative stress injury model and feeding of samples

[0087] Randomly collect W that has been in the embryonic stage for 3-5 days. 1118 Fruit flies were first anesthetized with CO2, and 15 male and 15 female fruit flies were placed in each tube, for a total of 30 flies, with 3 tubes collected from each group. The flies were fed freely for 3 days using the basal culture medium from section 1.2 and the experimental group culture medium. Then, the fruit flies were transferred to fruit fly tubes and starved for 2 hours in empty tubes. Next, they were transferred to fruit fly tubes with 5 circular filter papers at the bottom. For the blank control group, 400 μL of 5% sucrose solution was added to the filter paper; for the model group, 400 μL of 3% H2O2 (containing 5% sucrose) solution was added to the filter paper; for the experimental group, 3% H2O2 and the sample were simultaneously dissolved in 5% sucrose solution, adjusting the final sample concentration to 400 μL to fully moisten the filter paper. The filter paper was changed every 24 hours, and the feeding was continued for 6 days.

[0088] This experiment used the fruit fly survival curve to show the model establishment. Successful model establishment was indicated by the survival rate dropping to approximately 50% on day 3 after feeding with a 3% H₂O₂ solution (containing 5% sucrose) and to approximately 20% on day 5. The fruit flies were grown in a constant temperature and humidity environment: temperature 25.0 ± 0.5℃ and relative humidity 50–60%.

[0089] 1.4 Effects of different concentrations of 4'-GL and GMP on the release of SOD, CAT and MDA

[0090] Several fruit flies from the experimental group were frozen at -80℃ for 0.5 hours, then homogenized into a tissue slurry in an ice bath with pre-cooled sterile physiological saline, and centrifuged at 5000 r / min for 10 minutes at 4℃. After collecting the supernatant, the contents of total SOD, CAT, and MDA were determined using a superoxide dismutase (SOD) kit, a catalase (CAT) kit, and a malondialdehyde (MDA) kit. The experimental groups are shown in Table 1. In addition, a model group and a blank control group were also set up.

[0091] Table 1

[0092] Group Ingredients and concentration Group Ingredients and concentration Group 1 4'-GL: 0.025 mg / mL Group 4 GMP: 0.2 mg / mL Group 2 4'-GL: 0.375 mg / mL Group 5 GMP: 1.4 mg / mL Group 3 4'-GL: 0.75 mg / mL Group 6 GMP: 2.8 mg / mL

[0093] Experimental results:

[0094] The effects of different concentrations of 4'-galactosyllactose (4'-GL) and different concentrations of glycomacropeptide (GMP) on the release of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) in a H2O2-induced Drosophila intestinal oxidative stress injury model are shown in the appendix. Figures 1-6 And Table 2.

[0095] Table 2

[0096] SOD(U / mgprot) CAT(U / mgprot) MDA (nmol / mg) Blank control group 28.648 19.837 23.978 Model group 16.968 6.389 67.287 0.025 mg / mL 4'-GL 17.756 10.292 58.631 0.375 mg / mL 4'-GL 19.877 13.532 52.287 0.75 mg / mL 4'-GL 18.343 11.623 55.875 0.2 mg / mL GMP 18.056 9.169 60.290 1.4 mg / mL GMP 19.956 11.134 55.866 2.8 mg / mL GMP 20.835 13.326 51.658

[0097] Experimental results showed that 4'-GL at concentrations of 0.025–0.75 mg / mL could promote the release of SOD and CAT, and downregulate the release of the oxidative stress marker MDA. The experimental group treated with 0.375 mg / mL 4'-GL showed the most significant promotion of SOD and CAT release and inhibition of MDA release. Therefore, a concentration range of 0.025–0.75 mg / mL was selected as the optimal concentration range for the 4'-GL formulation.

[0098] GMP concentrations ranging from 0.2 to 2.8 mg / mL all promoted the release of SOD and CAT, and downregulated the release of the oxidative stress marker MDA. The experimental group treated with 2.8 mg / mL GMP showed the most significant promotion of SOD and CAT release and inhibition of MDA release. Therefore, a concentration range of 0.2–2.8 mg / mL was selected as the optimal concentration range for the combined GMP formulation.

[0099] Examples 1-3 Preparation of Compositions 1-3

[0100] Based on the results of Section 1.4 above regarding the effects of different concentrations of 4'-galactosyllactose (4'-GL) and different concentrations of glycomacropeptide (GMP) on the release of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) in a Drosophila intestinal oxidative stress injury model induced by H2O2, we further selected 0.025–0.75 mg / mL of 4'-GL and 0.2–2.8 mg / mL of GMP for compounding. The compounding method is shown in Section 1.3, and the compounding ratio is shown in Table 3, which are respectively designated as Composition 1, Composition 2, and Composition 3.

[0101] Table 3

[0102]

[0103] The following study used an H2O2-induced intestinal oxidative stress injury model in Drosophila to evaluate the ability of the compound composition to alleviate intestinal oxidative stress injury in Drosophila by assessing the release capacity of SOD, CAT, and MDA, survival rate, climbing ability, intestinal length, and permeability.

[0104] Data Statistics and Analysis: All statistical analysis and graphical display of data were performed using GraphPad Prism 8.0. T-tests were used for comparisons between groups, and one-way ANOVA was used for multiple groups. Significant differences were compared with the model group, with significance levels of P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***).

[0105] Effects of Compositions 1-3 in Test Examples 1 on the release of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA)

[0106] Test method:

[0107] The fruit flies and physiological saline were homogenized in an ice bath at a ratio of M1 (fly weight) : M2 (physiological saline) = 1 : 9, and then centrifuged at 4°C (2500 g / min, 15 min). The supernatant was then collected and the corresponding content was detected using the appropriate kit.

[0108] Experimental results: The effects of different concentrations of 4'-GL and GMP combinations on the release of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) are shown in Table 4. Figures 7-9 .

[0109] Table 4

[0110] SOD(U / mgprot) CAT(U / mgprot) MDA (nmol / mg) Blank control group 27.982 21.837 22.817 Model group 16.274 7.389 66.182 1.8 mg / mL 4'-GL 17.674 9.717 59.725 1.8 mg / mL GMP 19.543 12.148 53.357 Composition 1 23.598 16.123 42.445 Composition 2 24.898 17.964 39.822 Composition 3 26.29 19.951 36.905

[0111] Experimental results showed that different concentrations of the 4'-GL and GMP combination significantly increased the release of superoxide dismutase (SOD) and catalase (CAT) while significantly reducing the release of malondialdehyde (MDA). Figures 7-9 The experimental results show that the two have a synergistic effect. Compared with the same concentration of 4'-GL and GMP single substances, the composition significantly reduced the release of MDA and significantly increased the release of SOD and CAT.

[0112] Test Example 2: Fruit Fly Survival Rate Test

[0113] Test method: Thirty fruit flies were randomly selected from the blank control group and experimental group 3-5 days after emergence. Newly emerged fruit flies were collected and cultured for 3 days in ordinary culture medium and experimental group culture medium, respectively. Then, they were transferred to empty fruit fly tubes and starved for 2 hours. The required experimental solution (400 μl) was permeated into three layers of filter paper and supplied continuously for 6 days. The number of surviving fruit flies was recorded daily, and the experiment was repeated 3 times.

[0114] Experimental results: The effect of different concentrations of 4'-GL and GMP composition on the survival rate of fruit flies can be found in [reference needed]. Figure 10Experimental results showed that compositions of 4'-GL and GMP in different ratios could synergistically improve the survival rate of Drosophila subjected to H2O2-induced intestinal oxidative stress. Compared with the same concentration of 4'-GL and GMP, compositions 1, 2, and 3 significantly improved the survival rate of Drosophila, with compositions 1, 2, and 3 achieving survival rates of 39%, 59%, and 74%, respectively.

[0115] Test Example 3: Fruit Fly Climbing Ability Test

[0116] Test method: After inducing the fruit flies with a 3% H2O2 (containing 5% sucrose) solution for 48 hours, the fruit flies were anesthetized with ether and placed in the tracks of a climbing toolbox. Three fruit flies were placed in each track and the box was sealed. After all the fruit flies regained consciousness and adapted for ≥20 minutes, the box was gently shaken to place all the fruit flies at the bottom of the tracks. Due to negative tropism, the fruit flies climbed upwards along the inner wall. The number of fruit flies that climbed above 7 cm within 10 seconds was recorded (A), and the total number of fruit flies was recorded as (B). The climbing index was calculated using the formula: Climbing Index = A / B.

[0117] Experimental results: The effect of different concentrations of 4'-GL and GMP combinations on the climbing ability of fruit flies can be found in [reference needed]. Figure 11 Compared to the model group, the climbing ability of fruit flies fed with composition 1, composition 2 and composition 3 increased by 21.96%, 32.52% and 46.43%, respectively.

[0118] Test Example 4: Drosophila intestinal morphology indicators

[0119] Test method: After treating fruit flies according to the method in 1.3, the fruit fly intestines were dissected under a microscope and placed in chilled PBS. Then, 3-5 intestines from each group were taken out from the pre-chilled PBS and stored in sterile physiological saline for observation of morphological changes in the fruit fly intestines under a microscope.

[0120] Experimental Results: Drosophila were collected after induction, and their intestines were isolated. Morphological changes in the Drosophila intestine were observed under a microscope. Results regarding the prolongation of intestinal length in the H2O2-induced Drosophila intestinal oxidative stress model using a combination of 4'-GL and GMP are shown in [link to relevant documentation]. Figure 12 It can be seen that the intestinal length of the fruit flies in the model group fed with H2O2 was significantly shortened by 34.39%. 4'-GL and GMP and their combinations can increase the intestinal length to varying degrees, among which combination 3 has the most significant effect, with a length increase of 46.2% compared with the model group. This result also proves that 4'-GL and GMP and their combinations can protect intestinal epithelial cells from H2O2-induced intestinal oxidative stress damage and inhibit the shortening of intestinal length.

[0121] Test Example 5: Evaluation Test of Intestinal Barrier Integrity in Drosophila

[0122] Test Methods: Fruit flies possess good intestinal function and integrity, but under oxidative stress, intestinal homeostasis is disrupted, leading to impaired intestinal barrier function. Therefore, to evaluate the effect of the added composition on intestinal permeability, fruit flies were fed samples and 5% sucrose solution as experimental and blank control groups, respectively. 2.5% (w / w) of brilliant blue was added to the 5% sucrose solution. Newly emerged female flies in both groups were starved for 2 hours every day, treated with filter paper soaked in H2O2 or 5% sucrose solution for 6 hours, and then transferred to brilliant blue-stained food (sucrose). After 6 days of treatment, the body condition of the fruit flies was recorded by photography.

[0123] Experimental results: Figure 13 This figure shows the effects of 4'-GL, GMP, and their combinations on intestinal barrier function in a H2O2-induced Drosophila intestinal oxidative stress injury model. Loss of intestinal barrier function in Drosophila can be detected by a non-absorbable blue food dye called Brilliant Blue. When intestinal permeability increases, Brilliant Blue permeates through the intestinal epithelium and stains the entire body. Experimental results showed that the blank control group exhibited normal intestinal morphology, while the model group showed color diffusion throughout the abdomen and a dark color. Compared to the model group, the 4'-GL and GMP groups reduced the abdominal color in Drosophila. Combinations 1, 2, and 3 all reduced Brilliant Blue in the abdomen and decreased the extent of its diffusion. Combinations 2 and 3 significantly reduced the color diffusion in the abdominal region, thus reducing damage to the Drosophila intestinal barrier.

[0124] In summary, under the same concentration conditions, compared with the individual concentrations of 4'-GL and GMP, the combination of 4'-GL and GMP significantly promoted the release of superoxide dismutase (SOD) and catalase (CAT), while also significantly reducing the release of malondialdehyde (MDA), a marker of oxidative stress. The synergistic effect between the two substances enabled 4'-GL and GMP to better alleviate intestinal damage in Drosophila. Simultaneously, the combination improved the survival rate and climbing index of H2O2-induced Drosophila, prolonged the intestinal length of diseased Drosophila, enhanced the integrity of the intestinal barrier, and showed a trend towards recovery in the flies.

[0125] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A composition of human milk oligosaccharides, characterized in that, The composition comprises: 4'-galactosyl lactose and glycomacropeptide; The mass ratio of the 4'-galactosyl lactose and the glycomacropeptide is (1:30)~(8:112).

2. Use of a composition as described in claim 1 for the manufacture of a medicament having the following utility, characterized in that, The utility comprises relieving intestinal oxidative stress injury, The relieving intestinal oxidative stress injury comprises at least one of the following performances: increasing the release amount of superoxide dismutase; increasing the release amount of catalase; reducing the release amount of malondialdehyde; increasing the survival rate of fruit flies with H2O2-induced intestinal oxidative stress injury; increasing the climbing ability of fruit flies with H2O2-induced intestinal oxidative stress injury; prolonging the intestinal length of fruit flies with H2O2-induced intestinal oxidative stress injury; reducing the intestinal permeability of fruit flies with H2O2-induced intestinal oxidative stress injury; The application concentration of the composition is that the concentration of the 4'-galactosyl lactose is 0.025~0.75mg / mL, and the concentration of the glycomacropeptide is 0.2~2.8mg / mL.

3. The use of the composition in claim 1 in the preparation of a drug for treating and / or preventing intestinal infection and inflammatory bowel disease caused by intestinal oxidative stress.

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