A nutritional composition, products and uses thereof

Abstract

The present invention provides a nutritional composition comprising one or more of vitamin D, polydextrose and galacto-oligosaccharides in combination with a calcium source. The composition has the effect of improving the intestinal mucosal barrier including improving intestinal mucosal permeability, modulating expression of intercellular tight junction proteins, improving intestinal mucosal tissue structure and improving levels of markers of the intestinal mucosal barrier.

Description

Technical Field

[0001] This invention relates to the field of nutritional supplement technology, and in particular to a nutritional composition, its products, and applications. Background Technology

[0002] Besides forming bones and teeth, calcium maintains various physiological functions, such as regulating nerve and muscle excitability and mediating and regulating the contraction of muscles and intracellular microfilaments and microtubules; affecting capillary permeability and participating in the regulation of biological membrane integrity and plasma membrane permeability and their transformation processes; participating in the regulation of the release of various hormones and neurotransmitters, and participating in the regulation of lipase and ATPase activity; and being closely related to cellular activities such as phagocytosis, secretion, and division. Therefore, adequate calcium intake can not only improve bone health but may also reduce the occurrence of some chronic diseases, such as lowering blood pressure, reducing the risk of colon cancer, and improving diabetic bone loss and related symptoms.

[0003] The gut is not only a vital organ for digestion and nutrient absorption, but also a natural barrier protecting the body from antigens, pathogenic microorganisms, and their harmful metabolites in food, maintaining internal homeostasis. The intestinal mucosal barrier consists of physical, chemical, microbial, and immune barriers, each with distinct biological functions and molecular regulatory mechanisms. These barriers are organically combined through their respective signaling pathways to jointly defend against the invasion of external pathogens. When the intestinal mucosal barrier function is impaired, intestinal mucosal permeability increases, allowing harmful substances such as intestinal bacteria and endotoxins to escape uncontrollably into the extraluminal tissues, triggering a series of pathophysiological changes and even systemic inflammatory response syndrome and multiple organ dysfunction syndrome.

[0004] Therefore, it is essential to provide a nutritional composition that can improve the intestinal mucosal barrier. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a nutritional composition that can improve the intestinal mucosal barrier.

[0006] The present invention provides a nutritional composition comprising one or more of vitamin D, polydextrose and galactooligosaccharides, in combination with a calcium source.

[0007] Preferably, the raw materials include the following parts by weight:

[0008] Calcium source 100,000 to 800,000 parts, Vitamin D 0 to 10 parts, Polydextrose 0 to 2.65 million parts, Galacto-oligosaccharide 0 to 660,000 parts.

[0009] Preferably, the raw materials include the following parts by weight:

[0010] Calcium source 100,000 to 800,000 parts, vitamin D 1 to 10 parts, polydextrose 100,000 to 2,650,000 parts, galactooligosaccharide 100,000 to 660,000 parts.

[0011] Preferably, the weight ratio of the polydextrose to the galactooligosaccharide is (0.5-15):1.

[0012] Preferably, the calcium source includes one or more of the following: calcium carbonate, calcium gluconate, calcium citrate, calcium lactate, L-calcium lactate, calcium hydrogen phosphate, L-threonine calcium, glycine calcium, calcium aspartate, calcium citrate-malate, calcium acetate, calcium chloride, tricalcium phosphate, vitamin E succinate, calcium glycerophosphate, calcium oxide, calcium sulfate, calcium dihydrogen phosphate, milk mineral salts, casein calcium, calcium malate, and calcium ascorbate.

[0013] The present invention provides the use of the composition described in any of the above claims in the preparation of products that improve the intestinal mucosal barrier.

[0014] Preferably, the improvement of the intestinal mucosal barrier includes improving intestinal mucosal permeability, regulating the expression of intercellular tight junction proteins, improving intestinal mucosal tissue structure, and improving the levels of intestinal mucosal barrier markers.

[0015] Preferably, the improvement of intestinal mucosal permeability includes increasing the transmembrane resistance (TEER) and inhibiting the apparent permeability coefficient (Papp).

[0016] The intestinal mucosal barrier markers include D-lactic acid and sIgA.

[0017] The present invention provides a product for improving the intestinal mucosal barrier, comprising the composition described in any one of the above-mentioned technical solutions.

[0018] Preferably, the product includes food or health products; the food includes dairy products.

[0019] Compared with existing technologies, the present invention provides a nutritional composition comprising one or more of vitamin D, polydextrose, and galactooligosaccharides in combination with a calcium source. The above composition has the effects of improving the intestinal mucosal barrier, including improving intestinal mucosal permeability, regulating the expression of tight junction proteins between cells, improving intestinal mucosal tissue structure, and improving the levels of intestinal mucosal barrier markers. Attached Figure Description

[0020] Figure 1 The TEER ratio of each group of cells in Example 1 of this invention;

[0021] Figure 2 The Papp values ​​of cells in each group in Example 1 of this invention;

[0022] Figure 3 Immunofluorescence of cells in each group in Example 1 of this invention Figure 1(×200);

[0023] Figure 4 These are representative colon morphological images of each experimental group in Example 2 of the present invention. Detailed Implementation

[0024] This invention provides a nutritional composition, its product, and its application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and fall within the scope of protection of this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0025] The present invention provides a nutritional composition comprising one or more of vitamin D, polydextrose and galactooligosaccharides, in combination with a calcium source.

[0026] The nutritional composition described in this invention is a nutritional composition for improving the intestinal mucosal barrier.

[0027] The compositions of the present invention include combinations of vitamin D and calcium sources, combinations of calcium sources and polydextrose, combinations of calcium sources and galactooligosaccharides, and combinations of calcium sources, vitamin D, polydextrose, and galactooligosaccharides.

[0028] In a preferred embodiment of the present invention, the composition comprises the following raw materials in parts by weight:

[0029] Calcium source 100,000 to 800,000 parts, Vitamin D 0 to 10 parts, Polydextrose 0 to 2.65 million parts, Galacto-oligosaccharide 0 to 660,000 parts.

[0030] In a preferred embodiment of the present invention, the composition comprises the following raw materials in parts by weight:

[0031] Calcium source 100,000 to 800,000 parts, vitamin D 1 to 10 parts, polydextrose 100,000 to 2,650,000 parts, galactooligosaccharide 100,000 to 660,000 parts.

[0032] In a preferred embodiment of the present invention, the composition comprises the following raw materials in parts by weight:

[0033] Calcium source 200,000 to 600,000 parts, Vitamin D 2 to 8 parts, Polydextrose 1,000,000 to 2,600,000 parts, Galacto-oligosaccharide 200,000 to 650,000 parts.

[0034] In a preferred embodiment of the present invention, the composition comprises the following raw materials in parts by weight:

[0035] Calcium source 200,000 to 250,000 parts, Vitamin D 4 to 6 parts, Polydextrose 1.5 million to 2.5 million parts, and Galacto-oligosaccharides 350,000 to 600,000 parts.

[0036] In a preferred embodiment of the present invention, the dosage of the composition for improving the intestinal mucosal barrier is as follows:

[0037] Calcium source 400mg~500mg / day, vitamin D 8μg~12μg / day, polydextrose 3g~5g / day, galactooligosaccharide 0.7g~1.2g / day.

[0038] In a preferred embodiment of the present invention, the composition comprises 420,000 to 700,000 parts of a calcium source.

[0039] In one preferred embodiment of the present invention, the dosage of the composition is: 420mg-700mg / d of calcium source.

[0040] In a preferred embodiment of the present invention, the weight ratio of polydextrose to galactooligosaccharide is (1-15):1; more preferably (1-13):1; most preferably (1-10):1; particularly preferably (1-8):1; and may also be (2-7):1; specifically, it can be 2:1, 3:1, 4:1, 5:1, 6:1, 7:1; or any point value between the above two.

[0041] The polydextrose of this invention meets the requirements of GB 25541; the galactooligosaccharide meets the requirements of the Ministry of Health's Order No. 20 of 2008.

[0042] The calcium source described in this invention includes one or more of the following: calcium carbonate, calcium gluconate, calcium citrate, calcium lactate, L-calcium lactate, calcium hydrogen phosphate, L-threonine calcium, glycine calcium, calcium aspartate, calcium citrate-malate, calcium acetate, calcium chloride, tricalcium phosphate, vitamin E succinate, calcium glycerophosphate, calcium oxide, calcium sulfate, calcium dihydrogen phosphate, milk mineral salts, casein calcium, calcium malate, and calcium ascorbate. This invention does not limit the source of the above-mentioned calcium sources; commercially available calcium sources are acceptable.

[0043] The inventors have discovered that the nutritional composition provided by this invention has the effect of improving the intestinal mucosal barrier, including improving intestinal mucosal permeability, regulating the expression of intercellular tight junction proteins, improving intestinal mucosal tissue structure, and improving the level of intestinal mucosal barrier markers.

[0044] The components and proportions described above in this invention are functionally mutually supportive and have an interactive relationship; as long as the specific components and proportions described above are met, the technical effects of this invention can be achieved.

[0045] The present invention provides the use of the composition described in any of the above claims in the preparation of products that improve the intestinal mucosal barrier.

[0046] The improvement of the intestinal mucosal barrier described in this invention includes improving intestinal mucosal permeability, regulating the expression of tight junction proteins between cells, improving intestinal mucosal tissue structure, and improving the levels of intestinal mucosal barrier markers.

[0047] Specifically, the improvement of intestinal mucosal permeability includes increasing the transmembrane resistance (TEER) and inhibiting the apparent permeability coefficient (Papp).

[0048] The intestinal mucosal barrier markers described in this invention include D-lactic acid and sIgA.

[0049] This invention discovers that calcium deficiency can lead to increased intestinal cell permeability and damage to the intestinal mucosal barrier. Intervention with calcium and its compositions can improve the health benefits of intestinal mucosal barrier function. Specifically, this invention provides the application of calcium, particularly a calcium + vitamin D + galactooligosaccharide + polydextrose composition, in the preparation of foods for improving intestinal mucosal barrier function. The health benefits of improving the intestinal mucosal barrier have achieved unexpected results at both the cellular and animal experimental levels.

[0050] At the cellular level, an intestinal cell barrier damage model was established using calcium-free Caco-2 cells. The effects of calcium and its nutritional compositions on improving intestinal mucosal barrier damage were evaluated, yielding unexpected health benefits, including improved intestinal mucosal permeability (transmembrane resistance TEER, apparent permeability coefficient Papp value) and regulation of intercellular tight junction protein expression. Specifically: 1) Calcium-free culture caused the successfully established cell barrier to collapse within 4 days, with significantly reduced TEER and Papp values, leading to increased cell permeability and decreased compactness, resulting in cell barrier damage. 2) The damaged cell barrier in a calcium-free environment could be repaired by adding calcium; the addition of galactooligosaccharides or polydextrose alone could not repair the damaged intestinal cell barrier; the combined effect of calcium and vitamin D did not significantly enhance the intestinal cell barrier; the low-prebiotic ratio group and the high-prebiotic ratio group showed an enhancing trend, but the difference was not significant, and further evaluation was conducted using animal experiments.

[0051] At the animal experimental level, using a previously constructed growth animal model with calcium intake of only 1 / 3 of the recommended amount, the calcium nutrition status of Chinese children was simulated. After 3 months of low-calcium diet feeding, representing children aged 3-12 years, the model was used to evaluate the effects of calcium and its nutritional combinations on improving the intestinal mucosal barrier. Unexpected health effects were achieved, including improvement of intestinal mucosal tissue structure and intestinal mucosal barrier markers (such as D-lactic acid).

[0052] Specifically: 1) Histopathological results of colon tissue sections indicated that insufficient calcium intake exacerbated the inflammatory response of the lamina propria connective tissue in the colon of growing rats; rats with high calcium intake had intact colon tissue structure, with no obvious necrosis or large amount of inflammatory cell infiltration; suggesting the importance of sufficient calcium for the integrity of intestinal tissue structure; when calcium intake could not reach high calcium levels, appropriate amounts of calcium (75% of the high calcium group) and its combination alleviated the inflammatory cell infiltration of the lamina propria connective tissue to some extent, suggesting that when calcium intake is insufficient, the two prebiotics combined with calcium and vitamin D have a synergistic effect on improving the tissue morphology of the colon. 2) Long-term calcium deficiency leads to elevated serum D-lactate levels in growing rats, increased intestinal mucosal permeability, and damage to the intestinal mucosal barrier. Increasing calcium and vitamin D intake can lower serum D-lactate levels, alleviating intestinal mucosal barrier damage to some extent. High calcium intake can mitigate the effects of low calcium intake on intestinal mucosal damage. When calcium intake is insufficient, prebiotics and calcium + vitamin D can exert a synergistic effect, mitigating the adverse effects of low calcium intake on intestinal mucosal permeability. 3) Long-term calcium deficiency damages the intestinal mucosa in growing rats, causing the intestinal mucosa to secrete sIgA to resist pathogen invasion. Increasing calcium and vitamin D intake can lower sIgA levels, but the effect is not significant. High calcium intake and the combination of prebiotics and calcium + vitamin D can reduce sIgA levels, mitigating the adverse effects of low calcium intake on the intestinal mucosal barrier.

[0053] This invention has achieved technical effects beyond expectations. In cases of insufficient calcium intake, there is damage to the intestinal mucosal barrier. While supplementing with high calcium intake (calcium supplementation to the recommended intake level) has the best alleviating effect, it does not conform to the current dietary calcium intake situation in my country. Considering the actual situation of dietary calcium intake in my country, when calcium intake does not reach the recommended intake level or is at a high calcium level, the calcium + vitamin D + PDX + GOS combination can also improve the intestinal mucosal barrier, and its effect is significantly better than that of the calcium and vitamin D group.

[0054] The present invention provides a product for improving the intestinal mucosal barrier, comprising the composition described in any one of the above-mentioned technical solutions.

[0055] The products described in this invention include, but are not limited to, food and / or health products.

[0056] The food products described in this invention include, but are not limited to, snack foods, dairy products, and beverages.

[0057] The product described in this invention is a dairy product;

[0058] The dairy products described in this invention include liquid milk (pasteurized milk, sterilized milk, modified milk, fermented milk); milk powder (whole milk powder, skim milk powder, partially skimmed milk powder, modified milk powder, bovine colostrum powder); and other dairy products. Specifically, they can be categorized as follows: The first category is liquid milk, mainly including pasteurized milk, sterilized milk, yogurt, etc. The second category is milk powder, including whole milk powder, skim milk powder, sweetened whole milk powder, flavored milk powder, infant formula, and other formula milk powders. The third category is condensed milk. The fourth category is milk fat, including whipped cream for cakes, common butter for bread, etc. The fifth category is cheese and processed cheese. The sixth category is dairy ice cream. The seventh category is other dairy products, mainly including casein, lactose, milk tablets, etc.

[0059] The dairy products described in this invention preferably include one or more of the following: modified milk, modified milk powder, fermented milk, milk tablets, cheese, or dairy snacks. More preferably, the dairy products include infant formula, children's formula, and adult formula.

[0060] The components and proportions of the above-described composition have been clearly described above and will not be repeated here.

[0061] This invention does not limit the other ingredients or excipients in dairy products; those known to those skilled in the art are acceptable.

[0062] The present invention also provides a method for preparing a product that improves the intestinal mucosal barrier, which includes mixing the above-mentioned raw materials.

[0063] The intestinal cell barrier impairment model described in this invention is preferably constructed by culturing Caco-2 cells in a calcium-free medium.

[0064] The specific proportions of the compositions described in this invention can all repair damaged intestinal cells and improve the intestinal mucosal barrier function.

[0065] The technical effects of this invention are based on intestinal mucosal barrier damage caused by a calcium deficiency model. Calcium and its compositions can improve intestinal mucosal permeability (transmembrane resistance TEER, apparent permeability coefficient Papp value), regulate the expression of tight junction proteins between cells, reduce D-lactic acid and sIgA levels, and alleviate intestinal mucosal barrier damage. Although high calcium intake has the best effect in each comparison ratio, considering the actual situation of dietary calcium intake in my country, when calcium intake does not reach the recommended intake or is at a high calcium level, the calcium + vitamin D + PDX + GOS composition can also improve the intestinal mucosal barrier, and its effect is significantly better than that of the calcium and vitamin D group.

[0066] To further illustrate the present invention, the following detailed description of a nutritional composition, its products, and applications provided by the present invention is provided in conjunction with embodiments.

[0067] Example 1: Cell Experiment

[0068] 1.1 Establishment of the Caco-2 intestinal cell barrier model

[0069] Using Transwell chambers of the same specifications, cells were washed three times with PBS and inoculated with an appropriate concentration of Caco-2 cell suspension. They were then cultured in normal culture medium until the transepithelial electrical resistance (TEER) exceeded 200–400 Ω·cm. 2 If the Caco-2 intestinal cell barrier model is successfully established, then the chambers with similar TEER values ​​are selected and divided into a normal group and a calcium-free group. The normal group is cultured in normal medium without FBS (to prevent the presence of calcium in FBS). 2+ (This affects the experimental results). The calcium-free group was cultured in calcium-free medium and the relevant indicators were measured when the intestinal barrier was damaged. Three parallel wells were set up for each group in each experiment.

[0070] After the model was constructed, the transmembrane resistance of monolayer cells was measured using a Millicell-ERS 2 resistance meter; the apparent permeability coefficient (Papp) in the model was measured using Sigma-Aldrich fluorescein (free acid).

[0071] 1.2 Test substance concentration and grouping

[0072] After successfully constructing the Caco-2 intestinal cell injury barrier model, the model group continued to culture cells in normal culture medium without FBS; the other groups were cultured in calcium-free culture medium (without PBS) with the corresponding concentration of the test substance added according to the concentration of the test substance. The specific test substance grouping and concentration are shown in the table below.

[0073] Table 1. Grouping and Concentration of Test Substances

[0074]

[0075]

[0076] After 4 days of culture, the transmembrane resistance (TEER) and Papp values ​​of each group were measured. Immunofluorescence was used to determine the protein morphology of intercellular tight junction proteins ZO-1, Occludin, and Claudin-1, as well as the expression of immune factors in each group.

[0077] 1.3 Statistical Analysis

[0078] SPSS Statistics 26.0 software was used to process and analyze the raw data. Quantitative data were expressed as mean ± standard deviation (x±s). After testing for homogeneity of variance, one-way ANOVA was used to compare differences among multiple groups for data that met the homogeneity requirement. Dunnett's test was used for pairwise comparisons between the model group and each test group, and SNK-q test was used for comparisons among multiple groups. P < 0.05 or P < 0.01 was considered statistically significant.

[0079] 1.4 Experimental Results

[0080] (1) Establishment and evaluation of the Caco-2 intestinal cell barrier model

[0081] In the first two days after the barrier formed and the damage model was established, the TEER values ​​of the calcium-free group and the normal group showed similar trends over time and both increased. However, on the third day, the TEER value of the calcium-free group plummeted, falling below 100 Ω·cm. 2 The TEER in the normal group continued to decline, while the TEER in the control group steadily increased, but the increase was smaller. On day 4, the Papp value of cells in the control group was less than 1.0 × 10⁻⁶. - 6 The Papp value in the calcium-free group was significantly higher than that in the normal group (P<0.01). The results indicate that when the cell barrier is in a calcium-free environment, it can be destroyed in just 3-4 days, forming a damaged model.

[0082] Table 2. Trends in TEER values ​​of Caco-2 cells in the normal group and calcium-free group over time (Ω·cm) 2 , n=3)

[0083]

[0084] Table 3. Comparison of Papp values ​​(cm / s) of Caco-2 cells on day 4 between the normal group and the calcium-free group. n=3)

[0085]

[0086] Note: Compared with the normal group ** P<0.01.

[0087] (2) Repair effect of the test substance on the intestinal barrier injury model

[0088] 1) Comparison of TEER values ​​among the test groups

[0089] From Table 4 and Figure 1The results showed that the TEER ratios of the model group, GOS group, and PDX group were the lowest and significantly different from those of the other groups (P < 0.05). This indicates that culturing Caco-2 cells in a calcium-deficient environment can completely destroy the tight junction structure between cells, causing the TEER value to drop sharply to the initial level. The barrier formed by the monolayer structure between cells is completely broken down, and the effect of prebiotics alone cannot alleviate the continued damage to the barrier.

[0090] Contains Ca 2+ The TEER ratios of all the compound groups were significantly higher than those of the model group (P < 0.05). Among them, the TEER ratios of the GOS compound group and the PDX compound group were the highest, reaching 3.51 times and 3.28 times, respectively. Compared with the calcium-containing group, the TEER ratios of the GOS compound group and the PDX compound group were significantly increased (P < 0.05), while there were no statistically significant differences among the other groups (P > 0.05). Compared with the calcium-containing VD3 group, there were no statistically significant differences among the other groups (P > 0.05).

[0091] In terms of the TEER ratio, the GOS and PDX combination groups had significantly better effects on enhancing the intestinal barrier than Ca alone. 2+ The effects of the two prebiotics were not obvious compared to the calcium-containing VD3 group.

[0092] Table 4. TEER ratio of cells in each group ( n=3)

[0093]

[0094] Note: Different letters indicate statistical significance between groups, P<0.05.

[0095] 2) Comparison of Papp values ​​among the test groups

[0096] From Table 5 and Figure 2 It was found that the model group, GOS group, and PDX group had the highest Papp values, and these values ​​were significantly different from those of the other groups (P < 0.05). Among them, PDX had the highest Papp value, reaching 60.86 × 10⁻⁶. -7 The value of cm / s indicates that the tight junction structure between cells is disrupted in a calcium-deficient environment, and the increased permeability between cells leads to an increase in the Papp value, which is consistent with the TEER ratio result. This suggests that the effect of prebiotics alone cannot alleviate the continued damage to the barrier in a calcium-deficient environment.

[0097] Compared to the model group, the Ca-containing 2+ The Papp values ​​of all composite groups were significantly reduced (P < 0.05), with the calcium-containing group having the lowest value, at only 2.76 × 10⁻⁶. -7cm / s, and the Papp values of the high-prebiotic ratio group and the GOS composite group were also relatively low, but there were no statistical differences between them (P>0.05); compared with the calcium-containing group, there were no statistical differences in other groups (P>0.05); compared with the calcium + VD3 group, there were no statistical differences in the GOS composite group and the PDX composite group (P>0.05).

[0098] In summary, the calcium-free environment destroys the tight junction structure, resulting in larger intercellular spaces and increased permeability, while each composite group containing Ca 2+ can protect the damaged barrier and reduce the intercellular permeability.

[0099] Table 5 Papp values of cells in each group (×10 -7 cm / s, n = 3)

[0100]

[0101] Note: Different letters indicate statistical significance in pairwise comparisons between groups, P<0.05.

[0102] 3) Immunofluorescence images of tight junction proteins in each group

[0103] It can be Figure 3 seen that in the model group, the GOS group and the PDX group, the cell nuclei shrank and became smaller, and the three tight junction proteins also shrank into small rings, scattered and emitting weak green fluorescence. Some tight junction proteins were broken or missing, and there were obvious and large intercellular spaces, indicating a high degree of damage to the cell barrier and low compactness.

[0104] In other groups, the cell nuclei were large, round, with clear boundaries, emitting strong dark blue fluorescence; the three tight junction proteins showed a honeycomb network structure, with clear edges, emitting strong green fluorescence, tightly wrapping the cell nuclei, in a paving stone shape, and the intercellular spaces were small, with a high degree of compactness.

[0105] Example 2 Animal experiment

[0106] 2.1 Experimental animals and grouping:

[0107] Sixty SPF-grade weaned SD rats, male, weighing 45 g - 65 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., with the animal certificate number SCXK (Beijing) 2016-0006. The animals were housed in the barrier animal room of West China School of Public Health, Sichuan University, with the certificate number SYXK (Sichuan) 2018-011. During the feeding, the animals were ensured free access to water and sufficient food, and the drinking water was sterilized pure water. The rats were housed in cages with stainless steel wire bottoms. The animal room was kept quiet, clean, ventilated and in a suitable light state, at 20-26 °C, with a humidity of 40% - 70%, and a light-dark cycle of 12 h.

[0108] Rats were acclimatized for 2 weeks with AIN-93G standard feed (Nantong Tenofi Feed Technology Co., Ltd., Production License No.: Su Feed Certificate ((2019)06092). Rats were randomly divided into 5 groups of 12 rats each, based on body weight. Calcium, vitamin D3, galactooligosaccharides (GOS), and polydextrose (PDX) were administered by mixing with the feed. The feed formulations for each group are shown in Table 7. All treatments were administered continuously for 93 days after acclimatization. Animals were sacrificed on day 93, and all indicators were measured.

[0109] Table 6 Experimental Groups

[0110]

[0111] Table 7 Feed formulations for each experimental group

[0112]

[0113]

[0114] 2.2 Experimental Methods

[0115] (1) Animal handling and sample collection

[0116] After the final gavage, the rats were fasted for 16 hours, with free access to water. Blood was collected from the abdominal vein after anesthesia. The rats were euthanized by cervical dislocation. The blood samples were centrifuged at 3500 rpm for 10 minutes at 4°C to separate the serum, and the supernatant was collected. The serum was stored at -80°C until testing.

[0117] After euthanizing the rats by blood collection, the cecum and colon were quickly separated. Approximately 1-2 cm of the terminal colon was circumferentially removed 10 cm from the cecum. The intestinal contents were rinsed clean in physiological saline. A 1 cm segment from the middle of the colon was then taken and fully fixed in 4% paraformaldehyde at room temperature. The cecum was frozen at -80°C. Later, it was opened with a sterile scalpel and, under a clean bench, the contents of the cecum were scraped off with a disposable spatula and stored in cryovials at -80°C.

[0118] (2) Colonic histological examination

[0119] After fixation with 4% paraformaldehyde for 24 hours, the colon was dehydrated, hardened, cleared, embedded in paraffin, sectioned, and dewaxed before hematoxylin-eosin (HE) staining. Morphological changes were observed under a light microscope, and images were acquired using a digital medical image acquisition and processing system.

[0120] (3) Serum D-lactic acid and sIgA content in cecal contents

[0121] Serum D-lactic acid levels were determined following the ELISA kit's detection procedure.

[0122] (4) Data processing and result determination

[0123] All data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was performed using SPSS 26.0 statistical software. Homogeneity of variance was tested before the analysis; if the variances were homogeneous, the F-value was calculated. If the F-value...<F0.05,P> A p-value of 0.05 indicates that there is no statistically significant difference between the means of the groups; if the F-value is greater than F<0.05, the p-value is less than 0.05, indicating that there is a statistically significant difference between the means of the groups. Except for pairwise comparisons of means between multiple experimental groups and control groups using the LSD method, all other analyses were performed using LSD.

[0124] 2.3 Experimental Results

[0125] (1) Morphological features of rat intestinal mucosa

[0126] The results are as follows Figure 4 , Figure 4 Representative colonic morphological images from each experimental group; Note: Representative H&E stained sections of rat colons are shown under the same microscope (100* and 400*), scale bar = 100μm.

[0127] Low calcium group: The colon tissue structure is relatively intact, with a small number of intestinal glands undergoing degeneration and necrosis in local areas of the mucosa. The necrotic intestinal gland structure is blurred and the cells are disintegrating. In local areas of the lamina propria, inflammatory cells are infiltrated in small focal areas, mainly consisting of round, deeply stained lymphocytes.

[0128] High-calcium group: The colonic tissue mucosa is covered with a single layer of columnar epithelium that is smooth and without villi. No epithelial cell degeneration, necrosis or shedding is observed. The colonic glands in the lamina propria are densely arranged, and the glandular epithelium is distributed with a large number of goblet cells with unstained cytoplasm. No obvious necrosis or large amount of inflammatory cell infiltration is observed. The submucosa, muscularis propria and serosa are relatively intact in structure, and no obvious pathological changes are observed.

[0129] Calcium + Vitamin D: The colon tissue structure is relatively intact, with a small number of inflammatory cells infiltrating the connective tissue of the lamina propria, mainly lymphocytes.

[0130] In the calcium + vitamin D + PDX + GOS combination group, the colon morphology results were relatively complete. The mucosal surface of the intestinal villi was covered with a single layer of columnar epithelium, and the tubular intestinal gland structure in the lamina propria was clear. In the calcium + vitamin D + PDX + GOS combination group (PDX:GOS=1:1), the glandular epithelial cells were normal in morphology, but a small number of inflammatory cell infiltrations were visible. No inflammatory cell infiltrations were observed in the calcium + vitamin D + PDX + GOS combination group (PDX:GOS=4:1).

[0131] Colon morphology results indicated that insufficient calcium intake caused some inflammation in the colon of growing rats. In rats with normal calcium intake, the colon tissue structure was intact, with no obvious necrosis or large amount of inflammatory cell infiltration. The combination of calcium + vitamin D + PDX + GOS alleviated the inflammatory cell infiltration of the colonic lamina propria connective tissue to some extent, suggesting that the combined use of the two prebiotics with calcium and vitamin D can improve the tissue morphology of the colon.

[0132] (2) Serum D-lactic acid and sIgA levels in cecal contents

[0133] D-lactic acid (D-L-A) is a biomarker reflecting the degree of intestinal mucosal damage and changes in permeability. When intestinal mucosal permeability increases, a large amount of D-lactic acid produced by intestinal bacteria can enter the bloodstream, thus it can be used as a reliable indicator of increased intestinal permeability. The serum D-lactic acid levels are shown in the table below. The D-lactic acid level in the low-calcium group was significantly higher than that in the high-calcium group and the calcium + vitamin D group (P<0.05). The D-lactic acid level in the calcium + vitamin D group was significantly higher than that in the high-calcium group, but lower than that in the fortified group (P<0.05), suggesting that calcium intake affects serum D-lactic acid levels. The D-lactic acid levels in the calcium + vitamin D + PDX + GOS combination (PDX:GOS = 1:1) and the calcium + vitamin D + PDX + GOS combination (PDX:GOS = 4:1) were significantly lower than those in the calcium + vitamin D group (P<0.05 for both), indicating that prebiotics reduced the impact of insufficient calcium intake on serum D-lactic acid levels. The results showed that low calcium intake caused some damage to the intestinal mucosa, increased intestinal mucosal permeability, and allowed D-lactic acid produced by bacterial fermentation to enter the bloodstream through the damaged mucosa. High calcium intake could mitigate the effects of low calcium intake on intestinal mucosal damage. When calcium intake was not high enough, the intake of prebiotics alleviated the adverse effects of low calcium intake on intestinal mucosal permeability to some extent.

[0134] sIgA is the main antibody for local anti-infection action in the intestinal mucosa. When the intestinal mucosa is damaged, it produces sIgA to resist pathogen invasion. The sIgA levels in cecal contents are shown in the table below. The sIgA levels in the calcium + vitamin D group and the low-calcium group were significantly higher than those in the high-calcium group (P < 0.05). The sIgA levels in the cecal contents of the calcium + vitamin D + PDX + GOS combination (PDX:GOS = 1:1) and the calcium + vitamin D + PDX + GOS combination (PDX:GOS = 4:1) were lower than those in the calcium + vitamin D group, with the calcium + vitamin D + PDX + GOS combination (PDX:GOS = 4:1) being significantly lower than that in the high-calcium group (P < 0.05).

[0135] Table 8. Serum D-lactic acid and sIgA levels in cecal contents.

[0136]

[0137] Note: Different letters indicate statistical significance between groups, P<0.05.

[0138] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle 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 nutritional composition that helps improve the intestinal mucosal barrier, characterized in that, include: A combination of vitamin D, polydextrose, galactooligosaccharides, and calcium sources; The composition comprises the following raw materials in parts by weight: 200,000 to 250,000 parts of calcium source, 4 to 6 parts of vitamin D, 1,500,000 to 2,500,000 parts of polydextrose, and 350,000 to 600,000 parts of galactooligosaccharides. The weight ratio of the polydextrose to the galactooligosaccharides is 4:1; The improvements in the intestinal mucosal barrier include improving intestinal mucosal permeability, regulating the expression of tight junction proteins between cells, improving intestinal mucosal tissue structure, and improving the levels of intestinal mucosal barrier markers.

2. The composition according to claim 1, characterized in that, The calcium source includes one or more of the following: calcium carbonate, calcium gluconate, calcium citrate, calcium lactate, calcium hydrogen phosphate, L-threonine calcium, glycine calcium, aspartic acid calcium, citrate-malate calcium, calcium acetate, calcium chloride, tricalcium phosphate, vitamin E succinate calcium, glycerophosphate calcium, calcium oxide, calcium sulfate, calcium dihydrogen phosphate, milk mineral salts, casein calcium, calcium malate, and calcium ascorbate.

3. The use of the composition according to any one of claims 1 to 2 in the preparation of products that help improve the intestinal mucosal barrier; The improvements in the intestinal mucosal barrier include improving intestinal mucosal permeability, regulating the expression of tight junction proteins between cells, improving intestinal mucosal tissue structure, and improving the levels of intestinal mucosal barrier markers.

4. The application according to claim 3, characterized in that, The improvement of intestinal mucosal permeability includes increasing the transmembrane resistance (TEER) and inhibiting the apparent permeability coefficient (Papp). The intestinal mucosal barrier markers include D-lactic acid and sIgA.

5. A product that helps improve the intestinal mucosal barrier, characterized in that, Includes the composition according to any one of claims 1 to 2.

6. The product according to claim 5, characterized in that, The products include food or health products; the food includes dairy products.

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