Mineral composition as well as product and application thereof

Abstract

The invention provides a mineral composition. The mineral composition comprises calcium ions, magnesium ions and sulfate ions. The composition has the effects of improving intestinal barrier and permeability and regulating expression of intercellular tight junction protein.

Description

[0001] This invention is a divisional application with application number 202211230454.9, application date September 30, 2022, and invention name “A mineral composition, its products and applications”. Technical Field

[0002] The invention relates to the field of food technology, in particular to a mineral composition and a product and application thereof. Background Art

[0003] In addition to digestion and absorption, normal intestinal function also has a powerful barrier function to resist harmful intestinal organisms and the various toxins they produce. Ensuring intestinal health mainly depends on the three major barriers of the intestine, namely the intestinal mucosal epithelial barrier, the immune barrier formed by intestinal immune cells and their secretions, and the biological barrier formed by normal intestinal microbiota.

[0004] Existing scientists have done a lot of research on finding regulatory substances for the intestinal barrier. Some have started with functional sugars, such as chitosan, which improves the growth performance and humoral immunity of early weaned animals by upregulating the IGF-artificially induced growth axis, relieving matrix oxidative stress, and improving the matrix of the intestinal barrier. Some have started with probiotics, such as oral administration of Enterococcus faecium EF1 to protect the metabolic activity and membrane integrity of intestinal epithelial cells.

[0005] Therefore, it is very necessary to provide a mineral composition that can improve the intestinal barrier. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a mineral composition, and the mineral composition provided by the present invention can improve the intestinal barrier.

[0007] The invention provides a mineral composition comprising calcium ions, magnesium ions and sulfate ions.

[0008] Preferably, the mass ratio of the calcium ions, magnesium ions and sulfate ions is (1-25):1:(0.01-16).

[0009] Preferably, the mass ratio of the calcium ions, magnesium ions and sulfate ions is (1.5-22):1:(0.03-10).

[0010] Preferably, the source of calcium ions includes one or more of calcium acetate, calcium caseinate, calcium chloride, calcium citrate, calcium citrate, calcium citrate malate, calcium gluconate, calcium lactate, calcium malate, calcium hydrogen phosphate, calcium dihydrogen phosphate, calcium phosphate, calcium sulfate, calcium ascorbate, calcium glycerophosphate or milk calcium;

[0011] The source of the magnesium ion includes one or more of magnesium sulfate, magnesium chloride, magnesium carbonate, magnesium bicarbonate or magnesium gluconate;

[0012] The source of the sulfate ion includes: one or more of calcium sulfate, magnesium sulfate, zinc sulfate, ferrous sulfate, copper sulfate or manganese sulfate.

[0013] The present invention provides use of the composition described in any one of the above technical solutions in preparing a product for improving the intestinal barrier.

[0014] Preferably, the improvement of the intestinal barrier includes improving permeability, promoting tight junction protein expression and / or enhancing cell viability.

[0015] The present invention provides a product for improving the intestinal barrier, comprising the composition described in any one of the above technical solutions.

[0016] Preferably, the concentration of calcium ions in the product is 0.9 to 14.4 mmol / L;

[0017] The concentration of magnesium ions in the product is 0.1-1.6 mmol / L.

[0018] Preferably, the concentration of sulfate ions in the product is 0.1 to 1.6 mmol / L.

[0019] Preferably, the product is a dairy product; the dairy product includes infant formula milk powder, children's formula milk powder and adult formula milk powder.

[0020] Compared with the prior art, the present invention provides a mineral composition comprising calcium ions, magnesium ions and sulfate ions. The composition has the effects of improving intestinal barrier and permeability and regulating the expression of intercellular tight junction proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The morphological diagram of Caco-2 cells at different times in Example 2 of the present invention (X200) is shown.

[0022] Figure 2 (A) Changes of TEER values ​​of the normal group and the model group over time in Example 3 of the present invention; (B) Papp conditions of the normal group and the model group.

[0023] Figure 3 4 shows the Papp values ​​of different treatment groups in Example 4 of the present invention.

[0024] Figure 4 These are the immunofluorescence images of each group in Example 5 of the present invention (×200). DETAILED DESCRIPTION

[0025] The present invention provides a mineral composition and its products and applications. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they all fall within the scope of protection of the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0026] The invention provides a mineral composition comprising calcium ions, magnesium ions and sulfate ions.

[0027] According to the present invention, the mass ratio of calcium ions, magnesium ions and sulfate ions is (1-25):1:(0.01-16).

[0028] In some preferred embodiments of the present invention, the mass ratio of calcium ions, magnesium ions and sulfate ions is (1.5-22):1:(0.03-10).

[0029] In some preferred embodiments of the present invention, the mass ratio of calcium ions, magnesium ions and sulfate ions is (1.5-21.5):1:(0.04-8).

[0030] In some preferred embodiments of the present invention, the mass ratio of calcium ions, magnesium ions and sulfate ions is (1.7-21.5):1:(0.04-6).

[0031] In some preferred embodiments of the present invention, the mass ratio of calcium ions, magnesium ions and sulfate ions is (1.88-21.5):1:(0.04-5).

[0032] In the present invention, the source of calcium ions includes one or more of calcium acetate, calcium caseinate, calcium chloride, calcium citrate, calcium citrate, calcium citrate malate, calcium gluconate, calcium lactate, calcium malate, calcium hydrogen phosphate, calcium dihydrogen phosphate, calcium phosphate, calcium sulfate, calcium ascorbate, calcium glycerophosphate or milk calcium;

[0033] The source of the magnesium ion includes one or more of magnesium sulfate, magnesium chloride, magnesium carbonate, magnesium bicarbonate or magnesium gluconate;

[0034] The source of the sulfate ion includes: one or more of calcium sulfate, magnesium sulfate, zinc sulfate, ferrous sulfate, copper sulfate or manganese sulfate.

[0035] The inventors have found that the mineral composition provided by the present invention can be used to improve the intestinal barrier, including: repairing damaged intestinal barrier, improving intestinal permeability (TEER and Papp value), regulating the expression of tight protein junctions between cells, etc.

[0036] The above components and proportions of the present invention are technical features that functionally support each other and have an interactive relationship; as long as the above specific components and proportions are met, the technical effects of the present invention can be achieved.

[0037] The present invention provides use of the composition described in any one of the above technical solutions in preparing a product for improving the intestinal barrier.

[0038] Specifically, the improvement of the intestinal barrier includes improving permeability, promoting tight junction protein expression and / or enhancing cell viability.

[0039] The present invention provides a product for improving the intestinal barrier, comprising the composition described in any one of the above technical solutions.

[0040] The products of the present invention include but are not limited to food and / or health products.

[0041] The product of the present invention is a dairy product;

[0042] The dairy products described in the present invention include liquid milk (pasteurized milk, sterilized milk, modulated milk, fermented milk); milk powder (whole milk powder, skim milk powder, partially skimmed milk powder, modulated milk powder, colostrum powder); other dairy products. Specifically, the first category is liquid milk. Mainly including sterilized milk, sterilized milk, yogurt, etc. The second category is milk powder. Including whole milk powder, skim milk powder, whole fat sweetened milk powder, flavored milk powder, infant milk powder and other formula milk powder. The third category is condensed milk. The fourth category is milk fat. Including whipped cream for cakes, common cream for bread, etc. The fifth category is cheese and processed cheese. The sixth category is milk ice cream. The seventh category is other dairy products. Mainly including casein, lactose, milk flakes, etc.

[0043] The dairy product of the present invention preferably includes one or more of a formulated milk, a formulated milk powder, a fermented milk, a milk flake, a cheese or a milk snack. More preferably, the dairy product includes an infant formula milk powder, a children's formula milk powder and an adult formula milk powder.

[0044] The components and proportions of the composition of the present invention have been clearly described above and will not be repeated here.

[0045] The present invention does not limit the remaining ingredients or auxiliary materials of the dairy product, which are well known to those skilled in the art.

[0046] According to the present invention, the concentration of calcium ions in the product is preferably 0.9 to 14.4 mmol / L; more preferably 0.9 to 12 mmol / L; most preferably 0.9 to 10 mmol / L; particularly preferably 0.9 to 3.6 mmol / L.

[0047] The concentration of magnesium ions in the product of the present invention is preferably 0.1 to 1.6 mmol / L.

[0048] The concentration of sulfate ions in the product of the present invention is preferably 0.1 to 1.6 mmol / L.

[0049] In some preferred embodiments of the present invention, in the product:

[0050] The concentration of calcium ions is 0.9 to 14.4 mmol / L, the concentration of magnesium ions is 0.1 to 1.6 mmol / L, and the concentration of sulfate ions is 0.1 to 1.6 mmol / L.

[0051] In some preferred embodiments of the present invention, in the product:

[0052] The concentration of calcium ions is 0.9 to 3.6 mmol / L, the concentration of magnesium ions is 0.1 to 1.6 mmol / L, and the concentration of sulfate ions is 0.1 to 1.6 mmol / L.

[0053] In one preferred embodiment of the present invention, in the product:

[0054] The concentration of calcium ions was 1.8 mmol / L, the concentration of magnesium ions was 0.4 mmol / L, and the concentration of sulfate ions was 0.4 mmol / L.

[0055] In one preferred embodiment of the present invention, in the product:

[0056] The concentration of calcium ions was 1.8 mmol / L, the concentration of magnesium ions was 1.6 mmol / L, and the concentration of sulfate ions was 1.6 mmol / L.

[0057] The present invention also provides a method for preparing a product for improving the intestinal barrier, comprising mixing the above raw materials.

[0058] The intestinal barrier damage model of the present invention is preferably constructed by culturing Caco-2 cells in a calcium-free medium.

[0059] The compositions of the present invention with specific ratios and concentrations can repair damaged intestines and improve intestinal barrier function.

[0060] The composition of the present invention can make tight junction proteins present a honeycomb network structure with clearly visible edges and strong green fluorescence, tightly wrapping the cell nucleus in a paving stone shape, with smaller cell gaps and higher density; that is, it can regulate the expression of intercellular tight junction proteins; and at the same time improve cell viability.

[0061] In order to further illustrate the present invention, a mineral composition provided by the present invention and its products and applications are described in detail below in conjunction with examples.

[0062] Example 1 Effects of calcium ions, magnesium ions and sulfate on the intestinal barrier injury model

[0063] Caco-2 cells were cultured in calcium-free medium to construct an intestinal barrier damage model. After Caco-2 cells were revived, they were subcultured to grow into a dense monolayer of cells. When the transepithelial electrical resistance (TEER) of the monolayer cells exceeded 400 Ω·cm 2 , the intestinal barrier model was considered to be successfully established. In the Caco-2 model, the TEER value exceeded 400Ω·cm 2 Afterwards, the normal group was cultured with a change of medium (normal DMEM medium), and the calcium-free group was cultured with a calcium-free medium until the intestinal barrier was damaged. Each group was tested for relevant indicators. Three parallel wells were set up in each group. The culture medium information is as follows: DMEM high-glucose medium, DMEM calcium-free medium, calcium-free phosphate buffer (PBS), fetal bovine serum (FBS): Gibco, USA; 0.25% trypsin digestion solution (containing EDTA): Beijing Solebow Technology Co., Ltd.;

[0064] After the model was constructed, the cell morphology of the Caco-2 model was observed, and the transmembrane resistance of the monolayer cells was measured using a Millicell-ERS2 resistor. Then, the apparent permeability coefficient (Papp) value in the model was measured using fluorescein (free acid) from Sigma-Aldrich.

[0065] First, the CC-8 kit was used to detect the effects of the test substances (calcium ions, magnesium ions, and sulfate ions) on the survival rate of Caco-2 cells. The inoculation concentration in a 96-well plate was 1.0×10 5 The cell suspension was placed in an incubator and cultured. After the cells grew densely, different concentrations of Ca were added. 2+ The culture was continued for 4 days, and then CCK-8 solution was added and incubated for 2 h in the dark. OD 450nm , calculate the cell survival rate.

[0066] After constructing the Caco-2 intestinal barrier model, use a Ca-free 2+After the injury model was established by culturing Caco-2 cells in culture medium, a model group was set up (cultured in calcium-free culture medium), and the other groups were added with culture medium of corresponding test substance concentration, and relevant indicators were measured after culturing for 4 days.

[0067] Cell groups: model group, calcium group (Ca 2+ concentration: 1.8mmol / L), calcium-low magnesium-low sulfate group (Ca 2+ Concentration: 1.8mmol / L, Mg 2+ concentration: 0.4mmol / L, sulfate ion concentration: 0.4mmol / L) and calcium-high magnesium-high sulfate group (Ca 2 + Concentration: 1.8mmol / L, Mg 2+ concentration 1.6mmol / L, sulfate ion concentration 1.6mmol / L)

[0068] Then the transmembrane resistance TEER and Papp values ​​of each group were measured. Finally, immunofluorescence was used to determine the protein morphology of the tight junction proteins ZO-1, Occludin, and Claudin-1 and the expression of immune factors in each group.

[0069] Example 2: Caco-2 cell morphology observation

[0070] In the model of Example 1, the Caco-2 cells were smooth cobblestone-shaped, with vacuoles in the middle of a few cells, and tightly connected cell groups could be observed at 7 days. At 14 days, the cells proliferated rapidly, with a few dead cells falling off and floating, and the culture medium gradually became turbid. At 21 days, the cells were irregular polygonal and gradually fused into a single layer of cells with increased density, indicating that a single-layer intestinal barrier model had been formed at this time. Figure 1 .Caco-2 cell morphology at different times (X200).

[0071] Example 3: Construction of intestinal barrier damage model using calcium-free culture medium

[0072] The construction method of the normal group and the model group in Example 1 is as described above, and the changes in TEER and Papp values ​​of the normal group and the model group are tracked to determine that the intestinal barrier injury model is completed. In the first two days after the barrier is formed and the injury model is established, the TEER values ​​of the calcium-free group and the normal group have similar trends over time and show an upward trend. However, on the third day, the TEER value of the calcium-free group dropped sharply and was lower than 100Ω·cm 2 , and continued to decline; while the TEER of the normal group continued to rise steadily. On the 4th day, the Papp value of the cells in the normal group was less than 1.0×10-6cm / s, while the Papp value of the calcium-free group was significantly higher than that of the normal group (P<0.01); Figure 2.(A) Changes of TEER values ​​in the normal group and the model group over time; (B) Papp conditions in the normal group and the model group.

[0073] Example 4 Effects of calcium ions, magnesium ions and sulfate combinations on intestinal barrier damage model

[0074] Example 1 measured the effects of the normal group, model group, calcium-high magnesium-high sulfate, and calcium-low magnesium-low sulfate on the Papp value, and found that the Papp value of the model group was the highest, indicating that the intestinal permeability was the strongest and the density was the worst, while compared with the model group, the Papp of the other three groups was significantly reduced, and the Papp values ​​of the calcium-high magnesium-high sulfate group and the calcium-low magnesium-low sulfate group were not statistically different from those of the normal group (P>0.05). The experimental results show that both test ratios can repair damaged intestinal damage and improve intestinal barrier function. Figure 3 4 shows the Papp values ​​of different treatment groups in Example 4 of the present invention.

[0075] Example 5. Tight junction proteins in different groups

[0076] Fluorescence immunoassay was used to determine the expression of tight junction proteins between groups in different embodiments 1. The cell nuclei in the model group shrank and became smaller, and the three tight junction proteins also shrank into small rings, scattered and emitted weak green fluorescence. Some tight junction proteins were broken or missing, and there were large gaps between cells, indicating that the cell barrier was highly damaged and had low density. The cell nuclei in other groups were large and round, with clear boundaries and strong dark blue fluorescence; the three tight junction proteins were in a honeycomb network structure with clearly visible edges, emitting strong green fluorescence, tightly wrapped in the cell nucleus, shaped like paving stones, with small cell gaps and high density. Figure 4 These are the immunofluorescence images of each group in Example 5 of the present invention (×200).

[0077] Example 6. Effects of calcium ions, magnesium ions and sulfate ions on cell viability

[0078] In order to determine whether calcium ions and magnesium ions have an effect on cell activity, the effects of different concentrations of calcium ions and magnesium ions on cell viability were also measured in the experiment. 2+ With the increase of concentration gradient, the survival rate of Caco-2 cells showed a trend of first increasing and then decreasing. 2+ Under the environment of 1.8mmol / L Ca (cell survival rate 75.88%), the cell survival rate was significantly higher than that of the other concentration groups (P < 0.05), but there was no significant difference in the cell survival rate under these three concentrations (P > 0.05). 2+ The concentration was determined by the subsequent addition of Mg 2+ and sulfate ion concentration. Then 1.8mmol / L Ca 2+Concentration and different concentrations of Mg 2+ The cell survival rates of all dose groups exceeded 100%, among which Mg 2+ When the concentration of magnesium and sulfate ions was 1.6mmol / L, the cell survival rate was the lowest but still exceeded 100%. Based on this result, the concentrations of magnesium and sulfate in the calcium-low magnesium-low sulfate group and calcium-high magnesium-high sulfate group were set at 0.4mmol / L and 1.6mmol / L, respectively.

[0079] Table 1 Effects of different concentrations of calcium ions on cell activity

[0080] <![CDATA[Ca 2+ (mmol / L)]]> Cell viability(%) 0.9 <![CDATA[72.24±5.48 a ]]> 1.8 <![CDATA[75.88±5.90 a ]]> 3.6 <![CDATA[71.88±7.24 a ]]> 7.2 <![CDATA[59.04±2.97 b ]]> 14.4 <![CDATA[54.76±3.89 b ]]>

[0081] Table 2 Effects of different concentrations of magnesium ions on cell activity

[0082] <![CDATA[Mg 2+ (mmol / L)]]> Cell viability(%) 0.1 <![CDATA[173.72±24.57 ab ]]> 0.2 <![CDATA[190.91±8.70 a <!-- 5 -->]]> 0.4 <![CDATA[181.01±11.22 ab ]]> 0.6 <![CDATA[174.02±21.60 ab ]]> 0.8 <![CDATA[167.21±11.95 ab ]]> 1.0 <![CDATA[165.10±11.32 ab ]]> 1.2 <![CDATA[148.79±15.88 bc ]]> 1.6 <![CDATA[123.77±6.91 c ]]>

[0083] Table 3 Effects of different concentrations of sulfate ions on cell activity

[0084] Sulfate ion concentration (mmol / L) Cell viability(%) 0.1 <![CDATA[173.72±24.57 ab ]]> 0.2 <![CDATA[190.91±8.70 a ]]> 0.4 <![CDATA[181.01±11.22 ab ]]> 0.6 <![CDATA[174.02±21.60 ab ]]> 0.8 <![CDATA[167.21±11.95 ab ]]> 1.0 <![CDATA[165.10±11.32 ab ]]> 1.2 <![CDATA[148.79±15.88 bc ]]> 1.6 <![CDATA[123.77±6.91 c ]]>

[0085] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a mineral in the preparation of a product for improving the intestinal barrier, wherein: The mineral includes calcium ions, or the mineral includes calcium ions, magnesium ions and sulfate ions.

2. The use according to claim 1, wherein: In the product, the mass ratio of calcium ions, magnesium ions and sulfate ions is (1-25):1:(0.01-16).

3. The use according to claim 2, wherein: In the product, the mass ratio of calcium ions, magnesium ions and sulfate ions is (1.5-22):1:(0.03-10).

4. The use according to any one of claims 1 to 3, wherein: The source of calcium ions includes one or more of calcium acetate, calcium caseinate, calcium chloride, calcium citrate, calcium citrate, calcium citrate malate, calcium gluconate, calcium lactate, calcium malate, calcium hydrogen phosphate, calcium dihydrogen phosphate, calcium phosphate, calcium sulfate, calcium ascorbate, calcium glycerophosphate or milk calcium; The source of the magnesium ion includes one or more of magnesium sulfate, magnesium chloride, magnesium carbonate, magnesium bicarbonate or magnesium gluconate; The source of the sulfate ion includes: one or more of calcium sulfate, magnesium sulfate, zinc sulfate, ferrous sulfate, copper sulfate or manganese sulfate.

5. The use according to any one of claims 1 to 4, wherein: The improvement of the intestinal barrier includes improving permeability, promoting tight junction protein expression and / or enhancing cell viability; Preferably, the improving the intestinal barrier comprises: Improve the intestinal barrier transmembrane resistance TEER value; Reduce the intestinal barrier Papp value; Repair damaged intestines caused by calcium deficiency; Improve the protein morphology of intercellular tight junction proteins ZO-1, Occludin, and Claudin-1; and / or Enhances intestinal Caco-2 cell activity.

6. The use according to any one of claims 1 to 5, wherein: In the product, the calcium ion concentration is 0.9 to 14.4 mmol / L, preferably 0.9 to 3.6 mmol / L.

7. The use according to any one of claims 1 to 5, wherein: In the product, the concentration of calcium ions is 0.9 to 14.4 mmol / L, preferably 0.9 to 3.6 mmol / L, the concentration of magnesium ions is 0.1 to 1.6 mmol / L, and the concentration of sulfate ions is 0.1 to 1.6 mmol / L.

8. The use according to any one of claims 1 to 7, wherein: The product is a dairy product; preferably, the dairy product includes infant formula milk powder, children's formula milk powder or adult formula milk powder, wherein the concentrations of calcium ions, magnesium ions and sulfate ions are calculated based on the prepared state of the milk powder.

9. A product for improving intestinal barrier, comprising calcium ions, or calcium ions, magnesium ions and sulfate ions, in, The concentration of calcium ions in the product is 0.9 to 14.4 mmol / L, preferably 0.9 to 3.6 mmol / L; Preferably, the concentration of magnesium ions in the product is 0.1 to 1.6 mmol / L; Preferably, the concentration of sulfate ions in the product is 0.1 to 1.6 mmol / L.

10. The product for improving intestinal barrier according to claim 9, wherein the product is a dairy product; preferably, the dairy product comprises infant formula milk powder, children's formula milk powder or adult formula milk powder.

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