A nutritional composition rich in A2-β-casein that helps promote calcium absorption

Abstract

This invention provides a nutritional composition rich in A2-β-casein that promotes calcium absorption, belonging to the field of functional foods. The invention obtains A2-β-casein through genotyping and purification, and then combines it with colostrum basic protein and a small amount of milk-derived calcium to form compositions with different ratios. This invention utilizes a Caco-2 cell model to study the fluorescence imaging of calcium ion influx into Caco-2 cells, the calcium absorption-promoting activity, and the absorption pathway, thereby comprehensively evaluating the calcium absorption characteristics of this composition and obtaining a nutritional composition rich in A2-β-casein that promotes calcium absorption.

Description

Technical Field

[0001] This invention relates to the field of functional foods, and more particularly to a nutritional composition rich in A2-β-casein that helps promote calcium absorption. Background Technology

[0002] Calcium is the most abundant mineral element in the human body, playing an irreplaceable structural and regulatory role in key physiological processes such as bone mineralization, nerve signal transmission, muscle contraction coupling, and coagulation cascade reactions. Current traditional nutritional intervention strategies have long focused on increasing absolute calcium intake, such as through direct supplementation with inorganic or organic calcium salts like calcium carbonate and calcium citrate, or calcium fortification in food matrices. However, simply increasing the calcium loading dose cannot fundamentally solve the core problem of low absorption efficiency. In fact, excess calcium ions that are not effectively absorbed in the intestinal lumen can not only cause gastrointestinal discomfort such as constipation and bloating, but may also induce secondary micronutrient absorption disorders by competing with divalent cations such as iron and zinc for shared intestinal epithelial cell transport channels. Developing functional nutritional combinations that can effectively improve calcium bioavailability has become an important frontier direction in food nutrition and precision nutrition intervention.

[0003] Milk and its products are excellent sources of dietary calcium. Their advantage in promoting calcium absorption stems not only from their high calcium content but also from the unique complex structure formed between milk proteins and calcium. Approximately 80% of the protein in milk is casein, which is mainly divided into A1 and A2 types based on its genetic polymorphism. A2-β-casein is considered a naturally occurring variant found in the original dairy cow population, and its molecular structure is closer to that of casein in human milk. From a structural biology perspective, the only difference between A1 and A2 β-casein lies in the 67th amino acid residue of their peptide chain: histidine in A1 and proline in A2. This subtle difference alters the accessibility of digestive enzyme cleavage sites. During digestion, A1 β-casein is readily decomposed by enzymes to release the opioid-like heptapeptide—β-tyrophage-7, while A2, due to steric hindrance from proline, produces almost no of this peptide. Previous studies have suggested that β-casein-7 may be associated with intestinal inflammation, increased mucus secretion, and delayed transit time in some individuals after consuming milk, while A2-type β-casein exhibits superior gastrointestinal comfort. Furthermore, preliminary in vitro studies indicate that A2-type β-casein may indirectly facilitate calcium ion dissolution and retention in the intestinal lumen by maintaining a more stable casein micelle structure or by reducing damage to the absorptive epithelium caused by the intestinal inflammatory microenvironment, thereby creating favorable conditions for subsequent absorption.

[0004] However, calcium absorption is a multi-step, multi-factor regulated process involving intestinal dissolution, transmembrane transport, intracellular transport, and basolateral efflux. Optimization of a single component often fails to maximize absorption efficiency. In recent years, various targeted bioactive factors have been shown to synergistically promote calcium metabolism and utilization at different stages. Among them, colostrum basic proteins have attracted attention due to their origin in bovine colostrum and their rich content of various growth-promoting and anti-apoptotic active factors. Studies have shown that colostrum basic proteins have good biocompatibility and can enhance overall calcium absorption efficiency by upregulating the expression of calcium channel proteins on the surface of intestinal epithelial cells or by regulating the permeability of cellular bypass pathways.

[0005] However, current research on the synergistic effect of A2-β-casein and various nutrients in promoting calcium absorption still lacks systematicity. Therefore, the isolation and purification of A2-β-casein, the optimization of nutritional formulations, and research on the mechanisms by which Caco-2 cells promote calcium absorption are of significant theoretical value and application potential for improving children's bone health. Summary of the Invention

[0006] Based on the deficiencies of the existing technology, the present invention aims to provide a nutritional composition rich in A2-β-casein that helps promote calcium absorption, thereby solving the problem of low calcium absorption efficiency.

[0007] The first objective of this invention is to provide a nutritional composition comprising A2-β-casein, colostrum basic protein, and milk-derived calcium.

[0008] Preferably, the mass ratio of A2-β-casein to colostrum basic protein is 70-90:1-2.

[0009] Preferably, the mass ratio of A2-β-casein to milk-derived calcium is 70-90:8-16.

[0010] Preferably, the mass ratio of A2-β-casein, colostrum basic protein, and milk-derived calcium is 70-90:1-2:8-16.

[0011] Preferably, the mass ratio of A2-β-casein, colostrum basic protein, and milk-derived calcium is 70-90:1-2:12-16.

[0012] Preferably, the calcium transport rate of the nutritional composition is ≥50%.

[0013] A second objective of this invention is to provide a nutritional composition that promotes calcium absorption, comprising a compound of A2-β-casein and colostrum basic protein; wherein the mass ratio of A2-β-casein to colostrum basic protein is 70-90:1-2.

[0014] Preferably, the calcium-enhancing nutritional composition promotes calcium absorption by upregulating the expression of TRPV6 and / or Cav1.3 channel-related genes and proteins in Caco-2 cells.

[0015] A third objective of this invention is to provide a method for preparing the aforementioned A2-β-casein, comprising the following steps: S1, A2-β-casein genotype identification was performed on raw milk to screen out milk samples with positive A2 genotype; S2, homogenize and centrifuge the selected milk samples, adjust the pH to isoelectricity, and let stand to precipitate β-casein; to obtain crude casein. S3, crude casein was washed to neutral, dialyzed with water, and then freeze-dried; the freeze-dried crude casein was reconstituted and purified by anion exchange chromatography to obtain A2-β-casein.

[0016] The beneficial effects of this invention are as follows: (1) The present invention achieves a synergistic effect by combining A2-β-casein with colostrum basic protein and milk-derived calcium. This composition can significantly improve the calcium transport rate in the Caco-2 cell monolayer model, with the calcium transport rate of the specific ratio composition reaching more than 50%, which is superior to the effect of single components or simple addition.

[0017] (2) The mechanism of action of the nutritional composition provided by the present invention is clear. RT-qPCR and Western blot experiments have verified that the composition can upregulate the mRNA expression levels of TRPV6 and Cav1.3 calcium channel-related genes (TRPV6, CACNA1D) in Caco-2 cells and correspondingly increase the expression levels of channel proteins, indicating that it promotes calcium absorption by regulating the active calcium ion transport pathway in intestinal epithelial cells.

[0018] (3) The A2-β-casein used in this invention is derived from natural milk sources, and its molecular structure is closer to the casein form in breast milk. Compared with A1 casein, this component is more tolerant during digestion, which is beneficial to maintaining the stability of calcium peptide chelates, thereby improving the retention rate of calcium in the intestinal environment.

[0019] (4) The present invention selects dairy calcium as calcium fortifier. Compared with inorganic calcium salts such as calcium carbonate and calcium chloride, it provides calcium source while having a smaller impact on the stability of food system, which is conducive to maintaining the sensory quality and processing performance of the product.

[0020] (5) Through systematic in vitro cell model screening, the present invention has obtained a nutritional composition ratio with clear calcium absorption-promoting activity, providing a quantifiable technical solution for improving the calcium nutrition status of children and people at specific physiological stages. Attached Figure Description

[0021] Figure 1 This is an agarose gel electrophoresis image; specifically, it is an agarose gel electrophoresis pattern of ARMS-PCR products of the β-CN genotype; the first lane is a standard molecular weight marker (100~2000 bp); the second lane is the sample amplification product of primers specific to the A1 genotype; the third lane is the sample amplification product of primers specific to the A2 genotype. Figure 2 Image of purified protein; Figure 3 This is a polyacrylamide gel electrophoresis image; lane 1 is the standard molecular weight marker; lane 2 is the β-casein standard; lane 3 is the purified sample; Figure 4 This is a standard curve of BCA protein. Figure 5 The graph shows the cell viability of CCK-8 cells in different proportions of calcium, A2-β-casein, and CBP in the preliminary experimental composition for CCK-8 cell viability assay. Different letters in the figure represent significant differences. Figure 6 The graph shows the cell viability of CCK-8 cells with a drug concentration of 200 μg / mL and an A2-β-casein ratio of 30. Different letters in ag in the graph represent significant differences. Figure 7 The graph shows the cell viability of CCK-8 cells with a drug concentration of 200 μg / mL and an A2-β-casein ratio of 70% in the combination group. Different letters in af in the graph represent significant differences. Figure 8 The graph shows the cell viability of CCK-8 cells with a drug concentration of 200 μg / mL and an A2-β-casein ratio of 90% in the combination group. Different letters in ag in the graph represent significant differences. Figure 9 Microscopic images of the early, middle, and successful stages of Transwell model creation; the left side shows the early stage; the middle side shows the middle stage; and the right side shows the successful stage. Figure 10 A graph showing the transmembrane resistance of Caco-2 cells; Figure 11 This is a graph showing the ratio of alkaline phosphatase activities. Figure 12 Translocation rate graphs for 18 groups of compositions with different ratios; Figure 13 Fluorescence imaging of calcium ion influx into Caco-2 cells; Figure 14 A graph showing the relative mRNA expression levels of TRPV6 pathway-related genes by different compositions; Figure 15 A graph showing the relative mRNA expression levels of CAV1.3 pathway-related genes by different compositions; Figure 16 A graph showing the relative protein expression levels of TRPV6 pathway-related genes by different compositions; Figure 17 A graph showing the relative protein expression levels of CAV1.3 pathway-related genes by different compositions; Figure 18 This is a Western blotting image of proteins. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Unless otherwise specified, the experimental methods used in the specific implementation methods are all conventional methods, and the materials and reagents used are all commercially available unless otherwise specified.

[0024] In this invention, unless otherwise specified, "%" represents a percentage by mass; the raw materials and reagents used are all commercially available products.

[0025] Example 1: Extraction and content determination of A2-β-casein DNA Milk samples were collected, and genomic DNA was extracted from somatic cells using a DNA extraction kit (Takara Code No. 9178). Specific primers for the β-casein gene were designed, and the genotypes of the dairy cows were identified using allele-specific PCR. Milk from cows with the A2A2 homozygous genotype was selected as the raw material for subsequent extraction of A2-β-casein.

[0026] in: The amino acid sequence of A1-β-casein (position 67 = H) is shown in SEQ ID NO. 1: RELEELNVPG EIVESLSSSE ESITRINKKI EKFQSEEQQQ TEDELQDKIHPFAQTQSLVYPFPGPIHNSL PQNIPPLTQT PVVVPPFLQP EVMGVSKVKE AMAPKHKEMPFPKYPVEPFTESQSLTLTDV ENLHLLPPLL QSWMHQPHQP LPPTVMFPPQ SVLSLSQSKVLPVPQKAVPYPQRDMPIQAF LLYQEPVLGP VRGPFPIIV The amino acid sequence (position 67 = P) of A2-β-casein is shown in SEQ ID NO. 2: RELEELNVPG EIVESLSSSE ESITRINKKI EKFQSEEQQQ TEDELQDKIHPFAQTQSLVYPFPGPIPNSL PQNIPPLTQT PVVVPPFLQP EVMGVSKVKE AMAPKHKEMPFPKYPVEPFTESQSLTLTDV ENLHLLPPLL QSWMHQPHQP LPPTVMFPPQ SVLSLSQSKVLPVPQKAVPYPQRDMPIQAF LLYQEPVLGP VRGPFPIIV Example 2: Identification and typing of A2-β-casein genotype After numbering the raw milk, the genotype of A2-β-casein was identified using allele-specific PCR. Two independent reactions were set up: one group used primers CSNA1F / CSNAR to identify and amplify the 244 bp A1 variant (containing histidine), and the other group used primers CSNA2F / CSNAR to detect the 244 bp A2 variant. During amplification, the following steps were performed: pre-denaturation at 95℃ for 5 min, denaturation at 95℃ for 15 s, annealing at 58℃ for 15 s, extension at 72℃ for 60 s, and a final extension at 72℃ for 5 min after 35 cycles.

[0027] PCR amplification products from each reaction system were analyzed by 2% agarose gel electrophoresis (120 V). Genotyping was performed based on the electrophoresis results: if only the CSNA1F / CSNAR primer pair amplified a 244 bp band, it was identified as type A1; if only the CSNA2F / CSNAR primer pair amplified a 244 bp band, it was identified as type A2; if both primer pairs amplified bands, it was identified as A1 / A2 heterozygous. Type A2 milk samples were selected for subsequent extraction of A2-β-casein.

[0028] In primers CSNA1F / CSNAR: The sequence of primer CSNA1F (reverse primer) is shown in SEQ ID NO. 3: 5'-GATGTTTTGTGGGAGGCTGTTAT-3' The sequence of primer CSNAR (forward primer) is shown in SEQ ID NO. 4: 5'-GCCCAGATGAGAGAAGTGAGG-3' In primers CSNA2F / CSNAR: The sequence of primer CSNA2F (reverse primer) is shown in SEQ ID NO. 5: 5'-GATGTTTTGTGGGAGGCTGTTAG-3' The sequence of primer CSNAR (forward primer) is shown in SEQ ID NO. 6: 5'-GCCCAGATGAGAGAAGTGAGG-3' In this embodiment, the genotyping of the milk source before extracting A2-β-casein is based on the following technical considerations: The expression of A2-β-casein is controlled by the β-casein gene (CSN2) on bovine chromosome 6. This gene has two alleles, A1 and A2, which are co-dominantly inherited. A cow's genotype determines the type of β-casein in its milk: A1A1 homozygotes produce only A1-type β-casein, A1A2 heterozygotes produce both A1 and A2 types, and A2A2 homozygotes produce only A2-type β-casein. Approximately 70% of dairy cows worldwide are heterozygous or A1A1 homozygous. If β-casein is extracted directly from milk without genotyping, the product will be contaminated with A1-type β-casein, making it impossible to obtain high-purity A2-β-casein, thus affecting the accuracy of subsequent experiments and the efficacy of the resulting compounds.

[0029] Compared to directly detecting proteins in milk, DNA identification can be completed before the cows produce milk, allowing for the early screening of A2A2 homozygous cows and the establishment of dedicated milk sources, ensuring the purity and traceability of raw materials from the source. Gene detection technologies (such as allele-specific PCR) have high sensitivity and accuracy, avoiding interference from changes in milk storage and processing conditions on protein detection, while reducing ineffective inputs in subsequent purification processes and improving extraction efficiency.

[0030] A1 type β-casein may release β-casein-7 (BCM-7) during digestion, and some studies suggest that it may cause gastrointestinal discomfort in some people. A2 type β-casein, on the other hand, has a structure closer to breast milk and is more digestibly tolerable. Genotyping and screening for homozygous A2A2 milk sources can fundamentally ensure that the extracted A2 β-casein is free of A1 type impurities, thereby guaranteeing the safety, functional stability, and applicability of the final product in the functional food field.

[0031] Example 2: Setting up a genotyping step before extracting A2β-casein is an important technical means to ensure the purity of core components and improve product quality and efficacy.

[0032] In this embodiment, the 244 bp DNA fragment obtained by allele-specific PCR amplification is an intermediate product for genotyping. Its function is limited to serving as a marker for agarose gel electrophoresis to determine the β-casein genotype of the dairy cow from which the milk sample was obtained. This DNA fragment itself is not a component of the nutritional composition of this invention, nor is it used for subsequent protein extraction or any functional applications.

[0033] The sequence information of the bovine β-casein gene (CSN2) has been publicly available in databases such as GenBank. Based on this, only the primer sequences and reaction conditions for specifically amplifying the A1 and A2 alleles need to be disclosed. Those skilled in the art can expect to obtain a 244 bp specific amplification product based on the disclosed primer information and the known gene sequence, and perform genotyping identification by gel electrophoresis.

[0034] Example 3: Crude extraction of β-casein by isoelectric point precipitation Milk samples were homogenized for 1 min, centrifuged at 5000 r / min for 20 min at 4 ℃, and then the pH was adjusted to its isoelectric point of 4.7 and allowed to stand for 30 min to precipitate β-casein. The precipitate was washed with deionized water until neutral, and dialyzed with deionized water at 4 ℃ using a dialysis bag with a capacity of 8000-14000 D for 48 h. The dialysate was changed every 6-8 h, and the dialysis product was freeze-dried after dialysis. The obtained product was stored at -20 ℃.

[0035] Example 4: Anion exchange chromatography separation of β-casein β-casein in crude casein was purified by anion exchange chromatography. The lyophilized sample was reconstituted with mobile phase A to a concentration of 20 mg / mL, filtered through a 0.22 μm filter membrane, and then loaded into a protein purification system to achieve the separation and purification of β-casein.

[0036] The genotype of a sample can be identified by amplifying the corresponding A1-β-CN and A2-β-CN DNA fragments and then performing agarose gel electrophoresis. Kulibaba et al. performed ARMS-PCR on A1-β-CN and A2-β-CN DNA, obtaining corresponding 244 bp DNA amplification fragments, such as... Figure 1 As shown, this experiment also yielded a 244 bp band. No band appeared in the first lane; a band appeared in the second lane, therefore the genotype of β-CN in the sample is A2, and this sample can be used as the raw material for subsequent extraction of A2-β-CN.

[0037] Example 5: Identification of β-casein purity The purity of β-casein was determined by collecting the chromatographic peaks using SDS-PAGE. Lane 1 was used for the standard molecular weight marker; lane 2 for the β-casein standard; and lane 3 for A2-β-casein. 10 μL of sample and standard were mixed separately with an equal volume of 5× loading buffer, heated in a boiling water bath for 3–5 min, centrifuged at 12000 r / min for 2 min at 4℃, and then loaded and electrophoresed. The gels were then stained, and the destaining time was determined based on the gel's destaining properties.

[0038] The β-CN product obtained by anion exchange chromatography was subjected to SDS-PAGE to identify the product type. For example... Figure 3 As shown, the molecular weight of the β-CN product eluted by anion exchange chromatography is approximately 22–28 kDa, which is basically consistent with the molecular weight of β-CN obtained by Ken et al., and also consistent with the standard band in the first lane. Above the β-CN band is a narrower band, which, according to the results of Han et al., is α-CN. α-CN is difficult to separate from the casein system because it has the lowest isoelectric point range (4.9–5.4) in the casein fraction, and its content is relatively high. Therefore, compared to other caseins, α-CN exhibits a larger net charge in the buffer solution. Based on this, α-CN is not easily separated from the system under low ionic strength. However, compared to the total casein system, the purity of β-CN purified by this method is already relatively high.

[0039] The purpose of this embodiment is that commercially available milk comes from diverse sources, and different cows have different β-casein genotypes, including A1A1 homozygotes (producing only A1 type β-casein), A1A2 heterozygotes (producing both A1 and A2 types), and A2A2 homozygotes (producing only A2 type). If β-casein is extracted directly from milk that has not been genotype-identified, the resulting product will be mixed with A1 type β-casein, making it impossible to obtain high-purity A2-β-casein, thus affecting the accuracy of subsequent studies on its calcium absorption-promoting function.

[0040] Furthermore, there are currently no commercially available high-purity A2-β-casein standards available for direct use. Therefore, it is necessary to screen for A2A2 homozygous milk sources through genotyping and then isolate and purify high-purity A2-β-casein to ensure the purity and quality control of the core components used in subsequent efficacy verification experiments. The genotyping and purity identification steps in this invention are designed to achieve this purpose and are necessary prerequisites for ensuring the reliability of the entire research results.

[0041] Example 6: Desalting and concentration determination of β-casein.

[0042] The collected solution was desalted by dialysis using an 8000-14000 dialysis bag and then freeze-dried to prepare a solid sample, which was stored at -20°C. Subsequently, β-casein was reconstituted in PBS, with a final concentration of 1 mg / mL in the PBS buffer.

[0043] Example 7: Nutritional Combination Design Preliminary results of CCK-8 cell experiments are as follows: Figure 5 As shown, different drug concentrations have a significant impact on the viability of Caco-2 cells. To determine the optimal drug concentration for the entire cell experiment and eliminate cytotoxic effects, we selected four groups of compositions with low, medium, and high ratios and conducted cell viability experiments at different concentrations. Each composition was combined with 0.00008 wt% and 0.00045 wt% vitamin D3 and vitamin K2, respectively.

[0044] like Figure 5 As shown, overall, the survival rate of Caco-2 cells was above 80% under different concentrations and combinations of drug administration. Among them, the 70:1:8, 70:2:16, and 90:2:16 groups showed the highest cell survival rate at 200 μg / mL. Although the 30:1:8 group showed the highest cell survival rate at 100 μg / mL, the cell survival rate at 200 μg / mL was still above 100%, which is beneficial for cell survival, and there was no significant difference in cell survival rate between this concentration and 100 μg / mL. Therefore, 200 μg / mL was selected as the optimal drug administration concentration for subsequent cell experiments.

[0045] Formal experimental results for CCK-8 cells are as follows: Figure 6-9 As shown, when A2-β-casein was combined with other substances at a concentration of 3000 mg / 100g milk powder, its effect on cell proliferation was worse than that at concentrations of 7000 mg / 100g milk powder. Therefore, 18 combinations of A2-β-casein at concentrations of 7000 mg / 100g milk powder and 18 milk powder combinations were selected for subsequent transport and absorption experiments.

[0046] Example 8: Caco-2 cell resuscitation and passage culture Frozen cells were rapidly thawed in a 37°C water bath, transferred to complete culture medium, and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the cell pellet was resuspended in fresh culture medium and seeded into culture flasks. The flasks were incubated statically at 37°C with 5% CO2, and the culture medium was replaced the following day. Once cell confluence reached 80%, the cells were washed with PBS and then digested with trypsin for 2-5 minutes. Digestion was stopped with complete culture medium, followed by centrifugation to collect the cells. The cells were resuspended (in a medium containing 79% MEM, 20% serum, and 1% penicillin-dextrin) and passaged at a ratio of 1:2 to 1:3. The culture medium was changed every other day, and passages were performed every 3-4 days.

[0047] Example 9: Establishment of a Caco-2 cell monolayer model Take Caco-2 cells in the logarithmic growth phase, digest them, and then use 1×10⁻⁶ cells. 5 Cells were seeded at a density of 10 cells / mL in 12-well Transwell plates. 0.5 mL of culture medium was then added to the upper chamber (AP side) and 1.5 mL of complete culture medium to the lower chamber (BL side). The medium was changed every other day after seeding, and after 7 days of culture, the medium was changed daily. The cells were cultured for 21 to 27 days until a dense monolayer formed, at which point they were ready for subsequent experiments.

[0048] Example 10: Integrity evaluation of the Caco-2 cell monolayer model Transmembrane resistance (TEER) of Caco-2 monolayers was monitored using a cell-specific resistance meter. This indicator reflects the integrity of tight junctions between cells and is a key parameter for assessing cell barrier function. Measurements were taken periodically during culture.

[0049] The cell transmembrane resistance graph is shown below. Figure 10 As shown, the TEER value increased slightly within 7 days of modeling, because Caco-2 cells had just begun to adhere and cell division had not yet reached fusion; however, once Caco-2 cells began to fuse, the TEER value began to increase sharply from day 10, reaching 900 Ω·cm. 2 Around 400 Ω·cm². When the TEER value stably reaches above 400 Ω·cm², it indicates that the cells have fully differentiated and formed a dense monolayer, which can be used for subsequent experiments.

[0050] Example 11: Alkaline phosphatase activity assay Alkaline phosphatase (AKP) activity in Caco-2 cell monolayers was determined using a commercially available kit. Culture media from the AP and BL sides were collected on days 7, 9, 11, and 13 of the model culture for AKP activity analysis at each time point.

[0051] The alkaline phosphatase activity ratio graph is shown below. Figure 11 As shown, alkaline phosphatase activity is mainly expressed at the brush border of the cell monolayer and reflects the polarization of the cell monolayer membrane. If the AKP activity on the AP side is significantly higher than that on the BL side, it indicates that the Caco-2 cell monolayer membrane polarization is good. The figure shows that the activity ratio reached 1.18 on day 13, indicating that the polarization of the Caco-2 cell monolayer model was basically complete and it could be used for drug transport experiments.

[0052] Example 12: Determination of Calcium Content by Atomic Absorption Spectrometry Compositions containing different ratios of vitamin D3 and vitamin K2 were dissolved in HBSS to prepare a working solution of 200 μg / mL and equilibrated in a 37°C water bath. After establishing the Caco-2 monolayer model, the upper chamber was washed with HBSS and transferred to a new plate. Subsequently, preheated calcium-free HBSS was added to the lower chamber, and the sample solution was added to the upper chamber to initiate the transport experiment. After completion, the solution in the lower chamber was collected, and the calcium content was determined by atomic absorption spectrometry.

[0053] The calcium transport rates of 18 different compositions are as follows: Figure 12 As shown, overall, the calcium transport rates of different composition ratios were higher than those of the control group, indicating that A2-β-casein and CBP synergistically promote intestinal calcium absorption. Different composition ratios also showed significant differences in calcium transport and absorption. Among them, the transport rates of six compositions (70:1.5:16, 70:2:16, 90:1:12, 90:1:16, 90:2:8, and 90:2:12) were above 50%, suggesting further investigation into their transport and absorption mechanisms.

[0054] Example 13: Fluorescence imaging of calcium ion influx into Caco-2 cells 1.2×10 5 Caco-2 cells were seeded in 35 mm confocal culture dishes and cultured for 24 h to allow them to reach a sub-confluent state. Subsequently, the cells were washed twice with pre-warmed HBSS buffer, and 1 mL of fresh HBSS containing 2.5 μM Fluo-4 AMHBSS was added and incubated at 37°C for 30 min. After a second wash, the cells were equilibrated at room temperature for 30 min. Finally, the sample was added, and observation was performed using a Leica SP8 laser confocal microscope at an excitation wavelength of 488 nm, an emission wavelength of 510 nm, and an oil immersion depth of 40 ×.

[0055] Fluorescence imaging of calcium ion influx into Caco-2 cells, such as Figure 13As shown in the figure, compared with the blank group, the calcium ions in the combined group effectively flowed into Caco-2 cells and showed fluorescence; the blank group contained very little fluorescence, which may be because the Fluo-4 free ligand has very little fluorescence.

[0056] Example 14: Relative mRNA expression in calcium pathway genes The relative expression of TRPV6 and CACNA1D genes in the TRPV6 and Cav1.3 pathways in Caco-2 cells treated with different compositions was detected by RT-qPCR. Figure 14-15 The results showed that the relative mRNA expression levels of TRPV6 and Cav1.3 pathway genes in Caco-3 cells from different compositions were significantly different from those in the control group, and were all higher than those in the control group. Among the different compositions, the 70:2:16 group showed significantly higher relative mRNA expression levels of TRPV6 and Cav1.3 pathway genes than the other groups. This indicates that the combination of A2-β-CN and CBP can regulate the expression of calcium pathway genes. Furthermore, the specific ratio of A2-β-CN, CBP, and calcium can effectively promote calcium absorption.

[0057] Example 15: Relative protein expression in calcium pathway genes Western blot analysis was used to determine the effects of different compositions on the expression of TRPV6 and Cav1.3 proteins in Caco-2 cells. Figure 16-18 It was found that the expression of TRPV6 and CAV1.3 proteins in Caco-2 cells significantly increased after treatment with different compositions. Specifically, the 70:2:16 group showed significantly higher expression of TRPV6 and CAV1.3 proteins than other compositions. This result is consistent with the relative expression of TRPV6 and CACNA1D gene mRNA in Caco-2 cells treated with different compositions. This indicates that, from both gene expression and protein expression perspectives, the specific ratio of A2-β-CN, CBP, and calcium, represented primarily by the 70:2:16 group, can effectively improve intestinal calcium absorption efficiency. Currently, the 70:2:16 group can be identified as the optimal calcium absorption-promoting composition.

[0058] Test methods 1. PCR amplification AF1+AR was used as one group, and AF2+AR as another. The corresponding volumes of DNA template, 10 μL of PremixTaq™, and 0.8 μL each of the forward and reverse primers were added to each group, and the volume was adjusted to 20 μL with deionized water. The premix solution did not need to be diluted in the reaction system; however, the primers were diluted tenfold. The PCR amplification program was as follows: 95 ℃ pre-denaturation for 5 min, 95 ℃ denaturation for 15 s, 58 ℃ annealing for 15 s, 72 ℃ extension for 60 s, 35 cycles, followed by a final extension at 72 ℃ for 5 min.

[0059] 2. Agarose gel electrophoresis Take 0.8g of agarose and 40mL of 1×TAE solution, microwave for 1 minute until the agarose is completely dissolved, then add DNA dye for staining. During heating, avoid generating bubbles and ensure the solution is thoroughly mixed. Clean the gel casting tank of the electrophoresis apparatus, let it dry, place a glass plate inside, and seal the edges with transparent tape to form a mold. Prepare a comb in the gel casting tank. Cool the molten agarose solution to approximately 60°C, then gently pour it into the tank. Ensure no bubbles are generated during pouring and allow the solution to spread slowly to form a uniform gel layer. The gel thickness is generally between 3-5 mm. Wait approximately 20 minutes until the gel is completely solidified, then gently remove the comb. This creates spaced-apart sample wells on the gel plate. Add 5 μL of DNA Marker, and then add 5 μL of sample mixed with 1 μL of loading buffer. Use a micropipette to load the sample and DNA Marker into the wells of the gel. Place the gel in the electrophoresis tank and add an appropriate amount of electrophoresis buffer, ensuring the buffer fully covers the gel surface by approximately 1-2 mm. Connect the electrodes and start electrophoresis at 120 V. Use a gel imaging system to image and display the electrophoresis gel.

[0060] 3. SDS-PAGE identification of chromatographic peaks The collected solution was analyzed and identified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to determine the protein type and purity. First, staining solution, destaining solution, and electrode buffer were prepared. Then, the gels were prepared according to the SDS-PAGE kit instructions, using a 15% separating gel and a 5% stacking gel. The gels were added to the electrode wells, combs were inserted, and after the stacking gel solidified, the electrophoresis buffer was poured into the wells. The combs were removed, and the samples were loaded. Before loading, the collected solution and β-casein standard solution were mixed with 5× protein loading buffer, heated in a boiling water bath for 1-5 min, centrifuged at 12000 r / min for 1 min, and then loaded with a loading volume of 10 μL. The electrophoresis voltage was adjusted to 80 V, and after approximately 1 h, when the bromophenol blue indicator line reached the separating gel, the voltage was adjusted to 120 V. Finally, staining was performed for 1 h, and the destaining time was determined based on the destaining process of the gel itself. The gel was photographed and its purity was analyzed using a Bio-Rad GS800 optical density scanning analysis system.

[0061] 4. CCK-8 cell viability assay Add 100 μL of cell suspension to each well of a 96-well plate. After pre-culturing the plate in an incubator for 24 hours, add 100 μL of complete culture medium or complete culture medium containing different concentrations of the test sample. Continue culturing the plate in an incubator for an appropriate time, then add 10 μL of CCK-8 solution to each well and incubate the plate for 1–4 hours. Measure the absorbance at 450 nm using a microplate reader.

[0062] 5. TEER value determination method TEER (Ω·cm2)=(A-A0)×S In the formula: A is the measured resistance value, Ω; A0 is the blank resistance value, Ω; S is the area of ​​the filter membrane in the small chamber, cm². 2 .

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nutritional composition, characterized in that, It is composed of A2-β-casein, colostrum basic protein, and milk-derived calcium.

2. The nutritional composition according to claim 1, characterized in that, The mass ratio of A2-β-casein to colostrum basic protein is 70-90:1-2.

3. The nutritional composition according to claim 1, characterized in that, The mass ratio of A2-β-casein to milk-derived calcium is 70-90:8-16.

4. The nutritional composition according to claim 1, characterized in that, The mass ratio of A2-β-casein, colostrum basic protein, and milk-derived calcium is 70-90:1-2:8-16.

5. The nutritional composition according to claim 4, characterized in that, The mass ratio of A2-β-casein, colostrum basic protein, and milk-derived calcium is 70-90:1-2:12-16.

6. The nutritional composition according to any one of claims 1-5, characterized in that, The calcium transport rate of the nutritional composition is ≥50%.

7. A nutritional composition that promotes calcium absorption, characterized in that, It is composed of a combination of A2-β-casein and colostrum basic protein; the mass ratio of A2-β-casein to colostrum basic protein is 70-90:1-2.

8. The application of the nutritional composition for promoting calcium absorption according to claim 7, characterized in that, Calcium absorption is promoted by upregulating the expression of TRPV6 and / or Cav1.3 channel-related genes and proteins in Caco-2 cells.

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