Complex probiotic agent and its use in the preparation of products for promoting the absorption of vitamins and / or minerals

Abstract

The present application relates to the field of microbial technology, and particularly relates to a compound probiotic agent and application of the compound probiotic agent in preparation of a product for promoting absorption of vitamins and / or minerals. The compound probiotic agent according to the present application is composed of animal bifidobacterium lactis subspecies Bi66 and lactobacillus acidophilus LA15. The compound probiotic agent can significantly improve the absorption capacity of vitamin D (including D2 and D3), vitamin B (including B2, B6 and B12), calcium, zinc and other minerals, and compared with single animal bifidobacterium or single lactobacillus acidophilus, the compound probiotic agent has significant advantages when the amount of the bacteria used is consistent, that is, the animal bifidobacterium lactis subspecies Bi66 and the lactobacillus acidophilus LA15 can cooperate with each other, and have a significant synergistic effect in promoting absorption of vitamins and minerals.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more particularly to compound probiotic agents and their application in the preparation of products that promote the absorption of vitamins and / or minerals. Background Technology

[0002] Vitamins and minerals are essential nutrients for maintaining human physiological functions. Vitamin B complex (such as B2, B6, and B12) acts as coenzymes, widely participating in processes such as energy metabolism, neurotransmitter synthesis, and erythrocyte production; while vitamin D is a precursor to steroid hormones, playing a central role in calcium and phosphorus homeostasis, bone health, and immune regulation. Minerals are also indispensable elements for maintaining normal physiological functions: calcium is a major component of bones and teeth and participates in muscle contraction, nerve signal transmission, and blood clotting; iron is a core component of hemoglobin and myoglobin, responsible for oxygen transport and storage, and also participates in energy metabolism and immune function; zinc, as a cofactor for many enzymes, plays a crucial role in DNA synthesis, cell division, immune function, and wound healing. It is worth noting that the absorption of these nutrients is complex and easily interfered with.

[0003] As the primary site for nutrient absorption in the human body, the stability and functional integrity of the gut microenvironment directly determine the bioavailability of key nutrients such as vitamins and minerals. Probiotics, as live microorganisms, exert beneficial effects on host health when ingested in sufficient quantities. Currently, regulating and improving the gut microbiota structure through exogenous probiotic supplementation has become an important strategy for the prevention and adjunctive treatment of related diseases. The absorption of vitamin B depends on specific intestinal transport proteins, while vitamin D, as a fat-soluble vitamin, relies on micelles formed from bile salts for absorption, which is generally inefficient. The absorption of minerals such as calcium, iron, and zinc is subject to multiple regulation by intestinal pH, transport protein expression, competitive inhibition between ions, and anti-nutritional factors such as phytic acid, resulting in generally low bioavailability.

[0004] In recent years, although increasing evidence suggests that certain probiotic strains have the potential to promote vitamin and mineral absorption, overall effective studies remain relatively scarce, especially when it comes to combinations of multiple strains. There is a lack of arbitrariness in the combinations and a lack of synergistic effects; in particular, studies on promoting vitamin and mineral absorption through specific strain combinations are rarely reported. Therefore, there is an urgent need to develop a novel compound probiotic agent that, based on specific strain combinations, has been proven to have synergistic effects, effectively overcoming the shortcomings of existing technologies and achieving a more comprehensive and efficient nutritional balance. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a compound probiotic agent and its application in the preparation of products that promote the absorption of vitamins and / or minerals.

[0006] The compound probiotic provided by this invention consists of Bifidobacterium animalis subsp. lactis Bi66 and Lactobacillus acidophilus LA15.

[0007] This invention combines *Bifidobacterium animalis* subsp. lactis Bi66 with *Lactobacillus acidophilus* LA15, which can significantly enhance the absorption of vitamin D (including D2 and D3), vitamin B (including B2, B6, and B12), and minerals such as calcium and zinc. Experiments show that *Bifidobacterium animalis* subsp. lactis Bi66 and *Lactobacillus acidophilus* LA15 have a synergistic effect in the absorption of vitamins and minerals. Furthermore, compared to other strains of the same species, this combined strain shows more significant absorption of vitamins and minerals.

[0008] In this invention, the ratio of viable bacteria of Bifidobacterium animalis subsp. Lactobacillus Bi66 to Lactobacillus acidophilus LA15 is 1:5 to 5:1.

[0009] In some embodiments, the viable count ratio of Bifidobacterium animalis subsp. lactis Bi66 to Lactobacillus acidophilus LA15 is 1:5, 1:1, or 5:1.

[0010] This invention also provides the use of the compound probiotics as described above in the preparation of products that promote the absorption of vitamins and / or minerals.

[0011] In this invention, the vitamin is a B vitamin or vitamin D. Specifically, the B vitamin is at least one of vitamin B2, vitamin B6, or vitamin B12; and the vitamin D is at least one of vitamin D2 or vitamin D3.

[0012] In this invention, the mineral is at least one of calcium, iron, and zinc.

[0013] In this invention, the absorption-promoting effect includes enhancing the intestinal active transport capacity for vitamins and / or minerals, increasing passive diffusion efficiency, and reducing the inhibitory effect of anti-nutritional factors on mineral absorption in the intestine. Experimental data show that, compared with using any single strain or the control group alone, the compound probiotics of this invention exhibit significant synergistic effects in promoting the absorption of vitamins D2, D3, B2, B6, B12, as well as calcium, iron, and zinc. The composition has the most significant absorption-promoting effect on vitamin B12 and / or calcium.

[0014] Furthermore, the present invention also provides products that promote the absorption of vitamins and / or minerals, comprising the compound probiotics as described above.

[0015] The product described in this invention also includes at least one of a protective agent, a prebiotic, and an excipient.

[0016] In this invention, the dosage form of the product is preferably an oral preparation. Optionally, the oral preparation includes, but is not limited to, oral liquids, capsules, soft capsules, lyophilized powders, tablets, granules, or chewable tablets.

[0017] As a feasible example, the protective agent includes at least one of milk powder, starch, gelatin, dextrin, vegetable oil, sorbitol, sorbitol monostearate, sucrose, lactose, and polyvinylpyrrolidone.

[0018] As a feasible example, the prebiotics mentioned include at least one of fructooligosaccharides, galactooligosaccharides, isomaltooligosaccharides, xylooligosaccharides, stachyose, inulin, polydextrose, resistant dextrin, soybean oligosaccharides, and yeast β-glucan.

[0019] As a feasible example, the excipients include one or more of the following: fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, colorants, antioxidants, and flavoring agents. For example, the filler is at least one of microcrystalline cellulose, lactose, mannitol, and starch; the binder is at least one of hydroxypropyl methylcellulose, polyvinylpyrrolidone, starch paste, and gum arabic; the wetting agent is at least one of purified water and ethanol; the disintegrant is at least one of sodium carboxymethyl starch, crospovidone, crospomethyl cellulose sodium, and low-substituted hydroxypropyl cellulose; the emulsifier is at least one of Tween-80, Span-80, glyceryl monostearate, and soybean lecithin; the solubilizer is at least one of ethanol, propylene glycol, glycerin, and polyethylene glycol; the solubilizer is at least one of Tween-80, polysorbate-80, and poloxamer; the colorant is at least one of titanium dioxide, iron oxide red, iron oxide yellow, and food coloring; the antioxidant is at least one of vitamin C, vitamin E, sodium sulfite, sodium metabisulfite, and propyl gallate; and the flavoring agent is at least one of sucrose, stevia, aspartame, citric acid, malic acid, menthol, and flavoring.

[0020] As a feasibility example, the total number of live bacteria in the product is not less than 1×10⁻⁶. 8 CFU / ml or 1×10 8 CFU / g.

[0021] Furthermore, the present invention also provides a method for preparing the product as described above, comprising:

[0022] Bifidobacterium animalis subsp. lactis Bi66 and Lactobacillus acidophilus LA15 were activated and fermented to obtain bacterial broth. After centrifugation to obtain bacterial cells, the bacterial broth was mixed with the protective agent and freeze-dried to obtain the product.

[0023] In this invention, the activation medium is MRS medium; the activation conditions include incubation at 37℃±5℃ for 12~48 h, and continuous activation 1~3 times. In some embodiments, the activation conditions include incubation at 37℃ for 24 h, and continuous activation twice.

[0024] In this invention, the fermentation medium is MRS medium, and the fermentation conditions include: an inoculum size of 1% (v / v) to 5% (v / v), and incubation at 37℃ ± 5℃ for 20 to 24 hours. In some embodiments, the fermentation conditions include: an inoculum size of 3% (v / v), and incubation at 37℃ for 22 hours.

[0025] In this invention, the mass ratio of the protectant to the bacterial cells is (1~10):1. After mixing, the mixture is pre-cultured at 37℃±5℃ for 0.5~2 h before freeze-drying, with a freeze-drying time of 12h~48h. In some embodiments, the mass ratio of the protectant to the bacterial cells is 5:1, and the mixture is pre-cultured at 37℃ for 1 h before freeze-drying, with a freeze-drying time of 24h.

[0026] Furthermore, the present invention also provides a method for promoting the absorption of vitamins and / or minerals, the method comprising administering the product as described above. Specifically, the object of the absorption promotion method described in the present invention is a human or mammal. The mammals include, but are not limited to, mice, rats, rabbits, dogs, cats, cattle, sheep, horses, pigs, etc.

[0027] In this invention, the method of administration includes, but is not limited to, oral administration.

[0028] Furthermore, the present invention also provides a preparation for supplementing vitamins and / or minerals, which is at least one of the following:

[0029] I) Vitamins and the compound probiotics as described above, or vitamins and the product as described above, or vitamins and the product prepared by the method described above;

[0030] II) Minerals and the compound probiotics as described above, or minerals and the product as described above, or minerals and the product prepared by the method described above.

[0031] II) Vitamins, minerals and the compound probiotics as described above, or vitamins, minerals and the product as described above, or vitamins, minerals and the product prepared by the method described above.

[0032] The vitamin and / or mineral supplements include, but are not limited to, B vitamins or vitamin D. Specifically, the B vitamins are at least one of vitamin B2, vitamin B6, or vitamin B12; and the vitamin D is at least one of vitamin D2 or vitamin D3. The minerals include, but are not limited to, at least one of calcium, iron, and zinc.

[0033] The present invention also provides a method for supplementing vitamins and / or minerals, comprising administering the preparations as described above. In this invention, the administration method includes, but is not limited to, oral administration.

[0034] In this invention, the method is applied to humans or other mammals. These mammals include, but are not limited to, mice, rats, rabbits, dogs, cats, cattle, sheep, horses, and pigs. Oral administration of the preparation can effectively improve the body's absorption and utilization of vitamins and minerals, thereby alleviating health problems caused by vitamin and / or mineral deficiencies, such as poor bone development, anemia, and weakened immunity.

[0035] The compound probiotic agent involved in this invention is composed of Bifidobacterium animalis subsp. lactis Bi66 and Lactobacillus acidophilus LA15. This compound probiotic can significantly enhance the absorption of vitamin D (including D2 and D3), vitamin B (including B2, B6, and B12), and minerals such as calcium and zinc. Furthermore, while maintaining a consistent bacterial count, it exhibits a significant advantage compared to single Bifidobacterium animalis or single Lactobacillus acidophilus, meaning that Bifidobacterium animalis subsp. lactis Bi66 and Lactobacillus acidophilus LA15 can work synergistically to promote vitamin and mineral absorption. Attached Figure Description

[0036] Figure 1 Indicates the peak area of ​​vitamin D2;

[0037] Figure 2 This indicates the peak area of ​​propionic acid VD3;

[0038] Figure 3 Indicates the peak area of ​​VB2;

[0039] Figure 4 Indicates the peak area of ​​VB6;

[0040] Figure 5 Indicates the peak area of ​​VB12;

[0041] Figure 6 This indicates the intracellular calcium content of Caco-2 cells and the residual calcium concentration in the culture medium;

[0042] Figure 7 This indicates the intracellular iron content of Caco-2 cells and the residual iron concentration in the culture medium;

[0043] Figure 8 This indicates the zinc content in Caco-2 cells and the residual zinc concentration in the culture medium. Detailed Implementation

[0044] This invention provides a compound probiotic agent and its application in the preparation of products that promote the absorption of vitamins and / or minerals. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in 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.

[0045] Unless otherwise defined in this invention, scientific and technical terms related to this invention shall have the meanings understood by one of ordinary skill in the art.

[0046] The terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” herein also provides for schemes “consisting of…”.

[0047] The term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural.

[0048] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0049] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have a reasonable deviation within a certain range, such as ±10%, ±5%, ±1%, or ±0.5%.

[0050] The embodiments and comparative examples of this invention describe some examples. These embodiments illustrate certain implementations of the invention. However, this does not mean that the effects of the invention can only be achieved in these examples. In fact, any concentration of the components between the two endpoint values ​​shown in the embodiments can achieve a good effect in promoting the absorption of minerals and / or vitamins.

[0051] In addition, other supplementary bacterial strains were also tried in combination during the research and development, but the effects were not as good as those in the examples, so they will not be repeated here.

[0052] All test materials used in this invention are common commercially available products. Information on some test materials and instruments is shown in Table 1.

[0053] Table 1 Reagent / Instrument Information Sheet

[0054]

[0055] The preservation information of Bifidobacterium animalis subsp. lactis strain Bi66 of this invention is as follows: Preservation number: CCTCC NO. M 2023769; Classification name: Bifidobacterium animalis subsp. Lactis Bi66; Preservation date: May 17, 2023; Preservation institution: China Center for Type Culture Collection; Preservation address: Wuhan University, Wuhan, China.

[0056] The preservation information of the Lactobacillus acidophilus strain LA15 of this invention is as follows: Preservation number: CCTCC NO. M 2023768; Classification name: Lactobacillus acidophilus LA15; Preservation date: May 17, 2023; Preservation institution: China Center for Type Culture Collection; Preservation address: Wuhan University, Wuhan, China.

[0057] It should be understood that in the various embodiments of this application, the sequence numbers of the above processes do not imply the order of execution. Some or all steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application. The present invention is further illustrated below with reference to embodiments:

[0058] Example 1

[0059] Bifidobacterium animalis subsp. lactis Bi66 and Lactobacillus acidophilus LA15 were inoculated into MRS liquid medium and activated by culturing at 37°C for 24 h. This activation was repeated twice to obtain an activated solution. The activated solution was then inoculated into MRS liquid medium at a rate of 3% (v / v) and cultured at 37°C for 22 h to obtain a bacterial suspension. The bacterial suspension was centrifuged at 8000 r / min for 10 min at 4°C and filtered to obtain bacterial cells. The bacterial cells were mixed with 5 times their weight of skim milk powder to obtain a bacterial suspension. The bacterial suspension was pre-cultured at 37°C for 1 h and then freeze-dried for 24 h to obtain Bi66 (600 billion CFU / g) and Lactobacillus acidophilus LA15 (200 billion CFU / g). The obtained Bi66 and LA15 bacterial powders were then mixed into a bacterial powder at a viable cell ratio of 5:1.

[0060] Example 2

[0061] The preparation method is the same as in Example 1, with the ratio of Bi66:LA15 controlled at 1:1 (the ratio of viable bacteria).

[0062] Example 3

[0063] The preparation method is the same as in Example 1, with the ratio of Bi66:LA15 controlled at 1:5 (the ratio of viable bacteria).

[0064] Comparative Example 1

[0065] Bifidobacterium animalis subsp. lactis Bi66 was inoculated into MRS liquid medium and activated by culturing at 37°C for 24 h. This activation was repeated twice to obtain an activated solution. The activated solution was then inoculated into MRS liquid medium at a rate of 3% (v / v) and cultured at 37°C for 22 h to obtain a bacterial suspension. The bacterial suspension was centrifuged at 4°C and 8000 r / min for 10 min and filtered to obtain bacterial cells. The bacterial cells were mixed with 5 times their weight of skim milk powder to obtain a bacterial suspension. The bacterial suspension was pre-cultured at 37°C for 1 h and then freeze-dried for 24 h.

[0066] Comparative Example 2

[0067] The bacterial strain was Lactobacillus acidophilus LA15, and the preparation method was the same as that of Comparative Example 1.

[0068] Comparative Example 3

[0069] The bacterial strain was Bifidobacterium animalis subsp. lactis BB-12, and the preparation method was the same as that of Comparative Example 1.

[0070] Comparative Example 4

[0071] The bacterial strain was Lactobacillus acidophilus LA-5, and the preparation method was the same as that of Comparative Example 1.

[0072] Comparative Example 5

[0073] The bacterial strains were Bifidobacterium animalis subsp. lactis BB-12 and Lactobacillus acidophilus LA15. The preparation method was the same as in Example 1, and the ratio of BB-12:LA15 was controlled to be 1:1 (the ratio of viable bacteria).

[0074] Comparative Example 6

[0075] The bacterial strains were Bifidobacterium lactis subsp. Bi66 and Lactobacillus acidophilus LA-5. The preparation method was the same as in Example 1, with the ratio of Bi66:LA-5 controlled at 1:1 (the ratio of viable bacteria).

[0076] Comparative Example 7

[0077] The bacterial strains were Bifidobacterium animalis subsp. lactis BB-12 and Lactobacillus acidophilus LA-5. The preparation method was the same as in Example 1, and the ratio of BB-12:LA-5 was controlled to be 1:1 (the ratio of viable bacteria).

[0078] Comparative Example 8

[0079] The bacterial strains were Bifidobacterium animalis subsp. lactis Bi66 and Lactobacillus acidophilus LA15. The preparation method was the same as in Example 1, with the ratio of Bi66:LA15 controlled at 6:1 (the ratio of viable bacteria).

[0080] Experimental verification

[0081] 1. The effect of probiotics on the absorption of vitamins (VD2, VD3, VB2, VB6, VB12)

[0082] 1.1 Sample Collection

[0083] Caco-2 cells were seeded in 6-well plates and cultured at 37°C and 5% CO2 until 80% confluence. 20 μL of a mixed vitamin standard working solution (VD group: 4 μg / μL each of VD2 and VD3; VB group: 2 μg / μL VB2, 4 μg / μL VB6, 6 μg / μL VB12) was added to each well in both the experimental and control groups, and the cells were cultured for another 12 h. Subsequently, 20 μL of probiotic suspension (OD600 = 0.5) or mannose protein solution (1 mg / mL) was added to the experimental groups, while the control group received an equal volume of culture medium, and the cells were cultured for a total of 8 h. The supernatant from each well was collected; the cells were washed twice, sequentially with 200 μL of pure water and 200 μL of methanol, and the washes were combined. All samples were stored at -20°C, with three replicates per group.

[0084] 1.2 Preparation of the test solution

[0085] The collected cell washing solution samples were used as the test sample source. 400 μL of methanol was added to each EP tube containing the cell washing solution to be tested. After vortexing and centrifugation, the supernatant was collected and dried under nitrogen. The residue was reconstituted with 100 μL of methanol, vortexed, centrifuged again, and the supernatant was filtered through a 0.22 μm microporous membrane to obtain the test sample solution.

[0086] 1.3 Preparation of standard solutions

[0087] Accurately weigh 1.0 mg each of VD2, VD3, VB2, VB6, and VB12 standards, dissolve them in methanol (VD) or DMSO (VB), and dilute to 1 mL to prepare 1.0 mg / mL single-standard stock solutions. Prepare VD mixed working solutions (4 μg / μL each of VD2 and VD3) and VB mixed working solutions (2 μg / μL of VB2, 4 μg / μL of VB6, and 6 μg / μL of VB12) by diluting with methanol according to the required experimental proportions. Further dilute to obtain a series of standard curve solutions. Store all solutions at -20°C, protected from light.

[0088] 1.4 Chromatographic conditions

[0089] Vitamin D analysis was performed under the following chromatographic conditions: An Agilent ZORBAX SB-C18 column (4.6 × 150 mm, 5 μm); mobile phase: water (A)-acetonitrile (B), gradient elution: 0–2 min, 10% A; 2–10 min, 10%→0% A; 10–15 min, 0% A; 15–17 min, 0%→10% A; 17–22 min, 10% A. Flow rate: 1.0 mL / min; column temperature: 35℃; detection wavelength: 265 nm; injection volume: 10 μL.

[0090] Vitamin B analysis was performed under the following chromatographic conditions: An Agilent ZORBAX SB-C18 column (4.6 × 150 mm, 5 μm); mobile phase: water (A) - acetonitrile (B), gradient elution: 0–5 min, 95% A; 5–15 min, 95% → 60% A; 15–16 min, 60% → 50% A; 16–20 min, 50% → 95% A; 20–25 min, 95% A. The flow rate was 1.0 mL / min, column temperature was 28℃, UV detection wavelength was 265 nm, and injection volume was 10 μL.

[0091] 2. The effect of probiotics on the absorption of minerals (calcium, iron, zinc)

[0092] 2.1 Experimental Grouping

[0093] Using the Caco-2 cell model and atomic absorption spectrometry, this study investigated the effect of probiotics on calcium (Ca2+). 2+ ), iron (Fe) 2+ ), Zinc (Zn) 2+ The effects of mineral culture medium on absorption and transport were investigated. A blank control group (containing only mineral culture medium) and a probiotic treatment group (containing probiotics and mineral culture medium of the same concentration) were established, with three parallel wells in each group.

[0094] 2.2 Cell Culture and Treatment

[0095] Caco-2 cells were seeded in six-well plates and cultured in complete medium containing 10% fetal bovine serum until they fully differentiated and formed a dense monolayer. Before the experiment, cells were starved: the old medium was discarded, cells were washed twice with PBS, and then replaced with serum-free 1640 medium. Cells were incubated for 6 hours to deplete intracellular mineral reserves and eliminate serum interference.

[0096] 2.3 Co-culture of probiotics and minerals

[0097] A working solution containing the target minerals (1 mM CaCl2, 50 μM FeSO4, 100 μM ascorbic acid, 20 μM ZnSO4) was prepared using serum-free 1640 medium. The probiotics were adjusted to OD600 = 0.5. 20 μL of bacterial suspension was added to each well in the experimental group, while no bacteria were added to the control group. One hour after adding the bacterial suspension, an equal volume of the mineral-containing working solution was added to all wells, and the mixtures were incubated at 37°C for 2 hours.

[0098] 2.4 Sample Collection and Processing

[0099] Culture medium sample: Take culture medium from each well, centrifuge at 10,000 rpm for 5 minutes at 4°C, and store the supernatant at -20°C.

[0100] Cell lysis buffer: Wash cells three times with ice-bathed PBS, add 300 μL of pre-chilled RIPA lysis buffer (containing protease inhibitor) to each well, lyse on ice for 15 minutes, centrifuge at 12,000 rpm for 15 minutes at 4°C, and aliquot the supernatant.

[0101] Tube A (200 μL, stored at -80℃, for AAS detection);

[0102] Tube B (20 μL, store at 4℃ or -20℃ for BCA protein quantification).

[0103] 2.5 BCA protein quantification

[0104] To standardize intracellular mineral content data, the total protein concentration in cell lysates was determined using the BCA method. First, standard curve solutions with concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL were prepared using 5 mg / mL protein standards diluted with PBS. A suitable working solution was prepared by mixing BCA reagent A and reagent B at a 50:1 ratio. 10 µL of sample from tube B was diluted with 10 µL of PBS, and 20 µL of each of the standard solutions were added to 96-well plates. 200 µL of BCA working solution was then added to each well, gently vortexed, and incubated at 37°C for 30 minutes. After the reaction, the absorbance of each well at 562 nm was measured using a microplate reader. The protein concentration of each sample was calculated based on the standard curve for subsequent normalization calculations of mineral content.

[0105] 2.6 Microwave digestion and AAS detection

[0106] To thoroughly decompose the organic matrix in the samples and accurately release and bind minerals, all A-tube samples and culture medium supernatants underwent microwave digestion. 100 µL of sample was accurately measured and placed in a dedicated digestion vessel. 2.0 mL of high-purity concentrated nitric acid was added, and the vessel was sealed and placed in the microwave digester. After the program was completed (the default program), the digestion vessel was allowed to cool naturally to room temperature within the oven chamber (approximately 40 minutes). The vessel was carefully opened in a fume hood, and the digestion solution was completely transferred to a 50 mL acid-washed plastic volumetric flask. The inner vessel was washed several times with a small amount of ultrapure water, and the wash solutions were combined. Finally, the solution was diluted to the mark with ultrapure water to obtain a clear 2% HNO3 matrix test solution. The concentration of the target minerals in each sample was determined using atomic absorption spectrometry at the characteristic wavelengths of iron and zinc.

[0107] 3. Data Statistics and Analysis

[0108] The relevant data were statistically plotted using Excel and analyzed using Minitab software. Compared with the control group, ** represents P < 0.01, * represents P < 0.05, and NS represents no significant difference.

[0109] The relative percentages in the statistical tables represent the changes in each group's data relative to the control group. The calculation formula is: (Subject Group - Control Group) ÷ Control Group × 100% or (Control Group - Subject Group) ÷ Control Group × 100%.

[0110] The synergistic effect was calculated using the Bliss independence model to assess the synergistic / antagonistic effects of drug combinations. For special scenarios where the direction of single-drug effects is inconsistent (positive enhancement, negative inhibition), the Bliss score (BS) was used as the core criterion to eliminate the interference of effect sign on the results. The specific method is as follows:

[0111] ① Data preprocessing

[0112] The relative effect percentages of single-drug and combination groups measured in the experiment were converted to decimal form, while retaining the original effect sign.

[0113] ② Calculation of theoretical additive effect

[0114] Based on the core formula of the Bliss independence model, the theoretical additive effect (E) of the two-drug combination is calculated. add ):

[0115] E add =E A +E B -E A ×E B

[0116] In the formula:

[0117] E A E B These are the measured effect values ​​(decimal form) of the two single drugs, respectively.

[0118] E add This represents the theoretical superposition effect when the effects of the two drugs are completely independent.

[0119] ③ The Bliss score (BS) is used as the core indicator for determining synergy, and the calculation formula is as follows:

[0120] BS=E obs -E add

[0121] In the formula:

[0122] Eobs is the measured effect value (decimal form) of the two-drug combination group.

[0123] BS represents the difference between the measured effect and the theoretical additive effect; its sign and magnitude directly reflect the type of combined effect.

[0124] Synergistic effect: BS>0 indicates that the measured effect is significantly higher than the theoretical additive effect, and the combination of the two drugs has a synergistic effect;

[0125] Additive effect: BS≈0, indicating that the measured effect is basically consistent with the theoretical additive effect;

[0126] Antagonistic effect: BS<0 indicates that the measured effect is lower than the theoretical additive effect, and the combined use of the two drugs has a detrimental effect.

[0127] Test results

[0128] 1. Analysis of Results on Promoting Vitamin Absorption

[0129] The absorption of vitamins (D2, D3, B2, B6, B12) by Caco-2 cells treated with different probiotics was detected by high performance liquid chromatography (HPLC). The results are as follows: Figures 1-5 As shown in Tables 2-1 to 2-5. Figure 1 This indicates the peak area of ​​vitamin D2. Figure 2 This indicates the peak area of ​​propionic acid VD3. Figure 3 This represents the peak area of ​​VB2. Figure 4 This represents the peak area of ​​VB6. Figure 5 This represents the peak area of ​​VB12.

[0130] Table 2-1

[0131]

[0132] Table 2-2

[0133]

[0134] Table 2-3

[0135]

[0136] Table 2-4

[0137]

[0138] Table 2-5

[0139]

[0140] As shown in Tables 2-1 to 2-5, there are significant differences in the absorption-promoting effects of different probiotic single strains and compound strains on various vitamins.

[0141] For vitamin D2, the relative absorption percentages of each single-strain group (Bi66, LA15, BB-12, LA-5) were 5.47%, 8.16%, 4.85%, and 1.18%, respectively. Compared with the control group, none of these percentages reached a statistically significant level (P>0.05), indicating that the single probiotic had a weak effect on promoting vitamin D2 absorption. However, when Bi66 and LA15 were combined in a ratio of (1~5):(1~5), their relative absorption percentages significantly increased to 30.93%, 35.23%, and 34.18% (P<0.05), and their Bliss scores (BS values) were 0.177, 0.220, and 0.210, respectively, all greater than 0, showing a clear synergistic promoting effect. Other complex bacterial groups, such as BB-12+LA15 (1:1), Bi66+LA-5 (1:1), BB-12+LA-5 (1:1) and Bi66+LA15 (6:1), did not show a significant increase in relative absorption percentage, and their BS values ​​were less than or close to 0, indicating no synergistic effect.

[0142] Regarding vitamin D3 absorption, single-strain LA15 even showed a certain inhibitory effect (relative percentage -9.74%), while the promoting effects of Bi66, BB-12, and LA-5 were not significant (relative percentages of 5.62%, -2.22%, and 0.86%, respectively). However, the three compound ratios of Bi66 and LA15 (1:5, 1:1, and 5:1) significantly improved vitamin D3 absorption, with relative percentages of 27.05%, 28.75%, and 31.52% (P<0.05), and BS values ​​of 0.306, 0.323, and 0.351, respectively, indicating a significant synergistic effect. In addition, the BS values ​​of the BB-12+LA15 (1:1), BB-12+LA-5 (1:1), and Bi66+LA15 (6:1) groups were also greater than 0, indicating a synergistic effect, but because the BS values ​​were close to 0, the synergistic effect was very weak. Furthermore, the relative absorption percentage increase of these compound bacteria (e.g., BB-12+LA15 (1:1) was -3.54%) did not reach a significant level, which may be related to the inhibitory effect of LA15 single bacteria, but the combination showed an overall synergistic trend.

[0143] The results of vitamin B2 absorption studies showed that single strains Bi66 and LA15 significantly promoted its absorption, with relative percentages reaching 99.84% and 72.35%, respectively (P<0.05). The promoting effect was further enhanced when the two strains were combined. The relative percentages of the Bi66+LA15 (1:5), (1:1), and (5:1) groups reached 182.94%, 201.39%, and 218.48%, respectively (P<0.01), with BS values ​​of 0.830, 1.014, and 1.185, respectively, indicating a highly significant synergistic effect. The relative percentage of the Bi66+LA15 (6:1) group was 128.72% (P<0.05), with a BS value of 0.288, also showing a synergistic effect. However, the absorption-promoting effects of single strains BB-12 and LA-5, and their combinations with other strains, on vitamin B2 absorption were relatively weak or showed no significant synergistic effect.

[0144] For vitamin B6, single bacteria Bi66 and LA15 showed some inhibitory effects on absorption (relative percentages of -20.83% and -12.03%, respectively), while BB-12 and LA-5 showed slight promoting effects (3.66% and 0.78%, respectively). However, when Bi66 and LA15 were combined, the absorption was reversed. The relative percentages of the Bi66+LA15 (1:5), (1:1), and (5:1) groups were 24.03%, 21.76%, and 34.18%, respectively (P<0.05), with BS values ​​of 0.594, 0.571, and 0.695, respectively, showing a significant synergistic promoting effect. In addition, the BS values ​​of the BB-12+LA15 (1:1), Bi66+LA-5 (1:1), and Bi66+LA15 (6:1) groups were all greater than 0, indicating a synergistic effect. This suggests that specific probiotic combinations can effectively reverse the inhibitory effect of single bacteria and instead promote the absorption of vitamin B6.

[0145] Finally, regarding vitamin B12 absorption, single-strain Bi66 and LA15 significantly promoted its absorption, with relative percentages of 35.63% and 30.79%, respectively (P<0.05). The relative percentages of the Bi66+LA15 (1:5), (1:1), and (5:1) combinations further increased to 61.33%, 62.92%, and 72.17% (P<0.01), with BS values ​​of 0.059, 0.075, and 0.167, respectively, demonstrating a significant synergistic effect. The (5:1) group, with a higher proportion of Bi66, showed the best effect. While the relative percentage of the Bi66+LA15 (6:1) group was 27.09% (P<0.05), its BS value was -0.284, indicating no synergistic effect, suggesting that the ratio of the combinations may have a significant impact on the synergistic effect. Other complex bacterial groups, such as BB-12+LA15 (1:1), Bi66+LA-5 (1:1), and BB-12+LA-5 (1:1), did not show significant synergistic promoting effects.

[0146] In summary, the combined use of Bi66 and LA15 exhibits a general and significant synergistic effect in promoting the absorption of various vitamins (D2, D3, B2, B6, B12), especially within a certain ratio range (e.g., 1:5 to 5:1), where the effect is optimal. However, the synergistic effect of other probiotic combinations varies considerably depending on the types of vitamins and the ratio of the combinations.

[0147] 2. Analysis of Results on Promoting Mineral Absorption

[0148] This study used atomic absorption spectrometry (AAS) to determine the intracellular mineral content and corresponding residual mineral concentrations in the culture medium of Caco-2 cells treated with and untreated with probiotics. Intracellular mineral content was standardized using protein concentrations measured by the BCA method and is expressed as "μg mineral / mg protein". Residual minerals in the culture medium are directly expressed as the raw concentrations measured by AAS in "μg / L (ppb)". Specific results for calcium, iron, and zinc are as follows: Figure 6 A and B in the middle, Figure 7 A and B in the middle, Figure 8 A and B are shown in Tables 3-1 to 3-3 and Tables 4-1 to 4-3. Figure 6 In the text, A represents the intracellular calcium content of Caco-2 cells. Figure 6 In the text, B indicates the residual calcium concentration in the culture medium. Figure 7 In the figure, A represents the intracellular iron content of Caco-2 cells. Figure 7 In the middle B, the iron residual concentration in the culture medium is indicated. Figure 8 In the figure, A represents the intracellular zinc content of Caco-2 cells. Figure 8 B indicates the residual zinc concentration in the culture medium.

[0149] Table 3-1 Intracellular calcium content

[0150]

[0151] Table 3-2 Intracellular iron content

[0152]

[0153] Table 3-3 Intracellular Zinc Content

[0154]

[0155] As shown in Tables 3-1 to 3-3, there are significant differences in the effects of different probiotic single strains and compound strains on promoting mineral absorption.

[0156] Regarding calcium absorption, among the single bacterial groups, the intracellular calcium content in the Bi66 group (0.77±0.13 μg / mg protein) was 13.24% higher than that in the control group (0.68±0.04 μg / mg protein), the BB-12 group was 7.35% higher, while the LA15 and LA-5 groups were 20.59% and 1.47% lower, respectively. However, none of the single bacterial groups showed statistical significance compared with the control group (P>0.05). However, all three ratios of Bi66 to LA15 (1:5~5:1) significantly increased intracellular calcium content, reaching 1.15±0.09 μg / mg protein, 1.28±0.19 μg / mg protein, and 1.31±0.21 μg / mg protein, respectively (P<0.05). The relative absorption percentages significantly increased to 69.12%, 88.24%, and 92.65%, with BS values ​​of 0.737, 0.929, and 0.973, all well greater than 0, demonstrating a strong synergistic promoting effect. The 5:1 ratio showed the best effect. The relative absorption percentage of the BB-12+LA15 (1:1) group was 2.94%, with a BS value of 0.147, indicating a synergistic effect, but the promoting effect was not significant. Other complex bacterial groups, such as Bi66+LA-5 (1:1), BB-12+LA-5 (1:1) and Bi66+LA15 (6:1), showed a decrease or only a slight increase in relative absorption percentage, with BS values ​​less than 0, indicating no synergistic effect.

[0157] Regarding iron absorption, single strain Bi66 showed a certain promoting trend, with a relative absorption percentage of 18.75%, while LA15 and LA-5 both showed 6.25%, and BB-12 showed almost no change; none of the single strains reached significant levels. In the compound bacterial groups, the intracellular iron content of the Bi66+LA15 (1:1) group reached 0.024±0.002 μg / mg protein, and the relative absorption percentage was significantly increased to 50.00% (P<0.05), with a BS value of 0.262, indicating a significant synergistic effect. The relative absorption percentage of the Bi66+LA15 (5:1) group was 25.00%, with a BS value of 0.012, and the relative absorption percentage of the Bi66+LA15 (6:1) group was 31.25%, with a BS value of 0.074, all of which were determined to have a synergistic effect. Furthermore, the BB-12+LA15 (1:1) group had a relative absorption percentage of 12.50% and a BS value of 0.062, while the Bi66+LA-5 (1:1) group had a relative absorption percentage of 25.00% and a BS value of 0.012, indicating a synergistic effect. However, the increase in iron absorption in these groups was weaker than that in the Bi66+LA15 (1:1) group. The BB-12+LA-5 (1:1) group did not show a synergistic effect.

[0158] Regarding zinc absorption, the single strain Bi66 showed a significant promoting effect, with an intracellular zinc content of 0.52±0.08 μg / mg protein and a relative absorption percentage as high as 36.84% (P<0.05). The relative absorption percentages of the BB-12 group were 23.68%, the LA15 group was 13.16%, and the LA-5 group was 10.53%. Although the latter three showed an increase, it did not reach a significant level. The synergistic effect of Bi66 and LA15 was more significant when combined. In the Bi66+LA15 ratio range of 1:5 to 5:1, the intracellular zinc content reached 0.65±0.10 μg / mg protein, 0.69±0.09 μg / mg protein, and 0.73±0.05 μg / mg protein, respectively (P<0.01), with relative absorption percentages of 71.05%, 81.58%, and 92.11%, and BS values ​​of 0.259, 0.364, and 0.470, respectively. The synergistic effect increased with increasing Bi66 ratio, with the best effect observed at a 5:1 ratio. Although the relative absorption percentage of the Bi66+LA15 (6:1) group was 39.47% (P<0.05), the BS value was -0.057, indicating no synergistic effect. Other complex bacterial combinations such as BB-12+LA15 (1:1), Bi66+LA-5 (1:1), and BB-12+LA-5 (1:1) showed limited improvement in relative absorption percentage, with BS values ​​all less than 0, and no synergistic promoting effect was observed.

[0159] Based on the combined absorption results of calcium, iron, and zinc, the combined use of Bi66 and LA15, within a specific ratio range (especially 1:1 to 5:1), exhibited a significant synergistic effect on mineral absorption. The synergistic effect was particularly pronounced in the absorption of calcium and zinc. In contrast, other probiotic combinations showed weak or no synergistic effects, further confirming the superiority and ratio-dependent nature of the Bi66 and LA15 combination in promoting nutrient absorption.

[0160] Table 4-1 Residual calcium concentration in culture medium

[0161]

[0162] Table 4-2 Iron Residual Concentration in Culture Medium

[0163]

[0164] Table 4-3 Iron Residual Concentration in Culture Medium

[0165]

[0166] As shown in Tables 4-1 to 4-3, there are significant differences in the absorption-promoting effects of different probiotic single strains and compound strains on various minerals.

[0167] Regarding calcium absorption, among the single bacterial groups, the extracellular calcium content in the Bi66 group (5.32±1.33 μg / L) was 20.72% lower than that in the control group (6.71±1.01 μg / L), indicating that it may have promoted intracellular calcium absorption and reduced extracellular free calcium; the BB-12 group decreased by 5.81%, the LA-5 group decreased by 7.45%, and the LA15 group decreased by only 0.60%, but none of the single bacterial groups reached a statistically significant level compared with the control group (P>0.05). In the combined bacterial cultures, all three ratios of Bi66 to LA15 (1:5, 1:1, and 5:1) significantly reduced extracellular calcium levels, reaching 4.20±0.74 μg / L, 3.86±0.49 μg / L, and 3.85±0.53 μg / L, respectively (P<0.05). The relative reduction percentage (i.e., the percentage of calcium absorption promoted) significantly increased to 37.41%, 42.47%, and 42.62%, with BS values ​​of 0.162, 0.213, and 0.214, all greater than 0, indicating a synergistic effect in promoting calcium absorption. The 5:1 ratio showed the best effect, consistent with the trend of intracellular calcium content detection results. The Bi66+LA15 (6:1) group showed a relative reduction percentage of 17.44% and a BS value of -0.038, indicating no synergistic effect. Other complex bacterial groups, such as BB-12+LA15 (1:1), Bi66+LA-5 (1:1), and BB-12+LA-5 (1:1), showed relative reduction percentages of 1.79%, 5.51%, and -1.49%, respectively, with BS values ​​all less than 0. No synergistic effect on promoting calcium absorption was observed.

[0168] Regarding iron absorption, the extracellular liquid iron content in the single-strain Bi66, BB-12, and LA-5 groups decreased by 3.04%, 2.14%, and 3.57% respectively compared to the control group, while the LA15 group increased by 8.39%, but none of these results were statistically significant. In the combined bacterial groups, the extracellular liquid iron content in the Bi66+LA15 (1:5) group was 4.86±0.42 μg / L, with a relative decrease of 13.21% and a BS value of 0.183; the Bi66+LA15 (5:1) group showed a relative decrease of 15.18% and a BS value of 0.203; and the Bi66+LA15 (6:1) group showed a relative decrease of 10.18% and a BS value of 0.153, all indicating a synergistic effect. The Bi66+LA15 (1:1) group showed a relative decrease of -1.43% (i.e., a slight increase in iron content), but with a BS value of 0.037, it was still considered to have a synergistic effect. The BB-12+LA15 (1:1) group showed a relative reduction of -3.21% and a BS value of 0.029, indicating a synergistic effect. However, the Bi66+LA-5 (1:1) and BB-12+LA-5 (1:1) groups showed relative reductions of 1.96% and 3.75%, respectively, with BS values ​​of -0.045 and -0.019, respectively, showing no synergistic effect. Overall, the combination of Bi66 and LA15 at various ratios demonstrated a synergistic promoting trend of iron absorption by reducing extracellular iron content (or slowing its increase), with the 5:1 ratio showing a relatively better effect.

[0169] Regarding zinc absorption, the extracellular fluid zinc content in the Bi66 single-strain group (5.97±0.83 μg / L) was 8.58% lower than that in the control group (6.53±1.01 μg / L), and 3.06% lower in the BB-12 group. In contrast, the zinc content in the LA15 and LA-5 groups increased by 6.58% and 3.37%, respectively, but none of these results were statistically significant. The combination of Bi66 and LA15 significantly enhanced zinc absorption. The extracellular zinc levels in the Bi66+LA15 (1:5), (1:1), and (5:1) groups were 4.56±0.74 μg / L, 4.32±0.39 μg / L, and 4.19±0.51 μg / L, respectively (P<0.05), with relative reduction percentages of 30.17%, 33.84%, and 35.83%, and BS values ​​of 0.276, 0.313, and 0.333, respectively. The synergistic effect increased with the increase of the Bi66 ratio, with the best effect observed at a 5:1 ratio, which is highly consistent with the results of intracellular zinc content detection. Furthermore, the BS values ​​of the BB-12+LA15 (1:1), Bi66+LA-5 (1:1), BB-12+LA-5 (1:1), and Bi66+LA15 (6:1) groups were 0.016, 0.009, 0.036, and 0.036, respectively, all indicating a synergistic effect. However, their relative reduction percentages were -1.68%, 6.43%, 3.37%, and 6.13%, respectively, and their promoting effect was far less than that of the combinations of Bi66 and LA15 in the ratio range of 1:5 to 5:1.

[0170] The combined use of Bi66 and LA15, within a specific ratio range (especially 1:5 to 5:1), further validated the synergistic effect of Bi66 and LA15 in promoting mineral absorption by significantly reducing the mineral content in the extracellular fluid, with particularly significant effects on calcium and zinc absorption. This is corroborated by the analysis of intracellular mineral content, jointly indicating that Bi66 and LA15 are a probiotic combination with excellent synergistic effects in promoting mineral absorption.

[0171] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the 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 compound probiotic, characterized in that, It is composed of Bifidobacterium animalis subsp. lactis Bi66 and Lactobacillus acidophilus LA15; the viable count ratio of Bifidobacterium animalis subsp. lactis Bi66 and Lactobacillus acidophilus LA15 is 1:5 to 5:1; The preservation number of Bifidobacterium lactis subspecies Bi66 is CCTCC NO. M 2023769; The Lactobacillus acidophilus LA15 has the accession number CCTCC NO. M 2023768.

2. The compound probiotic according to claim 1, characterized in that, The viable count ratio of Bifidobacterium animalis subsp. Lactobacillus Bi66 to Lactobacillus acidophilus LA15 was 1:5, 1:1, or 5:

1.

3. The use of the compound probiotics of claim 1 or 2 in the preparation of products that promote the absorption of calcium, iron and / or zinc.

4. A product that promotes mineral absorption, characterized in that, It includes the compound probiotics as described in claim 1 or 2, wherein the minerals are calcium, iron and / or zinc.

5. The product according to claim 4, characterized in that, It also includes at least one of the following: protectants, prebiotics, and excipients.

6. The product according to claim 5, characterized in that, The protective agent includes at least one of milk powder, starch, gelatin, dextrin, vegetable oil, sorbitol, sorbitol monostearate, sucrose, lactose, and polyvinylpyrrolidone; The prebiotics mentioned include at least one of the following: fructooligosaccharides, galactooligosaccharides, isomaltooligosaccharides, xylooligosaccharides, stachyose, inulin, polydextrose, resistant dextrin, soybean oligosaccharides, and yeast β-glucan. The excipients include one or more of the following: fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, colorants, antioxidants, and flavoring agents.

7. The product according to any one of claims 4 to 6, characterized in that, The total number of viable bacteria is not less than 1×10⁻⁶. 8 CFU / ml or 1×10 8 CFU / g.

8. A method for preparing the product according to any one of claims 5 to 7, characterized in that, include: Bifidobacterium animalis subsp. lactis Bi66 and Lactobacillus acidophilus LA15 were activated and fermented to obtain bacterial broth. After centrifugation to obtain bacterial cells, the bacterial broth was mixed with the protective agent and freeze-dried to obtain the product.

9. The preparation method according to claim 8, characterized in that, The activated culture medium is MRS medium; the activation conditions include culturing at 37℃±5℃ for 12~48 h, and activating continuously 1~3 times.

10. The preparation method according to claim 8, characterized in that, The fermentation medium is MRS medium, and the fermentation conditions include: an inoculum size of 1% (v / v) to 5% (v / v) and incubation at 37℃±5℃ for 20 to 24 hours.

11. The preparation method according to claim 8, characterized in that, The mass ratio of the protectant to the bacterial cells is (1~10):

1. After mixing, the mixture is pre-cultured at 37℃±5℃ for 0.5~2 h before being freeze-dried for 12h~48h.

12. A mineral supplement formulation, characterized in that, It is a mineral and the compound probiotic as described in claim 1 or 2, or a mineral and the product as described in any one of claims 4 to 7, or a mineral and the product prepared by the method described in any one of claims 8 to 11; the mineral is calcium, iron and / or zinc.

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