Phytobacterium plantarum NuPlus-8 for synergistically promoting absorption of multiple minerals, product and application

Abstract

The invention belongs to the field of nutriology, and particularly relates to a plant lactobacillus NuPlus-8 capable of synergistically promoting absorption of multiple mineral substances, a product and application. The plant lactobacillus NuPlus-8 disclosed by the invention is derived from healthy breast milk, has good acid-resistant and cholate-resistant characteristics, and can survive and keep activity in related environments of intestinal tracts. The strain has the effect of synergistically promoting absorption of eight nutrient substances including calcium, magnesium, zinc, selenium, iron, phosphorus, vitamin D and protein, and meanwhile promotes skeletal development; phytase can be secreted, phytic acid-mineral compounds can be degraded, combined calcium, magnesium, zinc, selenium, iron and phosphorus can be released, and the solubility and the availability of mineral substances can be improved; and transmembrane transport and absorption of intestinal epithelial cells to various minerals, VD and amino acids are significantly improved. The strain has a unique physiological function of synergistically promoting multi-nutrient absorption, can be used in products for improving nutrient absorption and utilization efficiency, and has remarkable application value and industrial prospect.

Description

Technical Field

[0001] This invention belongs to the field of nutrition, specifically relating to a plant lactobacillus NuPlus-8 that synergistically promotes the absorption of multiple minerals, as well as its products and applications. Background Technology

[0002] Minerals are essential micronutrients for maintaining human life, participating in various key physiological processes such as bone formation, hemoglobin synthesis, nerve conduction, enzyme activity regulation, and immune response. Among them, the intake and absorption efficiency of minerals such as calcium, iron, zinc, and magnesium are closely related to an individual's nutritional status and health level. Children, pregnant women, and the elderly have higher mineral requirements, but their digestive and absorptive functions are relatively weak, making them highly susceptible to mineral deficiencies or poor utilization.

[0003] Currently, products on the market used to improve mineral deficiencies are mainly inorganic or organic mineral supplements, such as calcium lactate, zinc gluconate, and ferrous sulfate. However, these supplements have the following drawbacks: ① Limited absorption rate, affected by various factors such as intestinal pH, carrier transport capacity, and compatibility of ingredients; ② Significant side effects, with some products causing significant irritation in the gastrointestinal tract and easily leading to adverse reactions such as constipation, nausea, and a metallic taste; ③ Lack of synergistic effects, with different minerals exhibiting antagonistic or competitive absorption phenomena, such as the competitive inhibitory effect between iron and zinc, or calcium and magnesium; ④ Inability to achieve a "targeted, gentle, and continuous" absorption promotion process, resulting in limited intervention effects in chronic deficiency states.

[0004] In recent years, research on strains that promote mineral absorption has gradually attracted attention. Existing studies have reported that certain lactic acid bacteria, such as... Lactobacillus plantarum , Lactobacillus acidophilus and Bifidobacterium bifidum These strains can improve the bioavailability of minerals by regulating the gut microbiota, secreting organic acids, chelating metal ions, and activating the expression of mineral transport-related genes. For example: ① Regarding calcium absorption, some strains can increase calcium uptake by lowering intestinal pH, enhancing calcium ion solubility, and stimulating the expression of calcium channel proteins (such as TRPV6); ② Regarding zinc absorption, some strains possess metal chelating capabilities, which can form organic zinc complexes, improving their stability and absorption rate.

[0005] However, currently available strains generally face the following technical bottlenecks in promoting mineral absorption: First, they target only one mineral: most strains only work on one specific mineral, making it difficult to synergistically regulate the absorption of multiple minerals simultaneously; second, the mechanisms are unclear: many studies only focus on functional characterization, lacking a systematic analysis of molecular mechanisms and functional pathways; third, the extent of absorption enhancement is limited: the effect of some strains in enhancing mineral absorption is unstable and greatly affected by individual differences and the intestinal environment; fourth, there is a lack of industrially viable strain resources: currently, the types of strains with synergistic absorption-promoting functions that can be mass-produced are limited, and further development of efficient, safe, and stable strain resources is still needed.

[0006] Therefore, there is an urgent need to develop a strain that can synergistically promote the absorption of multiple minerals, has a clear mechanism of action, and has industrialization potential, in order to meet the multidimensional nutritional needs of the population, improve mineral utilization efficiency, and reduce the risk of adverse reactions caused by traditional supplements. Summary of the Invention

[0007] The purpose of this invention is to provide: A plant lactobacillus NuPlus-8 that synergistically promotes the absorption of multiple minerals, along with its products and applications, and related technologies, to address technical problems such as providing effective ways to promote nutrient absorption, or combinations thereof.

[0008] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0009] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0010] The definition of the standard chemical term can be found in the reference "Principles and Identification Techniques of Bacterial and Archaea Systematic Taxonomy", Higher Education Press, Chief Editors Li Wenjun, Liu Lan, Jiao Jianyu, and Fang Baozhu, 2025-01.

[0011] Unless otherwise stated, conventional methods within the scope of the art shall be used, such as methods for assessing the utilization of different carbohydrates, growth curve determination, antioxidant capacity, toxicity and safety testing, ability to inhibit pathogenic bacteria, and tolerance evaluation in artificial gastrointestinal acid and bile salt solutions.

[0012] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0013] The terms "optional / arbitrary" or "optionally / arbitrarily" mean that an event or situation subsequently described may or may not occur, including both the occurrence and non-occurrence of the event or situation. For example, according to the definition below: "The minerals described include any one or more of calcium, magnesium, zinc, selenium, iron, and phosphorus" means that the minerals may include calcium, or the minerals may include magnesium, or the minerals may include calcium, magnesium, zinc, selenium, iron, and phosphorus.

[0014] In this invention, the term "Lactiplantibacillus plantarum NuPlus-8" refers to a strain of Lactiplantibacillus plantarum screened and selectively domesticated in this invention, with the preservation number CGMCC No. 36325. This strain was isolated from healthy breast milk samples and, after multiple generations of domestication to withstand gastric juice, intestinal juice, and low-temperature freezing, exhibits excellent gastrointestinal tolerance and storage stability. It can synergistically promote the absorption of various minerals such as calcium, magnesium, zinc, and selenium, and also possesses phytase activity, vitamin D absorption-promoting activity, and amino acid absorption-promoting activity.

[0015] In this invention, the term "phytase activity" refers to a functional enzyme secreted by *Lactobacillus plantarum* NuPlus-8, which can degrade phytic acid molecules within the pH range of the gastrointestinal tract (pH 2.0-7.5), releasing phytic acid-chelated mineral ions such as calcium, magnesium, zinc, selenium, and iron, thereby improving the bioavailability of minerals in the diet. This activity was verified by the Malachite Green colorimetric method, and it exists in the form of supernatant and is sensitive to heat treatment.

[0016] In this invention, the term "epithelial permeability (P_app)" refers to an indicator characterizing the ability of substances to pass through the single-layer barrier of intestinal epithelial cells. The calculation formula is based on the concentration difference of substances in the upper and lower chambers of the Transwell chamber, the transport time, and the filter membrane area. In this invention, it is used to quantify the intestinal epithelial absorption efficiency of calcium, magnesium, zinc, selenium, and vitamin D3.

[0017] In this invention, the term "transepithelial resistance (TEER)" refers to an indicator used to assess the integrity of the Caco-2 cell monolayer barrier, with a TEER ≥ 500 Ω·cm. 2 This indicates the formation of tight junctions between cells, which can be used for in vitro mineral transmembrane transport experiments.

[0018] In a first aspect, the present invention provides a plant lactobacillus ( Lactiplantibacillus plantarum ).

[0019] Among its technical features is Lactobacillus plantarum.

[0020] Among them, the technical characteristic of *Lactobacillus plantarum* is *Lactobacillus plantarum* NuPlus-8, with the accession number CGMCCNo.36325.

[0021] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: Preferred solution: This invention provides a plant lactobacillus ( Lactiplantibacillus plantarum The *Lactobacillus plantarum* mentioned is *Lactobacillus plantarum* NuPlus-8, with the preservation number CGMCC No. 36325. This technical solution addresses the technical problem that "existing *Lactobacillus plantarum* species mostly only promote the absorption of single minerals, lacking the ability to synergistically regulate the absorption of multiple minerals such as calcium, magnesium, zinc, and selenium, thus failing to meet the human body's multidimensional mineral nutritional needs." Furthermore, it solves the technical problem that "existing *Lactobacillus plantarum* species with potential for promoting mineral absorption have poor gastrointestinal tolerance and insufficient phytase activity, making it difficult to stably colonize in the human gastrointestinal environment and effectively degrade phytic acid-mineral chelates to improve mineral bioavailability."

[0022] Secondly, the present invention provides a microbial agent.

[0023] Among its technical features is the use of microbial agents.

[0024] Among them, the technical characteristic microbial agent includes the aforementioned *Lactobacillus plantarum*.

[0025] Among them, the technically characteristic microbial agents include any one or more of the following: fermentation broth of *Lactobacillus plantarum*, fermentation broth precipitate, fermentation broth supernatant, live bacterial cells, inactivated bacterial cells, lyophilized powder, lysate, lysate, secondary metabolites, and exosomes.

[0026] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the second aspect of the present invention includes: Preferred Solution: This invention provides a microbial agent comprising the aforementioned *Lactobacillus plantarum*. This technical solution addresses the technical problem that "existing *Lactobacillus plantarum* products primarily promote the absorption of single minerals, lacking the ability to synergistically regulate the absorption of multiple minerals such as calcium, magnesium, zinc, and selenium, thus failing to meet the human body's multidimensional mineral nutritional needs." Furthermore, it solves the technical problem of "providing a suitable formulation for *Lactobacillus plantarum* NuPlus-8 to achieve large-scale production and convenient application."

[0027] Thirdly, the present invention provides the application of the above-mentioned *Lactobacillus plantarum* or its agent in the preparation of products that promote nutrient absorption.

[0028] Among them, the technical feature is: products that promote the absorption of nutrients.

[0029] Products with technical features that promote nutrient absorption include any one or more of the following: (1) Products that promote mineral absorption; (2) Products that promote vitamin absorption; (3) Products that promote the absorption of amino acids and / or proteins; (4) Products that promote bone growth and development.

[0030] Among them, products with technical features that promote nutrient absorption have any one or more of the following effects: (1) By upregulating the expression of mineral transport protein genes in intestinal epithelial cells, the transmembrane transport rate and bioavailability of minerals are increased, and mineral excretion is reduced; (2) By secreting phytase, phytic acid is degraded within the pH range of the gastrointestinal tract, releasing mineral ions chelated by phytic acid, thereby improving the availability of minerals; (3) Enhances the transmembrane absorption of vitamins in intestinal epithelial cells and promotes the absorption of minerals; (4) By upregulating the expression of amino acid transport proteins in intestinal epithelial cells, the absorption capacity of amino acids and / or proteins is enhanced; (5) Promotes the absorption and utilization of key osteogenic minerals and supports bone growth.

[0031] The minerals mentioned include any one or more of calcium, magnesium, zinc, selenium, iron, and phosphorus.

[0032] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the third aspect of the present invention includes: Preferred Solution: The nutrient absorption-promoting products provided by this invention include any one or more of the following: (1) products that promote mineral absorption; (2) products that promote vitamin absorption; (3) products that promote amino acid and / or protein absorption; and (4) products that promote bone growth and development. This technical solution, based on solving the technical problem that "existing *Lactobacillus plantarum* only promotes the absorption of a single mineral and lacks the ability to synergistically regulate the absorption of multiple minerals such as calcium, magnesium, zinc, and selenium, thus failing to meet the multi-dimensional mineral nutritional needs of the human body," further solves the technical problem of "providing a specific product form for *Lactobacillus plantarum* NuPlus-8."

[0033] Fourthly, the present invention provides a product that promotes the absorption of nutrients.

[0034] This includes technical features: product.

[0035] Among them, the technical features of the product include the aforementioned Lactobacillus plantarum or bacterial agent.

[0036] The dosage forms of products with technical characteristics include solid dosage forms, semi-solid dosage forms, or liquid dosage forms.

[0037] Among them, the technical feature products contain at least 1×10 8 CFU (Cytobacter plantarum)

[0038] Among them, products with technical features preferably contain ×10 8 -1×10 12 CFU (Cytobacter plantarum)

[0039] Among them, technical features of products also include auxiliary materials.

[0040] The excipients include any one or more of the following: diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, and buffers.

[0041] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the fourth aspect of the present invention includes: The first preferred embodiment: This invention provides a product that promotes the absorption of nutrients, the product comprising the aforementioned *Lactobacillus plantarum* or its agent, in a dosage form including solid, semi-solid, or liquid. This technical solution, while addressing the technical problem that "existing *Lactobacillus plantarum* products mostly only promote the absorption of single minerals and lack the ability to synergistically regulate the absorption of multiple minerals such as calcium, magnesium, zinc, and selenium, thus failing to meet the human body's multidimensional mineral nutritional needs," further addresses the technical problem of "providing a specific product dosage form for *Lactobacillus plantarum* NuPlus-8."

[0042] Second preferred embodiment: The present invention provides a product that promotes nutrient absorption, the product comprising the above-mentioned *Lactobacillus plantarum* or bacterial agent, containing at least 1×10⁻⁶. 8 CFU (Chemical Fumin) of Plant Lactobacillus. This technical solution not only addresses the technical problem that "existing plant lactobacillus products mostly only promote the absorption of a single mineral and lack the ability to synergistically regulate the absorption of multiple minerals such as calcium, magnesium, zinc, and selenium, thus failing to meet the human body's multidimensional mineral nutritional needs," but also further solves the technical problem that "limits the effective amount of plant lactobacillus NuPlus-8 in the product."

[0043] The third preferred embodiment: This invention provides a product that promotes nutrient absorption. The product comprises the aforementioned *Lactobacillus plantarum* or its agent, and further includes excipients, including any one or more of the following: diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, and buffers. This technical solution addresses the technical problem that "existing *Lactobacillus plantarum* products primarily promote the absorption of single minerals, lacking the ability to synergistically regulate the absorption of multiple minerals such as calcium, magnesium, zinc, and selenium, thus failing to meet the human body's multidimensional mineral nutritional needs." Furthermore, it solves the technical problem of "providing a suitable excipient system for *Lactobacillus plantarum* NuPlus-8."

[0044] In this invention, Examples 1-2 at least support the protection scope of the technical features “Lactobacillus plantarum” and “bacterial agent”.

[0045] Regarding the technical feature "Lactobacillus plantarum": The term "Lactobacillus plantarum" is derived from the foregoing explanation and / or the corresponding technical feature of NuPlus-8 in Examples 1-2.

[0046] Regarding the technical feature "microbial agent": The fermentation broth, fermentation broth precipitate, fermentation broth supernatant, live bacteria, inactivated bacteria, lyophilized powder, lysate, lysate, secondary metabolites, exosomes, etc., described in the foregoing explanation and / or Examples 1-2, are summarized by the common feature "a reagent containing *Lactobacillus plantarum* NuPlus-8". Therefore, those skilled in the art can reasonably infer that the technical feature of the bacterial agent, the subordinate concept of the bacterial agent, the technical means that are basically equivalent to the bacterial agent, and the technical means that can replace the bacterial agent based on the existing technical level within the scope of conventional technical means and common knowledge should all fall within the protection scope of the claims of this invention. For example, replacing the bacterial agent with lyophilized powder of *Lactobacillus plantarum*, fermentation supernatant of *Lactobacillus plantarum*, live bacteria of *Lactobacillus plantarum*, etc., while keeping other technical features unchanged, still falls within the protection scope of the claims of this invention.

[0047] Examples 3-7 of this invention at least support the protection scope of the technical feature "product that promotes nutrient absorption".

[0048] The technical feature "product that promotes nutrient absorption" is summarized from the common feature "promoting nutrient absorption," which includes the aforementioned explanations and / or the corresponding technical features in Examples 3-6, such as promoting mineral absorption, promoting vitamin absorption, promoting amino acid and / or protein absorption, and promoting bone growth and development. Therefore, those skilled in the art can reasonably presume that the technical feature "product that promotes nutrient absorption," the subordinate concept of "product that promotes nutrient absorption," the essentially equivalent technical means of "product that promotes nutrient absorption," and the technical means that can replace "product that promotes nutrient absorption" based on existing technology and common knowledge should all fall within the scope of protection of the claims of this invention. For example, replacing "product that promotes nutrient absorption" with "product that promotes calcium absorption" or "product that promotes iron absorption" while keeping other technical features unchanged still falls within the scope of protection of the claims of this invention.

[0049] In this invention, embodiments 3-7 at least support the protection scope of the technical feature "product".

[0050] The technical feature "product" is derived from the products containing *Lactobacillus plantarum*, products containing microbial agents, etc., as explained above and / or in Examples 3-7, through the generalization of the common feature "product". Therefore, those skilled in the art can reasonably presume that the technical feature product, the subordinate concept of product, the technical means that are basically equivalent to product, and the technical means that can replace product based on the existing level of technology and conventional technical means and common knowledge should all fall within the protection scope of the claims of this invention. For example, if other technical features remain unchanged, replacing the product with food, health products, pharmaceuticals, cosmetics, daily necessities, etc., still falls within the protection scope of the claims of this invention.

[0051] The beneficial effects of this invention are as follows: The present invention has at least the following beneficial effects: The *Lactobacillus plantarum* NuPlus-8 strain provided by this invention has been deposited at the China General Microbiological Culture Collection Center (CGMCC NO: 36325). This strain exhibits excellent acid and bile salt tolerance, enabling it to survive in the gastrointestinal environment. It possesses highly active phytase, and under intestinal-associated conditions of pH 2.0-7.5, it can significantly hydrolyze phytic acid, releasing inorganic phosphorus and bound calcium, magnesium, zinc, selenium, and iron ions, thereby improving its solubility and bioavailability. Further studies revealed that this strain upregulates the expression of mineral transport-related genes such as TRPV6, TRPM7, ZIP4, ZnT1, LAT1, and SLC26A6 at both cellular and animal levels, significantly promoting the transmembrane transport of minerals in intestinal epithelial cells and tissues. Simultaneously, in the Caco-2 cell model, *Lactobacillus plantarum* NuPlus-8 has been shown to promote vitamin D transmembrane permeability and absorption, providing a biological basis for its enhanced calcium ion absorption. In addition to the synergistic absorption of mineral elements, the plant lactobacillus NuPlus-8 of this invention exhibits an enhancing and promoting effect on the absorption of amino acids and dipeptides in intestinal epithelial cells, thereby promoting the absorption of amino acids and proteins.

[0052] The *Lactobacillus plantarum* NuPlus-8 of this invention has a unique physiological function of synergistically promoting the absorption of multiple nutrients. It can be applied in bacterial agents, mineral and vitamin supplements, and functional foods to improve nutrient absorption and utilization efficiency, prevent and alleviate mineral deficiency-related diseases, and has significant application value and industrial prospects.

[0053] Considering the possibility of this invention entering other countries, this invention also provides the following technical solutions: The present invention provides a method for promoting the absorption of nutrients, the method comprising using the above-mentioned *Lactobacillus plantarum*, bacterial agent or product.

[0054] This includes technical features: methods to promote the absorption of nutrients.

[0055] Among them, the technical features that promote nutrient absorption include administering an effective amount of *Lactobacillus plantarum*, bacterial agents, or products to the subject.

[0056] Specifically, the subjects included mammals.

[0057] Preferably, the subject is a human being.

[0058] Preservation Instructions Preserved strain: NuPlus-8; Classification and nomenclature: Lactobacillus plantarum Lactiplantibacillus plantarum ; Accession number: CGMCC No. 36325; Preservation period: October 23, 2025; Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections; Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0059] Figure 1 The colony morphology is NuPlus-8.

[0060] Figure 2 The morphology is obtained by Gram staining with NuPlus-8.

[0061] Figure 3 For NuPlus-8 hemolysis test.

[0062] Figure 4 The growth curve for NuPlus-8.

[0063] Figure 5 The effect of *Lactobacillus plantarum* NuPlus-8 on cell viability in the Caco2 cell model; a represents cell viability under single mineral element treatment, and b represents cell viability under mixed mineral element treatment.

[0064] Figure 6 P_app represents the transepithelial permeability (P_app) of *Lactobacillus plantarum* NuPlus-8 to calcium, magnesium, zinc, and selenium in the Caco2 cell model; there were no significant differences in ns between groups, *p<0.05, **p<0.01, ***p<0.001; #p<0.05, ##p<0.01, ###p<0.001.

[0065] Figure 7 The effect of *Lactobacillus plantarum* NuPlus-8 on the expression of genes related to the transport and absorption of calcium, magnesium, zinc, and selenium in a Caco 2 cell model; a: calbindin-D9K gene; b: TRPV6 gene; c: TRPM7 gene; d: ZnT1 gene; e: ZIP4 gene; f: SLC26A6 gene; g: LAT1 gene; vs. control group, ***p<0.001; between groups, ##p<0.01.

[0066] Figure 8 The effect of *Lactobacillus plantarum* NuPlus-8 on the expression of genes related to the transport and absorption of calcium, magnesium, zinc, and selenium in animal experiments; a is the calbindin-D9K gene; b is the TRPV6 gene; c is the TRPM7 gene; d is the ZnT1 gene; e is the ZIP4 gene; f is the LAT1 gene; g is the SLC26A6 gene; vs. control group, ***p<0.001; between groups, ##p<0.01.

[0067] Figure 9 The length of the rat femur was measured; a is a bar chart; b is an appearance chart; there was no significant difference in ns between groups, *p<0.05, **p<0.01.

[0068] Figure 10 Upregulation of amino acid transport-related proteins in intestinal epithelial cells by *Lactobacillus plantarum* NuPlus-8. *p<0.05, **p<0.01, ***p<0.001. Detailed Implementation

[0069] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0070] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0071] Example 1: Isolation, Identification and Domestication of Lactobacillus plantarum NuPlus-8 Lactobacillus plantarum was isolated from healthy breast milk samples. The samples were plated on MRS selective medium using a serial dilution method and anaerobically incubated at 37°C for 24 hours. Single colonies of yellowish, round, Gram-positive bacilli were picked and preliminarily identified as Lactobacillus plantarum. Subsequently, targeted acclimatization was carried out. Gastric juice acclimation: The strain was activated for three generations, centrifuged and the supernatant was discarded. The bacterial cells were washed once with PBS buffer and then resuspended in artificial gastric juice (NaCl 0.1g, pepsin 0.175g, water 50mL, fully dissolved, pH adjusted to 2.5 with dilute hydrochloric acid, mixed well and filtered through a 0.22μm filter under sterile conditions). After standing at 37℃ for 3h, 100μL of the bacterial suspension was spread on solid MRS medium and incubated at 37℃ for 24h. Strains with larger colonies were selected.

[0072] Screening for resistance to intestinal fluid: The obtained gastric fluid-resistant strains were further screened. After activation for three generations, the supernatant was discarded by centrifugation. The bacterial cells were washed once with PBS buffer and then resuspended in artificial intestinal fluid (0.68 g potassium dihydrogen phosphate, 1 g trypsin, 100 ml water, mixed and adjusted to pH 8, filtered through a 0.22 μm filter under sterile conditions). After standing at 37°C for 2 h, 100 μL of the bacterial suspension was spread on solid MRS medium and incubated at 37°C for 24 h. Strains with larger colonies were selected.

[0073] The results of gastric juice tolerance and intestinal juice tolerance screening are shown in Table 1: Table 1 Results of strains' acclimatization to gastrointestinal fluids

[0074] Low-temperature cryopreservation: Superior strains acclimatized to gastric and intestinal fluids were activated for three generations, then frozen at -80℃ for 2 hours, thawed at room temperature, and re-frozen at -80℃, repeated three times. The final thawed bacterial suspension was inoculated at 1% onto MRS liquid medium and cultured at 37℃ for 24 hours, constituting one acclimatization generation. After 30 generations of acclimatization, 100 μL of the bacterial suspension was spread onto solid MRS medium and cultured at 37℃ for 24 hours. Strains with larger colonies were selected. Bacterial powders were prepared from the original strain and acclimatized strains under the same conditions, stored at 37℃ for one month, and the survival rate was tested.

[0075] Table 2 Results of low-temperature acclimatization

[0076] As can be seen from Table 2 above, the survival rate of the strain after 30 generations of domestication is much higher than that of the original strain.

[0077] Finally, through the above domestication and screening, a strain with both high intestinal tolerance and high freeze resistance was obtained. It was identified as *Lactobacillus plantarum* by 16S rDNA sequence and named *Lactobacillus plantarum* NuPlus-8.

[0078] The 16S rRNA sequencing results are shown in SEQ ID NO.1:

[0079] Example 2: Detection of the physicochemical characteristics of the strain 2.1 Morphological and colony observation, hemolytic characteristics Pure bacterial culture was evenly spread onto a glass slide and fixed in the outer flame of an alcohol lamp. After Gram staining, the slide was slowly rinsed with deionized water to remove excess staining solution. After the slide dried, it was observed and photographed under an oil immersion microscope. One loopful of bacterial culture was streaked onto MRS agar medium using an inoculation loop. After incubation at 37°C for 24 hours, the colony morphology was observed.

[0080] The microbiological characteristics of Lactobacillus plantarum NuPlus-8 are as follows: (1) Colony morphology: such as Figure 1 As shown, the colonies grown in MRS agar medium are white with a slight yellow tinge, opaque, round, with a smooth and moist surface and neat edges.

[0081] (2) Gram staining morphology: such as Figure 2 As shown, the colonies are white, round, moist, opaque, and have neat edges.

[0082] (3) Hemolytic test: Colony characteristics on Columbia blood agar plates, such as Figure 3 The white, round colonies shown are moist with regular edges, and there is no hemolysis around the colonies.

[0083] 2.2 Utilization of different carbohydrates Preparation of colony plates: The strain is streaked on MRS agar plates. After colonies grow, single colonies are picked and streaked again until single colonies grow.

[0084] Preparation of bacterial test solution: Pick the colonies on the plate into 2 mL of physiological saline, shake and mix well, take an appropriate amount of bacterial solution (v) into 5 mL of physiological saline, measure the OD value, and then take (2v) from the original bacterial solution into 10 mL of API-matched culture medium and mix well.

[0085] Incubation reaction: Add sterile deionized water to the bottom plate of the incubation box to ensure a humid environment. Take out the test strips (0-19, 20-39, 40-49) from the packaging bag, separate them, place them in the bottom plate of the incubation box, and gently tilt them forward.

[0086] Using a pipette, draw 115 µL of bacterial culture, placing the pipette tip against the edge of the beaker to add it, avoiding air bubbles. Fill only the top of the tube (beaker) completely, and seal the top with sterile liquid paraffin to maintain an anaerobic environment. Incubate the tube at 37°C. Observe the color change of the reagent strips after 24 and 48 hours, with 48 hours as the final result. The results are shown in Table 3. *Lactobacillus plantarum* NuPlus-8 showed an absorbance of 0.90 at OD600 and could utilize 25 carbon sources after 48 hours of culture.

[0087] Table 3. Utilization of different carbohydrates by Lactobacillus plantarum NuPlus-8

[0088] Note: "V" in the table represents a variable reaction, and the results may fluctuate under different experimental conditions or batches. "+" indicates that the reaction is available, and "-" indicates that it is not available.

[0089] 2.3 Growth Curve Measurement Glyceryl spp. NuPlus-8 was inoculated into sterile MRS broth at a 1% inoculum and incubated at 37°C for 16 h. 1% of the inoculum was then transferred to a sterile 96-well plate containing 200 µl of MRS broth. After two generations of anaerobic culture at 37°C, the inoculum was again inoculated into 96-well plates containing 200 µl of MRS broth at a 1% inoculum. Each bacterium was replicated in three wells. The plates were then shaken and cultured, with OD600 measured every hour.

[0090] The results are as follows Figure 4 The results showed that *Lactobacillus plantarum* NuPlus-8 entered the logarithmic growth phase at 5 hours and the stationary phase at 15 hours, with an OD600 value of approximately 1.52 at the stationary phase.

[0091] 2.4 Antioxidant capacity NuPlus-8 cells were activated and cultured for three generations. Cell lysates and fermentation broth supernatants were prepared. The DPPH free radical scavenging capacity, hydroxyl free radical scavenging capacity, and total antioxidant capacity of the cell lysates and fermentation broth supernatants were tested using the Nanjing Jiancheng FRTP kit (catalog number: A015-3-1). The experimental procedures were performed in accordance with the kit instructions. The results are shown in Table 4.

[0092] Table 4 Antioxidant capacity of Lactobacillus plantarum NuPlus-8

[0093] 2.5 Toxicity testing and safety assessment The pathogenicity test method of food bacteria in Appendix A of GB 31615.2-2025 "National Food Safety Standard - Procedure for Safety Evaluation of Food-grade Microbial Strains" was used to test Lactobacillus plantarum NuPlus-8. The test animals showed no abnormalities or deaths, and their body weight was not statistically significant compared with the control group (p>0.05), indicating that this strain is non-pathogenic.

[0094] 2.6 Detection of ability to inhibit pathogenic bacteria Preparation of fermentation supernatant of the test bacteria: After three consecutive generations of activation, the third generation fermentation broth was centrifuged at 6000×g for 10min, the supernatant was collected, filtered through a 0.22μm micromembrane, and stored at -20℃ for later use.

[0095] After activating the pathogenic bacteria (Escherichia coli ATCC25922, Staphylococcus aureus ATCC25923, Pseudomonas aeruginosa ATCC27853, Enterococcus faecalis ATCC29212, Shigella flexneri CICC 21534, and Cronobacter sakazakii CICC 21560) three times in liquid culture medium, the third-generation culture medium was adjusted to a suitable absorbance value, so that the bacterial suspension concentration was 1×10⁻⁶. 8 CFU / mL - 5 × 10 8 CFU / mL.

[0096] Preparation of test plates: Heat and dissolve the prepared NA medium, cool to 45℃-50℃, add the prepared indicator bacterial suspension to the NA medium at an addition rate of 1%, mix thoroughly, measure 20mL and pour into a sterile Petri dish, gently shake the Petri dish to spread it evenly, and let it solidify before use.

[0097] Place 4-6 Oxford cups at equal intervals on a test plate containing indicator bacteria, press gently, and slowly add 200 μL of the fermentation supernatant of the test bacteria into the Oxford cups. Repeat each treatment three times. Place the plates in a refrigerator at 4℃-6℃ for pre-diffusion for 4-10 hours. Remove the plates and place them in a constant temperature incubator at 36℃±1℃, incubating upright until the inhibition zone is clear. Measure the diameter of the inhibition zone using calipers or an inhibition zone measuring instrument. Measure each inhibition zone three times along different directions and record the average value. Results are shown in Table 5.

[0098] Table 5. Diameter of the outer inhibition zone of *Lactobacillus plantarum* NuPlus-8 strain against pathogens.

[0099] The results showed that Lactobacillus plantarum NuPlus-8 had a good inhibitory effect on all the above pathogenic bacteria.

[0100] 2.7 Tolerance evaluation in artificial gastrointestinal acid and bile salt solutions NuPlus-8 strain was inoculated into liquid MRS and cultured overnight. 10 mL of culture medium was centrifuged at 8000 rpm for 5 min to obtain bacterial cells. The cells were washed twice with sterile PBS buffer, resuspended, and the bacterial concentration adjusted to 5 × 10⁻⁶. 8 CFU / mL. 1 mL of bacterial culture was mixed with 9 mL of simulated gastric fluid (MRS medium containing 1% pepsin, pH 2.5), simulated intestinal fluid (MRS medium containing 0.3% bovine bile salts, 1% trypsin, pH 8.0), and liquid MRS medium containing different bile salt concentrations (0, 0.1%, 0.2%, 0.3%, 0.5%). The mixtures were incubated anaerobically at 37°C. Samples were taken at different time points for plate colony counting to determine the viable count and calculate the survival rate. Survival rate = (logarithmic number of viable bacteria at sampling) / (logarithmic number of viable bacteria at h0) expressed as a percentage (%).

[0101] The results are shown in Table 6. After treatment in simulated gastric fluid for 3 hours and in intestinal fluid for 2 hours, the viable bacterial survival rates of the NuPlus-8 strain were 84.68% and 91.30%, respectively, indicating that the *Lactobacillus plantarum* NuPlus-8 strain of this invention has excellent resistance to gastric acid and intestinal fluid. The bile salt tolerance test results showed that the NuPlus-8 strain retained an 85.29% survival rate after culturing in a 0.3% bile salt solution for 3 hours. With increasing concentration, the survival rate of this strain in a 0.5% bile salt solution only decreased to 83.33%. These results demonstrate that the *Lactobacillus plantarum* NuPlus-8 strain of this invention has excellent ability to withstand the harsh environment of the gastrointestinal tract, and can successfully survive and colonize in the intestine, meeting the basic screening criteria.

[0102] Table 6. Acid-base and bile salt tolerance of Lactobacillus plantarum NuPlus-8

[0103] Example 3: Lactobacillus plantarum NuPlus-8 synergistically promotes the absorption of calcium, magnesium, zinc, and selenium in vitro. In this embodiment, an in vitro intestinal epithelial barrier model was established using the Caco-2 human colon adenocarcinoma cell line to evaluate the promoting effect of Lactobacillus plantarum NuPlus-8 on the absorption of four common minerals: calcium, magnesium, zinc, and selenium.

[0104] Caco-2 cells were passaged in a 37°C, 5% CO2 incubator. During the experiment, Caco-2 cells were seeded in Transwell chambers (polycarbonate membrane with 0.4 μm pore size). After the cells had completely fused and differentiated to form a stable monolayer epithelial barrier (usually more than 21 days), TEER (transepithelial resistance) was measured. A TEER ≥ 500 Ω·cm was observed. 2Once the integrity of tight junctions in cells is confirmed, subsequent experiments can proceed. The experiment is divided into two stages: single-element verification and mixed-element synergistic experiments.

[0105] (1) In the single-element verification stage, only one mineral solution (calcium, magnesium, zinc or selenium) was used to treat the cells each time, and a control group (mineral) and a strain treatment group (mineral + culture supernatant of Lactobacillus plantarum NuPlus-8, 10% (v / v)) were set up. During the single-element treatment, solutions containing Ca (CaCl2, final concentration 1.8mM), Mg (MgCl2, 1.0mM), Zn (ZnSO4, 50μM) or Se (SeMet 2μM or Na2SeO3 1μM, respectively) were prepared.

[0106] (2) In the mixed element synergistic experiment stage, four minerals were mixed at appropriate concentrations to form a composite solution. A control group (composite minerals) and a strain treatment group (composite minerals + culture supernatant of *Lactobacillus plantarum* NuPlus-8, 10% (v / v)) were set up respectively. The corresponding treatment solution was added to each group and treated for 2 hours. During the mixed element treatment, Ca 1.8mM, Mg 1.0mM, Zn 50μM, SeMet 2μM and Na2SeO3 1μM were added to the same working solution at the same time.

[0107] The culture supernatant of NuPlus-8 strain was obtained from its bacterial culture in MRS liquid medium for 24 hours. After sterilization by centrifugation at 8000 rpm for 10 min, the supernatant was collected and filtered through a 0.22 μm sterile filter membrane. After the experiment, the liquids from the upper and lower chambers of the Transwell were collected, and the mineral concentration was detected by inductively coupled plasma mass spectrometry (ICP-MS). The transmembrane transport and transepithelial permeability P_app were calculated to evaluate the mineral uptake efficiency.

[0108] CCK-8 experimental results showed that individual mineral elements and NuPlus-8 strain culture supernatant did not affect cell survival. Figure 5 The cell survival rate was above 85% after mixing with the element mixture or with the NuPlus-8 strain fermentation broth, ensuring that the treatment did not affect cell health. In cell experiments treated with single or mixed elements, compared with their respective control groups, *Lactobacillus plantarum* NuPlus-8 could increase the transmembrane transport of calcium, magnesium, zinc, and selenium (see Table 7); and under the mixed element background, the transmembrane transport of the above four elements by the NuPlus-8 strain was higher than that of the NuPlus-8 strain under the single element background. Similarly, the P_app transepithelial permeability index also showed a consistent trend ( Figure 6 Taking calcium as an example, compared with calcium treatment alone, the transepithelial permeability of calcium ions after treatment with NuPlus-8 strain increased from 2.9 × 10⁻⁶ to 2.9 × 10⁻⁶. 6cm / s increased to 4.8 × 10 6 Under a mixed elemental background, NuPlus-8 bacteria further increased P_app to 5.9 × 10 cm / s. 6 cm / s. Cellular transmembrane transport assays showed that *Lactobacillus plantarum* NuPlus-8 significantly enhanced the transmembrane transport and absorption of four elements: calcium, magnesium, zinc, and selenium by intestinal epithelial cells.

[0109] Table 7. In vitro transmembrane transport capacity (nmol / cm³) 2 / 2h)

[0110] qRT-PCR analysis of gene expression related to the regulation of calcium, magnesium, zinc, and selenium ion transport and absorption revealed ( Figure 7 Compared to the control group, in the experimental groups treated with *Lactobacillus plantarum* NuPlus-8 (calcium / magnesium / zinc / selenium alone + NuPlus-8 group, mixed mineral + NuPlus-8 group), the expression of calcium ion transport genes Calbindin-D9K and TRPV6 was upregulated by 1.9-2.3 times and 2.4-2.9 times, respectively; the expression of magnesium ion transport gene TRPM7 was upregulated by 1.8-2.3 times; the expression of zinc ion transport genes ZnT1 and ZIP4 was upregulated by 1.9-2.4 times and 2.2-2.5 times, respectively; and the expression of selenium transport genes LAT1 and SLC26A6 was upregulated by 1.8-2.3 times and 1.5-1.7 times, respectively. Mineral element treatment (including single-element and mixed-element treatments) also promoted the expression of corresponding element transport factors, with the gene expression increases ranging from 1.3-1.9 times, but the overall effect was significantly weaker than that of strain NuPlus-8. This indicates that the NuPlus-8 strain can regulate the intestinal mineral transport pathway to achieve synergistic absorption of multiple elements.

[0111] The results of this example show that the strain can not only promote the transport and absorption of calcium, magnesium, zinc and selenium ions individually, but also synergistically promote the absorption of the four elements.

[0112] Example 4: NuPlus-8 synergistically enhances the absorption of calcium, magnesium, zinc, and selenium minerals in vivo, promoting bone development. To further verify the promoting effect of Lactobacillus plantarum NuPlus-8 on the absorption of various minerals in vivo, animal experiments were conducted using a mouse model.

[0113] Three-week-old female C57BL / 6J mice, weighing approximately 10±1g, were selected as experimental animals. Mice were housed in an SPF-grade animal facility at a temperature maintained at 22±2℃ and a relative humidity of 50-60%, with a 12-hour light / dark cycle. Feed and water were provided freely, and no antibiotics were administered. The animals were randomly divided into four groups of 10 mice each: a control group, a mineral control group, a NuPlus-8 live bacterial suspension group, and a NuPlus-8 inactivated bacterial group. The control group was given normal feed and water. Mice in the mineral control group, NuPlus-8 live bacteria suspension group, and NuPlus-8 inactivated bacteria group were fed a mineral-fortified diet, in which calcium, magnesium, zinc, and selenium were added to the basal diet in the form of calcium carbonate, magnesium sulfate, zinc sulfate, and sodium selenite, respectively, so that the calcium content of the diet was 1.8 g / kg, magnesium content was 0.2 g / kg, zinc content was 50 mg / kg, and selenium content was 0.2 mg / kg. The NuPlus-8 live bacteria suspension group was given 10 mg / kg of Bacillus plantarum NuPlus-8 live bacteria suspension orally in addition to mineral supplementation. 8 CFU / animal / day, inactivated bacteria group, in addition to mineral supplementation, orally administered 10 doses of Bacillus plantarum NuPlus-8 inactivated bacterial suspension. 8 TFU / mouse / day, with a gavage volume of 0.2 mL each time. The entire treatment cycle lasted 6 weeks, and the weight of mice and their feed and water intake were recorded weekly. After the experiment, the mice were fasted for 12 hours and euthanized by cervical dislocation. Blood from the abdominal aorta was collected, and serum was separated. The concentrations of calcium, magnesium, zinc, and selenium in the serum were detected by atomic absorption spectrometry or ICP-MS. In addition, extracts from the small intestine, colon, and cecum were collected for the experimental detection in Example 4.

[0114] Table 8. Statistics on mineral element content in mice

[0115] The results are shown in Table 8. The serum calcium, magnesium, zinc, and selenium concentrations of the three groups of experimental mice were significantly higher than those of the blank control group, indicating that the mineral gavage was successful and could increase the element content in the blood. In the bacterial strain treatment groups (including the NuPlus-8 live bacteria group and the NuPlus-8 inactivated bacteria group), the serum calcium, magnesium, zinc, and selenium concentrations of mice were significantly higher than those of the mixed mineral group, and the effect of live bacteria was better than that of the inactivated bacteria group. After live bacteria treatment, the calcium, iron, and zinc concentrations increased by 31.07%, 21.74%, 27.62%, and 44% respectively compared with the mineral group. At the same time, the mineral content of the feces of mice in each group was detected. Table 8 shows that, contrary to the trend of serum mineral content, the amount of mineral excretion in the feces of the three groups of mice treated with minerals was higher than that of the blank control mice, presumably because the mice ingested more minerals. However, the amount of mineral excretion in the feces of mice in the bacterial strain group, especially the NuPlus-8 live bacteria treatment group, was significantly lower than that in the mineral group, and the results were not significantly different from those of the blank control.

[0116] Expression of Calbindin-D9K, TRPV6, TRPM7, ZnT1, ZIP4, LAT1, and SLC26A6 genes in mouse small intestinal tissue showed that ( Figure 8 Compared with the control group, the expression levels of the above genes were higher in the treatment group after adding live Lactobacillus plantarum NuPlus-8 bacteria (p<0.01). The results of the inactivated NuPlus-8 bacteria treatment were weaker than those of the live bacteria treatment and similar to those of the mineral treatment group. Specifically, after live bacteria treatment, the calcium ion-related transport genes Calbindin-D9K and TRPV6 were upregulated by about 2.3-fold and about 2.8-fold, respectively; the magnesium ion-related transport gene TRPM7 was upregulated by about 2.2-fold; the zinc ion-related transport genes ZnT1 and ZIP4 were upregulated by about 2.3-fold and about 2.8-fold, respectively; and the selenium-related transport genes LAT1 and SLC26A6 were upregulated by about 2.2-fold and about 2.4-fold, respectively.

[0117] Take the femur of a mouse and measure its length ( Figure 9 The results showed that the femur length in the NuPlus-8 live bacterial suspension group was significantly longer than that in the control group (approximately 5.46%), with a statistically significant difference (p < 0.01). The NuPlus-8 inactivated group also showed a significant difference compared to the control group (p < 0.05). The average femur length in the mineral control group was slightly longer than that in the control group, but the difference was not statistically significant.

[0118] Animal experiments have shown that Lactobacillus plantarum NuPlus-8 can significantly promote the absorption of minerals such as calcium, magnesium, zinc, and selenium in vivo, reduce the excretion of minerals in feces, and improve the absorption rate of mineral elements, thus having a significant promoting effect on bone development. The results of this embodiment provide experimental evidence for its application in the development of functional microbial agents, compound mineral supplements, and products that promote bone development.

[0119] Example 5: NuPlus-8 exhibits high phytase activity, promoting the release of various minerals. Phytic acid is the main form of phosphorus storage in plant seeds (grains, legumes). It chelates minerals (Ca, Mg, Zn, Se, and Fe, etc.) to form insoluble complexes, thereby affecting the absorption and utilization of mineral elements. This experiment can evaluate whether strain NuPlus-8 has phytase activity under intestinal-related pH conditions and whether it can release bound mineral elements by degrading phytic acid, thereby improving mineral availability.

[0120] The supernatant of *Lactobacillus plantarum* NuPlus-8 culture, live NuPlus-8 bacterial suspension, and heat-inactivated NuPlus-8 bacterial suspension of this invention were used as enzyme sources for phytase activity detection. Different buffer systems were prepared, including citrate-phosphate buffer (pH 2.0-4.0), acetate buffer (pH 4.5-5.5), MES (pH 6.0), and HEPES (pH 7.0-7.5). In the experiment, 5 mM sodium phytate was used as the substrate. Various bacterial samples were reacted in the buffer at 37°C for 30 min. After terminating the reaction, the amount of inorganic phosphorus released was detected using the Malachite Green colorimetric method to evaluate phytase activity. Commercially available phytase was used as a positive control, and blank culture medium was used as a negative control.

[0121] To assess the potential role of this strain in mineral uptake, a mixed solution containing 1 mM phytic acid and minerals (prepared in a phosphorus-free buffer (HEPES-NaCl, pH 7.4): sodium phytate 1 mM, CaCl2 1.8 mM, MgCl2 1.0 mM, ZnSO4 50 μM, Na2SeO3 1 μM, mixed and allowed to stand) was added to blank culture medium, NuPlus-8 culture supernatant, NuPlus-8 live bacterial suspension, and NuPlus-8 heat-inactivated bacterial suspension, and treated with commercial phytase. The mixture was incubated at 37 °C, and samples were taken after 12 h. The free mineral ion content was quantitatively determined by ICP-MS. In addition to Ca / Mg / Zn / Se mentioned above, Fe, which is essential for the human body, is often chelated by phytic acid to form insoluble complexes. Therefore, the mineral mixed solution in this experiment contained all five elements mentioned above, and the Fe solution was prepared using 100 µM FeCl3.

[0122] Table 9 shows that both the culture supernatant and live bacterial suspension of *Lactobacillus plantarum* NuPlus-8 significantly hydrolyzed and released inorganic phosphorus under four different acid-base conditions. The inorganic phosphorus levels at pH 4.5-5.5 and pH 6.0 were significantly higher than those at pH 2.0-4.0 and pH 7.0-7.5, indicating that the first two pH environments were close to the optimal acid-base conditions for enzyme activity. The NuPlus-8 strain maintained strong enzyme activity under simulated gastric and intestinal fluid acid-base conditions, with the inorganic phosphorus release from the NuPlus-8 supernatant group only slightly lower than that from commercial phytase. The live bacterial suspension was slightly less effective than the supernatant, while no significant activity was detected in the heat-inactivated bacterial suspension group, suggesting that phytase activity may have been lost due to heat treatment.

[0123] Table 9. Results of phytase activity assay

[0124] In the in vitro complex degradation experiment (Table 10), after treatment with NuPlus-8 strain (including three sample types: live bacterial suspension, inactivated suspension, and culture supernatant), the concentrations of free calcium, magnesium, zinc, and selenium ions all increased compared to the blank control group. The fermentation supernatant showed the highest increase, followed by the live bacterial suspension, while the inactivated bacterial suspension showed the weakest effect, only slightly higher than the blank group. Among them, in the experimental group with added supernatant, the concentrations of calcium, magnesium, zinc, selenium, and iron ions increased by 35.2%, 36.7%, 45.6%, 25%, and 45.8% respectively after 12 h compared to the control, which was comparable to the effect of commercial phytase.

[0125] Table 10 Mineral Release Concentration (μM)

[0126] The results of this embodiment demonstrate that *Lactobacillus plantarum* NuPlus-8 possesses phytase activity, which not only promotes the release of inorganic phosphorus and improves phosphorus bioavailability, but also degrades phytic acid-mineral complexes, releasing bound mineral ions, thereby improving the bioavailability of elements such as calcium, magnesium, zinc, selenium, and iron. This discovery reveals an important mechanistic basis for *Lactobacillus plantarum* NuPlus-8's promotion of mineral absorption.

[0127] Example 6: NuPlus-8 promotes the absorption of fat-soluble vitamin D. The above embodiments show that the *Lactobacillus plantarum* NuPlus-8 of the present invention can promote the absorption of calcium ions. Considering the promoting effect of vitamin D on calcium absorption, this embodiment tests whether the NuPlus-8 strain can promote the absorption and utilization of calcium ions by promoting the absorption of fat-soluble vitamin D.

[0128] This experiment used a human Caco-2 cell Transwell monolayer model to verify the promoting effect of Lactobacillus plantarum NuPlus-8 fermentation supernatant on the transepithelial uptake of vitamin D3 (cholecalciferol, hereinafter referred to as VD). Caco-2 cells were seeded on a 12-well Transwell scaffold (pore area approximately 1.12 cm²). 2The upper chamber had a volume of 0.5 mL, and the lower chamber had a volume of 1.5 mL. Vitamin D was first dissolved in a small amount of ethanol and then added to a standard mixed micelle solution (composition: sodium taurocholate 5 mM, phospholipids 0.5 mM, oil phase simulant 0.5 mM, HEPES-NaCl 10 mM, pH 7.4) to initially set the VD concentration in the upper chamber to 2 μM. The experimental group had *Lactobacillus plantarum* NuPlus-8 fermentation supernatant (10% v / v) added to the mixed micelles; the negative control did not include supernatant. After adding the treatment solution to the upper chamber and incubating for 2 h, 200 μL of liquid was removed from the lower chamber. The VD level was then quantitatively detected using LC-MS / MS, and the transmembrane permeability P_app was calculated.

[0129] The results are shown in Table 11. Under the 2-hour incubation condition, the P_app of VD3 in the control group was 1.0 × 10⁻⁶. -6 The P_app was 1.8 × 10 cm / s (total transmembrane volume 6.2 ng / well), while it increased to 1.8 × 10 cm / s after treatment with the fermentation supernatant of Lactobacillus plantarum NuPlus-8. -6 cm / s (total transmembrane absorption of approximately 11 ng / well), representing an increase of approximately 80%. This experiment demonstrates that *Lactobacillus plantarum* NuPlus-8 can directly promote the transmembrane absorption of vitamin D3 in an intestinal epithelial model, providing a biological basis for the role of NuPlus-8 in promoting calcium absorption.

[0130] Table 11. Vitamin D epithelial absorption assay

[0131] Example 7: Lactobacillus plantarum NuPlus-8 promotes amino acid uptake in Caco-2 cells. A human Caco-2 cell Transwell monolayer model was constructed following the methods described in the above embodiments. The experiment was divided into a blank control group and a *Lactobacillus plantarum* NuPlus-8 treatment group (supernatant, 10% v / v). Samples were added to the upper chamber of Caco-2 cells and co-cultured for 24 h. An equal volume of PBS was added to the control group. FITC-labeled L-lysine was added to the upper chamber of the cells as a tracer (final concentration 100 μM). After incubation for 30 min, 60 min, and 120 min, the lower chamber culture medium was collected, and the fluorescence intensity (excitation 485 nm, emission 520 nm) was detected using a fluorescence microplate reader. The amino acid fluorescence permeability was calculated. Simultaneously, cells were collected, and the expression of amino acid transporter-related genes, including SLC6A19 (neutral amino acid transporter), SLC38A2 (neutral amino acid sodium-dependent transporter), PEPT1 (small peptide transporter), and EAAT3 (glutamate transporter), was detected.

[0132] Table 12 Fluorescent amino acid permeability (%)

[0133] According to Table 12, compared with the blank control group, the *Lactobacillus plantarum* NuPlus-8 treatment group showed higher fluorescence permeability in the amino acid uptake experiment, and the permeability gradually increased with time, from 11±2% (30 min) to 36±4% (120 min), indicating that it can significantly enhance the transepithelial uptake of amino acids. qPCR results showed ( Figure 10 In the *Lactobacillus plantarum* NuPlus-8 group, the expression levels of SLC6A19, SLC38A2, PEPT1, and EAAT3 genes were all upregulated compared to the control group, with increases of approximately 1.8-fold, 1.5-fold, 2.4-fold, and 1.2-fold, respectively. These results indicate that *Lactobacillus plantarum* NuPlus-8 can enhance the cellular absorption capacity of amino acids and dipeptides by upregulating the expression of multiple amino acids and dipeptide transporters in intestinal epithelial cells, thereby promoting the absorption of amino acids and proteins.

[0134] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A Lactiplantibacillus (Lactobacillus) plantarum strain, characterized in that, Lactiplantibacillus plantarum The Lactobacillus plantarum mentioned is Lactobacillus plantarum NuPlus-8, with the accession number CGMCC No. 36325. ​ 2. An inoculant characterized in that, The bacterial agent comprises *Lactobacillus plantarum* as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The microbial agent includes any one or more of the following: fermentation broth of *Lactobacillus plantarum*, fermentation broth precipitate, fermentation broth supernatant, live bacterial cells, inactivated bacterial cells, lyophilized powder, lysate, lysate, secondary metabolites, and exosomes.

4. The use of the *Lactobacillus plantarum* as described in claim 1 or the bacterial agent as described in any one of claims 2-3 in the preparation of products that promote nutrient absorption.

5. The application according to claim 4, characterized in that, The aforementioned products that promote nutrient absorption include any one or more of the following: (1) Products that promote mineral absorption; (2) Products that promote vitamin absorption; (3) Products that promote the absorption of amino acids and / or proteins; (4) Products that promote bone growth and development.

6. The application according to claim 5, characterized in that, The product that promotes nutrient absorption has any one or more of the following effects: (1) By upregulating the expression of mineral transport protein genes in intestinal epithelial cells, the transmembrane transport rate and bioavailability of minerals are increased, and mineral excretion is reduced; (2) By secreting phytase, phytic acid is degraded within the pH range of the gastrointestinal tract, releasing mineral ions chelated by phytic acid, thereby improving the availability of minerals; (3) Enhances the transmembrane absorption of vitamins in intestinal epithelial cells and promotes the absorption of minerals; (4) By upregulating the expression of amino acid transport proteins in intestinal epithelial cells, the absorption capacity of amino acids and / or proteins is enhanced; (5) Promotes the absorption and utilization of key osteogenic minerals and supports bone growth.

7. The application according to claim 5, characterized in that, The minerals mentioned include any one or more of calcium, magnesium, zinc, selenium, iron, and phosphorus.

8. A product that promotes the absorption of nutrients, characterized in that, The product comprises *Lactobacillus plantarum* as described in claim 1 or the bacterial agent as described in any one of claims 2-3.

9. The product according to claim 8, characterized in that, The products contain at least 1 x 10 8 CFU of Lactobacillus plantarum.

10. The product according to claim 8, characterized in that, The dosage form of the product includes solid dosage form, semi-solid dosage form or liquid dosage form; the product also includes excipients, which include any one or more of the following: diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, and buffer.

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