A milk protein hydrolysate based on synergistic hydrolysis of enzymes and lactic acid bacteria, its preparation method and application in anti-osteoporosis

Abstract

The application discloses a milk protein hydrolysate based on enzyme and lactic acid bacteria synergistic hydrolysis, a preparation method thereof and application thereof in anti-osteoporosis. The method adopts an enzymolysis process to perform primary hydrolysis on milk protein, and then inoculates specific lactic acid bacteria to perform secondary hydrolysis. Experimental results show that, compared with single enzymolysis, the combined treatment reduces the molecular weight of the product, increases the proportion of small molecule peptides, and significantly enhances the activity of promoting osteoblast cell proliferation. The hydrolysate can relieve the damage of dexamethasone to osteoblast cells. In an in-vivo osteoporosis model, the hydrolysate shows the effect of improving bone metabolism related indexes, and has the potential of relieving osteoporosis. The method has controllable process and wide raw material sources, and the obtained milk protein hydrolysate has good application prospect in functional food, nutritional intervention and bone health related products. The application provides a natural active substance with wide sources and low price and a preparation technical path for relieving and treating osteoporosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a milk protein hydrolysate based on the synergistic hydrolysis of enzymes and lactic acid bacteria, its preparation method, and its application in anti-osteoporosis. Background Technology

[0002] Osteoporosis is a skeletal disease characterized by decreased bone mass, deterioration of bone microstructure, and increased bone fragility, and is particularly common in the elderly and postmenopausal women. The occurrence of osteoporosis is often closely related to bone metabolism disorders. Bone metabolism includes two key processes: bone formation and bone resorption. When the rate of bone resorption exceeds the rate of bone formation, it leads to an imbalance in bone remodeling, resulting in bone loss, decreased bone density, and ultimately osteoporosis. However, currently available osteoporosis medications can cause side effects with long-term use, such as gastrointestinal discomfort and cardiovascular risks. Therefore, developing bioactive substances with higher safety profiles suitable for long-term intake has become a research hotspot.

[0003] Food-derived bioactive peptides have attracted widespread attention due to their safe origin, low toxicity, and easy digestibility and absorption. Various protein peptides have been reported to play a positive role in bone metabolism regulation. Milk protein is an excellent source of protein and is rich in various amino acids. Milk protein peptides have been proven to possess a variety of biological activities, including antioxidant, blood pressure-lowering, immunomodulatory, and anti-inflammatory effects.

[0004] Enzymatic hydrolysis is currently the most commonly used method for producing peptides, characterized by mild reaction conditions and rapid reaction rates. However, protein hydrolysis can lead to bitterness, primarily due to the release of peptides containing hydrophobic amino acid residues. Microbial fermentation using lactic acid bacteria to produce endonucleases and exonucleases can further hydrolyze bitter peptides, reducing bitterness and increasing the content of free amino acids. However, because lactic acid bacteria have relatively low protease activity, the degree of protein hydrolysis during microbial fermentation alone is low. Therefore, a combined enzyme-lactic acid bacteria fermentation method can release more bioactive peptides from milk proteins with minimal impact on flavor.

[0005] Currently, research on the bone health effects of milk protein hydrolysates mainly focuses on fermented milk and the isolation and identification of specific functional peptides, while research on the anti-osteoporosis effects of whole milk protein hydrolysates remains relatively limited. Summary of the Invention

[0006] The purpose of this invention is to address the above-mentioned problems by providing a milk protein hydrolysate based on the synergistic hydrolysis of enzymes and lactic acid bacteria, its preparation method, and its application in the treatment of osteoporosis.

[0007] To achieve its objective, the present invention employs the following technical solution:

[0008] The first aspect of the present invention provides a strain of *Limosilactobacillus fermentum* DACN204, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026612.

[0009] A second aspect of the present invention provides a method for preparing milk protein hydrolysates based on the synergistic hydrolysis of enzymes and lactic acid bacteria, comprising the following steps:

[0010] (1) Single hydrolysis-enzymatic hydrolysis

[0011] Milk protein was dispersed in water, and flavor protease was added for enzymatic hydrolysis. After the reaction was completed, the enzyme was inactivated, the supernatant was collected by centrifugation, the pH was adjusted to 6.8-7.2, and the mixture was freeze-dried to obtain the freeze-dried powder of milk protein hydrolysis product.

[0012] (2) Secondary hydrolysis -- lactic acid bacteria fermentation enzymatic hydrolysis products

[0013] The lyophilized powder of the enzymatic hydrolysis product obtained in step (1) is dispersed in water, sterilized, inoculated with Lactobacillus fermentum DACN204, fermented, centrifuged after fermentation, the supernatant is collected, filtered through a filter membrane to remove residual bacteria and cell debris, and then freeze-dried to obtain the fermented milk protein hydrolysate.

[0014] Preferably, in step (1), the milk protein powder is dispersed in ultrapure water at a ratio of 2-6% (w / v), and flavor protease at a mass of 3-9% of the milk protein powder is added. The pH of the solution is adjusted to 7.2-7.8, and enzymatic hydrolysis is performed at 47-53°C for 2-5 hours.

[0015] More preferably, in step (1), the milk protein powder is dispersed in ultrapure water at a ratio of 3-5% (w / v), and flavor protease is added at a mass of 5-7% (preferably 7%) of the milk protein powder. The pH of the solution is adjusted to 7.4-7.6 (preferably 7.5), and the solution is enzymatically hydrolyzed at 49-51℃ (preferably 50℃) for 3-5 h (preferably 4 h).

[0016] Preferably, in step (2), the lyophilized powder of the enzymatic hydrolysis product obtained in step (1) is dispersed in deionized water at a ratio of (8~12) g / 100 mL, sterilized, inoculated with Lactobacillus fermentum DACN204 at an inoculation amount of 1.8~2.2%, fermented at 36~38℃ for 22~28 h, centrifuged after fermentation, the supernatant is collected, filtered through a filter membrane, and freeze-dried to obtain fermented milk protein hydrolysate.

[0017] A third aspect of the present invention provides a fermented milk protein hydrolysate prepared by any of the preparation methods described above.

[0018] A fourth aspect of the present invention provides the use of the above-described fermented milk protein hydrolysate in the preparation of a medicament for treating osteoporosis.

[0019] The fifth aspect of the present invention provides the use of the above-described fermented milk protein hydrolysate in the preparation of health foods that improve bone density.

[0020] A sixth aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of osteoporosis, comprising an effective dose of the above-described fermented milk protein hydrolysate and a pharmaceutically acceptable carrier; preferably, the pharmaceutically acceptable carrier comprises one or more of a filler, wetting agent, disintegrant, binder or lubricant.

[0021] A seventh aspect of the present invention provides a health food for improving bone density, comprising the above-mentioned fermented milk protein hydrolysate and food science acceptable excipients.

[0022] The beneficial effects of this invention are:

[0023] This invention provides a method for the synergistic hydrolysis of milk protein using enzymes and lactic acid bacteria, yielding a milk protein hydrolysate that can exert bone health benefits without the need to separate a single active peptide. The method employs an enzymatic hydrolysis process for the initial hydrolysis of milk protein, followed by a secondary hydrolysis using specific lactic acid bacteria. Experimental results show that the synergistic hydrolysis by enzymes and lactic acid bacteria produces a milk protein hydrolysate that differs from single enzymatic hydrolysis or single fermentation. Compared to single enzymatic hydrolysis, the combined treatment reduces the molecular weight of the product, increases the proportion of small peptides, significantly enhances osteoblast proliferation activity, and the hydrolysate can alleviate the damage to osteoblasts caused by dexamethasone. In an in vivo osteoporosis model, the hydrolysate exhibits the effect of improving bone metabolism-related indicators, showing potential for alleviating osteoporosis. This invention's method offers controllable processes and widely available raw materials, and the resulting milk protein hydrolysate shows promising application prospects in functional foods, nutritional interventions, and bone health-related products. This invention provides a widely available and inexpensive natural active substance and preparation technology pathway for the alleviation and treatment of osteoporosis. Attached Figure Description

[0024] Figure 1 To determine the degree of hydrolysis of milk protein under different conditions, (A) different types of enzymes, (B) different feed-to-liquid ratios, (C) different hydrolysis times, and (D) different amounts of enzyme added.

[0025] Figure 2 To illustrate the effect of different lactic acid bacteria fermentation products on the proliferation rate of MC3T3-E1 osteoblasts, 1-21 in the figure represents L-MPH prepared by fermenting MPH by strains numbered 1-21.

[0026] Figure 3The effect of different concentrations of FMPH treatment on the survival rate of MC3T3-E1 osteoblasts.

[0027] Figure 4 The images show the colony morphology (A) and microscopic morphology (B) of DACN204.

[0028] Figure 5 The effects of different concentrations of FMPH treatment for 24 h (A) and 48 h (B) on improving Dex-damaged osteoblasts.

[0029] Figure 6 The osteoproliferative activity of FMPH digestion products (A), and their ability to resist dexamethasone damage at 24 h (B) and 48 h (C).

[0030] Figure 7 This represents the molecular weight distribution of peptides in FMPH.

[0031] Figure 8 The weekly weight change of mice (A) and the final weight of mice (B) are shown.

[0032] Figure 9 The effects of FMPH on organ indices in mice: (A) liver index, (B) kidney index, (C) spleen index.

[0033] Figure 10 The effects of FMPH on mouse serum markers: (A) ALP, (B) Tracp-5b, (C) CTX-I.

[0034] Figure 11 To illustrate the effects of FMPH on bone microstructure in mice, (A) representative 2D and 3D images of femurs from each group using μCT, and (B) quantitative analysis of bone microstructure indices.

[0035] Figure 12 H&E staining of mouse femur.

[0036] Figure 13 Trap staining of mouse femur (A) and its quantitative analysis (B).

[0037] Figure 14 The effect of FMPH on the expression of bone formation-related genes in mice.

[0038] Figure 15 To investigate the effect of FMPH on the expression of osteogenic-related proteins in the mouse femur, (A) immunofluorescence detection of ALP protein, (B) relative fluorescence intensity of ALP protein, (C) immunofluorescence detection of OCN protein, (D) relative fluorescence intensity of OCN protein, (E) immunofluorescence detection of Runx2 protein, and (F) relative fluorescence intensity of Runx2 protein.

[0039] Figure 16 The effect of FMPH on the expression of genes related to the RANKL / OPG signaling pathway in mice.

[0040] Figure 17 To illustrate the effect of FMPH on RANKL protein expression in mouse femur, (A) immunofluorescence detection of RANKL protein, (B) relative fluorescence intensity of RANKL protein.

[0041] Figure 18 For key species analysis at the genus level, (A) correlation analysis between osteoporosis-related indicators and the top 20 species in terms of gut microbiota abundance, and (B) significance analysis of strains. Detailed Implementation

[0042] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0043] Unless otherwise specified, the experimental methods in the following examples are conventional methods; unless otherwise specified, the biological and chemical reagents used are conventional reagents in the art and can be obtained commercially.

[0044] Example 1: Preparation of milk protein hydrolysate by enzyme-fermentation method

[0045] 1. Experimental Methods

[0046] 1.1. Screening for suitable proteases

[0047] Milk protein powder (containing 80 wt% casein and 20 wt% whey protein) was dispersed in ultrapure water at a ratio of 5% (wt / vol). After complete dissolution, the pH of the solution was adjusted to the optimal pH for the enzyme, and the temperature was maintained at the optimal temperature for the enzyme. Alkaline protease, flavor protease, papain, trypsin, and neutral protease were added for hydrolysis for 3 h (enzyme to substrate ratio of 3%, wt / wt). The pH and temperature of the system were kept constant during hydrolysis, and the mixture was stirred continuously. After hydrolysis, the sample was heated in an 85℃ water bath for 20 min to inactivate the enzyme. The sample was centrifuged (2000 g, 20 min, 4℃), and the supernatant was collected. The pH of the solution was adjusted to 7.0, and the solution was freeze-dried under vacuum to obtain a lyophilized powder of the enzymatic hydrolysis product, which was stored at -20℃ for later use. The degree of hydrolysis of different enzymatic hydrolysis products was determined by the OPA method (Nielsen PM, Petersen D, Dambmann C. Improved method for determining food protein degree of hydrolysis [J]. Journal of Food Science, 2001, 66(5): 642-646) in order to screen suitable enzymatic hydrolytic proteases.

[0048] Table 1 Optimal conditions for enzymes

[0049]

[0050] 1.2. Single-factor experiments on enzymatic hydrolysis process

[0051] In experiments involving multiple factors, to investigate the impact of a single factor on the experiment, the degree of hydrolysis was determined using the OPA method to select suitable enzymatic hydrolysis conditions. This experiment examined the effects of three single factors—the material-to-liquid ratio, the amount of enzyme added, and the hydrolysis time—on the enzymatic hydrolysis of milk proteins under different experimental conditions. The product obtained from the optimal enzymatic hydrolysis process was named lactase hydrolysate (MPH).

[0052] 1.3. Lactic acid bacteria fermentation MPH

[0053] Disperse MPH lyophilized powder in deionized water at a ratio of 10 g / 100 mL (wt / vol) and sterilize at 115℃ for 15 min. Inoculate with bacterial cells at a ratio of 2% (vol / vol) (10 g / 100 mL ... 8 Fermentation was carried out at 37℃ for 24 h (CFU / mL). Sterile deionized water was used as a control instead of bacterial cells. After fermentation, the mixture was centrifuged (5000g, 20 min) to remove precipitate, filtered through a 0.22 μm filter membrane, and the fermentation supernatant was obtained. The supernatant was freeze-dried to obtain lactic acid bacteria fermented MPH lyophilized powder (L-MPH), and stored at -20℃. The osteocyte proliferation-promoting activity of different fermentation products was determined to screen suitable lactic acid bacteria. The fermentation product with the strongest osteocyte proliferation-promoting activity was named fermented milk protein hydrolysate (FMPH).

[0054] Table 2 Information on experimental strains

[0055]

[0056] 1.4. MTT assay for osteoblast proliferation-promoting activity

[0057] Take MC3T3-E1 osteoblasts in logarithmic growth, digest the cells, resuspend them in complete culture medium, count them, and adjust the cell suspension concentration to 5 × 10⁻⁶. 4Cells / mL were added to each well of a 96-well plate with 100 μL of cell suspension, while the control group received 100 μL of cell-free culture medium. After incubation at 37°C for 12 h, the culture medium was discarded. 100 μL of complete culture medium was added to each well of the control and control groups, while 100 μL of complete culture medium containing different lactic acid bacteria fermented MPH lyophilized powder L-MPH (1000 μg / mL) was added to each sample group. Incubation was continued for 48 h. The supernatant was discarded, and the sample was washed twice with PBS. 90 μL of basal culture medium and 10 μL of 5 mg / mL MTT solution were added to each well. After incubation at 37°C for 4 h, the culture medium and MTT in each well were removed, and 150 μL of dimethyl sulfoxide (DMSO) was added. The absorbance was then measured at 490 nm using a microplate reader. FMPH, the fermentation product with the strongest osteoblast proliferation-promoting activity, was obtained.

[0058]

[0059] 1.5. Effects of different concentrations of FMPH on osteoblast proliferation activity

[0060] Logarithmic osteoblasts (MC3T3-E1) were harvested, resuspended in complete culture medium, and counted. They were then seeded at a density of 5000 cells / well in 96-well plates. After incubation at 37°C for 12 h, osteoblasts were treated with different amounts of FMPH for 48 h. The final concentrations of FMPH in each well were 4000, 2000, 1000, 500, 250, and 125 μg / mL. Cell viability was determined using the MTT assay after cell culture. Each experiment was performed in five replicates.

[0061] 1.6. Effects of different concentrations of FMPH on osteoblast survival rate after dexamethasone injury

[0062] Logarithmic osteoblasts (MC3T3-E1) were harvested, resuspended, and counted. They were seeded at a density of 8000 cells / well in 96-well plates and cultured at 37°C for 12 h. The cells were then divided into three groups: a control group (chosen in complete culture medium for 24 h and 48 h); a model group (chosen in complete culture medium containing dexamethasone (final concentration 40 μmol / L per well) for 24 h and 48 h); and a sample group (chosen in complete culture medium containing dexamethasone (40 μmol / L) and different concentrations of FMPH (4000, 2000, 1000, 500, 250, and 125 μg / mL, respectively, for 24 h and 48 h). Cell viability was determined using the MTT assay after culture. Each experiment had five replicates.

[0063] 1.7. Simulating gastrointestinal digestion

[0064] 100 mg of FMPH lyophilized powder was weighed and resuspended in 10 mL of simulated gastric juice (SGF, 0.2% NaCl solution). The pH was adjusted to 2.0 using 1 mol / L HCl, and pepsin was added to the mixture at an enzyme-substrate ratio of 1:35 (w / w). The mixture was incubated at 37 °C with shaking (150 rpm / min) for 2 h. After gastric digestion, the pH was adjusted to 7.0 using 1 mol / L NaOH, and an equal volume (10 mL) of simulated intestinal fluid (SIF, 0.05 mol / L KH2PO4) was added. Trypsin was then added at an enzyme-substrate ratio of 1:25 (w / w), and the mixture was incubated for another 2 h under the same conditions. The reaction was terminated at 85 °C for 15 min. The mixture was centrifuged at 8000 g for 20 min at 4 °C, and the supernatant was lyophilized to obtain simulated gastrointestinal digest. The osteocyte proliferation-promoting activity of each component was determined using the MTT assay.

[0065] 1.8. Determination of molecular weight distribution

[0066] The molecular weight distribution of the samples was determined using an Agilent 1260 Infinity II GPC / SEC system equipped with a refractive index detector (RID). The chromatographic parameters were as follows: Ultrahydrogel tandem column (7.850 mm, 10 μm; 7.8300 mm, 10 μm); solvent flow rate 1 mL / min; test temperature 45℃; injection volume 50 μL; solvent: 0.1M NaNO3; standard sample: PEO / PEG.

[0067] 1.9. Determination of amino acid composition

[0068] The composition of hydrolyzed amino acids in FMPH was determined using ultra-high performance liquid chromatography-tandem high-resolution Orbitrap mass spectrometry.

[0069] 1.10. Data Statistics and Analysis

[0070] Data significance analysis was performed using the Tukey method in one-way ANOVA (P < 0.05) in SPSS 24.0 software, and graphs were plotted using Graphpad 7.0. Experimental results are expressed as mean ± standard deviation, and each experiment was performed in triplicate.

[0071] 2. Results and Analysis

[0072] 2.1. Lactase hydrolysis process

[0073] like Figure 1As shown, based on the changes in the degree of hydrolysis under various conditions, the optimal enzymatic hydrolysis process conditions for milk protein are: flavor protease as the enzyme type, a material-to-liquid ratio (i.e., the ratio of milk protein powder to ultrapure water) of 4% (wt / vol), an enzyme addition of 7%, and a hydrolysis time of 4 h.

[0074] 2.2. Screening for the most suitable fermentation strain

[0075] like Figure 2 As shown, the sample fermented by strain No. 2 exhibited the strongest osteocyte proliferation-promoting ability, with a cell viability rate reaching 121.19%. Therefore, strain No. 2 was selected as the subsequent fermentation strain, and its fermentation hydrolysate was named FMPH. Compared with MPH, FMPH significantly enhanced the osteocyte proliferation-promoting ability. Further investigation was conducted into the osteocyte proliferation-promoting activity of different concentrations of FMPH, such as... Figure 3 As shown, FMPH at concentrations of 250–4000 μg / mL significantly enhanced the ability to promote osteocyte proliferation (p < 0.05), with the highest cell survival rate observed at 4000 μg / mL FMPH intervention, reaching 135.72%.

[0076] Strain number 2 is *Lactobacillus fermentatus* (DACN204), and its identification information is as follows:

[0077] 1. Morphological identification

[0078] Morphological identification revealed that the target strain formed round, white, smooth colonies on solid culture medium. Microscopic observation showed that the bacteria were short rod-shaped, consistent with the morphological characteristics of Lactobacillus. The colony morphology and microscopic morphology of DACN204 are shown below. Figure 4 A and Figure 4 As shown in B.

[0079] 2. Molecular identification

[0080] The 16S rDNA gene sequencing results were identified by homology comparison analysis in the GenBank database using NCBI. The analysis showed that it had 99.93% homology with *Lactobacillus fermentum* 5LC (GenBank accession number: PX486746.1) in the database. The bidirectional sequence of the 16S rDNA gene amplification product of *Lactobacillus fermentum* DACN204 is shown in SEQ ID NO. 1.

[0081]

[0082] The preservation information for *Lactobacillus fermentatus* DACN204 is as follows:

[0083] The strain DACN204 was deposited at the China Center for Type Culture Collection (CCTCC) in April 2026. The address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposit date is April 7, 2026. The accession number is CCTCC NO: M 2026612. The classification name is: Limosilactobacillusfermentum DACN204.

[0084] 2.3. The anti-dexamethasone damage effect of FMPH

[0085] like Figure 5 As shown, when the treatment time was 24 h, FMPH concentrations in the range of 125–4000 μg / mL significantly alleviated Dex-induced osteoblast damage (p < 0.05). When the treatment time was 48 h, FMPH concentrations in the range of 1000–4000 μg / mL significantly alleviated Dex-induced osteoblast damage (p < 0.05).

[0086] 2.4. Effects of in vitro simulated gastrointestinal digestion on FMPH activity

[0087] like Figure 6 As shown, after simulated gastrointestinal digestion, although the bone proliferation-promoting and anti-Dex injury capabilities of FMPH decreased, they still maintained significant activity (p < 0.05), indicating that FMPH has good digestive stability in the gastrointestinal environment and can maintain certain functional activity during in vivo digestion.

[0088] 2.5. Molecular weight distribution

[0089] like Figure 7 As shown, the total percentage of components with a molecular weight <1 kDa in FMPH was 50.63%, higher than that in MPH (38.91%). The percentage of components with a molecular weight in the 1-2 kDa range in FMPH was 30.53%, lower than that in MPH (40.02%). These results indicate that, compared to MPH obtained through single enzymatic hydrolysis, FMPH obtained via enzyme-fermentation produces more small peptides and can further hydrolyze 1-2 kDa peptides into small peptides <1 kDa.

[0090] 2.6. Analysis of hydrolyzed amino acids

[0091] Table 3 shows the content and proportion of 24 amino acids. Compared with milk protein and MPH, FMPH shows an increase in the content and proportion of several amino acids, including L-histidine, L-arginine, L-asparagine, L-serine, glycine, and L-aspartic acid. Specifically, the content of L-histidine increased from 500.862 μg / g in milk protein and 782.106 μg / g in MPH to 1446.621 μg / g; L-arginine increased from 104.791 μg / g and 106.730 μg / g to 294.607 μg / g; L-glutamic acid increased from 168.938 μg / g and 196.239 μg / g to 274.686 μg / g; and L-leucine increased from 184.138 μg / g and 222.062 μg / g to 293.679 μg / g. FMPH-enriched amino acids, such as arginine, leucine, and glutamic acid, are believed to be related to bone metabolism regulation: Jennings et al. [Reference 1] found that the intake of alanine, arginine, glutamic acid, leucine, lysine, and proline was associated with increased bone mineral density in the spine and forearm of women. In addition, arginine is believed to have important effects on osteoblast differentiation, osteoclast activation, immune response, and angiogenesis [Reference 2].

[0092] References:

[0093] [Literature 1] Jennings A, MacGregor A, Spector T, et al. Amino acid intakes are associated with bone mineral density and prevalence of low bone mass in women: Evidence from discordant monozygotic twins [J]. Journal of Bone and Mineral Research, 2016, 31(2): 326-335.

[0094] [Literature 2] Wang SB, Ren Q, Huang YS, et al. Emerging roles of argininemetabolism in skeletal health and disease [J]. Metabolism, 2026, 175: 156451.

[0095] Table 3. Composition of hydrolyzed amino acids

[0096]

[0097] Example 2: The ameliorative effect and mechanism of FMPH on Dex-induced osteoporosis in mice.

[0098] 1. Experimental Methods

[0099] 1.1. Animal husbandry and model making

[0100] Male C57BL / 6J mice (8 weeks old) were housed under standard feeding conditions in laboratory cages at a temperature of 24-26℃ and a humidity of 50-60%, with a 12-hour dark / light cycle. After one week of acclimatization, 32 mice were randomly divided into 4 groups: (1) Control group (n=6): mice were injected intraperitoneally with saline every other day for 4 weeks and administered saline by gavage daily for 6 weeks. (2) Model group (Dex group (n=6): mice were injected intraperitoneally with 25 mg / kg dexamethasone (Dex) every other day for 4 weeks and administered saline by gavage daily for 6 weeks. (3) FMPH group (Dex+FMPH group (n=6): mice were injected intraperitoneally with 25 mg / kg dexamethasone every other day for 4 weeks and administered 200 mg / kg of the FMPH lyophilized powder in Example 1 by gavage daily for 6 weeks. (4) Positive control group (Dex+RIS, n=6): Mice were intraperitoneally injected with 25 mg / kg dexamethasone every other day for 4 weeks; and given risedronate sodium (RIS) by gavage every other day. Dexamethasone and risedronate sodium were administered on the same day for 6 weeks. The mice were weighed weekly during the period until the end of the experiment.

[0101] 1.2. Sample Collection

[0102] (1) Fecal collection

[0103] Each mouse was placed in a sterile disposable packaging box (sterilized under UV light for 40 min), and feces (6-8 pellets) from each mouse were collected. The pellets were then placed into sterile centrifuge tubes with tweezers, with 3-4 pellets in each of the two centrifuge tubes. The tubes were then immediately placed in liquid nitrogen and finally transferred to a -80°C freezer for storage.

[0104] (2) Blood sample collection

[0105] At the end of the experiment, the mice were fasted for 12 hours but allowed free access to water, and their final body weight was measured. Blood was collected from the mice by enucleation into 1.5 mL EP tubes, incubated at 4°C for 30 min, centrifuged (4°C, 1000g, 15 min), and the supernatant was collected into sterile EP tubes. The tubes were then stored at -80°C for later use.

[0106] (3) Skeleton collection

[0107] Both femurs and tibias were harvested, and after removing soft tissue, muscles, and ligaments, they were weighed. The left femur was fixed in an EP tube containing 4% paraformaldehyde and stored at room temperature for later H&E staining, TRAP staining, and immunofluorescence. The right femur was completely dissected, wrapped in clean gauze, and stored at -80°C for later Micro-CT analysis. Both tibias were completely dissected, placed in cryovials, and quickly stored at -80°C for later RT-qPCR analysis.

[0108] 1.3. Serum Biochemical Indicators Detection

[0109] Centrifuged mouse serum was frozen at -80°C and thawed at room temperature when needed for experiments. Serum biochemical parameters were detected using ELISA. ALP was used to evaluate bone formation status in mice, and CTX-I and TRACP-5b were used to evaluate bone resorption status.

[0110] 1.4. Organ Index

[0111] The liver, spleen, and kidneys of mice were removed, washed with physiological saline, dried, and weighed. Visceral parameters were calculated using the following formula:

[0112]

[0113] 1.5. Bone length and skeletal index

[0114] The left femur and tibia of the mouse were removed, and after soft tissue was removed, the lengths (mm) of the femur and tibia were measured using calipers. Then, they were placed in a ten-thousandths electronic balance, weighed accurately, and recorded as wet weight.

[0115]

[0116] 1.6. Micro-CT Detection of Various Indicators in Mouse Femurs

[0117] Three femurs were randomly selected from the femoral tissue of each group of mice, and Micro-CT equipment was used to detect the following related indicators of mouse femoral trabeculae: bone mineral density (BMD), tissue mineral density (TMD), bone surface area to bone volume ratio (BS / BV), trabecular thickness (Tb.Th), degree of anisotropy (DA), trabecular pattern factor (Tb.Pf), and structural pattern index (SMI).

[0118] 1.7. H&E staining and TRAP staining of mouse femur

[0119] H&E staining was performed to observe pathological changes in osteoporotic mice, and TRAP staining was performed to observe changes in the number of osteoclasts in the mouse femur. Mouse femoral tissue was fixed in a tissue fixative solution. After complete fixation, the femoral tissue was removed, air-dried, and then decalcified (using 10% EDTA for 2 weeks). It was then embedded in paraffin, sectioned, and the femoral structure was observed using H&E and TRAP staining. Images were observed and acquired using a microscope.

[0120] 1.8. Detection of bone tissue-related gene expression by real-time quantitative PCR.

[0121] Take 50 mg of bone tissue and place it in a 2 mL enzyme-free centrifuge tube. Add 1 mL of Trizol reagent and 3-4 enzyme-free grinding beads. Place the centrifuge tube in a homogenizer and homogenize until homogeneous. Recover the grinding beads and add 200 μL of chloroform to remove proteins. After mixing thoroughly, incubate at 4°C for 5 min, centrifuge (12000g, 15 min, 4°C), collect the supernatant, and add an equal volume of isopropanol to precipitate RNA. After mixing thoroughly, incubate at 4°C for 15 min, centrifuge (12000g, 15 min, 4°C), and discard the supernatant. Add an appropriate amount of 75% ethanol to wash the precipitate, centrifuge (12000g, 15 min, 4°C), discard the supernatant, and dry at room temperature for 15 min. Dissolve the RNA precipitate in an appropriate amount of RNase-free water, determine the RNA purity and concentration by absorbance ratio (260 / 280 nm), and reverse transcribe the RNA into cDNA using a reverse transcription kit.

[0122] qPCR was performed using SYBR Green Mater Mix in a 20 μL volume, containing 10 μL SYBR Green I, 6.4 μL RNase-free water, 0.8 μL each of forward and reverse primers (10 μmol / L), and 2.0 μL cDNA. The reaction conditions were: 95℃ pre-denaturation for 30 s; followed by 40 cycles at 95℃ for 5 s and 60.0℃ for 30 s. Finally, the Ct values ​​were normalized to the GADPH gene, and a 2... -ΔΔCt The relative expression level of the target gene was calculated using a method. Primer information is shown in the table below:

[0123] Table 4 Primer Sequences

[0124]

[0125] 1.9. Immunofluorescence analysis of mouse femur

[0126] Immunofluorescence staining was performed on histological sections of the femur. Paraffin sections were dewaxed to water and incubated with 3% hydrogen peroxide solution for 25 min to inhibit endogenous peroxidase activity. After incubation with 10% goat serum at 25°C for 30 min, the sections were then incubated with primary antibodies against TRAP and OCN at 4°C for 12 h. After washing with PBS, the sections were incubated with the corresponding species-specific secondary antibodies at 25°C for 50 min. Following immunostaining, the sections were stained with DAB and observed under an inverted fluorescence microscope.

[0127] 1.10.16S rRNA gene sequencing analysis of gut microbiota

[0128] Total DNA was extracted from mouse feces according to the kit instructions. PCR amplification of 16S rRNA was performed using upstream primer 338F (SEQ ID NO.22: 5'-ACTCCTACGGGAGGCAGCAG-3') and downstream primer 806R (SEQ ID NO.23: 5'-GGACTACHVGGGTWTCTAAT-3'). High-throughput sequencing was performed using an Illumina HiSeq™ 4000 sequencer. The raw data obtained from Illumina sequencing was split, and the paired-end reads were quality controlled and filtered. The reads were then spliced ​​based on the overlap relationship between the paired ends. Sequence noise reduction methods were used to process the optimized spliced ​​data, obtaining representative ASV sequences and abundance information. Analysis was performed based on the representative ASV sequences and abundance information. Data analysis and visualization were performed using the Shanghai Meiji Cloud Platform (https: / / cloud.majorbio.com).

[0129] 1.11. Data Statistics and Analysis

[0130] Data significance analysis was performed using the Tukey method in one-way ANOVA (P < 0.05) in SPSS 24.0 software, and graphs were plotted using Graphpad 7.0. Experimental results are expressed as mean ± standard deviation, and each experiment was performed in triplicate.

[0131] 2. Results and Analysis

[0132] 2.1. Effect of FMPH on body weight in osteoporotic mice

[0133] like Figure 8As shown, there was no significant difference in the initial body weight of the mice in each group. With the increase of feeding time, the body weight of the mice in the Control group increased accordingly. During the feeding period, the body weight of the mice in the Control group was always the highest, and the body weight of the mice in the Dex group was always the lowest. Dex treatment significantly reduced the final body weight of the mice (p < 0.05), while the use of FMPH and RIS interventions could significantly alleviate the abnormal reduction in body weight induced by Dex in the mice (p < 0.05).

[0134] 2.2. Effects of FMPH on organ indices in osteoporotic mice

[0135] like Figure 9 As shown, compared with the Control group, the liver and kidney indices of mice in the Dex group were significantly increased (p < 0.05), while the spleen index was significantly decreased (p < 0.05). FMPH and RIS interventions significantly alleviated the abnormally high liver and kidney indices induced by Dex in mice (p < 0.05), and also significantly alleviated the abnormally low spleen index induced by Dex (p < 0.05).

[0136] 2.3. Effects of FMPH on serum markers in osteoporotic mice

[0137] ALP is a common marker of bone formation, while TRACP-5b and CTX-I are common markers of bone resorption. Results of serum bone formation-related markers in mice are as follows: Figure 10 As shown in (A), compared with the Control group, the ALP level in the Dex group showed a decreasing trend, but the difference was not statistically significant (p > 0.05). However, compared with the Dex group, the serum ALP levels in the Dex + FMPH group and the Dex + RIS group were significantly increased (p < 0.05), indicating that FMPH and RIS intervention can promote bone formation in mice. Results of serum bone resorption-related indicators in mice are shown below. Figure 10 As shown in (BC), Dex treatment significantly increased the levels of TRACP-5b and CTX-I in mouse serum (p < 0.05), indicating that Dex exacerbates bone resorption in mice. However, after intervention with FMPH and RIS, the levels of TRACP-5b and CTX-I in mouse serum were significantly lower than those in the Dex group (p < 0.05), indicating that FMPH and RIS intervention can significantly inhibit the abnormal increase in Dex-induced bone resorption in mice.

[0138] 2.4. Effects of FMPH on bone microstructure in osteoporotic mice

[0139] Micro-CT can visually present the microstructural features of mouse bones. Representative 2D and 3D micro-CT images of the mouse femur are shown below. Figure 11As shown in (A), there were significant differences in trabecular bone structure among the groups. Compared with the Control group, the Dex group mice had larger and sparser trabecular bone gaps in the femur, exhibiting osteoporosis symptoms. However, after intervention with FMPH and RIS, the trabecular bone gaps in the femur of the mice decreased, the distribution tended to be normal, and the osteoporosis symptoms were alleviated.

[0140] After constructing the 3D structure of the femur, the relevant parameters were analyzed, and the results are as follows: Figure 11 As shown in (B), compared with the control group, the BMD and TMD of mice in the Dex group were significantly decreased (p < 0.05), indicating that Dex can significantly reduce bone mineral density in mice. Meanwhile, the BS / BV ratio in the Dex group was significantly higher than that in the control group (p < 0.05), indicating that Dex treatment leads to osteoporosis and porous bone tissue in mice. After intervention with FMPH and RIS, the BMD level in mice showed an upward trend, but the difference was not statistically significant (p > 0.05), while the TMD level significantly increased (p < 0.05), and the BS / BV level significantly decreased (p < 0.05), indicating that FMPH and RIS can effectively improve the abnormal bone microstructure index induced by Dex, thereby alleviating osteoporosis. Furthermore, compared with the control group, mice in the Dex group showed significantly decreased Tb.Th and DA (p < 0.05), and significantly increased Tb.Pf and SMI (p < 0.05), indicating that Dex narrows the trabecular bone in mice and transforms the trabecular structure from plate-like to rod-like, while also causing significant disruption in the spatial arrangement of the trabecular bone. However, FMPH intervention significantly inhibited the Dex-induced decrease in Tb.Th (p < 0.05) and brought DA, Tb.Pf, and SMI back to normal levels, indicating that FMPH can alleviate Dex-induced changes in trabecular microstructure to some extent.

[0141] 2.5. Effects of FMPH on bone morphology in osteoporotic mice

[0142] To investigate the effects of FMPH on the morphology of mouse bone tissue, H&E staining and trap staining were performed on the mouse femur. The results of H&E staining of the mouse femur are shown below. Figure 12 As shown, compared with the Control group, mice in the Dex group exhibited thinner trabecular bone thickness, larger gaps between trabeculae, and disruption of the continuity of the trabecular network structure in the femur. In contrast, compared with the Dex group, mice in the Dex + FMPH and Dex + RIS groups showed increased trabecular bone thickness, reduced fractures and gaps in the femur, consistent with the Micro-CT results, indicating that FMPH can alleviate osteoporosis-induced bone microstructural disorder to some extent.

[0143] Trap staining was used to specifically label osteoclasts (which appear purple-red) in the mouse femur. The results of Trap staining and quantitative analysis of the mouse femur are as follows: Figure 13 As shown in the figure, compared with the control group, the number of osteoclasts in the Dex group was significantly increased (p < 0.05), indicating that Dex can activate osteoclast-mediated bone resorption. After FMPH intervention, the number of osteoclasts in mouse bone tissue was significantly reduced (p < 0.05), indicating that FMPH can effectively inhibit Dex-induced abnormal osteoclast activation, thereby reducing bone resorption.

[0144] 2.6. Effects of FMPH on the expression of bone formation-related genes and proteins in osteoporotic mice

[0145] To further verify the osteogenic effect of FMPH, the expression of bone formation-related genes in the mouse tibia was detected by RT-qPCR. Figure 14 As shown, compared with the Control group, the Dex group significantly downregulated the relative mRNA expression levels of key osteogenic transcription factors Runx2 and Osterix, as well as the osteogenic marker OCN (p < 0.05), while FMPH and RIS interventions significantly inhibited these changes (p < 0.05). Furthermore, compared with the Dex group, the relative mRNA expression levels of the osteogenic markers ALP and Col1α1 were significantly upregulated in the Dex + FMPH and Dex + RIS groups (p < 0.05).

[0146] Simultaneously, the expression of ALP, OCN, and Runx2 proteins in the mouse femur was determined using immunofluorescence analysis, and the relative fluorescence intensity of each protein was quantitatively analyzed. The results are as follows: Figure 15 As shown, compared with the Control group, the expression levels of ALP and OCN proteins in the femur of mice in the Dex group were significantly decreased (p < 0.05), and the expression level of Runx2 protein showed a decreasing trend, but the difference was not statistically significant (p > 0.05). Compared with the Dex group, FMPH intervention significantly increased the expression levels of OCN and Runx2 proteins (p < 0.05) and restored the ALP protein expression level to a level that was not significantly different from that in the Control group (p > 0.05), consistent with the trend of RT-qPCR results. In addition, the expression levels of ALP and OCN proteins in the Dex + RIS group were significantly higher than those in the Dex group (p < 0.05). The results indicate that FMPH and RIS can promote osteoblast differentiation, enhance bone matrix synthesis, and accelerate osteogenic maturation by regulating the expression of bone formation-related genes and proteins in mice, thereby exerting potential osteogenic activity.

[0147] 2.7. Effects of FMPH on the expression of bone formation-related genes and proteins in osteoporotic mice

[0148] To further verify the osteogenic effect of FMPH, the expression of bone formation-related genes in the mouse tibia was detected by RT-qPCR. Figure 16 As shown, compared with the Control group, the Dex group significantly downregulated the relative mRNA expression levels of key osteogenic transcription factors Runx2 and Osterix, as well as the osteogenic marker OCN (p < 0.05), while FMPH and RIS interventions significantly inhibited these changes (p < 0.05). Furthermore, compared with the Dex group, the relative mRNA expression levels of the osteogenic markers ALP and Col1α1 were significantly upregulated in the Dex + FMPH and Dex + RIS groups (p < 0.05).

[0149] Simultaneously, the expression of ALP, OCN, and Runx2 proteins in the mouse femur was determined using immunofluorescence analysis, and the relative fluorescence intensity of each protein was quantitatively analyzed. The results are as follows: Figure 17 As shown, compared with the Control group, the expression levels of ALP and OCN proteins in the femur of mice in the Dex group were significantly decreased (p < 0.05), and the expression level of Runx2 protein showed a decreasing trend, but the difference was not statistically significant (p > 0.05). Compared with the Dex group, FMPH intervention significantly increased the expression levels of OCN and Runx2 proteins (p < 0.05) and restored the ALP protein expression level to a level that was not significantly different from that in the Control group (p > 0.05), consistent with the trend of RT-qPCR results. In addition, the expression levels of ALP and OCN proteins in the Dex + RIS group were significantly higher than those in the Dex group (p < 0.05). The results indicate that FMPH and RIS can promote osteoblast differentiation, enhance bone matrix synthesis, and accelerate osteogenic maturation by regulating the expression of bone formation-related genes and proteins in mice, thereby exerting potential osteogenic activity.

[0150] 2.8. Key Gut Microbiota Analysis

[0151] Correlation analysis was performed on the top 20 most abundant gut microbiota species and their association with osteoporosis, and the relative abundance changes of species with significant differences at the genus level were analyzed. Results are as follows: Figure 18As shown, FMPH intervention significantly modulates the relative abundance of bacteria such as Lactobacillus, Helicobacter, Bifidobacterium, norank_f__Muribaculaceae, and Desulfovibrio. The relative abundance of bacteria such as norank_f__[Eubacterium]_coprostanoligenes_group, norank_f__Muribaculaceae, Akkermansia, Lactobacillus, Helicobacter, and Bifidobacterium is significantly correlated with five or more biochemical indicators in osteoporotic mice, indicating that these bacteria are potential key bacteria for FMPH's anti-osteoporosis effect.

Claims

1. A strain of *Limosilactobacillus fermentum* DACN204 has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026612.

2. A method for preparing milk protein hydrolysates based on the synergistic hydrolysis of enzymes and lactic acid bacteria, characterized in that, Includes the following steps: (1) Single hydrolysis-enzymatic hydrolysis Milk protein was dispersed in water, and flavor protease was added for enzymatic hydrolysis. After the reaction was completed, the enzyme was inactivated, the supernatant was collected by centrifugation, the pH was adjusted to 6.8-7.2, and the mixture was freeze-dried to obtain the freeze-dried powder of milk protein hydrolysis product. (2) Secondary hydrolysis -- lactic acid bacteria fermentation enzymatic hydrolysis products The lyophilized powder of the enzymatic hydrolysis product obtained in step (1) is dispersed in water, sterilized, inoculated with the fermenting Lactobacillus mucinus DACN204 as described in claim 1, fermented, centrifuged after fermentation, the supernatant is collected, filtered through a filter membrane to remove residual bacteria and cell debris, and freeze-dried to obtain the fermented milk protein hydrolysate.

3. The preparation method according to claim 2, characterized in that, In step (1), milk protein powder is dispersed in ultrapure water at a ratio of 2-6% (w / v), flavor protease is added at a mass of 3-9% of the milk protein powder, the pH of the solution is adjusted to 7.2-7.8, and enzymatic hydrolysis is performed at 47-53℃ for 2-5 hours.

4. The preparation method according to claim 3, characterized in that, In step (1), milk protein powder is dispersed in ultrapure water at a ratio of 3-5% (w / v), flavor protease is added at a mass of 5-7% of the milk protein powder, the pH of the solution is adjusted to 7.4-7.6, and enzymatic hydrolysis is performed at 49-51℃ for 3-5 hours.

5. The preparation method according to claim 2, characterized in that, In step (2), the lyophilized powder of the enzymatic hydrolysis product obtained in step (1) is dispersed in deionized water at a ratio of (8~12) g / 100 mL, sterilized, and inoculated with the fermenting Lactobacillus mucinus DACN204 of claim 1 at an inoculation amount of 1.8~2.2%. Fermentation is carried out at 36~38℃ for 22~28 h. After fermentation, the supernatant is collected by centrifugation, filtered through a filter membrane, and freeze-dried to obtain the fermented milk protein hydrolysate.

6. A fermented milk protein hydrolysate, characterized in that: It is prepared by the preparation method according to any one of claims 2 to 5.

7. The use of the fermented milk protein hydrolysate according to claim 6 in the preparation of a medicament for treating osteoporosis.

8. The use of the fermented milk protein hydrolysate according to claim 6 in the preparation of health foods that improve bone density.

9. A pharmaceutical composition for the prevention or treatment of osteoporosis, characterized in that: The product includes an effective dose of the fermented milk protein hydrolysate of claim 6, and a pharmaceutically acceptable carrier; preferably, the pharmaceutically acceptable carrier includes one or more of fillers, wetting agents, disintegrants, binders, or lubricants.

10. A health food product for improving bone density, characterized in that: It includes the fermented milk protein hydrolysate as described in claim 6, as well as food-grade acceptable adjuvants.

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