Phytobacterium plantarum, sec-yellow leavening, preparation method thereof and application of sec-yellow leavening in improvement of bone health

Abstract

The invention provides a plant lactobacillus, and also discloses a secondary yellow fermented product obtained by fermenting eucommia ulmoides leaves, rehmannia, dried ginger and dendrobium officinale with the plant lactobacillus as well as a preparation method and application of the secondary yellow fermented product. The sec-yellow leavening can directionally activate a phenylpropane biosynthetic pathway and induce the generation of polyphenol characteristic small molecule metabolites, especially the generation of mulberry furan W. The invention also discloses a preparation method of the sec-yellow leavening. And compared with the raw materials, the fermented product has more active small molecule metabolites. Finally, zebra fish experiments after prednisone modeling prove that the sec-yellow yeast can significantly promote osteogenesis. The sec-yellow leavening is recovered to the normal zebrafish gene expression level mainly by regulating the related gene expression level. Therefore, the sec-yellow leavening can be applied to preparation of foods or medicines for improving bone health.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of probiotics, in particular to a plant lactobacillus, and a zonghuang fermentation product obtained by fermenting eucommia ulmoides leaves, rehmannia glutinosa, dried ginger and dendrobium officinale, and a preparation method thereof and its application in bone health. BACKGROUND

[0002] Bone, as the core tissue of human body movement support, metabolic regulation and hematopoietic microenvironment, bears multiple functions of mechanical support, calcium and phosphorus homeostasis regulation and bone marrow hematopoiesis, and plays an irreplaceable role in maintaining body activity, maintaining metabolic balance and stabilizing life health.

[0003] Osteoarthritis and osteoporosis are high-incidence bone degenerative diseases in the middle-aged and elderly population. Long-term illness not only induces chronic pain and loss of limb function, but also increases the risk of complications such as cardiovascular events and depression, causing a heavy burden on public health resources, and its prevention and treatment is urgent.

[0004] Although the commonly used anti-osteoporosis drugs (such as bisphosphonates) can delay bone loss, they have risks such as gastrointestinal irritation and mandibular necrosis; symptomatic treatment of osteoarthritis (such as non-steroidal anti-inflammatory drugs) can only relieve symptoms and cannot reverse cartilage damage, and long-term use can easily cause liver and kidney damage. Therefore, finding a safe, long-acting, and reversible pathological process intervention solution is the key pain point of the current prevention and treatment of bone degenerative diseases.

[0005] The present application adopts a food and medicine homologous group formula fermentation method. Traditional Chinese medicine theory believes that "the kidney governs the bone and marrow", and the ancient prescription for bone and muscle strengthening in "Qianjin Fang" uses eucommia ulmoides leaves, rehmannia glutinosa, dendrobium officinale and dried ginger as food and medicine substances, which has the effect of "tonifying liver and kidney and strengthening bones and muscles". Modern research has confirmed that through microbial fermentation of food and medicine substances, the transformation and enrichment of active substances can be achieved by means of microbial metabolism: first, the fermentation process can degrade macromolecular components in the raw materials to generate more easily absorbed metabolites such as phenolic acids (such as chlorogenic acid) and flavonoids (such as quercetin), thereby improving their bioavailability; second, fermentation can improve the flavor and remove the unpleasant flavor of traditional Chinese medicines; third, fermentation can degrade toxins and reduce anti-nutritional factors. However, most current products and patents focus on the enhancement of active ingredients and the improvement of bioavailability, without paying attention to detoxification and efficiency. Therefore, the present application develops a food and medicine substance fermentation product that has both food safety and pharmacological activity, can achieve the goal of "food and medicine homology" for the regulation of bone chronic diseases, and can also avoid the toxic and side effects of chemical drugs, providing a green and sustainable new strategy for the prevention and treatment of bone health (osteoporosis and osteoarthritis). SUMMARY

[0006] The first object of the present application is to provide a plant lactobacillus YS-GuLin18.

[0007] The second objective of this invention is to provide a fermented product obtained by fermenting the combination of Eucommia ulmoides leaves, Rehmannia glutinosa, dried ginger, and Dendrobium officinale with the probiotics.

[0008] The third objective of this invention is to provide a method for preparing the fermented product of *Zhonghuang*.

[0009] A fourth objective of this invention is to provide an application of the aforementioned fermented turmeric.

[0010] This invention is achieved through the following technical solution: A plant lactobacillus ( Lactiplantibacillus plantarum YS-GuLin18, with accession number CGMCC NO. 35529, was deposited on August 6, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China (Institute of Microbiology, Chinese Academy of Sciences). The strain was viable at the time of deposit.

[0011] A fermented product of Eucommia ulmoides, obtained by fermenting a food and medicine homologous composition consisting of 70wt% Eucommia ulmoides leaves, 15wt% Rehmannia glutinosa, 10wt% dried ginger, and 5wt% Dendrobium officinale by Lactobacillus plantarum YS-GuLin18.

[0012] The preparation method of the fermented product of Rheum palmatum includes the following steps: Seed culture of *Lactobacillus plantarum* YS-GuLin18 was inoculated into a sterilized substrate consisting of glucose, peptone, dipotassium hydrogen phosphate, a food-medicine homologous composition, and water. β-glucan hydrolase was then added to the substrate for the first fermentation step, followed by the addition of vitamin C for the second fermentation step, and then the addition of quinic acid for the third fermentation step. Finally, the substrate was inactivated, centrifuged, and the supernatant was collected.

[0013] The method for preparing the seed solution includes the following steps: Lactobacillus plantarum YS-GuLin18 was inoculated into MRS liquid medium and cultured to obtain bacterial suspension. Then, 2% of the bacterial suspension was inoculated into MRS liquid medium to obtain seed culture. The concentration of *Lactobacillus plantarum* YS-GuLin18 in the seed solution was 1×10⁻⁶. 9 CFU / mL.

[0014] The substrate comprises 4-6% glucose, 0.5-1% peptone, 0.10-0.25% dipotassium hydrogen phosphate, and 6-8% food and drug homologous composition.

[0015] The sterilization temperature is 115℃ and the time is 20 minutes; The inoculation amount of the seed liquid is 2%.

[0016] The enzyme activity of the beta-glucan hydrolytic enzyme is 20 U / g, and the fermentation temperature of the first step fermentation is 30-32 DEG C. The addition amount of the vitamin C is 0.1%, and the fermentation temperature of the second step fermentation is 35-37 DEG C. The addition amount of the quinic acid is 0.05%, and the fermentation temperature of the third step fermentation is 35-37 DEG C.

[0017] The end point pH of the third step fermentation is 3.8-4.0; The inactivation temperature is 115 DEG C, and the time is 30 min; The centrifugal speed is 6000 r / min, and the time is 3-5 min.

[0018] The application of the Zhonghuang fermentation product is applied to the preparation of food or medicine for increasing the head bone area. The application of the Zhonghuang fermentation product is applied to the preparation of food or medicine for down-regulating the expression level of runx2a and / or acp5a to improve bone health.

[0019] The application of the Zhonghuang fermentation product is applied to the preparation of food or medicine for improving bone health.

[0020] Compared with the prior art, the present application has the following beneficial effects: The plant lactobacillus YS-GuLin18 provided by the present application can directionally activate the phenylpropanoid biosynthesis pathway and induce the synthesis of polyphenol characteristic small molecule metabolites (chlorogenic acid, caffeic acid, mulberry furan W and other substances).

[0021] The abundance of trace toxic and harmful ingredients such as toxic alkaloids (dimethyl strychnine), carcinogens / environmental pollutants (1,6-dinitropyrene, imidacloprid), and toxic terpenes (taxane alcohol X, 2-amino benzothiazole) in the fermentation product provided by the present application is lower, and the fermentation product has higher food and drug safety, and can be used for the preparation of food and drugs.

[0022] The fermentation product prepared by the method provided by the present application can significantly up-regulate flavonoids such as farnesiferon, kaempferol and quercetin; up-regulate polyphenols such as chlorogenic acid, caffeic acid, thymol and mulberry furan W; up-regulate alkaloids such as berberine, piperine, trigonelline and betaine; and up-regulate terpenes such as onjisaponin, glycyrrhizic acid, hydroxyl asperosaponin and oleanolic acid.

[0023] Through zebrafish experiments, it is verified that the fermentation product of the present application can significantly promote zebrafish bone formation, improve bone health by increasing the head bone area and down-regulating the expression level of runx2a and acp5a, and especially for osteoporosis and osteoarthritis.

[0024] The method for preparing food and medicine homologous fermentation products provided by this invention is simple, controllable, and can be mass-produced. Attached Figure Description

[0025] Figure 1 The diagram shows an ontology of differentially classified metabolites in FD vs. D. Figure 2 The diagram shows the ontology of differentially classified metabolites in FD16 vs. D. Figure 3 The second-layer KEGG pathway category bar chart of FD16 vs. D is shown; Figure 4 The chemical structure of Mulberrofuran W is shown; Figure 5 The staining of zebrafish skeletons in the CK group is shown; Figure 6 The staining of the zebrafish skeletons in group M is shown; Figure 7 The staining of the zebrafish skeletons in group A is shown; Figure 8 The staining of the zebrafish skeletons in group B is shown; Figure 9 The staining of the zebrafish skeletons in group C is shown; Figure 10 The graph shows quantitative data of zebrafish skeletal staining from different groups. Figure 11 The effects of different groups on runx2a gene expression are shown.

[0026] Figure 12 The effects of different groups on acp5a gene expression were shown. Detailed Implementation

[0027] Example 1 The screening process for probiotics with high antioxidant activity is as follows: In this embodiment, strains with good probiotic properties (acid resistance, bile salt resistance, adhesion, drug sensitivity, etc.) were pre-screened from the longevity elderly lactic acid bacteria strain resource bank of Hunan Nutrition Tree Biotechnology Co., Ltd. These strains are BS906M7, BS88T4, BS11M9, BS619M14, BS683M16, and YS-GuLin18. Simultaneously, the antioxidant performance of the fermentation supernatant of these strains was evaluated from three dimensions: in vivo enzymatic antioxidants (SOD, CAT), in vitro free radical scavenging rate (DPPH), and overall antioxidant level (T-AOC).

[0028] SOD (total superoxide dismutase) is an important antioxidant enzyme in organisms and one of the common active ingredients in the supernatant of bacterial strains. It can specifically scavenge superoxide anion free radicals.

[0029] CAT (catalase) is another key antioxidant enzyme that catalyzes the breakdown of H2O2 into harmless water (H2O) and oxygen (O2), preventing H2O2 from accumulating in the body and transforming into more toxic hydroxyl radicals.

[0030] DPPH is a synthetically produced stable free radical (purple in color) and is a commonly used reagent for in vitro testing of the free radical scavenging ability of fermentation supernatants.

[0031] T-AOC (Total Antioxidant Capacity) is an indicator that comprehensively measures the overall antioxidant capacity of all antioxidant substances (enzymes + non-enzymes) in the fermentation supernatant of a bacterial strain, rather than targeting a specific free radical or enzyme.

[0032] The bacterial culture of the above-mentioned bacteria was adjusted to a concentration of 1×10⁻⁶. 9 Centrifuge at 4℃ and 6000 rpm for 5 min, and collect the supernatant as fermentation supernatant (CFS). The antioxidant properties of the fermentation supernatant are then determined according to the above indicators.

[0033] Table 1 Antioxidant capacity of different strains As shown in Table 1, strain YS-GuLin18 exhibited the best performance in terms of in vivo enzymatic antioxidant activity, in vitro free radical antioxidant activity, and overall antioxidant activity. Given its superior antioxidant activity, strain YS-GuLin18 was selected for further testing.

[0034] The identification of strain YS-GuLin18 is as follows: Strain YS-GuLin18 was selected from the intestines of long-lived elderly people.

[0035] After identification, strain YS-GuLin18 was found to be *Lactobacillus plantarum*.

[0036] The 16S sequence of the *Lactobacillus plantarum* YS-GuLin18 is as follows:

[0037] Example 2 Fermentation raw material screening To screen for compound raw materials with strong antioxidant properties and the ability to improve bone health, especially joint health, this experiment screened raw materials with good sales in domestic and international markets, as shown in Table 2, and measured the total antioxidant capacity (T-AOC) of the raw materials at a concentration of 0.1 g / mL.

[0038] Table 2 Total antioxidant capacity (T-AOC) of different raw materials As shown in Table 2, using T-AOC ≥ 30 μmol / g as the standard, high-quality food and medicinal raw materials were selected: Eucommia ulmoides leaves, Dendrobium officinale, Rehmannia glutinosa, and dried ginger as preferred raw materials. Based on the above raw materials, after consulting the pharmacopoeia, it was found that these four raw materials are similar to the components of Eucommia ulmoides pills in Qianjin Fang. Therefore, these four substances were combined for fermentation.

[0039] The formula uses a combination of "principal, assistant, adjuvant, and guide" herbs: Eucommia ulmoides leaf, Dendrobium officinale, Rehmannia glutinosa, and dried ginger. The principal herb, Eucommia ulmoides leaf (longevity leaf), primarily combats aging, nourishes the liver and kidneys, and strengthens bones and muscles. Its polyphenols (chlorogenic acid) provide antioxidant benefits and protect the bone microenvironment, while its flavonoids regulate bone metabolism pathways (bone breaking and bone growth). The assistant herb, Dendrobium officinale, primarily nourishes, clearing heat and nourishing the stomach and promoting fluid production. Its flavonoids promote bone differentiation and block bone resorption pathways, while its polysaccharides maintain the bone health barrier.

Assistant Herb - Rehmannia

Guide Herb - Dried Ginger

[0040] Example 3 Preparation of food and medicine homologous fermentation products In this embodiment, YS-GuLin18 was used to ferment the above-screened raw materials (Eucommia ulmoides leaves, Dendrobium officinale, Rehmannia glutinosa, and dried ginger) and the changes in their active ingredients before and after fermentation were compared.

[0041] The specific preparation process is as follows: ① Seed liquid preparation: After streaking the YS-GuLin18 glycerol tubes, pick single bacteria and culture them in MRS liquid medium. Then, inoculate 2% bacterial culture into the MRS liquid medium and adjust the seed culture concentration to 1×10⁻⁶. 9 CFU / mL. The MRS liquid culture medium was purchased from Guangdong Huankai Microbial Technology Co., Ltd.

[0042] ② Preparation of unfermented composition: A mixture of 5% glucose, 1% peptone, 0.25% dipotassium hydrogen phosphate, 8% food-medicine homologous composition, and water was sterilized at 115°C for 20 minutes to obtain an unfermented composition. The food-medicine homologous composition comprises 70 wt% Eucommia ulmoides leaf powder, 15 wt% Dendrobium officinale powder, 10 wt% Rehmannia glutinosa powder, and 5 wt% dried ginger powder.

[0043] ③ Seed liquid inoculation: YS-GuLin18 seed culture was inoculated into the unfermented composition, with the inoculation volume of seed culture being 2%; ④ Preparation of fermentation products: Fermentation was carried out using a low-temperature shallow-layer enzymatic hydrolysis coupled targeted fermentation method, and the specific steps are as follows: S1 initial stage (0-6h): Add β-glucan hydrolase (enzyme activity 20U / g) to the unfermented composition of the inoculated seed liquid and ferment at 30-32℃.

[0044] This stage is "substrate pretreatment + initial strain adaptation": β-glucan hydrolase can degrade difficult-to-use polysaccharides and polyphenols in the substrate, reducing substrate complexity and facilitating rapid utilization by the strain; 30-32℃ is the suitable temperature for the initial proliferation of the inoculated strain, which can help the strain adapt to the system quickly and accumulate population, laying the foundation for subsequent high-activity metabolism.

[0045] S2 mid-stage (6-8h): Add 0.1% vitamin C and ferment at 35-37℃.

[0046] This stage is "metabolic activation + optimal temperature matching for enzyme production": 35-37℃ is the optimal temperature for the strain to produce enzymes, which can maximize the activation of its metabolic pathways and promote enzyme synthesis; Vitamin C acts as a color protectant, which can preserve the color of food and medicinal substances and enhance the catalytic efficiency of enzymes, providing conditions for the subsequent conversion of active ingredients.

[0047] S3 late stage (8-16h): Add 0.05% quinic acid and ferment at 35-37℃ to obtain fermentation broth. The purpose of this step is to promote the conversion of caffeic acid to chlorogenic acid in the fermentation product.

[0048] This stage involves "targeted conversion + fermentation rhythm control": Quinic acid can directionally regulate metabolic pathways and promote the conversion of caffeic acid to chlorogenic acid (achieving enrichment of active ingredients); by using the above low-temperature shallow-enzymatic coupling targeted fermentation, the conversion of active ingredients can be completed while avoiding excessive consumption of active ingredients by the strain.

[0049] Throughout the fermentation process, the rotation speed was controlled at 130 r / min, and the final pH of the fermentation was 3.8-4.0. Fermentation was terminated after the corresponding fermentation time and final pH were reached.

[0050] ⑤ The above fermentation broth was sterilized at 115℃ for 30 min to obtain an inactivated fermentation broth. The inactivated fermentation broth was centrifuged at 6000 r / min for 5 min. The supernatant (i.e., food and medicine homologous fermentation product) was obtained by centrifugation.

[0051] Comparative Example 1 The difference from Example 1 lies in the use of a conventional fermentation process. The fermentation conditions are as follows: the temperature is maintained at 35-37℃ throughout the process, the rotation speed is 130 r / min, the fermentation time is 16 h, and the final pH of the fermentation is 3.8-4.0. Fermentation is terminated after reaching the corresponding fermentation time and final pH. The fermentation broth is sterilized at 115℃ for 30 min to obtain an inactivated fermentation broth. The inactivated fermentation broth is centrifuged at 6000 r / min for 5 min. The supernatant is obtained by centrifugation.

[0052] Comparative Example 2 The unfermented composition prepared according to step 2 in Example 3: The fermentation broth was prepared and inactivated using 5% glucose, 1% peptone, 0.25% dipotassium hydrogen phosphate, and 8% a food-medicinal homologous composition (the food-medicinal homologous composition was prepared in the following ratio: Eucommia ulmoides leaf powder: Dendrobium officinale powder: Rehmannia glutinosa powder: dried ginger powder in a ratio of 70wt%:15wt%:10wt%:5wt%) at 115℃ for 30 min. The inactivated fermentation broth was then centrifuged at 6000 r / min for 5 min. The supernatant was obtained after centrifugation.

[0053] The present invention collected the fermentation broth (i.e., the supernatant obtained by centrifugation, labeled as FD16) prepared in Example 3; the fermentation broth (i.e., the supernatant obtained by centrifugation, labeled as FD) prepared in Comparative Example 1 and the unfermented composition (i.e., the supernatant obtained by centrifugation, labeled as D) prepared in Comparative Example 2 and conducted polyphenol, flavonoid, antioxidant capacity and non-targeted omics determination.

[0054] The relevant indicators were measured as follows: 1. Detection of polyphenols, flavonoids, and antioxidant capacity: kit method As shown in Table 3, compared with groups D and FD, the fermentation broth (FD16) using low-temperature shallow-layer enzymatic hydrolysis coupled targeted fermentation significantly improved polyphenols, flavonoids, total antioxidant capacity (T-AOC), and DPPH scavenging rate. Compared with group D, polyphenols increased by 38.28%, flavonoids by 7.53%, total antioxidant capacity (T-AOC) by 67.01%, and DPPH free radical scavenging rate by 57.57%.

[0055] Table 3 Comparison of total phenols, flavonoids and antioxidant indicators among different groups 2. Non-targeted metabolomics The changes in overall metabolites in each group of samples were analyzed using liquid chromatography-mass spectrometry (LC-MS). The OPLS-DA model established using the experimental data showed R² and Q² ≥ 0.5, indicating that the model is stable and reliable, with good explanatory and predictive abilities. OPLS-DA VIP > 1 and P.value < 0.05, FC > 2 or FC < 1 / 2 were used as screening criteria to identify metabolites with significant differences, and their metabolite expression changes and functional pathways were analyzed.

[0056] ① Classification of differential metabolites The supernatants of D and FD were compared (FD vs. D), and a total of 15 significantly different metabolites were detected. The main categories include amino acids and their derivatives (7, 2.33%), carboxylic acids and their derivatives (8, 2.66%), dipeptides (6, 2.00%), fatty acyl groups (6, 2.00%), flavonoid O-glycosides (4, 1.33%), glycerophospholipids (4, 1.33%), glycosyl compounds (5, 1.66%), long-chain fatty acids (6, 1.99%), other categories (195, 64.78%), peptides (28, 9.33%), proberberine alkaloids and their derivatives (4, 1.33%), steroids and their derivatives (5, 1.66%), triterpenoid glycosides (4, 1.33%), and triterpenoids (12, 3.99%) (specifically as follows). Figure 1 (As shown).

[0057] The supernatants of D and FD16 were compared (FD16 vs. D), and a total of 15 significantly different metabolites were detected. The main categories include alkaloids and their derivatives (6, 0.9%), amino acids and their derivatives (23, 3.47%), carboxylic acids and their derivatives (13, 1.96%), fatty acyl groups (13, 1.96%), flavonoid O-glycosides (14, 2.11%), glycosyl compounds (11, 1.66%), organic oxygen compounds (7, 1.06%), other categories (478, 72.1%), peptides (38, 5.73%), phosphatidylcholine derivatives (9, 1.36%), purine nucleosides (7, 1.06%), steroids and their derivatives (8, 1.21%), triterpenoid glycosides (7, 1.06%), and triterpenoids (19, 2.87%) (details as follows). Figure 2 (As shown).

[0058] This shows that the FD16 group produced a greater variety of metabolites than the FD group, especially flavonoid O-glycosides and triterpenoid metabolites, which were more abundant and accounted for a higher proportion. In addition, the FD16 group also showed unique alkaloids and their derivatives.

[0059] ② Differential metabolites Different types of metabolites were observed after fermentation. We focused on the changes in flavonoids, polyphenols, alkaloids, and terpenoids in FD16 and FD fermentation products.

[0060] For FD vs. D, only vitexin-2-O-rhamnoside, piperine, and oleanolic acid showed significant upregulation after fermentation, while other substances showed no significant upregulation.

[0061] For FD16 vs. D, fermentation significantly upregulates flavonoids such as fisetin, kaempferol, and quercetin; polyphenols such as chlorogenic acid, caffeic acid, thymol, and sanguranol; alkaloids such as berberine, piperine, trigonelline, and betaine; and terpenoids such as aucubin, glycyrrhetinic acid, asiaticoside, and oleanolic acid.

[0062] After treatment with the low-temperature shallow-layer-enzymatic coupled targeted fermentation method of this application, FD16 showed the presence of sangfuran W in its metabolites, and the abundance of this substance was relatively high. Literature review indicates that sangfuran W is not an inherent component of the raw materials (Eucommia ulmoides leaves, Rehmannia glutinosa, dried ginger, and Dendrobium officinale), but rather a unique new substance generated through synergistic effects of the raw materials and microbial enzyme catalysis. Its structural formula is as follows: Figure 4 As shown. Other typical furan components include furanocoumarins such as psoralen, and morularans, especially morularan W, which has significant antiviral, anti-inflammatory, and osteoblast differentiation-promoting effects.

[0063] Therefore, it can be inferred that the FD16 group can effectively decompose macromolecules and release a greater number of significantly smaller molecules. Furthermore, these smaller molecules have a certain positive correlation with bone health. Among them, flavonoids (quercetin, fisetin, kaempferol), polyphenols (chlorogenic acid, sanguine W), alkaloids (berberine), and terpenes (glycyrrhetinic acid, aucubin) have the effect of inhibiting joint inflammation and relieving synovial congestion and swelling. Quercetin, fisetin, sanguine W, aucubin, and chlorogenic acid can reduce oxidative stress, promote chondrocyte proliferation and metabolism, delay chondrocyte apoptosis, and promote cartilage repair. Plumbagoside, glycyrrhetinic acid, and piperine can reduce inflammation-mediated pain sensitivity and relieve pain during joint movement. Therefore, subsequent experiments will be conducted to verify the efficacy of the fermentation broth in promoting bone health.

[0064] Table 4. Changes in beneficial metabolites among different groups Note: No significant difference: There was no significant difference in the active ingredient between the FD.vs.D group (P>0.05); Increase: The active ingredient was significantly upregulated between the FD16.vs.D or FD.vs.D group (P<0.05); Decrease: The active ingredient was significantly downregulated between the FD16.vs.D or FD.vs.D group (P<0.05).

[0065] ② Significant changes in metabolic pathways after fermentation of beneficial substances Regarding significantly altered metabolic pathways, the focus is on the biosynthesis of secondary metabolites (...). Figure 3 The following are the metabolic pathways that show significant changes in FD16 vs. D, including flavonoid biosynthesis, flavonone and flavonol biosynthesis, and phenylpropanoid biosynthesis. Figure 3 However, no significant changes were observed in the phenylpropanoid biosynthetic pathway in FD.vs.D. It is evident that FD16, through a low-temperature shallow-enzymatic coupled targeted fermentation process, may have activated the phenylpropanoid biosynthetic pathway, leading to the significant generation of chlorogenic acid, caffeic acid, and sanguran W. Chlorogenic acid is a core substance in Eucommia ulmoides leaves; its chemical nature is caffeoyl quinic acid, one of the core products of phenylpropanoid metabolism. It is generated by the ester bond between the phenylpropanoid precursor (caffeoyl-CoA) and quinic acid, and is a typical metabolite of the phenylpropanoid pathway. In summary, using a low-temperature shallow-enzymatic coupled targeted fermentation process, *Lactobacillus plantarum* YS-GuLin18 fermentation of *Eucommia ulmoides* leaf and *Rehmannia glutinosa* combination can directionally activate the phenylpropanoid biosynthetic pathway and induce the synthesis of polyphenolic characteristic small molecule metabolites (chlorogenic acid, caffeic acid, etc.).

[0066] Table 5. Significant pathways of change in beneficial substances Note: Yes: There is a significantly upregulated metabolic pathway between the FD16 vs. D or FD vs. D groups (P < 0.05); No: There is no significantly changed metabolic pathway between the FD16 vs. D or FD vs. D groups. ④ Reduced levels of toxic and harmful components In the fermentation process of food and medicine homology formulations, the trace amounts of toxic and harmful components originally contained in them can be significantly degraded. In particular, after fermentation by low-temperature shallow-enzymatic hydrolysis coupled targeted fermentation process (FD16 vs. D), the highly toxic alkaloids (dimethyl strychnine), carcinogenic / environmental pollutants (1,6-dinitropyrene, imidacloprid), and toxic terpenoids (paclitaxel X, 2-aminobenzothiazole) in the fermentation broth can be significantly reduced.

[0067] Table 6. Significantly Downgraded Toxic and Harmful Components Increase: The components were significantly upregulated in the FD16 vs. D or FD vs. D groups (P < 0.05); Decrease: The components were significantly downregulated in the FD16 vs. D or FD vs. D groups (P < 0.05).

[0068] To further verify the relationship between fermentation products and bone health, related in vivo verification experiments were conducted in zebrafish.

[0069] Example 4 The fermentation broth of the composition promotes bone formation in zebrafish. Healthy 4dpf juvenile fish were divided into six groups: control group (CK group), model group (M group), and sample group (A: conventional fermentation supernatant freeze-dried powder, 62.5 ug / mL; B: low-temperature shallow-enzymatic hydrolysis coupled targeted fermentation supernatant freeze-dried powder, 62.5 ug / mL; C: 62.5 ug / mL composition group (prepared according to the ratio of Eucommia ulmoides leaf powder: Dendrobium officinale powder: Rehmannia glutinosa powder: dried ginger powder in 70wt%:15wt%:10wt%:5wt%), with 10 fish in each group. They were continuously incubated in a constant temperature incubator at 28.5℃.

[0070] The total system was administered via exposure at 4 mL. The administration system is shown in Table 7.

[0071] Table 7. Drug administration system (unit: mL) Dosing cycle: From 4 dpf to 9 dpf, administer for 5 days, changing the incubation medium containing the drug daily at a 1 / 2 ratio (2 mL). The yolk sac of zebrafish juveniles contains abundant nutrients, sufficient for their survival to 9-10 dpf; therefore, feeding is unnecessary during the experiment. Alizarin red staining was used, and the staining results of the zebrafish juvenile head bones were observed and photographed under a biological microscope. Figures 5-9 As shown.

[0072] Compared with the control group (CK), the model group (M) showed blurred skull imaging and significantly underdeveloped spine, proving the modeling was successful. Five clear images of zebrafish with good posture were taken from each group and analyzed using software, such as... Figure 10 As shown.

[0073] Comparing CK and M, the quantitative data showed significant differences, indicating successful modeling. Group B demonstrated the best effect in improving bone health, significantly promoting bone formation in zebrafish. In terms of bone area, Group B was significantly superior to Groups C and A.

[0074] ②RT-PCR detection of related gene expression The specific testing method is as follows: RNA was extracted from zebrafish brain tissue using Trizol reagent. Total RNA was reverse transcribed into cDNA using a reverse transcription kit. The reaction was performed on a Real-time PCR instrument using the SYBR Premix Taq kit. (Following step 2) -ΔΔCt The relative mRNA expression levels of relevant genes in zebrafish were calculated using a method. Primers were designed, with β-actin selected as an internal control.

[0075] runx2a is a transcription factor that plays a crucial role in bone development and bone metabolism. It is a subtype of the runx2 gene, which plays a vital role in bone formation, especially in the differentiation and maturation of osteoblasts.

[0076] ACP5A is an important biomarker in the development of osteoporosis and osteoarthritis. Its role is focused on promoting bone resorption and it is used in clinical practice as an important indicator for assessing bone metabolism.

[0077] Depend on Figure 11 and Figure 12 It can be seen that after modeling, the gene expression levels of runx2a and acp5a increased, and their expression levels decreased after different group treatments. In particular, the expression levels of runx2a and acp5a in group B could be restored to normal.

[0078] In summary, the fermented product obtained after low-temperature shallow-enzymatic targeted fermentation has a significant effect on improving bone health, including osteoarthritis and osteoporosis. This is mainly reflected in increasing bone surface area, downregulating the gene expression of runx2a and acp5a, and restoring normal bone health levels.

Claims

1.A Lactiplantibacillus plantarum YS-GuLin18, characterized in that: The preservation number thereof is CGMCC NO. 35529, and the Latin name thereof is Lactiplantibacillus plantarum . 2.A Zhonghuang fermentation product, characterized in that: fermented from a food-medicine homologous composition by the Lactiplantibacillus plantarum YS-GuLin18 according to claim 1; the food-medicine homologous composition is composed of 70 wt% of Eucommia ulmoides leaves, 15 wt% of Rehmannia glutinosa, 10 wt% of dried ginger, and 5 wt% of Dendrobium officinale. 3.A preparation method of the Zhonghuang fermentation product according to claim 2, characterized in that: comprising the following steps: inoculating a seed liquid of the Lactiplantibacillus plantarum YS-GuLin18 into a sterilized substrate composed of glucose, peptone, dipotassium hydrogen phosphate, a food-medicine homologous composition, and water, then adding β-glucanase into the substrate for first-step fermentation, then adding vitamin C into the substrate for second-step fermentation, then adding quinic acid into the substrate for third-step fermentation, and finally inactivating, centrifuging, and collecting the supernatant. 4.A preparation method of the Zhonghuang fermentation product according to claim 3, characterized in that: the preparation method of the seed liquid comprises the following steps: inoculating the Lactiplantibacillus plantarum YS-GuLin18 into MRS liquid medium for culture to obtain a bacterial liquid, and then inoculating 2% of the bacterial liquid into MRS liquid medium to obtain the seed liquid; The concentration of Lactobacillus plantarum YS-GuLin18 in the seed liquid is 1 x 10 9 CFU / mL. 5.A preparation method of the Zhonghuang fermentation product according to claim 3, characterized in that: the substrate comprises 4-6% of glucose, 0.5-1% of peptone, 0.10-0.25% of dipotassium hydrogen phosphate, and 6-8% of the food-medicine homologous composition. 6.A preparation method of the Zhonghuang fermentation product according to claim 3, characterized in that: the sterilization temperature is 115℃, and the sterilization time is 20 min; the inoculation amount of the seed liquid is 2%. 7.A preparation method of the Zhonghuang fermentation product according to claim 3, characterized in that: the enzyme activity of the β-glucanase is 20 U / g, and the fermentation temperature of the first-step fermentation is 30-32℃; the addition amount of the vitamin C is 0.1%, and the fermentation temperature of the second-step fermentation is 35-37℃; the addition amount of the quinic acid is 0.05%, and the fermentation temperature of the third-step fermentation is 35-37℃. 8.A preparation method of the Zhonghuang fermentation product according to claim 3, characterized in that: the end-point pH of the third-step fermentation is 3.8-4.0; the inactivation temperature is 115℃, and the inactivation time is 30 min; the centrifugation speed is 6000 r / min, and the centrifugation time is 3-5 min. 9.An application of the Zhonghuang fermentation product according to claim 2, characterized in that: the Zhonghuang fermentation product is applied to the preparation of food or medicine for increasing the bone area of the head; or the Zhonghuang fermentation product is applied to the preparation of food or medicine for down-regulating the expression level of runx2a and / or acp5a to improve bone health. 10.An application of the Zhonghuang fermentation product according to claim 2, characterized in that: the Zhonghuang fermentation product is applied to the preparation of food or medicine for improving bone health; or the Zhonghuang fermentation product is applied to the preparation of food or medicine for improving osteoarthritis; or the Zhonghuang fermentation product is applied to the preparation of food or medicine for improving osteoporosis.

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