Lactobacillus plantarum, fermentation liquor of lycopus lucidus, preparation method thereof and application of improving kidney function and endurance

Abstract

The application discloses a plant lactobacillus. The plant lactobacillus has the characteristics of high tolerance to 30% (w / v) cistanche matrix. The application also discloses a homologous medicinal and edible formula which is composed of 85 parts of cistanche, 5 parts of rehmannia root, 5 parts of yizhi kernel and 5 parts of yam. The formula is fermented by the plant lactobacillus, and a cistanche and yam fermented liquid is prepared by adopting a multi-stage temperature fermentation process for 16 hours. Non-targeted metabolomics results show that the plant lactobacillus activates the upstream pathway of phenylethanoid glycoside synthesis, and the abundance of forsythoside B is up-regulated by 133 times compared with that before fermentation. The fermented liquid can play the functions of reproductive anti-aging, kidney protection and body resistance improvement by down-regulating the expressions of COX-2a and TGF-beta-1 genes.

Description

Technical Field

[0001] This invention belongs to the field of probiotic fermentation, specifically relating to a plant lactobacillus, a fermentation broth that improves kidney function and enhances endurance, its preparation method, and its application. Background Technology

[0002] In today's fast-paced, high-pressure society, chronic physical and mental fatigue and insufficient sustained endurance have become common health problems plaguing modern people. This fatigue is particularly pronounced and difficult to alleviate for patients with chronic diseases such as kidney failure. Current mainstream products on the market, such as energy drinks containing caffeine and other stimulants, focus primarily on short-term stimulation and immediate alertness, failing to fundamentally enhance the body's endurance reserves and sustained fatigue resistance. They may even exacerbate long-term fatigue by increasing the metabolic burden on the kidneys.

[0003] Traditional Chinese medicine preparations have accumulated rich experience in improving energy and enhancing endurance. Existing formulations often use tonifying herbs such as Cynomorium songaricum, Epimedium, ginseng, Astragalus membranaceus, Lycium barbarum, Angelica sinensis, and Cistanche deserticola as core ingredients, forming compound formulas based on the principles of monarch, minister, assistant, and guide in traditional Chinese medicine. However, these traditional preparations have several significant drawbacks: First, the processing methods mostly involve decoction, wine soaking, or traditional pill, powder, or paste preparations, which have limited damage to the cell walls of the herbs, resulting in low dissolution rates and poor bioavailability of key active ingredients such as polysaccharides and saponins. Second, the products usually have a slow onset of action, requiring long-term use to see results. More importantly, traditional preparation methods lack targeted conversion and enhancement techniques for the active ingredients, limiting the overall efficacy of the preparations.

[0004] Microbial fermentation technology offers a new approach to overcoming this bottleneck. However, existing technologies mostly employ a single fermentation process, making it difficult to simultaneously optimize cell growth, substrate decomposition, and the conversion of multiple target active ingredients.

[0005] Existing publicly available technologies often employ pretreatment methods such as crushing and enzymatic hydrolysis to improve the release and conversion rates of active ingredients in plants. This process is costly and requires subsequent enzyme inactivation. While fermentation can improve the bioavailability of active substances, the fermentation process is relatively simple and cannot maximize the utilization of strains and raw materials.

[0006] Among the existing published patents, Chinese patent application number CN202510637185.5 provides a fermentation product of Bifidobacterium longum subsp. infantis that enhances male fertility and vitality. However, this invention only uses a single Bifidobacterium longum subsp. infantis to ferment the composition, which results in a long production cycle and high process cost. Moreover, the composition in this invention contains as many as dozens of ingredients, which may lead to insufficient extraction of active ingredients and low utilization rate.

[0007] Although the Chinese patent application with application number CN202410188513.3 uses a single plant lactobacillus to ferment Cistanche deserticola slurry, it emphasizes the optimization of the enzymatic hydrolysis process before fermentation, which is costly to implement in industrial production. Summary of the Invention

[0008] The first objective of this invention is to provide a plant lactobacillus YS-Max09 that can efficiently ferment Cistanche deserticola substrate.

[0009] The second objective of this invention is to provide a Cistanche fermentation broth obtained by fermentation of the aforementioned Lactobacillus plantarum YS-Max09.

[0010] A third objective of this invention is to provide a method for preparing the fermentation broth.

[0011] A fourth objective of this invention is to provide a Cistanche deserticola freeze-dried formulation comprising the above-mentioned fermentation broth.

[0012] The fifth objective of this invention is to provide a method for preparing the Cistanche deserticola freeze-dried preparation.

[0013] The sixth object of the present invention is to provide an application of the aforementioned *Lactobacillus plantarum* YS-Max09.

[0014] The seventh object of the present invention is to provide an application of the Cistanche fermentation broth.

[0015] This invention is achieved through the following technical solution:

[0016] A strain of *Lactiplantibacillus plantarum*, YS-Max09, with accession number CGMCC No. 35530, was deposited on August 5, 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.

[0017] A Cistanche fermentation broth, obtained by fermenting Cistanche deserticola with the aforementioned Lactobacillus plantarum YS-Max09;

[0018] By weight, the Cistanche deserticola formula includes 85 parts of Cistanche deserticola, 5 parts of Rehmannia glutinosa, 5 parts of Alpinia oxyphylla, and 5 parts of Dioscorea opposita.

[0019] The Cistanche fermentation broth contains forsythoside B.

[0020] The preparation method of the Cistanche fermentation broth includes the following steps:

[0021] S1 mixes Cistanche deserticola, Rehmannia glutinosa, Alpinia oxyphylla, and Dioscorea opposita, then pulverizes the mixture to obtain a fine powder of the Cistanche deserticola formula.

[0022] S2 activates Lactobacillus plantarum YS-Max09, then inoculates the resulting bacterial solution into a compound fermentation medium for fermentation to obtain the product.

[0023] The compound fermentation medium includes 8 wt% fine powder of Cistanche deserticola and 92 wt% basal medium;

[0024] The basal culture medium comprises 3.0 w / v glucose, 2.0 w / v peptone, 0.25 w / v dipotassium hydrogen phosphate, and the remainder is water.

[0025] The preparation method of the composite fermentation medium includes the following steps:

[0026] Add the fine powder of the Cistanche deserticola formula to the basal culture medium, adjust the pH to 6.0, sterilize, and cool to room temperature to obtain the product;

[0027] The sterilization includes high-pressure steam sterilization;

[0028] The sterilization temperature is 120-125℃.

[0029] The fermentation includes the steps of inoculating a compound fermentation medium with 5% bacterial solution under aseptic conditions, fermenting at 40°C for 4 hours, fermenting at 36°C for 8 hours, and fermenting at 30°C for 4 hours.

[0030] The fermentation process also includes a step of supplementing with 0.2 w / v% glucose after fermentation at 36°C for 8 hours.

[0031] The method for preparing the Cistanche fermentation broth further includes the following steps:

[0032] After fermentation, the fermentation broth was placed in an environment of 4℃ to stop fermentation, heated at 85℃ for 30 minutes to sterilize, and centrifuged at 5000 rpm for 10 minutes to collect the supernatant, which is the final product.

[0033] The activation includes the following steps:

[0034] Lactobacillus plantarum YS-Max09 was streaked on MRS solid medium for activation, anaerobic culture at 37℃ for 48h, a single milky white colony was picked and inoculated into MRS liquid medium, cultured at 37℃ for 24h, and continuously subcultured for three generations. The OD600 of the bacterial culture was adjusted to 1 with physiological saline to obtain the final product.

[0035] The application of *Lactobacillus plantarum* YS-Max09 is in the preparation of reproductive anti-aging drugs; or

[0036] Used in the preparation of drugs for kidney protection; or

[0037] It can be used to prepare drugs that downregulate the expression of kidney inflammation-related genes COX-2a and TGF-β-1; or

[0038] It is used in the preparation of drugs that enhance the body's endurance.

[0039] The application of the Cistanche fermentation broth is in the preparation of reproductive anti-aging drugs; or

[0040] Used in the preparation of drugs for kidney protection; or

[0041] It can be used to prepare drugs that downregulate the expression of kidney inflammation-related genes COX-2a and TGF-β-1; or

[0042] It is used in the preparation of drugs that enhance the body's endurance.

[0043] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0044] 1. The *Lactobacillus plantarum* YS-Max09 of the present invention, after directed evolution, can tolerate 30% high concentration of *Cistanche deserticola* matrix. The cellulase activity increased from 12.93 U / mL to 35.29 U / mL, and the β-glucosidase activity increased from 19.77 U / mL to 52.60 U / mL. It can efficiently decompose the insoluble fiber of *Cistanche deserticola*, solving the problem of releasing active ingredients.

[0045] 2. The Cistanche deserticola formula of this invention originates from the Cistanche deserticola powder in the *Qianjin Fang* (Prescriptions Worth a Thousand Gold Pieces), a classic representative of kidney tonification and fatigue relief in traditional medicine. This invention optimizes and innovates upon this formula, adjusting the principal, assistant, and adjuvant relationships to form a formula with Cistanche deserticola as the principal herb, Rehmannia glutinosa as the assistant herb, and Alpinia oxyphylla and Dioscorea opposita as adjuvant herbs. These raw materials are rich in polysaccharides, saponins, flavonoids, and other effective components that can improve the body's oxygen utilization efficiency and clear lactic acid produced after exercise, exhibiting significant anti-fatigue and antioxidant effects. Based on the optimized Cistanche deserticola formula from *Qianjin Fang*, combined with fermentation using *Lactobacillus plantarum* strain YS-Max09, the abundance of phenylethanoid glycosides, a core component, in the fermentation broth is significantly increased, with Forsythoside B increasing by 133 times, resulting in a substantial improvement in bioavailability.

[0046] 3. The fermentation method provided by this invention, with its short-time multi-stage temperature fermentation process, increases β-glucosidase activity by 126.8% and polysaccharide content by 38.5% compared to traditional constant-temperature fermentation. This not only shortens the fermentation cycle but also avoids the over-utilization of active ingredients by the strain, thus optimizing both process efficiency and product quality.

[0047] 4. Zebrafish model verification shows that the high-concentration Cistanche fermentation broth provided by this invention can restore the creatinine and urea nitrogen levels of zebrafish with renal failure to near normal levels, which is 84% ​​lower than that of the model group. At the same time, it increases the content of reproductive anti-aging related components, and its functional advantages are significantly better than those of single probiotics. Attached Figure Description

[0048] Figure 1 The PCA score plots for the comparison group FR vs R are shown.

[0049] Figure 2 Volcano plots for the comparison group FR vs R are shown.

[0050] Figure 3 The comparison group FR vs R differential metabolite classification loop diagram (HMDB Super Class) is shown.

[0051] Figure 4 The hierarchical clustering results of differential metabolites in the comparison group FR vs R are shown (only the top 50 with VIP values ​​are plotted).

[0052] Figure 5 The structural formula of N-(p-Hydroxyphenethyl)actinidine is shown.

[0053] Figure 6 The structural formula of N-trans-feruloyltyramine is shown.

[0054] Figure 7 The structural formula of N-cis-Feruloyldopamine is shown.

[0055] Figure 8 The structural formula of Forsythoside B is shown.

[0056] Figure 9 The structural formula of Cistanoside F is shown.

[0057] Figure 10 The images of each group of zebrafish are shown.

[0058] Figure 11 The relative expression levels of COX-2a in the kidneys of zebrafish in each group are shown.

[0059] Figure 12 The relative expression levels of TGF-β-1 in the kidneys of zebrafish in each group are shown. Detailed Implementation

[0060] Example 1

[0061] Screening of Lactobacillus plantarum YS-Max09

[0062] (1) Isolation and identification of strains

[0063] Collect fecal samples from healthy, long-lived elderly individuals (age > 90 years), immediately place them in sterile sampling tubes, store at 4°C, and process as soon as possible. Weigh 1g of sample and add it to 9mL of sterile physiological saline, vortex thoroughly to prepare 10... -1 A homogeneous bacterial suspension was prepared. It was serially diluted 10-fold using physiological saline, and 10⁻⁶ filtrates were selected. -3 Up to 10 -6 100 μL of each dilution solution was spread on MRS solid medium plates containing 1% CaCO3 and incubated at 37°C under anaerobic conditions for 48 hours.

[0064] Single, milky-white, perfectly round colonies with a semi-transparent zone of dissolution were selected and purified by repeated streaking to obtain pure bacteria. The selected strains were evaluated for their probiotic function and safety, ultimately yielding a superior *Lactobacillus plantarum* BS17M3 strain.

[0065] (2) Evaluation of the probiotic function of the strain

[0066] The adhesiveness, gastrointestinal fluid tolerance, safety, and antioxidant activity of Lactobacillus plantarum BS17M3 were determined. The results are shown in the table below. BS17M3 exhibited excellent probiotic properties and safety, with a DPPH free radical scavenging capacity of up to 97.30%, demonstrating strong antioxidant activity.

[0067] Table 1 Evaluation results of BS17M3's probiotic functions, safety, and antioxidant activity

[0068]

[0069] (3) Adaptive evolution of strains

[0070] Through directed adaptive evolution technology, the original strain *Lactobacillus plantarum* BS17M3 was gradually made tolerant to and efficiently fermented with high concentrations of *Cistanche deserticola* substrate. Ultimately, a domesticated strain was obtained that could grow normally at a concentration of 30% (w / v) *Cistanche deserticola* and had enhanced metabolic activity, especially a strain with significantly improved cellulase and β-glucosidase activity, which was used for subsequent efficient decomposition of insoluble fiber in *Cistanche deserticola*.

[0071] First, BS17M3 was inoculated into commercially available MRS liquid medium (purchased from Guangdong Huankai Microbial Technology Co., Ltd.) and cultured at 37℃ for 24 hours, followed by three generations of activation subculturing. After stabilization, the BS17M3 strain was inoculated at 2% into a 5% concentration of Cistanche deserticola medium and cultured at 37℃. After three consecutive generations of stable growth, it was successively subcultured at 2% into higher concentrations (10%, 15%, 20%, 25%, and 30%) of Cistanche deserticola medium. Each concentration gradient underwent at least 3-5 generations of continuous subculturing. OD was monitored in real-time using a microplate reader during the culture process. 600 Based on the nm value, a domesticated strain was finally selected that could stably grow to OD600nm≥1.0 within 24 hours in 30% Cistanche deserticola medium, and whose cellulase and β-glucosidase activities were increased by more than 150% compared with the original strain in 5% substrate. This strain was named YS-Max09.

[0072] Table 2 Results of adaptive evolution of YS-Max09

[0073]

[0074] The strain was sequenced using 16S rDNA universal primers, and the gene sequence of Lactobacillus plantarum YS-Max09 is shown in SEQ ID NO. 1.

[0075] SEQ ID NO. 1

[0076]

[0077] Example 2

[0078] Preparation method of Cistanche fermentation broth

[0079] (1) Formulation preparation

[0080] The formulation of the Cistanche fermentation liquid of this invention is directly inspired by the Cistanche powder recorded in the Tang Dynasty classic of traditional Chinese medicine, *Qianjin Fang*. This formula inherits its core treatment principle of tonifying the kidneys and replenishing essence, while reducing the amount of herbs in the ancient formula that place a heavy burden on the kidneys. Based on the ancient formula, this invention strictly adheres to the principle of food and medicine sharing the same origin, making key optimizations and adjustments to the original formula, simplifying the formulation, and increasing the proportion of the principal herb, Cistanche deserticola. The final ingredients are determined to be Cistanche deserticola, Rehmannia glutinosa, Alpinia oxyphylla, and Dioscorea opposita, with a mass ratio of 85:5:5:5. The above mass ratio can have an error margin of plus or minus five percent.

[0081] In this innovative formulation:

[0082] As the principal herb, Cistanche deserticola's core functions are to tonify kidney yang and replenish essence and blood.

[0083] The core function of Rehmannia glutinosa is to nourish yin and blood to achieve yin-yang balance;

[0084] The core function of Alpinia oxyphylla is to warm the spleen and kidneys, consolidate essence and reduce urination, and enhance the warming and tonifying effect of the principal herb on kidney yang.

[0085] The core functions of yam are to invigorate the spleen and replenish qi, and to strengthen the kidneys and replenish essence.

[0086] The above four medicinal materials are screened, cleaned, and dried, and then accurately weighed according to the proportions. After being mixed evenly, they are pulverized through an 80-mesh sieve to obtain a finely mixed powder.

[0087] (2) Preparation of fermentation medium

[0088] This embodiment uses an optimized liquid basal culture medium with the following formula: glucose 3.0% (w / v), peptone 2.0% (w / v), and dipotassium hydrogen phosphate 0.25% (w / v). During preparation, each component is accurately weighed and dissolved in an appropriate amount of purified water, stirred thoroughly until completely dissolved. Then, 8% (w / v) of the Cistanche deserticola compound powder is added, mixed thoroughly, and then brought to the required total volume with purified water. The prepared culture medium is dispensed into fermenters, the pH is adjusted to 6.0, and autoclaved at 121°C for 20 minutes. After cooling to room temperature, the resulting composite culture medium is ready for subsequent gradient fermentation. This culture medium not only provides the basic carbon and nitrogen sources and buffer system required for cell growth, but more importantly, it directly uses the medicinal and edible compound as a fermentation substrate, laying the foundation for the targeted biotransformation of the core functional components.

[0089] (3) Short-term fermentation and preparation of Cistanche fermentation broth based on adaptive evolution strain YS-Max09

[0090] The core of this fermentation process lies in maximizing the metabolic and enzyme-producing potential of the microorganisms through precise temperature gradients within a very short time. The entire fermentation process is completed within 16 hours, and the specific operating steps are as follows:

[0091] ① Strains Preparation and Inoculation: The strain used in this example is *Lactobacillus plantarum* YS-Max09, obtained through directed adaptive evolution technology. Before inoculation into the Cistanche deserticola formulation medium, it was activated for 2-3 generations using commercially available ordinary MRS medium to enhance its activity. After activation, the culture was diluted to OD500. 600 =1, inoculate 5% of the Cistanche deserticola formula into the culture medium.

[0092] ② First stage (0-4h): During this stage, the fermentation temperature was controlled at 40℃, which is higher than the optimal growth temperature of the strain. This applied heat stress to the cells, effectively activating their stress response and significantly accelerating their metabolic initiation and the synthesis of primary enzymes, laying the foundation for rapid substrate decomposition. After this stage, the cellulase activity in the fermentation broth was measured to be 39.83 U / mL, providing a strong prerequisite for the subsequent conversion and release of active ingredients.

[0093] ③ Second stage (4h-12h): During this stage, the fermentation temperature is controlled at 36℃, which is the optimal range for enzyme production by the cells. After experiencing heat shock stress, the cells enter a high-speed synthesis and transformation stage at this temperature, efficiently converting glycosides in Cistanche deserticola that are difficult to function into active monomers. After this stage, the β-glucosidase activity and cellulase activity in the fermentation broth were measured to be 69.83 U / mL and 52.17 U / mL, respectively, reaching the fermentation peak.

[0094] ④ Third stage (12-16h): The fermentation temperature is controlled at 30℃ in this stage. This low temperature environment greatly slows down the growth and proliferation of the cells and prevents the strain from over-utilizing the active ingredients in the fermentation broth. At the same time, 0.2% glucose is added to the system at 12h to provide a rapid carbon source for the growth of the strain and further prevent the strain from utilizing the polysaccharides in Cistanche deserticola.

[0095] ⑤ Termination of fermentation: After 16 hours, the fermentation broth was placed at 4℃ to terminate fermentation. The fermentation broth was collected and sterilized at 85℃ for 30 minutes, and centrifuged at 5000 rpm for 10 minutes to obtain Cistanche fermentation broth.

[0096] Note: During the above fermentation stages, the fermentation speed should be maintained at 160 rpm.

[0097] Comparative Example 1: To verify the superiority of the multi-stage temperature fermentation in this embodiment, a single-temperature fermentation group (37℃ constant temperature fermentation for 16h) was set up as a comparative example, with other fermentation conditions remaining unchanged. The results are shown in the table below. Multi-stage fermentation improved the release of poorly soluble active ingredients in the formulation and prevented the over-utilization and decomposition of active ingredients in the fermentation broth by *Lactobacillus plantarum* YS-Max09.

[0098] Table 3 Comparison of 16h short-time fermentation effects

[0099]

[0100] Example 3

[0101] Non-targeted metabolomics analysis of Cistanche fermentation broth based on UPLC-Q-Exactive Plus MS technology

[0102] (1) Sample preparation

[0103] The fermented Cistanche broth was collected, with the unfermented Cistanche culture medium used as a control. Each sample was centrifuged at 12,000 rpm for 10 minutes at 4°C. 200 μL of the supernatant was precisely pipetted and mixed with 800 μL of pre-cooled methanol-acetonitrile mixture (1:1, v / v). The mixture was vortexed for 30 seconds and then allowed to stand at -20°C for 1 hour to precipitate the protein. Subsequently, the mixture was centrifuged again at 12,000 rpm for 15 minutes at 4°C. All the supernatant was precisely pipetted and gently dried using a nitrogen blower. The resulting residue was reconstituted with 100 μL of acetonitrile-water solution (1:1, v / v), vortexed for 2 minutes, and centrifuged under the same conditions. Finally, the supernatant was injected into the inner liner tube for analysis.

[0104] (2) On-machine testing

[0105] Liquid chromatography conditions: Throughout the analysis, the sample was placed in an autosampler at 4°C and analyzed using a SHIMADZU-LC30 ultra-high performance liquid chromatography (UHPLC) system. The chromatographic column used was an ACQUITY UPLC® HSS T3 (2.1 × 100 mm, 1.8 µm) (Waters, Milford, MA, USA). The injection volume was 16 μL, the column temperature was 40°C, and the flow rate was 0.3 mL / min. The mobile phases were: A: 0.1% formic acid aqueous solution, B: 0.1% formic acid acetonitrile solution; the gradient elution program was as follows:

[0106] 0.01–1 min, 2% B; 1–5 min, B changes linearly from 2% to 48%; 5–7 min, B changes linearly from 48% to 80%; 7–11 min, B changes linearly from 80% to 100%; 11–13 min, B remains at 100%; 13–13.01 min, B changes linearly from 100% to 2%; 13.01–15 min, B remains at 2%.

[0107] Mass spectrometry conditions: Acquisition time: 15 min. Precursor ion scan range: 75-1050 m / z, primary mass spectrometry resolution: 70,000 m / z 200, AGC target: 3e6, primary maximum IT: 100 ms. Secondary mass spectrometry analysis was performed using the following method: after each full scan, secondary mass spectra of the 10 highest intensity precursor ions were acquired (MS2 scan). Secondary mass spectrometry resolution: 17,500 m / z 200, AGC target: 1e5, secondary maximum IT: 50 ms, MS2 activation type: HCD, isolation window: 2 m / z, normalized collision energy (Setpped): 20, 30, 40.

[0108] (3) Results Analysis

[0109] The collected raw data were processed, including peak extraction, peak alignment, and normalization, and compounds were identified using a database. The principal component analysis plot is shown below. Figure 1 As shown. From Figure 1 It can be clearly seen that the Cistanche fermentation broth (FR group) showed significant material changes compared with before fermentation. The degree of separation of sample points within each group was small, indicating that the process had good repeatability.

[0110] The differential metabolites in the samples before and after fermentation were analyzed, and the results are as follows: Figure 2 and Figure 3 As shown, among the 670 differentially expressed metabolites detected, 423 were significantly upregulated and 247 were significantly downregulated. The differentially expressed metabolites were mainly organic acids and their derivatives, and phenylpropanes and polyketides. Organic acids and their derivatives accounted for 29.41%, while phenylpropanes and polyketides accounted for 25.18%.

[0111] The top 50 substances with the most significant upward and downward adjustments were analyzed, and the heatmap of substance changes is shown below. Figure 4As shown. Among the upregulated differential metabolites, those playing a key role in male reproductive anti-aging and improving renal failure include: Deoxyloganic acid tetraacetate, N-(p-Hydroxyphenethyl)actinidine, Saucerneol F, N-trans-feruloyltyramine, Spermine, Spermidine, N1-Acetylspermidine, N1,N8-Diacetylspermidine, Disinapoylputrescine, (-)-Epigallocatechin, Estriol, Ferulic acid 4-O-sulfate, Cordycepin, Palmatine, Corydalin, Forsythoside B, and 5'-Methylthioadenosine cover the core dimensions of anti-aging, including hormone regulation, anti-oxidative stress, germ cell protection, endocrine axis regulation, and kidney protection. Among them, Estriol, (-)-Epigallocatechin, and N-(p-Hydroxyphenethyl)actinidine are the highest priority core anti-aging substances. In addition, 5'-Methylthioadenosine is a purine nucleoside compound derived from the metabolism of the strain MTA. It has extremely strong antioxidant activity, scavenging reactive oxygen species (ROS) in the reproductive system and reducing DNA oxidative damage in sperm. This also indirectly demonstrates that the multi-stage temperature fermentation used in this invention successfully and efficiently activated the metabolic activity of the strain and improved the antioxidant activity of the fermentation broth.

[0112] Table 4 shows the phenylethyl glycosides and their derivatives with increased abundance. The accumulation of N-(p-Hydroxyphenethyl)actinidine, N-trans-feruloyltyramine, and N-cis-Feruloyldopamine collectively indicates the overall activation of the phenylethyl metabolic network. In the metabolite abundance identification, we found that the abundance of Forsythoside B was increased by 133 times compared with that before fermentation, and the abundance of Cistanoside F was increased by 29.54%. Phenylethyl glycosides with increased abundance but no significant difference included Tubuloside C and Acteoside, which fully demonstrate that the fermentation of YS-Max09 can increase the content of phenylethyl glycosides in the fermentation broth by synthesizing phenylethyl glycoside precursors.

[0113] The biosynthesis pathway of phenylethyl glycosides in Cistanche deserticola can be divided into two parts: an upstream pathway and a downstream pathway. The upstream pathway mainly involves the synthesis of phenylethyl groups. In this stage, under the action of microorganisms, tyrosine is gradually converted into tyrosol (phenylethyl skeleton) by tyrosine transaminase (CtTAT) and phenylpyruvate decarboxylase (CtPPDC). The downstream pathway involves the gradual modification of the functional groups on the phenylethyl group through enzymatic reactions (glucosylation, acylation, hydroxylation, etc.), ultimately synthesizing complex phenylethyl glycosides and their related metabolites. This invention identified five key compounds in the fermentation broth ( Figure 5-9 ): N-(p-Hydroxyphenethyl)actinidine (N-(p-hydroxyphenethyl)actinidine) Figure 5 ), which are phenethyl compounds; N-trans-feruloyltyramine (N-trans-feruloyltyramine) and N-cis-Feruloyldopamine (N-cis-Feruloyldopamine) Figure 6 , 7 ), are phenylacetamide compounds; Forsythoside B and Cistanoside F ( Figure 8 , 9These compounds are phenylethanol glycosides. All of them contain a phenylethyl core skeleton. Phenylacetamides are end products generated from phenylethylamine precursors via an independent amidation pathway, parallel to the main esterification / glycosylation pathway of phenylethanol glycosides, representing a downstream parallel pathway in phenylethanol glycoside biosynthesis. Cistanoside F and Forsythoside B share the same core structural unit, o-dihydroxyphenylethyl, exhibiting high homology and structural similarity, and are different products from the same phenylethanol glycoside core synthetic pathway. The above metabolographic profiles suggest that fermentation treatment may have highly activated the upstream pathway of phenylethanol glycoside synthesis, generating abundant phenylethylamine precursors, which are then fully utilized by multiple downstream parallel pathways.

[0114] To verify this hypothesis, the upstream rate-limiting enzyme tyrosine transaminase (CtTAT) was tested. The results showed that the activity of CtTAT increased by 281.69% compared to before fermentation. Simultaneously, differential metabolite analysis revealed a significant decrease in the abundance of the dipeptide Aspartyl-Tyrosine. These two results corroborate each other, confirming that the fermentation treatment of this invention specifically and efficiently activates the upstream biosynthetic pathway from tyrosine to the phenylethylamine backbone, accelerating the targeted conversion and supply of precursor substances.

[0115] Table 4. Substances in differentially metabolites related to the characteristic active components of Cistanche deserticola.

[0116]

[0117] Table 5. Tyrosine transaminase activity before and after fermentation

[0118]

[0119] Based on the results of non-targeted metabolomics research, it can be preliminarily determined that the Cistanche fermentation broth prepared in this invention may have potential reproductive anti-aging and kidney anti-inflammatory repair functions. The phenylethanoid glycosides with increased abundance in the fermentation broth have extremely strong antioxidant activity. They can activate the Nrf2 / HO-1 antioxidant pathway by scavenging free radicals, reducing the accumulation of oxidative damage products. Furthermore, they can reduce the release of pro-inflammatory factors (TNF-α, IL-6, IL-1β) by inhibiting the NκF-B / MAPK inflammatory signaling pathway, thereby alleviating tissue damage caused by inflammatory infiltration (such as renal interstitial inflammation and chronic inflammation of reproductive organs).

[0120] Example 4

[0121] Functional validation of Cistanche fermentation broth based on a zebrafish renal failure model

[0122] (1) Sample preparation

[0123] The Cistanche fermentation broth was prepared according to the method provided in Example 2. It was rotary evaporated at 45°C to 20% of its original volume. The concentrated broth was transferred to a freeze-drying bottle and pre-frozen at -80°C for 2 hours. Subsequently, it was transferred to a freeze dryer and freeze-dried for 24 hours at a cold trap temperature of -55°C and a vacuum degree <10 Pa, finally obtaining a brownish-yellow Cistanche fermentation broth freeze-dried powder. This freeze-dried powder was stored in a desiccator and dissolved and diluted to the required concentration using zebrafish culture water before use.

[0124] (2) Zebrafish rearing and grouping

[0125] Wild-type AB zebrafish (broodstock sourced from Hangzhou Huante Biotechnology) were raised in a standard environment. Under standard laboratory conditions (water temperature 28±1℃, conductivity 500-550 μS / cm, light / dark cycle 14 / 10 hours) in an automated circulating culture system, zebrafish embryos were incubated in a 28℃ constant-temperature programmable light incubator. 4-day-fly (dpf) wild-type AB zebrafish were randomly selected and placed in 6-well plates, with three replicates per well (30 fish / well), and a well volume of 8 mL. Specific groupings and treatments were as follows: ① Normal control group (Group C): cultured in standard water without any induction or drug treatment; ② Renal failure model group (Group M): zebrafish larvae were induced for 24 hours with 20 μmol / L aristolochic acid A added to the culture water, and then cultured in standard water after modeling; ③ YS-Max09 group: after modeling, resuspended YS-Max09 bacterial solution was added to the culture water to a final concentration of 10. 9 CFU / mL; ④ Low concentration Cistanche fermentation broth group (L-FR group): After modeling, Cistanche fermentation broth lyophilized powder was added to the aquaculture water, with a final concentration of 50 μg / mL; ⑤ High concentration Cistanche fermentation broth group (H-FR group): After modeling, Cistanche fermentation broth lyophilized powder was added to the aquaculture water, with a final concentration of 100 μg / mL.

[0126] (3) Swimming endurance test

[0127] Zebrafish were placed in a swimming tunnel with a water flow velocity starting at 9 cm / s and increasing by 2 cm / s per minute. The time to exhaustion (s) and the critical swimming velocity (Ucrit, cm / s) leading to exhaustion were recorded for each zebrafish. The results are shown in Table 6. Under the intervention of YS-Max09 and Cistanche fermentation broth, the critical swimming velocity and time to exhaustion of zebrafish were significantly increased and prolonged compared with the renal failure model group.

[0128] Table 6 Results of critical swimming speed and time to exhaustion for zebrafish in each group

[0129]

[0130] (4) Detection of renal function related indicators

[0131] After image acquisition, zebrafish were transferred to pre-chilled 1.5 mL centrifuge tubes (3 fish per tube, n=5), and 300 μL of pre-chilled RIPA lysis buffer (containing 1% PMSF protease inhibitor) was added. Mechanical homogenization was performed using a handheld electric homogenizer (IKA T10basic) under ice bath conditions (4000 rpm, 10-second intervals, 15-second intervals, repeated 5 times). The homogenate was incubated at 4°C for 30 minutes, followed by centrifugation at 12000 rpm for 15 minutes at 4°C. The supernatant was carefully aspirated, aliquoted, and stored at -80°C for subsequent biochemical assays.

[0132] Figure 10 Images of zebrafish from each group were collected. It was evident that YS-Max09 and Cistanche fermentation broth significantly improved aristolochic acid-induced swelling in zebrafish and alleviated abdominal edema caused by renal failure. The results of renal biochemical indicators are shown in Table 7. Compared with the normal control group (C), the renal failure model group (M) showed significantly increased levels of creatinine, blood urea nitrogen, and the inflammatory factor TNF-α, indicating the successful establishment of the aristolochic acid A-induced acute kidney injury model. Nitrogenous metabolic waste accumulated in the zebrafish, accompanied by a strong inflammatory response. After different interventions, the biochemical indicators of each treatment group showed regular changes. While the YS-Max09 probiotic group showed a certain protective trend, the effect was limited. The Cistanche fermentation broth intervention group, however, showed a significant, dose-dependent improvement in renal function. The high-dose group (H-FR) showed the most outstanding effect, with its creatinine and blood urea nitrogen levels recovering to levels close to the normal control group, a reduction of 84% compared to the model group.

[0133] Table 7 Results of biochemical indicators of zebrafish kidneys in each group

[0134]

[0135] To further explore the protective effect of Cistanche fermentation broth against renal failure at the molecular level, this invention used qPCR technology to detect the mRNA expression levels of cyclooxygenase-2a (COX-2a) gene, which is closely related to inflammatory response, and transforming growth factor-β-1 (TGF-β-1) gene, which is directly related to the initiation of renal fibrosis, in zebrafish kidney tissue. PRC results showed ( Figure 11 , 12 Treatment with aristolochic acid A increased the expression levels of COX-2a and TGF-β-1 in zebrafish kidneys, while treatment with YS-Max09 and Cistanche fermentation broth significantly reduced their expression levels.

[0136] Based on the comprehensive analysis of the above renal biochemical indicators, high-dose Cistanche fermentation broth (H-FR) exhibits excellent therapeutic effects. This biochemical analysis confirms the remarkable efficacy of the product of this invention in clearing uremic toxins and inhibiting renal inflammation, and also provides a solid mechanistic explanation for its ability to improve systemic fatigue and decreased endurance caused by renal failure.

[0137] Table 8 PCR Primer Sequences

[0138]

[0139] The above provides a detailed description of a pipeline safety monitoring method and device provided in this application. Specific examples have been used to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only intended to help understand the method and core ideas of this application. It should be noted that those skilled in the art can make various improvements and modifications to this application without departing from its principles, and these improvements and modifications also fall within the protection scope of this application.

Claims

1. A plant lactobacillus ( Lactiplantibacillus plantarum YS-Max09, characterized in that: Its accession number is CGMCC No.35530.

2. A Cistanche fermentation broth, characterized in that: The formula obtained by fermenting Cistanche deserticola with Lactobacillus plantarum YS-Max09 as described in claim 1; Based on the weight, the Cistanche deserticola formula consists of 85 parts Cistanche deserticola, 5 parts Rehmannia glutinosa, 5 parts Alpinia oxyphylla, and 5 parts Dioscorea opposita.

3. The Cistanche fermentation broth according to claim 2, characterized in that: The Cistanche fermentation broth contains forsythoside B.

4. The method for preparing the Cistanche fermentation broth according to claim 2, characterized in that, Includes the following steps: S1 mixes Cistanche deserticola, Rehmannia glutinosa, Alpinia oxyphylla, and Dioscorea opposita, then pulverizes the mixture to obtain a fine powder of the Cistanche deserticola formula. S2 activates Lactobacillus plantarum YS-Max09, then inoculates the resulting bacterial solution into a compound fermentation medium for fermentation to obtain the product. The compound fermentation medium includes 8 wt% fine powder of Cistanche deserticola and 92 wt% basal medium; The basal culture medium comprises 3.0 w / v glucose, 2.0 w / v peptone, 0.25 w / v dipotassium hydrogen phosphate, and the remainder is water.

5. The method for preparing the Cistanche fermentation broth according to claim 4, characterized in that: The preparation method of the composite fermentation medium includes the following steps: Add the fine powder of the Cistanche deserticola formula to the basal culture medium, adjust the pH to 6.0, sterilize, and cool to room temperature to obtain the product; The sterilization includes high-pressure steam sterilization; The sterilization temperature is 120-125℃.

6. The method for preparing the Cistanche fermentation broth according to claim 4, characterized in that: The fermentation includes the following steps: under aseptic conditions, inoculating a compound fermentation medium with 5% bacterial solution, fermenting at 160 rpm, fermenting at 40°C for 4 hours, fermenting at 36°C for 8 hours, and fermenting at 30°C for 4 hours. The fermentation process also includes a step of supplementing with 0.2 w / v% glucose after fermentation at 36°C for 8 hours.

7. The method for preparing the Cistanche fermentation broth according to claim 4, characterized in that: It also includes the following steps: After fermentation, the fermentation broth was placed in an environment of 4℃ to stop fermentation, heated at 85℃ for 30 minutes to sterilize, and centrifuged at 5000 rpm for 10 minutes to collect the supernatant, which is the final product.

8. The method for preparing the Cistanche fermentation broth according to claim 4, characterized in that: The activation includes the following steps: Lactobacillus plantarum YS-Max09 was streaked onto MRS solid medium for activation and cultured anaerobically at 37°C for 48 hours. A single milky-white colony was picked and inoculated into MRS liquid medium and cultured at 37°C for 24 hours. After three generations of continuous subculturing and activation, the OD600 of the bacterial culture was adjusted to 1 with physiological saline.

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