Lactococcus lactis CCFM1519 capable of converting EGCG (epigallocatechin gallate) to generate high-activity substances and enhance fatigue resistance
By fermenting EGCG with Lactococcus lactis subsp. CCFM1519, active substances such as 5-(4-hydroxyphenyl)valerate and 4-hydroxybenzoic acid are generated, which solves the problem of low absorption rate of EGCG in vivo and achieves significant anti-fatigue effect.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JIANGNAN UNIV
- Filing Date
- 2026-01-12
- Publication Date
- 2026-04-28
AI Technical Summary
In the existing technology, epigallocatechin gallate (EGCG) has low absorption rate and bioavailability in the body, making it difficult to achieve the best efficacy. Moreover, the bioavailability varies, which cannot effectively improve the anti-fatigue effect.
A strain of Lactococcus lactis subsp. lactis, CCFM1519, was provided, which can convert EGCG during fermentation to produce active substances such as 5-(4-hydroxyphenyl)valerate and 4-hydroxybenzoic acid, thereby enhancing the anti-fatigue effect of EGCG.
Fermentation of EGCG by Lactococcus lactis subsp. CCFM1519 significantly improved the degradation rate of EGCG. The generated active substances could significantly alleviate fatigue, improve the behavioral performance of mice, enhance the ability to clear serum metabolites, improve antioxidant capacity, and increase the expression of anti-fatigue related genes.
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Abstract
Description
Technical Field
[0001] This invention relates to a strain of Lactococcus lactis CCFM1519 that transforms EGCG to produce more active substances that enhance anti-fatigue properties, belonging to the field of microbial technology. Background Technology
[0002] Fatigue is a common symptom in the general population. Normal fatigue usually occurs after strenuous physical labor and can be relieved by rest or lifestyle changes. In contrast, pathological fatigue does not improve with rest and is experienced by many patients with chronic diseases. It is more intense, lasts longer, and severely impairs an individual's functional activities and quality of life. Fatigue is classified into central fatigue and peripheral fatigue based on its location. Central fatigue is caused by dysfunction of the central nervous system, while peripheral fatigue is usually caused by factors outside the central nervous system. Because the clinical manifestations of different types of fatigue are often mixed and the classification boundaries are not clear, researchers have gradually realized that fatigue cannot be completely separated for research based on its classification type. Fatigue cannot be considered solely as an effect produced by the central nervous system or the peripheral system; it is often the result of signal interaction. Fatigue not only refers to physical exhaustion and decreased physical ability, but also includes mental lethargy, depression, anxiety, memory loss, and diminished willpower, among other things.
[0003] Polysaccharides, flavonoids, and polyphenols isolated from natural drugs are potential anti-fatigue agents. Epigallocatechin gallate (EGCG) is a typical flavonoid-3-ol phenolic compound containing eight free hydroxyl groups and possesses various biological activities and physiological functions. However, EGCG has low absorption and bioavailability in vivo, making it difficult to reach concentrations that achieve optimal efficacy, and its bioavailability varies among different species. Therefore, obtaining strains capable of metabolizing EGCG and enhancing its anti-fatigue effects, and applying them to food, health products, or pharmaceuticals, is of great significance. Summary of the Invention
[0004] To address the shortcomings of existing technologies, this invention provides a strain of *Lactococcus lactis* subsp. *lactococcus* capable of converting EGCG to enhance its fatigue-relieving effects. Lactococcus lactis subsp. lactis CCFM1519 was mentioned, and its application was provided.
[0005] This invention provides a strain of Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. lactis CCFM1519, the Lactococcus lactis subsp. lactis, was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 1, 2025, with accession number GDMCC No: 66903.
[0006] This invention provides a microbial preparation containing the above-mentioned Lactococcus lactis subsp. CCFM1519.
[0007] In one embodiment, the content of *Lactococcus lactis* subsp. *CCFM1519* in the microbial preparation is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.
[0008] In one embodiment, the microbial preparation is a solid or liquid preparation.
[0009] This invention provides an EGCG fermentation product, which is a fermentation supernatant obtained by co-fermentation of Lactococcus lactis subsp. CCFM1519 and EGCG.
[0010] This invention provides a method for preparing the EGCG ferment, comprising the following steps: (1) The fermentation substrate containing EGCG was mixed with Lactococcus lactis subsp. CCFM1519 and fermented to obtain a fermentation mixture; (2) Centrifuge the fermentation mixture in step (1), collect the fermentation supernatant, and obtain EGCG fermentation product.
[0011] In one implementation, step (1) ensures a final concentration of not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 Fermentation was carried out after mixing CFU / g of Lactococcus lactis subsp. lactis CCFM1519 with the fermentation substrate.
[0012] In one implementation, the method includes the following steps: (1) Inoculate Lactococcus lactis subsp. CCFM1519 into the culture medium and culture at 35-40℃ for 48 h to obtain bacterial culture; (2) Add the bacterial solution obtained in step (1) to the fermentation substrate containing EGCG at an inoculation rate of 5% (v / v), ferment at 25-40℃ for 48h, and collect the fermentation supernatant.
[0013] In one embodiment, the EGCG-containing fermentation substrate comprises EGCG 150-250 mg / L, peptone 8-12 g / L, beef extract 8-12 g / L, glucose 15-25 g / L, sodium acetate 1.5-2.5 g / L, yeast powder 4-6 g / L, diammonium hydrogen citrate 1.5-2.5 g / L, K2PO4·3H2O 2-3 g / L, MgSO4·7H2O 0.08-0.15 g / L, MnSO4 0.04-0.06 g / L, and Tween 0.8-1.2 mL / L.
[0014] The present invention also provides a product containing the above-mentioned Lactococcus lactis subsp. CCFM1519, microbial preparations, or EGCG fermentation products.
[0015] In one embodiment, the product is food, medicine, or health product.
[0016] In one embodiment, the food is a dairy product, soy product, or fruit and vegetable product produced using at least one of the above-mentioned EGCG fermentation product, Lactococcus lactis subsp. CCFM1519, or microbial preparations.
[0017] In one embodiment, the dairy products include fermented milk, flavored fermented milk, fermented milk beverages, cream, cheese, milk-containing beverages, or milk powder; the soy products include soy milk and soy milk powder; and the fruit and vegetable products include fruit and vegetable products made from at least one of apple, grape, yellow peach, or bayberry products.
[0018] In one embodiment, the food is a fermented food, including solid food, liquid food, or semi-solid food.
[0019] In one embodiment, the food is a beverage or snack containing at least one of the above-mentioned EGCG fermentation product, Lactococcus lactis subsp. CCFM1519, or microbial preparations.
[0020] In one embodiment, the pharmaceutical product contains at least one of the above-mentioned EGCG fermentation product, Lactococcus lactis subsp. CCFM1519, or microbial preparation as a drug carrier and / or pharmaceutical excipient.
[0021] In one embodiment, the carrier includes one or more of the following commonly used in medicine: fillers, adhesives, wetting agents, disintegrants, lubricants, and flavoring agents.
[0022] In one embodiment, the dosage form of the drug is granules, capsules, tablets, pills, or oral liquid.
[0023] The present invention also provides the application of the above-mentioned Lactococcus lactis subsp. CCFM1519, or microbial preparations, or the EGCG fermentation product in the preparation of pharmaceuticals or health products for relieving physical fatigue.
[0024] In one embodiment, the medicine or health product has at least one of the following effects (1)-(4): (1) Enhance the body's water maze memory level; (2) Reduce the accumulation of serum metabolites (BLA, BUN) in the body; (3) Enhance the body's antioxidant capacity (SOD, GSH-PX, MDA); (4) Increase the expression of fatigue-related genes (MCT4, LDHB, BDNF, TrkB, DRD1).
[0025] This invention also provides the application of the above-mentioned Lactococcus lactis subsp. CCFM1519 in the transformation of EGCG.
[0026] Beneficial effects: 1. The Lactococcus lactis subsp. CCFM1519 provided by this invention has the ability to convert EGCG. After fermentation in a fermentation substrate containing EGCG, the degradation rate of EGCG reaches 40%, and active substances such as 5-(4-hydroxyphenyl)valerate and 4-hydroxybenzoic acid are generated.
[0027] 2. The *Lactococcus lactis* subsp. CCFM1519 provided by this invention can promote the efficacy of EGCG, and the transformed EGCG can enhance the ability to relieve fatigue, specifically manifested as follows: (1) Fermentation of EGCG by Lactococcus lactis subsp. CCFM1519 can improve the behavioral performance of mice. The latency in the water maze can be shortened from 55.52±7.97 seconds to 28.56±19.82 seconds, which is 13.56% shorter than that in the EGCG group; (2) Fermentation of EGCG by Lactococcus lactis subsp. CCFM1519 can enhance the ability to clear serum metabolites. The LA level was reduced by 23.68% and 4.42% compared with the model group and the EGCG group, respectively, and the BUN level was reduced by 16.97% and 7.90% compared with the model group and the EGCG group, respectively. (3) Fermentation of EGCG by Lactococcus lactis subsp. CCFM1519 can improve the antioxidant capacity of mice. Compared with the model group, SOD increased from 65.74±14.20 to 97.58±9.76, and GSH-PX activity increased from 1573.77±113.86 to 1868.85±66.71. Compared with the EGCG group, all indicators were significantly improved. (4) Fermentation of EGCG by Lactococcus lactis subsp. CCFM1519 can enhance the expression of anti-fatigue related genes. Compared with the model group, the expression level of BDNF increased from 0.54±0.14 to 0.86±0.14, the expression level of TrkB increased from 0.52±0.11 to 0.83±0.16, and the expression level of DRD1 increased from 0.54±0.19 to 0.91±0.28, which increased by 48.15%, 52.69%, and 40%, respectively. Compared with the EGCG group, the expression levels of each gene increased by 18.08%, 15.56%, and 21.33%, respectively.
[0028] Products with enhanced anti-fatigue effects can be produced using Lactococcus lactis subsp. CCFM1519 and EGCG. The production process, using EGCG as a raw material, enhances its efficacy. This process is safe, efficient, low-cost, and mild, making it suitable for large-scale industrial production. Therefore, the application of Lactococcus lactis subsp. CCFM1519 to EGCG-containing products has enormous application potential and a solid foundation.
[0029] Preservation of biological materials A strain of *Lactococcus lactis* subsp. CCFM1519, taxonomically named Lactococcus lactis subsp. lactis It was deposited on September 1, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66903, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description
[0030] Figure 1 High-performance liquid chromatograms of EGCG before and after fermentation; A: Lactococcus lactis subsp. CCFM1519 before fermentation; B: Lactococcus lactis subsp. CCFM1519 after fermentation; C: Lactococcus lactis subsp. FBJ3T3 after fermentation; Figure 2 LC-MS images of EGCG before and after fermentation; Figure 3 LC-MS chromatogram of 5-(4-hydroxyphenyl)valerate standard; Figure 4 LC-MS chromatogram of 4-hydroxybenzoic acid standard; Figure 5 The latency period of mice in different groups in the water maze; Figure 6 Serum metabolite levels in mice from different groups; Figure 7 Oxidative stress levels in mice of different groups; Figure 8 : Metabolite-related target levels in mice of different groups; Figure 9 Levels of oxidative stress-related targets in mice from different groups; Figure 10 Levels of neuroprotective targets in mice from different groups. Detailed Implementation
[0031] The SPF-grade male C57BL / 6J mice (6 weeks old, 20±2 g) used in the following examples were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; the EGCG used in the following examples was purchased from Maclean's Reagent Company; chloroform, isopropanol, ethanol, TRIzol, DEPC-treated water, and sodium chloride were from China National Pharmaceutical Reagent Co., Ltd.; lead wire was from Mingpinhui Hardware Store; grinding beads were from Huzhou Xiongsheng Grinding Co., Ltd.; reagent kits for blood urea nitrogen (BUN), blood lactate (BLA), malondialdehyde (MDA), superoxide dismutase (SOD) activity, and glutathione peroxidase (GSH-Px) were from Nanjing Jiancheng Bioengineering Institute; BCA protein concentration assay kit was from Shanghai Beyotime Biotechnology Co., Ltd.; reverse transcription kit and real-time fluorescence quantitative kit were from Nanjing Novizan Biotechnology Co., Ltd.; primers were from Shanghai Sangon Biotech Co., Ltd.
[0032] The culture media involved in the following examples are as follows: MRS solid medium: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, yeast extract 5 g / L, diammonium hydrogen citrate 2 g / L, K2PO4·3H2O 2.6 g / L, MgSO4·7H2O 0.1 g / L, MnSO4 0.05 g / L, Tween 80 1 mL / L, agar 20 g / L.
[0033] MRS liquid culture medium: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, yeast extract 5 g / L, diammonium hydrogen citrate 2 g / L, K2PO4·3H2O 2.6 g / L, MgSO4·7H2O 0.1 g / L, MnSO4 0.05 g / L, Tween 80 1 mL / L.
[0034] Fermentation substrate containing EGCG: EGCG 200 mg / L, peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, yeast powder 5 g / L, diammonium hydrogen citrate 2 g / L, K2PO4·3H2O 2.6 g / L, MgSO4·7H2O 0.1 g / L, MnSO4 0.05 g / L, Tween 80 mL / L.
[0035] The detection methods involved in the following embodiments are as follows: EGCG detection method: Qualitative and quantitative analysis was performed using a Waters 1525 high-performance liquid chromatograph (Waters Instruments, USA). The chromatographic column was a C18 (250 × 4.6 mm, 5 µm) column, with an autosampler performing gradient elution at a flow rate of 1.00 mL / min. The column temperature was 30℃, and the injection volume was 10 μL. The UV detector wavelength was 278 nm. The mobile phase was: A - 0.2% formic acid in water, B - acetonitrile; gradient elution: 0-3.5 min 5% B, 3.5-5 min 5%-10% B, 5-10 min 10% B, 10-30 min 10%-28% B, 30-35 min 28%-5% B.
[0036] Example 1: Screening, strain identification and preservation of Lactococcus lactis subsp. CCFM1519 1. Screening Using healthy adult feces as samples, the samples were serially diluted 10-fold with sterile saline to 10⁻⁶. -4 10 -5 10 -6 Then take 100 μL of each diluted by a factor of 10. -4 10 -5 10 -6 The diluted solution was plated on MRS solid medium and incubated at 37°C for 48 h. The colony morphology was observed and recorded. Colonies of different morphologies were picked from the MRS solid medium and streaked for isolation. After incubation at 37°C for 48 h, single colonies of different morphologies were picked from the MRS solid medium again and streaked for isolation until pure single colonies with consistent morphology were obtained. Pure colonies from the MRS solid medium were inoculated into 5 mL of MRS liquid medium and incubated at 37°C for 24 h. 1 mL of bacterial solution was taken into a sterile centrifuge tube, centrifuged at 8000 r / min for 3 min, and the upper medium was discarded. The obtained bacterial sludge was freeze-dried.
[0037] 2. Identification The isolated strain was subjected to PCR amplification of 16S rDNA. The PCR product was sent to Suzhou Genewise Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the nucleic acid sequence in NCBI. The results showed that it was Lactococcus lactis subsp. lactis and named Lactococcus lactis subsp. lactis CCFM1519.
[0038] 3. Save Lactococcus lactis subsp. CCFM1519 was inoculated into 5 mL of MRS liquid medium and cultured at 37 °C for 24 h. 1 mL of bacterial culture was taken into a sterile centrifuge tube, centrifuged at 8000 r / min for 3 min, and the upper culture medium was discarded. The bacterial sludge was resuspended in 30% glycerol solution and stored at -80 °C.
[0039] Example 2: Transformation of EGCG by Lactococcus lactis subsp. CCFM1519 1. Streaking of Lactococcus lactis subsp. CCFM1519 from Example 1 onto MRS solid medium, and incubating the plate upside down at 37°C for 48 h; picking a single colony and inoculating it into 5 mL of MRS liquid medium and incubating at 37°C for 48 h.
[0040] 2. Add 5% (v / v) of the bacterial culture of *Lactococcus lactis* subsp. *CCFM1519* obtained in step 1 to the fermentation substrate containing EGCG, and ferment at a constant temperature of 37℃ for 48 h. The initial concentration of the bacterial culture is 1×10⁻⁶. 6 CFU / mL.
[0041] 3. After fermentation, the fermentation supernatant was filtered through a 0.22 μm disposable microporous membrane to obtain the fermentation product. Before fermentation, a small amount of the fermentation supernatant was filtered through a 0.22 μm disposable microporous membrane to obtain the control solution. The fermentation product and the control solution were stored at 4℃ for later use.
[0042] 4. Accurately weigh 10 mg of EGCG reference standard into a 10 mL volumetric flask, accurately pipette the above solution and dilute to 250 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL, and 25 μg / mL, and plot a working curve under mobile phase.
[0043] 5. Take 2 mL of the fermentation product and control solution obtained in step (3), and analyze them by HPLC according to the chromatographic conditions before fermentation. Figure 1 A) and after fermentation ( Figure 1 B), after fermentation, the EGCG content in the supernatant was 124.4 μg / mL, and the reduction rate of EGCG was about 40%.
[0044] 6. Take 2 mL of the fermentation product obtained in step (3) and the control group solution, and analyze the fermentation process before and after fermentation by LC-MS. Figure 2 The results of the EGCG content analysis (AB) showed that while EGCG decreased, a product was generated. Compared with the standard, the product was 5-(4-hydroxyphenyl)valeric acid. Figure 3 ), 4-hydroxybenzoic acid ( Figure 4 The levels of metabolites were calculated to be 4.32 μg / mL and 10.94 μg / mL, respectively. This demonstrates that EGCG can be transformed by *Lactococcus lactis* subsp. *CCFM1002* under the above conditions.
[0045] Comparative Example 1: Transformation of EGCG by Lactococcus lactis subsp. lactis FBJ3T3 The specific implementation method is the same as in Example 2, except that Lactococcus lactis subsp. CCFM1519 was replaced with Lactococcus lactis subsp. FBJ3T3 obtained through self-screening and inoculated into a fermentation substrate containing EGCG for fermentation. The results before fermentation were measured. Figure 1 A) and after fermentation ( Figure 1 C) The EGCG content showed that Lactococcus lactis subsp. lactis FBJ3T3 did not have the ability to convert EGCG.
[0046] Example 3: Effects of EGCG fermentation by Lactococcus lactis subsp. CCFM1519 on the behavioral performance of fatigued mice Preparation of EGCG fermentation product of Lactococcus lactis subsp. lactis CCFM1519: 1) The lactococcus lactis subsp. CCFM1519 from Example 1 was streaked on MRS solid medium and incubated upside down at 37°C for 48 h; a single colony was picked and inoculated into 5 mL of MRS liquid medium and incubated at 37°C for 48 h to obtain the lactococcus lactis subsp. CCFM1519 bacterial suspension.
[0047] 2) Add 5% (v / v) of the bacterial culture of *Lactococcus lactis* subsp. *CCFM1519* obtained in step 1) to the fermentation substrate containing EGCG, and ferment at a constant temperature of 37℃ for 48 h. The initial concentration of the bacterial culture is 1×10⁻⁶. 6 CFU / mL. After fermentation, centrifuge at 8000 r / min for 20 min, collect the supernatant, filter it through a 0.22 μm disposable filter for sterilization, aliquot it, and store it at -20℃ to obtain the fermentation product of Lactococcus lactis subsp. lactis CCFM1519 EGCG.
[0048] Twenty-four healthy male C57BL / 6J mice aged 6 weeks were randomly divided into four groups of six mice each. The four groups were: control group (NC), model group (MC), EGCG intervention group (EGCG), and EGCG fermentation group (CCFM1519 fermented EGCG). After one week of environmental adaptation, weeks 2-4 were administered via gavage intervention, with consistent daily gavage times, and included forced swimming (6 min / day) and forced restraint (4 h / day). Behavioral training was conducted in week 5 for four days, with a water maze test performed on day 5 of week 5. The experiment ended at the end of week 5. Among them, the NC group and MC group were administered 200 μL of physiological saline by gavage; the EGCG group was administered 40 mg EGCG / kg body weight by gavage, with a gavage volume of 200 μL, based on the mouse body weight; the Lactococcus lactis subsp. lactis CCFM1519 fermented EGCG group was administered 200 μL of Lactococcus lactis subsp. lactis CCFM1519 EGCG fermentation product by gavage. The preparation method of Lactococcus lactis subsp. lactis CCFM1519 EGCG fermentation product was as follows: after obtaining the EGCG fermentation product according to the method of step (2), the amount of EGCG in the fermentation substrate containing EGCG before fermentation was used to calculate the EGCG fermentation product corresponding to 40 mg EGCG / kg body weight, and the volume was adjusted with physiological saline before being administered by gavage.
[0049] The behavioral training involved the Morris water maze test: the pool was divided into four quadrants, with a circular escape platform (12 cm in diameter) submerged 2 cm below the water surface in the second quadrant. When mice were trained to be placed in a specific quadrant, they were instructed to locate the escape platform. If the platform could not be found within 60 seconds, the mice were guided to its location for 10 seconds. The mice's swimming trajectory within the water maze was automatically recorded, and the experiment automatically stopped once the mouse successfully returned to the platform. If the mouse failed to find the platform within the set time, the experiment was considered a failure. The navigation process for each mouse was recorded, and the latency was calculated.
[0050] The experimental animals were grouped as shown in Table 1.
[0051] Table 1 Grouping of experimental animals
[0052] Experimental results are as follows Figure 5As shown, the latency to reach the plateau in the MC group (55.52±7.97s) was significantly longer than that in the NC group (9.72±1.27s). However, the latency in the EGCG group and the EGCG fermentation group using *Lactococcus lactis* subsp. CCFM1519 was significantly shorter than that in the MC group. Specifically, compared to the MC group, the latency in the EGCG group was shortened by 40.49%, while the latency in the EGCG fermentation group using *Lactococcus lactis* subsp. CCFM1519 was shortened by 48.56% (28.56±19.82s); compared to the EGCG group, the latency in the EGCG fermentation group using *Lactococcus lactis* subsp. CCFM1519 was shortened by 13.56%. These results indicate that EGCG fermentation using *Lactococcus lactis* subsp. CCFM1519 can effectively alleviate mental fatigue in mice, and the allergic effect is superior to that of EGCG alone.
[0053] Example 4: Effect of Lactococcus lactis subsp. lactis CCFM1519 fermentation and transformation of EGCG on serum metabolite accumulation in fatigued mice The grouping, modeling, and treatment methods for C57BL / 6J mice are the same as in Example 3.
[0054] After the experiment, the mice were anesthetized and blood was collected from the orbital cavity. 3000 ml of blood samples were collected. g Centrifuge for 15 min, collect the supernatant, and determine the levels of lactic acid (LA) and blood urea nitrogen (BUN) in mouse serum.
[0055] BUN (Brain Urine) is a byproduct of energy metabolism and a sensitive indicator related to fatigue. LA (Lactate) is a major byproduct of anaerobic metabolism, and the accumulation of lactate in serum is a significant cause of fatigue. Experimental results are as follows... Figure 6 As shown, serum LA levels were significantly higher in the MC group compared to the NC group. Compared to the MC group (12.06±1.08), the LA level in the EGCG group (9.63±1.65) decreased by 20.12%, while the LA level in the EGCG fermentation group (9.20±2.47) decreased by 23.68%. Compared to the NC group, BUN levels were significantly higher in the MC group. Compared to the MC group (11.62±1.39), the BUN level in the EGCG group (10.48±1.65) decreased by 9.85%, while the BUN level in the EGCG fermentation group (9.65±1.07) decreased by 16.97%. These results indicate that fatigue-induced exercise can significantly increase the levels of serum metabolites LA and BUN in the blood, while fermentation of EGCG by Lactococcus lactis subsp. CCFM1519 can alleviate the increase in blood LA and BUN levels caused by exercise fatigue.
[0056] Example 5: Effects of EGCG fermentation by Lactococcus lactis subsp. CCFM1519 on oxidative stress in fatigued mice The grouping, modeling, and treatment methods for C57BL / 6J mice are the same as in Example 3.
[0057] Mice were anesthetized and blood was collected from the orbital cavity. 3000 ml of blood samples were collected. g Centrifuge for 15 min and collect the supernatant. Detect superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) content according to the kit instructions.
[0058] Experimental results are as follows Figure 7 As shown, compared with the NC group (130.50±18.07), the SOD level in the MC group was significantly decreased to 65.74±14.20. Compared with the MC group, the SOD level in the EGCG group increased to 87.72±10.73, while the SOD level in the EGCG fermentation group of *Lactococcus lactis* subsp. CCFM1519 increased to 97.58±9.76. Compared with the NC group (7.52±1.30), the MDA content in the MC group was significantly increased to 10.56±0.79. Compared with the MC group, the MDA content in the EGCG group decreased to 9.20±0.78, while the MDA content in the EGCG fermentation group of *Lactococcus lactis* subsp. CCFM1519 decreased to 8.43±0.78. Compared with the NC group, the GSH-Px level in the MC group was significantly decreased to 1573.77±113.86. In contrast, the GSH-Px level in the EGCG group increased to 1888.52±219.06, and in the group fermented with *Lactococcus lactis* subsp. CCFM1519, it increased to 1868.85±66.71. These results indicate that serum MDA levels increase and SOD and GSH-Px enzyme activities decrease after fatigue in mice. Fermentation of EGCG with *Lactococcus lactis* subsp. CCFM1519 can increase SOD and GSH-Px enzyme activities, reduce MDA levels, and alleviate oxidative stress caused by fatigue.
[0059] Example 6: Effect of Lactococcus lactis subsp. lactis CCFM1519 fermentation and transformation of EGCG on the expression of serum metabolite clearance genes in fatigued mice The grouping, modeling, and treatment methods for C57BL / 6J mice are the same as in Example 3.
[0060] Skeletal muscle (gastrocnemius) tissues from both lower legs of mice were collected, washed with physiological saline, blotted dry with filter paper, weighed, and then added to physiological saline at a ratio of 1:9 (w / v). The tissues were homogenized in a high-throughput tissue homogenizer under ice bath conditions. The resulting homogenate was centrifuged at 12000 g for 15 min at 4°C, and the supernatant was collected. To correct for the degree of homogenization, the protein concentration of the supernatant was determined using a BCA protein concentration assay kit. Total RNA was extracted using TRIzol, and cDNA was obtained by reverse transcription using a reverse transcription kit. The expression level of anti-fatigue gene mRNA was detected by RT-qPCR using a real-time quantitative PCR kit. The primer sequences used are shown in the table.
[0061] Table 2 Primer Sequences
[0062] The results are as follows Figure 8 As shown, exercise increases lactate levels in the body, and lactate dehydrogenase (LDHB) and monocarboxylic acid transporter (MCT1) are important gene targets regulating lactate transport. Compared with the NC group, the expression level of LDHB in the MC group was significantly decreased. Compared with the MC group (0.48±0.13), the expression levels of LDHB in the EGCG group (0.67±0.08) and the EGCG fermentation group of *Lactococcus lactis* subsp. CCFM1519 (0.77±0.16) increased by 39.58% and 60.83%, respectively, with the EGCG fermentation group of *Lactococcus lactis* subsp. CCFM1519 being 15.22% higher than the EGCG group. Regarding the MCT1 gene expression level, the expression level of MCT1 in the MC group was significantly decreased compared with the NC group. Compared with the MC group (0.49±0.15), the MCT1 gene expression level in the EGCG group (0.72±0.09) increased by 47.35%, while the MCT1 expression level in the EGCG fermentation group of *Lactococcus lactis* subsp. CCFM1519 (0.81±0.26) increased by 64.90%, with the CCFM1519 fermentation group showing an 11.91% higher level than the EGCG group. These results indicate that *Lactococcus lactis* subsp. CCFM1519 fermentation of EGCG can upregulate the expression of LDHB and MCT1 to enhance the lactic acid clearance capacity of EGCG, thereby maintaining energy supply and delaying fatigue.
[0063] Example 7: Effects of EGCG fermentation by *Lactococcus lactis* subsp. *CCFM1519* on gene expression in oxidative stress regulatory pathways in fatigued mice The grouping, modeling, and treatment methods for C57BL / 6J mice were the same as in Example 3. The tissue processing and RT-qPCR methods were the same as in Example 6.
[0064] Nuclear factor E2-associated factor 2 (Nrf2) and heme oxygenase 1 (HO-1) are important regulators of oxidative stress. Experimental results are as follows... Figure 9 As shown, compared with the NC group, the Nrf2 gene expression level in the MC group was significantly reduced. Compared with the MC group (0.50±0.15), the Nrf2 gene expression level in the EGCG group (0.66±0.13) increased by 32.40%, while the Nrf2 gene expression level in the EGCG fermentation group of Lactococcus lactis subsp. CCFM1519 (0.76±0.11) increased by 51.60%, with the EGCG fermentation group of Lactococcus lactis subsp. CCFM1519 being 14.85% higher than the EGCG group. Compared with the NC group, the HO-1 gene expression in the MC group (0.58±0.14) was significantly decreased. Compared with the MC group, the HO-1 gene expression level in the EGCG group (0.81±0.08) increased by 38.97%, while the HO-1 gene expression level in the EGCG fermentation group of *Lactococcus lactis* subsp. CCFM1519 (0.91±0.27) increased by 57.24%, with the *Lactococcus lactis* subsp. CCFM1519 fermentation group showing a 13.15% higher level than the EGCG group. These results indicate that *Lactococcus lactis* subsp. CCFM1519 fermentation of EGCG can alleviate fatigue by upregulating the expression levels of Nrf2 and HO-1 genes.
[0065] Example 8: Effects of EGCG fermentation by Lactococcus lactis subsp. CCFM1519 on gene expression in neuroprotective signaling pathways in fatigued mice The grouping, modeling, and treatment methods for C57BL / 6J mice are the same as in Example 3.
[0066] The mice were euthanized by decapitation. The scalp was immediately cut open on ice, and the skull and dura mater were removed. The brain tissue was carefully separated and removed. After weighing, physiological saline was added at a ratio of 1:9 (w / v). The tissue was then placed in a high-throughput tissue homogenizer under ice bath conditions for tissue disruption. The resulting homogenate was centrifuged at 12,000 g for 15 min at 4°C, and the supernatant was collected.
[0067] The RT-qPCR method is the same as in Example 6.
[0068] Experimental results are as follows Figure 10As shown, compared with the NC group, the DRD1 gene expression level in the MC group was significantly reduced. Compared with the MC group (0.54±0.19), the EGCG group increased the DRD1 gene expression level to 0.75±0.15, while the EGCG group fermented with Lactococcus lactis subsp. CCFM1519 increased it to 0.91±0.28, which was 21.33% higher than the EGCG group. Compared with the NC group, the TPH gene expression level in the MC group was significantly increased. Compared with the MC group (1.86±0.36), the EGCG group and the EGCG group fermented with Lactococcus lactis subsp. CCFM1519 reduced the TPH gene expression level to 1.39±0.38 and 1.42±0.28, respectively. Compared with the NC group, the expression levels of BDNF and TrkB genes were significantly reduced in the MC group. In the EGCG group and the EGCG fermented with *Lactococcus lactis* subsp. CCFM1519, the BDNF gene expression level increased to 0.73±0.10 and 0.86±0.14, respectively, compared to the MC group (0.54±0.14). Similarly, the TrkB gene expression level increased to 0.72±0.09 and 0.83±0.16, respectively, in the EGCG group and the EGCG fermented with *Lactococcus lactis* subsp. CCFM1519, compared to the MC group (0.52±0.11). These results indicate that EGCG fermented with *Lactococcus lactis* subsp. CCFM1519 can alleviate fatigue by upregulating the expression levels of DRD1, BDNF, and TrkB genes.
[0069] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. A strain of Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. lactis CCFM1519 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 1, 2025, with accession number GDMCC No: 66903.
2. A microbial preparation containing the Lactococcus lactis subsp. CCFM1519 as described in claim 1.
3. The microbial preparation according to claim 2, characterized in that, In the aforementioned microbial preparation, the bacterial count of *Lactococcus lactis* subsp. *CCFM1519* is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.
4. A method for preparing EGCG ferment, characterized in that, The Lactococcus lactis subsp. CCFM1519 of claim 1 was inoculated into a fermentation substrate containing EGCG and fermented, and the fermentation supernatant was collected.
5. The method according to claim 4, characterized in that, The fermentation substrate containing EGCG includes EGCG 150~250mg / L, peptone 8~12g / L, beef extract 8~12g / L, glucose 15~25g / L, sodium acetate 1.5~2.5g / L, yeast powder 4~6g / L, diammonium hydrogen citrate 1.5~2.5g / L, K2PO4·3H2O 2~3g / L, MgSO4·7H2O 0.08~0.15g / L, MnSO4 0.04~0.06g / L, and Tween 0.8~1.2mL / L.
6. The method according to claim 4, characterized in that, In the fermentation substrate, the inoculum amount of *Lactococcus lactis* subsp. *CCFM1519* is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.
7. EGCG fermentation product prepared by any one of claims 4 to 6.
8. A product characterized in that, The product contains at least one of the following: Lactococcus lactis subsp. CCFM1519 as described in claim 1, or the microbial preparation as described in claim 2 or 3, or the EGCG ferment as described in claim 7. The product is a food, a drug, or a health product.
9. The use of Lactococcus lactis subsp. CCFM1519 as described in claim 1, or the microbial preparation as described in claim 2 or 3, or the method as described in any one of claims 4 to 6, or the EGCG ferment as described in claim 7 in the preparation of pharmaceuticals or health products for relieving physical fatigue.
10. The application of Lactococcus lactis subsp. CCFM1519 as described in claim 1 in the transformation of EGCG.