Lactobacillus delbrueckii subsp. Bulgaricus CCFM1520 for transforming curcumin to produce tetrahydrocurcumin to relieve neuroanxiety

Abstract

The invention discloses a lactobacillus delbrueckii subsp. Bulgaricus CCFM1520 for transforming curcumin to produce tetrahydrocurcumin to relieve nervous anxiety, and belongs to the technical field of microorganisms. The lactobacillus delbrueckii subsp. Bulgaricus CCFM1520 screened by the invention has the capability of converting curcumin, and can utilize curcumin and generate tetrahydrocurcumin. The invention further provides a synbiotic preparation prepared from the lactobacillus gasseri CCFM1481 and curcumin, the synbiotic preparation can be used as an anti-fatigue functional medicine, the individual behavioral memory level can be improved, the imbalance of body neurotransmitters (DA, 5-HT and NE) can be slowed down, the expression of body neuroprotection related genes (BDNF, TrkB, DRD1 and TH) can be improved, and the synbiotic preparation has a huge application prospect.

Description

A strain of *Lactobacillus denudata* subsp. Bulgaria (CCFM1520) that transforms curcumin to tetrahydrocurcumin to alleviate anxiety. Technical Field

[0001] This invention relates to a strain of Lactobacillus de Germanis subsp. bulgaricus CCFM1520 that transforms curcumin to produce tetrahydrocurcumin to relieve anxiety, belonging to the field of microbial technology. Background Technology

[0002] In today's fast-paced lifestyle, irregular schedules, insufficient sleep, mental stress, high psychological pressure, and chronic negative emotions are prevalent. Consequently, more and more people are in a sub-healthy state (a special state between health and disease), which often leads to anxiety and depression and is associated with cognitive impairment, physical dysfunction, and energy imbalance. In severe cases, it can lead to various diseases related to biological regulation and the immune system. Prolonged anxiety has become a major threat to human health, and its occurrence is a complex process involving multiple factors and systems, including neurotransmitter imbalances, abnormal neural circuits, and psychosocial factors. Therefore, finding potential anxiety-relieving drugs or preparations with clear efficacy and few side effects is crucial.

[0003] Curcumin, a lipophilic polyphenol contained in the rhizome of turmeric, has been used in traditional Asian medicine for centuries and is now widely used globally as a food flavoring. It has attracted considerable attention due to its pharmacological activity and potential neuroprotective properties. In fact, there are differences in the in vitro and in vivo activities of curcumin, along with its poor bioavailability and chemical instability. The gut microbiota is well known to play multiple important roles in normal human physiological functions, and its composition can be influenced by various environmental and lifestyle factors. Interestingly, curcumin and its metabolites have been shown to affect the gut microbiota. Notably, the interaction between curcumin and the gut microbiota produces two distinct phenomena: the first is the direct regulation of the gut microbiota by curcumin, and the second is the biotransformation of curcumin by the gut microbiota, producing active metabolites; both phenomena are crucial for the biological activity of curcumin. *Lactobacillus delbrueckii* subsp. bulgaricus is widely used worldwide in the production of fermented dairy products such as yogurt and cheese, and is one of the most economically valuable homofermenting lactic acid bacteria. Studies have shown that *Lactobacillus delbrueckii* subsp. bulgaricus possesses various probiotic effects, but it has not yet been found that *Lactobacillus delbrueckii* subsp. bulgaricus can efficiently utilize curcumin and exert an effect of relieving anxiety. Therefore, isolating and screening such probiotics to achieve synergistic effects with curcumin has broad application prospects. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a strain of Lactobacillus delbrueckii subsp bulgaricus (CCFM1520) that can convert curcumin to alleviate anxiety, and also provides the application of this strain.

[0005] This invention provides a strain of Lactobacillus delbrueckii subsp bulgaricus CCFM1520, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 1, 2025, with accession number GDMCC No.66904.

[0006] The present invention provides a microbial preparation containing the aforementioned Lactobacillus de Germanis subsp. Bulgaria CCFM1520.

[0007] In one embodiment, the content of *Lactobacillus delbrueckii subsp. bulgaricus* CCFM1520 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] The present invention also provides a synbiotic formulation containing the aforementioned Lactobacillus de Germanis subsp. Bulgaria CCFM1520.

[0010] In one embodiment, the synbiotic preparation contains the Lactobacillus deutschlandia subsp. bulgaricus CCFM1520 and its fermentation products.

[0011] In one embodiment, the concentration of *Lactobacillus denudata* subsp. bulgaricus CCFM1520 in the synbiotic preparation is ≥5 × 10⁻⁶. 9 CFU / mL.

[0012] This invention provides a method for preparing curcumin ferment, which involves culturing Lactobacillus bulgaricus subsp. CCFM1520 in a curcumin-containing culture medium to obtain curcumin ferment.

[0013] In one embodiment, the curcumin ferment contains tetrahydrocurcumin.

[0014] In one embodiment, the method includes the following steps: (1) fermenting Lactobacillus bulgaricus subsp. CCFM1520 in a curcumin-containing fermentation substrate; (2) centrifuging the fermentation product of step (1), collecting the fermentation supernatant, and obtaining curcumin fermentation product.

[0015] In one embodiment, the content of *Lactobacillus bulgaricus* subsp. *CCFM1520* in the fermentation during step (1) is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0016] In one embodiment, the method includes the following steps: (1) inoculating Lactobacillus bulgaricus subsp. CCFM1520 into a culture medium and culturing at 35-40℃ for 48 h to obtain a bacterial solution; (2) adding the bacterial solution obtained in step (1) to a curcumin-containing fermentation substrate at an inoculation rate of 5% (v / v), fermenting at 25-40℃ for 48 h, and collecting the fermentation supernatant.

[0017] The present invention also provides curcumin ferments prepared using the method described herein.

[0018] This invention provides a synbiotic preparation containing Lactobacillus denudata subsp. Bulgaria CCFM1520 and curcumin.

[0019] The present invention also provides a probiotic product containing the Lactobacillus de Germany subsp. bulgaricus CCFM1520 or the curcumin ferment.

[0020] In one embodiment, the product is food, medicine, or health product.

[0021] In one embodiment, the food is a dairy product, soy product, or fruit and vegetable product prepared using at least one of the curcumin ferment, synbiotic preparation, Lactobacillus bulgaricus subsp. CCFM1520, or microbial preparation.

[0022] 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 cabbage, white radish, cucumber, beet, yellow peach, or bayberry products.

[0023] In one embodiment, the food is a fermented food, including solid food, liquid food, or semi-solid food.

[0024] In one embodiment, the food is a beverage or snack containing at least one of the above-mentioned curcumin ferment, Lactobacillus bulgaricus subsp. CCFM1520, or a microbial preparation.

[0025] In one embodiment, the drug further comprises a drug carrier and / or pharmaceutical excipients.

[0026] 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.

[0027] In one embodiment, the dosage form of the drug is granules, capsules, tablets, pills, or oral liquid.

[0028] The present invention also provides the use of the above-mentioned Lactobacillus de Germanis subsp. Bulgaria CCFM1520, or microbial preparations, or the curcumin fermentation product in the preparation of medicaments for relieving nervous anxiety and / or anti-fatigue.

[0029] In one embodiment, the product has at least one of the following (1)-(4) effects: (1) improving individual behavioral empty field performance; (2) improving individual behavioral memory ability; (3) alleviating individual neurotransmitter (DA, 5-HT, NE) imbalance; (4) increasing the expression of individual neuroprotective related genes (BDNF, TrkB, DRD1, TH).

[0030] Beneficial effects: 1. The Lactobacillus bulgaricus subsp. CCFM1520 provided by this invention has the ability to convert curcumin. After fermentation in a curcumin-containing fermentation substrate, the degradation rate of curcumin is 84.12%, and tetrahydrocurcumin active substance is generated, with a conversion rate of 62.39%.

[0031] 2. The *Lactobacillus bulgaricus* subsp. *degenera* CCFM1520 provided by this invention can promote the efficacy of curcumin, and the converted curcumin has the ability to relieve anxiety, specifically: (1) Fermented curcumin from *Lactobacillus bulgaricus* subsp. *degenera* CCFM1520 can improve the behavioral open field performance of mice, with the movement distance increasing by 76.48% compared to the model group and by 26.55% compared to the curcumin group; (2) Fermented curcumin from *Lactobacillus bulgaricus* subsp. *degenera* CCFM1520 can improve the behavioral memory of mice, with the latency period shortening by 40.78% compared to the model group and increasing by 3.66% compared to the curcumin group; (3) *Lactobacillus bulgaricus* subsp. *degenera* CCFM1520 can improve the behavioral memory of mice, with the latency period shortening by 40.78% compared to the model group and increasing by 3.66% compared to the curcumin group; (4) Fermented curcumin from *Lactobacillus bulgaricus* subsp. *degenera* CCFM1520 can improve the behavioral memory of mice, with the latency period shortening by 40.78% compared to the model group and increasing by 3.66% compared to the curcumin group; (5) Fermented curcumin from *Lactobacillus bulgaricus* subsp. *degenera* CCFM1520 can improve the behavioral memory of mice, with the latency period shortening by 40.78% compared to the model group and increasing by 3.66% compared to the curcumin group; (6) Fermented curcumin from *Lactobacillus bulgaricus* subsp. *degenera* CCFM1520 can improve the behavioral memory of mice, with the latency The synergistic preparation of M1520 and curcumin and fermented curcumin can alleviate the imbalance of neurotransmitters in mice. Compared with the curcumin group, the NE content increased by 9.21% and 6.64%, respectively, and the 5-HT content decreased by 10.09% and 15.43%, respectively. The DA content of the fermented curcumin group increased by 10.97% compared with the curcumin group. (4) The synergistic preparation of Lactobacillus bulgaricus subsp. bulgaricus CCFM1520 and curcumin and fermented curcumin can improve the expression of neuroprotection-related genes. Compared with the curcumin group, the TrkB gene expression increased by 14.85% and 26.67%, respectively. The BDNF gene expression of the fermented curcumin group increased by 19.18% compared with the curcumin group.

[0032] Products that alleviate anxiety can be produced using *Lactobacillus deuterans* subsp. bulgaricus CCFM1520 and curcumin. The production process, using curcumin 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 *Lactobacillus deuterans* subsp. bulgaricus CCFM1520 to curcumin-containing products has significant application potential and a solid foundation.

[0033] A strain of Lactobacillus delbrueckii subsp. bulgaricus, CCFM1520, taxonomically named Lactobacillus delbrueckii subsp. bulgaricus, was deposited on September 1, 2025, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No. 66904), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0034] Figure 1 shows the high performance liquid chromatogram of curcumin before and after fermentation; Figure 2 shows the LC-MS chromatogram of curcumin before and after fermentation; Figure 3 shows the LC-MS chromatogram of tetrahydrocurcumin before and after fermentation; Figure 4 shows the open field test results of mice in different groups; Figure 5 shows the water maze test results of mice in different groups; Figure 6 shows the neurotransmitter levels of mice in different groups; Figure 7 shows the neuroprotective target levels of mice in different groups. Detailed Implementation

[0035] 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 curcumin 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.; 5-hydroxytryptamine (5-HT), dopamine (DA), and norepinephrine (NE) were from Shanghai Enzyme-Linked Biotechnology Co., Ltd.; the BCA protein concentration assay kit was from Shanghai Beyotime Biotechnology Co., Ltd.; the reverse transcription kit and real-time fluorescence quantitative kit were from Nanjing Novizan Biotechnology Co., Ltd.; and the primers were from Shanghai Sangon Biotech Co., Ltd.

[0036] 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 1 mL / L, agar 20 g / L.

[0037] 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 1 mL / L.

[0038] Curcumin 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 1 mL / L, curcumin 100 mg / L.

[0039] Fermentation substrate containing curcumin: curcumin 100 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 1 mL / L.

[0040] The detection methods involved in the following examples are as follows: Detection method for curcumin: Qualitative and quantitative analysis was performed using an Agilent 1260 Infinity III liquid chromatograph. 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 420 nm. The mobile phase was: A - 0.2% formic acid in water, B - acetonitrile; gradient elution: 0-6 min 20% B, 6-15 min 20%-35% B, 15-25 min 35%-70% B, 25-35 min 70%-40% B, 35-40 min 40%-20% B.

[0041] Example 1: Screening, identification and preservation of Lactobacillus deuterans subsp. bulgaricus CCFM1520 1. Screening: Using fecal samples from healthy adults, the samples were serially diluted 10-fold with sterile physiological 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.

[0042] 2. Identification: Morphological, physiological and biochemical and molecular biological identifications were performed on the selected strains to confirm that the biological classification of the strain is Lactobacillus de Germany subsp. bulgaricus, and it was named Lactobacillus de Germany subsp. bulgaricus CCFM1520.

[0043] 3. Preservation: Inoculate Lactobacillus bulgaricus subsp. CCFM1520 into 5 mL of MRS liquid medium and incubate at 37°C for 24 h; take 1 mL of bacterial suspension into a sterile centrifuge tube, centrifuge at 8000 r / min for 3 min, discard the upper culture medium, resuspend the bacterial sludge in 30% glycerol solution and store at -80°C.

[0044] Example 2: Preparation of a synergistic preparation of Lactobacillus bulgaricus subsp. bulgaricus CCFM1520 and curcumin and curcumin fermentation product 1. Preparation of Lactobacillus bulgaricus subsp. bulgaricus CCFM1520-curcumin fermentation product: (1) Lactobacillus bulgaricus subsp. bulgaricus CCFM1520 from Example 1 was streaked on MRS solid medium. The plate was 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 Lactobacillus bulgaricus subsp. bulgaricus CCFM1520 bacterial solution; (2) 5% (v / v) of the Lactobacillus bulgaricus subsp. bulgaricus CCFM1520 bacterial solution obtained in step (1) was added to the curcumin-containing fermentation substrate and fermented at 37°C for 48 h. The initial concentration of the bacterial solution was 1×10⁻⁶. 6 CFU / mL; (3) After fermentation, centrifuge at 8000 r / min for 20 min, take the supernatant, filter and sterilize with a 0.22 μm disposable filter, dispense and store at -20℃ to obtain curcumin fermentation product of Lactobacillus bulgaricus subsp. deuterans CCFM1520. The concentration of tetrahydrocurcumin in the fermentation product was 43.78 μg / mL.

[0045] 2. Preparation of the synergistic formulation of *Lactobacillus bulgaricus* subsp. *degenera* CCFM1520 and curcumin: *Lactobacillus bulgaricus* subsp. *degenera* CCFM1520 from Example 1 was streaked onto MRS solid medium. The plates were 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, yielding a bacterial concentration of 5 × 10⁻⁶. 9 A bacterial suspension of *Lactobacillus denudata* subsp. bulgaricus CCFM1520 (CFU / mL) was centrifuged to collect bacterial sludge. The sludge was then resuspended in 10 g / L curcumin to obtain a bacterial concentration of 5 × 10⁻⁶. 9 Synthetic preparations with CFU / mL.

[0046] Example 3: Transformation of curcumin by Lactobacillus bulgaricus subsp. 1520 from Example 1: Streaked Lactobacillus bulgaricus subsp. 1520 from Example 1 onto MRS solid medium and incubated upside down at 37°C for 48 h; single colonies were picked and inoculated into 5 mL of MRS liquid medium and incubated at 37°C for 48 h.

[0047] 2. Add 5% (v / v) of *Lactobacillus denudata* subsp. bulgaricus CCFM1520 obtained in step 1 to the curcumin-containing fermentation substrate, and ferment at 37℃ for 48 h. The initial concentration of the bacterial solution is 1×10⁻⁶. 6 CFU / mL.

[0048] 3. After fermentation, the fermentation supernatant was filtered through a 0.22 μm single-use microporous membrane and stored at 4℃ for later use.

[0049] 4. Accurately weigh 10 mg of curcumin reference standard into a 10 mL volumetric flask, accurately pipette the above solution and dilute to 150 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL and 5 μg / mL, and plot a working curve under mobile phase.

[0050] 5. Take 2 mL of the fermentation product obtained in step (3) and the control group solution (fermentation substrate containing curcumin). Detect the curcumin content before fermentation (Figure 1A) and after fermentation (Figure 1B) by HPLC according to the chromatographic conditions. The curcumin reduction rate was 84.12%.

[0051] 6. Take 2 mL of fermentation product and control solution obtained in step (3), and detect the content of curcumin before and after fermentation (Figure 2A-B) by LC-MS. The results show that while curcumin decreased, tetrahydrocurcumin was generated. Detect the content of tetrahydrocurcumin before and after fermentation (Figure 3A-B), and calculate the amount of metabolite generated as 43.78 μg / mL, with a conversion rate of 62.39%.

[0052] Example 4: Effects of the synergistic preparation of Lactobacillus deutschlandia subsp. bulgaricus CCFM1520 and curcumin and fermented curcumin on the open field behavior of mice. The fermented product of Lactobacillus deutschlandia subsp. bulgaricus CCFM1520-curcumin and the synergistic preparation of Lactobacillus deutschlandia subsp. bulgaricus CCFM1520-curcumin were prepared according to the steps in Example 2.

[0053] Thirty-six healthy male C57BL / 6J mice aged 6 weeks were randomly divided into 6 groups, with 6 mice in each group. The 6 groups were: blank control group (NC), model group (MC), curcumin group (CUR), *Lactobacillus delbrueckii* subsp. bulgaricus CCFM1520 (CCFM1520), *Lactobacillus delbrueckii* subsp. bulgaricus CCFM1520-curcumin synergistic preparation group (CCFM1520 compound CUR), and *Lactobacillus delbrueckii* subsp. bulgaricus CCFM1520 fermented curcumin group (CCFM1520 fermented CUR). After one week of environmental adaptation, weeks 2-4 were treated with gavage intervention, with consistent daily gavage times, and forced swimming (6 min / day) and forced restraint (4 h / day). Behavioral training was conducted in week 5. The NC and MC groups were administered saline by gavage, the CUR group was administered 100 mg / kg body weight by gavage, and the CCFM1520 group was administered a bacterial concentration of 5 × 10⁻⁶. 9 CCFM1520 bacterial suspension at CFU / mL, CCFM1520 combined with CUR group at 5×10 9The combined preparation of CFU / mL and 100 mg / kg curcumin was administered by gavage. The CCFM1520 fermentation CUR group was administered curcumin fermentation product of Lactobacillus bulgaricus subsp. bulgaricus CCFM1520 by gavage. The amount of curcumin in the fermentation substrate containing curcumin before fermentation was converted to the amount of curcumin fermentation product corresponding to 100 mg curcumin / kg body weight.

[0054] The experimental animals were grouped as shown in Table 1.

[0055] Table 1 Grouping of experimental animals

[0056] Open field test for mouse behavior: An open field test was conducted using an open field behavior detector with a four-grid layout. Movement of each mouse was recorded using an animal behavior video tracking system. The mice's movement trajectories within the activity box were recorded using OFT (Out-of-Flight Theory).

[0057] The experimental results are shown in Figure 4. Compared with the NC group (2143.83±390.24 cm), the open field movement distance of mice in the MC group (1245.08±323.86 cm) was significantly decreased. The open field movement distance of mice in the CUR group (1736.34±151.19 cm) and the CCFM1520 fermented CUR group (2197.26±522.25 cm) increased by 39.46% and 76.48% respectively compared with the MC group. Compared with the NC group (76.82±6.98%), the open field movement time ratio of mice in the MC group (41.21±6.18%) was significantly decreased. The open field movement time ratio of mice in the CUR group (54.19±5.18%) and the CCFM1520 compound CUR group (64.13±7.59%) increased by 31.49% and 55.62% respectively compared with the MC group. These results indicate that, compared with curcumin, the synergistic preparation of Lactobacillus delbrueckii subsp. bulgaricus CCFM1520 and curcumin, as well as fermented curcumin, are more effective in relieving anxiety in mice.

[0058] Example 5: Effects of a synergistic preparation of Lactobacillus deutschlandia subsp. bulgaricus CCFM1520 and curcumin and fermented curcumin on the water maze behavior of mice. The grouping, modeling and treatment methods of C57BL / 6J mice were the same as in Example 4.

[0059] The Morris water maze test for mouse behavior: A swimming pool was divided into four quadrants, with a circular escape platform submerged 2 cm below the water surface in the second quadrant. The mice were trained before the actual experiment. The mice's swimming trajectories within the water maze were automatically recorded, and the experiment automatically stopped when a mouse successfully returned to the platform. If a mouse failed to find the platform within the set time, the experiment was considered a failure. The navigation process of each mouse was recorded, and the latency was calculated. Finally, the escape platform was removed, and the number of times the mouse passed the platform's previous position within one minute was recorded.

[0060] As shown in Figure 5, compared with the NC group, the number of times mice in the MC group crossed the platform was significantly lower than that in the NC group. The number of times mice in the CUR group crossed the platform was not significantly different from that in the MC group. However, the number of times mice in the CCFM1520 combined with CUR group crossed the platform was significantly higher than that in the MC group, being 3.2 times that of the MC group. Compared with the NC group, the latency period of mice in the MC group (47.96±16.27s) was significantly prolonged. Compared with the MC group, the latency period of the CUR group (29.48±8.40s) was shortened by 38.53% compared to the MC group, while the latency period of the CCFM1520 fermented CUR group (20.50±16.65s) was shortened by 57.26%, and by 30.46% compared to curcumin. These results indicate that the synergistic preparation of *Lactobacillus delbrueckii* subsp. bulgaricus CCFM1520 and curcumin, as well as fermented curcumin, have the ability to effectively alleviate behavioral memory impairment in mice.

[0061] Example 6: Effects of a synergistic preparation of Lactobacillus deutschlandia subsp. bulgaricus CCFM1520 and curcumin and fermented curcumin on neurotransmitters in mice. The grouping, modeling, and treatment methods for C57BL / 6J mice were the same as in Example 4.

[0062] 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 extracted, and the levels of serotonin (5-HT), dopamine (DA), and norepinephrine (NE) were measured according to the kit instructions.

[0063] The experimental results are shown in Figure 6. Compared with the NC group (15.67±2.73 pg / mg), the DA content in the MC group (10.24±1.20 pg / mg) was significantly lower. Compared with the MC group, the DA content in the CCFM1520 fermented CUR group (13.13±3.28 pg / mg) was increased by 28.20%. Compared with the NC group (1.11±0.16 ng / mg), the NE content in the MC group (0.59±0.19 ng / mg) was significantly lower. Compared with the MC group, the NE content in the CUR group increased to 0.78±0.13 ng / mg, while the NE content in the CCFM1520 compound CUR group and the CCFM1520 fermented CUR group increased to 0.82±0.09 ng / mg and 0.89±0.14 ng / mg, respectively. Compared with the NC group (0.49±0.09 ng / mg), the 5-HT content in the MC group (0.80±0.09 ng / mg) was significantly increased. Compared with the MC group, the 5-HT content in the CUR group decreased to 0.70±0.09 ng / mg, while the 5-HT content in the CCFM1520 compound CUR group and the CCFM1520 fermented CUR group decreased to 0.63±0.10 ng / mg and 0.54±0.06 ng / mg, respectively. These results indicate that the synergistic preparation of Lactobacillus delbrueckii subsp. bulgaricus CCFM1520 and curcumin, as well as fermented curcumin, are more effective than curcumin in alleviating neurotransmitter imbalance in mice.

[0064] Example 7: Effects of a synergistic preparation of *Lactobacillus denudata* subsp. bulgaricus CCFM1520 and curcumin, and fermented curcumin, on gene expression in mouse neuroprotective signaling pathways. The grouping, modeling, and treatment methods for C57BL / 6J mice were the same as in Example 4. Tissue treatment methods were the same as in Example 6.

[0065] To correct for the degree of tissue homogenization, the protein concentration in 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 mRNA expression level of the gene was then detected by RT-qPCR using a real-time quantitative PCR kit. The primer sequences used are shown in the table.

[0066] Table 2 Primer Sequences

[0067] Brain-derived neurotrophic factor (BDNF) binds to tyrosine kinase receptor B (TrkB), activating multiple intracellular signaling cascades. These responses regulate cellular responses required for neurogenesis, memory, and learning. The experimental results are shown in Figure 7. Compared to the NC group, the BDNF gene expression level in the MC group (0.53±0.09) was significantly decreased. Compared to the MC group, the BDNF gene expression level in the CUR group (0.73±0.10) increased by 37.36%, while the curcumin fermented by *Lactobacillus deuterans* subsp. bulgaricus CCFM1520 increased by 64.15% (0.87±0.17). Compared with the NC group, the TrkB gene expression level in the MC group was significantly decreased. Compared with the MC group (0.56±0.06), the TrkB gene expression level in the CUR group (0.66±0.05) increased by 17.86%, while the expression levels of *Lactobacillus delbrueckii* subsp. bulgaricus CCFM1520 combined with curcumin (0.76±0.07) and fermented curcumin (0.84±0.12) increased by 35.36% and 49.29%, respectively. Compared with the NC group, the expression levels of DRD1 and TH genes in the MC group were significantly decreased, while the gene expression levels of *Lactobacillus delbrueckii* subsp. bulgaricus CCFM1520 fermented curcumin increased by 52.31% and 55%, respectively, compared with the MC group. These results indicate that, compared with curcumin alone, the synergistic preparation of *Lactobacillus delbrueckii* subsp. bulgaricus CCFM1520 and curcumin, as well as fermented curcumin, are more likely to alleviate anxiety by upregulating the expression levels of genes related to neuroprotective signaling pathways.

[0068] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone 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. Lactobacillus delbrueckii subsp bulgaricus CCFM1520 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 1, 2025, with accession number GDMCCNo.66904.

2. A microbial preparation containing the Lactobacillus deutschlandiae subsp. bulgaricus CCFM1520 as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The content of *Lactobacillus denudata* subsp. bulgaricus CCFM1520 in the microbial preparation is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

4. A synbiotic preparation containing the Lactobacillus deutschlandia subsp. bulgaricus CCFM1520 as described in claim 1.

5. The synthetic preparation according to claim 4, characterized in that, Contains the Lactobacillus bulgaricus subsp. CCFM1520 of claim 1 and its fermentation products.

6. The synthetic preparation according to claim 5, characterized in that, The preparation of the fermentation product includes the following steps: (1) fermenting Lactobacillus bulgaricus subsp. CCFM1520 in a fermentation substrate containing curcumin; (2) centrifuging the fermentation product of step (1), collecting the fermentation supernatant, and obtaining curcumin fermentation product.

7. The composition, characterized in that, It contains Lactobacillus deutschlandiae subsp. bulgaricus 1520 as described in claim 1 and curcumin.

8. The composition according to claim 7, characterized in that, The composition is a food, medicine, or health product.

9. The use of Lactobacillus deuterans subsp. bulgaricus CCFM1520 as described in claim 1, the curcumin ferment, or the synbiotic preparation in the preparation of a medicament for relieving anxiety and / or combating fatigue.

10. The application according to claim 9, characterized in that, The drug has at least one of the following (1)-(4) effects: (1) improving individual behavioral empty field performance; (2) improving individual behavioral memory ability; (3) alleviating individual neurotransmitter imbalance; (4) increasing the expression of individual neuroprotective genes.

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