Pediococcus acidilactici GOLDGUT-EZMind and application thereof in preparation of anti-depression and anti-anxiety products

Abstract

The invention discloses a strain of pediococcus acidilactici GOLDGUT-EZMind and an application of the strain of pediococcus acidilactici GOLDGUT-EZMind in preparation of a product for resisting depression and anxiety. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.35671, the preservation date is August 18, 2025, and the strain is named as pediococcus acidilactici in taxonomy. According to the invention, a brand new strategy that the strain can relieve neuroinflammation and improve depression and anxiety through dual ways of resisting neuroinflammation and regulating neurotransmitters is found for the first time, and the strain can be used for preparing drugs, foods and nutritional supplements, and can be used for non-drug intervention or adjuvant treatment of depression, anxiety and other central nervous system diseases related to neuroinflammation. Good application prospects and industrialization values are realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological bacterial agents, in particular to a Pediococcus acidilactici strain GOLDGUT-EZMind and application thereof in preparation of an anti-depression and anti-anxiety product. BACKGROUND

[0002] Depression is a serious mood disorder characterized by persistent low mood, anhedonia, impaired social functioning, and decreased cognitive ability. Its etiology is complex, involving multiple factors such as neuroinflammation, neurotransmitter imbalance, and hypothalamic-pituitary-adrenal axis abnormalities.

[0003] Currently, drug intervention for depression mainly relies on antidepressants aimed at increasing the availability of monoamine neurotransmitters in the brain, such as paroxetine, sertraline, and fluoxetine. However, these treatments often come with significant side effects, including headaches, insomnia, and weight changes, highlighting the urgent need for alternative treatment strategies. The limitations of existing drug treatments for mental health disorders such as depression and anxiety have prompted the scientific community to actively seek safer and more effective alternative therapies.

[0004] Probiotics, as a class of active microorganisms beneficial to the host, have gained attention in recent years for their potential in regulating the "gut-brain axis" function and improving emotional state. Existing research has preliminarily revealed that specific probiotic strains have the efficacy of relieving depression and anxiety symptoms, and their effects are highly strain-dependent. Animal experimental evidence shows that L. reuteri 3 can exert its effects by affecting host tryptophan metabolism; while Bifidobacterium breve CCFM1025 mainly regulates intestinal microbial community structure, thereby affecting the function of the brain 5-hydroxytryptamine system to improve depression-like behavior. Meanwhile, in human population studies, L. helveticus and B. longum have also been confirmed to have positive intervention effects on depression symptoms.

[0005] Although the above studies provide a theoretical basis for probiotic intervention in mental health, existing technology is not only limited by the small number of effective strains, but also lacks a deep understanding of how these strains affect brain function and behavior through specific molecular and biochemical pathways. Elucidating the mechanism of action is crucial for driving development in this field and achieving precise applications. China has a unique microbial resource library, providing a great opportunity for screening indigenous lactic acid bacteria with clear depression-improving efficacy and novel mechanisms of action.

[0006] Therefore, how to develop a new probiotic strain that can effectively alleviate depression and anxiety symptoms is a problem that needs to be solved by those skilled in the art. SUMMARY

[0007] Therefore, the present application aims to provide a Pediococcus acidilactici strain GOLDGUT-EZMind and its application in the preparation of products for resisting depression and anxiety, so as to solve the problems of large side effects of anti-depression drugs and unclear intervention mechanism of probiotics in the prior art.

[0008] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0009] The Pediococcus acidilactici strain GOLDGUT-EZMind is obtained by screening, isolation and purification from a characteristic fermented food sweet pickled in the Qinghai-Tibet Plateau region, and is preserved in the China General Microbiological Culture Collection Center (CGMCC) located at No. 1, Beichen West Road, Chaoyang District, Beijing, with a preservation number of CGMCC NO. 35671 and a preservation date of August 18, 2025, and is taxonomically named as Pediococcus acidilactici.

[0010] The present application also claims the application of the above-mentioned Pediococcus acidilactici strain GOLDGUT-EZMind in the preparation of products for preventing, improving or treating mood disorders.

[0011] The present application clarifies that its mechanism of action is:

[0012] a) reducing neuroinflammation, particularly inhibiting the overactivation of microglia in the brain;

[0013] b) reducing the expression levels of pro-inflammatory factors TNF-α, IL-1β and IL-6;

[0014] c) restoring the balance of neurotransmitters in the brain, particularly increasing the levels of 5-hydroxytryptamine (5-HT) and dopamine (DA);

[0015] so as to improve depression and anxiety-like behaviors induced by chronic unpredictable mild stress.

[0016] Animal experiments show that oral administration of a bacterial suspension with a dose of 1x10 9 CFU / day can significantly relieve the depression and anxiety-like behaviors of model mice.

[0017] Further, the above-mentioned Pediococcus acidilactici strain GOLDGUT-EZMind includes the bacterial bodies, metabolites and fermentation broth of the Pediococcus acidilactici strain GOLDGUT-EZMind.

[0018] Further, the above-mentioned mood disorders include depression and anxiety and other central nervous system diseases related to neuroinflammation.

[0019] Further, the anxiety includes acute anxiety, chronic anxiety, nervous function regulation disorder, cardiovascular and cerebrovascular diseases, and digestive system diseases.

[0020] Further, the depression includes chronic stress-induced depression.

[0021] Further, the products include drugs, foods, and nutritional supplements, etc., for preventing, improving, or treating mood disorders, especially depression and anxiety, and other central nervous system diseases related to neuroinflammation.

[0022] Further, the drugs are administered by oral route, and the dosage forms include capsules, tablets, granules, oral solutions, and lyophilized powders, etc.

[0023] Further, the foods include general foods, special medical purpose formula foods, functional foods, and health foods, etc.

[0024] Further, the foods are consumed by oral route, and the dosage forms include yogurt and milk beverages, etc.

[0025] According to the technical solutions, compared with the prior art, the present application has the following beneficial effects:

[0026] 1. The present application screens and identifies a Pediococcus acidilactici strain GOLDGUT-EZMind that can effectively relieve depression and anxiety symptoms from abundant microbial resources, further explores and clarifies the internal biological mechanism of regulating host health, and applies the strain with clear efficacy and mechanism to functional food development, thereby providing an innovative solution with clearer target and more sufficient scientific basis for preventing and improving depression through dietary approach.

[0027] 2. The Pediococcus acidilactici strain GOLDGUT-EZMind in the present application shows significant effects in improving chronic stress-induced anxiety and depression-like behaviors, such as significantly improving anxiety-like behaviors, reducing despair behaviors, and improving hedonic experience in animal models.

[0028] 3. The present application first discloses and confirms that the Pediococcus acidilactici strain GOLDGUT-EZMind plays an antidepressant role through the "improving activation of neural microglial cells, relieving neuroinflammation, and restoring neurotransmitter levels in the brain" pathway. The strain efficiently produces ILA in the intestinal tract, and ILA enters the brain and activates the AHR signaling pathway, thereby inhibiting the overactivation of microglial cells, down-regulating pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, and effectively reducing neuroinflammation. The elucidation of this mechanism provides a solid scientific basis for the application of the present application, and distinguishes it from the probiotic products in the prior art with unknown mechanisms.

[0029] 4. The P. acidilactici GOLDGUT-EZMind of the present application can significantly increase the levels of 5-HT and DA in the cerebral cortex of model mice with depression, restore neurotransmitter balance, and correct the neurotransmitter imbalance caused by chronic stress, which is the core target of many first-line antidepressants.

[0030] 5. The present application first discovered that P. acidilactici GOLDGUT-EZMind can relieve neuroinflammation and improve depression and anxiety through the dual pathways of anti-neuroinflammation and neurotransmitter regulation, providing a new strategy for the preparation of pharmaceuticals, food and nutritional supplements, non-pharmacological intervention or adjuvant therapy for depression, anxiety and other central nervous system diseases related to neuroinflammation, and has good application prospects and industrial value.

[0031] 6. The P. acidilactici GOLDGUT-EZMind of the present application has a reasonable dosage and significant efficacy, effectively avoiding the adverse effects caused by the unclear dosage of existing probiotic preparations. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 It is a real picture of the colony morphological characteristics of P. acidilactici GOLDGUT-EZMind;

[0033] Figure 2 It is a morphological picture of P. acidilactici GOLDGUT-EZMind observed under a microscope;

[0034] Figure 3 It is a phylogenetic tree constructed by homology comparison of the sequence of P. acidilactici GOLDGUT-EZMind and similar strains included in GenBank;

[0035] Figure 4 It is an experiment of P. acidilactici GOLDGUT-EZMind improving anxiety-like behavior, wherein (A) is the center area stay time in the open field, and (B) is the open arm stay time in the elevated plus maze, and the results are expressed as mean ± standard error (n=8);

[0036] Figure 5 It is an experiment of P. acidilactici GOLDGUT-EZMind improving depression-like behavior, wherein (A) is the tail suspension immobility time, and (B) is the sucrose preference, and the results are expressed as mean ± standard error (n=8);

[0037] Figure 6 It is an experiment of P. acidilactici GOLDGUT-EZMind improving the imbalance of cortical neurotransmitter levels, wherein (A) is 5-HT, and (B) is DA, and the results are expressed as mean ± standard error (n=6);

[0038] Figure 7Effects of P. acidilactici GOLDGUT-EZMind on improving inflammatory cytokines expression in brain, (A)-(C) relative mRNA expression of inflammatory cytokines in hippocampus; (D)-(F) inflammatory cytokines levels in cortex, results were expressed as mean ± standard error (n = 6);

[0039] Figure 8 Effects of P. acidilactici GOLDGUT-EZMind on improving excessive activation of brain microglia, (A) representative images of IBA-1 protein immunofluorescence staining in hippocampal DG region and cortex (200x); (B)&(C) quantification of IBA-1 positive area in hippocampal DG region and cortex; (D)&(E) quantification of IBA-1 fluorescence intensity in hippocampal DG region and cortex; results were expressed as mean ± standard error (n = 3). DETAILED DESCRIPTION

[0040] The technical solutions in the embodiments of the present application will be described below in a clear and complete manner. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present application.

[0041] Embodiment 1

[0042] Isolation, identification and preservation of P. acidilactici GOLDGUT-EZMind

[0043] 1. Sample collection

[0044] 50-100 g of samples of characteristic fermented food sweet glutinous rice were collected in sterile self-sealing bags from the Qinghai-Tibet Plateau region, 2-3 samples were collected for each sample, sealed and transported at low temperature, and temporarily stored in a 4°C refrigerator (for isolation and culture within one week).

[0045] 2. Preparation of culture medium

[0046] ① Preparation of MRS solid plate: accurately weigh 48 g of MRS broth medium, 1 mL of resazurin, 1000 mL of distilled water, and 15.00 g of agar powder in a beaker, dissolve thoroughly, then transfer to anaerobic bottles, pass nitrogen for 3 min, then put into a sterilization pot for 115°C sterilization for 15 min. After sterilization, transfer to an anaerobic box after slight cooling, pour into disposable culture dishes one by one, about 20 mL each, after complete cooling and solidification, turn over and move to a culture box, use after one day without contamination.

[0047] ② Preparation of MRS liquid medium: accurately weigh 48 g of MRS broth medium, 1 mL of resazurin, and 1000 mL of distilled water in a beaker, dissolve thoroughly, then transfer to anaerobic bottles, pass nitrogen for 3 min, then put into a sterilizer for 115°C sterilization for 15 min. After sterilization, quickly transfer to an anaerobic box, cool, then dispense 1.4 mL of MRS liquid medium into 2.0 mL round-bottom centrifuge tubes, and move to a culture box. After one day, if there is no contamination, it can be used.

[0048] 3. Isolation and culture of strains

[0049] ① Sample dilution: transfer fresh samples to an anaerobic box, carefully wipe the bag opening or bottle opening with alcohol cotton balls twice, about 0.2 g in 1.0 mL PBS solution, thoroughly blow to suspension. Take 100 μL of the suspension in 900 μL of PBS solution, mix thoroughly, and gradually dilute by 10 times to 10 -8 .

[0050] ② Sample plating culture: take the diluted sample of ①, select 10 -5 , 10 -6 , 10 -7 , and 10 -8 times dilution gradient of the bacterial solution for plate coating. Take 100 μL of the diluted solution on the plate, and coat with a coating rod, 2 plates for each sample and each dilution gradient. Place the evenly coated plates in a 37°C anaerobic incubator for 48 h, and place blank plates as controls.

[0051] ③ Colony purification and passage: take out the culture dishes cultured in ②, carefully observe the morphological characteristics of the colonies (colony diameter, morphology, color, water content, edge, transparency, and elevation) in an anaerobic box. Observe with a magnifying glass, and distinguish different colonies according to morphology. After roughly distinguishing, use a inoculating loop to pick different morphological colonies (3-5 strains of the same morphological colonies are picked repeatedly), plate streak culture for 24-48 h, repeat 2-3 times to obtain pure single colonies. Then use a gun head to pick them and place them in 1.5 mL of MRS liquid medium, culture for 16-24 h, then take 100 μL of the bacterial solution in a new 1.5 mL of MRS liquid medium, and repeat the passage 2-3 times.

[0052] 4. Strain preservation

[0053] After subculture, the purified strains were preserved at the end of the logarithmic phase. 200 μL of bacterial solution was mixed with 200 μL of 40% glycerol in a 2.0 mL cryovial, and the strain number and information were labeled. At least 3 vials of each strain were preserved. The labeled strains were placed in a pre-cooled cryovial, and the strain information and growth conditions were recorded on the cover. Then, the cryovial was placed in a -80°C refrigerator. One of the backup vials was used for subsequent DNA sequencing, probiotic function evaluation, and metabolite analysis.

[0054] 5. 16S rDNA identification

[0055] ① PCR amplification of 16S rRNA gene of single strain screened and cultured: 1 mL of bacterial solution cultured for 24 h was centrifuged (10000 r / min, 5 min, 4°C) to discard the supernatant, and 200 μL of PBS was used for suspension. The full-length universal primer of bacterial 16S rRNA gene was used, the upstream primer was 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', and the downstream primer was 1492R: 5'-CTACGGCTACCTTGTTACGA-3'. The PCR amplification system (25 μL) included 1 μL of each of the universal primers 27F and 1492R, 1 μL of bacterial solution template, 12 μL of 2×Taq PCR Master Mix, and finally ddH2O was added to 25 μL. The PCR reaction conditions were as follows: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 55°C annealing for 45 s, 72°C extension for 1 min, a total of 35 cycles; 72°C extension for 10 min.

[0056] ② Electrophoresis detection: 100 mL of 1.5% agarose gel was prepared, heated in a microwave oven until completely dissolved, and after slight cooling, 6 μL of GelRed nucleic acid gel dye was added, mixed well, and poured into a gel plate with a gel holder and a gel comb. After cooling until it solidifies, the gel comb was removed, and the gel was taken out together with the gel holder and placed in an electrophoresis tank. The PCR product after amplification was spotted 5 μL, and DL 2000 DNA Marker was spotted 5 μL. Electrophoresis detection was performed at 200 V for 15 min. After electrophoresis, gel imaging was performed to check the banding in the 1500 bp fragment region. In the absence of a blank control band, the PCR product with positive amplification results was selected for purification and Sanger sequencing.

[0057]

[0058] This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35671 and deposit date of August 18, 2025. Its taxonomic name is Pediococcus acidilactici.

[0059] 6. Test Results

[0060] like Figure 1 As shown, the morphology of the plate colonies is as follows: approximately 0.5-2 mm in diameter, round, with neat edges, opaque, milky white or grayish white in color, with a raised center, smooth surface, moist texture, and easy to pick up.

[0061] like Figure 2 As shown, under a microscope, this strain is observed to be Gram-positive, spherical cells, usually arranged in pairs, chains, or clusters, and does not form spores or move.

[0062] like Figure 3 As shown, the 16S identification results indicate that, according to the phylogenetic tree of the model bacteria, this bacterium is a strain of Pediococcus acidilactici.

[0063] Example 2

[0064] Pediococcus lactis GOLDGUT-EZMind can improve depression-anxiety-like behavior in mice.

[0065] 1. Grouping of experimental animals

[0066] Eight-week-old male C57BL / 6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animal procedures were approved by the Animal Ethics Committee of Northwest A&F University (Ethics No.: IACUC2024-1110).

[0067] After one week of acclimatization, mice were randomly divided into three groups: control group (CON), model group (CUMS), and GOLDGUT-EZMind intervention group (CUMS+GOLDGUT-EZMind), with eight mice in each group. Mice in the model and intervention groups received a 6-week chronic, unpredictable, mild stress regimen. Stressors included 5 minutes of swimming in 4°C cold water, 3 hours of restraint, 24 hours of fasting or water deprivation, damp bedding, cage tilting, and 5 minutes of tail clamping, with the sequence randomly assigned weekly to reduce predictability. The CON group received no stress treatment.

[0068] 2. Processing methods for each group

[0069] Control group: Normal drinking water, supplemented with 100μL PBS by gavage daily.

[0070] Model group: received a 6-week chronic unpredictable mild stress protocol and were supplemented with PBS 100 μL by gavage every day.

[0071] GOLDGUT-EZ Mind intervention group: received a 6-week chronic unpredictable mild stress protocol and were supplemented with 100 μL of a suspension of cultured Lactobacillus acidophilus GOLDGUT-EZ Mind in PBS to a viable count of 1 x 10 10 CFU / mL, prepared fresh daily. The bacterial suspension was supplemented by gavage every day.

[0072] 3. Behavioral testing methods

[0073] After the 6-week intervention, mice were assessed for anxiety- and depression-like behaviors by a series of behavioral tests.

[0074] 3.1 Open field test

[0075] The open field test was used to assess locomotor activity and anxiety-like behavior in mice. The test was performed in an opaque, uncovered monitoring box (50 cm x 50 cm x 50 cm) with a floor divided into 25 equal squares. A video camera positioned above the arena recorded the locomotor trajectory of the mice for 5 min. The total distance traveled and the time spent in the central area were analyzed to assess the level of locomotor activity and anxiety. The open field was wiped with 75% alcohol before testing to eliminate odor interference from other mice.

[0076] 3.2 Elevated plus maze test

[0077] The elevated plus maze was used to assess the level of anxiety in mice and consisted of two open arms (30 cm x 8 cm), two closed arms (30 cm x 8 cm x 15 cm), and a central area (8 cm x 8 cm). The mice were placed in the central area and the time spent in the open arms was recorded to assess the level of anxiety in the mice.

[0078] 3.3 Tail suspension test

[0079] The tail suspension test was used to assess the level of depression in mice. The tail of the mouse was taped to the tail tip at a distance of 1-2 cm, and the mouse was placed at a distance of about 20 cm from the ground. The time spent immobile by the mouse in the last 4 min of a 6-min test was recorded to assess the level of despair in the mouse as a reflection of its depression.

[0080] 3.4 Sucrose preference test

[0081] Sucrose preference is often used to assess the degree of anhedonia and depression in rodents. The experiment was divided into two parts: two days of adaptation training and one day of testing. On the first day of adaptation training, mice were given two bottles of 1% sucrose solution (w / v) to adapt. On the second day, one of the sucrose solutions was replaced with drinking water, and the positions of the two bottles were exchanged in the middle to avoid positional interference. Before the test, the mice were deprived of food and water for 12 h. Then the mice were provided with one bottle of 1% sucrose solution (w / v) and drinking water for 12 h, and weighed after the end to calculate the liquid consumption. The ratio of sucrose solution consumption to total liquid consumption was used to evaluate sucrose preference.

[0082] 4. Data and statistical analysis

[0083] Data and statistical analysis were performed using GraphPad Prism 8.0. The results were expressed as mean ± standard error (Mean ± SEM), and one-way ANOVA was used to determine significant differences between groups, and Tukey's multiple comparison test was used as post-hoc analysis. p < 0.05 was considered statistically significant, where * represents p < 0.05, with significant difference, and ** represents p < 0.01, with extremely significant difference.

[0084] 5. Test results

[0085] It can be seen from Figure 4 that compared with the CON group, the CUMS group mice had significantly reduced stay time in the center of the open field and exploration time in the open arm of the elevated plus maze, showing typical anxiety-like behavior, while after 6 weeks of probiotic intervention, the mice had significantly increased stay time in the center of the open field (p < 0.01) and exploration time in the open arm of the elevated plus maze (p < 0.01), and significantly reduced anxiety-like behavior. In addition, it can be seen from Figure 5 that compared with the CON group, the CUMS group mice had significantly increased immobility time in the tail suspension test and significantly reduced sucrose preference, showing typical despair and anhedonic depression-like behavior. After probiotic intervention, the mice had significantly reduced immobility time in the tail suspension test (p < 0.01) and significantly increased sucrose preference index (p < 0.01). The above results show that P. acidilactici GOLDGUT-EZ Mind intervention can significantly improve the anxiety and depression-like behavior of mice.

[0086] Example 3

[0087] P. acidilactici GOLDGUT-EZ Mind improves the imbalance of neurotransmitters in the cortex

[0088] 1. Experimental grouping and treatment were the same as in Example 2.

[0089] 2. Experimental method

[0090] At the end of the 6-week intervention, the mice were sacrificed and the cortical tissues were collected. The levels of neurotransmitters in the cortical tissues were detected by high-performance liquid chromatography. The specific method is as follows:

[0091] The cortical tissues were homogenized in physiological saline to 10% (w / v), and centrifuged (12000 r / min, 15 min, 4°C) to obtain the supernatant. The precipitant (0.60 mol / L perchloric acid, 1.20 mol / L hydrogen peroxide, 2.00 mmol / L ethylenediaminetetraacetic acid) was added to the supernatant at a ratio of 1:1. The mixture was incubated on ice for 10 min, and then centrifuged again under the same conditions. The resulting supernatant was filtered through a 0.22 μm microporous membrane to remove particulate matter and prepare the sample for analysis. Immediately after sample preparation, the 5-HT and DA levels in the supernatant were analyzed by HPLC. 20 μL of each sample was injected into the chromatograph, and the excitation wavelength of the RF-20A fluorescence detector was 290 nm and the emission wavelength was 330 nm. Chromatographic separation was performed at 30°C at a flow rate of 1.0 mL / min. The mobile phase used was chromatographic grade methanol (mobile phase A) and 0.10 mol / L sodium acetate buffer (pH 5.1, containing 0.10 mmol / L ethylenediaminetetraacetic acid disodium, mobile phase B), with an elution ratio of 1:9, and a C18 chromatographic column was used for isocratic elution for 25 min.

[0092] 3. Data and statistical analysis

[0093] Data and statistical analysis were performed using GraphPad Prism 8.0. The results are expressed as mean ± standard error (Mean ± SEM), and one-way ANOVA was used to determine significant differences between groups, and Tukey's multiple comparison test was used as post-hoc analysis. p<0.05 was considered statistically significant, where * indicates p<0.05, with a significant difference, and ** indicates p<0.01, with a highly significant difference.

[0094] 4. Test results

[0095] The occurrence of depression is associated with abnormalities in neurotransmitter levels. It is known that Figure 6 Compared with the CON group, the levels of 5-HT and DA in the cortex of mice in the CUMS group were significantly reduced, and the 6-week probiotic intervention significantly improved the levels of 5-HT and DA in mice (p<0.05, p<0.01), returning to normal levels. This indicates that Lactococcus lactis GOLDGUT-EZMind improves the reduction of neurotransmitter (5-HT and DA) levels in the cortex of mice induced by chronic stress.

[0096] Example 4

[0097] Pediococcus acidilactici GOLDGUT-EZ Mind reduces expression of inflammatory factors in brain

[0098] 1. Experimental grouping and treatment were the same as in Example 1.

[0099] 2. Experimental method

[0100] After the end of the 6-week intervention, the mice were sacrificed and their cortical and hippocampal tissues were collected. The cortical tissues were used to detect the levels of inflammatory factors using an ELISA kit, and the hippocampal tissues were used to detect the gene expression levels of inflammatory factors using qRT-PCR. The specific methods are as follows:

[0101] 2.1 Real-time quantitative PCR

[0102] RNA extraction and reverse transcription: Weigh about 50 mg of tissue sample on an ice brick and transfer it to a 1.5 mL enzyme-free centrifuge tube according to the sequence number. Add 700 μL of BIOZOL reagent to the centrifuge tube, mix well and centrifuge (12000 r / min, 5 min, 4℃), transfer the upper aqueous phase to another empty tube, add 350 μL of isopropanol, mix well and centrifuge (12000 r / min, 10 min, 4℃), discard the supernatant, add 75% ethanol and gently shake, air dry at room temperature, then dissolve the mRNA sample with sterile enzyme-free water; use a nucleic acid quantifier to measure the absorbance value (260 nm / 280 nm) and concentration of the mRNA sample, select samples with an absorbance value between 1.8 and 2.0 for subsequent operations; calculate and dilute all mRNA samples to the same concentration; prepare the reverse transcription system according to the instructions of the reverse transcription kit, and use a PCR amplifier to reverse transcribe the mRNA to cDNA.

[0103] qRT-PCR: Continue to dilute the cDNA after reverse transcription to the required concentration, prepare the PCR reaction system, and use a PCR instrument 5 to quantify, the PCR reaction conditions are: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 15 s, 60℃ for 1 min, a total of 40 cycles; 60℃ extension for 10 min. Use the endogenous gene GAPDH as a control and record the threshold value (Ct), use the 2 -ΔΔCT method to calculate the relative content of related gene expression.

[0104] The PCR amplification primer sequences are as follows:

[0105] Table 1 Primer sequences

[0106]

[0107] 2.2 ELISA detection

[0108] The levels of inflammatory cytokines (TNF-a, IL-1 b and IL-6) in the cortex were determined using a double antibody sandwich method according to the method of the enzyme-linked immunosorbent assay (ELISA) kit instructions.

[0109] 3. Data and statistical analysis

[0110] Data and statistical analysis were performed using GraphPad Prism 8.0. The results were expressed as mean ± standard error (Mean ± SEM), and one-way ANOVA was used to determine significant differences between groups, and Tukey's multiple comparison test was used as post-hoc analysis. p < 0.05 was considered statistically significant, where * represents p < 0.05, with a significant difference, and ** represents p < 0.01, with a highly significant difference.

[0111] 4. Test results

[0112] Abnormal expression of inflammatory cytokines in the central nervous system can disrupt neuronal function, leading to neurotransmitter imbalance and abnormal neural network function, thereby triggering or exacerbating depressive symptoms. The CUMS model is a widely used animal model of depression, which can induce depressive symptoms in mice. Figure 7 It was found that the mRNA expression levels of pro-inflammatory cytokines TNF-a, IL-1 b and IL-6 in the hippocampus were significantly up-regulated in CUMS group compared with CON group mice, and the levels of inflammatory cytokines in the cortex also had the same trend. The intervention of Lactococcus GOLDGUT-EZMind significantly reduced the mRNA expression of pro-inflammatory cytokines TNF-a, IL-1 b and IL-6 in the hippocampus (p < 0.05), and reduced the levels of pro-inflammatory cytokines TNF-a and IL-1 b in the cortex (p < 0.01). The above results showed that the intervention of Lactococcus GOLDGUT-EZMind can significantly improve the abnormal expression of inflammatory cytokines in the brain of CUMS-induced mice.

[0113] Example 5

[0114] Effect of Lactococcus GOLDGUT-EZMind on excessive activation of brain microglial cells

[0115] 1. Experimental grouping and treatment The same as Example 2.

[0116] 2. Experimental method

[0117] After the mice were sacrificed, half of the brain of the mice was dissected and put into 4% (v / v) paraformaldehyde for 24 h. Then the specimen was embedded in paraffin, cut into 5 pm sections for subsequent staining. The specific steps of staining are as follows: ① The section was dried in a 37 °C oven overnight; ② xylene immersion for dewaxing (5 min x 2 times), gradient ethanol immersion for 5 min for rehydration, PBS washing (5 min x 4 times); ③ the tissue section was immersed in 0.5% Triton X-100 permeation liquid for 15 min, PBS washing (3 min x 6 times); ④ the section was boiled in 100 °C water bath for 15 min after microwave heating in sodium citrate repair solution for 8 min, and then cooled to room temperature, PBS washing (5 min x 6 times); ⑤ 3% H2O2 immersion for 15 min to block endogenous peroxidase, PBS washing (3 min x 5 times); ⑥ goat serum was added on the tissue section to block non-specific sites, incubated at room temperature for 20 min, then the section was wiped dry and covered with primary antibody (anti-IBA-1, 1:1000), incubated in a wet box at 4 °C overnight; ⑦ the section was taken out the next day and restored to room temperature, PBS washing (3 min x 6 times); ⑧ fluorescent secondary antibody was incubated at room temperature for 2 h in the dark, PBS washing 3 min x 6 times; ⑨ DAPI mounting. Immunofluorescence images were obtained using an inverted fluorescence microscope, and Image J software was used to quantify positive areas.

[0118] 3. Data and statistical analysis

[0119] Data and statistical analysis were performed using GraphPad Prism 8.0. The results were expressed as mean ± standard error (Mean ± SEM), and one-way ANOVA was used to determine the significant differences between groups, and Tukey's multiple comparison test was used as post-hoc analysis. p < 0.05 was considered statistically significant, where * represents p < 0.05, with significant difference, and ** represents p < 0.01, with extremely significant difference.

[0120] 4. Test results

[0121] The over-activation of microglia is one of the pathological features of depression, and IBA-1 is a protein that specifically marks neural microglia and is usually used as an activation marker. The results showed that the number of IBA-1 positive cells in the hippocampus of the mice in the model group was significantly increased compared with that in the control group, and the number of IBA-1 positive cells in the hippocampus of the mice in the treatment group was significantly reduced compared with that in the model group. Figure 8It can be seen that compared with the CON group, the positive area and fluorescence intensity of microglia in the hippocampal dentate gyrus (DG) and cortex of the CUMS group mice were significantly increased, indicating that the microglia in the hippocampus and cortex were over-activated. After the intervention of GOLDGUT-EZMind, the positive area and fluorescence intensity of microglia in the DG and cortex of the mice were significantly reduced (p<0.05, p<0.01). This shows that the intervention of Lactococcus lactis GOLDGUT-EZMind can significantly improve the over-activation of microglia in the hippocampal DG and cortex of CUMS-induced mice.

[0122] The above description of disclosed embodiments enables one of ordinary skill in the art to make or use the application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Thus, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of *Pediococcus lactis* GOLDGUT-EZMind, characterized in that, This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35671 and deposit date of August 18, 2025. Its taxonomic name is Pediococcus acidilactici.

2. The use of the lactic acid porphyria GOLDGUT-EZMind as described in claim 1 in the preparation of products for the prevention, improvement or treatment of mood disorders.

3. The application according to claim 2, characterized in that, The *Pediococcus lactis* GOLDGUT-EZMind includes at least one of the *Pediococcus lactis* GOLDGUT-EZMind cells, metabolites, and fermentation broth.

4. The application according to claim 2, characterized in that, The mood disorders mentioned include depression and anxiety.

5. The application according to claim 4, characterized in that, The anxiety disorders mentioned include acute anxiety, chronic anxiety, neurological dysfunction, cardiovascular and cerebrovascular diseases, and digestive system diseases.

6. The application according to claim 4, characterized in that, The depression mentioned includes depression induced by chronic stress.

7. The application according to claim 2, characterized in that, The products include pharmaceuticals, food, and nutritional supplements.

8. The application according to claim 7, characterized in that, The drug is administered orally, and its dosage forms include capsules, tablets, granules, oral liquids, and lyophilized powders.

9. The application according to claim 7, characterized in that, The food products mentioned include general food, food for special medical purposes, functional food, and health food.

10. The application according to claim 7 or 8, characterized in that, The food is to be consumed orally, and the dosage forms include yogurt and dairy beverages.

Images