Streptococcus lactis capable of reducing activation of hippocampal microglia cells and application thereof

Abstract

The present application relates to a Pediococcus acidilactici capable of reducing the activation of hippocampal microglia cells and its application, belonging to the technical field of microorganisms. The Pediococcus acidilactici described in the present application is Pediococcus acidilactici CCFM1344, which was preserved in the Guangdong Microbial Culture Collection Center on September 12, 2023, with the preservation number GDMCC NO.63800. The preservation address is Building 59, 5th Floor, Guangdong Microbial Institute, 100 Middle Martyrs Road, Guangzhou. The Pediococcus acidilactici described in the present application can inhibit the over-activation of microglia cells caused by chronic stress, reduce the concentration of pro-inflammatory factors in the hippocampus of the brain, thereby relieving the activation of microglia cells in the hippocampus of the brain caused by chronic stress, and has excellent application prospects.

Description

Technical Field

[0001] This invention relates to a strain of *Pediococcus lactis* that can reduce the activation of microglia in the hippocampus and its applications, belonging to the field of microbial technology. Background Technology

[0002] Microglia are the most common innate immune cells of the central nervous system (CNS), accounting for 10%-20% of the total number of CNS cells. They are the most abundant immune cells in the CNS, playing a crucial role in maintaining CNS homeostasis and inducing neuroinflammation. During early development, microglia promote neuronal circuit formation and growth by releasing trophic factors, a function essential for normal brain development. However, when CNS lesions occur, microglia are abnormally activated and secrete various cytokines and inflammatory factors, thereby inducing immune and inflammatory responses and further exacerbating CNS damage.

[0003] Probiotics are live microorganisms that, when ingested in sufficient quantities, have beneficial effects on the host's longevity. Existing research has revealed that probiotic intake can inhibit microglia; for example, intervention with *Clostridium butyricum* in mice with Parkinson's disease can improve motor function, inhibit microglia activation, and improve synaptic dysfunction. However, long-term excessive use of *Clostridium butyricum* has been found to potentially cause diarrhea and bloody feces in farmed animals. *Pediococcus lactis*, as a probiotic, is a generally recognized safe (GRAS) strain. Current research on lactobacilli mainly focuses on their inhibitory effects on harmful intestinal microorganisms and their role in enhancing the gastrointestinal barrier. However, there are no reports on *Pediococcus lactis* alleviating abnormal microglia activation and related symptoms. Summary of the Invention

[0004] [Technical Issues]

[0005] The technical problem to be solved by the present invention is the lack of Pyrococcus lactis in the prior art that can effectively alleviate the activation of microglia in the hippocampus and related symptoms.

[0006] [Technical Solution]

[0007] To address the aforementioned technical problems, this invention provides a strain of *Pediococcus acidilactici* CCFM1344. This strain can reduce serum LPS levels and the expression of downstream LPS receptors TLR4 and NF-κB in the hippocampus, as well as reduce the expression of pro-inflammatory factors TNF-α and IL-1β in the hippocampus. This strain can also reduce the expression levels of the microglia marker Iba1 gene and Iba1 protein, thereby alleviating the activation of microglia in the hippocampus caused by chronic stress.

[0008] This invention provides a strain of Pediococcus acidilactici CCFM1344, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO.63800.

[0009] The Pediococcus acidilactici strain was derived from fecal samples of healthy adults and identified as Pediococcus acidilactici by 16S rDNA.

[0010] The *Pediococcus lactis* CCFM1344 is a Gram-positive bacterium that appears spherical under a microscope. When grown on De Man-Rogosa and Sharpe (MRS) agar, it forms smooth, translucent, round colonies that are white with neat edges. In MRS liquid medium, it grows in a uniformly turbid manner, and the cells precipitate white after prolonged exposure. The optimal growth temperature is 37°C.

[0011] The present invention also provides a microbial preparation containing the above-mentioned Pediococcus acidilactici CCFM1344, or its lyophilized powder.

[0012] In one embodiment, the amount of Pediococcus acidilactici CCFM1344 added to the microbial preparation is not less than 1×10⁻⁶. 8 CFU / g or 1×10 8 CFU / mL.

[0013] In one embodiment, the microbial preparation is a solid preparation or a liquid preparation.

[0014] The present invention also provides products containing the above-mentioned Pediococcus acidilactici CCFM1344, or products containing the above-mentioned microbial preparations.

[0015] The present invention also provides the use of the above-mentioned Pediococcus acidilactici CCFM1344, or the above-mentioned microbial preparation, in the preparation of products for the prevention and / or treatment of hippocampal mitochondrial activation and / or mental illnesses associated with hippocampal mitochondrial activation.

[0016] In one embodiment, the amount of live *Pediococcus lactis* CCFM1344 bacteria added to the product is not less than 1×10⁻⁶. 8 CFU / g or 1×10 8 CFU / mL.

[0017] In one embodiment, the product includes food, medicine, or health products.

[0018] In one embodiment, the food product includes dairy products, soy products, or fruit and vegetable products.

[0019] In one embodiment, the dairy product includes fermented milk, flavored fermented milk, fermented milk beverages, cream, cheese, milk-containing beverages, or milk powder.

[0020] In one embodiment, the soy products include soy milk and soy milk powder.

[0021] In one embodiment, the fruit and vegetable products include those made from at least one of cabbage, white radish, cucumber, beet, yellow peach, or bayberry products.

[0022] In one embodiment, the food includes solid food, liquid food, or semi-solid food.

[0023] In one implementation, the food includes beverages or snacks.

[0024] In one embodiment, the pharmaceutical product includes a drug carrier and / or pharmaceutical excipients.

[0025] In one embodiment, the drug carrier comprises one or more of microcapsules, microspheres, nanoparticles, and liposomes.

[0026] In one embodiment, the pharmaceutical excipient includes one or more of the following: medically acceptable fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents.

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

[0028] In one embodiment, the prevention and / or treatment of hippocampal mitral activating cells and / or mental illnesses associated with hippocampal mitral activating cells includes at least one of the following (a)-(g) effects:

[0029] (a) Restore intestinal lactic acid levels in a chronic stress model;

[0030] (b) Inhibits the proliferation of intestinal pathogens caused by chronic stress;

[0031] (c) Increase gene expression of intestinal tight junction proteins ZO-1, and / or Occludin, and / or Claudin-1;

[0032] (d) Reduce serum LPS levels;

[0033] (e) Reduce the expression of LPS downstream receptors TLR4 and / or NF-κB in the hippocampus of the brain;

[0034] (f) Reduce gene expression of pro-inflammatory factors TNF-α and / or IL-1β in the hippocampus of the brain;

[0035] (g) Reduce the expression of ion-calcium binding linker 1 (Iba1).

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0037] The *Pediococcus lactis* CCFM1344 provided by this invention can alleviate hippocampal microglia activation and associated mental illnesses, specifically in the following ways:

[0038] 1. Significantly upregulated the content of lactic acid in intestinal metabolites; the CCFM1344 treatment group was 2.96 times that of the model group and 2.91 times that of the P. FSDLZ42M3 group.

[0039] 2. It inhibits the abnormal increase of pathogenic bacteria in the intestinal flora and regulates the homeostasis of the intestinal microecology. Compared with the model group, the relative abundance of Escherichia coli and Staphylococcus aureus in the CCFM1344 treatment group decreased by 76.87% (P<0.001) and 91.66% (P=0.004), respectively; compared with the Pediococcus lactis FSDLZ42M3 treatment group, the relative abundance of Escherichia coli and Staphylococcus aureus decreased by 77.26% (P<0.001) and 97.59% (P<0.001), respectively.

[0040] 3. Upregulation of intestinal tight junction proteins ZO-1, Occludin, and Claudin-1 gene expression: Compared with the model group, the relative expression level of ZO-1 gene expressing ZO-1 protein increased by 2.63 times (P<0.001), the relative expression level of Ocln1 gene expressing Occludin protein increased by 2.87 times, and the relative expression level of Cldn1 gene expressing Claudin-1 protein increased by 2.28 times (P<0.001) in the CCFM1344 treatment group; compared with Pediococcus lactis FSDLZ42M3 (P<0.001), the relative expression level of ZO-1 gene increased by 2.75 times (P<0.001), the relative expression level of Ocln1 gene increased by 4.53 times (P<0.001), and the relative expression level of Cldn1 gene increased by 0.5 times (P=0.007);

[0041] 4. Reduced serum LPS levels: Compared with the model group, the LPS level in the CCFM1344 treatment group decreased by 31.08% (P = 0.0011); compared with the Lactococcus FSDLZ42M3 treatment group, the LPS level decreased by 32.42% (P = 0.0006).

[0042] 5. Reduce the relative expression levels of genes expressing LPS downstream receptor TLR4 and NF-κB proteins in the hippocampus of the brain, as well as the expression levels of LPS downstream receptor TLR4 and NF-κB proteins:

[0043] Compared with the model group, the relative expression level of the Tlr4 gene expressing TLR4 protein was downregulated by 58.35% in the CCFM1344 treatment group (P<0.001), and the relative expression level of the Nfkb1 gene expressing NF-κB protein was downregulated by 71.8% (P=0.008); compared with the Pediococcus lactis FSDLZ42M3 treatment group, the relative expression level of the Tlr4 gene was downregulated by 46.48% (P<0.001), and the relative expression level of the Nfkb1 gene was downregulated by 61.75% (P<0.001).

[0044] Compared with the model group, the TLR4 and NF-κB protein levels in the CCFM1344 treatment group decreased by 43.3% and 56.6%, respectively (the relative expression levels of TLR4 and NF-κB protein in the model group were 1.905 and 2.21, respectively; the relative expression levels of TLR4 and NF-κB protein in the CCFM1344 treatment group were 1.08 and 0.96, respectively); compared with the *Pediococcus lactis* FSDLZ42M3 treatment group, the TLR4 and NF-κB protein levels decreased by 46.3% and 66.3%, respectively (the relative expression levels of TLR4 and NF-κB protein in the *Pediococcus lactis* FSDLZ42M3 treatment group were 2.01 and 2.845, respectively).

[0045] 6. Reduced expression of pro-inflammatory factors TNF-α and IL-1β in the hippocampus of the brain. Compared with the model group, the relative expression level of the TNF-α gene Tnf in the CCFM1344 treatment group was downregulated by 55.32% (P<0.001), and the relative expression level of the IL-1β gene Il1b was downregulated by 44.63% (P=0.01); compared with the Pediococcus lactis FSDLZ42M3 treatment group, the relative expression level of Tnf gene was downregulated by 44.15% (P=0.01), and the relative expression level of Il1b gene was downregulated by 50.56% (P<0.001).

[0046] 7. Inhibits excessive activation of microglia caused by chronic stress. The relative expression level of Iba1 mRNA, a marker of microglia, was 0.784. Compared with the model group (relative expression level of Iba1 mRNA was 1.86), the relative expression level of Iba1 gene in the CCFM1344 treatment group was downregulated by 57.85% (P<0.001); compared with the Pediococcus lactis FSDLZ42M3 treatment group (relative expression level of Iba1 mRNA was 1.64), the relative expression level of Iba1 gene was downregulated by 52.2% (P=0.006).

[0047] Compared with the model group, the expression of Iba1 protein in the CCFM1344 treatment group decreased by 36.5% (the relative expression level of Iba1 protein in the model group was 2; the relative expression level of Iba1 protein in the CCFM1344 treatment group was 1.27, P = 0.013); compared with the Pediococcus lactis FSDLZ42M3 treatment group, it decreased by 46.5% (the relative expression level of Iba1 protein in the FSDLZ42M3 treatment group was 2.375, P = 0.003).

[0048] This invention expands the application scope of *Pediococcus lactis* as a probiotic and provides theoretical guidance for using probiotics to prevent, alleviate, and treat nervous system dysfunction. The *Pediococcus lactis* described in this invention can be used to prepare functional foods, health products, and pharmaceuticals that reduce hippocampal microglia activation, inhibit intestinal pathogens, and alleviate intestinal barrier damage, showing very broad application prospects.

[0049] Preservation of biological materials

[0050] A strain of *Pediococcus acidilactici*, CCFM1344, taxonomically named *Pediococcus acidilactici*, was deposited on September 12, 2023, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC NO. 63800), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology. Attached Figure Description

[0051] Figure 1 Lactic acid production and relative lactic acid content in intestinal metabolites of different Pediococcus acidilactici strains: (A) Lactic acid production of different Pediococcus acidilactici strains; (B) Lactic acid content in intestinal metabolites of Pediococcus acidilactici CCFM1344 and Pediococcus acidilactici FSDLZ42M3 mice; where *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0052] Figure 2 Schematic diagram of changes in intestinal pathogenic bacteria in experimental mice after 4 weeks of intervention with *Pediococcus lactis* CCFM1344 and *Pediococcus lactis* FSDLZ42M3: (A) Relative abundance of *Escherichia coli*; (B) Relative abundance of *Staphylococcus*; where *P<0.05, **P<0.01, ***P<0.001;

[0053] Figure 3Schematic diagram of intestinal permeability and serum LPS content in mice after 4 weeks of intervention with *Pediococcus lactis* CCFM1344 and *Pediococcus lactis* FSDLZ42M3: (A) Expression level of ZO-1 gene expressing ZO-1 protein in colon; (B) Expression level of Ocln1 gene expressing Occludin protein in colon; (C) Expression level of Cldn1 gene expressing Claudin-1 protein in colon; (D) Serum LPS content; where *P<0.05, **P<0.01, ***P<0.001;

[0054] Figure 4 Schematic diagram of the expression levels of genes Tlr4 (expressing the downstream receptor TLR4 protein of LPS) and Nfkb1 (expressing the NF-κB protein), as well as genes Tnf (expressing the inflammatory cytokine TNF-α) and Il1b (expressing the inflammatory cytokine IL-1β) in the hippocampus of experimental mice after 4 weeks of intervention with *Pediococcus lactis* CCFM1344 and *Pediococcus lactis* FSDLZ42M3: (A) Expression level of Tlr4 gene in hippocampus; (B) Expression level of Nfkb1 gene in hippocampus; (C) Expression level of Tnf gene in hippocampus; (D) Expression level of Il1b gene in hippocampus; where *P<0.05, **P<0.01, ***P<0.001;

[0055] Figure 5 Schematic diagram of the expression levels of downstream LPS receptor proteins in the hippocampus of experimental mice after 4 weeks of intervention with P. coli CCFM1344 and P. coli FSDLZ42M3: (A) TLR4 in hippocampus; (B) NF-κB in hippocampus; where *P<0.05, **P<0.01, ***P<0.001;

[0056] Figure 6 The expression levels of the Iba1 gene and protein, markers of microglia, in the hippocampus of mice were determined after 4 weeks of intervention with *Pediococcus lactis* CCFM1344 and *Pediococcus lactis* FSDLZ42M3. *P<0.05, **P<0.01, ***P<0.001. (A) Iba1 gene expression level; (B) Iba1 protein expression level; (C) Iba1 protein expression gel. Detailed Implementation

[0057] Preparation method of *Pediococcus lactis* CCFM1344 bacterial agent: Activated second-generation *Pediococcus lactis* CCFM1344 was cultured in MRS liquid medium at 37℃ for 12 h. The bacterial cells were collected by centrifugation at 6000×g for 3 min at 4℃. The supernatant was discarded, and the bacterial cells were resuspended in 10% sterile defatted emulsion to achieve a bacterial concentration of 5×10⁻⁶. 9 CFU / mL.

[0058] MRS culture medium formula: 10g peptone, 10g beef extract, 5g yeast extract, 2g diammonium citrate, 5g sodium acetate, 20g glucose, 1mL Tween, 0.5g magnesium sulfate, 0.25g manganese sulfate, 15g agar powder, pH adjusted to 6.2-6.4, autoclaved for 15min (101Kpa, 121℃).

[0059] Liquid chromatography detection conditions: Acquity HPLC: column, Inert Sustain C18 (150mm×4.6mm, 5μm); mobile phase: 100% 0.02mol / L KH2PO4, pH=2.3, filtered through a 0.22μm filter membrane; detection wavelength 210nm, column oven 30℃, mobile phase flow rate 0.1mL / min, injection volume 10μL.

[0060] Catalase analysis method: Pick colonies from MRS solid medium, place them on a clean glass slide, and then add 1-2 drops of 3% hydrogen peroxide solution. Strains that produce a large number of bubbles within 1 minute are catalase-positive; those that do not produce bubbles are catalase-negative.

[0061] Example 1: Screening, isolation, and identification of Pyotrophic Lactococcus CCFM1344

[0062] (I) Isolation and screening of Pediococcus lactis

[0063] Take 1g of fresh feces from a healthy adult, dilute it serially, spread the fecal sample on MRS solid medium, and incubate it in an anaerobic workstation at a temperature of 37℃ for 48h.

[0064] Observe and record colony morphology, and pick colonies for streaking and purification;

[0065] The colonies were cultured in MRS liquid medium at 37°C for 48 hours, and the resulting colonies were Gram-stained to record their morphology.

[0066] Gram-negative bacterial strains and Gram-positive cocci were discarded from the colonies, and Gram-positive bacilli were selected.

[0067] After catalase analysis, catalase-positive strains were discarded, and catalase-negative strains were retained.

[0068] (II) Molecular biological identification of probiotics:

[0069] (l) Single-strain genome extraction: 1 mL of the obtained probiotic culture was transferred to a 1.5 mL centrifuge tube and centrifuged at 10000×g for 2 min. The supernatant was discarded to obtain the bacterial cells. The bacterial cells were washed with 1 mL of sterile water and centrifuged at 10000×g for 2 min. The supernatant was discarded to obtain the bacterial cells. 200 μL of the probiotic culture was added to the centrifuge tube. Incubate SDS lysis buffer at 80°C for 30 min. Then, add a phenol-chloroform mixture (Tris-saturated phenol:chloroform:isoamyl alcohol = 25:24:1, invert and mix thoroughly. Centrifuge at 12000×g for 5 min and collect 200 μL of the supernatant. Add 400 μL of ice-cold ethanol or ice-cold isopropanol to the 200 μL of supernatant and incubate at -20°C for 1 h. Centrifuge at 12000×g for 5 min and discard the supernatant. Resuspend the precipitate in 500 μL of 70% (v / v) ice-cold ethanol and centrifuge at 12000×g for 2 min. Discard the supernatant. Dry the precipitate in a 60°C oven or air dry. Redissolve the precipitate in 50 μL of ddH2O to obtain template DNA for PCR.

[0070] (2) 16S rDNA PCR:

[0071] A. Bacterial 16S rDNA 50μL PCR reaction system: 10×Taq buffer, 5μL; dNTP, 5μL; 27F, 0.5μL; 1492R, 0.5μL; Taq enzyme, 0.5μL; template, 0.5μL; ddH2O, 38μL.

[0072] B. PCR conditions: 95℃, 5 min; 95℃, 10 s; 55℃, 30 s; 72℃, 30 s; step 2-4 30×; 72℃, 5 min; 12℃, 2 min;

[0073] C. Prepare a 1% agarose gel, then mix the PCR product with 10000× loading buffer, load 2 μL, run at 120V for 30 min, and then perform gel imaging;

[0074] D. The obtained PCR product was sent to a professional sequencing company. The sequencing results were compared with those obtained by searching and similarity analysis in GeneBank using BLAST, and the product was identified as Pediococcus lactis.

[0075] (3) Whole genome sequencing

[0076] The extracted whole genome was sent to a professional sequencing company, where it was sequenced using a next-generation sequencer. The resulting sequences were then searched and compared for similarity in GenBank using BLAST. The sequencing results identified CCFM1344 as a newly discovered strain of *Pediococcus lactis*. The strain was stored at -80°C for future use.

[0077] The preserved bacterial culture was used to pick up the original bacterial culture through an inoculation loop, and purified by streaking on MRS medium. The culture was then incubated at 37°C for 48 hours. A single colony was picked up with the inoculation loop and inoculated into MRS liquid medium, and incubated at 37°C for 8 hours.

[0078] The bacterial culture was collected, and the fermentation broth was centrifuged (12000×g, 5 min, 4℃). The supernatant was collected, filtered through a 0.22 μm filter membrane, and the lactic acid content in the extract was determined by liquid chromatography-mass spectrometry. Qualitative analysis was performed using the retention time of the liquid chromatography peak and the mass spectrum, and quantitative analysis was performed using the external standard method to measure the peak area.

[0079] Most strains of the genus *Pediococcus* can ferment glucose, xylose, and fructose to produce lactic acid. The results (Table 1) show that CCFM1344 produced 23.143 mg / mL of lactic acid, which is the highest among *Pediococcus* strains of the same genus.

[0080] Table 1. Lactic acid production from *Pediococcus lactis* fermentation.

[0081] strain name Lactic acid production (mg / mL) CCFM1344 23.143 FXJKS33M10 21.1065 FYNLJ9L2 20.767 HN26 20.3305 DYNDL11M51 19.133 CCFM18 17.589 FXJSW10M1 17.074 FQHXN47L1 17.058 DYNDL1151 17.0575 FXJKS25M4 16.464 FSDLZ42M3 15.581

[0082] Example 2: Preparation of Pediococcus lactis inoculum

[0083] Activated second-generation *Pediococcus lactis* CCFM1344 and FSDLZ42M3 were inoculated at 2.5% in MRS liquid medium and cultured at 37°C for 8 hours for subculturing and expansion. The cells were collected by centrifugation at 6000×g for 3 minutes at 4°C, the supernatant was discarded, and the cells were resuspended in 10% sterile defatted emulsion to achieve a bacterial concentration of 5×10⁻⁶. 9 CFU / mL yields either the Porphyromonas lactis CCFM1344 inoculum or the Porphyromonas FSDLZ42M3 inoculum.

[0084] Example 3: Pediococcus lactis CCFM1344 has the ability to regulate intestinal lactic acid levels.

[0085] Twenty-four male C57BL / 6J mice, 4 weeks old, weighing 18-20g, SPF grade, were selected. After one week of acclimatization, the mice were randomly divided into four groups according to their weight: a normal control group, a chronic stress model group, and treatment groups with CCFM1344 and FSDLZ42M3, with 6 mice in each group. The environmental conditions for the mice were: a temperature of 21-25℃, a relative humidity of 40-60%, and a diurnal light-dark cycle of 12h light-12h darkness. The grouping and feeding protocols for the mice are shown in the table below:

[0086] Table 2 Grouping and Feeding Protocols for Laboratory Mice

[0087] Group Name Daily processing method Blank control group (N=6) Gavage 200 μL of 10% sterile skim milk Model group (N=6) CUMS+ 200μL 10% sterile skim milk administered by gavage CCFM1344 processing group (N=6) <![CDATA[CUMS + intragastric administration of 200 μL of CCFM1344 bacterial agent (5×10 9 CFU / mL)]]> FSDLZ42M3 processing group (N=6) <![CDATA[CUMS + intragastric administration of 200 μL of FSDLZ42M3 bacterial agent (5×10 9 CFU / mL)]]>

[0088] The specific steps of the feeding program are as follows:

[0089] (1) The first week is the adaptation period. No intervention is performed during the adaptation period, and the experimental mice are fed a normal diet.

[0090] (2) The chronic unpredictable stress mouse (CUMS) model was established and administered via gavage during weeks two to five:

[0091] A chronic unpredictable stress (CUMS) mouse model was established by subjecting mice to the following random stimuli: 1) fasting for 24 hours; 2) water deprivation plus empty bottle stimulation for 24 hours; 3) tail clamping for 3 minutes; 4) moist bedding for 24 hours; 5) immobilization for 1-2 hours; 6) cage tilting at 45° for 24 hours; 7) continuous light exposure for 24 hours; 8) no bedding for 24 hours; 9) forced swimming for 15 minutes; and 10) isolation for 24 hours. One to two stimuli were randomly selected each day, with the timing of each stimulation determined randomly to avoid circadian rhythms. Each method was repeated no more than four times for a period of four weeks.

[0092] Gavage: The experimental mice were administered 200 μL / mouse / day via gavage.

[0093] In week six, after euthanizing mice by collecting blood from their eyeballs, 20 mg of cecal contents were weighed, added to 200 μL of H2O, vortexed for 30 s, and homogenized three times at 70 Hz for 30 s each time. Then, 800 μL of methanol:acetonitrile = (1:1, v / v) (pre-cooled at -20℃) was added to precipitate the protein. The mixture was centrifuged at 12000 × g for 15 min at 4℃, and the supernatant was collected and concentrated under vacuum. Then, 200 μL of acetonitrile:water (1:1) was added to reconstitute the supernatant, and after centrifugation again, the supernatant was analyzed by GC-MS. The results showed that ( Figure 1 CUMS caused significant changes in the relative content of multiple substances in the mouse intestine. Specifically, compared to the normal control group, CUMS modeling (model group) resulted in a 72.02% decrease in the relative concentration of lactic acid in intestinal metabolites (relative lactic acid content 0.2798). CCFM1344 intervention (relative lactic acid content 0.8267) increased the lactic acid content in mouse intestinal metabolites by 1.95 times compared to the model group (P<0.001), returning to near-normal levels. However, after intervention with *Pediococcus lactis* FSDLZ42M3, the relative lactic acid content in mouse intestinal metabolites was 0.2854, an increase of 2% compared to the model group (P=0.97), which was not statistically significant. This indicates that CCFM1344 intervention can increase the lactic acid content in intestinal metabolites.

[0094] Example 4: Pietrococcus lactis CCFM1344 can inhibit intestinal pathogens.

[0095] The mouse grouping and treatment procedures were the same as in Example 3. Fresh feces were collected from mice at the end of the sixth week. Total genome was extracted from mouse feces using the MP DNASpin kit for Faces (MP Corporation, USA). The V3-V4 region of the bacterial template DNA was amplified using primers (341F: 5'-CCTAYGGGRBGCASCAG-3'; 806R: 5'-GGACTACNNGGGTATCTAAT-3'). PCR products were purified by agarose gel electrophoresis and recovered using the Qubit dsDNA BR gel recovery kit. Sample sequencing was performed on an Illumina Miseq PE300 platform. After obtaining the initial library data, QIIME software was used for quality control, assembly, annotation, and other analyses of the subsequent data.

[0096] The results are as follows Figure 2 As shown, compared to the normal control group (relative abundances of Escherichia coli and Staphylococcus aureus were 0.0000546 and 0.0000062, respectively), CUMS modeling (model group) significantly increased the abundance of pathogenic bacteria such as Escherichia coli and Staphylococcus aureus (relative abundances of 0.000288 and 0.00151, respectively) in the mouse intestines; compared to the model group, the relative abundances of Escherichia coli and Staphylococcus aureus after CCFM1344 intervention (relative abundances of 0.0000546 and 0.0000062, respectively) were significantly increased. The levels of pathogenic bacteria in the *Pediococcus lactis* FSDLZ42M3 treatment group decreased by 76.87% (P<0.001) and 91.66% (P=0.004), respectively, effectively alleviating this phenomenon. However, compared with the model group, the relative abundances of *Escherichia coli* and *Staphylococcus* in the *Pediococcus lactis* FSDLZ42M3 treatment group were 0.0000504 (P=0.79) and 0.001194 (P=0.4651), respectively, which were not statistically significant. Therefore, CUMS causes an abnormal increase in the abundance of pathogenic bacteria and intestinal microecological imbalance in mice. CCFM1344 intervention can regulate intestinal metabolites and intestinal flora, inhibiting the abnormal increase of pathogenic bacteria, while the control strain FSDLZ42M3 did not have this effect.

[0097] Example 5: Pyrococcus lactis CCFM1344 can repair intestinal barrier damage caused by chronic stress.

[0098] The mouse grouping and processing steps were the same as in Example 3. After euthanizing the mice by collecting blood from their eyeballs, the thoracic cavity was opened, and a colonic tissue (20 mg) was removed. Trizol reagent was added to lyse the colonic tissue to extract RNA from the sample, and its concentration and purity were tested (A260 / A280 should be greater than 2.0). cDNA was obtained using the Vazyme reverse transcription kit and detected on a quantitative real-time amplification instrument. Three parallel wells were set for each sample. After the reaction, 2 -ΔΔCtThe threshold cycling method was used to analyze the results and calculate the expression of each gene among different groups. The sequences of the target genes used were obtained from Primer Bank, and the primers were synthesized by Shanghai Sangon Biotech Co., Ltd. Detailed gene sequences are shown in the table below:

[0099] Table 3 Primer gene sequences

[0100]

[0101]

[0102] Tight junction proteins ZO-1, Occludin, and Claudin-1 are closely related to intestinal barrier permeability. The intestinal barrier protects intestinal cells from intestinal microorganisms and antigens. However, changes in the gut microbiota, intestinal immune system, and metabolism can also lead to intestinal barrier damage and the phenomenon of "leaky gut".

[0103] The results are as follows Figure 3 As shown, gene ZO-1 expresses ZO-1 protein, gene Ocln1 expresses Occludin protein, and gene Cldn1 expresses Claudin-1 protein. Compared with the CUMS group (model group) (ZO-1 mRNA relative expression level was 0.399; Occludin mRNA relative expression level was 0.269; Claudin-1 mRNA relative expression level was 0.494), CCFM1320 intervention (ZO-1 mRNA relative expression level was 1.45; Occludin mRNA relative expression level was 0.269; Claudin-1 mRNA relative expression level was 0.494) resulted in a lower relative expression level of ZO-1 mRNA (ZO-1 mRNA relative expression level was 1.45; Occludin-1 mRNA relative expression level was 0.269; Claudin-1 mRNA relative expression level was 0.494) resulted in a lower relative expression level of Claudin-1 mRNA (ZO-1 The relative expression levels of ZO-1 mRNA (1.04) and Claudin-1 mRNA (1.62) in mouse colon increased the relative expression levels of tight junction proteins ZO-1, Occludin, and Claudin-1 by 2.63-fold (P<0.001), 2.87-fold (P<0.001), and 2.28-fold (P<0.001), respectively, before returning to near-normal levels. Compared with the control bacterium FSDLZ42M3 (ZO-1 mRNA relative expression level 0.387; Occludin-1 mRNA relative expression level 1.62), the relative expression levels of these proteins were significantly higher. The relative expression levels of ZO-1 mRNA were 0.188 and Claudin-1 mRNA were 1.02. CCFM1344 intervention increased the relative expression levels of tight junction proteins ZO-1 by 2.75-fold (P<0.001), Occludin by 4.53-fold (P<0.001), and Claudin-1 by 0.5-fold (P=0.007) in the colon of mice. The expression of tight junction proteins ZO-1, Occludin, and Claudin-1 was significantly reduced in the colon tissue of the model group mice, but CCFM1344 intervention restored the normal expression of these protein-related genes, thereby repairing intestinal barrier damage.

[0104] Example 6: Pyrococcus lactis CCFM1344 can alleviate abnormally elevated serum LPS levels.

[0105] The grouping and treatment procedures for mice were the same as in Example 3. Mice were sacrificed at the end of the sixth week by collecting blood from their eyeballs. The obtained plasma was allowed to stand for 1 hour, then centrifuged at 3000×g for 15 minutes, and the supernatant serum was collected. LPS was measured using an enzyme-linked immunosorbent assay kit (Sempercapto, Nanjing).

[0106] Lipopolysaccharide (LPS) is a major component of the bacterial membrane of Gram-negative bacteria and can promote inflammatory responses by activating the TLR4 pathway. When TLR4 receives stimulation from LPS or pro-inflammatory factors, it promotes the activation and release of nuclear factor-kappaB (NF-κB) into the cell nucleus. This further activates NF-κB target genes, producing pro-inflammatory factors such as tumor necrosis factor (TNF-α), interleukin (IL-1β), and IL-6, while inhibiting the production of anti-inflammatory factors such as IL-10, thus participating in the pathological process of the inflammatory response.

[0107] Results of detecting LPS in serum ( Figure 3 D) showed that, compared to the blank control group (serum LPS level 26.58 mg / mL), CUMS (serum LPS level 38.23 mg / mL) led to a 43.83% increase in serum LPS in mice (P = 0.0013). After CCFM1344 intervention, serum LPS levels returned to normal (LPS level 26.35 mg / mL, P = 0.9995), reversing the phenomenon caused by the modeling. However, the FSDLZ42M3 control group (serum LPS level 38.99 mg / mL, P = 0.0007) did not exhibit this effect.

[0108] The above results indicate that CUMS leads to abnormal LPS levels in serum, while CCFM1344 intervention can inhibit the abnormal increase in LPS levels.

[0109] Example 7: Pyrococcus lactis CCFM1344 can reduce central nervous system inflammatory response caused by chronic stress.

[0110] The mouse grouping and processing procedures were the same as in Example 3. After blood was collected from the eyeballs of the mice, the brain tissue was quickly dissected, and the hippocampus tissue was isolated on ice. The hippocampus tissue was processed using the same method as in Example 5 for determining the expression of tight junction protein-related genes. Primers were synthesized by Shanghai Sangon Biotech Co., Ltd. Detailed gene sequences are shown in the table below:

[0111] Table 4 Primer gene sequences

[0112]

[0113] LPS can promote inflammatory responses by activating the TLR4 / NF-κB signaling pathway and release a series of cytokines and chemokines to trigger immune responses and respond to the microenvironment after injury or pathological events.

[0114] The results are as follows Figure 4 As shown, the Tlr4 gene expresses TLR4 protein, the Nfkb1 gene expresses NF-κB protein, the Tnf gene expresses the pro-inflammatory cytokine TNF-α, and the Il1b gene expresses the pro-inflammatory cytokine IL-1β. Compared with the control group, the relative expression level of TLR4 mRNA in the model group was 2.296 (P = 0.002); the relative expression level of NF-κB mRNA was 2.173 (P = 0.007). CUMS led to the upregulation of TLR4 and NF-κB gene expression in the mouse hippocampus and promoted the expression of downstream pro-inflammatory cytokines TNF-α (relative mRNA expression level 2.354, P < 0.001) and IL-1β (relative mRNA expression level 1.749, P = 0.02). CCFM1344 intervention can downregulate the abnormally elevated levels of TLR4, NF-κB, and downstream inflammatory factors (the relative expression level of TLR4 mRNA was 0.958, P = 0.001; the relative expression level of NF-κB mRNA was 0.612, P < 0.001). Compared to the FSDLZ42M3 group (TLR4 mRNA relative expression level 1.79, P = 0.008; NF-κB mRNA relative expression level 1.6, P < 0.001), the CCFM1344 treatment group showed a 46.48% and 61.75% downregulation of TLR4 and NF-κB, respectively. Furthermore, the levels of inflammatory factors TNF-α (mRNA relative expression level 1.88, P = 0.02) and IL-1β (mRNA relative expression level 1.96, P < 0.001) after FSDLZ42M3 intervention were not significantly different from those in the model group. This indicates that FSDLZ42M3 intervention cannot alleviate the inflammatory response of the central nervous system in mice induced by CUMS modeling.

[0115] Weigh 20 mg of the hippocampal tissue mentioned above, add RIPA lysis buffer (Beyotime, P0013B) containing a protease inhibitor (Beyotime, P1005), and homogenize the tissue using a tissue homogenizer at 55-65 Hz, intermittently, until no obvious precipitation occurs. Centrifuge at 12000×g, 4℃ for 15 min, collect the supernatant, and determine the protein using the BCA (Bicinchoninnc acid) method, referring to the kit method (Beyotime, Enhanced P0010). Quantitative analysis of the target protein is performed using Western blotting and polyacrylamide gel electrophoresis, with the specific steps as follows:

[0116] (1) Sample preparation: Boil the denatured protein sample after protein quantification for 10 minutes and set aside. Prepare a 12% acrylamide separating gel according to the molecular weight of the target protein. Calculate the loading volume based on a loading amount of 20 μg.

[0117] (2) Electrophoresis: Maintain a constant voltage of 80V to make the sample run until it forms a straight line parallel to the boundary between the stacking gel and the separating gel; then adjust the voltage to 120V and run for 1.5-2.0h until the marker corresponding to the target protein band is clearly separated and the loading is complete.

[0118] (3) Transfer: Soak a PVDF membrane (6mm × 8.5mm) in anhydrous methanol for 20s. On a white plate, layer one sponge, three layers of filter paper, the membrane, separating gel, three layers of filter paper, and one layer of sponge in sequence. Then, gently press out the air bubbles, close the lid, and install it on the transfer apparatus. Add transfer solution pre-cooled to 4℃ to the transfer tank (transfer conditions: constant current 300mA, 1.5h).

[0119] (4) Blocking: Place the PVDF membrane in the blocking buffer prepared by BSA for blocking and gently shake it on a shaker at room temperature for 1 hour.

[0120] (5) Immunoblot reaction: Prepare antibody at an appropriate dilution according to the primary antibody instructions, gently shake in a closed container, incubate at 4°C for 12 hours, then add TBST to wash the membrane 4 times (10 min / wash), and then incubate with secondary antibody. Prepare antibody at an appropriate dilution according to the secondary antibody instructions, gently shake in a closed container, and incubate at room temperature for 1-2 hours. Wash the membrane 4 times with TBST (10 min / wash).

[0121] (6) Development: Place the above PVDF membrane in a dark room, add commercial developer for 30 seconds, and then perform chemiluminescence and detection under a protein imager for autoradiography. Scan or photograph the film, and analyze the molecular weight and net optical density value of the target band using a gel image processing system.

[0122] (7) Quantitative analysis: The protein imager captures images, and the ImageJ software is used to perform grayscale analysis to calculate the relative expression level of the target protein.

[0123] Consistent results were obtained by measuring the protein expression of TLR4 and NF-κB in the hippocampus using Western blotting. Figure 5 Compared to the blank control group, CUMS led to overexpression of TLR4 and NF-κB (the relative expression levels of TLR4 and NF-κB proteins in the model group were 1.905 and 2.21, respectively), while the relative expression levels of TLR4 and NF-κB proteins in the CCFM1344 treatment group were 1.08 and 0.96, respectively. Compared with the model group, TLR4 and NF-κB proteins decreased by 43.3% (P = 0.008) and 56.6% (P = 0.03), respectively.

[0124] CCFM1344 intervention significantly alleviated this phenomenon. Therefore, the above results indicate that CCFM1344 intervention in mice can alleviate the overexpression of pro-inflammatory factors in the hippocampus.

[0125] Example 8: Pyrococcus lactis CCFM1344 can inhibit excessive activation of microglia caused by chronic stress.

[0126] The mouse grouping and processing procedures were the same as in Example 3. After blood was collected from the eyeballs of the mice, the brain tissue was quickly dissected, and the hippocampus tissue was isolated on ice. The hippocampus tissue was processed according to the method for determining the expression of related genes in the colon tissue in Example 3. Primers were synthesized by Shanghai Sangon Biotech Co., Ltd. Detailed gene sequences are shown in the table below:

[0127] Table 5 Primer sequences for detecting Iba1 gene expression level

[0128]

[0129] Microglia play a crucial role in the brain's immune regulatory system, serving as key regulators of the brain's innate immune response. Originating from primitive macrophages in the yolk sac, they act as primary phagocytes in the brain. Microglia detect risk-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) by expressing pattern recognition receptors (PRRs). In cases of central nervous system inflammation, microglia become activated, functioning as immune presenting cells (APCs) and influencing the homeostasis of Th1 and Th2 cell-mediated immune responses. Therefore, microglia activation is a significant indicator of central nervous system inflammation. Activated microglia produce and release a series of cytokines and chemokines to trigger immune responses and react to the microenvironment following injury or pathological events. Ionized calcium binding adapter molecule 1 (Iba1) is a calcium-binding protein specifically expressed in microglia. Iba1 antibodies are currently widely used as markers for microglia. When microglia are abnormally activated, the Iba1 protein is abnormally expressed.

[0130] The results are as follows Figure 6 As shown, compared with the control group, CUMS modeling promoted abnormal expression of Iba1 gene in mouse hippocampus (relative expression level of Iba1 mRNA was 2, P<0.001), while CCFM1344 intervention significantly reduced the activation of microglia caused by modeling (relative expression level of Iba1 mRNA was 1.27, P>0.99).

[0131] Weigh 20 mg of the hippocampal tissue described above, add RIPA lysis buffer (Beyotime, P0013B) containing a protease inhibitor (Beyotime, P1005), and homogenize the tissue using a tissue homogenizer at 55-65 Hz, intermittently, until no obvious precipitation occurs. Centrifuge at 12000×g, 4℃ for 15 min, collect the supernatant, and determine the protein using the BCA (Bicinchoninnc acid) method, referring to the kit method (Beyotime, Enhanced P0010). Quantitatively analyze the target protein using Western blotting with polyacrylamide gel electrophoresis; refer to Example 4 for specific operating procedures.

[0132] Consistent results were obtained by measuring Iba1 protein expression in the hippocampus using Western blotting. Figure 6CUMS induced Iba1 overexpression (the relative expression level of Iba1 protein in the model group was 2). Compared with the model group, CCFM1344 intervention reduced Iba1 protein expression by 36.5% (the relative expression level of Iba1 protein in the CCFM1344 treatment group was 1.27, P = 0.013), significantly alleviating this phenomenon. Therefore, the above results indicate that CCFM1344 intervention in mice can inhibit chronic stress-induced microglial cell activation in the hippocampus.

[0133] Example 9: Manufacturing of food containing Pediococcus lactis CCFM1344

[0134] Select fresh vegetables (such as cucumbers, carrots, beets, celery, cabbage products, or a mixture of several), wash them, juice them, and then perform high-temperature instantaneous sterilization. After sterilizing at 140℃ for 2 seconds, immediately cool to 37℃, and then inoculate with a bacterial agent or fermentation agent containing *Pediococcus lactis* CCFM1344 to achieve a *Pediococcus lactis* CCFM1344 concentration of 1×10⁻⁶. 8 Fruit and vegetable beverages containing live P. lactococcus CCFM1344 bacteria were obtained by refrigerating at 4°C with a concentration of CFU / mL or higher.

[0135] 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 Pediococcus lactis ( Pediococcus acidilactici CCFM1344, characterized in that, The *Pediococcus lactis* strain is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO.63800, deposit date of September 12, 2023, and address of Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. A microbial preparation, characterized in that, The microbial preparation contains the *Pediococcus lactis* as described in claim 1. Pediococcus acidilactici CCFM1344 or its freeze-dried powder.

3. A product containing the *Pediococcus lactis* of claim 1, or containing the microbial preparation of claim 2, characterized in that, The product in question is a pharmaceutical product.

4. The product according to claim 3, characterized in that, The drug includes drug carriers and / or pharmaceutical excipients.

5. The product according to claim 4, characterized in that, The drug carrier comprises one or more of the following: microcapsules, microspheres, nanoparticles, and liposomes.

6. The microbial preparation according to claim 2, or the product according to any one of claims 3-5, characterized in that, The amount of viable Pediococcus lactis added to the microbial preparation or product shall not be less than 1×10⁻⁶. 8 CFU / g or 1×10 8 CFU / mL.

7. The use of the *Pediococcus lactis* of claim 1, or the microbial preparation of claim 2, in the preparation of a medicament for the prevention and / or treatment of hippocampal microglia activation and / or mental illness associated with hippocampal microglia activation.

8. The application according to claim 7, characterized in that, The application includes at least one aspect of the following (a)-(g): (a) Restoring intestinal lactate levels in a chronic stress model; (b) Inhibits the proliferation of intestinal pathogens caused by chronic stress; (c) Increase gene expression of intestinal tight junction proteins ZO-1, and / or Occludin, and / or Claudin-1; (d) Reduce serum LPS levels; (e) Reduce the expression of LPS downstream receptors TLR4 and / or NF-κB in the hippocampus of the brain; (f) Reduce gene expression of pro-inflammatory factors TNF-α and / or IL-1β in the hippocampus of the brain; (g) Reduce the expression of ion-calcium binding linker 1 Iba1.

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