Lactococcus lactis gJ-11 for regulating intestinal homeostasis and application thereof

Abstract

The application discloses a lactococcus lactis GJ-11 for regulating intestinal homeostasis and application thereof. The lactococcus lactis GJ-11 is isolated and cultured from dendrobium officinale, and the preservation number is CCTCC NO: M 20253062. Through tracking and multi-omics analysis on refractory IBS-D patients receiving FMT treatment, it is found that the long-term efficacy of FMT is related to the colonization level of the donor-derived flora. Further combined with metagenomic and metabolomic analysis, and through species tracing and functional annotation, a key effector bacterium, lactococcus lactis expressing CCR, is accurately locked. Experiments prove that the strain not only has excellent intestinal colonization ability, but also can specifically express cinnamoyl coenzyme A reductase, and promote the synthesis of cinnamyl aldehyde in the intestine. The application provides a new probiotic and an effective solution for the precise treatment of various intestinal homeostasis imbalance related diseases.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to a lactococcus lactis GJ-11 that regulates intestinal homeostasis and its applications. Background Technology

[0002] Gut homeostasis refers to the dynamic balance among the gut microbiota, intestinal barrier, immune system, and neuroendocrine system. Its imbalance is not only associated with irritable bowel syndrome (IBS), but also participates in the development of inflammatory bowel disease, chronic constipation, metabolic syndrome, and even neuropsychiatric disorders. Diarrhea-predominant IBS-D is particularly prevalent in my country, with patients often experiencing recurrent abdominal pain, diarrhea, and visceral hypersensitivity. Current clinical treatment is mostly symptomatic, lacking radical treatments that target the core pathogenesis.

[0003] In recent years, gut microbiota imbalance has been confirmed as a key factor in the development and progression of IBS-D. Current microbiota intervention methods mainly include: ① Traditional probiotics: such as Bifidobacterium and Lactobacillus. Some strains have been shown to have some efficacy against IBS, but the overall effect is limited, individual differences are significant, and most strains are derived from fermented foods, lacking clinical evidence targeting the pathological mechanisms of IBS. ② Fecal microbiota transplantation (FMT): By reconstructing the patient's gut microbiota, it has shown good efficacy in some patients with refractory IBS, but it faces problems such as complex donor selection, high cost, and unclear mechanisms of action, making widespread application difficult. Therefore, identifying specific probiotics with clear therapeutic efficacy from clinically effective donors has become an urgent need for the development of microbiota-based drugs. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies, such as the limited treatment options for intestinal homeostasis-related diseases (e.g., IBS-D), insufficient clinical targeting of traditional probiotics, and difficulty in controlling the safety and standardization of fecal microbiota transplantation (FMT), by providing a strain of Lactococcus lactis with significant intestinal protective effects, as well as inactivated bacterial cells, lysates, fermentation products, and active metabolites (e.g., cinnamaldehyde) derived therefrom with equivalent or synergistic effects.

[0005] To achieve the above objectives, this application adopts the following technical solution:

[0006] In a first aspect, this application provides a *Lactococcus lactis* GJ-11, wherein the *Lactococcus lactis* GJ-11 is classified and named as follows: Lactococcus lactis subsp. lactisGJ-11 has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20253062, on December 31, 2025, at Wuhan University, Wuhan, Hubei Province.

[0007] In a second aspect, this application provides a biological agent comprising Lactococcus lactis GJ-11 as described in the first aspect or comprising a bacterial agent prepared from Lactococcus lactis GJ-11 as described in the first aspect.

[0008] Furthermore, the preparation method of the biological agent includes: inoculating the Lactococcus lactis GJ-11 described in the first aspect into a fermentation medium for fermentation culture, collecting the fermentation broth after the culture is completed, centrifuging to remove the supernatant to obtain bacterial cells, freeze-drying the bacterial cells to prepare lyophilized powder, and adding or not adding excipients, other probiotics or probiotic agents to obtain the product.

[0009] Furthermore, the fermentation medium is a modified MRS medium, the formulation of which includes: 10 g / L soybean peptone, 5 g / L yeast extract, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 ml Tween 80, 0.58 g / L magnesium sulfate heptahydrate, and 0.25 g / L manganese sulfate tetrahydrate. And / or, the fermentation culture is carried out at a temperature of 36-38°C for 24-48 hours; And / or, the excipients are selected from carriers, adsorbents, cosolvents, dispersants, stabilizers, antioxidants, At least one of pH adjuster, preservative, defoamer, slow-release agent and binder; And / or, the other probiotics are selected from Lactobacillus acidophilus ( Lactobacillus acidophilus Lactobacillus rhamnosus ( Lactobacillus rhamnosus Lactobacillus plantarum ( Lactobacillus plantarum Lactobacillus reuteri ( Lactobacillus reuteri Lactobacillus casei ( Lactobacillus casei Lactobacillus paracasei ( Lactobacillus paracasei Bifidobacterium lactis (Bifidobacterium animalis subsp. lactis) Bifidobacterium animalis subsp. lactis Bifidobacterium longum ( Bifidobacterium longum ), Bifidobacterium breve Bifidobacterium breve Any one or more of the following: Bifidobacterium bifidum.

[0010] Thirdly, this application provides the use of Lactococcus lactis expressing CCR or its culture, lysate, fermentation product, active metabolite, or biological agent as an active ingredient in the preparation of therapeutic drugs for diseases related to intestinal homeostasis imbalance.

[0011] Furthermore, the diseases related to intestinal homeostasis imbalance include irritable bowel syndrome, inflammatory bowel disease, intestinal injury, metabolic endocrine diseases related to intestinal imbalance, autoimmune and allergic diseases, cardiovascular diseases, or neuropsychiatric diseases; the lysate is cinnamyl-CoA reductase (CCR). Furthermore, the drug comprises an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient is one or more of Lactococcus lactis GJ-11 or its culture, lysate, fermentation product, and active metabolite as described in the first aspect.

[0012] Furthermore, the pharmaceutically acceptable excipient is selected from at least one of the following: carrier, adsorbent, solubilizer, dispersant, stabilizer, antioxidant, pH adjuster, preservative, defoamer, sustained-release agent, and binder.

[0013] Furthermore, the drug includes an active ingredient, which is a biological agent as described in the second aspect.

[0014] Furthermore, the dosage form of the drug is an oral formulation or an injectable formulation.

[0015] Compared with the prior art, this application has the following beneficial effects: This application is the first to discover and demonstrate a *Lactococcus lactis* strain with significant intestinal protective effects. This strain not only possesses excellent intestinal colonization ability but also specifically expresses cinnamyl-CoA reductase (CCR), promoting the synthesis of cinnamaldehyde in the intestine. Furthermore, a *Lactococcus lactis* strain GJ-11 expressing CCR was isolated and cultured from *Dendrobium* plants. Compared to currently available traditional probiotics, which are mostly derived from fermented foods and have unclear mechanisms, the *Lactococcus lactis* GJ-11 provided in this application was obtained by identifying and screening based on the metabolic pathway characteristics of a clinically effective cohort, exhibiting more specific pathophysiological targeting. This provides a novel biological agent for the precision treatment of various diseases related to intestinal homeostasis imbalance. Attached Figure Description

[0016] Figure 1Differences in donor microbiota colonization were observed between FMT-responsive and non-responsive donors, including: (A) Bray-curtis distances between responders (R) and non-responders (NR) and their corresponding donor transplant samples before treatment; (B) Changes in Bray-curtis distances between responders (R) and non-responders (NR) before and after treatment and their corresponding donor transplant samples; (C) Linear regression analysis of changes in IBS-SSS scores and Bray-curtis distances; (D) PCoA analysis based on the Bray-curtis distance algorithm: showing the differences in gut microbiota composition between FMT-responsive and healthy donors before treatment, after treatment, 1 month after treatment, and 4 months after the last treatment, from left to right; (E) PCoA analysis based on the Bray-curtis distance algorithm: showing the differences in gut microbiota composition between FMT-non-responsive and healthy donors before treatment, after treatment, 1 month after treatment, and 4 months after the last treatment, from left to right. p<0.05, p<0.01, p<0.001, ns: no significant difference.

[0017] Figure 2 The clinical cohort identified *Lactococcus lactis*; among them: (A) a heatmap showing the correlation between metabolite content and IBS-D symptoms, mood, and quality of life scores: the metabolites in the figure are differentially expressed metabolites included in the phenylpropane biosynthesis pathway, red represents positive correlation, blue represents negative correlation, and the correspondence between color and correlation coefficient is shown in the legend, with asterisks in the small squares representing significance p-values; (B) the correlation between cinnamaldehyde content and bacterial abundance; (C) the correlation between the expression abundance of the catalytic enzyme required for cinnamaldehyde synthesis and bacterial abundance; (DE) a bar chart comparing the abundance of *Lactococcus lactis* in healthy donors and after FMT treatment with IBS-D patients; p<0.05, p<0.01, p<0.001, ns: no significant difference.

[0018] Figure 3This clinical validation cohort confirmed changes in Lactococcus lactis and related pathways; including: (A) changes in the relative abundance (log10 transformation) of Lactococcus lactis in the gut of patients before and after FMT treatment; and (B) comparison of the absolute abundance of fecal phenylpropanoid biosynthesis pathway before and after FMT treatment in the validation cohort. Data are expressed as mean ± SEM and paired t-tests were used. p<0.05, p<0.01, p<0.001, ns: no significant difference.

[0019] Figure 4 Isolation, culture and identification of Lactococcus lactis GJ-11; (A) strain development tree; (B) expression of CCR gene by Lactococcus lactis GJ-11, lanes 1-3 are CCR gene amplification products using GJ-11 genomic DNA as template, a clear specific band can be seen at about 1000 bp, consistent with the size of the expected target fragment.

[0020] Figure 5 Lactococcus lactis GJ-11 improved the symptoms of IBS-D mice; among them: (A) Bristol score of feces in each group of mice; (B) number of fecal particles (FPO) in each group of mice within 30 minutes; (C) fecal water content (FWC) in each group of mice; (D) intestinal transit time (GI) in each group of mice. p<0.05, p<0.01, p<0.001, ns: no significant difference.

[0021] Figure 6 Effects of Lactococcus lactis GJ-11 on the expression of histamine synthesis-related genes in P815 mast cells.

[0022] Figure 7 Effect of Lactococcus lactis GJ-11 on the expression of the inflammatory cytokine IL-6 in HaCaT keratinocytes.

[0023] Figure 8 Effect of Lactococcus lactis GJ-11 postbiotic on the expression of the inflammatory cytokine IL-6 in HaCaT cells.

[0024] Figure 9 Effects of cinnamaldehyde on histamine release-related indicators in P815 mast cells.

[0025] Preservation Instructions The classification and naming of Lactococcus lactis GJ-11 Lactococcus lactis subsp. lactis GJ-11 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20253062, deposit date December 31, 2025, at Wuhan University, Wuhan, Hubei Province. Detailed Implementation

[0026] To make the technical solution of this application clearer and easier to understand, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0027] Unless otherwise specified, the test methods or experimental methods described in the following examples are conventional methods; unless otherwise stated, the experimental materials (including but not limited to reagents and test kits) involved in the following examples are commercially available standards or commercial reagents.

[0028] Example 1: Discovery of Lactococcus lactis Efficacy correlation in clinical cohorts: This embodiment, through in-depth clinical follow-up of refractory IBS-D patients receiving fecal microbiota transplantation (FMT), found significant heterogeneity in the long-term efficacy of FMT. Comparative analysis of gut microbiota colonization characteristics between the effective and ineffective groups revealed a significant reduction in the Bray-curtis distance between the effective group and their corresponding healthy donors after treatment. Figure 1 As shown in Figure A, the change in distance before and after treatment was significantly greater than that in the FMT ineffective group, such as... Figure 1 As shown in Figure B, correlation analysis revealed a positive correlation between changes in IBS-SSS scores before and after FMT treatment and changes in Bray-curtis distance between fecal samples and corresponding donor fecal microbiota samples. Figure 1 As shown in Figure C, the shift in the gut microbiota structure towards that of the donor is a crucial factor in symptom relief after FMT treatment, and the gut microbiota of patients in the effective group significantly tended towards the donor after treatment. Figure 1 As shown in DE, this suggests that colonization of certain strains of bacteria from healthy donors in the recipient's gut is a prerequisite for achieving clinical remission.

[0029] Multi-omics approach identifies key effector bacteria, *Lactococcus lactis*: Combined metagenomic and metabolomics analysis revealed significant enrichment of the phenylpropanoid biosynthesis pathway in the gut of patients who responded to FMT. The levels of the key differentially expressed metabolite cinnamaldehyde significantly increased after treatment and were negatively correlated with disease severity scores. Figure 2 As shown in A. Further gene species and functional annotation revealed that *Lactococcus lactis* highly expresses cinnamyl-CoA reductase (CCR), the key rate-limiting enzyme in the synthesis of cinnamaldehyde in this pathway, such as... Figure 2As shown in BC. Clinical data showed that the abundance of Lactococcus lactis and the CCR gene in the intestines of patients in the effective group and healthy donors was significantly higher than that in patients before treatment and in the ineffective group, such as Figure 2 As shown in DE. The above findings demonstrate that this *Lactococcus lactis* is a major contributor to the in situ synthesis of cinnamaldehyde in the intestine and a core functional bacterium regulating the "bacteria-metabolite-target" axis.

[0030] Furthermore, validation was performed by constructing an independent validation cohort (n=20). The results showed that the abundance of *Lactococcus lactis* and the activity of the phenylpropane biosynthesis pathway in IBS-D patients at baseline were significantly lower than in healthy donors; after FMT treatment, the abundance of *Lactococcus lactis* in the effective group patients significantly increased, such as... Figure 3 As shown in AB, this result further confirms in an independent population that Lactococcus lactis and its metabolic pathways are key factors mediating the efficacy of FMT.

[0031] Example 2: Acquisition and identification of Lactococcus lactis and preparation of its inoculum Given that Example 1 confirmed that Lactococcus lactis and its metabolic pathway are key factors mediating the efficacy of FMT, and that the Lactococcus lactis expresses CCR, the inventors attempted to isolate and culture Lactococcus lactis strains expressing CCR from human feces and plants. After several attempts, the inventors isolated and cultured a Lactococcus lactis strain GJ-11 expressing the CCR gene from Dendrobium officinale. The specific isolation, culture, and identification process is as follows: 1) Source of strain Take fresh Dendrobium officinale stems (gifted by Tong Hanchuntang, origin: Yunnan), surface treat with 75% alcohol, grind, then take 0.5g to 4.5mL of sterile physiological saline, shake thoroughly to disperse the sample, and then serially dilute 100μL of the sample.

[0032] Select an appropriate gradient (10) -1 10 -2 The mixture was evenly spread on MRS screening medium (10g peptone, 5g beef extract, 1mL Tween 80, 2g dipotassium hydrogen phosphate, 4g yeast extract, 0.2g magnesium sulfate, 2g triammonium citrate, 20g glucose, 0.05g manganese sulfate, 5g sodium acetate, 16g agar, 1L distilled water, pH 6.2±0.2, sterilized at 121℃ for 25min) and incubated under anaerobic conditions at 37℃ for 36–48h.

[0033] 2) Isolation and purification of strains Select single colonies that produce a milky white color, purify them repeatedly until no other bacteria are present, preserve the purified strains in the appropriate isolation medium, add 30% glycerol as a protectant, and freeze at -20℃.

[0034] When the GJ-11 strain was cultured on MRS solid medium for 24 hours, the colonies were white, opaque, raised, moist, with smooth and glossy edges, and the colony diameter was 0.8–1.0 mm.

[0035] The preparation method of MRS solid culture medium is as follows: 10g peptone, 5g beef extract powder, 1mL Tween 80, 2g dipotassium hydrogen phosphate, 4g yeast extract powder, 0.2g magnesium sulfate, 2g triammonium citrate, 20g glucose, 0.05g manganese sulfate, 5g sodium acetate, 16g agar, 1L distilled water, pH 6.2±0.2, sterilized at 121℃ for 25min.

[0036] 3) Identification of the 16S rRNA gene of the strain The genomes of lactic acid bacteria strains were extracted using a rapid bacterial genomic DNA extraction kit, and PCR amplification was performed using these samples as templates. The successfully amplified PCR products were sequenced and then assembled to obtain DNA sequences. Sequence homology analysis was performed using NCBI's BLAST search system.

[0037] Select the known strain with the highest similarity and analyze its similarity.

[0038] A phylogenetic tree was constructed using the Neighbor-Joining (NJ) method in MEGA6 software. For example... Figure 4 As shown in A, the phylogenetic tree suggests that GJ-11 is a newly identified subsp. lactis of Lactococcus lactis, with the closest relative being Lactococcus lactis subsp. lactis strain UL8, showing 100% homology.

[0039] The gene sequence of the 16S rRNA of strain GJ-11, as shown below (SEQ ID NO: 1), was obtained through sequencing:

[0040] The above-mentioned isolated and identified lactococcus lactis GJ-11 ( Lactococcus lactis subsp. lactis GJ-11 was deposited at the China Center for Type Culture Collection (CCTCC) on December 31, 2025, with accession number CCTCC NO: M 20253062, at Wuhan University, Wuhan, Hubei Province.

[0041] 4) Identification of CCR gene expression in Lactococcus lactis: To verify whether *Lactococcus lactis* GJ-11 carries the cinnamyl-CoA reductase (CCR) gene, this study retrieved conserved CCR gene sequences from known *Lactococcus lactis* strains by searching the NCBI database and relevant previous literature. Specific primers were designed (upstream primer CCR-F, SEQ ID NO: 2: 5'-CTTGTAACAGGTGGTTCTG-3'; downstream primer CCR-R, SEQ ID NO: 3: 5'-CTCCCAAATCCAACATAGTTT-3'). PCR amplification was performed using GJ-11 genomic DNA as a template. The amplification products were detected by agarose gel electrophoresis. The results showed that a specific band of the expected size could be amplified in the GJ-11 genome. Figure 4 B) indicates that the strain carries the CCR gene in its genome, which confirms at the molecular level that Lactococcus lactis GJ-11 has the enzymatic basis for catalyzing the synthesis of cinnamaldehyde.

[0042] 5) A bacterial agent (GJ-11 lyophilized bacterial powder) was prepared based on the isolated GJ-11: The modified MRS broth culture medium was selected with the following formula: 10 g / L soybean peptone, 5 g / L yeast extract, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 ml Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate tetrahydrate, pH 6.2±0.2, and 1000 ml sterile water. The medium was sterilized at 121°C for 25 min before use.

[0043] Remove the glycerol cryovials containing GJ-11, thaw them, and inoculate them into modified MRS broth (without animal protein) in a biosafety cabinet. Incubate at 37°C for 24-48 hours. After incubation, assess bacterial concentration using the OD600 value. Centrifuge at 10000 rpm, then wash the precipitated bacteria three times with an equal volume of physiological saline (NS) to remove culture medium components. The bacterial density of the suspension will be measured using the OD600 value. Centrifuge the suspension again at 10000 rpm, freeze at -80°C, freeze-dry under vacuum to prepare a lyophilized powder, and store in a cool, dry place. Take samples and perform viable counts using serial dilutions to determine the viable bacterial concentration of the lyophilized powder.

[0044] Example 3: Effects of Lactococcus lactis on IBS-D model mice Experimental Objective: To establish a mouse model of chronic restraint stress-induced IBS-D and evaluate the effects of Lactococcus lactis GJ-11 intervention on IBS-D symptoms in mice by using indicators such as the number of fecal particles per unit time, fecal water content, fecal Bristol classification, and intestinal transit time, so as to provide in vivo validation of Lactococcus lactis for the treatment of IBS-D.

[0045] Experimental reagents: Lactococcus lactis GJ-11, PBS buffer, etc.

[0046] Experimental Groups: a) Control group: After 3 weeks of chronic restraint stress modeling, PBS was administered by gavage at a rate of 200 μL / day for 7 days. b) Lactococcus lactis GJ-11 intervention group: After 3 weeks of chronic restraint stress modeling, 1×10^9 CFU of Lactococcus lactis GJ-11 was administered by gavage at a dose of 200 μL / day for 7 days.

[0047] Experimental Methods: This embodiment uses the chronic restraint stress method to construct an IBS-D mouse model: Mice were placed in specially designed 50 mL centrifuge tubes with vents in the wall to restrict their movement for 3 hours daily for 3 consecutive weeks to simulate chronic stress-induced intestinal dysfunction. During the intervention phase, Lactococcus lactis GJ-11 was inoculated into MRS liquid medium and cultured at 37°C until the logarithmic growth phase. After centrifugation, the bacteria were collected, washed twice with sterile PBS buffer, resuspended, and the viable bacterial concentration was adjusted to 5 × 10^9 CFU / mL. After modeling, mice in the Lactococcus lactis intervention group were administered 200 μL of bacterial solution by gavage daily for 7 consecutive days; mice in the control group were simultaneously administered an equal volume of sterile PBS by gavage as a control.

[0048] Statistical analysis: Six mice were used in each group. Data are expressed as mean ± SEM. One-way ANOVA was used to compare differences between groups. P < 0.05 was considered statistically significant.

[0049] Experimental results: After 7 days of intervention with Lactococcus lactis (1×10^9 CFU), the diarrhea symptoms in mice were significantly alleviated, such as... Figure 5 As shown, the intervention group mice exhibited a decrease in Bristol scores (…). Figure 5 A) The number of fecal particles (FPO) per unit time decreases ( Figure 5 B) Decreased fecal water content (FWC) Figure 5 C) and intestinal transit time (GI) were significantly prolonged. Figure 5 D).

[0050] Example 4: Effects of Lactococcus lactis GJ-11 on the expression of histamine synthesis-related genes in P815 mast cells Experimental Objective: To establish a P815 mast cell activation model induced by PMA combined with ionomycin, and to evaluate the intervention effect of Lactococcus lactis GJ-11 on mast cell activation and histamine synthesis by detecting the mRNA level of histidine decarboxylase (HDC), a key rate-limiting enzyme in histamine synthesis, so as to provide in vitro cellular evidence for its anti-allergic efficacy.

[0051] Cells and strains: mouse mast cell tumor cells P815; Lactococcus lactis GJ-11.

[0052] Main reagents and instruments: PMA, Ionomycin, DMEM high glucose medium, fetal bovine serum, PBS buffer, TRIzol reagent, reverse transcription kit, qPCR kit; real-time quantitative PCR instrument, cell culture incubator, high-speed refrigerated centrifuge.

[0053] Experimental Groups: a) Normal control group: P815 cells were cultured routinely without any inducing agents or bacterial strains.

[0054] b) Allergy model group: P815 cells were stimulated with 50 nM PMA + 500 nM Ionomycin.

[0055] c) Lactococcus lactis GJ-11 intervention group: After pretreatment with Lactococcus lactis GJ-11, the same dose of PMA + Ionomycin was administered for stimulation.

[0056] Experimental methods: a) Cell culture: P815 cells were placed in DMEM medium containing 10% fetal bovine serum and cultured at 37 ℃ with 5% CO2 saturated humidity. Cells in the logarithmic growth phase were used for experiments.

[0057] b) Cell preparation: Select a single colony of Lactococcus lactis GJ-11 and inoculate it into MRS liquid medium. Incubate at 37 ℃ until the logarithmic growth phase. Collect the cells by centrifugation at 4 ℃ and 8000 r / min for 10 min. Wash twice with sterile PBS and resuspend to adjust the concentration to the appropriate titer.

[0058] c) Intervention and modeling: P815 cells were seeded in culture plates. The intervention group was incubated with washed Lactococcus lactis GJ-11 bacterial suspension at a ratio of 100 CFU / cell for 4 h. The model group and the control group were incubated with an equal volume of PBS. Subsequently, the model group and the intervention group were incubated with a final concentration of 50 nM PMA + 500 nM Ionomycin to induce activation, while the control group was incubated with an equal volume of culture medium.

[0059] d) Sample collection: After induction, cells were collected, washed twice with PBS, lysed by pipetting with TRIzol reagent, and total RNA was collected.

[0060] e) qPCR detection: Total RNA was extracted and cDNA was synthesized by reverse transcription according to the kit instructions. β-actin was used as an internal control, and the relative expression level of HDC mRNA was detected by qPCR.

[0061] Statistical analysis: The experiment was repeated 3 times. Data are expressed as mean ± SEM. One-way ANOVA was used to compare differences between groups. P < 0.05 was considered statistically significant.

[0062] Experimental results: Compared with the normal control group, the HDC mRNA level in P815 cells of the model group was significantly increased; compared with the model group, the HDC mRNA expression in the Lactococcus lactis GJ-11 intervention group was significantly downregulated, such as... Figure 6 As shown, this strain can inhibit the expression of histamine synthesis-related genes in mast cells, thus exerting an anti-allergic effect.

[0063] Example 5: Effect of Lactococcus lactis GJ-11 on the expression of the inflammatory cytokine IL-6 in HaCaT keratinocytes Experimental objective: To establish a skin inflammation model by inducing immortalized human keratinocytes (HaCaT) with TNF-α combined with IFN-γ, to evaluate the regulatory effect of Lactococcus lactis GJ-11 on the inflammatory factor IL-6, and to clarify its in vitro activity in alleviating skin inflammation.

[0064] Cells and strains: Human immortalized keratinocytes HaCaT; Lactococcus lactis GJ-11.

[0065] Main reagents: TNF-α, IFN-γ, DMEM medium, fetal bovine serum, TRIzol reagent, qPCR kit.

[0066] Experimental Groups: a) Control group: HaCaT cells were cultured in a routine manner.

[0067] b) Inflammation model group: Stimulated with 10 ng / ml TNF-α + 10 ng / ml IFN-γ for 24 h.

[0068] c) Lactococcus lactis GJ-11 intervention group: Lactococcus lactis GJ-11 lysate + 10 ng / ml TNF-α + 10 ng / ml IFN-γ were co-treated for 24 h.

[0069] Experimental Methods: HaCaT cells were cultured in DMEM medium containing 10% fetal bovine serum to the logarithmic growth phase and then inoculated into culture plates. Preparation of Lactococcus lactis GJ-11 lysate: Cells were collected, sonicated, centrifuged, and the supernatant was collected, lyophilized, weighed, and filtered through a filter membrane for sterilization. Corresponding reagents were added according to the groups, and the cells were incubated at 37 ℃ in a 5% CO2 incubator for 24 h. Cells were collected, total RNA was extracted and reverse transcribed, and the relative expression level of IL-6 mRNA was detected by qPCR.

[0070] Statistical analysis: The experiment was repeated 3 times. Data are expressed as mean ± SEM. One-way ANOVA was used to compare differences between groups. P < 0.05 was considered statistically significant.

[0071] Experimental results: The IL-6 mRNA level in HaCaT cells of the inflammation model group was significantly higher than that of the normal control group; compared with the model group, the IL-6 mRNA level in the Lactococcus lactis GJ-11 lysate intervention group was significantly decreased, such as... Figure 7 As shown, this indicates that it can effectively inhibit the inflammatory response of keratinocytes.

[0072] Example 6: Effect of Lactococcus lactis GJ-11 postbiotic on the expression of the inflammatory cytokine IL-6 in HaCaT cells Experimental objective: To clarify the alleviating effect of postbiotics (fermentation metabolites, bacterial components, and other non-live bacterial active components) of Lactococcus lactis GJ-11 on inflammation of HaCaT cells, and to provide experimental evidence for the development of stable anti-inflammatory functional components.

[0073] Experimental Groups: a) Normal control group: cells were cultured routinely without inducing agents.

[0074] b) Inflammation model group: Stimulated with 10 ng / ml TNF-α + 10 ng / ml IFN-γ for 24 h.

[0075] c) Lactococcus lactis GJ-11 postbiotic intervention group: GJ-11 postbiotic + inflammatory factors were treated together.

[0076] Experimental methods: a) Preparation of post-biotics: Lactococcus lactis GJ-11 fermentation culture, centrifugation, inactivation, concentration, and filtration sterilization are used to obtain GJ-11 post-biotic samples.

[0077] b) HaCaT cell culture, grouping, and culture conditions are the same as in Example 5.

[0078] c) Cell collection, RNA extraction, reverse transcription, and qPCR detection of IL-6 mRNA expression levels.

[0079] Statistical analysis: The experiment was repeated 3 times. Data are expressed as mean ± SEM. One-way ANOVA was used to compare differences between groups. P < 0.05 was considered statistically significant.

[0080] Experimental results: Lactococcus lactis GJ-11 postbiotic significantly inhibited TNF-α / IFN-γ-induced IL-6 overexpression in HaCaT cells, such as Figure 8 As shown, this indicates that GJ-11 post-biotic has stable and significant anti-inflammatory activity in the skin.

[0081] Example 7: Effects of cinnamaldehyde on histamine release-related indicators in P815 mast cells Experimental objective: To investigate the inhibitory effects of different concentrations of cinnamaldehyde on PMA + ionomycin-induced activation of P815 cells and histamine synthesis-related pathways, and to determine its effective concentration range for anti-allergy effects.

[0082] Experimental reagents: Cinnamaldehyde, PMA, Ionomycin, cell culture medium, etc.

[0083] Experimental Groups: a) Normal control group: cells were cultured routinely without inducing agents.

[0084] b) Allergy model group: Stimulation with 50 nM PMA + 500 nM Ionomycin for 5 h.

[0085] c) Low-dose cinnamaldehyde intervention group: 5 μM cinnamaldehyde + stimulating factor co-treatment.

[0086] d) Cinnamaldehyde medium-dose intervention group: 10 μM cinnamaldehyde + stimulating factor co-treatment.

[0087] e) High-dose cinnamaldehyde intervention group: 25 μM cinnamaldehyde + stimulating factor co-treatment.

[0088] Experimental methods: a) P815 cell culture and inoculation are the same as in Example 4.

[0089] b) The intervention group was pre-treated with the corresponding concentration of cinnamaldehyde, then PMA + Ionomycin, and incubated together for 5 h.

[0090] c) Collect cells, extract RNA, and use qPCR to detect the expression of histamine synthesis and release-related genes.

[0091] Statistical analysis: The experiment was repeated 3 times. Data are expressed as mean ± SEM. One-way ANOVA was used to compare differences between groups. P < 0.05 was considered statistically significant.

[0092] Experimental results: The expression of histamine synthesis-related genes was significantly increased in the model group; compared with the model group, both 10 and 25 μM cinnamaldehyde inhibited the expression of histamine synthesis-related genes in a concentration-dependent manner, with the 25 μM group showing the most significant inhibitory effect. Figure 9 As shown, cinnamaldehyde can effectively inhibit mast cell activation and histamine synthesis, and has in vitro anti-allergic activity.

[0093] The above description is merely a preferred embodiment of this application and is not intended to limit this application in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from this application, and these improvements and additions should also be considered within the scope of protection of this application.

Claims

1. A type of lactococcus lactis GJ-11, characterized in that, The classification name of the lactococcus GJ-11 is... Lactococcus lactis subsp. lactis GJ-11 has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20253062, on December 31, 2025, at Wuhan University, Wuhan, Hubei Province.

2. A biological agent, characterized in that, The biological agent comprises Lactococcus lactis GJ-11 as described in claim 1 or comprises a bacterial agent prepared from Lactococcus lactis GJ-11 as described in claim 1.

3. The biological agent according to claim 2, characterized in that, The preparation method of the biological agent The method includes: inoculating the Lactococcus lactis GJ-11 of claim 1 into a fermentation medium for fermentation culture, collecting the fermentation broth after the culture is completed, centrifuging to remove the supernatant to obtain bacterial cells, freeze-drying the bacterial cells to make freeze-dried powder, and adding or not adding excipients, other probiotics or probiotic agents to obtain the final product.

4. The biological agent according to claim 3, characterized in that, The fermentation medium used is a modified MRS medium, the formulation of which includes: 10 g / L soybean peptone, 5 g / L yeast extract, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 ml Tween 80, 0.58 g / L magnesium sulfate heptahydrate, and 0.25 g / L manganese sulfate tetrahydrate. And / or, the fermentation culture is carried out at a temperature of 36-38°C for 24-48 hours; And / or, the excipients are selected from carriers, adsorbents, cosolvents, dispersants, stabilizers, antioxidants, At least one of pH adjuster, preservative, defoamer, slow-release agent and binder; And / or, the other probiotics are selected from Lactobacillus acidophilus ( Lactobacillus acidophilus Lactobacillus rhamnosus ( Lactobacillus rhamnosus Lactobacillus plantarum ( Lactobacillus plantarum Lactobacillus reuteri ( Lactobacillus reuteri Lactobacillus casei ( Lactobacillus casei Lactobacillus paracasei ( Lactobacillus paracasei Bifidobacterium lactis (Bifidobacterium animalis subsp. lactis) Bifidobacterium animalis subsp. lactis Bifidobacterium longum ( Bifidobacterium longum ), Bifidobacterium breve Bifidobacterium breve Any one or more of the following: Bifidobacterium bifidum.

5. Application of Lactococcus lactis expressing CCR or its culture, lysate, fermentation product, active metabolite, and biological agent as active ingredients in the preparation of therapeutic drugs for diseases related to intestinal homeostasis imbalance.

6. The application according to claim 5, characterized in that, The diseases related to intestinal homeostasis imbalance include irritable bowel syndrome, inflammatory bowel disease, intestinal injury, metabolic endocrine diseases related to intestinal imbalance, autoimmune and allergic diseases, cardiovascular diseases or neuropsychiatric diseases; the lysate is cinnamyl-CoA reductase (CCR), and the active metabolite is cinnamaldehyde.

7. The application according to claim 5, characterized in that, The drug comprises an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient is one or more of the following: Lactococcus lactis GJ-11 as described in claim 1 or its culture, lysate, fermentation product, and active metabolite.

8. The application according to claim 7, characterized in that, The pharmaceutically acceptable excipients are selected from at least one of the following: carriers, adsorbents, solubilizers, dispersants, stabilizers, antioxidants, pH adjusters, preservatives, defoamers, sustained-release agents, and binders.

9. The application according to claim 5, characterized in that, The drug comprises an active ingredient, which is a biological agent as described in any one of claims 2-4.

10. The application according to any one of claims 5-8, characterized in that, The drug is available in oral or injectable form.

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