Phytobacterium plantarum DACNJS22 and application thereof in preventing / relieving / treating biological metabolic toxicity of benzopyrene

Abstract

The invention discloses a plant lactobacillus DACNJS22 strain and application thereof in preventing / relieving / treating biological metabolic toxicity of benzopyrene. The lactobacillus plantarum DACNJS22 is preserved in the China Center for Type Culture Collection, and the preservation number of the lactobacillus plantarum DACNJS22 is CCTCC (China Center for Type Culture Collection) NO: M20241957. The plant lactobacillus DACNJS22 disclosed by the invention can be used for efficiently reducing in-vivo bioconversion of benzopyrene into a key carcinogen (benzo (a) pyrene-7, 8-dihydro-diol-9, 10-epoxide) by inhibiting the expression level of cytochrome P450 family genes, and can be used for reducing the biological metabolic toxicity of benzopyrene from the source, so that the application of the plant lactobacillus DACNJS22 in the field of bioconversion of benzopyrene into the key carcinogen is realized. The intake of benzopyrene-polluted food and the damage of benzopyrene exposed to the environment to animal bodies or human bodies can be reduced; the compound can effectively prevent and / or relieve and / or treat tissue and organ injuries caused by oral exposure of benzopyrene and related cognitive diseases.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Lactobacillus plantarum DACNJS22 and application thereof in preventing / alleviating / treating benzopyrene biological metabolic toxicity. Background Art

[0002] Benzo(a)pyrene is the most toxic and representative of the PAH family. It has been classified as a Class 1 human carcinogen by the International Agency for Research on Cancer (IARC) and can cause a variety of cancers, including gastrointestinal cancer, bladder cancer, lung cancer, and other cancers. In addition to its recognized carcinogenicity, benzo(a)pyrene has a range of other toxicological properties, including cytotoxicity, genotoxicity, neurotoxicity, and endocrine disrupting activity. Worryingly, an increasing number of studies have shown that PAHs, especially benzo(a)pyrene, appear to be a class of compounds with neurotoxic properties. For example, a cohort study conducted by the Columbia Center for Children's Environmental Health (CCCEH) found that prenatal exposure to PAHs was associated with developmental delay, decreased IQ, anxiety / depression symptoms, and attention problems in children aged 3-9 years. A large number of epidemiological studies have also shown that environmental benzo(a)pyrene levels are negatively correlated with neurobehavioral function, learning ability, and autonomic nervous system function in coke oven workers. In addition, the inventor's preliminary experiments showed that oral administration of 50 mg / kg / body weight or 100 mg / kg / body weight of benzopyrene for 30 consecutive days can significantly induce cognitive impairment in C57BL / 6 mice and significantly reduce learning and memory behaviors. It is worth noting that the biological metabolic toxicity (including neurotoxicity) produced by benzopyrene in the body is mainly caused by the metabolic conversion of the cytochrome P450 family into the key carcinogen benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide. However, to date, there is still a lack of dietary strategies or suitable drug treatment plans that effectively inhibit the conversion of benzopyrene to benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide, thereby reducing the biological metabolic toxicity of benzopyrene.

[0003] Lactobacillus plantarum is a very important lactic acid-producing strain and an important probiotic that can have beneficial effects on intestinal health, metabolic disorders, and brain health. Previous studies (including animal models and human clinical trials) have shown that Lactobacillus plantarum can mitigate the risk of exposure to exogenous toxic substances, such as mycotoxins, heavy metals, and polycyclic aromatic hydrocarbons, by binding to or affecting the host's metabolism. For example, Lactobacillus plantarum C88 can prevent aflatoxin B1-induced liver damage in mice by inhibiting NF-κB-mediated inflammation and excessive cell apoptosis. Lactobacillus plantarum CCFM8610 can significantly reduce the cadmium (Cd) content in the blood of volunteers; animal experiments further showed that this strain can alleviate Cd-induced intestinal and liver damage by regulating enterohepatic circulation and intestinal microbiota. Excitingly, in addition to reducing the risk of exposure to exogenous toxins, Lactobacillus plantarum has the potential to reduce the occurrence of neurodegenerative diseases. For example, Lactobacillus plantarum WSJ-06 can alleviate lead exposure-induced neuroinflammation and memory impairment by regulating intestinal microbiota and metabolites. Lactobacillus plantarum DP189 can delay neurodegeneration induced by α-synuclein (α-SYN) accumulation in the substantia nigra of Parkinson's disease (PD) mice by inhibiting oxidative stress, suppressing proinflammatory responses, and regulating intestinal microbiota. In addition, Lactobacillus plantarum may also have therapeutic effects on depression, Alzheimer's disease (AD), and other neurodegenerative diseases, which has been widely reported. Therefore, given its potential to resist neuronal damage and its probiotic properties, Lactobacillus plantarum may be a candidate probiotic strain for alleviating the biological metabolic toxicity caused by benzopyrene.

[0004] According to the available data and literature, there are only a few data to prove that Lactobacillus plantarum or other lactobacilli can reduce the content of benzopyrene in food or animal bodies by adsorption, thereby reducing the impact of oral exposure to benzopyrene. However, to date, no Lactobacillus plantarum that effectively inhibits the conversion of benzopyrene into benzopyrene (a) pyrene-7,8-dihydrodiol-9,10-epoxide in animal bodies has been reported. In addition, Lactobacillus plantarum with good probiotic properties can be beneficial to human health, such as good gastrointestinal fluid tolerance, antioxidant, anti-inflammatory, and promoting the enrichment of intestinal short-chain fatty acid-producing bacteria and increasing the level of short-chain fatty acids produced by the body. Therefore, it is urgent to develop probiotic Lactobacillus plantarum with independent intellectual property rights that inhibit the biological metabolic conversion of benzopyrene and reduce the biological metabolic toxicity of benzopyrene to effectively reduce the exposure risk of benzopyrene and promote the development of public health. Summary of the invention

[0005] The purpose of the present invention is to solve the above problems and provide a strain of Lactobacillus plantarum DACNJS22 and its application in preventing / alleviating / treating benzopyrene biological metabolic toxicity.

[0006] In order to achieve its purpose, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides a strain of Lactobacillus plantarum DACNJS22 (Lactiplantibacillus plantarum DACNJS22), which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M20241957.

[0008] A second aspect of the present invention provides a bacterial agent, the active ingredient of which comprises the above-mentioned Lactobacillus plantarum DACNJS22 or its fermentation liquid.

[0009] Preferably, the bacterial agent is a freeze-dried powder.

[0010] The third aspect of the present invention provides a method for preparing the above-mentioned bacterial agent, which is obtained by inoculating the above-mentioned Lactobacillus plantarum DACNJS22 into a culture medium for cultivation.

[0011] The method for preparing the fermentation liquid of DACNJS22 is as follows: after activating the Lactobacillus plantarum DACNJS22 strain, the strain is cultured in a liquid culture medium at 30-37° C. for 18-30 hours to obtain the fermentation liquid of Lactobacillus plantarum DACNJS22; preferably, the liquid culture medium is an MRS liquid culture medium.

[0012] The fourth aspect of the present invention provides the use of the above-mentioned Lactobacillus plantarum DACNJS22 or the above-mentioned bacterial agent in the preparation of a product for any of the following purposes:

[0013] (1) Prevent and / or mitigate and / or treat benzopyrene exposure;

[0014] (2) Reduce the biological metabolic toxicity of benzopyrene;

[0015] (3) inhibiting the conversion of benzopyrene to benzopyrene(a)pyrene-7,8-dihydrodiol-9,10-epoxide;

[0016] (4) Preventing and / or alleviating and / or treating tissue and organ damage caused by benzopyrene biometabolism;

[0017] (5) Preventing and / or alleviating and / or treating cognitive diseases related to benzopyrene biometabolism.

[0018] The application (4) prevents and / or alleviates and / or treats tissue and organ damage caused by the biological metabolism of benzopyrene; wherein, Lactobacillus plantarum DACNJS22 prevents and / or alleviates and / or treats pathological damage, inflammatory response or oxidative stress level caused by exposure to benzopyrene.

[0019] The application (5) prevents and / or alleviates and / or treats cognitive diseases related to the biological metabolism of benzopyrene; wherein, Lactobacillus plantarum DACNJS22 prevents and / or alleviates and / or treats cognitive disorders caused by the neurotoxin of benzopyrene.

[0020] The product is selected from food, food additives, health products, feed, feed additives, reagents or medicines.

[0021] The medicine further contains a pharmaceutically acceptable carrier; the food or health product or feed further contains auxiliary materials.

[0022] The beneficial effects of the present invention are:

[0023] 1. The plant lactobacillus DACNJS22 of the present invention is of natural origin, has good in vitro probiotic properties and safety, including gastrointestinal fluid tolerance, self-aggregation and hydrophobicity, as well as no antibiotic resistance and hemolytic activity. The strain has safety and multiple probiotic properties. The strain itself and its fermentation are applied to food / drugs, etc., which can be beneficial to the health of consuming animals or humans, and can be suitable for the development and application of probiotic preparations, pills, powders and probiotic fermentation series products.

[0024] 2. The plant lactobacillus DACNJS22 of the present invention can effectively reduce the bioconversion of benzopyrene into key carcinogens (benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide) in the body by inhibiting the expression level of cytochrome P450 family genes, and can reduce the biological metabolic toxicity of benzopyrene from the source, which is beneficial to reducing the damage to animals or humans caused by the intake of foods contaminated with benzopyrene and environmental exposure to benzopyrene.

[0025] 3. The plant lactobacillus DACNJS22 of the present invention can effectively enrich intestinal short-chain fatty acid-producing bacteria and increase the levels of acetate and butyrate in feces, which can be beneficial to the intestinal health and body homeostasis of the host. When used as food or medicine, it can produce multiple benefits for consumers, including but not limited to anti-inflammation, anti-oxidation, maintenance of intestinal health and reduction of the occurrence of cognitive impairment.

[0026] 4. The plant lactobacillus DACNJS22 of the present invention can effectively prevent and / or alleviate and / or treat tissue and organ damage caused by oral exposure to benzopyrene (for example, disease characteristics such as colon and hippocampal pathological damage, inflammatory response, oxidative stress levels, etc.), as well as the occurrence of related cognitive diseases (cognitive impairment caused by the neurotoxins of benzopyrene). BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 These are the results of gastric acid and bile salt tolerance and hemolytic activity of Lactobacillus plantarum DACNJS22.

[0028] Figure 2This is the result of Lactobacillus plantarum DACNJS22 reducing the biometabolic transformation of benzopyrene.

[0029] Figure 3 Results show that Lactobacillus plantarum DACNJS22 alleviates benzopyrene-induced colonic injury.

[0030] Figure 4 The results show that Lactobacillus plantarum DACNJS22 alleviates the pathological characteristics of benzopyrene-induced hippocampal damage.

[0031] Figure 5 Results show that Lactobacillus plantarum DACNJS22 alleviates benzopyrene-induced neuroinflammation and synaptic defects.

[0032] Figure 6 Results show that Lactobacillus plantarum DACNJS22 alleviates benzopyrene-induced cognitive behavioral deficits.

[0033] Figure 7 The results showed that Lactobacillus plantarum DACNJS22 enriched intestinal short-chain fatty acids.

[0034] Figure 8 These are the results of correlation analysis between the level of short-chain fatty acids enriched by Lactobacillus plantarum DACNJS22 and intestinal flora. DETAILED DESCRIPTION

[0035] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0036] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0037] Example 1: Identification of Lactobacillus plantarum DACNJS22

[0038] (1) The plant lactobacillus DACNJS22 isolated from yak milk obtained from the strain library of the Specialty Fermented Food Innovation Team of the School of Food Science of Southwest University was activated for three generations in MRS broth and then streaked to obtain a single colony. The purified single colony was re-inoculated into MRS broth and incubated at 37°C for 24 hours.

[0039] (2) Molecular identification: The genomic DNA of lactic acid bacteria was extracted by the cetyltrimethylammonium bromide (CTAB) method. The universal primers 27F (SEQ ID NO.1: 5'-AGAGTTTGATCCTGGCTC AG-3') and 1492R (SEQ ID NO.2: 5'-TACGGATACCTTGTTACGACTT-3') for amplifying bacterial 16S rDNA were used to perform PCR amplification of the full-length sequence of 16S rDNA of the target lactic acid bacteria and then sent to Sangon Biotech (Shanghai) Co., Ltd. for bidirectional sequencing until the sequence was complete. The 16S rDNA sequence was submitted to the GenBank database on the website of the National Center for Biotechnology Information of the United States and compared using the BLAST online server (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The target species name was determined based on the known lactic acid bacteria species with the highest comparison score and a sequence identity of 100%.

[0040] (3) After morphological and molecular identification, it was confirmed that the colony morphology and microscopic characteristics of the target Lactobacillus plantarum showed that its surface was smooth, and the cells were white, spherical and short-chain rod-shaped, which was consistent with the morphological characteristics of lactobacillus. The 16S rRNA gene sequence amplified from Lactobacillus plantarum DACNJS22 is shown in SEQ ID NO.3, and the similarity with Lactobacillus plantarum 3748 (GenBank accession number: MT538602.1) in BLAST comparison was 99.73%.

[0041] The 16S rDNA sequence of the target lactic acid bacteria is as follows (SEQ ID NO.3):

[0042]

[0043] The strain DACNJS22 was sent to the China Center for Type Culture Collection (CCTCC) for preservation in September 2024. The address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The preservation date is September 11, 2024; the preservation number is CCTCC NO: M 20241957; the classification name is: Lactobacillus plantarum DACNJS22 Lactiplantibacillus plantarm DACNJS22.

[0044] Example 2: In vitro probiotic properties and safety evaluation of Lactobacillus plantarum DACNJS22

[0045] (1) For the bile salt tolerance test, different concentrations (0.1%, 0.3% and 0.5% (w / v)) of bile salt were added to the MRS liquid culture medium; for the acid resistance test, 2 mol / mL HCl was used to adjust the MRS liquid culture medium to different pH values ​​(pH 2.0, 3.0 and 4.0). 8 CFU / mL) was added to the above-mentioned MRS liquid culture medium with different treatments and incubated at 37°C for 4 hours. Then, 10 μL of each bacterial suspension was coated on an MRS solid plate by a 10-fold serial dilution method and cultured at 37°C for 24 hours. The number of viable bacteria of 30 to 300 single colonies was selected for counting, and the untreated Lactobacillus plantarum DACNJS22 was used as a control.

[0046] (2) The hemolytic activity of Lactobacillus plantarum DACNJS222 was evaluated as follows: Lactobacillus plantarum DACNJS22 and Escherichia coli O157:H7 cultures were first spread on Columbia blood agar containing fresh sheep blood and cultured at 28°C for 24 hours. The hemolytic activity was determined according to the following rules: if the colonies on the plate did not change, the strain was determined to be non-hemolytic; if the colonies showed a completely clear hemolytic ring on the plate, the strain was determined to be a β-hemolytic strain.

[0047] (3) The results showed that when treated with pH values ​​of 2, 3 and 4 for 4 h, the survival rates of Lactobacillus plantarum DACNJS22 remained at 98.97%, 98.36% and 72.26% respectively compared with the control ( Figure 1 A). When treated with bile salts at concentrations of 0.1%, 0.3%, and 0.5% for 4 h, the survival rates of Lactobacillus plantarum DACNJS22 remained at 97.89%, 87.22%, and 63.93%, respectively, compared with the control group (Figure B). In addition, the Lactobacillus plantarum DACNJS22 strain did not show any hemolytic activity ( Figure 1C), a transparent ring is shown near the positive control bacteria (Escherichia coli O157:H7) ( Figure 1 D), which was identified as beta-hemolytic. Therefore, these findings confirm that Lactobacillus plantarum DACNJS22 has good in vitro probiotic properties and a good safety profile.

[0048] Example 3: Biometabolism activity of Lactobacillus plantarum DACNJS22 on benzopyrene-exposed mice

[0049] The three strains of Lactobacillus plantarum (DACNJS22, LPP95, e3) were cultured in MRS liquid medium at 37°C for 24 h, and the cells were collected by centrifugation and resuspended in sterile saline to prepare a concentration of about 1×10 9 CFU / mL bacterial suspension. Lactobacillus plantarum strains LPP95 and e3 were isolated from yak yogurt and stored in the bacterial strain bank of the Specialty Fermented Food Innovation Team of the School of Food Science, Southwest University.

[0050] After a 7-day acclimation period, the mice were randomly divided into the following 5 groups:

[0051] Control group: mice received normal saline and olive oil at a dose of 10 mL / kg / body weight.

[0052] Model group: mice received normal saline at a volume of 10 mL / kg / body weight; 2 hours later, these mice received 50 mg / kg / body weight of benzopyrene (dissolved in olive oil).

[0053] The mice in the e3, LP33, and S22 groups received 10 mL / kg / body weight of normal saline, which contained 1×10 9 CFU of Lactobacillus plantarum (e3 / LP33 / DACNJS22); 2 hours later, these mice also received 50 mg / kg / body weight of benzopyrene (dissolved in olive oil).

[0054] Each group had 10 mice, and oral gavage was performed for 30 consecutive days. The body weight of the mice was measured every 6 days, and the experimental period lasted for 30 days. The collected serum was used to determine the content of benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE-DNA); part of the colon and hippocampus tissues were used to determine the mRNA expression levels of CYP1A1 and CYP1B1 ( Figure 2 A).

[0055] like Figure 2As shown in B, after 30 days of administration, the weight of mice in the model group increased slowly and was significantly lower than that of the control group on the 24th and 30th days (P<0.05). In contrast, administration of Lactobacillus plantarum e3, LP33, and DACNJS22 (especially DACNJS22 (S22 group) attenuated the weight loss caused by benzopyrene. In addition, compared with the control group, the mRNA expression levels of CYP1A1 and CYP1B1 in the colon and hippocampus of mice in the model group continued to be significantly upregulated, while the S22 group significantly reversed this situation. In addition, the LP33 group also significantly reduced the increase in the expression levels of CYP1A1 and CYP1B1 in the hippocampus induced by benzopyrene (P<0.05; Figure 2 CF). In addition, the levels of BPDE-DNA adducts in serum were reduced in the e3, LP33 and S22 groups to varying degrees (P < 0.05), especially in the S22 group, which had a higher effect than the LP33 and e3 groups ( Figure 2 G). These results indicate that Lactobacillus plantarum DACNJS22 is the strain with the strongest ability to biotransform benzo[a]pyrene among the three tested Lactobacillus plantarum strains and can effectively inhibit the conversion of benzo[a]pyrene to BPDE-DNA in vivo.

[0056] Example 4: Effect of Lactobacillus plantarum DACNJS22 on the mitigation of tissue and organ damage in mice exposed to benzopyrene

[0057] After the C57BL / 6 mice were grouped, modeled and treated according to Example 3, parts of the colon and hippocampus tissues were fixed in 4% paraformaldehyde solution for hematoxylin and eosin staining, immunofluorescence analysis, antioxidant enzyme activity test, and expression level test of inflammatory and barrier factors.

[0058] Hematoxylin and eosin staining showed that the colon tissues of the mice in the model group showed severe pathological damage and colitis symptoms, including ulcers, crypt destruction, and partial inflammatory cell infiltration ( Figure 3 A, P < 0.05). In contrast, these symptoms were partially reversed in the e3, LP33, and S22 groups. The characteristics of mice in the S22 group were very similar to those in the control group, with relatively intact colonic mucosal epithelium, normal crypts, orderly glandular tissue, and no obvious ulcers. In addition, the expression levels of pyroptotic factors (NLRP3, ASC, caspase-1, and GSDMD) and inflammatory factors (TNF-α, IL-1β, IL-6, and CoX-2) in the model group were significantly upregulated (except CoX-2) ( Figure 5B and 5C, P < 0.05). Similarly, compared with the model group, the S22 group significantly reversed these up-regulated levels, especially the up-regulated levels of NLRP3, caspase-1, GSDMD, TNF-α, IL-1β, and IL-6. However, the e3 or LP33 group only significantly reversed the expression levels of ASC, caspase-1, GSDMD, and IL-6. In addition, IF staining analysis also confirmed the changes in their expression levels. Figure 3 As shown in D and E, compared with the model group, the mean fluorescence intensity of anti-F4 / 80 and NLRP3 positive cells in the S22 group was significantly downregulated (P<0.05). These results strongly indicate that Lactobacillus plantarum DACNJS22 can effectively alleviate benzopyrene-induced colon inflammation.

[0059] In addition to the BaP-induced colonic mucosal damage revealed by hematoxylin and eosin staining, benzopyrene administration induced the abnormal expression of TJ proteins, as reflected by the significant downregulation of the mRNA expression levels of ZO-1, occludin, and claudin-1 ( Figure 3 F, P < 0.05). It is noteworthy that the S22 group also significantly reversed the down-regulated mRNA levels of these genes. In addition, except for Claudin-1, which was significantly up-regulated in the LP33 group, there was no significant change in the mRNA expression levels of these genes between the model group and the LP33 and e3 groups. Similarly, IF staining analysis also showed that the mean fluorescence intensity of anti-ZO-1 positive cells in the S22 group was significantly higher than that in the model group ( Figure 3 G, P<0.05). These results indicate that strain DACNJS22 can alleviate BaP-induced colonic injury, which is related to the recovery of colonic inflammation and the maintenance of intestinal barrier function.

[0060] Considering that benzopyrene is also neurotoxic, oxidative stress is one of the most important factors in benzopyrene-induced neurological damage. Hematoxylin and eosin staining showed that the dentate gyrus (DG) and miliary 1 (CA1) regions of the hippocampus of the model group mice showed severe neuronal degeneration, sparse cell arrangement, neuronal necrosis and mild edema ( Figure 4 A). In contrast, the e3, LP33, and S22 groups alleviated these neuronal injuries to varying degrees. Among them, the characteristics of the S22 group were very similar to those of the control group, with no obvious neuronal necrosis, no inflammatory cell infiltration, and mild edema. Consistent with the pathological results, the GSH-Px and SOD activities in the model group were significantly reduced, while the MDA and H2O2 contents were significantly increased ( Figure 4BE, P < 0.05). However, the S22 group reversed the changes in these oxidative indicators. In addition, the LP33 group only slowed down the increase in SOD activity and the decrease in H2O2 content induced by benzopyrene, while there was no significant difference between the e3 group and the model group. These results indicate that the DACNJS22 strain can effectively alleviate BaP-induced oxidative neurological damage. In addition, the effects of Lactobacillus plantarum DACNJS22 on the expression levels of pyroptotic factors (NLRP3, ASC, Caspase-1 and GSDMD), inflammatory factors (TNF-α, IL-1β, Il-6 and CoX-2) and synaptic proteins (BDNF, PSD and SYN) in hippocampal tissue induced by benzopyrene were further studied. As expected, the mRNA expression levels of NLRP3, caspase-1, TNF-α, IL-1β and IL-6 in the hippocampal tissue of the model group were significantly upregulated compared with those of the control group ( Figure 5 A and B, P < 0.05), showing a pyroptotic neuroinflammatory response. In contrast, administration of the S22 group significantly reduced the mRNA expression levels of these benzopyrene-induced up-regulated factors except IL-1β, while the e3 and LP33 groups only significantly downregulated the mRNA expression level of IL-6 (P < 0.05). Similarly, the mRNA expression levels of synaptic plasticity markers (BDNF, presynaptic protein SYN, and postsynaptic protein PSD95) in the model group also decreased significantly. However, among the three treatment groups, only the S22 group effectively restored the mRNA expression levels of these downregulated synaptic proteins ( Figure 4 C). In addition, IF staining analysis also confirmed these results. Figure 5 As shown in DF, compared with the control group, the mean fluorescence intensity of anti-Iba-1 and NLRP3 positive cells in the model group was significantly increased, while the mean fluorescence intensity of BDNF positive cells was decreased (P<0.05). In contrast, the S22 group reversed these abnormal changes. These results confirmed that the DACNJS22 strain can alleviate brain tissue damage induced by benzopyrene neurotoxicity, which is reflected in the effective inhibition of oxidative stress damage, pyroptotic neuroinflammation and synaptic defects.

[0061] Example 5: Effect of Lactobacillus plantarum DACNJS22 on the alleviation of cognitive impairment in mice exposed to benzopyrene

[0062] After the C57BL / 6 mice were grouped, modeled and treated according to Example 3, the mice were subjected to behavioral assessments on the last three days of the treatment period, including nesting behavior, novel object and location recognition tests.

[0063] The nesting behavior test showed that the nesting ability of mice in the model group was significantly lower than that in the control group, as shown by a significant decrease in the nesting score and a significant increase in the weight of the unbroken nest ( Figure 6AC, P < 0.05). In contrast, the nest scores of mice in the LP33 and S22 groups increased significantly, and the weight of unbroken nests decreased significantly (P < 0.05). In addition, the ability of mice in the model group to recognize new objects and new locations was also affected. Figure 6 As shown in DF, the place discrimination index (PDI) and novel object discrimination index (NODI) of the model group mice were significantly reduced compared with the control group (P<0.05). However, only the S22 group mice effectively reversed these behavioral indices. These results indicate that strain DACNJS22 can effectively alleviate the cognitive behavioral deficits induced by benzopyrene.

[0064] Example 6: Regulatory effects of Lactobacillus plantarum DACNJS22 on intestinal flora and short-chain fatty acids

[0065] After the C57BL / 6 mice were grouped, modeled and treated according to Example 3, the feces of the mice were collected for 16r SRNA sequencing and short-chain fatty acid content determination.

[0066] Gut microbiota plays a crucial role in probiotics alleviating gut-brain damage. To explore the regulatory effects of Lactobacillus plantarum DACNJS22 on gut microbiota, 16S rRNA gene sequencing was performed on the fecal samples of each group using the Illumina sequencing platform. Figure 7 As shown in A, there were no significant differences in the five alpha diversity indices (Sob, Ace, Chao, Shannon, and Simpson indices) between the control group and the model group; however, a significant increase in the Sob, Ace, and Chao indices was observed in the S22 group, and a significant increase in the Shannon and Simpson indices was observed in the e3 group. Principal coordinate analysis (PCoA) and nonmetric multidimensional scaling analysis (NMDS) showed that there were significant differences between the model group and the control group, and the characteristics of the S22 group were closest to the Con group ( Figure 7 B and C). In addition, the petal clustering plot also showed that the total ASV content of all treatment groups was significantly increased or decreased compared with the model group ( Figure 7 D) Therefore, all Lactobacillus plantarum strains were able to specifically modulate the gut microbial profile of mice.

[0067] In addition, the effect of Lactobacillus plantarum DACNJS22 strain on the composition of intestinal microbiota was further analyzed. The main dominant bacterial phyla in each group were Fungi, Bacteroidetes, Ascomycota, and Actinobacteria ( Figure 7 E). However, there was no significant difference in the composition of the intestinal microbiota between the control group and the model group. In contrast, in the e3 group, the relative abundance of sessile bacteria, spirochetes, and cyanobacteria increased significantly, while the relative abundance of Bacteroidetes decreased significantly. In the LP33 group and S22 group, the relative abundance of Proteobacteria and Verrucomicrobia increased significantly ( Figure 6 F, P < 0.05). At the genus level, the relative abundance of unclassified __Puccinella, Desulfovibrio, Candidatus_Saccharimonas, and Bacteroides in the model group decreased significantly compared with the control group, indicating that benzopyrene treatment caused changes in the characteristics of the intestinal microbiota. In contrast, the relative abundance of Lactobacillus and Bacillus in the e3 group increased significantly, while the relative abundance of Prevotellaceae_UCG-001 and norank_f__Muribaculaceae decreased significantly. The abundance of Enterobacteriaceae increased significantly and the abundance of Desulfovibrio decreased in the LP33 group, while the abundance of unclassified __f__ Clostridium in the S22 group mice increased significantly ( Figure 7 G and 7H, P < 0.05). In addition, Ruminococcus, Lachnospiraceae_UCG-006, and Allobaculum were characteristic microbiota of the S22 group. In general, the characteristic intestinal microbiota of each group may be potential biomarkers affecting host health. Therefore, Lachnospiraceae_UCG-006, Allobaculum, and Ruminococcus may be biomarkers for the DACNJS22 strain to alleviate benzopyrene-induced neurotoxicity, and these microorganisms are all short-chain lipid-producing bacteria.

[0068] In addition, the interference effect of strain DACNJS22 on promoting fecal SCFA production was further investigated. Figure 8 As shown in A, only the fecal butyrate level in the model group decreased significantly, while only the S22 group effectively reversed the fecal butyrate level and further increased the fecal acetate and isobutyrate levels. Spearman correlation analysis of SCFAs and gut microbiota significantly regulated by the S22 group showed that the relative abundance of unclassified _f_ Lachnospiraceae and Lachnospiraceae was positively correlated with butyrate and isobutyrate levels ( Figure 8 B and 7C). Therefore, these results fully confirmed that the DACNJS22 strain not only alleviated the biotoxicity induced by BPA by regulating SCFA-producing bacteria and increasing the levels of SCFAs (especially butyrate), but also beneficially regulated intestinal microorganisms and promoted the production of short-chain fatty acids.

Claims

1. Lactobacillus plantarum DACNJS22 (Lactiplantibacillus plantarm DACNJS22), deposited in China Center for Type Culture Collection, with the deposit number being CCTCC NO: M 20241957.

2. A bacterial agent, the active ingredient of which comprises the plant lactobacillus DACNJS22 or its fermentation liquid according to claim 1.

3. The bacterial agent according to claim 2, characterized in that: The bacterial agent is freeze-dried powder.

4. A method for preparing the bacterial agent according to claim 2 or 3, characterized in that: The plant lactobacillus DACNJS22 according to claim 1 is inoculated into a culture medium and cultured to obtain the product.

5. The preparation method according to claim 4, characterized in that: The method for preparing the fermentation liquid of DACNJS22 is as follows: after activating the Lactobacillus plantarum DACNJS22 strain, the strain is cultured in a liquid culture medium at 30-37° C. for 18-30 hours to obtain the fermentation liquid of Lactobacillus plantarum DACNJS22; preferably, the liquid culture medium is an MRS liquid culture medium.

6. Use of the plant lactobacillus DACNJS22 according to claim 1 or the bacterial agent according to claim 2 in the preparation of any of the following products: (1) Prevent and / or mitigate and / or treat benzopyrene exposure; (2) Reduce the biological metabolic toxicity of benzopyrene; (3) inhibiting the conversion of benzopyrene to benzopyrene(a)pyrene-7,8-dihydrodiol-9,10-epoxide; (4) Preventing and / or alleviating and / or treating tissue and organ damage caused by benzopyrene biometabolism; (5) Preventing and / or alleviating and / or treating cognitive diseases related to benzopyrene biometabolism.

7. The use according to claim 6, characterized in that: The application (4) prevents and / or alleviates and / or treats tissue and organ damage caused by the biological metabolism of benzopyrene; wherein, Lactobacillus plantarum DACNJS22 prevents and / or alleviates and / or treats pathological damage, inflammatory response or oxidative stress level caused by exposure to benzopyrene.

8. The use according to claim 6, characterized in that: The application (5) prevents and / or alleviates and / or treats cognitive diseases related to the biological metabolism of benzopyrene; wherein, Lactobacillus plantarum DACNJS22 prevents and / or alleviates and / or treats cognitive disorders caused by the neurotoxin of benzopyrene.

9. The use according to claim 6, characterized in that: The product is selected from food, food additives, health products, feed, feed additives, reagents or medicines.

10. The use according to claim 9, characterized in that: The medicine further contains a pharmaceutically acceptable carrier; the food or health product or feed further contains auxiliary materials.

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