Pediococcus acidilactici and application of pediococcus acidilactici in preparation of brain-intestinal axis regulation efficacy postbiotic product

Abstract

The invention relates to the technical field of microbiology and food processing, in particular to pediococcus acidilactici and application of the pediococcus acidilactici in preparation of a brain-intestinal axis regulating efficacy metagen product.The high-activity pediococcus acidilactici DY-1 is separated out from natural fermentation pickles of super-polyselenium plant cardamine violifolia for the first time; the strain can be widely applied to fermentation of high-selenium plant-based food, can significantly promote glycolysis release of selenium-containing active substances, has good acid resistance, high protease activity and high lipase activity, can fully ferment plant-based fermentation raw materials, and has high yield. Glycolysis of macromolecular substances such as protein, saccharides and fat is facilitated, accumulation of active ingredients such as essential amino acid, unsaturated fatty acid, alkaloid and phenolic substances is promoted, and the edibility and functionality of the product are improved; and the organic selenium-rich metagen product prepared by sterilizing the fermentation mixture can synergistically improve human body cognition and emotion regulation regulated by microorganism-intestinal-brain axis.

Description

Technical Field

[0001] This invention relates to the technical fields of microbiology and food processing, specifically to a type of *Pediococcus lactis* and its application in the preparation of postbiotic products that regulate the brain-gut axis. Background Technology

[0002] The gut-brain axis refers to the complex bidirectional communication network formed between the gut microbiota and the gut and brain through neural, endocrine, immune, and metabolic pathways. This system not only regulates digestion, metabolism, and immunity but also profoundly affects mood, cognition, behavior, and nervous system health. Microbes can bidirectionally influence the central nervous system via the vagus nerve and regulate the activity of the HPA axis through the immune system, including plasma levels of glucocorticoids, tryptophan metabolism, the production, expression, and conversion of neurotransmitters and neurotrophic factors, and the production of neuroactive metabolites, such as short-chain fatty acids (SCFAs). SCAs can regulate neurotrophic factor levels, promote neurogenesis, affect glial cell morphology and function, promote serotonin formation, and improve neuronal homeostasis and function, directly or indirectly altering the pathophysiology of cognitive, emotional, and mental disorders. Gut dysbiosis exacerbates neuroimmune responses and worsens pathological behaviors in the brain.

[0003] Selenium, as an essential trace element, plays a vital role in human health. Selenoproteins, in which selenium is a key component, possess excellent histological and enzymatic activities, including glutathione peroxidase (GPx), thioredoxin reductase (TRxR), and iodothyronine deiodinase (DIO). These are well-known antioxidants and catalysts for the secretion of active thyroid hormones. Selenium is also essential for maintaining a normal immune system, effectively resisting viral infections and the development of AIDS, while also improving sperm motility and reducing the risk of miscarriage. Furthermore, selenium has been shown to have significant effects in preventing cardiovascular disease and fighting cancer. Artificial selenium supplementation has become a major research focus in related health food research both domestically and internationally.

[0004] Viola yezoensis, a novel super-aggregated cruciferous leafy vegetable, was discovered in the selenium-rich Yutangba area of ​​Enshi City, Hubei Province. Due to its centuries-long history of safe consumption and medicinal use in my country, the National Health Commission officially approved its management in 2021, referencing the standards for common leafy vegetables. Besides high levels of organic selenium, including selenocysteine, methylselenocysteine, and selenomethionine, Viola yezoensis also contains abundant protein, soluble sugars, polysaccharides, flavonoids, vitamins, and minerals. Previous studies have shown that selenium-containing active extracts from Viola yezoensis have multiple therapeutic effects on various human diseases, including inflammation, cancer, liver disease, diabetes, and neurodegenerative diseases.

[0005] Currently, food processing utilization of *Vigna arvensis* edible resources is limited to traditional processing techniques such as crushing, extraction, and mixing. Even though it has sufficient nitrogen and carbon sources for microbial growth, its application through fermentation is rarely reported. Research on superior strains suitable for fermentation processing of high-value selenium-rich vegetables, including *Vigna arvensis*, is also lacking. Summary of the Invention

[0006] To solve the above-mentioned technical problems, the first aspect of the present invention provides Pediococcus acidilactici, whose scientific name is Pediococcus acidilactici, strain number is DY-1, and the preservation number of the Pediococcus acidilactici is CCTCC No: M2025908.

[0007] Preferably, the lactic acid cocci are isolated from naturally fermented kimchi made from wild rice.

[0008] Preferably, the 16S rDNA sequence of the *Pediococcus lactis* is shown in SEQ ID NO: 1.

[0009] A second aspect of the present invention provides a fermentation agent, the fermentation agent comprising: The fermentation broth obtained by fermenting the aforementioned Pediococcus lactis; Alternatively, the fermentation broth can be spray-dried to obtain a dry powder inoculum.

[0010] A third aspect of the present invention provides an application of the aforementioned Pediococcus lactis, wherein the Pediococcus lactis is applied to the fermentation of high-selenium cruciferous vegetables.

[0011] The high-selenium cruciferous vegetables mentioned include: violet-leafed watercress, rapeseed, cabbage, broccoli, etc.

[0012] The fourth aspect of this invention provides an organic selenium-rich postbiotic product with brain-gut axis regulating effects, which is obtained by fermentation of the aforementioned Pediococcus lactis.

[0013] The fifth aspect of this invention provides a method for preparing the organic selenium-rich postbiotic product with brain-gut axis regulating effects, comprising the following steps: (1) The lactic acid cocci were cultured in MRS medium to obtain a lactic acid cocci solution, and the solution was resuspended to obtain a seed solution; (2) After crushing the high-selenium plant-based fresh raw material, add 0.2wt%-0.5wt% salt and 0.2wt%-0.5wt% vitamin C and mix thoroughly to obtain the high-selenium plant-based raw material; (3) The seed solution was prepared to a final concentration of 1×10⁻⁶. 6 -1×10 7 CFU / g is inoculated into high-selenium plant-based raw materials for anaerobic fermentation; (4) The fermentation product obtained in step (3) is treated at 100-121°C for 15-40 min, dried and pulverized to obtain organic selenium-enriched post-vitamin powder of Pyrococcus lactis. (5) Add organic selenium-rich postbiotic powder of Pycnococcus lactis to a carrier or excipient to produce a postbiotic product with brain-gut axis regulation effect.

[0014] Preferably, in step (1), the culture time is 24-48h and the culture temperature is 30-38℃.

[0015] Preferably, in step (2), the high-selenium plant-based fresh raw material is one or more of high-selenium cruciferous vegetables, with a selenium content of 50-2000 mg / kg and an organic selenium content of more than 80%.

[0016] Specifically, high-selenium cruciferous vegetables include one or more of the following: violet cress, broccoli, cabbage, rapeseed shoots, and soybeans.

[0017] Preferably, in step (3), the fermentation time is 1-14 days and the fermentation temperature is 25-38℃.

[0018] Preferably, in step (5), the product type is food, medicine or health product, and the product dosage form is powder, granules, capsules, tablets, pills or oral liquid.

[0019] The beneficial effects of this invention are as follows: (1) Pediococcus lactis is a type of lactic acid bacteria that is widely used in the food fermentation industry and is beneficial to food and human body. This invention has for the first time isolated a highly active strain of Pediococcus lactis DY-1 from the naturally fermented kimchi of the super-selenophora plant Viola yedoensis. It can be widely used in the fermentation of high-selenium plant-based foods and can significantly promote the fermentation and release of selenium-containing active substances.

[0020] (2) The lactic acid cocci DY-1 disclosed in this invention has good acid resistance, high protease activity and high lipase activity, which can fully ferment plant-based fermentation raw materials, and help the fermentation of macromolecules such as proteins, sugars and fats, as well as promote the accumulation of active ingredients such as essential amino acids, unsaturated fatty acids, alkaloids and phenolic substances, thereby improving the edibility and functionality of the product. (3) The lactic acid cocci DY-1 disclosed in this invention can be used in the fermentation of high-selenium cruciferous vegetables to synthesize tryptamines, amides and active dipeptides. These substances can improve intestinal inflammation conditions, help regulate the human brain-gut axis, improve cognition, regulate rhythm, and improve mood, sleep and appetite.

[0021] (4) The Lactococcus lactis DY-1 disclosed in this invention is a dominant strain for high-selenium plant-based fermentation. It can rapidly produce acid in its fermentation system and establish a dominant position, promoting the degradation of macromolecules in high-selenium plant-based raw materials and the synthesis of active substances. The organic selenium-rich post-biotic products prepared by sterilizing its fermentation mixture can synergistically enhance the improvement of human cognition and mood regulation regulated by the microbial-gut-brain axis. Its application and product development market is unprecedented. Attached Figure Description

[0022] Figure 1 This is a phylogenetic tree diagram of the *Pediococcus lactis* DY-1 strain of the present invention; Figure 2 The results of the acid resistance test for Pyotrophic Lateral Communicum DY-1 in Example 3; Figure 3 This is a graph showing the changes in the content of metabolites in the two raw material powders in Example 3; Figure 4 The graph shows the changes in the content of the top 10 metabolites in the two raw material powders in Example 3; Figure 5 This is the composition of the mouse gut microbiota at the mid-phylum level in Example 4. Specific Implementation

[0023] The present invention will now be described in detail with reference to the embodiments and accompanying drawings. The following embodiments are implemented based on the technical solution of the present invention, and detailed implementation methods and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0024] Unless otherwise specified, the methods described in the following examples are conventional; the reagents and materials described are commercially available unless otherwise specified.

[0025] Implementation Example 1: Isolation, Identification, and Preservation of Strains In this embodiment, Pyotrophic cytokine DY-1 was isolated, identified, and preserved.

[0026] (1) Strains Isolation: Fresh leaves of *Violetia violacea*, a wild hyperselenophora plant growing around Shuanghe Fishpond Dam in Enshi, were thoroughly rinsed with water and slightly dried. The mixture was then pulped and transferred to a sealed solid fermentation bag. Negative pressure was applied to remove air, and the mixture was allowed to undergo natural anaerobic fermentation at room temperature for 7 days to obtain naturally fermented *Violetia violacea* pickles. 1g of sample was thoroughly vortexed in 10mL of sterile phosphate-buffered saline (PBS) and then serially diluted. The dilution was from 10... 5 Take 100 μL of the sample diluted 1:1 and plate it on MRS agar medium, then incubate at 37°C for 24 to 48 hours. Then, select a single colony for streak isolation to obtain a pure culture of the isolated strain.

[0027] (2) Strain identification: DNA was extracted and isolated from the pure culture strain using a bacterial genomic DNA extraction kit. The extracted genomic DNA was used as a template for PCR amplification of the 16S rDNA gene (universal primers 27F / 1492R) to obtain the 16S rDNA sequence of the strain. Comparison with the NCBI database revealed that the strain with the highest homology was *Pediococcus acidilactici* strain HDM5 (OR485709.1), with a similarity of 98.75%. A phylogenetic tree constructed based on their 16S rRNA gene sequences is shown below. Figure 1 As shown. Based on the experimental results, this strain was preliminarily identified as *Pediococcus acidilactici* and named *Pediococcus acidilactici* DY-1. Its 16S rDNA sequence is shown in SEQ ID NO: 1: (3) Preservation of strain: Pediococcus acidilactici DY-1 has been deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China. The strain is classified as Pediococcus acidilactici DY-1, with accession number CCTCC No: M2025908, and the deposit date is April 27, 2025.

[0028] Example 2 Preparation of Selenium-Enriched Postbiotics (SE-Postbiotics): (1) Incubate *Pediococcus lactis* DY-1 in MRS medium for 24-48 h at a temperature of 30-38 °C to obtain a *Pediococcus lactis* solution. Centrifuge the solution and resuspend it in sterile water to obtain a concentration of 1×10⁻⁶. 9 Seed liquid at CFU / ml.

[0029] (2) After cleaning the high-selenium plant-based fresh raw materials, air-dry them naturally in a clean environment at 20℃ for 4 hours, then crush them using a high-speed blender for 20 seconds. Immediately afterward, add 0.5% (w / w) salt and 0.5% (w / w) vitamin C and mix thoroughly. The high-selenium plant-based raw materials are fresh stems and leaves of Viola yedoensis, with a selenium content of 180 mg / kg and an organic selenium content of more than 80%.

[0030] (3) The seed solution was prepared to a final concentration of 1×10⁻⁶. 6 CFU / ml was inoculated into the high-selenium plant-based raw material, and then aseptically transferred to a sterile one-way breathable fermentation bag to remove air before anaerobic fermentation for 5 days at a fermentation temperature of 32℃.

[0031] (4) The fermentation product was treated at 121°C for 15 min, freeze-dried and pulverized to obtain organic selenium-enriched post-vitamin powder of Pycnococcus lactis.

[0032] The salt used in step (2) of this embodiment is commercially available ordinary table salt (mainly using the NaCl component in the table salt; other trace elements do not affect the results of this experiment).

[0033] Implementation Example 3: Acid Resistance Test of Pediococcus lactis DY-1 and Analysis of Postbiotic Product Components (1) Acid resistance test of Pediococcus acidilactici DY-1: The pH of the MRS culture medium was adjusted to 3.0, 4.0, 5.0, 6.8 with 1M HCl, and then autoclaved at 115℃ for 20 min for later use. Pediococcus acidilactici DY-1 was activated and inoculated into the above culture medium at an inoculum rate of 2%. The model strain of Pediococcus acidilactici CICC 25166 was used as a control. The absorbance at OD600nm was measured after incubation at 30℃ for 24 h. The results are as follows. Figure 2 As shown.

[0034] (2) Analysis of the composition of the post-genetic product: The post-genetic product rich in organic selenium (DY-1) prepared in Example 2, the post-genetic freeze-dried powder obtained by fermentation with the same raw materials and processing technology as in Example 2 using the model strain Pediococcus acidilactici CICC25166 (CICC-25166), and the freeze-dried raw material powder of Viola yedoensis plant culture medium without fermentation treatment (CK) were subjected to total selenium, selenium speciation and broad-targeted metabolomics assays, respectively.

[0035] Total selenium and selenium speciation were determined by weighing approximately 0.1 g of lyophilized powder and digesting it with a mixture of HNO3 and HClO4 (volume ratio 4:1), followed by reduction with 6 mol / L HCl. The selenium content was determined by atomic fluorescence spectrometry. The content of each selenium speciation was determined by HPLC-AFS. Table 1 shows the selenium content and speciation data of post-organic selenium enrichment selenium.

[0036] Table 1. Selenium content and speciation in selenium-rich post-selenium metageners

[0037] Depend on Figure 1 As shown in Table 1, *Pediococcus lactis* DY-1 exhibits good acid resistance and can grow normally at pH 4.0 compared to the model strain CICC-25166. Furthermore, fermentation of high-selenium plant-based raw material (violet leaf mint) promoted the release of selenocysteine, demonstrating its promising potential as a fermentation strain for high-selenium plant-based raw materials.

[0038] Broadly targeted metabolomics assays: 50 mg of the selenium-enriched post-biotic product prepared in Example 2 (DY-1), the post-biotic lyophilized powder obtained by fermentation with the same raw materials and process parameters as in Example 2 using the Pediococcus acidilactici CICC 25166 (CICC-25166), and the lyophilized raw material powder of Viola yedoensis plant culture medium without fermentation treatment (CK) were mixed with 1.2 mL of 70% methanol solution. The solution was vortexed for 30 s every 30 min for a total of 6 times. The mixture was centrifuged at 12000 rpm for 3 min at 4 °C, and the supernatant was filtered through a 0.22 µm microporous membrane for UPLC-MS / MS analysis. Figure 3 The figure shown is a graph illustrating the changes in the content of metabolites in two raw material powders. Figure 4 The graph shows the changes in the content of the top 10 metabolites in the two raw material powders.

[0039] Broad-target metabolomics data from each treatment group showed that, compared to the control (CK) and model strain CICC-25166, *Pediococcus lactis* DY-1 fermentation promoted the fermentation of macromolecules such as proteins, carbohydrates, and fats in *Corydalis yanhusuo*, with the contents of active metabolites such as amino acid derivatives, terpenes, lipids, phenolic acids, and flavonoids increasing several times or even exponentially. Figure 3 The increased abundance of organic post-biotics significantly enhances the edibility and nutritional value of the metabolites. Meanwhile, the graph shows the changes in the content of the 10 most abundant metabolites. Figure 4 The study showed that *Pediococcus lactis* DY-1 synthesized tryptophan, serotonin, 2-phenylethylamine, α-linolenic acid, and dipeptides—essentially important active substances proven to regulate gut microbiota, the human peripheral nervous system, and the gut microbiota-brain axis. These substances can effectively improve mood and cognitive function and have a protective effect against various diseases, including depression. This indicates that selenium-rich post-biotics have potential brain-gut axis regulatory effects.

[0040] Example 4: Establishment of a Cortisol-Induced Mouse Model of Depression and Intervention with Selenium-Rich Postbiotics (1) The experimental animals used were 4-week-old male mice. The mice were kept in a constant temperature (25℃) animal room with a 12-hour light / 12-hour dark cycle. There were 5 mice per cage in each group, and a total of 20 mice were set up in 4 groups.

[0041] (2) Mice were given Cortisol at a dose of 20 mg / kg (0.1 mL) via subcutaneous injection in the groin to induce depression. The control group was injected with 1% dimethyl sulfoxide saline. The drugs were administered randomly between 9:00 and 16:00 every day. Treatment group 1 was fed a regular diet. Treatment group 2 mice were fed with 0.2% of post-biotic freeze-dried powder obtained by fermentation with the same raw materials and processing technology as in Example 2 using the Pediococcus acidilactici CICC 25166. Treatment group 3 mice were fed with 0.2% of the organic selenium-enriched post-biotic freeze-dried powder prepared in Example 2. The treatment lasted for 3 weeks.

[0042] (3) Feces from mice in the three treatment groups were collected for 16S rDNA microbial diversity assay to examine the intestinal flora at the phylum level. The specific results are as follows: Figure 5 As shown in the figure, CK is the control group, and T1-T3 are treatment groups 1-3, respectively.

[0043] (3) Blood was collected using the ocular blood sampling method. After collection, the blood was left at room temperature for 2 hours, and then centrifuged at 3500 r / min for 15 min at 4℃. The upper serum was collected and stored at −80℃. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) inflammatory factors were measured using a kit.

[0044] (4) The mice were given sugar water adaptation training for 48 h. At the same time, the mice were given water and 1% sugar water. The positions of water and sugar water were interchanged every 12 h. After the sugar water adaptation training was completed, the mice were fasted and deprived of water for 22 h. Then, the amount of water and sugar water consumed by each mouse within 2 h was measured, and the sugar water preference was calculated.

[0045] Table 2 shows the differences in validation factor indicators and sucrose preference among mice in the control group and treatment groups 1-3.

[0046] Table 2. Differences in validation factor indices and sucrose preference among mice in the control group and treatment groups 1-3.

[0047] in, .

[0048] Sugar water preference is a classic behavioral test for assessing anhedonia in mice, commonly used in studies of depression, chronic stress, and neurodegenerative diseases. As shown in Table 2, compared to the control group, Cortisol injection significantly reduced sugar water preference in mice, indicating the successful establishment of a Cortisol-induced mouse depression model. Dietary supplementation with *Viola yedoensis* powder and selenium-enriched post-biotic extract both improved preference in mice. The selenium-enriched post-biotic treatment group almost restored sugar water preference to control levels, indicating that the selenium-enriched post-biotic fermented with *Pediococcus lactis* DY-1 significantly improved Cortisol-induced anhedonia symptoms. Consistent with widespread reports, mouse depression was accompanied by an increase in serum inflammatory factors. The Cortisol-induced group showed an increase of more than twofold in two key inflammatory factors, TNF-α and IL-6, while the selenium-enriched post-biotic supplementation group almost offset the pro-inflammatory effect of Cortisol, with inflammatory factor indicators similar to the control group (Table 2). Furthermore, the results of the mouse gut microbiota composition showed… Figure 5 Cortisol treatment led to gut microbiota dysbiosis in mice, with a significant decrease in the proportion of beneficial Firmicutes. Treatment with selenium-enriched prebiotics not only restored the gut microbiota but also effectively increased the proportion of Firmicutes.

[0049] The above conclusions indicate that selenium-rich prebiotics have brain-gut axis regulatory effects and the potential to prevent and treat depression, chronic stress, and neurodegeneration. This is mainly related to the selenium-containing active substances released from high-selenium plant-based raw materials fermented by Pediococcus lactis, as well as the synthesized tryptophan, serotonin, 2-phenylethylamine, α-linolenic acid, dipeptides, and other substances. Their mechanism of action in living organisms may be related to the metabolic mechanisms of regulating intestinal flora and inhibiting the production of inflammatory factors.

[0050] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of lactic acid cocci, scientifically named *Pediococcus acidilactici*, strain number DY-1, characterized in that... The preservation number of the lactic acid porphyria is CCTCC No: M2025908.

2. The *Pediococcus lactis* according to claim 1, characterized in that, The lactic acid cocci were isolated from naturally fermented kimchi made from wild rice.

3. The *Pediococcus lactis* according to claim 1, characterized in that, The 16S rDNA sequence of the *Pediococcus lactis* is shown in SEQ ID NO:

1.

4. A fermentation agent, characterized in that, The fermentation agent includes: Fermentation broth obtained by fermenting Pediococcus lactis as described in any one of claims 1-3; Alternatively, the fermentation broth can be spray-dried to obtain a dry powder inoculum.

5. An application of *Pediococcus lactis* as described in any one of claims 1-3, characterized in that, The lactic acid porphyria was applied to the fermentation of high-selenium cruciferous vegetables.

6. A selenium-rich postbiotic product with brain-gut axis regulating effects, characterized in that, It is prepared by fermentation with *Pediococcus lactis* as described in any one of claims 1-3.

7. A method for preparing an organic selenium-rich postbiotic product with brain-gut axis regulating effects as described in claim 6, characterized in that, Includes the following steps: (1) The lactic acid cocci were cultured in MRS medium to obtain a lactic acid cocci solution, and the solution was resuspended to obtain a seed solution; (2) After crushing the high-selenium plant-based fresh raw material, add 0.2wt%-0.5wt% salt and 0.2wt%-0.5wt% vitamin C and mix thoroughly to obtain the high-selenium plant-based raw material; (3) The seed solution was prepared to a final concentration of 1×10⁻⁶. 6 -1×10 7 CFU / g is inoculated into high-selenium plant-based raw materials for anaerobic fermentation; (4) Sterilize, dry and pulverize the fermentation product obtained in step (3) to obtain organic selenium-rich post-biotic powder of Pyrococcus lactis; (5) Add organic selenium-rich postbiotic powder of Pycnococcus lactis to a carrier or excipient to produce a postbiotic product with brain-gut axis regulation effect.

8. The preparation method according to claim 7, characterized in that, In step (1), the culture time is 24-48h and the culture temperature is 30-38℃.

9. The preparation method according to claim 7, characterized in that, In step (2), the high-selenium plant-based fresh raw material is one or more of high-selenium cruciferous vegetables, with a selenium content of 50-2000 mg / kg and an organic selenium content of more than 80%; in step (3), the fermentation time is 1-14 days and the fermentation temperature is 25-38℃.

10. The preparation method according to claim 7, characterized in that, In step (5), the product type is food, medicine or health product, and the product dosage form is powder, granules, capsules, tablets, pills or oral liquid.

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