Pediococcus pentosaceus metagen and application thereof in resisting oxidative inflammation and improving intestinal flora

Abstract

The invention relates to the technical field of microbial preparations, and discloses a pediococcus pentosaceus metagen and application of the pediococcus pentosaceus metagen in resisting oxidative inflammation and improving intestinal flora, and the pediococcus pentosaceus metagen is a preparation with a substance obtained by inactivating pediococcus pentosaceus CQFP202447 as an active ingredient. The preservation number of the pediococcus pentosaceus CQFP202447 is CGMCC (China General Microbiological Culture Collection Center) NO.29615. The pediococcus The pediococcus pentosaceus metagen disclosed by the invention has a remarkable effect in the aspects of resisting oxidative inflammation and improving intestinal flora. The unique biological activity of the pediococcus pentosaceus CQFP202447 not only comes from the excellent characteristics of the pediococcus pentosaceus CQFP202447, but also benefits from the special conversion after high-temperature inactivation treatment, so that the active ingredients of the pediococcus pentosaceus CQFP202447 are more stable and are easily absorbed and utilized by a human body.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial preparations, and in particular to a Pediococcus pentosaceus postbiotic and its application in resisting oxidative inflammation and improving intestinal flora. Background Art

[0002] With the accelerated pace of modern life and changes in dietary habits, people are facing an increasing number of health issues, two of which are oxidative stress and intestinal flora imbalance. Oxidative stress is caused by an imbalance between oxidation and antioxidant activity in the body, leading to the accumulation of reactive oxygen species and free radicals in the body or cells, which in turn triggers a series of damaging reactions. Intestinal flora imbalance is closely related to various chronic diseases such as obesity, diabetes, and cardiovascular disease. Therefore, finding effective methods to combat oxidative inflammation and improve intestinal flora has become a hot topic of research.

[0003] In recent years, probiotics and their postbiotics have garnered significant attention for their health benefits. Pediococcus pentosaceus, a common probiotic, has been shown to possess diverse biological activities, including antioxidant, anti-inflammatory, and gut microbiome regulation.

[0004] However, there are relatively few studies on postbiotics from Pediococcus pentosaceus, and its application potential in anti-oxidative inflammation and improving intestinal flora needs to be explored. Summary of the Invention

[0005] In view of this, the present invention proposes a Pediococcus pentosaceus postbiotic and its application in anti-oxidative inflammation and improving intestinal flora, aiming to explore its specific application in anti-oxidative inflammation and improving intestinal flora through in-depth research on its biological activity and mechanism of action.

[0006] The present invention provides a Pediococcus pentosaceus postbiotic, which is a preparation with a substance obtained by inactivating Pediococcus pentosaceus CQFP202447 as an active ingredient;

[0007] The preservation number of the Pediococcus pentosaceus CQFP202447 is CGMCC NO.29615.

[0008] The present invention also provides an application of the Pediococcus pentosaceus postbiotic in at least one of the following:

[0009] A1) Application in the preparation of anti-oxidative and anti-inflammatory products;

[0010] A2) Application in improving sports endurance;

[0011] A3) Application in the preparation of products for improving exercise endurance and inflammation;

[0012] A4) Application in changing intestinal flora;

[0013] A5) Application in the preparation of products for altering intestinal flora inflammation;

[0014] A6) Use in the preparation of products for preventing and / or treating intestinal barrier damage and inflammation;

[0015] A7) Use in the preparation of products for preventing and / or treating brain injury and inflammation;

[0016] A8) Use in the preparation of a product for preventing and / or treating inflammation of brain cell nucleus pyknosis.

[0017] The present invention also provides a product containing the above-mentioned Pediococcus pentosaceus postbiotic.

[0018] Preferably, the product is a liquid bacterial agent or a solid bacterial agent.

[0019] The present invention also provides a method for preparing a Pediococcus pentosaceus postbiotic, comprising:

[0020] The Pediococcus pentosaceus postbiotic is obtained by inactivating Pediococcus pentosaceus CQFP202447 at high temperature;

[0021] The preservation number of the Pediococcus pentosaceus CQFP202447 is CGMCC NO.29615.

[0022] The present invention also provides a method for preparing a Pediococcus pentosaceus postbiotic, comprising:

[0023] Fermenting polysaccharides by Pediococcus pentosaceus CQFP202447 to obtain the Pediococcus pentosaceus postbiotics;

[0024] The preservation number of the Pediococcus pentosaceus CQFP202447 is CGMCC NO.29615.

[0025] Preferably, the polysaccharide is seaweed polysaccharide.

[0026] Preferably, the seaweed polysaccharide is blood coral polysaccharide

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The Pediococcus pentosaceus postbiotics of the present invention have significant effects in resisting oxidative inflammation and improving intestinal flora. Its unique biological activity is derived not only from the excellent properties of Pediococcus pentosaceus CQFP202447 itself, but also from the special transformation after high-temperature inactivation treatment, making its active ingredients more stable and easily absorbed and utilized by the human body.

[0029] In terms of anti-oxidative inflammation, the Pediococcus pentosaceus postbiotic of the present invention can effectively scavenge free radicals in the body, reduce oxidative stress, and thus alleviate the degree of inflammation. In addition, it can also promote the function of the immune system and enhance the body's resistance to inflammation, thereby achieving the purpose of preventing and treating inflammatory diseases.

[0030] In terms of improving intestinal flora, the Pediococcus pentosaceus postbiotics of the present invention can regulate the intestinal microecological balance, promote the proliferation of beneficial bacteria, and inhibit the growth of harmful bacteria. Simultaneously, it can also improve the intestinal mucosal barrier function, enhance the intestinal resistance to harmful substances, and reduce the occurrence of intestinal diseases. Furthermore, the Pediococcus pentosaceus postbiotics of the present invention can also promote the absorption and utilization of nutrients, improving the body's nutritional level.

[0031] The product of the present invention has various forms and can be used as a liquid inoculant for direct drinking or as a solid inoculant for adding to food or medicine. This variety of product forms is not only convenient for consumers but also can meet the needs of different groups of people.

[0032] The present invention utilizes a high-temperature inactivation method during the preparation of Pediococcus pentosaceus postbiotics. This method is simple, cost-effective, and capable of maintaining the stability of the active ingredients of Pediococcus pentosaceus CQFP202447. Furthermore, the present invention improves the purity and activity of the Pediococcus pentosaceus postbiotics by optimizing the preparation process, resulting in improved application effects.

[0033] In summary, the Pediococcus pentosaceus postbiotics of the present invention have significant effects in anti-oxidative inflammation and improving intestinal flora, and the product form is flexible and diverse, and the preparation process is simple and feasible. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Various other advantages and benefits will become apparent to those skilled in the art upon reading the detailed description of the preferred embodiment below. The accompanying drawings are for illustration purposes only and are not to be considered as limiting the present invention. The same reference symbols are used throughout the drawings to represent the same components. In the drawings:

[0035] Figure 1 This is the result of the change of weight-bearing swimming time of mice;

[0036] Figure 2 This is the result of the change in running time of mice;

[0037] Figure 3 The results are the pathological changes of mouse brain tissue;

[0038] Figure 4 Results of serum oxidation and inflammatory indicators in mice;

[0039] Figure 5These are the results of oxidation and inflammation indicators in mouse brain tissue;

[0040] Figure 6 This is the original data result of the mouse intestinal flora change experiment;

[0041] Figure 7 This is the α-diversity analysis result of the mouse intestinal flora change experiment;

[0042] Figure 8 This is the β-diversity analysis result of the mouse intestinal flora change experiment;

[0043] Figure 9 This is the community composition analysis result of the mouse intestinal flora change experiment;

[0044] Figure 10 These are the results of species difference analysis of the mouse intestinal flora change experiment. DETAILED DESCRIPTION

[0045] Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art. It should be noted that, unless there is a conflict, the embodiments of the present disclosure and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with the embodiments.

[0046] All experimental data (including the bacterial colony analysis section) were statistically analyzed using SPSS 20.0 software (SPSS Inc, Chicago, IL, USA) with one-way analysis of variance and Tukey's multiple comparison method; p < 0.05 was considered statistically significant. Graphs were generated using GraphPad Prism 9.3. Data are presented as mean ± standard deviation (SD) and were repeated three times.

[0047] Example 1

[0048] 1. Material Preparation

[0049] A new strain of Pediococcus pentosaceus was isolated from pickle juice in the Zhendong area of ​​Kaixian County, Chongqing, China, and named Pediococcus pentosaceus CQFP202447. It was registered and deposited in the China Center for General Culture Collection of Microorganisms on January 12, 2024, with the accession number CGMCC NO.29615.

[0050] Recovery of strains: Take out the frozen tube of the experimental strain from the -80°C environment, thaw it, and then inoculate (200 μL) into MRS liquid culture medium (total volume 8 mL). Resuscitation culture was carried out at a constant temperature of 37°C for 24 hours. Subsequently, mix evenly, take 3% inoculum, transfer it to another MRS liquid culture medium, and continue to culture for 18 hours. Then, collect the bottom Pentosaceae CQFP202447 by centrifugation (conditions are 4°C, 4000rpm, time 10 minutes). After collection, resuspend with physiological saline until the cell concentration is adjusted to 1.0×10 9 The bacterial stock solution was obtained at the order of CFU / kg·bw.

[0051] Preparation of blood coral polysaccharide: Blood coral seaweed powder (obtained after freeze-drying and crushing of blood coral seaweed) was diluted with double distilled water at a ratio of 1:20 (W / V). Subsequently, it was extracted three times by means of a water bath (set temperature at 95°C for 40 minutes), and the filtrates were combined by vacuum filtration. To ensure the purity of the solution, it was filtered to remove impurities and evaporated and concentrated on a rotary evaporator until the volume was approximately 100 mL. Then, anhydrous ethanol was added to the solution until its final concentration reached 80%, and the solution was allowed to stand at 4°C overnight. Afterwards, the sample was centrifuged (at a speed of 4000 r / min for 10 minutes) and the supernatant was discarded. The precipitate was repeatedly dissolved with hot water (100 mL) and then precipitated with 80% ethanol for 12 hours. After precipitation, centrifugation was performed again (conditions were 4°C, 4000 r / min, and continued for 10 minutes), and the obtained product was freeze-dried (for 48 hours) to finally obtain refined blood coral polysaccharide, which was stored at 4°C for later use.

[0052] Preparation of seaweed polysaccharides fermented by Pediococcus pentosaceus CQFP202447: The refined blood coral polysaccharide was diluted into a conical flask with boiling water at a ratio of 1:10 (mass / volume), and then placed in a high-pressure sterilizer for sterilization under strictly controlled conditions of 121°C for 15 minutes. The sterilized polysaccharide liquid was cooled in an ultra-clean workbench and divided into pre-sterilized 10mL centrifuge tubes. 5mL of polysaccharide liquid was injected into each centrifuge tube and inoculated with 50uL of activated bacterial stock solution. Subsequently, the centrifuge tube was sealed and placed in a constant temperature incubator for overnight culture at 37°C in preparation for the mouse gavage experiment the next day.

[0053] The remaining experimental materials and reagents are shown in Table 1, and the instruments and equipment are shown in Table 2.

[0054] Table 1

[0055]

[0056]

[0057] Table 2

[0058] name Model / Article Number factory Youpu series ultrapure water purifier UPH-II-20T Sichuan Youpu Ultrapure Technology Co., Ltd. Multifunctional microplate reader VarioskanLUX Thermofisher Biotechnology Co., Ltd. of the United States water bath HHW21·600AII Tianjin Test Instrument Co., Ltd. Refrigerated centrifuge Centrifuge5418R Eppendorf, Germany centrifuge BY-80C Beijing Baiyang Medical Equipment Co., Ltd. drying oven A0-80A Shidukai Instrument Equipment (Shanghai) Co., Ltd. electronic balance BSA124S Sartorius Scientific Instruments (Beijing) Co., Ltd. Mouse experimental treadmill ZH-PT Anhui Zhenghua Biological Instrument Equipment Co., Ltd. electron microscope BX43 Olympus Corporation of Japan Biological sample homogenizer Bioprep-24 Hangzhou Aosheng Instrument Co., Ltd. Automatic PCR amplification instrument SteponePlus Thermofisher Biotechnology Co., Ltd. of the United States

[0059] 2. Experimental Methods

[0060] 1. Establishment of experimental animal models

[0061] For this experiment, 30 SPF-grade male C57BL / 6J mice, all 7-8 weeks old, were purchased from Hunan Slake Jingda Laboratory Animal Co., Ltd. Animal production license number: SCXK(Xiang)2019-0004. The mice were housed in a standard laboratory animal room, maintaining a constant room temperature of 23±2°C and a relative humidity of 55±5%. They were housed under a strict 12-hour light cycle and provided with ample food and water to acclimate to the experimental environment for one week.

[0062] The experimental mice were divided into six experimental groups, as follows:

[0063] (1) Normal group (NRM): mice in this group did not receive any therapeutic intervention;

[0064] (2) Model group (ABX): intraperitoneal injection of mixed antibiotic preparations and gavage with normal saline;

[0065] (3) Caffeic acid group (ABX+CA): caffeic acid (10 mg / kg) was administered by gavage after injection of the mixed antibiotic preparation;

[0066] (4) Pediococcus pentosaceus CQFP202447 group (ABX+PP): After the injection of mixed antibiotic preparation, Pediococcus pentosaceus CQFP202447 (10 9 CFU / kg·bw);

[0067] (5) High-temperature inactivated Pediococcus pentosaceus CQFP202447 group (ABX+DPP): After the injection of mixed antibiotic preparation, Pediococcus pentosaceus CQFP202447 (10 9 CFU / kg·bw);

[0068] (6) Pediococcus pentosaceus CQFP202447 fermented seaweed polysaccharide group (ABX+PP-PS): After intraperitoneal injection of mixed antibiotic preparation, seaweed polysaccharide fermented by Pediococcus pentosaceus CQFP202447 was given by gavage.

[0069] During the experiment, injections / gavages were performed every 12 hours, ensuring that the dose for gavage and intraperitoneal injection was accurate to 0.1 mL / 10 g bw. The experimental period lasted two weeks. The antibiotic mixture consisted of neomycin 5 mg / mL, vancomycin 25 mg / mL, amphotericin B 0.1 mg / mL, ampicillin 10 mg / mL, metronidazole 5 mg / mL, and lipopolysaccharide 1.5 μg / mL.

[0070] 2. Mouse exhaustion experiment

[0071] 2.1 Weighted swimming test

[0072] Two weeks later, after the 14th injection / oral gavage, a weighted swimming test was performed. For the weighted swimming test, a 60cm x 50cm trough with a depth of approximately 35cm was used, and the water temperature was maintained at 23±2°C. Thirty minutes after administration, mice in the normal, model, caffeic acid, Pediococcus pentosaceus CQFP202447, heat-killed Pediococcus pentosaceus CQFP202447, and Pediococcus pentosaceus CQFP202447 fermented seaweed polysaccharide groups were treated. Specifically, a lead wire equivalent to 2% of the mouse's body weight was tied to its tail and clearly marked. The mice were then placed in the water one by one in sequence for the weighted swimming test. A timer was used to accurately record the time. Exhaustion was determined when the mouse's head remained submerged in the water for more than 8 seconds and was unable to resurface. Following the experiment, the mice were carefully wiped dry with a dry towel.

[0073] The results of the mouse weighted swimming experiment (changes in mouse weighted swimming time) are as follows Figure 1 As shown in the figure, the values ​​shown in ac are mean ± standard deviation. According to Tukey's test, the mean values ​​with different letters in the same figure are significantly different (P < 0.05). Excessive use of antibiotics will seriously affect the endurance performance of mice, while the introduction of probiotics can effectively improve this situation. Figure 1 As shown, compared with NRM, ABX mice had significantly reduced weight-bearing swimming time (P < 0.05), clearly demonstrating the negative impact of antibiotic abuse on mouse endurance. However, after prior oral administration of probiotics such as CA, PP, DPP, and PP-PS, the weight-bearing swimming time of mice significantly improved, reaching a level between NRM and ABX. While the differences between the PP and DPP groups were not significant, the PP-PS group achieved a superior effect compared to the former two and was similar to that of the CA group, demonstrating the significant efficacy of specific probiotic combinations in improving mouse endurance.

[0074] 2.2 Running experiment

[0075] The endurance of mice was further investigated through running experiments. This experiment used a ZH-PT mouse treadmill, which has a length, width, and height of 565mm, 630mm, and 310mm, respectively. It has 8 single runways, each 55mm wide. In the experiment, the following parameters were set: the treadmill inclination was fixed at 5°, the stimulation current was 0.6mA, and the acceleration was 2m / s. 2 The mice were trained at a speed of 20 m / min for 30 minutes, with a one-minute rest period between each exercise. Four days before the formal experiment, the mice underwent adaptive training, conducted once daily for five minutes each. Finally, on the final day of the experimental animal model establishment trial, formal testing was conducted, and the experimental results were recorded in detail.

[0076] like Figure 2 As shown, the changes in running time and swimming performance of mice showed a high degree of synchronization. Compared with the NRM control group, the running time of ABX mice showed a significant decrease (P < 0.05). After undergoing different pre-gavage intervention measures of CA, PP, DPP, and PP-PS, the mice's weighted running time was significantly improved, with the intervention effects of the PP and DPP groups being relatively similar. Notably, the running time of mice in the PP-PS group was significantly longer than that of the PP and DPP groups, and its effect was similar to that of the CA group.

[0077] 3. Tissue processing and testing

[0078] 3.1 Tissue processing

[0079] After the final exercise test, feces were collected, mice were weighed, and blood was collected from the eyeballs under ether anesthesia. After cervical dissection, the brain, liver, and cecum were dissected and collected. The liver was dissected and weighed. A portion of the brain tissue was immersed in tissue fixative for sectioning. The remaining tissue was stored at -80°C for subsequent experiments.

[0080] 3.2 Pathological observation

[0081] The brain tissue (0.5 cm 2 The tissues were fixed in a tissue fixative for more than 48 hours, then embedded in paraffin and sectioned (4 μm). Hematoxylin and eosin staining was used to observe pathological changes under a BX43 microscope (Olympus, Tokyo, Japan).

[0082] Figure 3 The HE staining results of mouse brain tissue are shown in Figure 2. Figure 3As shown, the morphology of neurons in the cerebral cortex of mice in the NRM group was normal, with neatly arranged and uniform cells and prominent, larger nuclei. In contrast, the number of cells in the brain tissue of mice in the ABX group was significantly reduced, with darker nuclei and significant pyknosis. Some cells showed punctate necrosis and disorganized arrangement. Cortical neurons atrophied and shrank, indicating severe cell damage. Pretreatment with CA, PP, DPP, and PP-PS alleviated the pyknosis of the nuclei in the mouse brain tissue, increased the number of cells, and restored some neurons to normal morphology, but a few neurons still showed signs of atrophy. PP-PS pretreatment had the most significant effect, followed by PP pretreatment, which was similar to the effect of caffeic acid pretreatment.

[0083] 3.3 Changes in mouse body weight, liver weight, and liver index

[0084] Mouse body weight, liver weight and liver index are important indicators for evaluating mouse health status and drug effects.

[0085] As shown in Table 3, when comparing the body weights of mice in the NRM and ABX groups, it was found that after 14 days of intervention, the body weight of mice in the ABX group showed a downward trend, but this did not reach a statistically significant level (P>0.05). Similarly, the body weights of mice treated with various interventions, including CA, PP, DPP, and PP-PS, did not show significant changes.

[0086] On the other hand, we observed an increase in liver weight in the combined antibiotic-induced model group, but this increase also did not reach statistical significance (P>0.05). Increased liver weight often reflects a state of liver congestion and acute inflammation. Notably, after treatment with CA, PP, DPP, and PP-PS, the liver weights of mice decreased, indicating that these treatments are effective in alleviating liver congestion and acute inflammation. These phenomena were also reflected in the liver index, which, although fluctuating between groups, did not show significant differences.

[0087] Table 3

[0088] Grouping Weight (g) Liver weight (g) Liver index NRM 20.92±2.21a 0.80±0.09ab 3.87±0.12a ABX 18.50±1.13a 0.92±0.11a 4.72±0.61a ABX+CA 18.84±0.77a 0.74±0.05ab 3.94±0.27a ABX+PP 19.62±1.09a 0.70±0.09b 3.59±0.65a ABXs+DPP 19.25±1.18a 0.87±0.02ab 4.55±0.16a ABXs+PP-PS 18.84±1.08a 0.88±0.07ab 4.73±0.50a

[0089] 3.4 Detection of oxidative and inflammatory indicators in mouse serum and brain tissue

[0090] 3.4.1 Detection process

[0091] Serum preparation: Mouse blood was placed at 4°C for 2 hours, centrifuged at 3000 rpm and 4°C for 15 minutes, and then the upper serum was obtained and frozen at -80°C.

[0092] Preparation of brain tissue homogenate: After adding 0.1 g of mouse brain tissue to 0.9 mL of normal saline, the tissue was homogenized (Bioprep-24; Hangzhou Aosheng Instrument Co., Ltd., Hangzhou, China) at a speed of 6 m / s for three times for 30 seconds.

[0093] Index detection: The levels of malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) in mouse serum and brain homogenate were measured using kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China); the levels of cytokines tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and -10 in serum and brain tissue were measured using enzyme-linked immunosorbent assay kits (Shanghai ELISA Biotechnology Co., Ltd., Shanghai, China).

[0094] 3.4.2 Detection results of mouse serum oxidation and inflammatory indicators

[0095] Mouse serum oxidation and inflammatory indicators such as Figure 4 shown.

[0096] MDA, GSH, and SOD can reflect the body's oxidative damage and antioxidant capacity. Figure 4 Data analysis of A-4C revealed that compared with the NRM group, serum MDA levels in the ABX group were significantly increased, while GSH and CuZn-SOD levels were significantly decreased. This difference was statistically significant (P < 0.05). Further comparison revealed that after pretreatment with CA, PP, DPP, and PP-PS, serum MDA levels in the ABX group were significantly decreased, while GSH and CuZn-SOD levels were significantly increased. This difference was also statistically significant (P < 0.05). PP-PS was most effective in reducing MDA levels, followed by PP, DPP, and CA. Regarding increasing CuZn-SOD activity, PP demonstrated the best effect, similar to that of the NRM group, followed by PP-PS, DPP, and CA, which were similar in effect. Regarding the increase in GSH levels, there were no significant differences among the CA, PP, DPP, and PP-PS groups, all of which were similar to those of the NRM group.

[0097] Cytokines such as IL-6, IL-10, and TNF-α are important indicators reflecting the degree of inflammation in the body. Figure 4As shown in D-4F, compared with the NRM group, the expression of pro-inflammatory factors TNF-α and IL-6 in the ABX group was significantly increased, while the expression of anti-inflammatory factor IL-10 was significantly decreased (P < 0.05). After pretreatment with CA, PP, DPP, and PP-PS, the expression of TNF-α and IL-6 was downregulated, while the expression of IL-10 was upregulated. Specifically, DPP had the most significant effect in downregulating IL-6 expression, followed by PP, PP-PS, and CA; in terms of downregulating TNF-α expression, PP-PS performed best, followed by DPP and CA, which was similar to the NRM group, and the effect of PP was relatively weak. As for the upregulation effect of IL-10, no significant difference was shown among the four groups of DPP, PP, PP-PS, and CA.

[0098] 3.4.3 Detection results of oxidative and inflammatory indicators in mouse brain tissue

[0099] like Figure 5 As shown in Figure A-5C, compared with the NRM group, the ABX group showed a significant increase in brain tissue MDA content, while GSH and CuZn-SOD levels decreased significantly (P < 0.05). Pretreatment with CA, PP, DPP, and PP-PS resulted in a downward trend in brain MDA content, while GSH and CuZn-SOD levels increased. PP-PS and DPP were similarly effective in reducing MDA content, both outperforming PP and CA, and falling between NRM and ABX. PP-PS was most effective in increasing T-SOD activity, followed by PP, and DPP in third place. CA even surpassed NRM in terms of its effect. PP was slightly more effective than CA, DPP, and PP-PS in increasing GSH content, with the latter three showing similar effects.

[0100] like Figure 5 As shown in Figures D-5F, compared with the NRM group, the expression of the proinflammatory cytokines TNF-α and IL-6 was significantly increased in the ABX group, while the expression of the anti-inflammatory cytokine IL-10 was significantly decreased (P < 0.05). After pretreatment with CA, PP, DPP, and PP-PS, the expression of TNF-α and IL-6 was downregulated, while the expression of IL-10 was upregulated. In terms of reducing IL-6 expression, CA was slightly more effective than PP, DPP, and PP-PS, with the latter three having similar effects. In terms of reducing TNF-α expression, there was no significant difference between PP-PS, PP, CA, and NRM, with DPP having the most significant effect. In terms of increasing IL-10 expression, PP-PS and CA had similar effects, both being superior to PP and DPP, and falling between NRM and ABX.

[0101] 3.5 Real-time fluorescence quantitative PCR analysis

[0102] 3.5.1 Analysis process

[0103] After homogenization of brain and cecal tissues, total RNA was extracted using TRIzol reagent (Beijing Solebao Technology Co., Ltd., Beijing, China) and diluted to a concentration of 1 μg / μL. Total RNA was reverse transcribed into cDNA (Thermo Fisher Biotechnology Co., Ltd., USA). Then, 1.0 μL of cDNA, 2.0 μL of forward and reverse primers (10 μM), and 10.0 μL of premix (Thermo Fisher Biotechnology Co., Ltd., USA) were mixed and reacted for 40 cycles under the following conditions: 95°C for 3 minutes, 60°C for 30 seconds, and 95°C for 1 minute. β-actin was used as an internal reference gene, and data were calculated using the 2-ΔΔCt method. Primer sequences are shown in Table 4.

[0104] Table 4

[0105] Primer ForwardPrimer(3'to5') ReversePrimer(3'to5') ZO-1 5′-GCTTTAGCGAACAGAAGGAGC-3′ 5′-TTCATTTTTCCGAGACTTCACCA-3′ Occludin-1 5′-TTGAAAGTCCACCTCCTTACAGA-3′ 5′-CCGGATAAAAAGAGTACGCTGG-3′ Claudin-1 5′-GCCTTGATGGTAATTGGCATCC-3′ 5′-GGCCACTAATGTCGCCAGAC-3′ BDNF 5′-TTACCTGGATGCCGCAAACAT-3′ 5′-TGACCCACTCGCTAATACTGTC-3′ CREB 5′-CAGGGGGTGCCAAGGATTGAAG-3′ 5′-ACTGCTAGTTTGGTAAATGGGG-3′ ERK1 5′-TCCGCCATGAGAATGTTATAGGC-3′ 5′-GGTGGTGTTGATAAGCAGATTGG-3′ AKT 5′-AGAAGAGACGATGGACTTCCG-3′ 5′-TCAAACTCGTTCATGGTCACAC-3′ β-actin 5′-GGCTGTATTCCCCTCCATCG-3′ 5′-CCAGTTGGTAACAATGCCATGT-3′

[0106] 3.5.2 Expression of AKT, BDNF, CREB, and ERK1 genes in mouse brain tissue

[0107] AKT, CREB, BDNF, ERK1, and IL-6 play important roles in brain nerve injury. The results of real-time fluorescence quantitative PCR are shown in Table 5. Compared with the NRM group, the ABX group showed a significant decrease in AKT, CREB, BDNF, and ERK1 gene expression. However, after CA, PP, DPP, and PP-PS intervention, the expression levels of these genes recovered to a statistically significant level (P < 0.05).

[0108] In terms of BDNF gene upregulation, PP-PS was more effective than PP and DPP, and its effect was close to that of CA. In terms of CREB gene upregulation, PP was the most effective, followed by PP-PS, while DPP and CA had similar effects.

[0109] Regarding AKT gene upregulation, PP-PS was significantly more effective than PP, DPP, and CA, while the latter three showed no significant differences in their effects. Regarding ERK1 gene upregulation, PP was the most effective, similar to CA, while PP-PS and DPP were relatively weaker, with no significant differences in their effects.

[0110] Table 5

[0111]

[0112]

[0113] 3.5.3 ZO-1, occludin-1, and claudin-1 gene expression in mouse cecum

[0114] ZO-1, occludin-1, and claudin-1, as classic tight junction proteins, can reflect the mechanical barrier function of intestinal epithelial cells and the degree of damage to the tight junction structure. As shown in Table 6, compared with the NRM group, the ABX group showed a significant decrease in the expression of ZO-1, occludin-1, and claudin-1 genes (P < 0.05). After multiple interventions such as CA, PP, DPP, and PP-PS, the expression of these genes showed varying degrees of improvement. Specifically, in the expression of ZO-1, the effect of PP-PS was slightly higher than that of PP, and both were significantly higher than CA and DPP. For the expression of occludin-1, PP-PS and PP showed the best effects, followed by DPP, and CA showed the weakest performance. In the expression of claudin-1, PP-PS showed the best effect, followed by PP, and the effect of DPP was relatively weak, similar to that of CA.

[0115] Table 6

[0116] Grouping ZO-1 occludin-1 claudin-1 NRM 1.00±0.09a 1.00±0.06b 1.01±0.18a ABX 0.08±0.01c 0.52±0.15c 0.43±0.12c ABX+CA 0.17±0.01c 0.83±0.15bc 0.52±0.10bc ABX+PP 0.49±0.09b 1.54±0.21a 0.61±0.16bc ABX+DPP 0.16±0.07c 1.11±0.24b 0.52±0.17bc ABX+PP-PS 0.60±0.07b 1.49±0.18a 0.76±0.16ab

[0117] 3.6 Microbial flora analysis

[0118] 3.6.1 Main reagents and instruments

[0119] PF Mag-Bind Stool DNA Extraction Kit (Omega, USA); FastPfu Polymerase (Beijing Quanshijin Biotechnology Co., Ltd.); ABI 9700 PCR instrument (ABI, USA); DYY-6C electrophoresis instrument (Beijing Liuyi Biotechnology Co., Ltd.).

[0120] High-throughput sequencing was performed by Shanghai Meiji Biotechnology Co., Ltd.

[0121] 3.6.2 Methods

[0122] 3.6.2.1 Sample DNA extraction

[0123] The total genomic DNA of the microbial community was extracted according to the instructions of the PF Mag-Bind Stool DNA Kit (Omega Bio-tek, Georgia, US). The integrity of the extracted genomic DNA was detected by 1% agarose gel electrophoresis, and the DNA concentration and purity were determined using NanoDrop2000 (Thermo Scientific, USA).

[0124] 3.6.2.2 PCR amplification and sequencing library construction

[0125] The DNA extracted above was used as a template to amplify the V3-V4 variable region of the 16S rRNA gene using the upstream primer 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and the downstream primer 806R (5'-GGACTACHVGGGTWTCTAAT-3') carrying the barcode sequence. The amplification procedure was as follows: 95°C pre-denaturation for 3 min, 27 cycles (95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s), followed by 72°C stable extension for 10 min, and finally stored at 4°C (PCR instrument: ABI 9700). The PCR reaction system consisted of 4 μL of 5× TransStart FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of upstream primer (5 μM), 0.8 μL of downstream primer (5 μM), 0.4 μL of TransStart FastPfu DNA polymerase, and 10 ng of template DNA, made up to 20 μL. Three replicates were performed for each sample. PCR products from the same sample were pooled and recovered on a 2% agarose gel and purified. Band sizes were determined by electrophoresis on a 2% agarose gel, and the recovered products were quantified using a Quantus™ Fluorometer (Promega, USA).

[0126] use The purified PCR products were subjected to library construction using the Rapid DNA-Seq Kit (Bioo Scientific, Austin, Texas, USA): (1) adapter ligation; (2) removal of adapter self-ligated fragments using magnetic bead screening; (3) library template enrichment using PCR amplification; and (4) magnetic bead recovery of the PCR products to obtain the final library. Sequencing was performed using the Illumina PE300 platform (Shanghai Meiji Biopharmaceutical Technology Co., Ltd.). Raw data were uploaded to the NCBI SRA database.

[0127] 3.6.2.3 High-throughput sequencing data analysis

[0128] Fastp (https: / / github.com / OpenGene / fastp, version 0.19.6) software was used for quality control of the double-end raw sequencing sequences, and FLASH (http: / / www.cbcb.umd.edu / software / flash, version 1.2.11) software was used for splicing: (1) The bases with a quality value of less than 20 at the end of the reads were filtered, and a 50-bp window was set. If the average quality value in the window was less than 20, the bases at the end of the window were truncated, and reads less than 50 bp after quality control were filtered, and reads containing N bases were removed; (2) Based on the overlap relationship between PE reads, paired reads were spliced ​​(merged) into a sequence with a minimum overlap length of 10 bp; (3) The maximum mismatch ratio allowed in the overlap region of the spliced ​​sequence was 0.2, and non-compliant sequences were filtered out; (4) Samples were distinguished based on the barcodes and primers at the beginning and end of the sequence, and the sequence direction was adjusted. The number of mismatches allowed for the barcode was 0, and the maximum number of primer mismatches was 2.

[0129] Using default parameters, the DADA2 plugin (or Deblur plugin) in the Qiime2 pipeline was used to de-noise the optimized sequences after quality control assembly. Sequences de-noised with DADA2 (or Deblur) are often referred to as ASVs (amplicon sequence variants). Sequences annotated to chloroplasts and mitochondria were removed from all samples (recommended if samples contain chloroplast or mitochondrial contamination). To minimize the impact of sequencing depth on subsequent alpha and beta diversity data analysis, the number of sequences in all samples was truncated to 20,000 (recommended). After truncating, the average goodness of coverage (Good's coverage) for each sample still reached 99.09%. ASVs were classified according to species taxonomy using the Naive Bayes (or Vsearch, or Blast) classifier in Qiime2 based on the Sliva 16S rRNA gene database (v138). 16S rRNA function prediction was performed using PICRUSt2 (version 2.2.0) software.

[0130] 3.6.2.4 Statistical analysis

[0131] All data analyses were performed on the MajorBio cloud platform (https: / / cloud.majorbio.com). Alpha diversity indices such as Chao 1 and the Shannon index were calculated using Mothur software (http: / / www.mothur.org / wiki / Calculators, version 1.30.2). Intergroup differences in alpha diversity were analyzed using the Wilcoxon rank sum test. Principal coordinate analysis (PCoA) using the Bray-Curtis distance algorithm was used to examine the similarity of microbial community structure between samples, and the nonparametric PERMANOVA test was used to analyze whether differences in microbial community structure between sample groups were significant. Linear discriminant analysis (LEfSe) effect size analysis (http: / / huttenhower.sph.harvard.edu / LEfSe) (LDA > 2, P < 0.05) was used to identify bacterial taxa with significant differences in abundance at the phylum to genus level between groups. Distance-based redundancy analysis (db-RDA) was used to investigate the impact of clinical indicators on gut bacterial community structure. Linear regression analysis was used to assess the impact of the main clinical indicators identified in the db-RDA analysis on the microbial alpha diversity index. Species were selected for correlation network analysis based on Spearman correlations (|r|>0.6p<0.05).

[0132] 3.6.2.5 Changes in Mouse Intestinal Microflora

[0133] like Figure 6 As shown, the Pan / Core species analysis curves are relatively flat, indicating that the sample size of this experiment is sufficient. The Rank-Abundance curve has a large range on the horizontal axis, indicating that the samples in this experiment have high microbial species richness and uniformity.

[0134] α-diversity analysis. The Sobs index, Shannon index, and Coverage index intergroup difference test analysis (Kruskal-Wallis rank sum test) can reflect the richness, diversity, and coverage of the microbial community. Figure 7As shown, compared with the NRM group, the Sobs index, Shannon index, and Coverge index of the intestinal flora in the model group were significantly reduced (P < 0.05). Compared with the ABX group, the Sobs index and Shannon index of the intestinal flora in the CA, PP, DPP, and PP-PS groups were all increased. In terms of the Shannon index, CA and PP-PS showed significant differences compared with ABX (P < 0.01), and DPP also showed a significant difference compared with CA (P < 0.05). These results indicate that CA, PP, DPP, and PP-PS can improve the richness and diversity of the intestinal flora in antibiotic-induced mice, especially PP-PS.

[0135] β-diversity analysis. The results are as follows Figure 8 As shown, a weighted-unifrac PCoA analysis revealed that the NRM group and other treatment groups were completely separated along the PC1 axis, with all treatment groups clustering together. The PP and DPP groups had a high degree of overlap; the PP-PS group was clearly separated from the PP and DPP groups and closest to the NRM group; PP-PS, PP, and DPP were significantly further away from the ABX group. Furthermore, CA was intermediate among the other treatment groups. These results indicate that the gut microbiome structure of treated mice shifted, and that PP-PS corrected the structure (approaching that of healthy mice).

[0136] Community composition analysis. The results of the ASV level Venn diagram are as follows Figure 9 The results showed that there were 65 ASV units in total, 5037 of which were unique to the NRM group, 1039 to the ABX group, 1559 to the ABX+CA group, 1876 to the ABX+PP group, 1351 to the ABX+DPP group, and 1344 to the ABX+PP-PS group. To further analyze the differences in the microbial communities among the groups, the community structure bar plot was used to compare the relative abundance of the microbial community components of the groups. The dominant ASV units in each group were as follows: Figure 9 shown.

[0137] Species difference analysis. Linear discriminant analysis effect size (LEfSe) multi-level species difference discriminant analysis can perform linear discriminant analysis on samples according to different grouping conditions based on taxonomic composition to find out the communities or species that have a significant difference in sample division. LEfSe analysis was used to verify species differences (LDA threshold was set to 4, filtering species except for phylum and genus levels). Compared with the NRM group, the species differences in each treatment group were different. The specific results are as follows: Figure 10 shown.

[0138] 3. Conclusion

[0139] This study used a mixed antibiotic formulation (neomycin 5 mg / mL, vancomycin 25 mg / mL, amphotericin B 0.1 mg / mL, ampicillin 10 mg / mL, metronidazole 5 mg / mL, and lipopolysaccharide 1.5 μg / mL) to administer intraperitoneally to mice to investigate its effects on motor function, brain tissue oxidation, inflammation, intestinal barrier damage, and changes in microbial flora. Experimental verification showed that the formulation significantly reduced motor function, increased brain tissue oxidative stress, a pronounced inflammatory response, intestinal barrier damage, and significant changes in microbial flora.

[0140] To improve this situation, the present invention used Pediococcus pentosaceus CQFP202447 and its fermentation preparation for intervention. Results showed that the mice's endurance was significantly improved, with prolonged times to exhaustion in both weighted swimming and running. Furthermore, this intervention significantly alleviated antibiotic-induced oxidative stress and inflammatory responses in mice, reducing serum and brain levels of oxidative and inflammatory markers such as MDA, IL-6, and TNF-α, while increasing levels of antioxidant and anti-inflammatory markers such as SOD, GSH, and IL-10.

[0141] HE staining results showed that intervention with Pediococcus pentosaceus CQFP202447 and its fermentation preparation improved cell morphology in mouse brain tissue, with reduced pyknosis and increased cell number. Real-time PCR analysis further confirmed that this intervention activated and enhanced the expression of genes in the AKT / CREB / BDNF / ERK1 pathway in the brain, thereby playing a positive role in protecting the central nervous system. It also enhanced the expression of ZO-1, occludin-1, and claudin-1 genes in cecal tissue, effectively ameliorating antibiotic-induced intestinal epithelial cell barrier damage.

[0142] In this study, we observed that the richness, uniformity, and diversity of the gut microbiota in ABX mice were significantly reduced compared to healthy mice. However, after intervention with Pediococcus pentosaceus CQFP202447 and its fermentation preparation, these indicators were restored, demonstrating the positive effect of this intervention in improving the structure of the mouse gut microbiota.

[0143] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered by the scope of protection of the claims of the present invention.

Claims

1. A Pediococcus pentosaceus postbiotic, characterized in that: The Pediococcus pentosaceus postbiotic is a preparation with a substance obtained by inactivating Pediococcus pentosaceus CQFP202447 as an active ingredient; The preservation number of the Pediococcus pentosaceus CQFP202447 is CGMCC NO.29615.

2. Use of the Pediococcus pentosaceus postbiotic according to claim 1 in at least one of the following: A1) Application in the preparation of anti-oxidative inflammation products; A2) Application in improving sports endurance; A3) Application in the preparation of products for improving sports endurance and inflammation; A4) Application in changing intestinal flora; A5) Application in the preparation of products for altering intestinal flora inflammation; A6) Application in the preparation of products for preventing and / or treating intestinal barrier damage and inflammation; A7) Use in the preparation of products for preventing and / or treating brain injury and inflammation; A8) Use in the preparation of products for preventing and / or treating brain cell nuclear pyknosis and inflammation.

3. A product, characterized in that: The product contains the Pediococcus pentosaceus postbiotic according to claim 1.

4. The product according to claim 4, characterized in that The product is a liquid bacterial agent or a solid bacterial agent.

5. A method for preparing a postbiotic of Pediococcus pentosaceus, characterized in that: include: The Pediococcus pentosaceus postbiotics are obtained by inactivating Pediococcus pentosaceus CQFP202447 at high temperature; The preservation number of the Pediococcus pentosaceus CQFP202447 is CGMCC NO.29615.

6. A method for preparing a postbiotic of Pediococcus pentosaceus, characterized in that: include: Fermenting polysaccharides by Pediococcus pentosaceus CQFP202447 to obtain the Pediococcus pentosaceus postbiotics; The preservation number of the Pediococcus pentosaceus CQFP202447 is CGMCC NO.29615.

7. The preparation method according to claim 6, characterized in that: The polysaccharide is seaweed polysaccharide.

8. The preparation method according to claim 6, characterized in that: The seaweed polysaccharide is blood coral polysaccharide.

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