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

Abstract

The invention relates to the technical field of microorganisms, and discloses pediococcus pentosaceus and application thereof in resisting oxidative inflammation and improving intestinal flora. The pediococcus pentosaceus is named as pediococcus pentosaceus CQFP202447, and the preservation number of the pediococcus pentosaceus CQFP202447 is CGMCC (China General Microbiological Culture Collection Center) NO.29615. The pediococcus pentosaceus has the advantages that the pediococcus The pediococcus pentosaceus CQFP202447 disclosed by the invention can effectively improve the intestinal environment and enhance the antioxidant and anti-inflammatory capacities of the body through the unique physiological functions and metabolic characteristics, thereby enhancing the overall health level.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to Pediococcus pentosaceus and application thereof in resisting oxidation inflammation and improving intestinal flora. Background Art

[0002] Excessive use of antibiotics has led to an imbalance in intestinal flora, which in turn has led to a series of health problems such as intestinal inflammation and decreased immunity.

[0003] To meet this challenge, the intervention of probiotics is particularly important, as it can effectively improve the various adverse reactions caused by the abuse of antibiotics. The mechanism of action of probiotics is mainly reflected in the following aspects: first, it protects the integrity of the intestinal barrier function and prevents the adhesion of harmful bacteria; second, it provides nutrition and energy to the intestine by fermenting dietary fiber; third, it stimulates the immune system and enhances immune function, thereby reducing the occurrence of inflammation. In addition, probiotics play an important role in improving human immunity. It can not only adjust the endogenous microbial system in the intestine by secreting enzymes and nutrients, but also prevent related diseases, thereby directly or indirectly improving the physiological functions of the intestine and central nervous system.

[0004] Therefore, finding a microbial strain that can improve intestinal flora and have antioxidant and anti-inflammatory functions is of great significance for maintaining human health. Summary of the invention

[0005] In view of this, the present invention proposes a Pediococcus pentosaceus and its application in anti-oxidative inflammation and improving intestinal flora, aiming to provide a strain with significant probiotic properties to solve the imbalance of intestinal flora and the accompanying health problems in the prior art.

[0006] The invention provides Pediococcus pentosaceus CQFP202447, and the preservation number of the Pediococcus pentosaceus CQFP202447 is CGMCC NO.29615.

[0007] The present invention also provides a use of the above-mentioned Pediococcus pentosaceus CQFP202447 or its bacterial suspension in at least one of the following:

[0008] A1) Application in the preparation of anti-oxidative inflammation products;

[0009] A2) Application in improving sports endurance;

[0010] A3) Application in the preparation of products for improving sports endurance and inflammation;

[0011] A4) Application in changing intestinal flora;

[0012] A5) Application in the preparation of products for altering intestinal flora inflammation;

[0013] A6) Application in the preparation of products for preventing and / or treating intestinal barrier damage and inflammation;

[0014] A7) Use in the preparation of products for preventing and / or treating brain injury and inflammation;

[0015] A8) Use in the preparation of products for preventing and / or treating brain cell nuclear pyknosis and inflammation.

[0016] The present invention also provides a product, which contains the above-mentioned Pediococcus pentosaceus CQFP202447.

[0017] The present invention also provides a method for preparing the product, comprising the steps of using the Pediococcus pentosaceus CQFP202447 as a component of the product to obtain the product.

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

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The Pediococcus pentosaceus CQFP202447 of the present invention exhibits significant biological activity and great potential in many applications. Specifically, the wide application of the strain in the fields of food, health care products and medicine not only greatly enriches the functionality of the products, but also provides consumers with more abundant health choices.

[0021] In the food field, Pediococcus pentosaceus CQFP202447 can be used as a functional food additive to produce foods with antioxidant and anti-inflammatory functions. For example, it can be added to yogurt, juice, dairy products and other foods to effectively improve the intestinal environment, enhance human immunity, and thus prevent the occurrence of chronic diseases.

[0022] In the field of health products, this strain can be used to develop products with multiple health benefits, such as improving sports endurance, preventing intestinal barrier damage and brain damage. These products can meet the needs of different groups of people, especially athletes, the elderly and other groups that need special care.

[0023] In the medical field, the application prospects of Pediococcus pentosaceus CQFP202447 are particularly broad. Compared with traditional drug treatments, probiotics have attracted extensive attention and high attention from the medical community due to their small side effects and high safety. As an important component of probiotics, Pediococcus pentosaceus CQFP202447 can be used to prevent and treat intestinal diseases, brain diseases, etc., showing its unique medical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. Moreover, the same reference symbols are used throughout the accompanying drawings to represent the same components. In the accompanying drawings:

[0025] Figure 1 These are the results of the changes in weighted swimming time of mice;

[0026] Figure 2 This is the result of the change in running time of mice;

[0027] Figure 3 The results are the pathological changes of mouse brain tissue;

[0028] Figure 4 Results of serum oxidation and inflammatory indicators in mice;

[0029] Figure 5 Results of oxidation and inflammation indexes in mouse brain tissue;

[0030] Figure 6 This is the original data result of the mouse intestinal flora change experiment;

[0031] Figure 7 This is the α-diversity analysis result of the mouse intestinal flora change experiment;

[0032] Figure 8 The results of β-diversity analysis of the mouse intestinal flora change experiment;

[0033] Fig. 9 This is the community composition analysis result of the mouse intestinal flora change experiment;

[0034] Fig.10 These are the results of species difference analysis of the mouse intestinal flora change experiment. DETAILED DESCRIPTION

[0035] 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 described herein. On the contrary, these embodiments are provided in order 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, in the absence of conflict, the embodiments of the present invention and the features described 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 combination with the embodiments.

[0036] All experimental data below (bacteria removal analysis section) were statistically analyzed using SPSS 20.0 software (SPSS Inc, Chicago, IL, USA), combined with one-way ANOVA and Tukey's multiple comparisons; p < 0.05 was considered statistically significant. GraphPad Prism 9.3 was used for drawing. Data are expressed as mean ± standard deviation (SD), repeated 3 times.

[0037] Example 1

[0038] 1. Material preparation

[0039] A new strain of Pediococcus pentosaceus was screened from kimchi juice in the Zhendong area of ​​Kaixian County, Chongqing, China, and was named Pediococcus pentosaceus CQFP202447. It was registered and stored in the General Microbiology Center of China National Microbiological Culture Collection on January 12, 2024, with the accession number CGMCC NO.29615.

[0040] Strain recovery: Take out the cryopreservation 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). Perform recovery culture at a constant temperature of 37°C for 24 hours. Then, mix evenly, take 3% of the inoculum, transfer it to another MRS liquid culture medium, and continue to culture for 18 hours. Then, collect the bottom Pediococcus pentose CQFP202447 by centrifugation (conditions are 4°C, 4000rpm, and duration is 10 minutes). After collection, resuspend it in 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.

[0041] 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 water bath (set temperature at 95°C for 40 minutes), and the filtrate was 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 about 100 mL. Then, anhydrous ethanol was added to the solution until its final concentration reached 80%, and the solution was left to stand at 4°C overnight. After that, the sample was centrifuged (speed of 4000r / min for 10 minutes) and the supernatant was discarded. The precipitate was repeatedly dissolved with hot water (100mL), and then precipitated with 80% ethanol, and the precipitation lasted 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.

[0042] 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 at a strictly controlled 121°C for 15 minutes. The sterilized polysaccharide liquid was cooled in an ultra-clean workbench and dispensed 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.

[0043] The remaining experimental materials and reagents are shown in Table 1, and the instruments and equipment are shown in Table 2.

[0044] Table 1

[0045]

[0046] Table 2

[0047]

[0048] 2. Experimental Methods

[0049] 1. Establishment of experimental animal model

[0050] A total of 30 SPF-grade male C57BL / 6J mice, all 7-8 weeks old, were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. Animal production license number: SCXK (Xiang) 2019-0004. The experimental mice were raised in a standard laboratory animal room environment, ensuring that the room temperature was constant at 23±2°C and the relative humidity was maintained at 55±5%. The feeding conditions strictly followed a 12-hour light cycle and provided adequate food and water to adapt to the experimental environment for one week.

[0051] The experimental mice were divided into six experimental groups, as follows:

[0052] (1) Normal group (NRM): mice in this group did not receive any therapeutic intervention;

[0053] (2) Model group (ABX): intraperitoneal injection of mixed antibiotic preparations and gavage of normal saline;

[0054] (3) Caffeic acid group (ABX+CA): caffeic acid (10 mg / kg) was administered by oral gavage after injection of the mixed antibiotic preparation;

[0055] (4) Pediococcus pentosaceus CQFP202447 group (ABX+PP): After injection of the mixed antibiotic preparation, Pediococcus pentosaceus CQFP202447 (10 9 CFU / kg·bw);

[0056] (5) High-temperature inactivated Pediococcus pentosaceus CQFP202447 group (ABX+DPP): After injection of the mixed antibiotic preparation, the mice were intragastrically administered with high-temperature inactivated Pediococcus pentosaceus CQFP202447 (10 9 CFU / kg·bw);

[0057] (6) Pediococcus pentosaceus CQFP202447 fermented seaweed polysaccharide group (ABX+PP-PS): After intraperitoneal injection of a mixed antibiotic preparation, seaweed polysaccharide fermented by Pediococcus pentosaceus CQFP202447 was administered orally.

[0058] During the experiment, injection / gavage was performed every 12 hours to ensure that the dose of gavage and intraperitoneal injection was accurate to 0.1 mL / 10 g·bw. The experimental period was two weeks. The mixed antibiotic preparation included 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.

[0059] 2. Mouse exhaustion experiment

[0060] 2.1 Weighted swimming test

[0061] Two weeks later, a weighted swimming test was performed after the 14th injection / oral gavage. In the weighted swimming test, a Φ60cm×50cm water tank was selected, the water depth was about 35cm, and the water temperature was maintained within the range of 23±2℃. 30 minutes after administration, the mice in the normal group, model group, caffeic acid group, Pentosaceae CQFP202447 group, high temperature inactivated Pentosaceae CQFP202447 group, and Pentosaceae CQFP202447 fermented seaweed polysaccharide group were treated respectively. The specific operation is: tie a lead wire equivalent to 2% of its body weight to the tail of the mouse, and mark it clearly, and then put it into the water one by one in order for the weighted swimming test. The timer accurately records that when the mouse head sinks into the water for more than 8 seconds and cannot float to the surface by itself, the mouse is judged to be exhausted. After the experiment, the mouse is carefully wiped with a dry towel to ensure that its body is dry.

[0062] 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 ​​of different letters in the same figure are significantly different (P < 0.05). Excessive use of antibiotics will seriously affect the endurance performance of mice, and the introduction of probiotics can effectively improve this situation. Figure 1 As shown in the figure, compared with NRM, the duration of ABX mice in the weighted swimming test was significantly reduced (P < 0.05), which clearly reflects the negative impact of antibiotic abuse on mouse endurance. However, after the advance oral intervention of probiotics such as CA, PP, DPP, and PP-PS, the weighted swimming time of mice was significantly restored, and its performance was between NRM and ABX. Among them, the difference between the PP and DPP groups was not significant, but the effect of the PP-PS group was better than the former two, and was similar to the CA group, which fully demonstrated the significant effect of a specific probiotic combination in improving mouse endurance.

[0063] 2.2 Running experiment

[0064] The endurance of mice was further investigated through running experiments. This experiment used the ZH-PT mouse experimental treadmill, which has a length, width, and height of 565mm, 630mm, and 310mm, respectively, and has 8 single tracks, 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 speed was controlled at 20m / min, the mice exercised for 30 minutes, and rested for 1 minute after each exercise. Four days before the formal experiment, the mice were given adaptive training once a day, and each training lasted for 5 minutes. Finally, on the last day of the experimental animal model establishment experiment, formal testing was carried out and the experimental results were recorded in detail.

[0065] like Figure 2 As shown in the figure, the running time of mice was highly synchronized with the swimming situation. Compared with the NRM control group, the running time of ABX mice showed a significant decrease trend (P < 0.05). After experiencing different advance gavage intervention measures of CA, PP, DPP, and PP-PS, the weighted running time of mice was significantly restored, among which the intervention effects of PP and DPP groups were relatively close. It is worth noting that 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.

[0066] 3. Tissue processing and testing

[0067] 3.1 Tissue processing

[0068] After the last exercise test, feces were collected, mice were weighed, and blood was collected from the eyeballs after ether anesthesia. After cervical dissection, brain, liver, and cecum tissues were dissected and collected. The liver was separated and weighed. Part of the brain tissue was soaked in tissue fixative for slice preparation. The remaining tissues were stored at -80°C for subsequent experiments.

[0069] 3.2 Pathological observation

[0070] Brain tissue (0.5 cm 2 ) were fixed in tissue fixative for more than 48 h, 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).

[0071] Figure 3 The HE staining results of mouse brain tissue. Figure 3 As shown in the figure, the morphology of the cerebral cortex neurons in the NRM group mice was normal, the cells were neatly arranged, the morphology was uniform, the nuclei were prominent and large in size. In contrast, the number of brain tissue cells in the ABX group mice was significantly reduced, the color of the nuclei deepened, and significant pyknosis occurred. Some cells showed punctate necrosis and disordered arrangement. The cortical neurons atrophied and became smaller, and the cells were severely damaged. After pretreatment with CA, PP, DPP and PP-PS, the pyknosis of the nuclei in the brain tissue of mice was alleviated, the number of cells also increased, and some neurons recovered to normal morphology, but a few neurons still showed atrophy. Among them, the effect of PP-PS pretreatment was the most significant, followed by PP pretreatment, which was similar to the effect of caffeic acid pretreatment.

[0072] 3.3 Changes in body weight, liver weight and liver index of mice

[0073] Mouse body weight, liver weight and liver index are important indicators for assessing the health status of mice and the effects of drugs.

[0074] As shown in Table 3, when comparing the body weight data of mice in the NRM group and the ABX group, it was found that after 14 days of intervention, the body weight of mice in the ABX group showed a downward trend, but it did not reach a statistically significant level (P>0.05). Similarly, the body weight of mice treated with CA, PP, DPP, PP-PS and other intervention methods did not show significant changes.

[0075] On the other hand, in the model group induced by combined antibiotics, we observed an increase in liver weight, but this increase also did not reach a statistically significant level (P>0.05). The increase in liver weight often reflects the state of liver congestion and acute inflammation. It is worth noting that after CA, PP, DPP, PP-PS and other treatments, the liver weight of mice decreased, indicating that these treatments have a certain effect on relieving liver congestion and acute inflammation. These phenomena are also reflected in the liver index. Although there are fluctuations between the groups, there is no significant difference.

[0076] Table 3

[0077] 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

[0078] 3.4 Detection of oxidative and inflammatory indicators in mouse serum and brain tissue

[0079] 3.4.1 Detection process

[0080] 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.

[0081] Preparation of brain tissue homogenate: After 0.1 g of mouse brain tissue was added to 0.9 mL of normal saline, the tissue was homogenized (Bioprep-24; Hangzhou Aosheng Instrument Co., Ltd., Hangzhou, China) three times at a rate of 6 m / s for 30 seconds.

[0082] Index detection: The levels of malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) in mouse serum and brain homogenate were measured by 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 by enzyme-linked immunosorbent assay kits (Shanghai ELISA Biotechnology Co., Ltd., Shanghai, China).

[0083] 3.4.2 Detection results of mouse serum oxidation and inflammatory indicators

[0084] Mouse serum oxidation and inflammatory indicators such as Figure 4 shown.

[0085] MDA, GSH, and SOD can reflect the body's oxidative damage and antioxidant capacity. Figure 4 Data analysis of A-4C showed that compared with the NRM group, the MDA level in the serum of the ABX group increased significantly, while the GSH and CuZn-SOD levels decreased significantly, and this difference was statistically significant (P < 0.05). Further comparison showed that after pretreatment with CA, PP, DPP, and PP-PS, the MDA level in the serum of the ABX group decreased significantly, while the GSH and CuZn-SOD levels increased significantly, and this difference was also statistically significant (P < 0.05). Among them, PP-PS had the most significant effect in reducing the MDA content, followed by PP, DPP, and CA; in terms of increasing the activity of CuZn-SOD, PP showed the best effect similar to that of the NRM group, and PP-PS, DPP, and CA had the second and similar effects; as for the increase in GSH content, there was no significant difference among the four groups of CA, PP, DPP, and PP-PS, all of which were similar to the NRM group.

[0086] Cytokines such as IL-6, IL-10, and TNF-α are important indicators of the degree of inflammation in the body. Figure 4 As shown in D-4F, compared with the NRM group, the expression of pro-inflammatory factors TNF-α and IL-6 in the ABX group increased significantly, while the expression of anti-inflammatory factor IL-10 decreased significantly (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, there was no significant difference between the four groups of DPP, PP, PP-PS, and CA.

[0087] 3.4.3 Detection results of oxidative and inflammatory indicators in mouse brain tissue

[0088] like Figure 5As shown in A-5C, compared with the NRM group, the MDA content in the brain tissue of the ABX group was significantly increased, while the levels of GSH and CuZn-SOD were significantly decreased, and the differences were statistically significant (P < 0.05). After pretreatment with CA, PP, DPP, and PP-PS, the MDA content in the brain tissue showed a downward trend, while the levels of GSH and CuZn-SOD increased. Among them, in terms of reducing the MDA content, PP-PS and DPP had similar effects, both better than PP and CA, and both between NRM and ABX; in terms of increasing T-SOD activity, PP-PS had the best effect, followed by PP, and DPP was third, and the effect of CA even surpassed NRM; in terms of increasing GSH content, PP was slightly better than CA, DPP, and PP-PS, and the latter three had similar effects.

[0089] like Figure 5 As shown in D-5F, 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. In terms of reducing the expression of IL-6, CA was slightly better than PP, DPP, and PP-PS, and the effects of the latter three were similar; in terms of reducing the expression of TNF-α, there was no significant difference between PP-PS, PP, CA and NRM, and the effect of DPP was the most significant. In terms of increasing the expression of IL-10, PP-PS and CA had similar effects, both better than PP and DPP, and both were between NRM and ABX.

[0090] 3.5 Real-time fluorescence quantitative PCR analysis

[0091] 3.5.1 Analysis process

[0092] After homogenization of brain and cecal tissues, total RNA from brain and cecal tissues was extracted using TRI zo l 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 cDNA, 2.0 μL forward and reverse primers (10 μm) and 10.0 μL premix (Thermo Fisher Biotechnology Co., Ltd., USA) were mixed and reacted for 40 cycles under the following conditions: 3 min at 95 °C, 30 s at 60 °C, and 1 min at 95 °C. β-Actin was an internal reference gene, and the data were calculated by the 2-ΔΔCt method. The primer sequences are shown in Table 4.

[0093] Table 4

[0094]

[0095] 3.5.2 Expression of AKT, BDNF, CREB and ERK1 genes in mouse brain tissue

[0096] AKT, CREB, BDNF, ERK1, and IL-6 play an important role in cranial nerve injury. The results of real-time fluorescence quantitative PCR are shown in Table 5. Compared with the NRM group, the gene expressions of AKT, CREB, BDNF, and ERK1 in the ABX group were significantly decreased. However, after the intervention of CA, PP, DPP, and PP-PS, the expression levels of these genes recovered and reached a statistically significant level (P < 0.05).

[0097] In terms of BDNF gene upregulation, PP-PS intervention effect was better than PP and DPP, and its effect was close to CA. In terms of CREB gene upregulation, PP intervention effect was the most significant, followed by PP-PS, and the intervention effects of DPP and CA were relatively close.

[0098] For the upregulation of AKT gene, the intervention effect of PP-PS was significantly better than that of PP, DPP and CA, while the latter three showed no significant difference in effect. As for the upregulation of ERK1 gene, the intervention effect of PP was the most prominent, similar to that of CA, while the intervention effects of PP-PS and DPP were relatively weak, and there was no significant difference in effect between the two.

[0099] Table 5

[0100] Grouping AKT BDNF CREB ERK1 NRM 1.03±0.28bc 1.00±0.08b 1.03±0.27b 1.06±0.36abc ABX 0.58±0.10c 0.47±0.09c 0.22±0.14c 0.83±0.23c ABX+CA 0.99±0.48bc 1.81±0.32a 0.89±0.14b 1.35±0.06ab ABX+PP 1.41±0.06ab 1.26±0.19b 1.85±0.29a 1.46±0.25a ABX+DPP 1.77±0.23b 1.03±0.10b 0.99±0.15b 0.81±0.10c ABX+PP-PS 1.90±0.58a 1.43±0.46ab 1.52±0.25a 0.97±0.23bc

[0101] 3.5.3 ZO-1, occludin-1 and claudin-1 gene expression in mouse cecum

[0102] ZO-1, occludin-1 and claudin-1 are classic tight junction proteins that 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 different 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 effect, followed by DPP, and CA was the weakest. 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.

[0103] Table 6

[0104]

[0105] 3.6 Microbial flora analysis

[0106] 3.6.1 Main reagents and instruments

[0107] PF Mag-Bind Stool DNA 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.).

[0108] High-throughput sequencing was completed by Shanghai Meiji Biotechnology Co., Ltd.

[0109] 3.6.2 Methods

[0110] 3.6.2.1 Sample DNA extraction

[0111] 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).

[0112] 3.6.2.2 PCR amplification and sequencing library construction

[0113] The DNA extracted above was used as a template, and the upstream primer 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and the downstream primer 806R (5'-GGACTACHVGGGTWTCTAAT-3') carrying the Barcode sequence were used to perform PCR amplification of the V3-V4 variable region of the 16S rRNA gene. 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), then 72°C stable extension for 10 min, and finally stored at 4°C (PCR instrument: ABI 9700 model). The PCR reaction system was: 4 μL of 5× TransStart FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of upstream primer (5 uM), 0.8 μL of downstream primer (5 uM), 0.4 μL of TransStart FastPfu DNA polymerase, 10 ng of template DNA, and the total volume was 20 μL. Three replicates were performed for each sample. The PCR products of the same sample were mixed and recovered using 2% agarose gel and purified. The size of the band fragments was detected by 2% agarose gel electrophoresis and the size of the band fragments was detected by Quantus PCR. TM The recovered products were detected and quantified using a Fluorometer (Promega, USA).

[0114] use The purified PCR products were used for library construction using Rapid DNA-Seq Kit (Bioo Scientific, Austin, Texas, USA): (1) linker ligation; (2) using magnetic beads to screen and remove linker self-ligated fragments; (3) using PCR amplification to enrich the library template; (4) using magnetic beads to recover the PCR products to obtain the final library. Sequencing was performed using Illumina's PE300 platform (Shanghai Meiji Biopharmaceutical Technology Co., Ltd.). The original data were uploaded to the NCBI SRA database.

[0115] 3.6.2.3 High-throughput sequencing data analysis

[0116] 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 back-end bases were cut from the window, and the reads less than 50 bp after quality control were filtered, and the reads containing N bases were removed; (2) According to the overlap relationship between PE reads, the paired reads were spliced ​​(merged) into a sequence, and the minimum overlap length was 10 bp; (3) The maximum mismatch ratio allowed in the overlap region of the spliced ​​sequence was 0.2, and the non-compliant sequences were screened; (4) The samples were distinguished according to 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 mismatches for the primers was 2.

[0117] Based on the default parameters, the optimized sequences after quality control splicing were denoised using the DADA2 plug-in (or Deblur plug-in) in the Qiime2 process. The sequences after DADA2 (or Deblur) denoising are usually called ASVs (i.e., amplicon sequence variants). The chloroplast and mitochondrial sequences annotated to all samples were removed (if the samples were contaminated with chloroplasts and mitochondria, it is recommended to remove them). In order to minimize the impact of sequencing depth on subsequent Alpha diversity and Beta diversity data analysis, the number of sequences of all samples was flattened to 20,000 (it is recommended to flatten). After flattening, the average sequence coverage (Good's coverage) of each sample can still reach 99.09%. Based on the Sliva 16S rRNA gene database (v138), the Naive bayes (or Vsearch, or Blast) classifier in Qiime2 was used to perform species taxonomy analysis on ASVs. PICRUSt2 (version 2.2.0) software was used for 16S function prediction analysis.

[0118] 3.6.2.4 Statistical analysis

[0119] All data analyses were performed on the MajorBio cloud platform (https: / / cloud.majorbio.com), as follows: mothur software (http: / / www.mothur.org / wiki / Calculators, version 1.30.2) was used to calculate the alpha diversity index Chao 1, Shannon index, etc., and the Wilxocon rank sum test was used to analyze the inter-group differences in alpha diversity; PCoA analysis (principal coordinate analysis) based on the Bray-Curtis distance algorithm was used to test the similarity of the microbial community structure between samples, and the PERMANOVA non-parametric test was combined to analyze whether the microbial community structure between sample groups was significant; LEfSe analysis (Linear discriminant analysis Effect Size) (http: / / huttenhower.sph.harvard.edu / LEfSe) (LDA>2, P<0.05) was used to determine the bacterial groups with significant differences in abundance from the phylum to the genus level between different groups. Distance-based redundancy analysis (db-RDA) was used to investigate the impact of clinical indicators on the intestinal bacterial community structure. Linear regression analysis was used to evaluate 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 correlation |r|>0.6p<0.05.

[0120] 3.6.2.5 Results of changes in intestinal flora of mice

[0121] like Figure 6 As shown in the figure, 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 a high microbial species richness and uniformity.

[0122] α-Diversity analysis. The Sobs index, Shannon index and coverage index inter-group difference test analysis (Kruskal-Wallis rank sum test) can reflect the richness, diversity and coverage of the microbial community. Figure 7As shown in the results, 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 increased. Among them, in the Shannon index, CA and PP-PS had significant differences relative to ABX (P<0.01), and DPP also had significant differences relative to CA (P<0.05). The above results show that CA, PP, DPP and PP-PS can improve the richness and diversity of the intestinal flora of antibiotic-induced mice, especially PP-PS.

[0123] β-diversity analysis. The results are as follows Figure 8 As shown in the figure, the PCoA study based on weighted-unifrac found that the NRM group and other treatment groups were completely separated in the PC1 axis direction, and the treatment groups were clustered together. Among them, the PP and DPP groups had a high degree of overlap; the PP-PS group was significantly separated from PP and DPP, and was closest to the NRM group; PP-PS, PP, and DPP were significantly away from the ABX group. In addition, CA was in the middle of the other treatment groups. The above results show that the community structure of the intestinal flora of mice in the treatment group has changed, and PP-PS can correct the community structure of the intestinal flora of mice (tending to the community structure of the intestinal flora of healthy mice).

[0124] Community composition analysis. The results of the ASV level Venn diagram are shown in Fig. 9 The data showed that there were 65 ASV units in total, 5037 unique ASV units in the NRM group, 1039 unique ASV units in the ABX group, 1559 unique ASV units in the ABX+CA group, 1876 unique ASV units in the ABX+PP group, 1351 unique ASV units in the ABX+DPP group, and 1344 unique ASV units in the ABX+PP-PS group. In order to further analyze the differences in the microbial communities of each group, the community structure Bar diagram was used to compare the relative abundance of the microbial community composition of each group. The dominant ASV units of each group are shown in Figure 2. Fig. 9 shown.

[0125] 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: Fig.10 shown.

[0126] Conclusion

[0127] The present invention adopts a mixed antibiotic preparation (neomycin 5mg / mL, vancomycin 25mg / mL, amphotericin B 0.1mg / mL, ampicillin 10mg / mL, metronidazole 5mg / mL, lipopolysaccharide 1.5μg / mL) and acts on mice by intraperitoneal injection to explore its effects on the motor function, brain tissue oxidation, inflammation, intestinal barrier damage and flora changes of mice. After experimental verification, the preparation caused a significant decrease in the motor function of mice, aggravated oxidative stress in brain tissue, obvious inflammatory reaction, intestinal barrier damage and significant changes in flora structure.

[0128] To improve the above situation, the present invention uses Pediococcus pentosaceus CQFP202447 and fermentation preparations for intervention. The results show that the endurance of mice is significantly improved, and the exhaustion time of weighted swimming and running is prolonged. In addition, the intervention measures also significantly alleviate the oxidative stress and inflammatory response of mice caused by antibiotics, reduce the levels of oxidative and inflammatory indicators such as MDA, IL-6, TNF-α in serum and brain, and increase the levels of antioxidant and anti-inflammatory indicators such as SOD, GSH and IL-10.

[0129] HE staining results showed that after intervention with Pediococcus pentosaceus CQFP202447 and fermentation preparations, the cell morphology of mouse brain tissue was improved, the shrinkage phenomenon was reduced and the number increased. Real-time PCR analysis further confirmed that the intervention activated and enhanced the expression of AKT / CREB / BDNF / ERK1 pathway genes in the brain, thus playing a positive role in protecting the central nervous system of the brain; at the same time, it also enhanced the expression of ZO-1, occludin-1 and claudin-1 genes in cecal tissue, effectively improving the intestinal epithelial cell barrier damage caused by antibiotics.

[0130] In the present invention, we observed that the richness, uniformity and diversity of the intestinal flora of ABX mice were significantly reduced compared with healthy mice. However, after the intervention of Pediococcus pentosaceus CQFP202447 and fermentation preparations, these indicators were adjusted back, showing the positive effect of the intervention measures in improving the intestinal flora structure of mice.

[0131] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant 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 within the scope of protection of the claims of the present invention.

Claims

1. Pediococcus pentosaceus CQFP202447, characterized in that The accession number of the Pediococcus pentosaceus CQFP202447 is CGMCCNO.29615.

2. Use of the Pediococcus pentosaceus CQFP202447 or its bacterial suspension 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 CQFP202447 described in claim 1.

4. A method for preparing the product according to claim 3, characterized in that: The method comprises the steps of using the Pediococcus pentosaceus CQFP202447 as a component of a product to obtain the product.

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

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