Pediococcus pentosaceus strain a20001 with anti-inflammatory and 5-ht secreting function, and derivative products and applications thereof

Abstract

The application provides a Pediococcus pentosaceus strain A20001 with anti-inflammatory and 5-HT secretion functions, and a derivative product and application thereof, and belongs to the technical field of functional microorganisms.The strain A20001 is isolated from the excrement of a healthy centenarian in Guangxi, and is identified as Pediococcus pentosaceus through molecular identification and morphological identification.The Pediococcus pentosaceus strain A20001 has the functions of anti-inflammation and 5-HT secretion, and provides a new idea for the treatment of diseases related to intestinal inflammation and / or depression caused by insufficient 5-HT secretion.

Description

Technical Field

[0001] This invention belongs to the field of functional microbial technology, specifically relating to a Pediococcus pentosacchari strain A20001 with anti-inflammatory and 5-HT secretion functions, its derivatives and applications. Background Technology

[0002] In recent years, the role of gut microbiota in various diseases has become increasingly prominent. Numerous studies on the gut-brain axis have shown that probiotics can influence the expression of neurotrophic factors in the brain by regulating the abundance of the gut microbiota and the levels of its related metabolic secretions, thereby affecting central nervous system diseases. Developing diverse and adaptable probiotics is of great significance to human health.

[0003] Serotonin (5-HT), also known as 5-hydroxytryptamine, is an important neurotransmitter and signaling molecule that plays a significant role in both the central and peripheral nervous systems. In the central nervous system, it participates in regulating mood, sleep, feeding, body temperature, activity, and sexual activity. In the peripheral nervous system, it participates in regulating vascular tone and gastrointestinal motility. For example, gut microbiota 5-HT is associated with irritable bowel syndrome (IBS). Studies have shown that gut microbiota can stimulate monocytes / macrophages and mast cells to release 5-HT, producing a series of pro-inflammatory factors, inhibiting intestinal motility, mediating dysregulation between gut microbiota and the mucosal immune system, and ultimately leading to visceral hypersensitivity. Furthermore, gut microbiota 5-HT is also associated with Alzheimer's disease. Studies have demonstrated that using SSRIs or directly increasing extracellular 5-HT levels can reduce Aβ levels in mouse brain tissue fluid by 25%. Simultaneously, SSRIs can inhibit 5-HT reuptake, increase 5-HT concentration in the brain, and reduce Aβ through receptor interactions. In addition, existing technologies have also revealed an association between serotonin and depression. Patent publication CN114717148A discloses a strain of *Lactobacillus paracasei* CCFM1229 that alleviates depressive symptoms and its applications. This strain can increase the concentrations of 5-HT and BDNF in the mouse brain, thus alleviating depressive symptoms. Another example is patent publication CN116004455A, which discloses a fermenting *Lactobacillus mucinus* strain that secretes serotonin (5-HT). Limosilactobacillus fermentum) Strains A21196 and their applications belong to the field of functional microbial technology. Strains A21196 not only possess antibacterial capabilities but also the ability to secrete 5-HT.

[0004] Pediococcus pentosaceus is a Gram-positive bacterium that, as a commensal bacterium of the human body, plays a role in preventing pathogenic bacterial infections and gastrointestinal inflammation. Patent publication CN115851551A discloses a strain of Pediococcus pentosaceus A21358 with anti-Helicobacter pylori activity, as well as the ability to reduce gastrointestinal inflammation and maintain intestinal homeostasis. However, there are currently no reports on Pediococcus pentosaceus simultaneously possessing anti-inflammatory properties and secreting 5-HT. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a novel Pediococcus pentosaceus strain A20001, which has anti-inflammatory and 5-HT secretion functions.

[0006] This invention provides a Pentosaccharidococcus ( Pediococcus pentosaceus The strain is A20001, with the accession number GDMCC No: 64674.

[0007] The present invention provides a probiotic agent, the active ingredient of which includes the Pediococcus pentosaceus strain A20001.

[0008] Preferably, the morphology of the Pediococcus pentosaceus strain A20001 includes Pediococcus pentosaceus cells and / or Pediococcus pentosaceus spores.

[0009] Preferably, the concentration of the Pediococcus pentosaceus strain A20001 includes 1×10⁻⁶. 9 CFU / mg ~ 1×10 11 CFU / mg or 1×10 9 CFU / mL ~ 1×10 11 CFU / mL.

[0010] Preferably, the dosage form of the probiotic agent includes at least one of the following: tablets, drops, capsules, and powders.

[0011] This invention provides the application of the Pediococcus pentosaceus strain A20001 or the probiotic agent in the preparation of products rich in 5-hydroxytryptamine.

[0012] This invention provides a method for determining the secretion of 5-HT by the *Pediococcus pentosaceus* strain A20001, comprising the following steps:

[0013] Pediococcus pentosaceus strain A20001 was inoculated into MRS liquid medium and cultured with shaking. The bacterial cells were collected, resuspended in Hanks salt balance solution, and then cultured statically. The culture supernatant was collected and loaded onto a high performance liquid chromatograph for the detection of 5-HT.

[0014] Preferably, the detection conditions of the high performance liquid chromatograph are as follows: the C18 reversed-phase column has a specification of 200 mm × 4.6 mm and a diameter of 5 μm; the mobile phase is 0.02 mol / L potassium dihydrogen phosphate-methanol solution; the flow rate is 0.5 ml / min; and the ultraviolet detector has a detection wavelength of 275 nm.

[0015] Preferably, the temperature for the oscillation culture is 36~38℃;

[0016] The shaking culture time is 10-14 hours;

[0017] The rotation speed of the oscillation culture is 120~220 rpm;

[0018] The static incubation time is 1.5~2.5h.

[0019] This invention provides the use of the Pediococcus pentosaceus strain A20001 or the probiotic agent in the preparation of drugs for anti-inflammatory and / or diseases caused by 5-HT deficiency.

[0020] This invention provides a *Pediococcus pentosaceus* strain A20001, with accession number GDMCC No: 64674. Strain A20001 was isolated from the feces of a healthy centenarian in Guangxi Province and, after molecular and morphological identification, belongs to *Pediococcus pentosaceus*. Pediococcus pentosaceus The strain A20001 exhibits rapid growth, entering the logarithmic growth phase within 2 hours of inoculation and reaching the stationary phase at 14 hours. The strain maintains stable growth without showing a decline phase within 24 hours. Qualitative analysis using high-performance liquid chromatography (HPLC) showed that strain A20001 possesses the ability to self-secrete 5-HT, and the secretion amount is superior to that of the control strain A21196 selected from the same strain. 9 The CFU / mL CFU / mL Pediococcus pentosaceus strain A20001 secreted 2.898 μg / mL of 5-HT, while the control strain A21196 secreted 2.535 μg / mL. Simultaneously, this invention also conducted in vitro inflammation model detection experiments. Treatment with Pediococcus pentosaceus strain A20001 effectively reduced the expression of pro-inflammatory factors, and its inhibitory effect on the inflammatory response was comparable to or better than that of the commercial strain Lactobacillus rhamnosaceus strain GG (LGG). Therefore, the Pediococcus pentosaceus strain A20001 provided by this invention possesses both anti-inflammatory and 5-HT secretion functions, offering a new approach for the treatment of intestinal inflammation-related diseases and / or depression caused by insufficient 5-HT secretion.

[0021] This invention provides a method for determining the secretion of 5-HT by *Pediococcus pentosaceus* strain A20001, comprising the following steps: inoculating *Pediococcus pentosaceus* strain A20001 into MRS liquid medium and culturing with shaking; collecting the bacterial cells; resuspending the bacterial cells in Hanks' salt equilibrium solution and then incubating statically; collecting the culture supernatant and loading it onto a high-performance liquid chromatograph for 5-HT detection. The determination method provided by this invention is characterized by high efficiency, simplicity, sensitivity, stability, and good reproducibility, and is suitable for rapid qualitative and quantitative analysis of 5-HT. Attached Figure Description

[0022] Figure 1 Morphological diagram of Pediococcus pentosaceus strain A20001;

[0023] Figure 2 This is a growth curve diagram of Pediococcus pentosaceus A20001;

[0024] Figure 3 The graph shows the anti-inflammatory effects of Pediococcus pentosaceus A20001, where A represents the IL-6 detection result and B represents the TNF-α detection result.

[0025] Figure 4 A standard curve for the qualitative detection of 5-HT by high performance liquid chromatography (HPLC);

[0026] Figure 5 The image shows the results of 5-HT secretion by Pediococcus pentosaceus strain A20001.

[0027] Figure 6 This is a diagram showing the results of 5-HT secretion by the control strain A21196.

[0028] Information on the preservation of biological materials

[0029] Pediococcus pentosaceus ( Pediococcus pentosaceus Strain A20001 was deposited on May 23, 2024 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 64674. Detailed Implementation

[0030] This invention provides a Pentosaccharidococcus ( Pediococcus pentosaceus The strain is A20001, with the accession number GDMCC No: 64674.

[0031] In this invention, strain A20001 was isolated from the feces of healthy centenarians in Guangxi. The colonies of strain A20001 are round, yellowish, smooth, raised, with regular edges, and opaque, belonging to Gram-positive cocci. The 16S rDNA sequence of strain A20001 is shown in SEQ ID NO:1. Comparison with the NCBI database showed that strain A20001 had the highest sequence similarity to *Pediococcus pentosaceus*, with a sequence similarity of 99.86%. Molecular identification and morphological identification confirm its classification as *Pediococcus pentosaceus*. Pediococcus pentosaceusThe strain A20001 exhibits rapid growth, entering the logarithmic growth phase within 2 hours of inoculation and reaching the stationary phase at 14 hours. The strain maintains stable growth without showing a decline phase within 24 hours. In vitro inflammatory model treatment with strain A20001 showed that it effectively reduced the expression of inflammatory factors (IL-6, TNF-α), indicating that strain A20001 possesses anti-inflammatory properties, with an inhibitory effect comparable to that of the commercial strain *Lactobacillus rhamnosus* strain GG (LGG strain). Furthermore, this invention also conducted qualitative and quantitative detection of the secretions from strain A20001. Results showed that strain A20001 possesses the ability to self-secrete 5-HT, which was quantitatively detected using the internal standard method. 9 The self-secreted 5-HT yield of strain A20001 (CFU / mL) was 2.898 μg / mL, while that of the control strain A21196 (fermenting Lactobacillus mucilaginosus) was 2.535 μg / mL. This indicates that strain A20001 has a good 5-HT secretion function.

[0032] In this invention, the method for expanding the culture of strain A20001 preferably includes the following steps:

[0033] The activated strain A20001 is used to prepare a seed culture, which is then inoculated into MRS liquid medium for expansion culture. The activation method for strain A20001 is preferably to streak the strain A20001 onto an MRS plate and pick single colonies. The streak culture temperature is preferably 36-38℃, more preferably 37℃. The inoculum size during streak culture is preferably 0.5%-2%, more preferably 1%. The method for preparing the seed culture for strain A20001 is preferably to inoculate the picked single colonies into MRS liquid medium and culture them to obtain the seed culture. The culture temperature is preferably 36-38℃, more preferably 37℃. The culture time is preferably 2-4 hours, more preferably 3 hours. The inoculum size during culture is preferably 3%-6%, more preferably 4%-5%. The expansion culture temperature is 36-38℃, more preferably 37℃. The inoculum size during expansion culture is preferably 5%-10%, more preferably 8%. This invention does not impose any special restrictions on the formulation of MRS liquid culture medium and MRS plates; well-known MRS liquid culture medium and MRS plate formulations can be used. For example, the MRS liquid culture medium formulation consists of 20 g / L glucose, 10 g / L beef extract, 10 g / L peptone, 5 g / L yeast extract, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium citrate, 5 g / L sodium acetate, 0.58 g / L magnesium sulfate, 0.25 g / L manganese sulfate, and 0.1% Tween-80, with a pH of 6.6-6.8. The only difference between the MRS plates and the MRS liquid culture medium is the addition of 1.5%-2% agar.

[0034] The present invention provides a probiotic agent, the active ingredient of which includes the Pediococcus pentosaceus strain A20001.

[0035] In this invention, the probiotic agent refers to supplementing the intestinal flora with beneficial bacteria, ensuring a sufficient number of these bacteria to exert unique biological effects, thereby achieving the purpose of health care, disease prevention, and treatment. The morphology of the *Pediococcus pentosaceus* strain A20001 preferably includes *Pediococcus pentosaceus* cells and / or *Pediococcus pentosaceus* spores. The *Pediococcus pentosaceus* cells are the solid phase obtained by solid-liquid separation from the culture medium after expanded culture. The *Pediococcus pentosaceus* spores are round or oval, stress-resistant dormant bodies formed within the cells of *Pediococcus pentosaceus* after it has grown and developed to a certain stage.

[0036] In this invention, the concentration of the Pediococcus pentosaceus strain A20001 is preferably 1×10⁻⁶. 9 CFU / mg ~ 1×10 11 CFU / mg or 1×10 9 CFU / mL ~ 1×10 11 CFU / mL, more preferably 1×10⁻⁶ 10 CFU / mg or 1×10 10 CFU / mL. The dosage form of the probiotic agent preferably includes at least one of the following: tablets, drops, capsules, and powder. The powder is processed using low-temperature freeze-drying technology to ensure the activity of the probiotics. The excipients of the powder preferably include freeze-drying agents, such as skim milk powder. The powder is suitable for dissolving in warm water and is suitable for infants. The tablets are prepared by mixing Pediococcus pentosaccharide strain A20001 with excipients and compressing them into tablets, suitable for chewing. The excipients preferably include sweeteners or cocoa powder. The capsules preferably contain Pediococcus pentosaccharide strain A20001 alone or mixed with excipients as a core packaged in a capsule shell. The drops are prepared by mixing Pediococcus pentosaccharide strain A20001 with edible oil to form air- and moisture-proof drops; when taking the drops, the oily substance is squeezed into the mouth through the dropper opening.

[0037] This invention provides the application of the Pediococcus pentosaceus strain A20001 or the probiotic agent in the preparation of products rich in 5-HT.

[0038] In this invention, the product is preferably a pharmaceutical product. Existing technology reports that 5-HT can be further converted into melatonin in the human body, and melatonin has been proven to have sedative and sleep-inducing effects. Simultaneously, 5-HT can enhance memory and protect neurons from damage caused by "excitoxins." Therefore, sufficient 5-HT can prevent brain damage during aging. Therefore, the product prepared by this invention has sedative, sleep-promoting, memory-enhancing, and neuroprotective functions. The preferred method for preparing the 5-HT-rich product involves culturing *Pediococcus pentosaceus* strain A20001 or the probiotic agent, collecting the culture medium, and adding 5-HT, with or without separation, to the product excipients to prepare the corresponding pharmaceutical product. Experiments have shown that *Pediococcus pentosaceus* strain A20001 has the function of extracellularly secreting 5-HT. Through cultivation, 5-HT is secreted into the culture medium, which contains not only *Pediococcus pentosaceus* cells but also the secreted 5-HT, thereby improving the pathological or sub-healthy state caused by insufficient 5-HT in the body.

[0039] This invention provides a method for determining the secretion of 5-HT by the *Pediococcus pentosaceus* strain A20001, comprising the following steps:

[0040] Pediococcus pentosaceus strain A20001 was inoculated into MRS liquid medium and cultured with shaking. The bacterial cells were collected, resuspended in Hanks salt balance solution, and then cultured statically. The culture supernatant was collected and loaded onto a high performance liquid chromatograph for the detection of 5-HT.

[0041] In this invention, the culture method for the *Pediococcus pentosaceus* strain A20001 is the same as that described in the above-mentioned technical solution, and will not be repeated here. The preferred temperature for the shaking culture is 36-38°C, more preferably 37°C. The preferred time for the shaking culture is 10-14 hours, more preferably 12 hours. The preferred rotation speed for the shaking culture is 120-220 rpm, more preferably 150-200 rpm, and most preferably 180 rpm.

[0042] In this invention, the method for collecting bacterial cells preferably involves centrifuging the culture medium. The centrifugation speed is preferably 5000-12000 rpm, more preferably 10000-12000 rpm. The centrifugation time is preferably 5-10 min, more preferably 8-10 min. The bacterial cells are preferably washed and resuspended. The solution used for washing the bacterial cells is preferably Hanks' salt balance solution. The number of washes is preferably 2-4 times, more preferably 3 times. During resuspension, the amount of Hanks' salt balance solution added is 0.5-1 times the volume of the culture medium discarded, more preferably 1 time. Separating the bacterial cells facilitates the removal of various impurities while obtaining cells with good 5-HT secretion state. The static culture time is preferably 1.5-2.5 h, more preferably 2 h. The purpose of static culture is to allow cells with good 5-HT secretion state to secrete 5-HT in a clean background solution, which is beneficial for 5-HT detection and reduces the interference of culture medium components on the detection results. After collecting the culture supernatant, it is preferably filtered to remove impurities. The preferred method for filtration and impurity removal is membrane filtration. The preferred pore size of the membrane is 0.22 μm. Membrane filtration facilitates the removal of impurities and prevents them from clogging the pipeline during HPLC detection.

[0043] In this invention, the preferred detection conditions for the high-performance liquid chromatograph are: a C18 reversed-phase column with dimensions of 200 mm × 4.6 mm and a diameter of 5 μm; a mobile phase of 0.02 mol / L potassium dihydrogen phosphate-methanol solution; a flow rate of 0.5 ml / min; and an ultraviolet detector with a detection wavelength of 275 nm. The content of 5-HT is quantitatively determined using the internal standard method. The regression equation is y = 21.12x - 2.2888, and the correlation coefficient R0 is [value missing]. 2 =0.9959, where y represents the peak area and x represents the concentration of 5-HT.

[0044] This invention provides the use of the Pediococcus pentosaceus strain A20001 or the probiotic agent in the preparation of drugs for anti-inflammatory and / or diseases caused by 5-HT deficiency.

[0045] In this invention, the anti-inflammatory effect is preferably defined as the inhibition of the expression of pro-inflammatory factors. The pro-inflammatory factors preferably include IL-6 and / or TNF-α. In this embodiment of the invention, the anti-inflammatory function of *Pediococcus pentosaceus* strain A20001 is verified using an in vitro inflammation model. The in vitro inflammation model is preferably the lipopolysaccharide (LPS)-induced mouse mononuclear macrophage cell line RAW264.7. The concentration of the LPS is preferably 100 ng / mL. The treatment concentration of *Pediococcus pentosaceus* strain A20001 is the OD of the bacterial suspension. 600nmThe value was 0.3. After treatment with the Pediococcus pentosaceus strain A20001 for 24 h, compared with the LPS model group, Pediococcus pentosaceus strain A20001 could significantly reduce the expression of pro-inflammatory factors IL-6 and TNF-α, and inhibit inflammatory function. The inhibitory effect was comparable to that of the commercial strain Lactobacillus rhamnosus strain GG (LGG strain).

[0046] In this invention, the diseases caused by 5-HT deficiency preferably include at least one of the following diseases: depression, anxiety, and obsessive-compulsive disorder. Existing research shows that depression is closely related to 5-HT. The development of modern antidepressants is mostly related to increasing the concentration of 5-HT in the brain or inhibiting platelet uptake of 5-HT, because the structure-activity relationship of drugs inhibiting platelet uptake of 5-HT is consistent with the inhibition of uptake by the central nervous system synaptosome. For example, fluoxetine increases the concentration of 5-HT in the synaptic cleft by selectively inhibiting the reuptake of 5-HT by central neurons, thereby playing an antidepressant and mood-improving therapeutic role (Research progress on 5-HT and gut microbiota and its role in gut-brain related diseases, Li Xiaoxiao et al., Chinese Journal of Rehabilitation Medicine, 2019:34(1), 116-118.). In another embodiment of the present invention, an experiment was conducted to detect the ability of Pediococcus pentosacchari A20001 to secrete 5-HT. The results showed that Pediococcus pentosacchari strain A20001 not only has the characteristic of extracellular secretion of 5-HT, but also the amount of 5-HT secreted is better than that of the control strain Lactobacillus fermentum strain A21196 isolated from the same sample.

[0047] In this invention, the effective concentration of Pediococcus pentosaceus strain A20001 in the drug is preferably 1×10⁻⁶. 9 CFU / mg ~ 1×10 11 CFU / mg or 1×10 9 CFU / mL ~ 1×10 11 CFU / mL, more preferably 1×10⁻⁶ 10 CFU / mg or 1×10 10 CFU / mL. The drug is a probiotic. The preferred dosage form of the probiotic is an oral dosage form, such as tablets, capsules, powders, and drops. The drug is taken orally. The dosage is 1×10⁻⁶. 9 CFU / mg / dose ~ 1×10 11 CFU / mg / dose, taken 1-2 times daily.

[0048] The following detailed description, in conjunction with embodiments, illustrates a Pediococcus pentosacchari strain A20001 with anti-inflammatory and 5-HT secretion functions, its derivatives, and applications, but these should not be construed as limiting the scope of protection of this invention.

[0049] Example 1

[0050] Isolation and identification of Pediococcus pentosaceus strain A20001

[0051] Fecal samples from healthy centenarians in Guangxi were serially diluted and spread onto MRS plates. The plates were incubated at 37°C for 48 hours to obtain single colonies. Single colonies were picked, inoculated onto MRS liquid medium, and incubated at 37°C on a shaker for 24 hours. Bacterial culture was then streaked onto MRS plates to continue culturing and obtaining single colonies. This process was repeated to obtain pure isolates of *Lactobacillus*. Single colonies of the pure isolates were then subjected to Gram staining and PCR detection using universal 16S rDNA amplification primers.

[0052] The morphology of Pediococcus pentosaceus strain A20001 is as follows: Figure 1 As shown in Figure A, the colonies are round, yellowish, smooth, raised, with neat edges, and opaque. They belong to Gram-positive bacteria, specifically cocci.

[0053] Specific identification results such as Figure 1 As shown in Figure B. The 16S rDNA sequence of strain A20001:

[0054]

[0055] The 16S rDNA sequence of strain A20001 was compared with the NCBI database. The results showed that strain A20001 is related to Pediococcus pentosaceus (…). Pediococcus pentosaceus The sequence similarity was the highest, reaching 99.86%. Therefore, based on the molecular and morphological identification results, strain A20001 was identified as Pediococcus pentosaceus.

[0056] Example 2

[0057] Growth curve of Pediococcus pentosaceus strain A20001

[0058] Strain A20001 was activated by streaking from a glycerol storage tube, and single clones were picked and cultured in MRS liquid medium. The growth was detected using a microplate reader, and graphs were plotted after the assay was completed.

[0059] Figure 2 The growth curve of Pediococcus pentosaceus strain A20001 is shown. It can be seen that Pediococcus pentosaceus strain A20001 grows extremely rapidly, entering the logarithmic growth phase within 2 hours and reaching the stationary phase at 14 hours. Strain A20001 exhibits stable growth and does not show a decline phase within 24 hours.

[0060] Example 3

[0061] Pediococcus pentosaceus strain A20001 has anti-inflammatory function.

[0062] The mouse mononuclear macrophage cell line RAW264.7 was used as an in vitro inflammation model after induction with lipopolysaccharide (LPS). RAW264.7 cells were cultured overnight in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibiotics at 37°C in a 5% CO2 incubator. The next day, the cells were seeded into 12-well plates at 3 × 10⁶ cells per well. 5 After cell adhesion, four groups were set up: control group (Con), LPS modeling group, LPS+A20001 group, and LPS+LGG group, with 3 wells per group. LPS (final concentration 100 ng / ml) was added sequentially to establish the model, and bacterial suspension (OD) was prepared. 600nm =0.3) co-incubated with cells, and after 24 h, the cell supernatant was collected by centrifugation. The expression levels of inflammatory factors IL-6 and TNF-α were detected according to the instructions of the ELISA kit (purchased from Jianglai Biotechnology).

[0063] The results are as follows Figure 3 As shown, compared with the LPS model group, the addition of Pediococcus pentosaceus strain A20001 significantly reduced the pro-inflammatory factor IL-6 (IL-6). p <0.001), TNF-α ( pThe expression of <0.0001) indicates that it has a significant anti-inflammatory function, and the inhibitory effect is comparable to that of the commercial strain Lactobacillus rhamnosus GG (LGG).

[0064] Example 4

[0065] Detection of 5-HT secreted by Pediococcus pentosaceus A20001

[0066] Single colonies of purified strain A20001 and control strain A21196 (publication number CN116004455A) were picked and inoculated into MRS liquid medium and cultured overnight at 37°C in a shaker. After centrifugation, the bacterial cells were washed three times with HBSS (Hanks salt balance solution); then resuspended in HBSS solution (formula shown in Table 1) and incubated at 37°C for 2 hours. The supernatant was collected by centrifugation and filtered through a 0.22 μm filter into a liquid chromatography bottle for later use.

[0067] Table 1 HBSS solution formulation

[0068]

[0069] Preparation of 5-HT standard solution: Dissolve 5-HT standard in HBSS in the dark to concentrations of 1.0 μg / mL, 2.0 μg / mL, 3.0 μg / mL, 4.0 μg / mL, and 5.0 μg / mL, and filter into a liquid chromatography bottle using a 0.22 μm filter.

[0070] 5-HT was qualitatively determined by high-performance liquid chromatography (HPLC) using a Chromai Leaps HPLC system. The chromatographic conditions were as follows: C18 reversed-phase column (200 mm × 4.6 mm, 5 μm); mobile phase: 0.02 mol / L potassium dihydrogen phosphate-methanol (25:2 v / v); initial gradient program; flow rate: 0.5 mL / min; UV detector; detection wavelength: 275 nm. The content of 5-HT secreted by strain A20001 was quantitatively evaluated using the internal standard method. Standard curve: Within the concentration range of 1.0–5 μg / mL, the peak area showed a good linear relationship with the 5-HT concentration. The regression equation was y = 21.12x - 2.2888, where the correlation coefficient R0 was 1. 2 =0.9959, y represents the peak area, and x represents the 5-HT concentration (see...) Figure 4 ).

[0071] Figure 5 This demonstrates that the 5-HT secreted by the *Pediococcus pentosaceus* strain A20001 of this invention is completely separated from the internal standard within 13-14 minutes. 9The self-secreted 5-HT yield of strain A20001 (CFU / mL) was 2.898 μg / mL, while that of control strain A21196 was 2.535 μg / mL. Figure 6 As can be seen, the Pediococcus pentosaceus strain A20001 provided by this invention has a strong ability to secrete 5-HT, and its ability to secrete 5-HT is stronger than that of the control strain.

[0072] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of Pediococcus pentosaceus ( Pediococcus pentosaceus strain A20001, characterized in that, It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 64674.

2. A probiotic agent, characterized in that, The active ingredient includes the Pediococcus pentosaceus strain A20001 as described in claim 1.

3. The probiotic agent according to claim 2, characterized in that, The concentration of the *Pediococcus pentosaceus* strain A20001 includes 1×10⁻⁶. 9 CFU / mg ~ 1×10 11 CFU / mg or 1×10 9 CFU / mL ~ 1×10 11 CFU / mL.

4. The probiotic agent according to claim 2 or 3, characterized in that, The dosage form of the probiotic agent includes at least one of the following: tablets, drops, capsules, and powders.

5. The use of the Pediococcus pentosaceus strain A20001 of claim 1 or the probiotic agent of claim 2 or 3 in the preparation of a product rich in 5-hydroxytryptamine, wherein the product is a drug.

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