Phytobacterium plantarum strain A21337 with functions of inhibiting bacteria and secreting 5-hydroxytryptamine as well as probiotic agent and application thereof

Abstract

The invention provides a lactobacillus plantarum strain A21337 with the functions of inhibiting bacteria and secreting 5-hydroxytryptamine, a probiotic agent of the lactobacillus plantarum strain A21337 and application of the lactobacillus plantarum strain A21337, and belongs to the technical field of functional microorganisms. The invention provides application of phytobacterium plantarum or a microbial agent containing the phytobacterium plantarum in preparation of a product resisting pathogenic bacteria and / or rich in 5-hydroxytryptamine. According to the invention, a plant lactobacillus strain A21337 is separated from intestinal feces of healthy 100-year old people in Guangxi province, has the characteristics of secreting 5-hydroxytryptamine and inhibiting pathogenic bacteria, and has good tolerance to gastrointestinal fluid. Therefore, the strain A21337 or the probiotic agent containing the strain A21337 provides a new means for preparing a product which is resistant to pathogenic bacteria and / or rich in 5-hydroxytryptamine.

Description

Technical Field

[0001] The invention belongs to the technical field of functional microorganisms, and in particular relates to a Lactobacillus plantarum strain A21337 with the functions of inhibiting bacteria and secreting 5-hydroxytryptamine, a probiotic agent and an application thereof. Background Art

[0002] 5-hydroxytryptamine (5-HT), also known as serotonin, is an important neurotransmitter and signal molecule that plays a major role in both the central nervous system and the periphery. In the central nervous system, it participates in regulating emotions, sleep, food intake, body temperature, activity and sexual activity. In the peripheral nervous system, it participates in the regulation of vascular tone and gastrointestinal motility. For example, intestinal flora 5-HT is related to irritable bowel syndrome. Studies have shown that intestinal flora can stimulate mononuclear macrophages and mast cells, release 5-HT, produce a series of proinflammatory factors, inhibit intestinal motility, mediate the imbalance of intestinal flora and mucosal immune system, and ultimately produce visceral hypersensitivity. In addition, the prior art also discloses that 5-hydroxytryptamine is related to depression. The patent with publication number CN114717148A discloses a strain of Lactobacillus paracasei CCFM1229 and its application for relieving depression. The strain can increase the concentration of 5-HT and BDNF in the mouse brain, and has the effect of relieving depression.

[0003] Lactobacillus plantarum is a Gram-positive bacterium that, as a commensal bacterium in the human body, plays a role in anti-oxidation and inhibiting gastrointestinal inflammation. However, there are currently no reports on Lactobacillus plantarum having both antibacterial and 5-HT secretion functions. Summary of the invention

[0004] In view of this, an object of the present invention is to provide an application of Lactobacillus plantarum or a microbial agent containing Lactobacillus plantarum in the preparation of products that are anti-pathogenic and / or rich in 5-hydroxytryptamine, providing a new choice for the preparation of intestinal probiotic drugs.

[0005] The invention provides an application of Lactobacillus plantarum in preparing a product that resists pathogens and / or is rich in 5-hydroxytryptamine.

[0006] Preferably, the Lactobacillus plantarum includes Lactobacillus plantarum cells and / or Lactobacillus plantarum spores.

[0007] Preferably, the strain of Lactobacillus plantarum includes strain A21337;

[0008] The deposit number of the strain A21337 is GDMCC No: 64677.

[0009] Preferably, the product rich in serotonin includes at least one of the following: medicine, food and health care product.

[0010] The invention provides a plant lactobacillus strain A21337, whose preservation number is GDMCC No: 64677.

[0011] The invention provides a probiotic, wherein the active ingredient comprises the Lactobacillus plantarum strain A21337.

[0012] Preferably, the effective viable bacterial concentration of the Lactobacillus plantarum strain A21337 is 1×10 9 CFU / mg~1×10 11 CFU / mg or 1×10 9 CFU / mL~1×10 11 CFU / mL.

[0013] Preferably, the dosage form of the probiotic includes at least one of the following: liquid, tablet, drop, capsule and powder.

[0014] The invention provides application of the Lactobacillus plantarum strain A21337 or the probiotic agent in preparing a medicine for resisting pathogenic bacteria.

[0015] Preferably, the pathogenic bacteria include Escherichia coli.

[0016] The present invention provides an application of Lactobacillus plantarum or a microbial agent containing Lactobacillus plantarum in preparing a product rich in 5-HT. The present invention uses Lactobacillus plantarum as a microbial research object, separates the bacterial body through liquid culture, places the separated bacterial body in a Hanks salt balance solution to secrete metabolites for a short time, uses high performance liquid chromatography (HPLC) to qualitatively detect 5-HT, and uses an internal standard method to quantitatively detect the secretion amount of 5-HT. The results show that Lactobacillus plantarum has the characteristic of secreting 5-HT, and 10 9 Under CFU concentration, the output of plant lactobacillus autosecreting 5-HT is 1.061 μg / mL. As can be seen, the plant lactobacillus is used in the product rich in 5-HT in fermentation production. Meanwhile, the in vitro antibacterial experiment carried out by the present invention shows that plant lactobacillus can significantly inhibit the growth of pathogenic bacteria (Escherichia coli) and has a good effect of inhibiting intestinal pathogens, for preparing antibacterial products, new approach is provided.

[0017] The invention provides a plant lactobacillus strain A21337. The plant lactobacillus strain A21337 is used as an object for cultivation, and the bacterial body is separated and placed in a Hanks salt balance solution to secrete metabolites for a short time, and a high performance liquid chromatography (HPLC) method is used to qualitatively detect 5-HT, and an internal standard method is used to quantitatively detect the secretion amount of 5-HT. The results show that the plant lactobacillus has the characteristic of secreting 5-HT, and 10 9 Under CFU concentration, the output of plant lactobacillus autosecreting 5-HT is 1.061 μg / mL. Plant lactobacillus A21337 can significantly inhibit the growth of pathogenic bacteria (Escherichia coli) at the same time, and its inhibitory effect is suitable with the control strain plant lactobacillus P8. It can be seen that the plant lactobacillus strain A21337 provided by the present invention and its probiotic agent are used in the preparation of products that resist pathogens and / or are rich in 5-hydroxytryptamine. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the morphological diagram of Lactobacillus plantarum strain A21337;

[0019] Figure 2 This is a graph showing the test results of the resistance of Lactobacillus plantarum strain A21337 to artificial gastrointestinal fluid;

[0020] Figure 3 This is the result of the inhibition of pathogens by Lactobacillus plantarum strain A21337;

[0021] Figure 4 This is the detection result of 5-HT secretion by Lactobacillus plantarum strain A21337.

[0022] Biomaterial Deposit Information

[0023] Lactobacillus plantarum strain A21337 was deposited in Guangdong Provincial Microbiological Culture Collection Center on May 23, 2024. The unit is abbreviated as GDMCC. The address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The deposit number is GDMCC No: 64677. DETAILED DESCRIPTION

[0024] The invention provides an application of Lactobacillus plantarum in preparing a product that resists pathogens and / or is rich in 5-hydroxytryptamine.

[0025] In the present invention, the plant lactobacillus preferably includes plant lactobacillus cells and / or plant lactobacillus spores. The plant lactobacillus cell refers to the solid-phase cell collected by expanding the activated plant lactobacillus, separating solid and liquid. The plant lactobacillus spore refers to a round or oval stress-resistant dormant body formed in the cell after plant lactobacillus grows and develops to a certain stage. In one embodiment of the present invention, the plant lactobacillus strain preferably includes strain A21337. The deposit number of the strain A21337 is preferably GDMCC No: 64677.

[0026] In the present invention, the product rich in 5-hydroxytryptamine preferably includes at least one of the following: medicine, food and health care products. According to prior art reports, 5-HT can be further converted into melatonin in the human body, and melatonin has been proven to have sedative and sleep-inducing effects. At the same time, 5-HT can enhance memory and protect neurons from damage by "excitatory neurotoxins". Therefore, sufficient 5-HT can prevent brain damage during aging. Therefore, the product prepared by the present invention has the functions of sedation, sleep promotion, memory enhancement and neuroprotection. The preparation method of the product rich in 5-HT preferably cultivates plant lactobacillus or the microbial agent, collects the culture solution to separate or not separate 5-HT and adds it to the product excipients to prepare the corresponding medicine, food or health care product. Experiments have shown that the plant lactobacillus has the function of secreting 5-HT extracellularly, and 5-HT is secreted into the culture solution by cultivation. The culture solution not only contains the plant lactobacillus bacteria, but also includes the secreted 5-HT, thereby improving the morbid or sub-healthy state caused by insufficient 5-HT in the body.

[0027] The invention provides a plant lactobacillus strain A21337, whose preservation number is GDMCC No: 64677.

[0028] In the present invention, the strain A21337 is isolated from the feces of healthy centenarians in Guangxi. The surface colony diameter of the strain A21337 is about 3mm, convex, round, smooth and light yellow. The 16S rDNA of the strain A21337 is shown in SEQ ID NO: 1. After comparison with the NCBI database, the sequence similarity of the 16S rDNA of the strain A21337 and Lactobacillus plantarum strain 6632 is 99.79%. After morphological identification and molecular identification, the strain A21337 is Lactobacillus plantarum. The strain A21337 has good gastrointestinal fluid resistance, especially when the pH is 2.5 artificial gastric fluid for 3h, the survival rate is significantly higher than that of the control strain P8, and has good gastrointestinal fluid tolerance, so that it can reach the intestine to play a role.

[0029] The invention provides a probiotic, wherein the active ingredient comprises the Lactobacillus plantarum strain A21337.

[0030] In the present invention, the probiotic agent refers to supplementing the beneficial bacteria in the intestine so that the beneficial bacteria have sufficient quantity and exert unique biological effects, thereby achieving the purpose of health care or disease prevention and treatment. The effective live bacteria concentration of the Lactobacillus plantarum strain A21337 in the probiotic agent is preferably 1×10 9 CFU / mg~1×10 11 CFU / mg or 1×10 9 CFU / mL~1×10 11 CFU / mL, more preferably 1×10 10 CFU / mg or 1×10 10 CFU / mL. The dosage form of the probiotic preferably includes at least one of the following: water, tablets, drops, capsules and powders. The powder is processed by low-temperature freeze-drying technology to ensure the activity of the probiotics. The auxiliary materials of the powder preferably include freeze-drying agents, such as skimmed milk powder, etc. The powder is suitable for warm water and is suitable for infants. The tablet is prepared by mixing plant lactobacillus strain A21337 and auxiliary materials and pressing them into tablets, which are suitable for chewing and taking. The auxiliary materials preferably include sweeteners or cocoa powder, etc. The capsule is preferably Lactobacillus plantarum strain A21337 alone or mixed with auxiliary materials as a capsule core in the capsule shell. The drops are made by mixing plant lactobacillus strain A21337 with edible oil to make drops that isolate air and moisture. When taking, open the dropper port to squeeze the encapsulated oil into the mouth. The preparation method of the water is preferably to cultivate plant lactobacillus, and add antifreeze to the obtained culture solution to obtain the water. The method for culturing the plant lactobacillus preferably inoculates the activated plant lactobacillus into an MRS liquid culture medium for culturing. The temperature of the culturing is preferably 36-38°C, more preferably 37°C. The culturing time is preferably 22-26h, more preferably 24h. The antifreeze agent is preferably glycerol. The amount of the antifreeze agent added accounts for 20%-80% of the total volume of the aqueous solution, more preferably 40%-50%. The method for preparing the powder is preferably culturing plant lactobacillus, separating the obtained culture solution into solid and liquid, collecting the solid phase bacteria, mixing the bacteria with a freeze-drying protective agent, freeze-drying, and obtaining a powder. The method for solid-liquid separation is preferably centrifugation. The speed of the centrifugation is preferably 5000-12000rpm, more preferably 10000-12000rpm. The centrifugation time is preferably 5-10min, more preferably 8-10min. The freeze-drying protective agent is preferably selected from one or more of glucose, mannitol, skim milk, etc. The amount of the freeze-drying protective agent added is 5%-15% of the system before freeze-drying, more preferably 10%.

[0031] The invention provides application of the Lactobacillus plantarum strain A21337 or the probiotic agent in preparing a medicine for resisting pathogenic bacteria.

[0032] In the present invention, the pathogenic bacteria preferably include Escherichia coli. In one embodiment of the present invention, Escherichia coli O157:H7 (ATCC 35150) and Escherichia coli (ATCC 25922) were used as pathogenic bacteria, and in vitro antibacterial experiments were carried out. The results showed that Lactobacillus plantarum A21337 can significantly inhibit the growth of pathogenic bacteria, and its inhibitory effect is equivalent to that of the control strain P8.

[0033] The following is a detailed description of the Lactobacillus plantarum strain A21337 with antibacterial and 5-hydroxytryptamine secretion functions and its probiotic agent and application provided by the present invention in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1

[0035] Isolation and identification of Lactobacillus plantarum strain A21337

[0036] The intestinal feces of a healthy centenarian was used as a sample of microbial flora. After multiple dilutions, it was spread on an MRS plate and cultured in a biochemical incubator at 37°C for 48 hours to obtain a single colony. Pick a single colony, inoculate it in an MRS liquid culture medium, and culture it in a 37°C shaker for 24 hours. Take the bacterial liquid and streak it on the MRS plate, and continue to culture a single colony. Pick a single colony and streak it again, and continue to culture a single colony, which is the pure isolated strain obtained, named A21337. The pure isolated strain A21337 was picked for Gram staining, PCR amplification of 1616S rDNA, and sequencing of the PCR product. The sequencing results were compared with the NCBI database.

[0037] The growth state of the strain A21337 in the MRS plate is as follows Figure 1 As shown, the surface colonies are about 3 mm in diameter, raised, round in shape, smooth in surface, and light yellow in color.

[0038]

[0039] Example 2

[0040] Resistance of Lactobacillus plantarum A21337 to artificial gastrointestinal fluid

[0041] The monoclonal colonies of plant lactobacillus strain A21337 and control strain plant lactobacillus P8 were selected and inoculated in MRS liquid medium, and cultured overnight in a shaking table at 37°C. According to the inoculum volume of 10%, the bacterial liquid was inoculated in artificial gastric juice with a pH of 2.5, 3 time points (0h, 1h, 3h) were set, 100 μl was respectively taken and coated on the MRS plate, and after culturing in a biochemical incubator at 37°C for 48h, the number of single colonies was counted; the bacterial liquid treated with artificial gastric juice for 3h was inoculated in artificial intestinal juice with a pH of 6.8 according to the inoculum volume of 10%, 4 time points (0h, 2h, 4h, 8h) were set, 100 μl was respectively taken and coated on the MRS plate, and after culturing in a biochemical incubator at 37°C for 48h, the number of single colonies was counted, and the survival rate was calculated according to formula I.

[0042] Survival rate (%) = (number of single colonies at different time points / number of single colonies at 0 h) × 100% Formula I

[0043] The results are as follows Figure 2 The results showed that the survival rate of strain A21337 was significantly higher than that of the control strain P8 in artificial gastric juice with a pH of 2.5 for 3 hours, but the survival rate in intestinal juice was equivalent to that of the control strain P8, indicating that strain A21337 can successfully pass through the stimulation of gastrointestinal juice and reach the intestine to exert its effect.

[0044] Example 3

[0045] Detection of Inhibition of Pathogens by Lactobacillus plantarum A21337

[0046] The pathogenic bacteria Escherichia coli O157:H7 (ATCC 35150) and Escherichia coli (ATCC25922) in the logarithmic growth phase were spread on the LB plate and placed in an Oxford cup. The activated A21337 strain was prepared into a bacterial suspension and the OD value of the bacterial suspension was measured. 600 Adjust to 0.5, take 200 μl of the adjusted bacterial suspension and inoculate it into an Oxford cup, leave overnight, and observe its effect on pathogenic bacteria.

[0047] The results are shown in Table 1 below.

[0048] Table 1 Detection results of the two strains inhibiting pathogens

[0049]

[0050] The results showed that the Lactobacillus plantarum A21337 could significantly inhibit the growth of pathogenic bacteria, and its inhibitory effect was comparable to that of the control strain P8.

[0051] Example 4

[0052] Lactobacillus plantarum A21337 secretes 5-HT

[0053] Pick a single colony of the purified strain A21337, inoculate it in MRS liquid medium, and culture it in a shaking incubator at 37°C overnight. Collect the bacterial liquid, centrifuge it, and wash it three times with HBSS (Hanks salt balance solution); then resuspend the bacteria with HBSS solution (see Table 2) and let it stand in a 37°C biochemical incubator for 2 hours. Centrifuge and collect the supernatant, filter it into a liquid phase bottle with a 0.22μm filter head, and set aside.

[0054] Table 2 HBSS solution formula

[0055] Element Content (g / L) NaCI 8.0 <![CDATA[MgSO 4 ·7H 2 Oh]]> 0.1 KCl 0.4 <![CDATA[MgCl 2 ·6H 2 O]]> 0.1 <![CDATA[CaCl 2 ]]> 0.14 <![CDATA[NaHPO 4 ·12H 2 The]]> 0.048 <![CDATA[KH 2 AFTER 4 ]]> 0.06 glucose 1.0 Double distilled water -

[0056] Preparation of HT standard solution: Dissolve the 5-HT standard in HBSS in the dark to a concentration of 1.0 μg / mL, 2.0 μg / mL, 3.0 μg / mL, 4.0 μg / mL, and 5.0 μg / mL. Filter with a 0.22 μm filter into a liquid phase bottle for later use.

[0057] High performance liquid chromatography (HPLC) was used to qualitatively detect 5-HT. The HPLC instrument model was Chromai Leaps (Konomi), and the chromatographic conditions were: C18 reverse phase chromatographic column (200mm×4.6mm, 5μm), the mobile phase was 0.02mol / L potassium dihydrogen phosphate-methanol (volume ratio of 25:2) solution, the instrument default gradient detection was used, the flow rate was 0.5ml / min, the ultraviolet detector, the detection wavelength was 275nm, and the internal standard method was used to quantitatively evaluate the content of A21337 autocrine 5-HT.

[0058] Standard working curve: In the concentration range of 1.0 to 5 μg / mL, the peak area has a good linear relationship with the 5-HT concentration. The regression equation is Y=21.12x-2.2888, and the correlation coefficient is R 2 = 0.9959, Y represents the peak area, x represents the 5-HT concentration (e.g. Figure 3 ).

[0059] Figure 4 It was shown that the 5-HT secreted by Lactobacillus plantarum strain A21337 was completely separated from the internal standard within 13-14 min. 9 The production of autocrine 5-HT by strain A21337 at CFU / mL was 1.061 μg / mL.

[0060] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of Lactobacillus plantarum in preparing products that resist pathogens and / or are rich in 5-hydroxytryptamine.

2. The application according to claim 1, characterized in that: The Lactobacillus plantarum comprises Lactobacillus plantarum cells and / or Lactobacillus plantarum spores.

3. The application according to claim 1, characterized in that: The strains of Lactobacillus plantarum include strain A21337; The deposit number of the strain A21337 is GDMCC No: 64677.

4. The use according to any one of claims 1 to 3, characterized in that: The product rich in 5-hydroxytryptamine includes at least one of the following: medicine, food and health care product.

5. A Lactobacillus plantarum strain A21337, characterized in that The deposit number is GDMCC No:64677.

6. A probiotic, characterized in that: The active ingredient comprises the Lactobacillus plantarum strain A21337 as claimed in claim 5.

7. The probiotic according to claim 6, characterized in that: The dosage form of the probiotics includes at least one of the following: liquid, tablet, drop, capsule and powder.

8. The probiotic according to claim 7, characterized in that: The effective live bacteria concentration of Lactobacillus plantarum strain A21337 in the probiotic agent is 1×10 9 CFU / mg~1×10 11 CFU / mg or 1×10 9 CFU / mL~1×10 11 CFU / mL.

9. Use of the probiotic according to any one of claims 6 to 8 in the preparation of products that resist pathogens and / or are rich in 5-hydroxytryptamine.

10. The use according to claim 9, characterized in that: The pathogenic bacteria include Escherichia coli.

Images