Preparation method and application of lactic acid bacteria postbiotics

Abstract

The present invention belongs to the field of microbial technology, and specifically relates to the probiotic properties of lactic acid bacteria and the alleviating effect of their postbiotics on pulmonary fibrosis. Pediococcus pentosaceus ) ZFML001, whose deposit number is CCTCC NO: M 2024601. The present invention also provides a method for using the above-mentioned Pediococcus pentosaceus ( Pediococcus pentosaceus ) A postbiotic prepared by ZFML001, and use of the postbiotic in preparing a drug for alleviating or assisting in the treatment of pulmonary fibrosis.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to the probiotic properties of lactic acid bacteria and the alleviating effect of their postbiotics on pulmonary fibrosis. Background Art

[0002] Pulmonary fibrosis (PF) is the main pathological process of most lung diseases. The pathogenesis includes imbalanced inflammatory response, extracellular matrix deposition and abnormal myofibroblast proliferation, which destroy the normal structure of alveoli, leading to respiratory failure and death. The triggering factors of PF include allergens, smoking, radiation and drug treatment. For example, bleomycin, which is used to fight squamous cell carcinoma, testicular cancer, malignant lymphoma and other diseases, has severe pulmonary toxicity and can induce the progression of pulmonary fibrosis (gut-lung axis). The current treatment for pulmonary fibrosis relies solely on slowing its progression. There is no effective treatment in clinical practice, so the mortality rate of some fibrotic lung diseases is still high.

[0003] Probiotics have received great attention as a promising method to improve intestinal health. Among them, lactic acid bacteria are "generally regarded as safe", and many lactic acid bacteria have been reported to have probiotic potential. For example, Lactobacillus plantarum ZJ316 has been shown to inhibit colitis and regulate intestinal microecology in mice. Lactic acid bacteria (LAB) need to remain alive and colonize when they reach the intestine to exert their health-enhancing properties, which requires them to have a certain tolerance to the harsh environment of the gastrointestinal tract. In addition, there may be certain risks in administering live microorganisms to organisms, such as increasing the risk of bacteremia in individuals with impaired gastrointestinal flora. One way to avoid these limitations is to prepare LAB into postbiotics, that is, inanimate microorganisms or their components. Extensive studies have found beneficial effects of postbiotics on the host, including immunomodulatory effects and regulation of intestinal microbiota. Although these characteristics are similar to probiotics, the stability and safety of postbiotics make them more attractive.

[0004] The invention of CN118126908A, "A strain of Pediococcus pentosaceus for improving fatigue and enhancing motor function and its products", states that Pediococcus pentosaceus YF01 (CGMCC No. 29920) can regulate oxidative stress and muscle fiber expression to improve the body's antioxidant capacity, thereby reducing the damage caused by sports fatigue and improving the body's motor function.

[0005] The invention of CN116549491A "Application of Pediococcus pentosaceus SMM914 in the preparation of drugs for the treatment of chronic obstructive pulmonary disease" states that Pediococcus pentosaceus SMM914 can exert an antioxidant effect in chronic obstructive pulmonary disease model mice and improve the composition of intestinal flora, overcoming the problem that existing antioxidants cannot efficiently improve endogenous oxidizability. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a preparation method of lactic acid bacteria postbiotics and its alleviating effect on pulmonary fibrosis.

[0007] In order to solve the above technical problems, the present invention provides a Pediococcus pentosaceus ( Pediococcus pentosaceus ) ZFML001, its deposit number is CCTCC NO:M 2024601.

[0008] The present invention also provides a method for preparing ... Pediococcus pentosaceus ) Postbiotics prepared by ZFML001.

[0009] The present invention also provides a method for preparing the above-mentioned postbiotics, comprising the following steps:

[0010] 1) Pediococcus pentosaceus Pediococcus pentosaceus ) ZFML001 is inoculated into a fermentation medium for culture to obtain a bacterial liquid;

[0011] Pediococcus pentosaceus ( Pediococcus pentosaceus ) ZFML001 was inoculated into a fermentation medium for activation and passaged twice to obtain a bacterial solution;

[0012] 2) Heat-treat the bacterial solution and then resuspend it with physiological saline to obtain postbiotics.

[0013] As an improvement to the preparation method of the postbiotics of the present invention:

[0014] After activation of Pediococcus pentosaceus ZFML001, a single colony was picked and inoculated into 10 mL of MRS liquid medium, cultured at 37°C for 12 h, and then inoculated into MRS liquid medium at a 1% (volume %) inoculation rate, and cultured at 37°C for 12 h;

[0015] The bacterial solution obtained from the above culture was treated in a hot water bath (95°C for 20 min to inactivate) and then resuspended in physiological saline to a concentration of 1×10 10 cfu / mL, and obtain postbiotics.

[0016] As a further improvement of the preparation method of the postbiotics of the present invention:

[0017] The activation culture temperature of Pediococcus pentosaceus ZFML001 was 37°C and the culture time was 48 h.

[0018] The present invention also provides the use of postbiotics in preparing drugs for alleviating or assisting in the treatment of pulmonary fibrosis.

[0019] In the above-mentioned method for preparing postbiotics:

[0020] During the activation stage of the strain, the culture conditions were 37°C for 48 h; during the subculture stage, the inoculation amount on the culture medium was 1%; during the subculture stage, the culture conditions were 37°C for 12 h.

[0021] The culture medium for strain activation and subculture is MRS culture medium; MRS solid culture medium is used for activation, and MRS liquid culture medium is used for subculture.

[0022] The deposit information of Pediococcus pentosaceus ZFML001 of the present invention is as follows:

[0023] Deposit name: Pediococcus pentosaceus ZFML001 Pediococcus pentosaceus ZFML001, deposit unit: China Center for Type Culture Collection, deposit address: Wuhan University, Wuhan, China, deposit number: CCTCC NO:M2024601, deposit date April 1, 2024.

[0024] The Pediococcus pentosaceus ZFML001 of the present invention is separated from fermented sour radish, and after sequencing, its 16SrRNA sequence is compared with the nucleic acid sequence in NCBI, and the comparison result is obtained to identify it as Pediococcus pentosaceus.

[0025] The Pediococcus pentosaceus ZFML001 of the present invention has good probiotic properties, and exhibits good capabilities in environmental stress tolerance, gastrointestinal tolerance, surface hydrophobicity and self-aggregation. The postbiotics prepared by the lactic acid bacteria of the present invention can effectively increase the weight of mice with pulmonary fibrosis and alleviate pulmonary fibrosis, and therefore have great application prospects in alleviating or assisting the treatment of pulmonary fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0027] Figure 1 This is a graph showing the growth of Pediococcus pentosaceus ZFML001 under environmental stress;

[0028] Figure 1 Middle: The left picture shows the growth of Pediococcus pentosaceus ZFML001 under different sodium chloride concentrations, and the right picture shows the growth of Pediococcus pentosaceus ZFML001 under different ethanol concentrations; MRS stands for this culture medium only (without any bacterial solution added).

[0029] Figure 2 This is a graph showing the growth of Pediococcus pentosaceus ZFML001 in simulated gastric fluid.

[0030] Figure 3 This is a graph showing the surface hydrophobicity and self-aggregation ability of Pediococcus pentosaceus ZFML001;

[0031] Figure 3Middle: A is a surface hydrophobicity diagram of Pediococcus pentosaceus ZFML001, and B is a self-aggregation ability diagram of Pediococcus pentosaceus ZFML001.

[0032] Figure 4 This is a graph showing the weight changes of mice treated with postbiotics from Pediococcus pentosaceus ZFML001;

[0033] Figure 5 H&E staining of lung tissue sections of mice treated with postbiotics of Pediococcus pentosaceus ZFML001. A to D correspond to the Control group, PF group, Alive group, and Inactivated group, respectively.

[0034] Figure 6 RT-qPCR images of lung tissues of mice with pulmonary fibrosis and mice treated with Pediococcus pentosaceus ZFM001 postbiotic.

[0035] Figure 7 The graph shows the content of intestinal short-chain fatty acids (acetate, propionate, and butyrate) in mice with pulmonary fibrosis and mice treated with the postbiotic Pediococcus pentosaceus ZFM001. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0037] In the following examples, unless otherwise specified, the operating methods used are all conventional operating methods, the equipment used are all conventional equipment, and the equipment and materials used in each example are the same.

[0038] Acquisition of Pediococcus pentosaceus ZFML001:

[0039] The fermented pickled radish from farmers in Zhejiang Province was used as the sample. 0.1 g of pickled radish was cut into pieces and added into 1 mL of PBS buffer (0.01 M, pH 7.3-7.5) to make the pickled radish initial solution. According to the 10-fold dilution method, the pickled radish initial solution was diluted to 10 with PBS buffer. -4 , 10 -5 , 10 -6 As bacterial suspension, 100 μL of bacterial suspension was spread on MRS solid medium containing calcium carbonate (2%), and incubated in a 37°C incubator for 48 h, and then the colony with the largest calcium dissolution circle was selected for identification. The identification result of its 16S rDNA sequence is shown in SEQ ID NO: 1.

[0040] It was subsequently identified as Pediococcus pentosaceus through comparison with the official website of the National Center of Biotechnology Information (NCBI) and named Pediococcus pentosaceus ( Pediococcus pentosaceus )ZFML001.

[0041] The preservation information is as follows:

[0042] Deposit name: Pediococcus pentosaceus ZFML001 Pediococcus pentosaceus ZFML001, deposit unit: China Center for Type Culture Collection, deposit address: Wuhan University, Wuhan, China, deposit number: CCTCC NO:M2024601, deposit date April 1, 2024.

[0043] Example 1: Environmental stress tolerance of Pediococcus pentosaceus ZFML001

[0044] The growth of Pediococcus pentosaceus ZFML001 was observed under different concentrations of sodium chloride (0, 3%, 6%, 8%, 10%) and ethanol (0, 2.5%, 5%, 10%, 10%, 15%).

[0045] The activation method of Pediococcus pentosaceus ZFML001 is as follows: use an inoculation loop to dip the preserved bacterial solution of Pediococcus pentosaceus ZFML001, streak it on MRS solid medium and incubate it in a 37 ℃ incubator for 48 hours. Then pick a single colony and inoculate it twice at a 1% inoculation amount in 10 mL of MRS liquid medium for shaking culture (37 ℃, 12 hours; 180 rpm / min), so as to obtain two generations of activated Pediococcus pentosaceus ZFML001.

[0046] Take the activated two-generation Pediococcus pentosaceus ZFML001 and centrifuge it at 8000 r / min for 10 min, and dissolve the bacteria in an equal volume (ie, 10 mL) of PBS buffer (0.01 M, pH 7.3-7.5) to prepare a bacterial suspension.

[0047] 0, 3, 6, 8, and 10 g of sodium chloride were added to 100 mL of MRS liquid culture medium, respectively, to obtain MRS liquid culture medium containing sodium chloride with a mass concentration of sodium chloride of 0, 3%, 6%, 8%, and 10%, respectively.

[0048] 0, 2.5, 5, 7.5, 10, and 15 mL of ethanol were added to different volumes of MRS liquid culture medium and the volume was fixed to 100 mL to obtain ethanol-containing MRS liquid culture medium with ethanol volume concentrations of 0, 2.5%, 5%, 7.5%, 10%, and 15%, respectively.

[0049] 30 μL of bacterial suspension was inoculated into 5 mL of MRS liquid medium containing different concentrations of sodium chloride or ethanol and incubated at 37 °C. After culturing for 24 h without shaking, the OD 600 Measure absorbance.

[0050] The results are as follows Figure 1 As shown, Pediococcus pentosaceus ZFML001 can tolerate higher concentrations of sodium chloride and ethanol, and Pediococcus pentosaceus ZFML001 has good environmental stress resistance.

[0051] Example 2: Growth of Pediococcus pentosaceus ZFML001 in simulated gastric fluid

[0052] Preparation of simulated gastric fluid: simulated gastric fluid is prepared by adjusting the pH of physiological saline to 3.0 with 6 mol / L HCL, sterilizing it at 121°C for 15 min, and then adding 0.3% (w / v) protease.

[0053] Take a 5 mL centrifuge tube and add 0.2 mL of bacterial suspension, 0.3 mL of 0.5% sodium chloride solution, and 1 mL of simulated gastric fluid as the reaction system. Incubate in a 37 °C shaker (180 rpm / min). Take 100 μL of the solution at 0, 1, 2, and 3 h, respectively, and dilute to 10 with PBS buffer (0.01 M, pH 7.3-7.5). -4 , 10 -5 , 10 -6 Then, 100 μL of bacterial suspension was taken from each of the three gradients and added to MRS solid culture medium for coating, and then placed in a 37 ℃ incubator. Plate counts were performed after 48 h of incubation.

[0054] The results are as follows Figure 2 As shown, Pediococcus pentosaceus ZFML001 can withstand the erosion of simulated gastric juice and can survive in human gastric juice, which has practical significance.

[0055] Example 3: Surface hydrophobicity and self-aggregation of Pediococcus pentosaceus ZFML001

[0056] Take the activated two generations of Pediococcus pentosaceus ZFML001 and centrifuge at 8000 r / min for 10 min. Add an appropriate amount of PBS buffer (0.01 M, pH 7.3-7.5) to the bacteria to prepare the OD 600 = 1.0 of bacterial suspension.

[0057] 1. Determination of the surface hydrophobicity of Pediococcus pentosaceus ZFML001 using the microbial adhesive hydrocarbon method:

[0058] Dichloromethane, ethyl acetate, and 1-butanol were selected as hydrophobic organic solvents. 600= 1.0) and a hydrophobic organic solvent in a volume ratio of 3:1 and incubated at 37 °C for 3 h. After the incubation, the aqueous phase (located at the bottom) was taken.

[0059] The absorbance of the aqueous phase after incubation was measured at 600 nm; the hydrophobicity was calculated as follows:

[0060] Hydrophobicity (%) = [1- (A 1 / A 0 )]×100%;

[0061] Among them, A 0 is the absorbance of the bacterial suspension before mixing, A 1 is the absorbance of the aqueous phase after incubation.

[0062] 2. Determination of self-aggregation ability:

[0063] Take 4 mL of bacterial suspension (OD 600 = 1.0), incubated at 37 °C, and the upper suspension was taken at 0, 1, 2, 3, 4, and 5 h to measure OD 600 , calculate the percentage of automatic aggregation. The formula for calculating the self-aggregation ability is as follows:

[0064] Self-aggregation (%) = [1-(A t / A 0 )]×100%

[0065] t refers to the absorbance at time “t” and 0 refers to the absorbance at t = 0.

[0066] The results are as follows Figure 3 As shown in the figure, the hydrophobicity of Pediococcus pentosaceus ZFML001 to the three organic solvents was higher than 40%, and the self-aggregation rate gradually increased from 0 to 5 h, with the self-aggregation rate at 5 h being greater than 60%. Hydrophobicity and self-aggregation ability are necessary conditions for probiotics to adhere to gastrointestinal epithelial cells and host cells, respectively.

[0067] In summary, it was shown that Pediococcus pentosaceus ZFML001 had good hydrophobicity and self-aggregation ability, and had good probiotic properties.

[0068] Example 4. Preparation of postbiotics from Pediococcus pentosaceus ZFML001

[0069] After activation of Pediococcus pentosaceus ZFML001, a single colony was picked and inoculated into 10 mL of MRS liquid culture medium, cultured at 37°C for 12 h, and then inoculated into new MRS liquid culture medium at a 1% inoculum volume and cultured at 37°C for 12 h.

[0070] The cultured bacterial solution was heat treated (95°C, 20 min) and then resuspended in saline to a concentration of 1×10 10 cfu / mL, and obtain postbiotics.

[0071] Example 5: Effect of postbiotic treatment of Pediococcus pentosaceus ZFML001 on pulmonary fibrosis mice

[0072] Four groups of six-week-old C57BL / 6 mice were selected, with 12 mice in each group. Before the experiment, all mice were fed with conventional adaptive feeding for 1 week and kept in cages with constant temperature (23°C), constant humidity (55%±5%), and 12 / 12-hour light / dark cycle, with free access to food and water. The four groups of mice were set as Control, PF, Alive, and Inactivated.

[0073] All mice were anesthetized with 2.5% Avertin in proportion to their weight, and then they were hung vertically on a fixator for modeling. The modeling method for mice in the Control group was to slowly infuse 30 μL of normal saline into the mouse lungs after anesthesia; while the modeling method for mice in the PF group, Alive group, and Inactivated group was to slowly infuse 30 μL of bleomycin (3U / kg) into the mouse lungs after anesthesia. This day was recorded as day 0, and then the mice in the Control group and PF group were gavaged with 200 μL of normal saline every day; while the mice in the Alive and Inactivated groups were gavaged with 200 μL of Pediococcus pentosaceus ZFML001 (1×10 9 cfu / mL), Pediococcus pentosaceus ZFML001 postbiotics (2.5×10 9 cfu / mL). The gavage operation was continued for 21 days, and the weight of mice was recorded before gavage every day; during the experiment, mice had free access to food and water.

[0074] The results are as follows Figure 4~Figure 7 As shown, first, compared with the treatment with bleomycin alone (PF group), feeding Pediococcus pentosaceus ZFML001 and its postbiotics (Alive and Inactivated groups) alleviated the weight loss of mice, and their body weight on day 21 was significantly higher than that of mice in the PF group (p< 0.05). It should be noted that the weight of mice in the Inactivated group was significantly higher than that in the Alive group on day 21 (p< 0.05), which indicates that the ability of supplementing ZFML001 postbiotics to alleviate the weight loss of mice is better than that of live bacteria ( Figure 4 ). In addition, the lung tissue sections treated with postbiotics showed stronger improvement in damage and more obvious reduction in inflammatory cell infiltration ( Figure 5 ).

[0075] The quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) method was used to detect the gene expression of inflammatory factors related to pulmonary fibrosis in the lung tissue of mice 21 days later. The total RNA of the lung was extracted using a total RNA isolation kit (Foregene, ChenduChina). cDNA was synthesized by a PrimeScript RT reagent kit (Takara Bio, Beijing, China). qRT-PCR was performed using a SYBG Green® PCR kit (Takara Bio, Beijing, China) and followed the following thermal cycle program: preamplification at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control and was expressed by 2 -ΔΔCT Gene expression relative to GAPDH was calculated. The primers used were synthesized by Sanguang Biotechnology Co., Ltd. The results are shown in Figure 6 As shown. Therefore, it can be concluded that ZFML001 postbiotics can significantly inhibit the expression of three proinflammatory cytokines (IL1-β, TNF-α, TGF-β) associated with the formation of pulmonary fibrosis. Therefore, ZFML001 postbiotics may alleviate pulmonary fibrosis by inhibiting lung inflammation and the formation of fibroblasts.

[0076] The content of short-chain fatty acids (mainly acetic acid, propionic acid and butyric acid) in the cecal feces of mice after 21 days was detected by gas chromatography-mass spectrometry (GC-MS). GC analysis was performed on a trace 1300 gas chromatograph (Thermo Fisher Scientific, USA). The GC was equipped with an Agilent HP-INNOWAX capillary column, helium as the carrier gas, and the flow rate was 1 mL / min. The injection was carried out in a 10:1 split mode with an injection volume of 1 μL and an injection temperature of 250 °C. The temperatures of the ion source and MS transfer line were 300 °C and 250 °C, respectively. The column temperature was programmed to increase from an initial temperature of 90 °C, then to 120 °C at a rate of 10 °C / min, to 150 °C at a rate of 5 °C / min, and finally to 250 °C at a rate of 25 °C / min and maintained for 2 minutes. The metabolites were detected by mass spectrometry in electron impact ionization mode on ISQ 7000 (Thermo Fisher Scientific, USA). Single ion monitoring (SIM) mode was used with an electron energy of 70 eV. The results are shown in Figure 7 As shown. Short-chain fatty acids (SCFAs), especially acetate, propionate and butyrate, are produced by intestinal flora and can directly provide energy to the intestinal epithelium and improve immunity. Figure 7It can be seen that bleomycin treatment led to a significant decrease in the content of acetic acid and butyric acid in the intestine of mice, while the content of acetic acid, propionic acid and butyric acid in the group fed with ZFML001 postbiotics was significantly higher than that in the PF group. Propionic acid and butyric acid are related to the pro-inflammatory / anti-inflammatory balance of the body and are involved in the body's immune regulation, which shows that ZFML001 postbiotics improve pulmonary fibrosis by increasing the content of short-chain fatty acids in the intestine of mice, especially propionic acid and butyric acid.

[0077] It should be noted that the postbiotics prepared by Pediococcus pentosaceus YF01 and Pediococcus pentosaceus SSM914 described in the background technology cannot show the corresponding regulatory effects on proinflammatory cytokines (IL1-β, TNF-α, TGF-β) related to pulmonary fibrosis and short-chain fatty acids (acetic acid, propionic acid, butyric acid) related to immunity.

[0078] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. Pediococcus pentosaceus ( Pediococcus pentosaceus ) The use of postbiotics prepared by ZFML001 in the preparation of drugs for alleviating or assisting the treatment of pulmonary fibrosis, characterized in that: The deposit number of Pediococcus pentosaceus ZFML001 is CCTCCNO:M 2024601.

2. The use according to claim 1, characterized in that The preparation method of postbiotics comprises the following steps: 1) Pediococcus pentosaceus ZFML001 is inoculated into a fermentation medium for culture to obtain a bacterial liquid; 2) Heat-treat the bacterial solution and then resuspend it with physiological saline to obtain postbiotics.

3. The use according to claim 2, characterized in that: After activating Pediococcus pentosaceus ZFML001, a single colony was picked and inoculated into 10 mL of MRS liquid medium and cultured at 37°C for 12 h; Then inoculate into MRS liquid medium at 1% inoculum and culture at 37°C for 12 h; The bacterial solution obtained from the above culture was treated in a hot water bath and then resuspended in physiological saline to a concentration of 1×10 10 cfu / mL, and obtain postbiotics.

4. The use according to claim 3, characterized in that: The activation culture temperature of Pediococcus pentosaceus ZFML001 was 37 °C and the culture time was 48 h.

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