Composite platycodon grandiflorum fermentation inoculant and platycodon grandiflorum fermentation method thereof

Abstract

The invention discloses a composite platycodon grandiflorum fermentation inoculant and a platycodon grandiflorum fermentation method thereof, and relates to a fermentation inoculant and a fermentation method thereof. The composite platycodon grandiflorum fermentation inoculant is formed by combining two or three of lactobacillus plantarum M3, lactobacillus brevis JW12 and leuconostoc mesenteroides ZS. The composite platycodon grandiflorum fermentation inoculant is used for platycodon grandiflorum fermentation, the content of platycodon grandiflorum total saponins and platycodon grandiflorum saponin D of the fermented platycodon grandiflorum decoction pieces is increased by more than one time, the mycoplasma pneumonia treatment effect of the platycodon grandiflorum decoction pieces is improved, and particularly, the composite platycodon grandiflorum fermentation inoculant has a remarkable callback effect on Treg / Th17 unbalance caused by mycoplasma pneumonia and promotes Treg / Th17 cell balance.

Description

Technical Field

[0001] The present invention relates to a fermentation bacterial agent and a fermentation method thereof. Background Art

[0002] Platycodon grandiflorum is a perennial herb, also known as Monk's Cap, Radix Pseudostellariae, Chinese Bellflower, and Lantern Flower. It is the dried root of Platycodon grandiflorum (Jacq.) A.D of the genus Platycodon in the family Campanulaceae. It tastes sweet and bitter, and is neutral in nature. It is often used as medicine with its root, belonging to the liver meridian and lung meridian, and is one of the 40 commonly used Chinese medicinal materials in large quantities. At the same time, Platycodon grandiflorum is also a product with both medicinal and edible uses.

[0003] There have been many studies on fermenting Platycodon grandiflorum with microorganisms, and the effects produced by fermenting Platycodon grandiflorum with different microorganisms may be different. In-depth research on the fermentation of Platycodon grandiflorum by microorganisms is of great significance for improving the effective active ingredients of Platycodon grandiflorum and the therapeutic effects on specific diseases. Summary of the Invention

[0004] The present invention provides a compound Platycodon grandiflorum fermentation bacterial agent and a method for fermenting Platycodon grandiflorum, which can greatly increase the contents of total saponins and platycodin D in Platycodon grandiflorum and improve its therapeutic effect on mycoplasma pneumonia.

[0005] The compound Platycodon grandiflorum fermentation bacterial agent of the present invention is composed of a combination of two or three of Lactobacillus plantarum M3 (deposit number CGMCC NO.26065), Lactobacillus brevis JW12 (deposit number CGMCC NO.33957), and Leuconostoc mesenteroides ZS (deposit number CGMCC NO.33958).

[0006] Furthermore, the quantitative ratio between different strains in the fermentation bacterial agent is 1:1.

[0007] The method for fermenting Platycodon grandiflorum with the above compound Platycodon grandiflorum fermentation bacterial agent of the present invention: Platycodon grandiflorum is removed of impurities, washed, moistened thoroughly, and sliced, then soaked in water for 8 - 12 h, and then sterilized at high temperature. After that, the compound Platycodon grandiflorum fermentation bacterial agent liquid is inoculated at an inoculation amount of 2 - 5% (v / w), and fermented in an environment of 30°C ± 2°C for 6 d - 8 d, and then dried at 60°C ± 2°C to complete the fermentation of Platycodon grandiflorum; wherein, the compound Platycodon grandiflorum fermentation bacterial agent liquid is composed of a combination of the bacterial liquids of two or three of Lactobacillus plantarum M3, Lactobacillus brevis JW12, and Leuconostoc mesenteroides ZS.

[0008] Furthermore, the thickness of the sliced Platycodon grandiflorum is 3 - 5 mm.

[0009] Furthermore, the amount of added water is 2 times the mass of Platycodon grandiflorum.

[0010] Furthermore, sterilize at 121°C for 15 minutes.

[0011] Furthermore, the quantity ratio between different strains in the compound Platycodon grandiflorum fermentation bacterial agent liquid is 1:1.

[0012] Furthermore, the concentration of microorganisms in the compound Platycodon grandiflorum fermentation bacterial agent liquid is 1×10 8 ~5×10 8 cfu / ml.

[0013] The compound Platycodon grandiflorum fermentation bacterial agent of the present invention is used for Platycodon grandiflorum fermentation. The contents of total Platycodon grandiflorum saponins and Platycodon grandiflorum saponin D in the fermented Platycodon grandiflorum decoction pieces are increased by more than 1 time. It also improves the therapeutic effect on mycoplasma pneumonia. Especially for the Treg / Th17 imbalance caused by mycoplasma pneumonia, it has a significant callback effect and promotes the balance of Treg / Th17 cells.

[0014] Lactobacillus plantarum M3 (deposit number CGMCC NO.26065) is deposited in the China General Microbiological Culture Collection Center (CGMCC) and is published in the bulletin of the patent "Application of Lactobacillus plantarum M3 in improving the content of secondary saponins of Platycodon grandiflorum and lipid-lowering activity" (CN202410206570.X).

[0015] Lactobacillus brevis JW12 is Lactobacillus brevis and belongs to the genus Lactobacillus brevis; it is deposited in the China General Microbiological Culture Collection Center (CGMCC), the deposit address is No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, the deposit number is CGMCC No. 33957, and the deposit date is March 24, 2025.

[0016] Leuconostoc mesenteroides ZS is Leuconostoc mesenteroides and belongs to the genus Leucanostoc; it is deposited in the China General Microbiological Culture Collection Center (CGMCC), the deposit address is No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, the deposit number is CGMCC No. 33958, and the deposit date is March 24, 2025. Description of the Drawings

[0017] Figure 1 It is the observation diagram of the lung tissues of rats in each experimental group in Example 7;

[0018] Figure 2 It is the ELISA detection result diagram of the serum and lung tissues of rats in each experimental group in Example 7;

[0019] Figure 3 It is a detection chart of the expression levels of RORγt and Foxp3 mRNAs in the intestinal and lung tissues of rats in each experimental group in Example 7;

[0020] Figure 4 It is a detection chart of the expression levels of RORγt and Foxp3 proteins in the intestinal and lung tissues of rats in each experimental group in Example 7. Detailed implementation manners

[0021] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0022] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments may be combined with each other.

[0023] Detailed implementation manner one: The compound Platycodon grandiflorum fermentation bacterium agent in this implementation manner is composed of two or three combinations of Lactobacillus plantarum M3, Lactobacillus brevis JW12, and Leuconostoc mesenteroides ZS.

[0024] Detailed implementation manner two: The difference between this implementation manner and detailed implementation manner one is that the quantity ratio between different strains in the fermentation bacterium agent is 1:1. Other steps and parameters are the same as those in detailed implementation manner one.

[0025] Detailed implementation manner three: The difference between this implementation manner and detailed implementation manner one or two is that the fermentation bacterium agent is composed of Lactobacillus plantarum M3, Lactobacillus brevis JW12, and Leuconostoc mesenteroides ZS. Other steps and parameters are the same as those in detailed implementation manner one or two.

[0026] Detailed implementation manner four: The difference between this implementation manner and detailed implementation manner one or two is that the fermentation bacterium agent is composed of Lactobacillus plantarum M3 and Lactobacillus brevis JW12. Other steps and parameters are the same as those in detailed implementation manner one or two.

[0027] Detailed implementation manner five: The difference between this implementation manner and detailed implementation manner one or two is that the fermentation bacterium agent is composed of Lactobacillus brevis JW12 and Leuconostoc mesenteroides ZS. Other steps and parameters are the same as those in detailed implementation manner one or two.

[0028] Specific Embodiment Six: The difference between this embodiment and Specific Embodiment One or Two lies in that the fermentation inoculant is composed of Lactobacillus plantarum M3 and Leuconostoc mesenteroides ZS. Other steps and parameters are the same as those in Specific Embodiment One or Two.

[0029] Specific Embodiment Seven: Method for fermenting platycodon grandiflorum with the compound platycodon grandiflorum fermentation inoculant: Remove impurities from platycodon grandiflorum, wash, moisten thoroughly, and slice, then soak in water for 8 - 12 h, followed by high-temperature sterilization. Then inoculate the compound platycodon grandiflorum fermentation inoculant liquid at an inoculation amount of 2 - 5% (v / w), ferment at 30°C ± 2°C for 6 d - 8 d, and then dry at 60°C ± 2°C to complete the fermentation of platycodon grandiflorum; among them, the compound platycodon grandiflorum fermentation inoculant liquid is composed of the liquid combinations of two or three kinds of bacteria among Lactobacillus plantarum M3, Lactobacillus brevis JW12, and Leuconostoc mesenteroides ZS.

[0030] As a pro-inflammatory effector cell, Th17 cells, together with Treg cells, a type of cells with immunosuppressive functions, jointly maintain immune homeostasis. An increase in the proportion of Th17 cells and a decrease in the number or function of Treg cells will lead to an imbalance between immunosuppression and pro-inflammatory responses.

[0031] Specific Embodiment Eight: The difference between this embodiment and Specific Embodiment Seven lies in that the thickness of the sliced platycodon grandiflorum is 3 - 5 mm. Other steps and parameters are the same as those in Specific Embodiment Seven.

[0032] Specific Embodiment Nine: The difference between this embodiment and Specific Embodiment Seven or Eight lies in that the water addition amount is 2 times the mass of platycodon grandiflorum; sterilize at 121°C for 15 min. Other steps and parameters are the same as those in Specific Embodiment Seven or Eight.

[0033] Specific Embodiment Ten: The difference between this embodiment and Specific Embodiment Seven, Eight, or Nine lies in that the quantity ratio between different strains in the compound platycodon grandiflorum fermentation inoculant liquid is 1:1. Other steps and parameters are the same as those in Specific Embodiment Seven, Eight, or Nine.

[0034] Specific Embodiment Eleven: The difference between this embodiment and any one of Specific Embodiments Seven to Ten lies in that the concentration of microorganisms in the compound platycodon grandiflorum fermentation inoculant liquid is 1×10 8 ~5×10 8 cfu / ml. Other steps and parameters are the same as those in any one of Specific Embodiments Seven to Ten.

[0035] Example 1

[0036] Method for fermenting platycodon grandiflorum with compound platycodon grandiflorum fermentation starter: Remove impurities from platycodon grandiflorum, wash, moisten thoroughly, slice, then soak in water for 8 h, followed by high-temperature sterilization. Then inoculate the compound platycodon grandiflorum fermentation starter liquid at an inoculation amount of 3% (v / w), ferment at 30 °C for 6.5 d, and then dry at 60 °C to complete the fermentation of platycodon grandiflorum. Among them, the compound platycodon grandiflorum fermentation starter liquid is composed of the liquid of Lactobacillus plantarum M3 and Lactobacillus brevis JW12 (A + B).

[0037] The thickness of the sliced platycodon grandiflorum is 3 mm. The water addition amount is 2 times the mass of platycodon grandiflorum; sterilize at 121 °C for 15 min. The quantity ratio between different strains in the compound platycodon grandiflorum fermentation starter liquid is 1:1. The concentration of microorganisms in the compound platycodon grandiflorum fermentation starter liquid is 2×10 8 ~3×10 8 cfu / ml.

[0038] Example 2

[0039] Method for fermenting platycodon grandiflorum with compound platycodon grandiflorum fermentation starter: Remove impurities from platycodon grandiflorum, wash, moisten thoroughly, slice, then soak in water for 10 h, followed by high-temperature sterilization. Then inoculate the compound platycodon grandiflorum fermentation starter liquid at an inoculation amount of 5% (v / w), ferment at 30 °C for 7 d, and then dry at 60 °C to complete the fermentation of platycodon grandiflorum. Among them, the compound platycodon grandiflorum fermentation starter liquid is composed of the liquid of Lactobacillus brevis JW12 and Leuconostoc mesenteroides ZS (B + C).

[0040] The thickness of the sliced platycodon grandiflorum is 4 mm. The water addition amount is 2 times the mass of platycodon grandiflorum; sterilize at 121 °C for 15 min. The quantity ratio between different strains in the compound platycodon grandiflorum fermentation starter liquid is 1:1. The concentration of microorganisms in the compound platycodon grandiflorum fermentation starter liquid is 3×10 8 ~4×10 8 cfu / ml.

[0041] Example 3

[0042] Method for fermenting platycodon grandiflorum with compound platycodon grandiflorum fermentation starter: Remove impurities from platycodon grandiflorum, wash, moisten thoroughly, slice, then soak in water for 12 h, followed by high-temperature sterilization. Then inoculate the compound platycodon grandiflorum fermentation starter liquid at an inoculation amount of 4% (v / w), ferment at 30 °C for 8 d, and then dry at 60 °C to complete the fermentation of platycodon grandiflorum. Among them, the compound platycodon grandiflorum fermentation starter liquid is composed of the liquid of Lactobacillus plantarum M3 and Leuconostoc mesenteroides ZS (A + C).

[0043] The thickness of the sliced platycodon grandiflorum is 5 mm. The water addition amount is 2 times the mass of platycodon grandiflorum; sterilize at 121 °C for 15 min. The quantity ratio between different strains in the compound platycodon grandiflorum fermentation starter liquid is 1:1. The concentration of microorganisms in the compound platycodon grandiflorum fermentation starter liquid is 3×10 8 ~5×10 8cfu / ml.

[0044] Example 4

[0045] Method for fermenting Platycodon grandiflorum with a compound Platycodon grandiflorum fermentation bacterium agent: Remove impurities from Platycodon grandiflorum, wash, moisten thoroughly, and slice. Then soak in water for 9 h, followed by high-temperature sterilization. After that, inoculate the compound Platycodon grandiflorum fermentation bacterium agent solution at an inoculation amount of 2% (v / w), and ferment at 30 °C for 7 d, and then dry at 60 °C to complete the fermentation of Platycodon grandiflorum. Among them, the compound Platycodon grandiflorum fermentation bacterium agent solution is composed of the bacterial solutions of Lactobacillus plantarum M3, Lactobacillus brevis JW12, and Leuconostoc mesenteroides ZS (A + B + C).

[0046] The thickness of the sliced Platycodon grandiflorum is 3 mm. The amount of added water is 2 times the mass of Platycodon grandiflorum; sterilize at 121 °C for 15 min. The quantity ratio between different strains in the compound Platycodon grandiflorum fermentation bacterium agent solution is 1:1:1. The concentration of microorganisms in the compound Platycodon grandiflorum fermentation bacterium agent solution is 2×10 8 ~4×10 8 cfu / ml.

[0047] Example 5

[0048] 1. Total saponin determination method

[0049] Preparation of reference substance solution: Take an appropriate amount of platycodin D reference substance, accurately weigh it, and make a solution containing 1 mg of platycodin D per 1 ml with methanol.

[0050] Preparation of standard curve: Accurately measure 40, 80, 120, 160, 200, 240 μl of the reference substance solution and place them in test tubes, evaporate to dryness, add 0.5 ml of 8% vanillin ethanol test solution and 5 ml of 72% sulfuric acid solution, shake well, heat in a water bath at 60 °C for 10 minutes, immediately cool in an ice-water bath for 10 minutes, and shake well. Using the corresponding reagent as the blank, according to the ultraviolet-visible spectrophotometry (General Principles 0401 of the Chinese Pharmacopoeia 2020 Edition), measure the absorbance at a wavelength of 470 nm. Taking the absorbance as the ordinate and the concentration as the abscissa, plot the standard curve.

[0051] Determination method: Take 0.1 g of the powder of the sample to be tested (passing through No. 2 sieve), accurately weigh it, add 25 ml of methanol, reflux and extract 2 times, each time for 1.5 hours, filter, wash the medicinal residues with an appropriate amount of methanol, combine the filtrates, and evaporate to dryness. Dissolve the residue in methanol and transfer it to a 5 ml volumetric flask, dilute to the mark with methanol, shake well, and use it as the test solution. Accurately pipette 40 μl of the test solution into a test tube, and according to the method under the preparation of the standard curve, starting from "evaporate to dryness", determine the absorbance according to law. Read the amount of platycodin D in the test solution from the standard curve and calculate.

[0052] 2. Determination of platycodin D

[0053] Determined by high performance liquid chromatography (General Principles 0512 of Chinese Pharmacopoeia 2020 Edition).

[0054] Chromatographic conditions and system suitability test: Using octadecylsilane chemically bonded silica gel as the filler; using acetonitrile - water (25:75) as the mobile phase; detected by evaporative light scattering detector; the number of theoretical plates calculated by platycodin D peak should be not less than 3000.

[0055] Preparation of reference solution: Take an appropriate amount of platycodin D reference substance, weigh accurately, and make a solution containing 0.1 mg of platycodin D per 1 ml with methanol.

[0056] Preparation of test solution: Take about 1.0 g of the powder of the sample to be tested (passed through No. 2 sieve), weigh accurately, place it in a stoppered conical flask, accurately add 50 ml of 50% methanol, weigh, ultrasonically treat (power 250 W, frequency 40 kHz) for 45 minutes, cool, weigh again, make up the lost weight with 50% methanol, shake well, filter; accurately measure 25 ml of the subsequent filtrate, evaporate to dryness, add 20 ml of water to the residue, heat slightly to dissolve, extract with water - saturated n - butanol by shaking 3 times, 20 ml each time, combine the n - butanol layers, wash with 50 ml of ammonia test solution, discard the ammonia layer, then wash with 50 ml of water saturated with n - butanol, discard the water layer, recover the solvent of the n - butanol layer to dryness, dissolve the residue with an appropriate amount of methanol, transfer it to a 5 - ml volumetric flask, add methanol to the scale, shake well, filter, and take the subsequent filtrate.

[0057] Assay method: Accurately pipette 10 μl and 20 μl of the reference solution respectively, and 20 μl of the test solution, inject into the liquid chromatograph for determination, and calculate by the logarithmic equation of external standard two - point method.

[0058] Comparative experiment of Example 6

[0059] Control 1: Remove impurities from platycodon grandiflorum, wash, moisten thoroughly, slice, then soak in water for 9 h, then sterilize at high temperature, store in an environment of 30 °C for 7 d and then dry at 60 °C. The thickness of the platycodon grandiflorum slices after slicing is 3 mm. The amount of water added is 2 times the mass of platycodon grandiflorum; sterilize at 121 °C for 15 min.

[0060] Control 2: Remove impurities from platycodon grandiflorum, wash, moisten thoroughly, slice, then soak in water for 8 h, then sterilize at high temperature, and then inoculate with Lactobacillus plantarum M3 bacterial liquid (A) at an inoculation amount of 3% (v / w), ferment at 30 °C for 6.5 d and then dry at 60 °C, thus completing the fermentation of platycodon grandiflorum. The thickness of the platycodon grandiflorum slices after slicing is 3 mm. The amount of water added is 2 times the mass of platycodon grandiflorum; sterilize at 121 °C for 15 min. The concentration of microorganisms in the Lactobacillus plantarum M3 is 2×10 8 ~3×10 8 cfu / ml.

[0061] Control 3: Remove impurities from platycodon root, wash, moisten thoroughly, slice, then soak in water for 8 h, followed by high-temperature sterilization. After that, inoculate with Lactobacillus brevis JW12 bacterial liquid (B) at an inoculation amount of 5% (v / w), ferment at 30 °C for 7 d, and then dry at 60 °C to complete the fermentation of platycodon root. The thickness of the sliced platycodon root is 4 mm. The water addition amount is 2 times the mass of platycodon root; sterilize at 121 °C for 15 min. The concentration of microorganisms in Lactobacillus brevis JW12 is 2×10 8 ~3×10 8 cfu / ml.

[0062] Control 4: Remove impurities from platycodon root, wash, moisten thoroughly, slice, then soak in water for 8 h, followed by high-temperature sterilization. After that, inoculate with Leuconostoc mesenteroides ZS bacterial liquid (C) at an inoculation amount of 4% (v / w), ferment at 30 °C for 8 d, and then dry at 60 °C to complete the fermentation of platycodon root. The thickness of the sliced platycodon root is 5 mm. The water addition amount is 2 times the mass of platycodon root; sterilize at 121 °C for 15 min. The concentration of microorganisms in Leuconostoc mesenteroides ZS is 2×10 8 ~3×10 8 cfu / ml.

[0063] In this example, the method of Example 5 was used to detect Examples 1 - 4 and Controls 1 - 4 of this example. The detection data are shown in Table 1.

[0064] Table 1 Determination of platycodin D and total platycodon saponins (X±S, n = 3)

[0065]

[0066] Example 7

[0067] As pro-inflammatory effector cells, Th17 cells, together with Treg cells, a type of cells with immunosuppressive functions, jointly maintain immune homeostasis. An increase in the proportion of Th17 cells and a decrease in the number or function of Treg cells will lead to an imbalance between immunosuppression and pro-inflammatory responses. Studies have confirmed that the imbalance of Treg / Th17 cells is an important feature of patients with Mycoplasma pneumoniae infection. There is evidence that the Treg function remains impaired in patients with mycoplasma pneumonia 1 year after infection, while the Treg function has recovered in patients with pneumonia caused by other microbial infections. Therefore, regulating the balance of Treg / Th17 cells and repairing Treg function are the keys to treating Mycoplasma pneumoniae infection.

[0068] The fermented Platycodon grandiflorum prepared in Example 3 was used for extraction: Take the fermented Platycodon grandiflorum of Example 3, add 8 times the volume of 75% ethanol, heat under reflux for extraction 3 times, 2 hours each time, filter, and combine the filtrates; The filtrate was concentrated under reduced pressure to recover ethanol to a thick paste to obtain the fermented Platycodon grandiflorum extract; Then the fermented Platycodon grandiflorum extract was dissolved in water to form a solution containing 1 g of crude drug per 1 mL of crude drug amount. AB-8 macroporous adsorption resin was used, the sample loading amount was such that the mass ratio of dry resin to medicinal material was 1:4, the column diameter to height ratio was 1:20, the sample loading flow rate was 2 BV / h, and it was eluted with 5 BV of water, 25% ethanol, and 50% ethanol respectively, controlling the elution rate at 1.5 BV / h. The 50% ethanol eluate was collected, concentrated under reduced pressure, and dried to obtain the fermented Platycodon grandiflorum extract (FPGS).

[0069] Take the unfermented Platycodon grandiflorum and extract it according to the above method to obtain the Platycodon grandiflorum extract (PGS).

[0070] Establishment, administration, and pharmacodynamic observation of the MPP (Mycoplasma pneumoniae pneumonia) rat model:

[0071] 50 SPF-grade Wistar rat pups, half male and half female, were raised in a constant temperature and humidity environment, with a body weight of 100 - 120 g, and adaptively raised for 7 days. Then the rats were divided into 5 groups, namely the blank group (KB), the model group (MX), the azithromycin group (AQ), the PGS group, and the FPGS group, with 10 rats in each group. They were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. Except for the blank group, 10 6 CCU·mL -1 50 μL of Mycoplasma pneumoniae (MP) bacterial liquid was slowly dropped into the nostrils of the rats in the other groups. Using their natural inspiration action, the MP bacterial liquid was inhaled into the airway to the lungs, and the blank control group was dropped with an equal amount of culture medium (culture medium without MP). The drug administration groups were given the medicinal liquid by gavage every day. The azithromycin group was administered for 5 days (45 mg·kg -1 ·d -1 for the first time, 22.5 mg·kg -1 ·d -1 ) for the 2nd - 5th days); The PGS and FPGS groups were administered according to the crude drug amount of 0.9 g·kg -1 ·d -1 for 10 days; The blank group and the model group were given an equal amount of pure water.

[0072] The experimental results are as Figure 1As shown, in the KB group, the alveolar structure was clear, the alveolar wall was thin, there was no edema fluid in the alveolar cavity, and no inflammatory cell infiltration was seen in the lung tissue; in the MX group, the lung tissue of the rats showed slightly thickened alveolar walls, atrophy of a large number of alveoli, compensatory enlargement of the surrounding alveolar cavities, widened alveolar septa, and accompanied by a small amount of lymphocyte infiltration (blue arrow), degeneration of a small number of bronchial epithelial cells, occasional hemorrhage, focal lymphocyte infiltration around a small number of blood vessels (yellow arrow), and congestion of a small number of blood vessels (green arrow); in the AQ group, the lung tissue showed slightly thickened alveolar walls, atrophy of a small number of alveoli, compensatory enlargement of the surrounding alveolar cavities, widened alveolar septa, and accompanied by a small amount of lymphocyte infiltration (blue arrow), and occasional very small amounts of lymphocyte infiltration around blood vessels (yellow arrow); in the PGS group, the lung tissue showed degeneration of a small number of bronchial epithelial cells, slightly thickened alveolar walls, widened alveolar septa, accompanied by a small amount of lymphocyte infiltration (blue arrow), and no obvious abnormalities in the interstitial blood vessels; in the FPGS group, the lung tissue showed slightly thickened alveolar walls, atrophy of a small number of alveoli, compensatory enlargement of the surrounding alveolar cavities, widened alveolar septa, and accompanied by extremely small amounts of lymphocytic infiltration. The above results indicate that the inflammation in each drug-administered group was significantly alleviated.

[0073] Detection of inflammatory factors in rats:

[0074] Serum was drawn from the rats in each experimental group, stored frozen, thawed, centrifuged at 3500 r / min for 5 min at 4°C, and the supernatant was taken. After the lung tissue was thawed, the weight of the lung tissue was accurately weighed and 9 times the volume of homogenization medium (0.9% normal saline) was added at a ratio of weight (mg): volume (μL) = 1:9. Under ice-water bath conditions, mechanical homogenization was performed to prepare a 10% homogenate, centrifuged at 2500 - 3000 revolutions per minute for 10 minutes, and the supernatant was taken. The contents of inflammatory factors IL-10 and IL-17 in the serum and lung tissue of the rats were measured according to the ELISA kit.

[0075] By measuring the inflammatory factors IL-10 and IL-17, the effect of FPGS in treating MPP was evaluated (the ELISA test results of the serum and lung tissue of the rats in each experimental group are shown in Table 2 and Figure 2 as follows). Compared with the blank group, the level of IL-10 in the model group decreased significantly, and the level of IL-17 increased significantly (P < 0.01). Compared with the model group, the level of IL-10 in the serum and lung tissue of each drug-administered group increased significantly (P < 0.05, P < 0.01), and the level of IL-17 decreased significantly (P < 0.05, P < 0.01). Compared with the PGS group, the release of the inflammatory factor IL-17 in the serum and lung tissue of the FPGS group decreased by 10.40% and 4.22% respectively, and the release of the anti-inflammatory factor IL-10 increased by 5.26% and 8.28% respectively.

[0076] Table 2 Detection of inflammatory factors (X±S, n = 10)

[0077]

[0078] Compared with KB: ##: P < 0.01, #: P < 0.05. Compared with MX: **: P < 0.01, *: P < 0.05.

[0079] In this example, through pharmacodynamic experiments, it is proved that after the platycodon grandiflorum of the present invention is fermented, it can effectively improve the improvement effect of its extract on the lung tissue pathology and inflammatory factors of rats with mycoplasma pneumoniae. FPGS can improve the pathological manifestations of interstitial pneumonia caused by mycoplasma pneumoniae, restore the thickening of the alveolar wall, and reduce the infiltration of inflammatory cells. It inhibits the secretion of the pro-inflammatory cytokine IL-17 and increases the expression of the anti-inflammatory factor IL-10.

[0080] RORγt is a key transcription factor for the specific differentiation of Th17, and Foxp3 is a specific transcription factor for the maturation and function maintenance of peripheral Tregs. The expressions of RORγt, Foxp3 mRNA and protein in the intestinal and lung tissues of rats were measured by PCR and Western Blot methods, and the effects of FPGS on their expressions were analyzed to clarify the effects of FPGS on immune cells.

[0081] The expressions of RORγt mRNA and Foxp3 mRNA were measured by PCR. Compared with the blank group, Foxp3 decreased and RORγt increased in the lung and intestinal tissues of the model group rats (P < 0.01); compared with the model group, Foxp3 increased and RORγT decreased in the lung and intestinal tissues of each drug administration group (P < 0.05, P < 0.01), as Figure 3 and Table 3 show. Compared with the PGS group, the expression levels of RORγt mRNA in the lung and intestinal tissues of FPGS decreased by 5.01% and 15.40% respectively, and the expression levels of Foxp3 mRNA increased by 11.33% and 8.46% respectively.

[0082] Table 3 Determination of Foxp3 and RORγt mRNA (X±S, n = 10)

[0083]

[0084] Compared with KB: ## : P < 0.01, # : P < 0.05. Compared with MX: ** : P < 0.01, * : P < 0.05

[0085] The expression of RORγt and Foxp3 proteins was detected by Western Blot. Compared with the blank group, the level of Foxp3 decreased while the level of RORγt increased in the lung and intestinal tissues of the model group rats (P < 0.01). Compared with the model group, the level of Foxp3 increased while the level of RORγt decreased in the lung and intestinal tissues of each drug administration group (P < 0.05, P < 0.01), as Figure 4 shown in Table 4. Compared with the PGS group, the expression levels of RORγt protein in the lung and intestinal tissues of the FPGS group decreased by 11.45% and 6.35% respectively, while the expression levels of Foxp3 protein increased by 8.55% and 12.14% respectively.

[0086] Table 4 Determination of Foxp3 and RORγt proteins (X±S, n = 10)

[0087]

[0088] Compared with KB: ## : P < 0.01, # : P < 0.05. Compared with MX: ** : P < 0.01, * : P < 0.05

[0089] Based on the above experimental results, the expressions of RORγt mRNA and protein in the lung and intestinal tissues of MPP rats increased, while the expressions of Foxp3 mRNA and protein decreased, indicating enhanced Th17 cell-related inflammatory responses and weakened Treg cell functions. While reducing the expressions of RORγt mRNA and protein, PGS and FPGS could increase the expressions of Foxp3 mRNA and protein, suggesting that the drugs inhibit the expression of RORγt to reduce the differentiation and function of Th17 cells, thereby reducing inflammatory responses, and promote the expression of Foxp3 to enhance the function of Treg cells, thus inhibiting excessive immune responses, regulating the balance between Th17 and Treg cells, repairing Treg functions, and achieving the effect of rapid treatment of Mycoplasma pneumoniae infection. Simultaneous determination of the lung and intestinal tissues also re-verified the traditional Chinese medicine theory that "the lung and the large intestine are interior-exteriorly related" and the treatment principle of treating the lung and the large intestine simultaneously, and the effect of FPGS was better than that of PGS.

Claims

1. A composite platycodon fermentation agent, characterized in that: The fermentation bacterial agent is composed of two or three of plant lactobacillus M3, Lactobacillus brevis JW12 and Leuconostoc mesenteroides ZS.

2. The composite platycodon fermentation agent according to claim 1, characterized in that The quantity ratios of different strains in the fermentation bacterial agent are all 1:

1.

3. A method for fermenting Platycodon grandiflorum using the composite Platycodon grandiflorum fermentation agent according to claim 1, characterized in that: The method proceeds as follows: The Platycodon grandiflorum is cleaned, soaked, sliced, and then soaked in water for 8-12 hours, sterilized at high temperature, and then inoculated with a 2-5% (v / w) inoculum of a composite Platycodon grandiflorum fermentation agent, fermented in an environment of 30°C±2°C for 6-8 days, and then dried at 60°C±2°C, thereby completing the fermentation of Platycodon grandiflorum; The bacterial liquid of the composite platycodon fermentation agent is composed of the bacterial liquid of two or three bacteria selected from Lactobacillus plantarum M3, Lactobacillus brevis JW12, and Leuconostoc mesenteroides ZS.

4. The method for fermenting Platycodon grandiflorum according to claim 3, characterized in that: The thickness of Platycodon grandiflorum after slicing is 3 to 5 mm; the amount of water added is twice the mass of Platycodon grandiflorum.

5. The method for fermenting Platycodon grandiflorum according to claim 3, characterized in that: Sterilize at 121℃ for 15min.

6. The method for fermenting Platycodon grandiflorum according to claim 3, characterized in that: The quantity ratios of different strains in the bacterial liquid of the composite platycodon fermentation agent are all 1:

1.

7. The method for fermenting Platycodon grandiflorum according to claim 3, 4, 5 or 6, characterized in that: The concentration of microorganisms in the bacterial solution of the composite platycodon fermentation agent is 1×10 8 ~5×10 8 cfu / ml.

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