Composition capable of resisting bacterial pneumonia, preparation method and application

Abstract

The invention belongs to the technical field of traditional Chinese medicine, and particularly relates to a composition capable of resisting bacterial pneumonia, a preparation method and application. The composition is prepared by fermenting a mixture of a traditional Chinese medicine composition, isomaltooligosacharide and mint powder through compound probiotics, and the traditional Chinese medicine composition comprises the following raw materials: 10-25 parts of loquat leaves, 5-15 parts of momordica grosvenori, 5-15 parts of liquorice, 2-10 parts of orange peel and 1-8 parts of scaphium scaphigerum. The compound probiotics are prepared from the following components in parts by mass: 1 to 2 parts of plant lactobacillus HCS03-001, 1 to 1 part of lactobacillus reuteri HCS02-001 and 1 part of lactobacillus rhamnosus HCS01-013. The medicinal and edible raw material extracting solution is used as a culture medium for fermentation, and the composition generated by combining specific lactic acid bacteria can be used for preparing bacterial pneumonia resisting products.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and particularly relates to a composition capable of resisting bacterial pneumonia, a preparation method and an application thereof. Background Art

[0002] Cough is one of the most common symptoms seen in respiratory medicine clinics, especially chronic cough, which is often accompanied by dry throat, cough, a foreign body sensation in the throat, and hoarseness. This includes acute and chronic pharyngitis (hoarseness and foreign body sensation), as well as seasonal and environmental pharyngeal discomfort (dry throat, cough, a foreign body sensation in the throat, and hoarseness).

[0003] The factors that cause throat and lung discomfort can be roughly divided into the following categories: 1. External factors: If the climate in the living area is cold and dry, the working environment is polluted by dust and chemical gases, or the throat is irritated by tobacco, alcohol, spicy food for a long time. Bacterial infection, viral infection such as pneumonia, mycoplasma infection, etc.

[0004] 2. Physical factors: Pharyngitis can also be a local manifestation of certain systemic diseases, such as anemia, indigestion, long-term constipation, heart disease, bronchitis, asthma, liver disease, diabetes and chronic nephritis.

[0005] 3. Occupational factors: This mainly occurs in people who work with the voice, such as teachers, actors, etc. Long-term multilingual singing and performing can irritate the pharynx, causing chronic congestion and leading to the disease.

[0006] The formula for treating cough, pharyngitis and pneumonia is mainly based on the principles of clearing the lungs, resolving phlegm and relieving cough, moistening the lungs and nourishing yin, moistening dryness and relieving cough.

[0007] Chinese invention patent application CN202410267996.6 discloses a throat-clearing and throat-moistening product, comprising a main drug and auxiliary materials, wherein the main drug is composed of the following components in parts by weight: 25-30 parts of loquat leaves, 10-15 parts of tangerine peel, 6-8 parts of green fruit, 6-8 parts of platycodon, 5-7 parts of licorice, 6-8 parts of momordica grosvenori, 10-12 parts of sterculia lychnophora, 3-5 parts of lily, 10-12 parts of mint, 6-8 parts of chrysanthemum, 10-15 parts of snow pear, 8-10 parts of tangerine peel, 8-10 parts of cimicifuga and 12-15 parts of bupleurum; also disclosed is a method for preparing the throat-clearing and throat-moistening product, comprising the following steps: S1, slicing the main drug and soaking it; S2, boiling and filtering the slices; S3, concentrating, drying and crushing the filtrate; S4, whole-granulating; S5, tableting, sterilizing and packaging. The invention eliminates the clinical symptoms of cough, sore throat, foreign body sensation in the throat, itchy and dry throat, and dry mucosa quickly and effectively; it can significantly improve throat discomfort and throat inflammation, and has the effect of clearing and moistening the throat. Another Chinese invention patent application, CN201510886390.1, discloses a traditional Chinese medicine composition for treating cough. The composition comprises the following ingredients and dosages by weight: 3-18 parts of chrysanthemum, 2-12 parts of ephedra, 2-12 parts of reed rhizome, 2-12 parts of poria, 12-25 parts of liquorice, 10-21 parts of stemona, 2-12 parts of capillaris, 5-17 parts of salvia miltiorrhiza, 10-22 parts of houttuynia cordata, 11-27 parts of dalbergia odorifera, 7-22 parts of pistachios, 1-2 momordica grosvenori, 9-15 parts of mulberry leaf, 3-9 parts of tangerine peel, 15-31 parts of mint, 11-19 parts of loquat leaf, 7-15 parts of artemisia annua, 11-23 parts of panax notoginseng, 5-10 parts of scutellaria baicalensis, 10-20 parts of immature bitter orange, and 10-25 parts of aster flower. The traditional Chinese medicine composition for treating cough of the invention has the effects of promoting lung function and clearing away heat, resolving phlegm and relieving cough, moistening the lung and relieving asthma, strengthening the spleen and replenishing qi, regulating the middle and promoting diuresis. It has significant efficacy in treating cough, reliable effect, simple preparation, mild medicinal properties and no toxic side effects.

[0008] Currently, the traditional Chinese medicine compound prescriptions that can treat pharyngitis and pneumonia either have too many medicinal flavors, which is not convenient for pharmacies or patients to decoct, have poor clinical compliance, or their effectiveness needs to be further improved. Summary of the Invention

[0009] In view of the deficiencies in the prior art, the present invention provides a composition capable of resisting bacterial pneumonia, a preparation method and application.

[0010] In order to achieve the purpose of the present invention, the technical solutions adopted are as follows: On the one hand, the present invention provides a composition capable of resisting bacterial pneumonia. The composition is fermented into a traditional Chinese medicine composition by composite probiotics. The raw materials of the traditional Chinese medicine composition are 10-25 parts of loquat leaves, 5-15 parts of momordica grosvenori, 5-15 parts of licorice, 2-10 parts of orange peel and 1-8 parts of sterculia lychnophora. The composite probiotics are Lactobacillus plantarum HCS03-001, Lactobacillus reuteri HCS02-001 and Lactobacillus rhamnosus HCS01-013 in a mass ratio of 1-2:1:1.

[0011] Preferably, the composition capable of resisting bacterial pneumonia further comprises isomaltooligosaccharide and mint powder, and the mass ratio of the traditional Chinese medicine composition, isomaltooligosaccharide and mint powder is 1000:10-40:3-20.

[0012] In a second aspect, the present invention provides a method for preparing a composition capable of resisting bacterial pneumonia, comprising the following steps: (1) extracting the Chinese herbal medicine composition to obtain an extract, adding isomaltooligosaccharide and mint flavor powder to the mixture, and sterilizing to obtain a fermentation substrate; (2) Preparing the composite probiotics into a bacterial solution and activating it; (3) The activated bacterial liquid is inoculated into the fermentation substrate and fermentation is completed.

[0013] Preferably, in step (1), the raw materials of the traditional Chinese medicine composition are mixed, soaked in water, extracted, and filtered to obtain an extract.

[0014] Preferably, the extract contains 23-73 g crude drug / L.

[0015] Preferably, the extraction in step (1) includes two extractions, the ratio of the total mass of the added water to the total mass of the raw materials of the Chinese medicine composition is 45-55:1, the extraction temperature is 95-105°C, the time of the first extraction is 50-70 minutes, the time of the second extraction is 10-30 minutes, and the filtration is through 80-120 mesh.

[0016] Preferably, the sterilization in step (1) is performed at 110-120° C. for 20-40 minutes.

[0017] Preferably, in step (2), the composite probiotics are added with water to prepare a bacterial solution, and the mass volume ratio of the composite probiotics to water is 1 kg:8-12 L.

[0018] Preferably, the activation in step (2) comprises inoculating the bacterial solution into the activation medium at an inoculum size of 0.03-0.08%.

[0019] Preferably, the activation medium in step (2) comprises 900-910 g / L of water, 30-55 g / L of anhydrous glucose, 15-35 g / L of yeast extract, 8-12 g / L of yeast peptone, 2-4 g / L of L-malic acid, 1-3 g / L of citric acid monohydrate, 1-3 g / L of potassium dihydrogen phosphate, 0.3-0.7 g / L of calcium chloride, 0.04-0.08 g / L of magnesium sulfate, and 0.005-0.015 g / L of manganese sulfate.

[0020] Preferably, the culturing in step (2) includes agitation culturing with a stirring speed of 95-105 r / min and an air pressure of 0.02-0.03 MPa; and culturing at 37±0.2°C for 16 h±1 h.

[0021] Preferably, in step (3), the activated bacterial liquid is inoculated into the fermentation substrate at an inoculum amount of 2-8%, and fermented at 37±0.2°C for 48-72 h with stirring at a stirring speed of 35-45 r / min.

[0022] Preferably, after the fermentation in step (3), the fermentation liquid is passed through an 80-120 mesh filter and then centrifuged at a speed of 8000-11000 r / min for 3-5 min. After the centrifugation in step (3), the fermentation liquid is concentrated 1-3 times and sterilized.

[0023] In a third aspect, the present invention provides use of the above-mentioned composition capable of resisting bacterial pneumonia in the preparation of products for resisting bacterial pneumonia and / or clearing the throat and relieving cough.

[0024] Compared with the prior art, the present invention has the following beneficial effects: (1) The extract of edible and medicinal raw materials is used as a culture medium for fermentation, and combined with the postbiotics produced by several specific lactic acid bacteria, it produces substances that are beneficial to the throat and lungs, while combining the effects of probiotics and edible raw materials.

[0025] (2) The present invention uses a specific Chinese medicine composition, which has a significantly better anti-pneumonia effect than other large prescriptions after fermentation.

[0026] (3) The present invention uses a composite probiotic of Lactobacillus plantarum HCS03-001: Lactobacillus mucilaginosus HCS02-001: Lactobacillus rhamnosus HCS01-013 in a specific ratio, and the fermentation liquid produced is significantly more effective than other probiotic strains.

[0027] (4) The activation culture medium of the present invention can better activate the bacteria and achieve a more outstanding fermentation effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a picture of neutrophils in the swim bladder of zebrafish after sample treatment. Note: Green fluorescent dots are neutrophils. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with specific embodiments. The following raw materials are all commercially available conventional raw materials.

[0030] Example 1 A composition capable of resisting bacterial pneumonia is prepared by fermenting a mixture of a traditional Chinese medicine composition, isomaltooligosaccharide, and mint powder with composite probiotics. The raw materials of the traditional Chinese medicine composition are loquat leaves, momordica grosvenori, liquorice, tangerine peel, and puffed sea. The composite probiotics are powders of Lactobacillus plantarum HCS03-001, Lactobacillus mucilaginosus HCS02-001, and Lactobacillus rhamnosus HCS01-013 in a mass ratio of 2:1:1.

[0031] The preparation method of the composition comprises the following steps: (1) Place the raw materials of the Chinese medicine composition (formula see Table 1) directly into the multifunctional extraction tank and soak them completely with water; Primary extraction: Mix the raw materials of the Chinese medicine composition, add 75% of the total water amount (see Table 1 for the total water amount), and steam and extract for 50 min; filter through 100 mesh to obtain filtrate 1, and pump the material into a secondary fermentation tank for temporary storage; Secondary extraction: add water to the extraction tank again, the amount of water added is 25% of the total water volume, and boil and extract for 20 minutes; after the extraction is completed, filter through 100 mesh to obtain filtrate 2, and pump it into the secondary fermentation tank through a pump. Start stirring (stirring speed 40 rpm), mix the two filtrates evenly, and obtain the extract; The extract was mixed with isomaltooligosaccharide and mint powder according to the formula in Table 2, sterilized at 115°C for 30 min, and cooled to 37°C to obtain a fermentation substrate.

[0032] (2) Dissolve the composite probiotics (see Table 3) in sterile water at a ratio of 1:10 (kg:L) and stir thoroughly to remove lumps to obtain a bacterial solution. Inoculate the bacterial solution into the activation culture medium (formula see Table 4) at a 0.05% inoculum (v / v) and incubate at 37°C for 16 h. The culture was agitated at 100 r / min and maintained at a pressure of 0.02-0.03 MPa without ventilation.

[0033] (3) Aseptically inoculate the activated bacterial solution into the fermentation substrate at an inoculum volume of 5% (v / v), and ferment at 37±0.2℃ for 60h with stirring at a speed of 40r / min; culture without ventilation and maintain the air pressure at 0.02-0.03MPa.

[0034] Table 1

[0035] Table 2

[0036] Table 3

[0037] Table 4

[0038] Example 2 A composition capable of resisting bacterial pneumonia is prepared by fermenting a mixture of a traditional Chinese medicine composition, isomaltooligosaccharide, and mint powder with composite probiotics. The raw materials of the traditional Chinese medicine composition are loquat leaves, momordica grosvenori, liquorice, tangerine peel, and puffed sea cucumber. The composite probiotics are powders of Lactobacillus plantarum HCS03-001, Lactobacillus mucilaginosus HCS02-001, and Lactobacillus rhamnosus HCS01-013 in a mass ratio of 1:1:1.

[0039] The preparation method of the composition comprises the following steps: (1) Place the raw materials of the Chinese medicine composition (formula see Table 5) directly into the multifunctional extraction tank and soak them completely with water; Primary extraction: add 75% of the total water volume (see Table 5 for total water volume), steam and extract at 95°C for 60 min; filter through 100 mesh to obtain filtrate 1, pump it into a secondary fermentation tank, and store it temporarily; Secondary extraction: add water to the extraction tank again, the amount of water added is 25% of the total water volume, the temperature is 95°C, and the steam extraction is carried out for 10 minutes; after the extraction is completed, filter it through 100 mesh to obtain filtrate 2, and pump it into the secondary fermentation tank through a pump. Start stirring (stirring speed 40 rpm) and mix the two filtrates evenly to obtain the extract; The extract was mixed with isomaltooligosaccharide and mint powder according to the formula in Table 6, sterilized at 120°C for 20 min, and cooled to 37°C to obtain a fermentation substrate.

[0040] (2) Dissolve the composite probiotic powder (see Table 7 for details) in sterile water at a ratio of 1:12 (kg:L) and stir until smooth and free of lumps to obtain a bacterial solution. Inoculate the bacterial solution into the activation culture medium (see Table 8 for the formula) at a 0.08% inoculum (v / v) and incubate at 37 ± 0.2°C for 17 h with agitation at 100 r / min. Maintain aeration at a pressure of 0.02 MPa.

[0041] (3) The activated bacterial liquid was aseptically inoculated into the fermentation substrate at an inoculum size of 5%, and fermented at 37°C for 48 h with stirring at a speed of 40 r / min and an air pressure of 0.02 MPa.

[0042] Table 5

[0043] Table 6

[0044] Table 7

[0045] Table 8

[0046] Comparative Example 1 The difference between this comparative example and Example 1 is that the raw materials of the Chinese medicine composition are different, as shown in Table 9, and the rest are the same as Example 1.

[0047] Table 9

[0048] Comparative Example 2 The only difference between this comparative example and Example 1 is that the composite probiotic strains are different, as shown in Table 10 below.

[0049] Table 10

[0050] Comparative Example 3 The difference between this comparative example and Example 1 is that the ratio of the composite probiotic strains is different, as shown in Table 11, and the rest is consistent with Example 1.

[0051] Table 11

[0052] Comparative Example 4 The only difference between this comparative example and Example 1 is that the ratio of the activation culture medium is different, as shown in Table 12.

[0053] Table 12

[0054] Experimental example: Anti-bacterial pneumonia efficacy experiment 1. Maximum Detectable Concentration (MTC) Determination Transgenic neutrophil green fluorescent MPX zebrafish (5 days post-fertilization (dpf)) were randomly selected and plated in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples (concentrations shown in Table 13) were administered in water. A normal control group and a model control group were also established, with a volume of 3 mL per well. Except for the normal control group, all other experimental groups received LPS injected into the swim bladder to establish a zebrafish bacterial pneumonia model. After treatment at 28°C for 4 hours, the MTC of the samples in the model zebrafish was measured.

[0055] Under the experimental conditions, the anti-bacterial pneumonia efficacy of the composition of the present invention is 12.5 μL / mL in zebrafish. The results of the concentration exploration experiment on the anti-bacterial pneumonia efficacy of the composition of Example 1 are shown in Table 13.

[0056] Table 13 Results of the experimental study on the concentration of samples for the efficacy against bacterial pneumonia (n = 30)

[0057] 2. Evaluation of efficacy against bacterial pneumonia 5-day-old zebrafish of the MPX strain, transgenic for green neutrophil fluorescence, were randomly selected and plated in 6-well plates. Thirty zebrafish were treated in each well (experimental group). Samples were administered in water (concentrations shown in Table 14). A positive control group received dexamethasone acetate at a concentration of 43.5 μg / mL. A normal control group and a model control group were also established. Each well contained 3 mL of solution. Except for the normal control group, all other experimental groups received LPS intra-swim injections to establish a zebrafish bacterial pneumonia model. After treatment at 28°C for 4 hours, ten randomly selected zebrafish from each experimental group were photographed under a fluorescence microscope. Data were acquired using NIS-Elements D3.20 advanced image processing software. Neutrophil counts in the swim bladder were analyzed, and statistical analysis of this indicator was used to evaluate the anti-bacterial pneumonia efficacy of the samples. Statistical analysis was performed using SPSS 26.0 software. P < 0.05 indicated a statistically significant difference.

[0058] Under the experimental conditions, the composition of the present invention has the effect of resisting bacterial pneumonia, as shown in Table 14. The neutrophils in the swim bladder of zebrafish treated with different concentrations of the composition in representative Example 1 are shown in Table 14. Figure 1 .

[0059] Table 14 Results of the experimental evaluation of the efficacy of samples against bacterial pneumonia (n = 10)

[0060] Note: Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001.

[0061] 3. Evaluation of efficacy against bacterial pneumonia 5dpf transgenic neutrophil green fluorescent MPX strain zebrafish were randomly selected in a 6-well plate, and 30 zebrafish were treated in each well. The experimental group was given 3.12μg / mL, 6.25μg / mL, and 12.5μg / mL of the composition of Example 1, and the positive control group was given 43.5μg / mL of dexamethasone acetate. A normal control group and a model control group were also set up, with a capacity of 3mL per well. Except for the normal control group, the rest of the experimental groups were injected with LPS into the swim bladder to establish a zebrafish bacterial pneumonia model. Three experiments were set up in parallel. After treatment at 28°C for 4h, total RNA from each group of zebrafish was extracted using a pre-loaded magnetic bead method universal RNA extraction kit C, and the total RNA concentration and purity were measured using a UV-visible spectrophotometer. 2.00μg of total RNA from the zebrafish sample was taken, and 20.0μL of cDNA was synthesized according to the instructions of the cDNA first-strand synthesis kit. The expression of β-actin and tgfβ-1 genes was detected by q-PCR. β-actin was used as an internal control for gene expression to calculate the relative RNA expression of the tgfβ-1 gene. Statistical analysis was performed using SPSS 26.0 software. P < 0.05 indicated statistical significance.

[0062] Test results: 1) RNA extraction results At the end of the experiment, zebrafish total RNA was extracted, and the RNA concentration and A260 / A280 ratio were measured using a UV-visible spectrophotometer (Table 15). The A260 / A280 ratios were both between 1.8 and 2.2, indicating that the extracted zebrafish total RNA was of good quality and could be used for subsequent q-PCR experiments.

[0063] Table 15 Total RNA concentration and A260 / A280 ratio (n = 3)

[0064] 2) Evaluation of efficacy against bacterial pneumonia When LPS induces acute lung injury, inflammatory cells such as neutrophils are recruited and activated. After activation, inflammatory cells infiltrate the lung tissue and release inflammatory mediators, causing an increase in the number of neutrophils and macrophages in zebrafish, and an increase in the relative expression levels of tgfb1a, il1b, and tnfa genes.

[0065] Under the experimental conditions, the composition of the present invention has an anti-bacterial pneumonia effect, which is specifically manifested by down-regulating the relative expression level of the tgfβ-1 gene, as shown in Table 16 for details.

[0066] Table 16 Sample anti-bacterial pneumonia efficacy evaluation (gene) experimental results

[0067] Note: Compared with the model control group, *p<0.05, **p<0.01.

[0068] The above detailed description is a specific description of one feasible embodiment of the present invention. This embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not depart from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A composition capable of resisting bacterial pneumonia, characterized in that: The composition is a traditional Chinese medicine composition fermented by composite probiotics. The raw materials of the traditional Chinese medicine composition are 10-25 parts of loquat leaves, 5-15 parts of momordica grosvenori, 5-15 parts of licorice, 2-10 parts of orange peel and 1-8 parts of pistachio tataricus. The composite probiotics are Lactobacillus plantarum HCS03-001, Lactobacillus mucilaginosus HCS02-001 and Lactobacillus rhamnosus HCS01-013 in a mass ratio of 1-2:1:

1.

2. The composition capable of resisting bacterial pneumonia according to claim 1, characterized in that The composition further comprises isomaltooligosaccharide and mint flavor powder, and the mass ratio of the traditional Chinese medicine composition, isomaltooligosaccharide and mint flavor powder is 1000:10-40:3-20.

3. A method for preparing the composition capable of resisting bacterial pneumonia according to claim 2, characterized in that: The steps include: (1) extracting the Chinese herbal medicine composition to obtain an extract, adding isomaltooligosaccharide and mint flavor powder to the mixture, and sterilizing to obtain a fermentation substrate; (2) Preparing the composite probiotics into a bacterial solution and activating it; (3) The activated bacterial liquid is inoculated into the fermentation substrate and fermentation is completed.

4. The preparation method according to claim 3, characterized in that In step (1), the raw materials of the Chinese medicine composition are mixed, soaked in water, extracted, and filtered to obtain an extract.

5. The preparation method according to claim 4, characterized in that The extract contains 23-73g crude drug / L; The extraction in step (1) includes two steaming extractions, the ratio of the total mass of water added to the total mass of the raw materials of the Chinese medicine composition is 45-55:1, the time of the first extraction is 50-70 minutes, the time of the second extraction is 10-30 minutes, and the filtration is through 80-120 mesh; The sterilization in step (1) is performed at 110-120°C for 20-40 minutes.

6. The preparation method according to claim 3, characterized in that In step (2), the composite probiotics are added with water to prepare a bacterial solution, and the mass volume ratio of the composite probiotics to water is 1 kg:8-12 L; the activation in step (2) includes inoculating the bacterial solution into the activation culture medium at an inoculum amount of 0.03-0.08%.

7. The preparation method according to claim 6, characterized in that The activation culture medium in step (2) comprises 900-910 g / L of water, 30-55 g / L of anhydrous glucose, 15-35 g / L of yeast extract, 8-12 g / L of yeast peptone, 2-4 g / L of L-malic acid, 1-3 g / L of citric acid monohydrate, 1-3 g / L of potassium dihydrogen phosphate, 0.3-0.7 g / L of calcium chloride, 0.04-0.08 g / L of magnesium sulfate, and 0.005-0.015 g / L of manganese sulfate. The activation in step (2) comprises agitation culture at a stirring speed of 95-105 r / min, an air pressure of 0.02-0.03 MPa, and culture at 37±0.2°C for 16 h±1 h.

8. The preparation method according to claim 3, characterized in that In step (3), the activated bacterial liquid is inoculated into the fermentation substrate at an inoculum amount of 2-8%, and fermented at 37±0.2°C for 48-72h with stirring at a stirring speed of 35-45 r / min.

9. The preparation method according to claim 3, characterized in that After the fermentation in step (3), the fermentation liquid is passed through an 80-120 mesh filter and then centrifuged at a speed of 8000-11000 r / min for 3-5 min. After the centrifugation in step (3), the fermentation liquid is concentrated 1-3 times and sterilized.

10. Use of the composition capable of resisting bacterial pneumonia according to any one of claims 1 to 2 in the preparation of a product for resisting bacterial pneumonia and / or clearing the throat and relieving cough.

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