A composition, method of making and use capable of combating bacterial pneumonia

Abstract

The application 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, oligoisomaltose and a peppermint-flavored powder through complex probiotics. The raw materials of the traditional Chinese medicine composition are 10-25 parts of leaves of loquat, 5-15 parts of momordica grosvenori, 5-15 parts of licorice, 2-10 parts of orange peel and 1-8 parts of fat sea. The complex probiotics are plant lactobacillus HCS03-001, royi mucous lactobacillus HCS02-001 and rhamnose lactobacillus HCS01-013 with a mass ratio of 1-2:1:1. The application uses an extract of homologous raw materials of food and medicine as a culture medium for fermentation, and a composition produced by specific lactic acid bacteria can be used for preparing an anti-bacterial pneumonia product.

Description

TECHNICAL FIELD

[0001] The present application 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. BACKGROUND

[0002] Cough is one of the most common symptoms in the outpatient clinic of respiratory medicine, especially chronic cough, dry throat, cough, foreign body sensation in the throat, hoarseness. Including acute and chronic pharyngitis: hoarseness, foreign body sensation; Seasonal, environmental discomfort in the throat: dry throat, cough, foreign body sensation in the throat, hoarseness.

[0003] The factors of throat and lung discomfort are roughly as follows:

[0004] 1. External factors: If the living area is cold and dry, the working environment is polluted by dust and chemical gas, or the throat is long-term stimulated by alcohol, spicy food, bacterial infection, viral infection such as pneumonia, mycoplasma infection, etc.

[0005] 2. Physical factors: Pharyngitis can also be a local manifestation of some systemic diseases, such as anemia, indigestion, long-term constipation, heart disease, bronchitis, asthma, liver disease, diabetes and chronic nephritis, etc.

[0006] 3. Occupational factors: mainly occur in voice workers, such as teachers, actors, etc. Because of long-term language and singing, it can stimulate the throat, cause chronic congestion and cause disease.

[0007] The formula for treating cough, pharyngitis and pneumonia is mainly based on the principle of clearing the lung, reducing phlegm and relieving cough, moistening the lung and nourishing yin, and relieving dry cough.

[0008] Chinese invention patent application CN202410267996.6 discloses a product for clearing the throat and moistening the throat, which comprises main drugs and auxiliary materials, and the main drugs are composed of 25-30 parts of loquat leaves, 10-15 parts of orange peel, 6-8 parts of green fruit, 6-8 parts of platycodon grandiflorum, 5-7 parts of licorice, 6-8 parts of momordica grosvenori, 10-12 parts of fat sea, 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 orange red, 8-10 parts of rhizoma cimicifugae and 12-15 parts of bupleurum. A preparation method of the product for clearing the throat and moistening the throat is also disclosed, which comprises the following steps: S1, soaking the sliced main drugs; S2, boiling and filtering the sliced main drugs; S3, concentrating, drying and crushing the filtrate; S4, granulating; S5, tabletting, sterilizing and packaging. The present application can quickly eliminate the clinical symptoms of cough, sore throat, foreign body sensation in the throat, itchy throat and dry mucosa, has good efficacy, can significantly improve the discomfort of the throat and throat inflammation, and has the effect of clearing the throat and moistening the throat.

[0009] Another Chinese invention patent application CN201510886390.1 discloses a traditional Chinese medicine composition for treating cough. By weight parts, it contains the following ingredients and amounts: chrysanthemum 3-18 parts, Ephedra 2-12 parts, root of reed 2-12 parts, Poria cocos 2-12 parts, licorice 12-25 parts, 100-21 parts, Artemisia capillaris 2-12 parts, Salvia miltiorrhiza 5-17 parts, Houttuynia cordata 10-22 parts, 11-27 parts of Danxiang, 7-22 parts of Pangdahai, 1-2 of Fructus Momordicae, 9-15 parts of mulberry leaves, 3-9 parts of orange peel, 15-31 parts of mint, 11-19 parts of loquat leaves, 7-15 parts of Artemisia annua, 11-23 parts of Sanqi, 5-10 parts of Radix Bistortae, 10-20 parts of Citrus aurantium, 10-25 parts of Ziyuanhua. The traditional Chinese medicine composition for treating cough has the effects of promoting lung and clearing heat, resolving phlegm and relieving cough, moistening lung and relieving asthma, invigorating spleen and tonifying qi, and regulating middle and promoting diuresis, and has significant effect on treating cough, reliable effect, simple preparation, mild drug nature and no toxic side effects.

[0010] At present, the traditional Chinese medicine compound capable of treating pharyngitis and pneumonia either has many medicinal herbs, which is not conducive to decoction in pharmacy or by patients, and has poor clinical compliance, or the effect needs to be further improved. SUMMARY

[0011] In view of the deficiencies in the prior art, the present application provides a composition capable of resisting bacterial pneumonia, a preparation method and application.

[0012] In order to achieve the object of the present application, the technical scheme adopted is as follows:

[0013] On the one hand, the present application provides a composition capable of resisting bacterial pneumonia, which is prepared by fermenting a traditional Chinese medicine composition with complex probiotics. The raw materials of the traditional Chinese medicine composition are 10-25 parts of loquat leaves, 5-15 parts of Fructus Momordicae, 5-15 parts of licorice, 2-10 parts of orange peel and 1-8 parts of Pangdahai. The complex probiotics are Lactobacillus plantarum HCS03-001, Lactobacillus rhamnosus HCS02-001 and Lactobacillus rhamnosus HCS01-013 in a mass ratio of 1-2:1:1.

[0014] Preferably, the composition capable of resisting bacterial pneumonia further comprises isomaltooligosaccharide and mint-flavored powder, and the mass ratio of the traditional Chinese medicine composition, isomaltooligosaccharide and mint-flavored powder is 1000:10-40:3-20.

[0015] On the second aspect, the present application provides a preparation method of a composition capable of resisting bacterial pneumonia, which comprises the following steps:

[0016] (1) Extracting a traditional Chinese medicine composition to obtain an extract, mixing isomaltooligosaccharide and mint-flavored powder, and sterilizing to obtain a fermentation substrate;

[0017] (2) Preparing complex probiotics into a bacterial solution and activating;

[0018] (3) inoculate the activated bacteria liquid into the fermentation substrate, and ferment to obtain.

[0019] Preferably, in step (1), the raw materials of the traditional Chinese medicine composition are mixed, soaked with water, extracted, filtered, and an extract is obtained.

[0020] Preferably, the extract contains 23-73 g of crude drug per liter.

[0021] Preferably, in step (1), the extraction includes two extractions, the ratio of the total mass of water to the total mass of the raw materials of the traditional Chinese medicine composition is 45-55:1, the temperature of the extraction is 95-105°C, the first extraction is performed for 50-70 min, the second extraction is performed for 10-30 min, and the filtration is performed through 80-120 mesh.

[0022] Preferably, in step (1), the sterilization is performed at 110-120°C for 20-40 min.

[0023] Preferably, in step (2), the probiotic bacteria are mixed with water to obtain a bacteria liquid, and the mass-volume ratio of the probiotic bacteria to water is 1 kg:8-12 L.

[0024] Preferably, in step (2), the activation includes inoculating the bacteria liquid into the activation culture medium at an inoculation amount of 0.03-0.08%.

[0025] Preferably, in step (2), the activation culture medium comprises water 900-910 g / L, anhydrous glucose 30-55 g / L, yeast extract 15-35 g / L, yeast peptone 8-12 g / L, L-malic acid 2-4 g / L, citric acid monohydrate 1-3 g / L, potassium dihydrogen phosphate 1-3 g / L, calcium chloride 0.3-0.7 g / L, magnesium sulfate 0.04-0.08 g / L, and manganese sulfate 0.005-0.015 g / L.

[0026] Preferably, in step (2), the culture includes agitated culture, the stirring speed is 95-105 r / min, the air pressure is 0.02-0.03 MPa, and the culture is performed at 37±0.2°C for 16 h±1 h.

[0027] Preferably, in step (3), the activated bacteria liquid is inoculated into the fermentation substrate at an inoculation amount of 2-8%, and the fermentation is performed at 37±0.2°C for 48-72 h, the agitated culture is performed at a stirring speed of 35-45 r / min.

[0028] Preferably, in step (3), after the fermentation, the fermentation liquid is filtered through 80-120 mesh, 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 by 1-3 times, and then sterilized to obtain.

[0029] In a third aspect, the present application provides the use of the composition capable of resisting bacterial pneumonia in the preparation of a product for resisting bacterial pneumonia and / or clearing the throat and relieving cough.

[0030] Compared with the prior art, the present application has the following beneficial effects:

[0031] (1) The fermentation is carried out by using the extract of the medicinal and edible material as the culture medium, and the postbiotics produced by the specific lactic acid bacteria, so that the substances beneficial to the throat and the lungs are produced, and the effects of the probiotics and the medicinal and edible material are combined.

[0032] (2) The specific traditional Chinese medicine composition is used in the present application, and the effect of resisting pneumonia after fermentation is significantly better than that of other large prescriptions.

[0033] (3) The specific ratio of the plant lactobacillus HCS03-001, the rohde's mucous lactobacillus HCS02-001 and the rhamnose lactobacillus HCS01-013 complex probiotics is used in the present application, and the effect of the fermentation liquor prepared by the complex probiotics is significantly better than that of other probiotic strains.

[0034] (4) The activated culture medium can better activate the strains, and the fermentation effect is more prominent. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 It is a neutrophil diagram in the swim bladder position of the sample after treatment. Note: the green fluorescent points are neutrophils. DETAILED DESCRIPTION

[0036] The present application will be further described below in combination with specific embodiments. The following raw materials are all conventional commercially available raw materials.

[0037] Example 1

[0038] A composition capable of resisting bacterial pneumonia is prepared by fermenting a mixture of a traditional Chinese medicine composition, oligomaltose and a peppermint-flavored powder, wherein the raw materials of the traditional Chinese medicine composition are loquat leaves, momordica grosvenori, licorice, orange peel and fat sea cucumber, and the complex probiotics are the bacterial powder of plant lactobacillus HCS03-001, rohde's mucous lactobacillus HCS02-001 and rhamnose lactobacillus HCS01-013 in a mass ratio of 2:1:1.

[0039] The preparation method of the composition is as follows:

[0040] (1) The traditional Chinese medicine composition raw materials (see Table 1 for the formula) are directly put into a multifunctional extraction tank and soaked with water;

[0041] Primary extraction: the raw materials of the traditional Chinese medicine composition were mixed, and the water amount was 75% of the total water amount (the total water amount is shown in Table 1), and decocting extraction was performed for 50 min; 100 mesh filtration was performed to obtain filtrate 1, which was pumped to a secondary fermentation tank for temporary storage;

[0042] Secondary extraction: water was added to the extraction tank again, and the water amount was 25% of the total water amount, and decocting extraction was performed for 20 min; after the extraction was completed, 100 mesh filtration was performed to obtain filtrate 2, which was pumped to the secondary fermentation tank, and stirring was started (stirring speed: 40 rpm), and the two filtrates were mixed uniformly to obtain an extraction liquid;

[0043] The extraction liquid, oligoisomaltose and peppermint flavor powder were mixed according to the formulation in Table 2, sterilized at 115°C for 30 min, and then cooled to 37°C to obtain a fermentation substrate.

[0044] (2) The compound probiotic bacteria (see Table 3) were dissolved in sterile water, and the bacteria powder: sterile water (kg: L) = 1: 10, and stirring was performed until the bacteria powder was uniformly dissolved without clumping to obtain a bacteria liquid. The bacteria liquid was inoculated into the activated culture medium (formulation shown in Table 4) at an inoculation amount (v / v) of 0.05%, and the culture was performed at 37°C for 16 h. The culture was a stirring culture, and the stirring speed was 100 r / min; the culture was not aerated, and the gas pressure was maintained at 0.02-0.03 MPa.

[0045] (3) The activated bacteria liquid was inoculated into the fermentation substrate at an inoculation amount of 5% (v / v), and the fermentation was performed at 37±0.2°C for 60 h, and the culture was a stirring culture, and the stirring speed was 40 r / min; the culture was not aerated, and the gas pressure was maintained at 0.02-0.03 MPa.

[0046] Table 1

[0047]

[0048] Table 2

[0049]

[0050] Table 3

[0051]

[0052] Table 4

[0053]

[0054] Example 2

[0055] A composition capable of resisting bacterial pneumonia, the composition is formed by fermenting a mixture of a traditional Chinese medicine composition, isomaltooligosaccharide and mint-flavored powder, the raw materials of the traditional Chinese medicine composition are loquat leaves, momordica grosvenori, licorice, orange peel and fat sea, the composite probiotics are lactobacillus plantarum HCS03-001, lactobacillus rhamnosus HCS01-013 and lactobacillus johnsonii HCS02-001 in a mass ratio of 1:1:1.

[0056] The preparation method of the composition, the steps are as follows:

[0057] (1) The raw materials of the traditional Chinese medicine composition (see Table 5 for formula) are directly put into a multifunctional extraction tank and fully soaked with water;

[0058] First extraction: the water amount is 75% of the total water amount (see Table 5 for total water amount), the temperature is 95℃, and the extraction is cooked for 60 min; 100 mesh filtration is performed to obtain filtrate 1, which is pumped to a secondary fermentation tank for temporary storage;

[0059] Second extraction: water is added to the extraction tank again, the water amount is 25% of the total water amount, the temperature is 95℃, and the extraction is cooked for 10 min; after the extraction is completed, 100 mesh filtration is performed to obtain filtrate 2, which is pumped to the secondary fermentation tank and stirred (stirring speed 40 rpm); the two filtrates are mixed uniformly to obtain an extraction liquid;

[0060] The extraction liquid, isomaltooligosaccharide and mint-flavored powder are mixed according to the formula in Table 6, sterilized at 120℃ for 20 min; cooled to 37℃ to obtain a fermentation substrate.

[0061] (2) The composite probiotic powder (see Table 7 for specific information) is dissolved in sterile water, the powder:sterile water (kg:L) =1:12, and stirred uniformly without clumping to obtain a bacterial solution. The bacterial powder solution is inoculated into the activated culture medium (see Table 8 for formula) at an inoculation amount of 0.08% (v / v), and cultured at 37±0.2℃ for 17h, with stirring at a stirring speed of 100r / min; the culture is not aerated, and the gas pressure is maintained at 0.02MPa.

[0062] (3) The activated bacterial solution is inoculated into the fermentation substrate at an inoculation amount of 5%, and fermented at 37℃ for 48h, with stirring at a stirring speed of 40r / min, and the gas pressure is maintained at 0.02MPa.

[0063] Table 5

[0064]

[0065] Table 6

[0066]

[0067] Table 7

[0068]

[0069] Table 8

[0070]

[0071] Comparative Example 1

[0072] The present comparative example differs from Example 1 only in that the raw materials of the traditional Chinese medicine composition are different, as shown in Table 9, and the rest is the same as Example 1.

[0073] Table 9

[0074]

[0075] Comparative Example 2

[0076] The present comparative example differs from Example 1 only in that the strains of the compound probiotic bacteria are different, as shown in Table 10.

[0077] Table 10

[0078]

[0079] Comparative Example 3

[0080] The present comparative example differs from Example 1 only in that the ratio of the strains of the compound probiotic bacteria is different, as shown in Table 11, and the rest is the same as Example 1.

[0081] Table 11

[0082]

[0083] Comparative Example 4

[0084] The present comparative example differs from Example 1 only in that the ratio of the activated culture medium is different, as shown in Table 12.

[0085] Table 12

[0086]

[0087] Experimental Example: Anti-bacterial pneumonia efficacy experiment

[0088] 1. Maximum test concentration (MTC) determination

[0089] Randomly selected 5dpf transgenic neutrophil green fluorescence MPX strain zebrafish in 6-well plates, 30 zebrafish in each well (experimental group) were treated. The sample was given in water (the concentration is shown in Table 13), and the normal control group and the model control group were set up, and the volume in each well was 3 mL. Except for the normal control group, the rest of the experimental groups were given LPS by fish swim bladder injection to establish a zebrafish bacterial pneumonia model. After 4h treatment at 28℃, the MTC of the sample on the model zebrafish was determined.

[0090] Under the conditions of this experiment, the efficacy of the composition of the present application against bacterial pneumonia zebrafish MTC was 12.5 μL / mL. The results of the concentration groping experiment of the composition of Example 1 against bacterial pneumonia are shown in Table 13.

[0091] Table 13 Results of concentration groping experiment of sample against bacterial pneumonia (n = 30)

[0092]

[0093] 2. Evaluation of efficacy against bacterial pneumonia

[0094] Randomly selected 5dpf transgenic neutrophil green fluorescence MPX strain zebrafish in 6-well plates, 30 zebrafish in each well (experimental group) were treated. The sample was given in water (the concentration is shown in Table 13), and the normal control group and the model control group were set up, and the volume in each well was 3 mL. Except for the normal control group, the rest of the experimental groups were given LPS by fish swim bladder injection to establish a zebrafish bacterial pneumonia model. After 4h treatment at 28℃, the MTC of the sample on the model zebrafish was determined.

[0095] The statistical processing results are expressed by mean ± SE. Statistical analysis was performed using SPSS 26.0 software, and p<0.05 indicates that the difference is statistically significant.

[0096] Under the conditions of this experiment, the composition of the present application has the efficacy against bacterial pneumonia, which is shown in detail in Table 14, and the representative neutrophils in the swim bladder of zebrafish after treatment with the composition of Example 1 at different concentrations are shown in Figure 1 .

[0097] Table 14 Results of evaluation experiment of sample against bacterial pneumonia (n = 10)

[0098]

[0099] Note: Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001.

[0100] 3. Anti-bacterial pneumonia efficacy evaluation

[0101] Randomly selected 5 dpf transgenic neutrophil green fluorescent MPX strain zebrafish in 6-well plates, 30 zebrafish were treated in each well. The experimental group was given 3.12 μg / mL, 6.25 μg / mL, 12.5 μg / mL of Example 1 composition, and the positive control group was given 43.5 μg / mL of dexamethasone acetate, while setting up a normal control group and a model control group, and the volume in each well was 3 mL. Except for the normal control group, the rest of the experimental groups were given LPS by fish swim bladder injection to establish a zebrafish bacterial pneumonia model. Three parallel experiments were set up. After 4 h of treatment at 28°C, total RNA of each group of zebrafish was extracted using a preloaded magnetic bead method universal RNA extraction kit C, and the concentration and purity of total RNA were determined using a UV-visible spectrophotometer. 2.00 μg of zebrafish sample total RNA was taken, and 20.0 μL of cDNA was synthesized according to the cDNA first strand synthesis kit instructions, and the expression of β-actin and tgfβ-1 genes was detected by q-PCR. Use β-actin as the internal reference for gene expression, and calculate the RNA relative expression of tgfβ-1 gene. The results were statistically processed and expressed as mean ± SE, and statistical analysis was performed using SPSS 26.0 software, p<0.05 indicating that the difference was statistically significant.

[0102] Test results:

[0103] 1) RNA extraction results

[0104] At the end of the experiment, total RNA of zebrafish was extracted, and the concentration and A260 / A280 ratio of RNA were determined by UV-visible spectrophotometer (Table 15). The A260 / A280 ratio was between 1.8 and 2.2, indicating that the quality of the extracted zebrafish total RNA was good and could be used for subsequent q-PCR experiments.

[0105] Table 15 Concentration and A260 / A280 ratio of total RNA (n = 3)

[0106]

[0107] 2) Anti-bacterial pneumonia efficacy evaluation

[0108] When LPS induces acute lung injury, inflammatory cells such as neutrophils are recruited and activated. After activation, inflammatory cells infiltrate lung tissue and release inflammatory mediators, causing an increase in zebrafish neutrophils and macrophages, and an increase in the relative expression of tgfb1a, il1b, and tnfa genes.

[0109] Under the experimental conditions, the composition of the present application has the efficacy of anti-bacterial pneumonia, which is specifically manifested as down-regulating the relative expression amount of tgfβ-1 gene, and the details are shown in Table 16.

[0110] Table 16 Experimental results of anti-bacterial pneumonia efficacy evaluation (gene) of samples

[0111]

[0112] Note: Compared with the model control group, *p<0.05, **p<0.01.

[0113] The above detailed description is a specific description of one of the feasible embodiments of the present application, which is not used to limit the patent scope of the present application, and any equivalent implementation or change without departing from the present application shall be included in the scope of the technical scheme of the present application.

Claims

1. A composition capable of fighting bacterial pneumonia, characterized in that, The composition is fermented by complex probiotics, and raw materials of the traditional Chinese medicine composition are 10-25 parts of leaves of loquat, 5-15 parts of momordica grosvenori swingle, 5-15 parts of licorice, 2-10 parts of orange peel and 1-8 parts of fat sea cucumber; the complex probiotics are lactobacillus plantarum HCS03-001, lactobacillus rhamnosus HCS02-001 and lactobacillus rhamnosus HCS01-013 with a mass ratio of 1-2:1:

1.

2. The composition capable of anti-bacterial pneumonia according to claim 1, characterized by, The composition further comprises oligoisomaltose and peppermint flavor powder, and the mass ratio of the traditional Chinese medicine composition, the oligoisomaltose and the peppermint flavor powder is 1000:10-40:3-20.

3. A method of preparing the composition capable of resisting bacterial pneumonia according to claim 2, characterized by, The method comprises the following steps: (1) mixing raw materials of the traditional Chinese medicine composition, soaking in water, extracting, filtering and obtaining an extract; (2) preparing a bacterial solution of the complex probiotics and activating the complex probiotics; (3) inoculating the activated bacterial solution into the fermentation substrate to obtain the fermented product.

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

5. The preparation method according to claim 4, characterized in that, The extract contains 23-73 g of crude drug per liter; In step (1), the extraction comprises two times of boiling extraction, and the total mass of water added is 45-55 times the total mass of the raw materials of the traditional Chinese medicine composition; the first extraction is performed for 50-70 min, and the second extraction is performed for 10-30 min; the filtering is performed through a 80-120 mesh screen; In step (1), the sterilization is performed at 110-120 °C for 20-40 min.

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

7. The production method according to claim 6, wherein In step (2), the activation culture medium comprises water 900-910 g / L, anhydrous glucose 30-55 g / L, yeast extract 15-35 g / L, yeast peptone 8-12 g / L, L-malic acid 2-4 g / L, citric acid monohydrate 1-3 g / L, potassium dihydrogen phosphate 1-3 g / L, calcium chloride 0.3-0.7 g / L, magnesium sulfate 0.04-0.08 g / L and manganese sulfate 0.005-0.015 g / L; in step (2), the activation comprises stirring culture, and the stirring speed is 95-105 r / min; the air pressure is 0.02-0.03 MPa; and the culture is performed 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 solution is inoculated into the fermentation substrate at an inoculation amount of 2-8%, and the fermentation is performed at 37±0.2 °C for 48-72 h under stirring culture at a stirring speed of 35-45 r / min.

9. The preparation method according to claim 3, characterized in that, In step (3), after the fermentation, the fermentation liquid is filtered through a 80-120 mesh screen and then centrifuged at a speed of 8000-11000 r / min for 3-5 min; after the centrifugation, the fermentation liquid is concentrated by 1-3 times and then sterilized to obtain the fermented product.

10. Use of the composition capable of resisting bacterial pneumonia according to any one of claims 1-2 in the preparation of a medicine for resisting bacterial pneumonia, and / or a health food or medicine for clearing the throat.

Images