CN113577128A

CN113577128A - Astragalus membranaceus fermentation liquor as well as preparation method and application thereof

Info

Publication number: CN113577128A
Application number: CN202111022767.0A
Authority: CN
Inventors: 张春江; 李玉玺; 刘冠兰; 霍泽琪
Original assignee: Lanzhou University
Current assignee: Lanzhou University
Priority date: 2021-09-01
Filing date: 2021-09-01
Publication date: 2021-11-02
Anticipated expiration: 2041-09-01
Also published as: CN113577128B

Abstract

The invention belongs to the technical field of traditional Chinese medicine fermentation, and provides astragalus membranaceus fermentation liquor as well as a preparation method and application thereof. The preparation process of the invention comprises the following steps: slicing radix astragali, soaking in water, decocting, and filtering to obtain residue and first medicinal liquid; adding water into the dregs of a decoction, and decocting to obtain a second liquid medicine; mixing the first liquid medicine and the second liquid medicine, and concentrating to obtain radix astragali water extractive solution; mixing Lactobacillus plantarum LZU-S-ZCJ and LZU-J-TSL6, inoculating into radix astragali water extractive solution, and fermenting to obtain radix astragali fermentation broth. The astragalus fermentation liquor obtained by the method can relieve the edema degree of the mouse lung, relieve the inflammatory cell infiltration state caused by Lipopolysaccharide (LPS), reduce the accumulation of protein liquid in the mouse lung, reduce the concentration of inflammatory factors in the acute lung injury mouse BALF, regulate the frontal intestinal flora of the acute lung injury mouse and the like, and has good effect of relieving the acute lung injury of the mouse.

Description

Astragalus membranaceus fermentation liquor as well as preparation method and application thereof

Technical Field

The invention relates to the technical field of traditional Chinese medicine fermentation, in particular to astragalus membranaceus fermentation liquor as well as a preparation method and application thereof.

Background

The Chinese medicine can relieve diseases by inhibiting colonization of pathogens and balancing intestinal flora. The probiotic fermentation can solve the problem that the polysaccharide in the traditional Chinese medicine preparation is not easy to be digested and absorbed by human bodies, and the utilization rate of the polysaccharide after the traditional Chinese medicine is fermented by microorganisms is greatly improved. The fermentation product needs to improve the number of live bacteria in the product to play a role, and a reasonable fermentation process is one of important factors for improving the number of the live bacteria in the fermentation product.

In recent years, a great deal of research on the treatment of acute lung injury by traditional Chinese medicines is carried out at home and abroad, and the research shows that the effective components of the traditional Chinese medicines have the advantages of multiple targets, multiple ways, small adverse reaction and the like, and can play a role in preventing and treating the acute lung injury. Research proves that emodin can obviously inhibit the expression of E-selectin (E-selectin) of ALI mice and improve acute lung injury induced by Lipopolysaccharide (LPS), and Crocin (Crocin) can reduce lung tissue injury induced by LPS and reduce the expression of phosphorylated I kappa B and the activity of NF-kappa B. The astragalus root has a plurality of pharmacological activities of regulating immunity, resisting tumor, resisting inflammation and the like, and researches show that flavone and saponin substances in the astragalus root water extract have an anti-inflammatory effect. However, few reports have been made on the research of using microorganisms to ferment the astragalus membranaceus water extract in relieving acute lung injury of mice.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide astragalus fermentation broth which has the effect of relieving acute lung injury of mice and is prepared by mixing Lactobacillus plantarum LZU-S-ZCJ and Lactobacillus plantarum LZU-J-TSL6 and fermenting astragalus aqueous extract, a preparation method and application thereof.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a preparation method of astragalus fermentation liquor, which comprises the following steps:

(1) slicing radix astragali, soaking in water, decocting, and filtering to obtain residue and first medicinal liquid;

(2) adding water into the dregs of a decoction, and decocting to obtain a second liquid medicine;

(3) mixing the first liquid medicine and the second liquid medicine, and concentrating to obtain radix astragali water extractive solution;

(4) mixing Lactobacillus plantarum LZU-S-ZCJ and Lactobacillus plantarum LZU-J-TSL6, inoculating into radix astragali water extract, and fermenting to obtain radix astragali fermentation liquid.

Preferably, in the step (1), the volume ratio of the astragalus slice to water is 1: 8-12, the soaking time is 3-5 h, the decocting temperature is 94-97 ℃, the decocting time is 0.5-1.5 h, and the filtering times are 1-3 times.

Preferably, in the step (2), the volume ratio of the medicine residues to the water is 1: 6-10, the decoction temperature is 94-97 ℃, and the decoction time is 0.5-1.5 h.

Preferably, in the step (3), when the astragalus slices are 0.2-0.8 g, the volume of the astragalus water extract is 1 mL.

Preferably, in the step (4), the total viable count of the lactobacillus plantarum LZU-S-ZCJ and the lactobacillus plantarum LZU-J-TSL6 is 14.5-15.5 Log10CFU/mL, and the viable count ratio of the lactobacillus plantarum LZU-S-ZCJ to the lactobacillus plantarum LZU-J-TSL6 is 1: 2-4;

the inoculation amount of the mixed strain accounts for 2-4% of the volume of the astragalus water extract;

the fermentation temperature is 35-38 ℃, and the fermentation time is 35-40 h.

The invention also provides astragalus fermentation liquor.

The invention also provides application of the astragalus fermentation liquor in preparing a medicine for relieving acute lung injury.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a preparation method of astragalus membranaceus fermentation liquor, and the astragalus membranaceus fermentation liquor obtained by the preparation method has a good effect of relieving acute lung injury of mice. The concrete expression is as follows: the astragalus fermentation liquor prepared by the method effectively relieves the edema degree of the mouse lung, relieves the inflammatory cell infiltration state caused by Lipopolysaccharide (LPS), reduces the accumulation of protein liquid in the mouse lung, reduces the concentration of inflammatory factors in the acute lung injury mouse BALF, and can also regulate the frontal intestinal flora and the like of the acute lung injury mouse.

Drawings

FIG. 1 shows the growth of Lactobacillus plantarum LZU-S-ZCJ, LZU-J-TSL6 fermented alone or after mixing the two according to the number ratio of viable bacteria;

FIG. 2 is a graph showing the effect of a single factor on fermentation broth of Astragalus membranaceus;

FIG. 3 is an experimental flow chart of the effect of alleviating acute lung injury before and after fermentation of an aqueous extract of Astragalus membranaceus;

FIG. 4 shows the effect of groups on the ratio of the dry weight to the wet weight of lung tissue in mice with acute lung injury (note: C: normal group; M: model group; P: dexamethasone group; A: water extract of Astragalus membranaceus group; FA: fermentation broth of Astragalus membranaceus group; LP: Lactobacillus plantarum group; LA: water extract of Astragalus membranaceus and mixed solution of Lactobacillus plantarum);

FIG. 5 shows the influence of groups on lung tissue pathology of mice with acute lung injury (note: C: normal group; M: model group; P: dexamethasone group; A: radix astragali water extract group; FA: radix astragali fermentation broth group; LP: Lactobacillus plantarum group; LA: radix astragali water extract and Lactobacillus plantarum mixed solution group);

FIG. 6 shows the effect of MPO activity in lung tissue of mice with acute lung injury by groups (Note: C: normal group; M: model group; P: dexamethasone group; A: water extract of Astragalus membranaceus group; FA: fermentation broth of Astragalus membranaceus group; LP: Lactobacillus plantarum group; LA: water extract of Astragalus membranaceus and mixed solution of Lactobacillus plantarum);

FIG. 7 shows the effect of groups on the protein concentration in BALF of mice with acute lung injury (note: C: normal group; M: model group; P: dexamethasone group; A: water extract of Astragalus membranaceus group; FA: fermentation broth of Astragalus membranaceus group; LP: Lactobacillus plantarum group; LA: mixed solution of water extract of Astragalus membranaceus and Lactobacillus plantarum group);

FIG. 8 shows the effect of groups on the concentration of inflammatory factors in BALF of mice with acute lung injury (note: C: normal group; M: model group; P: dexamethasone group; A: water extract of Astragalus membranaceus group; FA: fermentation broth of Astragalus membranaceus group; LP: Lactobacillus plantarum group; LA: mixed solution of water extract of Astragalus membranaceus and Lactobacillus plantarum group);

FIG. 9 is a bar graph of the relative abundance changes of the intestinal flora of mice in the genus before and after modeling in each experimental group (note: BC: normal group after the completion of preventive drug administration; BL: model group, BD: dexamethasone group, BA: radix astragali water extract group, BFA: radix astragali fermentation broth group, AC: normal group after the completion of modeling; AL: model group, AD: dexamethasone, AA: radix astragali water extract group, AFA: radix astragali fermentation broth group);

FIG. 10 shows significantly different bacterial colonies in the mouse intestinal tract before and after molding (note: AC: normal group after molding completion; AL: model group; AD: dexamethasone; AA: radix astragali water extract group; AFA: radix astragali fermentation broth group);

FIG. 11 is a heat map of relative abundance changes at genus level before and after modeling of intestinal flora in mice of each experimental group;

fig. 12 is a graph showing the pathway analysis of various groups of stool samples after molding.

Deposit description

Lactobacillus plantarum LZU-S-ZCJ, Latin, named Lactobacillus plantarum LZU-S-ZCJ, with the preservation unit name of Guangdong province center for microbial bacterial culture, the address of No. 5 th of No. 59 college of Michelia Tokyo No. 100, Guangzhou city, the preservation date of 2020, 12 and 31 days, and the preservation number of GDMCC NO: 61402;

lactobacillus plantarum LZU-J-TSL6 with the Latin name of Lactobacillus plantarum LZU-J-TSL6 and the preservation unit name of Guangdong province microbial bacterial breed conservation center, the address of No. 59 building 5 of Michelia Tokyo No. 100 of Guangzhou city, the preservation date of 2020, 10, 23 days and the preservation number of GDMCC NO: 61242.

Detailed Description

In the invention, in the step (1), the volume ratio of the astragalus slice to water is preferably 1: 8-12, and more preferably 1: 10; the soaking time is preferably 3-5 h, and further preferably 4 h; the preferable decocting temperature is 94-97 ℃, the further preferable temperature is 96 ℃, and the preferable time for decocting is 0.5-1.5 h, the further preferable time is 1 h; the number of filtration is preferably 1 to 3, and more preferably 2.

In the invention, in the step (2), the volume ratio of the medicine residue to the water is preferably 1: 6-10, and more preferably 1: 8; the decoction temperature is preferably 94-97 ℃, more preferably 96 ℃, and the decoction time is preferably 0.5-1.5 h, more preferably 1 h.

In the present invention, in the step (3), preferably, the volume of the astragalus aqueous extract is 1mL when the astragalus section is 0.2 to 0.8g, more preferably, the volume of the astragalus aqueous extract is 1mL when the astragalus section is 0.4 to 0.6g, and still more preferably, the volume of the astragalus aqueous extract is 1mL when the astragalus section is 0.5 g.

In the invention, in the step (4), the total viable count of the Lactobacillus plantarum LZU-S-ZCJ and the Lactobacillus plantarum LZU-J-TSL6 is preferably 14.5-15.5 Log10CFU/mL, more preferably 14.76Log10CFU/mL, and the viable count ratio of the Lactobacillus plantarum LZU-S-ZCJ-TSL 6 is preferably 1: 2-4, more preferably 1: 3;

the inoculation amount of the mixed strain is preferably 2-4% of the volume of the astragalus water extract, and is further preferably 3% of the volume of the astragalus water extract;

the fermentation temperature is preferably 35-38 ℃, more preferably 36 ℃, and the fermentation time is preferably 35-40 h, more preferably 36 h.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

The L.plantarum LZU-S-ZCJ and LZU-J-TSL6 used in the examples below were both isolated from Gansu specialty food, Bonus Water.

Example 1

Placing radix astragali slices in a round-bottom flask, adding 8 times of sterile water, soaking for 3h, decocting at 94 deg.C for 0.5h, and filtering the medicinal liquid with gauze into a beaker to obtain residue and first medicinal liquid;

adding 6 times volume of sterile water into the residue, and decocting at 94 deg.C for 0.5h to obtain second medicinal liquid;

mixing the first liquid medicine and the second liquid medicine, and concentrating to obtain radix astragali water extractive solution;

mixing Lactobacillus plantarum LZU-S-ZCJ and LZU-J-TSL6 at viable bacteria number ratio of 1:2, inoculating 2% of the mixture into the radix astragali water extractive solution, and fermenting at 35 deg.C for 35h to obtain radix astragali fermentation broth.

Example 2

Placing radix astragali slices in a round-bottom flask, adding 10 times of sterile water, soaking for 4h, decocting at 96 deg.C for 1h, filtering the medicinal liquid with gauze into a beaker, and filtering twice to obtain residue and first medicinal liquid;

adding 8 times of sterile water into the residue, and decocting at 96 deg.C for 1 hr to obtain a second medicinal liquid;

mixing Lactobacillus plantarum LZU-S-ZCJ and LZU-J-TSL6 at viable count ratio of 1:3, inoculating into radix astragali water extractive solution at inoculation amount of 3%, and fermenting at 36 deg.C for 36h to obtain radix astragali fermentation broth.

Example 3

Placing radix astragali slices in a round-bottom flask, adding 12 times of sterile water, soaking for 5h, decocting at 97 deg.C for 1.5h, and filtering the medicinal liquid with gauze into a beaker to obtain residue and first medicinal liquid;

adding 10 times volume of sterile water into the residue, and decocting at 97 deg.C for 1.5 hr to obtain second medicinal liquid;

mixing Lactobacillus plantarum LZU-S-ZCJ and LZU-J-TSL6 at viable bacteria number ratio of 1:4, inoculating 4% of the mixture into the radix astragali water extractive solution, and fermenting at 38 deg.C for 40h to obtain radix astragali fermentation broth.

Comparative example 1

Placing radix astragali slices in a round-bottom flask, adding 10 times of sterile water, soaking for 4h, decocting for 1h, filtering the liquid medicine with gauze into a beaker, and filtering twice to obtain residue and first liquid medicine;

adding 8 times of sterile water into the residues, and continuously decocting for 1h to obtain a second liquid medicine;

inoculating Lactobacillus plantarum LZU-S-ZCJ with 3% of inoculation amount in the radix astragali water extract, and fermenting at 36 deg.C for 36h to obtain radix astragali fermentation broth.

Comparative example 2

inoculating Lactobacillus plantarum LZU-J-TSL6 in an inoculum size of 3% into the radix astragali water extract, and fermenting at 36 deg.C for 36h to obtain radix astragali fermentation broth.

Comparative example 3

mixing Lactobacillus plantarum LZU-S-ZCJ and LZU-J-TSL6 at viable bacteria number ratio of 1:1, inoculating into radix astragali water extractive solution at inoculation amount of 3%, and fermenting at 36 deg.C for 36h to obtain radix astragali fermentation broth.

Comparative example 4

mixing Lactobacillus plantarum LZU-S-ZCJ and LZU-J-TSL6 at viable count ratio of 3:1, inoculating into radix astragali water extractive solution at 3%, and fermenting at 36 deg.C for 36h to obtain radix astragali fermentation broth.

Comparative example 5

mixing Lactobacillus plantarum LZU-S-ZCJ and LZU-J-TSL6 at viable count ratio of 1:3, inoculating into radix astragali water extractive solution at inoculation amount of 3%, and fermenting at 36 deg.C for 24 hr to obtain radix astragali fermentation broth.

Comparative example 6

mixing Lactobacillus plantarum LZU-S-ZCJ and LZU-J-TSL6 at viable bacteria number ratio of 1:3, inoculating 5% of the mixture into the radix astragali water extractive solution, and fermenting at 38 deg.C for 48h to obtain radix astragali fermentation broth.

Comparative example 7

mixing Lactobacillus plantarum LZU-S-ZCJ and LZU-J-TSL6 at viable bacteria number ratio of 1:3, inoculating into radix astragali water extractive solution at 3%, and fermenting at 37 deg.C for 48 hr to obtain radix astragali fermentation broth.

Comparative example 8

mixing Lactobacillus plantarum LZU-S-ZCJ and LZU-J-TSL6 at viable bacteria number ratio of 1:3, inoculating 4% of the mixture into the radix astragali water extractive solution, and fermenting at 38 deg.C for 24 hr to obtain radix astragali fermentation broth.

Comparative example 9

mixing Lactobacillus plantarum LZU-S-ZCJ and LZU-J-TSL6 at viable bacteria number ratio of 1:3, inoculating 5% of the mixture into the radix astragali water extractive solution, and fermenting at 36 deg.C for 36h to obtain radix astragali fermentation broth.

Comparative example 10

mixing Lactobacillus plantarum LZU-S-ZCJ and LZU-J-TSL6 at viable bacteria number ratio of 1:3, inoculating 4% of the mixture into the radix astragali water extractive solution, and fermenting at 36 deg.C for 48h to obtain radix astragali fermentation broth.

Comparative example 11

mixing Lactobacillus plantarum LZU-S-ZCJ and LZU-J-TSL6 at viable bacteria number ratio of 1:3, inoculating 5% of the mixture into the radix astragali water extractive solution, and fermenting at 37 deg.C for 24 hr to obtain radix astragali fermentation broth.

Experimental example 1

The experimental group of example 2, the control group of comparative examples 1 to 4 and the MRS medium of the blank control group were used to study the growth of the Lactobacillus plantarum LZU-S-ZCJ, LZU-J-TSL6 after single fermentation and the mixture of the two according to the number ratio of viable bacteria, and the results are shown in FIG. 1.

As can be seen from FIG. 1, when the ratio of viable bacteria of Lactobacillus plantarum LZU-S-ZCJ and LZU-J-TSL6 was 1:3, the growth in the aqueous extract of Astragalus was better than that of the other combinations of strains. It can be seen that the fermentation effect was better with the strain volume ratio of example 2 of the present application compared to the control group.

Experimental example 2

The method takes the number of viable bacteria in the astragalus fermentation liquor as an index to explore the optimum astragalus water extract concentration, strain inoculation amount, fermentation temperature and fermentation time for fermentation.

(1) Concentration of the water extract of astragalus: adding radix astragali water extractive solutions with different concentrations into a conical flask, mixing Lactobacillus plantarum LZU-S-ZCJ and LZU-J-TSL6 according to the ratio of viable bacteria number of 1:3, inoculating 4% of the mixture into the radix astragali water extractive solution, culturing at 37 deg.C for 24h, coating a flat plate to calculate viable bacteria number of the fermentation broth, and determining the optimal radix astragali water extractive solution concentration.

(2) Inoculating strains: adding radix astragali water extractive solution with concentration of 0.5g/mL into conical flask, inoculating bacterial solutions with different proportions, culturing at 37 deg.C for 24 hr, coating plate to calculate viable count of fermentation broth, and determining optimal strain inoculation amount.

(3) Fermentation time: adding 0.5g/mL astragalus aqueous extract into a conical flask, mixing lactobacillus plantarum LZU-S-ZCJ and LZU-J-TSL6 according to the ratio of viable bacteria number to number of 1:3, inoculating the mixture into the astragalus aqueous extract by 4 percent of inoculation amount, fermenting at 37 ℃ for different time, coating a flat plate to calculate the viable bacteria number of the fermentation liquid, and determining the optimal fermentation time.

(4) Fermentation temperature: adding 0.5g/mL radix astragali water extract into a conical flask, mixing Lactobacillus plantarum LZU-S-ZCJ and LZU-J-TSL6 according to the ratio of viable bacteria number of 1:3, inoculating 4% of the mixture into the radix astragali water extract, fermenting at different temperatures for 36h, coating a flat plate to calculate the viable bacteria number of the fermentation liquid, and determining the optimal fermentation temperature.

The results are shown in FIG. 2. As can be seen from fig. 2, the number of viable bacteria in the fermentation broth continuously increases with the increasing concentration of the water extract of astragalus membranaceus, and when the concentration of the water extract of astragalus membranaceus reaches 0.5g/mL, the number of viable bacteria in the fermentation broth is the largest, so that the concentration of the water extract of astragalus membranaceus is continuously increased, and the number of viable bacteria in the fermentation broth is decreased on the contrary, which may be caused by the change of the osmotic pressure of bacteria or the change of the pH of the fermentation broth with the increasing concentration of the water extract of astragalus membranaceus, so that the fermentation environment is not favorable for the growth of bacteria, and the optimal concentration of the water extract of astragalus membranaceus is 0.5 g/mL;

along with the continuous increase of the strain inoculation rate, the number of the live bacteria in the astragalus fermentation liquor also rises, when the strain inoculation rate is 4%, the number of the live bacteria in the fermentation liquor is the largest, the strain inoculation rate is continuously increased, and the number of the live bacteria in the fermentation liquor begins to decrease, which may be caused by the fact that the initial strain inoculation amount is too large, the substrate consumption is accelerated, and the fermentation product accumulation is caused, so that the 4% strain inoculation amount is the optimal strain inoculation amount;

under different fermentation temperatures, the number of live bacteria in the fermentation liquor is different, under the condition of 37 ℃, the number of live bacteria in the fermentation liquor is the largest, and the number of live bacteria in the fermentation liquor is reduced when the number of live bacteria in the fermentation liquor is higher or lower than the temperature, so that the temperature of 37 ℃ is the optimal fermentation temperature, under the fermentation temperature, the thalli grow more fully, and the number of live bacteria in the fermentation liquor is the largest;

the number of the living bacteria in the fermentation liquid is increased continuously along with the increase of the fermentation time, when the fermentation time reaches 36h, the number of the living bacteria in the fermentation liquid is the largest, if the fermentation time is prolonged continuously, the number of the living bacteria begins to decrease, which is probably that the nutrient substances in the fermentation liquid can not continuously meet the requirement of the growth and the propagation of the thalli along with the extension of the fermentation time, so the fermentation time is 36h, which is the optimal fermentation time.

Experimental example 3

The example 2 is taken as an experimental group, the comparative examples 5-11 are taken as a control group, the viable count in the fermentation liquor is taken as an index, and the fermentation effect under different process conditions is researched. The results are shown in Table 1.

TABLE 1 fermentation results under different process conditions

Group of	Viable count (Log10CFU/mL)
		Example 2	14.76
Comparative example 5	13.61
		Comparative example 6	13.18
Comparative example 7	13.98
		Comparative example 8	13.86
Comparative example 9	14.03
		Comparative example 10	13.97
Comparative example 11	13.83

As can be seen from Table 1, compared with the control group, the fermentation effect is the best by using the process conditions of the example 2 of the present application, and the viable count in the astragalus fermentation liquid can be as high as 14.76Log10 CFU/mL.

The mice used in the following experiments are purchased from the medical experiment center of Lanzhou university, are healthy SPF (specific pathogen free) Balb/c male mice with the age of about 7 weeks, have the weight of 20 +/-2 g, are raised under the conditions of 20 +/-2 ℃ and the relative humidity of 35-55 percent, simulate the alternate circadian rhythm of illumination/darkness for 12 hours, are fed with normal sterilized feed, and are drunk with sterile distilled water, and the adaptation period is 7 days.

Grouping experiments:

placing 56 Balb/c male mice into 7 mouse cages randomly, interchanging the mice every three days (the dung eating behavior of the mice), keeping the intestinal flora of all the mice at a uniform level as far as possible, and carrying out adaptive breeding for 7 days to ensure that the mice adapt to the environment. After the adaptation period, each group of mice was subjected to gavage treatment from day 8 to day 21 (prevention period) and day 22 (molding period), and on day 22 (molding period), the mice were sacrificed after 24 hours of reaction by nasal administration with a physiological saline or LPS solution, and the bronchial lavage fluid and lung tissue of the mice were taken for measurement of various indices. The whole experimental process is shown in fig. 3, and is specifically grouped as follows:

(1) normal group (C): the mice were fed with normal feed and water throughout the experimental period. In the prevention period, 100 mu L of sterile water is filled into the stomach of each mouse every day, the stomach filling treatment is carried out in the same molding period, and physiological saline is instilled into the nasal cavities of the mice, wherein the nasal cavities of the left and the right of each mouse are respectively 10 mu L.

(2) Model group (M): the mice were fed with normal feed and water throughout the experimental period. In the prevention period, 100 mu L of sterile water is filled into the stomach of each mouse every day, the stomach filling treatment is carried out in the same molding period, and LPS solution with the concentration of 12mg/mL (the administration dose is 12mg/Kg) is instilled into the nasal cavity of each mouse, and 10 mu L of LPS solution is respectively filled into the left nasal cavity and the right nasal cavity of each mouse.

(3) Dexamethasone group (P): the mice were fed with normal feed and water throughout the experimental period. 100 mu L of sterile water is perfused into each mouse every day in the prevention period, 100 mu L of the gastric lavage dexamethasone solution is perfused into the nasal cavity of the mouse in the molding period (the administration dose is 0.107mg/Kg), and LPS solution with the concentration of 12mg/mL is instilled into the nasal cavity of the mouse (the administration dose is 12mg/Kg), and 10 mu L of each nasal cavity is separately irrigated into the left nasal cavity and the right nasal cavity of each mouse. The dexamethasone dosage is converted according to the conversion method of body surface area of human body and mouse, and the dosage of dexamethasone is 0.75mg per day for adult according to the recommended dosage of the drug instruction.

(4) Astragalus aqueous extract group (a): the mice were fed with normal feed and water throughout the experimental period. In the prevention period, 100 mu L of astragalus mongholicus water extract with the gavage concentration of 0.5g/mL (the administration dose is 2.5g/kg) is administered to each mouse every day; the same gastric lavage treatment is carried out during the molding period, and LPS solution with the concentration of 12mg/mL (the administration dose is 12mg/Kg) is instilled into the nasal cavity of the mouse, and the nasal cavity of each mouse is respectively provided with 10 mu L of the left nasal cavity and the right nasal cavity. The administration dose of the astragalus aqueous extract is 2.5g/kg, the common dose of the astragalus which is regulated by 2020 edition pharmacopoeia is 9-30g, and the administration dose range (1.26g/kg-4.26g/kg) is converted according to the body surface area of a human body and a mouse.

(5) Astragalus fermentation broth group (FA): take the astragalus fermentation broth obtained in example 2 of this application as an example. In the whole experimental period, the feed and drinking water of the mice are normally supplied, and 100 mu L of astragalus fermentation liquor with the gastric gavage concentration of 0.5g/mL is fed to each mouse every day in the prevention period (the administration dose is 2.5g/kg, and the bacteria concentration is 14.76Log10 CFU/mL). The same gastric lavage was performed during the molding, and LPS solution with concentration of 12mg/mL (administration dose of 12mg/Kg) was instilled into the nasal cavity of each mouse, 10. mu.L of each mouse left and right nasal cavity. The administration dosage of the astragalus fermentation liquor is 2.5g/kg, the administration dosage range (1.26g/kg-4.26g/kg) of the daily recommended dosage of the astragalus in 2020 pharmacopoeia, which is converted by referring to the surface areas of human bodies and mouse bodies, is met, and the concentration of lactobacillus plantarum is 14.76Log10CFU/mL of the bacteria contained in the astragalus fermentation liquor under the optimal fermentation condition.

(6) Lactobacillus plantarum group (LP): the mice were fed with normal feed and water throughout the experimental period. In the prevention period, 100 mu L of lactobacillus plantarum suspension prepared by each mouse by stomach filling every day (after the astragalus fermentation liquor is centrifuged, the obtained bacteria are suspended and washed by PBS for three times, after centrifugation, the supernatant is discarded, 100 mu L of normal saline is added and mixed evenly, and the bacteria concentration is 14.76Log10 CFU/mL). The same gastric lavage treatment is carried out during the molding period, and LPS solution with the concentration of 12mg/mL (the administration dose is 12mg/Kg) is instilled into the nasal cavity of the mouse, and the nasal cavity of each mouse is respectively provided with 10 mu L of the left nasal cavity and the right nasal cavity.

(7) Astragalus aqueous extract and lactobacillus plantarum mixed liquor group (LA): the mice were fed with normal feed and water throughout the experimental period. In the prevention period, 100 mu L of the mixed solution of the astragalus mongholicus water extract and lactobacillus plantarum is prepared by intragastric administration of each mouse every day (100 mu L of astragalus mongholicus fermentation liquor is centrifuged, the obtained bacteria are suspended and washed three times by PBS (phosphate buffer solution), supernatant is removed after centrifugation, 100 mu L of astragalus mongholicus water extract is added and mixed uniformly, the administration dose is 2.5g/kg, and the bacteria concentration is 14.76Log10 CFU/mL). The same gastric lavage treatment is carried out during the molding period, and LPS solution with the concentration of 12mg/mL (the administration dose is 12mg/Kg) is instilled into the nasal cavity of the mouse, and the nasal cavity of each mouse is respectively provided with 10 mu L of the left nasal cavity and the right nasal cavity.

Establishing an animal model:

and (3) centrifuging the medicine tube filled with the LPS at low temperature, fully dissolving the LPS by using sterile physiological saline after ensuring that the powder is completely adhered to the tube wall, and preparing LPS solutions with different concentrations. After the mice are fixed, the drug is administrated by nasal drip, the left nasal cavity and the right nasal cavity of each mouse are respectively 10 mu L, the mice are killed after the drug administration of the nasal cavities is carried out for 24h, and samples such as bronchial lavage fluid, lung tissues and the like are collected for detecting various indexes.

Preparation of bronchial lavage:

after a mouse is killed by a cervical dislocation method, gently dissecting the neck skin and the chest cavity of the mouse, exposing an air outlet pipe and the chest cavity, ligating the right lung portal, gently pricking a small hole on the trachea by using a needle head, inserting a self-made trocar-gun head combination, fixing by using silk threads and forceps, slowly injecting 0.5mL of PBS (phosphate buffered saline) buffer solution precooled to 4 ℃ in advance into the left lung, irrigating the left lung tissue, slightly kneading and withdrawing by using an injector after the buffer solution stays in the lung for 1min, and repeatedly sucking for 3 times to obtain the mouse bronchial lavage fluid (BALF), wherein the liquid withdrawal rate is required to be more than 70% in the whole experiment. And (3) placing the BALF in a low-temperature centrifuge, centrifuging for 15min at 3000r/min, subpackaging the obtained supernatant into a sterile centrifuge tube, and freezing and storing at-80 ℃ for detecting the concentration of inflammatory factors and proteins.

Experimental example 4

The influence of each group on the ratio of the dry weight to the wet weight of the lung tissue of the acute lung injury mouse is researched:

collecting right lung tissue of a mouse, removing thymus gland, residual blood and fat tissue covering the surface of the lung, sucking water on the surface of the lung tissue by using filter paper, accurately weighing and recording the wet weight (W) of the lung tissue, then placing the lung tissue in a drying box at 60 ℃, drying to constant weight, weighing and recording the dry weight (D) of the lung tissue, and calculating the W/D to evaluate the edema degree of the lung tissue. The results are shown in FIG. 4.

As can be seen from fig. 4, compared with the model group, the lung wet-dry weight ratio of the mice in the dexamethasone group, the radix astragali water extract group, the radix astragali fermentation liquid group, and the radix astragali water extract and lactobacillus plantarum mixed liquid group was significantly reduced (P <0.01), and the lung wet-dry weight ratio of the mice in the lactobacillus plantarum group was not significantly changed. Compared with other administration groups, the W/D of the radix astragali fermentation liquid group in the application example 2 is closer to that of the normal group, which shows that the radix astragali fermentation liquid obtained in the application example 2 has relatively good relieving effect on the pulmonary edema degree of the mice.

Experimental example 5

Study of the effects of groups on lung pathology in mice with acute lung injury:

the method comprises the following steps of taking out the right lung middle lobe of a mouse after the mouse is sacrificed, washing the right lung middle lobe by a pre-precooled 4 ℃ PBS buffer solution, placing lung tissues in a 10% neutral formalin solution for fixation, placing the lung tissues in room temperature for 24 hours, then carrying out ethanol dehydration treatment, xylene transparency treatment and conventional paraffin embedding, cutting the lung tissues into 4 mu m slices, carrying out anti-dewaxing treatment, slice taking, tablet baking and other treatment, staining the lung tissues by hematoxylin and eosin, then carrying out dehydration, transparency and sealing treatment, observing the pathological change condition of the lung tissues under a common optical microscope, scoring the lung tissues by damage, and referring to the scoring standard of Mikawa and the like. The results are shown in FIG. 5.

As can be seen from fig. 5, after the mice instilled LPS via the nasal cavities, the alveolar space is increased, a large number of erythrocytes and neutrophils are accumulated in the alveoli and the pulmonary interstitium, the alveolar damage is severe, and the lung damage of the mice is relieved to different degrees in each administration group, the degree of lung tissue damage of the mice with the astragalus membranaceus fermentation broth group of example 2 of the application is light, and the pathological score of the lung tissue is relatively lowest.

Experimental example 6

The effect of various groups on MPO activity in lung tissue of acute lung injury mice was studied:

after sacrifice of the mice, a portion of the right lung tissue was filled into sterile EP tubes, snap frozen with liquid nitrogen and immediately frozen in a-80 ℃ freezer. The MPO content in the mouse lung tissue is detected by using a Myeloperoxidase (MPO) kit, and the operation process is strictly carried out according to the kit instruction. The results are shown in FIG. 6.

As can be seen from FIG. 6, MPO enzyme activities of the Lactobacillus plantarum group, the radix astragali water extract and the Lactobacillus plantarum mixed solution group did not significantly change; compared with other administration groups, the MPO enzyme activity of the radix astragali fermentation broth group in the embodiment 2 is lower, which shows that the inflammatory cell infiltration state in the radix astragali fermentation broth group in the embodiment 2 is lighter, and the inflammatory cell infiltration state caused by LPS is relieved.

Experimental example 7

The effect of each group on protein concentration in BALF in mice with acute lung injury was studied:

unfreezing the frozen BALF supernatant, and determining the protein concentration in BALF by using a BCA protein concentration kit according to the kit instruction. The results are shown in FIG. 7.

As can be seen from fig. 7, the protein concentration in the astragalus fermentation broth group mouse BALF is closer to that in the normal group compared to the other administration groups, which indicates that the astragalus fermentation broth of example 2 of the present application has a better effect of reducing the accumulation of protein fluid in the lung of the mouse.

Experimental example 8

The influence of each group on the concentration of inflammatory factors in BALF of acute lung injury mice was studied:

unfreezing BALF supernatant, detecting the contents of TNF-alpha, IL-1 beta and IL-6 in mouse BALF by using an ELISA kit, and strictly performing the operation process according to the kit specification.

The results are shown in fig. 8, compared with the model group, the concentrations of IL-1 β in dexamethasone, the radix astragali water extract group, the radix astragali fermentation broth, the lactobacillus plantarum group, the radix astragali water extract group and the lactobacillus plantarum mixed solution group BALF are all significantly reduced (P < 0.01); the astragalus fermentation liquor administration reduces the concentrations of IL-6, IL-1 beta and TNF-alpha in mouse BALF, and relieves the inflammation state of ALI mouse. Therefore, the astragalus fermentation liquid of the application example 2 has the best effect of reducing inflammatory factors.

Experimental example 9

Research on the regulating effect of the astragalus membranaceus fermentation liquid on the intestinal flora of the mice with acute lung injury in example 2 of the application:

the experimental mouse feces were collected, and the feces samples were filled in sterile EP tubes and immediately frozen in a-80 ℃ refrigerator. Extracting genome DNA contained in the fecal sample by SDS method, detecting the purity and concentration of the genome DNA by agarose gel electrophoresis, and carrying out PCR by using specific primers and high-efficiency fidelity enzyme. And constructing a library, performing computer sequencing and basic data analysis on the PCR product, processing sequencing original data to obtain an optimized sequence, and uploading the data to a cloud platform for flora analysis. Data were divided by OUT, and species abundance and depth were analyzed on the basis of OTU classification, and species differences among groups were analyzed. The results are shown in FIGS. 9 and 10.

As is clear from fig. 9 and 10, after the model was created, the abundance of Candidatus _ sacchara in mice with acute lung injury was significantly increased (P <0.05), the content of Helicobacter (Helicobacter pylori) was also increased, and the content of lactobacillus and bifidobacterium was decreased, as compared with the normal group. Helicobacter pylori is extremely involved in the development of gastric cancer and gastric polyps. The astragalus fermentation liquor can reduce the abundance of acute lung injury mice Corynebacterium (Corynebacterium), Staphylococcus (Staphylococcus), and GCA-900066575 (P <0.05), and increase the abundance of Eubacterium _ xylophilum _ group (P < 0.05); in addition, Akkermansia, bifidobacterium and ruminococcus abundances of the astragalus mongholicus fermentation group are remarkably increased. Corynebacterium species are enriched in bacterial communities of ALI survivors and correspond to specific immune markers and T cells in plasma. Staphylococcus is a common opportunistic pathogen that can cause serious infections and even fatality. Akkermansia can utilize substrate which can not be metabolized by the host to produce short chain fatty acid, and can be connected with the host through Gpr43 receptor, and has anti-inflammatory effect.

Experimental example 10

Study of the effect of the fermentation broth of astragalus membranaceus on the regulation of intestinal metabolism of mice with acute lung injury:

mouse stool samples were collected and frozen in a-80 ℃ freezer. Weighing 50mg of excrement sample, adding 1000 mu L of extracting solution, carrying out ultrasonic treatment for 5min (ice water bath) after grinding treatment for 4min at 35Hz, standing the sample in a refrigerator at the temperature of-40 ℃ for 1h, then centrifuging for 15min at 12000r/min, taking supernate, filtering the supernate with a 0.22 mu m filter membrane, transferring the supernate into a sample injection bottle for UHPLC-MS/MS analysis, introducing original mass spectrum data into XCMS software, carrying out the work of correcting retention time, identifying peaks, extracting peaks, integrating peaks, aligning peaks and the like, and identifying the data peaks containing the tandem mass spectrum by using a secondary mass spectrum database (provided by Shanghai Baiqu) and a cracking rule matching method. The results are shown in FIGS. 11 and 12.

As can be seen from FIGS. 11 and 12, after modeling, the fermented milkvetch root solution group has reduced content of the metabolites Indoxyl (Indoxyl), 1-methylnicotinamine (1-Methylnicotinamide), Aminoadipic acid (Aminoadipic acid), Pyridine (Pyridine), Santene (santalene), N-acetyldopamide, Acylaminophen, 2-O-methylytosin (2-O-Methylcytosine), Imidazoleacetic acid riboside (riboside imidazoleacetate), N-nitrosyldimethyl amine (dimethylamine nitroso), Nicotinate and aminonicotinamide methylecolide (Nicotinate and nicotinamide), Lysine biosynthesis (biosynthesis of Lysine), Lysine degradation (Lysine degradation), Arginine and proline (Arginine and proline), wherein the metabolic contribution of the metabolites is high, and the metabolic contribution of the metabolites is high, compared with the fermented milkvetch root solution group, wherein the fermented milkvetch root solution group has reduced content of the metabolites of Indoxyl (Indoxyl), 1-methylnicotinate and nicotinamide 3-methylnicotinate and nicotinamide (nicotinamide-methylnicotinate and nicotinamide), and the metabolic contribution of the metabolite of nicotinamide and nicotinamide is high metabolite of nicotinamide and nicotinamide 3-methylnicotinate .

In conclusion, the astragalus membranaceus fermentation liquor obtained by the preparation method has a good effect of relieving acute lung injury of mice. The concrete expression is as follows: the astragalus fermentation liquor prepared by the method effectively relieves the edema degree of the mouse lung, relieves the inflammatory cell infiltration state caused by Lipopolysaccharide (LPS), reduces the accumulation of protein liquid in the mouse lung, reduces the concentration of inflammatory factors in the acute lung injury mouse BALF, and can also regulate the frontal intestinal flora and the like of the acute lung injury mouse.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of astragalus fermentation liquor is characterized by comprising the following steps:

2. The preparation method of the astragalus fermentation broth according to claim 1, wherein in the step (1), the volume ratio of the sliced astragalus to water is 1: 8-12, the soaking time is 3-5 h, the decoction temperature is 94-97 ℃, the decoction time is 0.5-1.5 h, and the filtration times are 1-3 times.

3. The preparation method of the astragalus membranaceus fermentation liquid according to claim 1 or 2, wherein in the step (2), the volume ratio of the medicine residues to water is 1: 6-10, the decocting temperature is 94-97 ℃, and the decocting time is 0.5-1.5 hours.

4. The method for preparing astragalus fermentation broth according to claim 3, wherein in the step (3), when the sliced astragalus is 0.2-0.8 g, the volume of the astragalus water extract is 1 mL.

5. The method for preparing astragalus membranaceus fermentation liquor according to claim 4, wherein in the step (4), the total viable count of lactobacillus plantarum LZU-S-ZCJ and lactobacillus plantarum LZU-J-TSL6 is 14.5-15.5 Log10CFU/mL, and the viable count ratio of lactobacillus plantarum LZU-S-ZCJ-TSL 6 is 1: 2-4;

6. The fermentation broth of Astragalus membranaceus obtained by the method of any one of claims 1 to 5.

7. Use of the astragalus membranaceus fermentation broth of claim 6 in the preparation of a medicament for relieving acute lung injury.