CN107312730B

CN107312730B - Compound Chinese herbal medicine microecological preparation and rapid production method thereof based on solid state fermentation

Info

Publication number: CN107312730B
Application number: CN201710559527.1A
Authority: CN
Inventors: 张炎达; 潘慧青; 肖建设
Original assignee: Fujian Brady Pharmaceutical Co ltd
Current assignee: Fujian Brady Pharmaceutical Co ltd
Priority date: 2017-07-11
Filing date: 2017-07-11
Publication date: 2023-02-28
Anticipated expiration: 2037-07-11
Also published as: CN107312730A

Abstract

The invention belongs to the technical field of production and preparation of microecologics, and particularly discloses a compound Chinese herbal medicine microecologics and a rapid production method based on solid state fermentation. The method comprises the following steps: (1) activating four fermentation strains; (2) Preparing a mixed seed solution A of enterococcus faecalis BD-1 and Clostridium butyricum RCM-2; (3) preparing a saccharomyces cerevisiae seed solution B; (4) preparing a Clostridium ljunii seed solution C; (5) preparing a fermentation seed solution by proportioning A, B and C; (6) uniformly mixing the fermented seed liquid and the fermentation substrate matrix; and (7) fermenting. The method provided by the invention can obviously accelerate the fermentation rate of the solid matrix, is easy for inner bag shaping of the fermented product, reduces the fermentation production time, is beneficial to batch production, can reduce the mould pollution probability, and is suitable for popularization and use of bagged solid fermentation operation.

Description

Compound Chinese herbal medicine microecological preparation and rapid production method thereof based on solid state fermentation

Technical Field

The invention belongs to the technical field of production and preparation of microecologics, and particularly relates to a compound Chinese herbal medicine microecologics and a solid fermentation-based rapid production method thereof.

Background

The livestock industry in China is rapidly developed since the last century, the total amount of main livestock products in China is the first world, the total yield of meat products in China exceeds 10000 ten thousand tons, the quantity of eggs is about 3500 ten thousand tons, and the first world total quantity of Cicado is the first China. In addition, according to the recent supply side reform of China, the livestock scale and the development thereof in China will greatly drive the industrial development, so that the industrialization, scale and standardization development of the livestock industry in China will be further promoted globally, and the livestock breeding industry in China has huge development potential, huge space and great significance in the future. However, the phenomenon of antibiotic abuse in the livestock and poultry industry is very serious in China, in recent years, the management work of antibiotic abuse in the domestic livestock and poultry industry is increased in China, and part of antibiotic banning notices are issued by the Ministry of agriculture in 2016 and 2017 respectively. Meanwhile, domestic and even global call for antibiotic-free breeding is increasing, different antibiotic substitutes are brought forward, scientists find that the alternative potential of probiotics, chinese herbal medicines, antibacterial peptides and the like is great in the future at present, and especially the microecologics which are compatible products of the probiotics and the Chinese herbal medicines are developed rapidly. Scientific researches prove that the microecological preparation can improve the drug effect, is nontoxic and residue-free, can actively supplement intestinal probiotics, is beneficial to the repair and rebalancing of intestinal flora, and has pharmacological synergy.

At present, the effect of a fermentation type microecological preparation product in the dosage form of the microecological preparation is better, more and more domestic mobile security enterprises begin to increase the production and sale of the preparation, however, two conventional problems always restrict the further development of the industry, firstly, the fermentation speed of the microecological preparation needs longer time, the fermentation time of the solid microecological preparation is generally 2 weeks to 2 months, the long fermentation time not only reduces the goods delivery rate but also increases the cost, and the pollution probability of external pollution bacteria is increased along with the increase of time; and secondly, the microbial ecological agent has the condition of mixed bacteria pollution in the fermentation process and the shelf life, so that the management cost is increased, and the product quality is obviously influenced.

Disclosure of Invention

In order to overcome the problems that the efficient fermentation type tea microecological preparation is difficult to prepare due to the lack of tea fermentation probiotic strains capable of tolerating tea polyphenol and the shelf life of a finished product of the microecological preparation is short in the prior art, the invention aims to provide the compound Chinese herbal medicine microecological preparation and a rapid production method based on solid state fermentation.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a method for rapidly producing a compound Chinese herbal medicine microecological preparation based on solid state fermentation comprises the following steps:

(1) Activation of four fermentation strains:

enterococcus faecalis: (A)Enterococcus faecalis) Activation of BD-1: washing enterococcus faecalis BD-1 test tube slant strains with sterile normal saline in an aseptic environment, then inoculating the washed enterococcus faecalis BD-1 test tube slant strains into an MRS liquid culture medium containing 0.2 to 0.5 percent of radix pseudostellariae fibrous extract, and statically culturing for 42 to 48h at the temperature of 35 to 37 ℃; wherein the percentage of the radix pseudostellariae fibrous extract is calculated by mass volume percentage g/mL (namely, 100mL of MRS liquid culture medium contains 0.2 to 0.5g of the radix pseudostellariae fibrous extract); enterococcus faecalis BD-1 is a new strain that applicant has deposited in the chinese collection of type cultures at the address: wuhan city Wuchang Lojia mountain, preservation date: 7 months and 7 days in 2017; the preservation number is: CCTCC NO: m2017416;

clostridium butyricum (C)Clostridium butyricum) Activation of RCM-2: washing a clostridium butyricum RCM-2 test tube slant strain with sterile normal saline in an aseptic environment, then inoculating the strain into an MRS liquid culture medium containing 0.2 to 0.5 percent of radix pseudostellariae fibrous extract, and statically culturing the strain for 42 to 48h at the temperature of 35 to 37 ℃; wherein the percentage of the radix pseudostellariae fibrous extract is measured by mass volume percentage g/mL (namely 100mL MRS liquid culture medium contains 0.2 to 0.5g of radix pseudostellariae fibrous extract); clostridium butyricum RCM-2 is a new strain that applicants have deposited in the chinese type culture collection, at address: wuhan city Wuchang Lojia mountain, preservation date: 2017, 6 and 29 months; the preservation number is as follows: CCTCC NO M2017396;

saccharomyces cerevisiae (C.,)Saccharomyces cerevisiae) Activation of (2): commercial edible brewing yeast powder is inoculated into a liquid culture medium, and static culture is carried out at 30 to 37 ℃ for 24 to 32h; the viable count of the brewing yeast powder is more than or equal to 1.0 multiplied by 10 ⁸ CFU/g; the liquid culture medium comprises: 40 to 60 percent of wort, 5 to 10 percent of glucose, 0.2 to 0.5 percent of radix pseudostellariae fibrous extract and the balance of water, and the natural pH value; wherein, the percentage of the wort is calculated by volume percentage, the percentage of the glucose and the radix pseudostellariae fibrous extract is calculated by g/mL (namely 100mL of liquid culture medium contains 40 to 60mL of the wort, 5 to 10g of the glucose and 0.2 to 0.5g of the radix pseudostellariae fibrous extract);

young's clostridium (Clostridium: (II)Clostridium ljungdahlii) Activation of (2): inoculating the young clostridium liquid preserved in a frozen storage tube into an improved MRS liquid culture medium (with the same amount of fructose in the culture medium replacing glucose) containing 0.2-0.5% of radix pseudostellariae fibrous extract at one time, and statically culturing for 42-48h at 35-37 ℃; wherein the percentage of the radix pseudostellariae fibrous extract is calculated by mass volume percentage g/mL (namely, 100mL of MRS liquid culture medium contains 0.2 to 0.5g of the radix pseudostellariae fibrous extract);

(2) And preparing mixed fermentation seed liquid of enterococcus faecalis BD-1 and clostridium butyricum RCM-2, wherein the seed liquid A is:

under an aseptic environment, mixing an activated liquid of the enterococcus faecalis BD-1 obtained in the step (1) and an activated liquid of the clostridium butyricum RCM-2 according to a volume ratio of 1:1 to 5: 1 to form a mixed activated seed liquid, inoculating the mixed activated seed liquid into an MRS liquid culture medium containing 0.2 to 0.5 percent of the radix pseudostellariae fibrous extract according to a volume percentage of 3 to 5 percent, and performing static fermentation at 35 to 37 ℃ for 36 to 48h to prepare a seed liquid A; wherein the percentage of the radix pseudostellariae fibrous extract is measured by mass volume percentage g/mL (namely 100mL MRS liquid culture medium contains 0.2 to 0.5g of radix pseudostellariae fibrous extract);

(3) Preparing a saccharomyces cerevisiae seed solution, namely a seed solution B:

under an aseptic environment, inoculating the activated liquid of the saccharomyces cerevisiae obtained in the step (1) into a liquid culture medium according to the volume percentage of 5-10%, and performing static culture at the temperature of 30-37 ℃ for 30-36h to prepare a seed liquid B; the liquid culture medium consists of: 40 to 60 percent of wort, 5 to 10 percent of glucose, 0.2 to 0.5 percent of radix pseudostellariae fibrous extract and the balance of water, and the natural pH value; wherein the percentage of the wort is calculated by volume percentage, and the percentage of the glucose and the radix pseudostellariae fibrous extract is calculated by g/mL;

(4) Preparing a young clostridium seed solution, namely a seed solution C:

under an aseptic environment, inoculating 3-7% by volume of the activated solution of the young clostridium obtained in the step (1) into an improved MRS liquid culture medium containing 0.2-0.5% of a radix pseudostellariae fibrous extract, and performing static fermentation for 32-36h at the temperature of 35-37 ℃ to obtain a seed solution C; wherein the percentage of the radix pseudostellariae fibrous extract is calculated by mass volume percentage g/mL (namely, 100mL of MRS liquid culture medium contains 0.2 to 0.5g of the radix pseudostellariae fibrous extract);

(5) And preparing a fermented seed solution by proportioning the seed solutions A, B and C:

mixing the seed liquids A, B and C obtained in the steps (2), (3) and (4) according to the volume ratio of A to B to C = 1:1 to 5: 4: 3 to obtain a fermentation seed liquid;

(6) Preparing a fermentation substrate:

the fermentation substrate is prepared by crushing and mixing the following raw materials in the composition (1) or the composition (2) in parts by mass;

composition (1): 20 to 60 parts of radix pseudostellariae rootlets, 10 to 30 parts of dried orange peel, 10 to 30 parts of hawthorn, 10 to 30 parts of medicated leaven, 5 to 15 parts of green tea and 0.05 to 0.1 part of allicin;

composition (2): 20 to 30 parts of radix pseudostellariae, 20 to 25 parts of astragalus mongholicus, 10 to 15 parts of codonopsis pilosula, 10 to 15 parts of radix isatidis, 10 to 15 parts of folium isatidis, 5 to 10 parts of honeysuckle, 10 to 15 parts of dried orange peel, 10 to 15 parts of Chinese yam, 5 to 15 parts of green tea and 0.05 to 0.1 part of allicin;

(7) And fermenting:

adding the fermentation seed liquid obtained in the step (5) into the fermentation substrate matrix obtained in the step (6), uniformly stirring, filling into a fermentation bag, sealing, and fermenting the fermentation bag at the temperature of 32 to 37 ℃ for 4 to 7d under the condition of keeping out of the sun to prepare a compound Chinese herbal medicine microecological preparation; wherein the percentage of the fermentation seed liquid in the fermentation substrate matrix is 25 to 35 percent in terms of volume mass percent (L/kg).

In the present invention, the fibrous root extract of radix pseudostellariae can be obtained by the conventional techniques, for example, by referring to, but not limited to, the method disclosed in the patent entitled "a method for preparing fibrous root extract of radix pseudostellariae" filed in 2016, 8, 30 (application number 201610758305.8) by the applicant.

Has the advantages that:

1. in the invention, enterococcus faecalis BD-1 is separated from village peasant households (coordinate: 119) in Cudrania tricuspidata county ^。 51′59.7〞E，27 ^。 13 '34.0N') autotrophic healthy adult pig cecal mucosa, the obtained enterococcus faecalis BD-1 is safe and nontoxic, the surface of a bacterial colony is smooth and convex after being cultured in an MRS culture medium at 37 ℃ for 48 hours, the periphery is neat, the color of the bacterial colony is off-white, the diameter of the bacterial colony is 0.5 to 1.2mm, G + is examined under a microscope, no spore exists, and the length of the bacterial body is about 0.5 to 1 mu m; the clostridium butyricum RCM-2 of the invention is separated from village farmers (coordinate: 119) in Cudrania tricuspidata county ^。 52′04.1〞E，27 ^。 13'32.7 "" N) of a caecal mucosa segment of a healthy adult cock bred from a circled mountain forest land, the obtained clostridium butyricum RCM-2 is safe and nontoxic, the surface of a bacterial colony cultured in an RCM culture medium at 37 ℃ for 48h is smooth and convex, the periphery is neat, the color of the bacterial colony is milk yellow and opaque and has slight acid odor, the diameter of the bacterial colony is 0.5 to 1.5cm, the bacterial colony is inspected by a microscope G +, spores exist, the spore is oval, the spore is grown or is grown secondarily, the thallus of the spore is spindle-shaped, and the thallus length is about 3.5 to 7.0 mu m; enterococcus faecalis BD-1 and Clostridium acidocaldarium RCM-2 have good tea polyphenol and allicin resistance, and radix Pseudostellariae fibrous extract can remarkably promote the growth of two strains;

2. in the fermentation method of the invention, the saccharomyces cerevisiae can firstly grow rapidly to generate a large amount of CO ₂ Gas, so that the sealed fermentation bag can be quickly inflated in a short time to dilute the residual air in the bag and continuously discharge the residual air through a one-way valve to produce CO ₂ A gaseous environment; then enterococcus faecalis BD-1 and Clostridium butyricum RCM-2 can be synergistically fermented to continuously produce acid and H ₂ Gas, make the fermentation bag continuously aerated, and CO ₂ Gas and H ₂ Mixing the gases; the young clostridium grows continuously along with the reduction of the pH value of the matrix, and when organic sugar in the solid matrix is reduced (the growth of other bacteria is reduced continuously), the growth of the young clostridium is accelerated continuously to be beneficial to CO ₂ And H ₂ The gas generates organic acid and alcohol, further reduces the gas content in the sealed fermentation bag, causes the fermentation bag to be continuously shriveled and shaped, and the shape of the finished product preparation is the same as that of the materialThe result of the bag suction machine is the same after bag suction, but the bag suction effect caused by the method is better along with the time extension, the product storage is greatly facilitated, the organic acid, the sub-vacuum and the garlicin in the fermented finished product can also obviously reduce the pollution, the transportation is facilitated, and the shelf life is prolonged;

3. the method provided by the invention can obviously accelerate the fermentation rate of the fermentation substrate, is easy for inner bag shaping of the fermentation product, reduces the fermentation production time, is beneficial to batch production, can reduce the mould pollution probability, and is suitable for popularization and application of bagged solid fermentation operation.

Drawings

FIG. 1: phylogenetic tree of enterococcus faecalis BD-1.

FIG. 2: a phylogenetic tree of clostridium butyricum RCM-2.

Detailed Description

In the following examples, clostridium ljunii was purchased from the American type culture Collection (ATCC 55380); the radix pseudostellariae fibrous extract is prepared by the method disclosed in the embodiment 1 in the patent application No. CN 201610758305.8; the formula of each culture medium is as follows:

MRS liquid medium (1L): 10g of peptone, 5g of beef powder, 20g of glucose, 4g of yeast powder, 5g of sodium acetate, 2g of dipotassium phosphate, 0.2g of magnesium sulfate, 2g of ammonium citrate tribasic, 0.05g of manganese sulfate, 1mL of tween 80 and 1L of purified water, wherein the pH value is 6.2 +/-0.2;

modified MRS liquid medium (1L): the difference from the MRS liquid culture medium is that fructose in the culture medium is equivalent to replace glucose;

MRS agar plate or MRS slant medium (1L): 10g of peptone, 5g of beef powder, 20g of glucose, 4g of yeast powder, 5g of sodium acetate, 2g of dipotassium phosphate, 0.2g of magnesium sulfate, 2g of triammonium citrate, 0.05g of manganese sulfate, 80 mL of tween, 15g of agar, 1L of purified water, and the pH value of the mixture is 6.2 +/-0.2;

RCM agar plate or RCM slant medium (1L): 3g of yeast extract, 10g of beef extract, 10g of tryptone, 5g of glucose, 1g of soluble starch, 5g of sodium chloride, 3g of sodium acetate trihydrate, 0.15g of cysteine hydrochloride, 15g of agar, 1L of purified water and 6.8 +/-0.2 of pH value, and sterilizing for later use;

starch medium (1L): 5g of beef extract, 5g of peptone, 0.5g of sodium chloride, 20g of soluble starch, 18g of agar and 1L of purified water, wherein the pH value is not less than 7.2 +/-0.1, and the beef extract is sterilized for later use;

wherein, MRS liquid culture medium containing radix Pseudostellariae extract, tea polyphenol or allicin is prepared by adding radix Pseudostellariae extract, tea polyphenol or allicin on the basis of the above MRS liquid culture medium; the MRS/RCM agar plates containing bromcresol purple indicator and tea polyphenol or allicin are added with bromcresol purple indicator, tea polyphenol or allicin correspondingly on the basis of the formula of the MRS/RCM agar plates.

Example 1 isolation and identification of enterococcus faecalis BD-1

1. Separation, enrichment and purification of enterococcus faecalis BD-1

Under the aseptic operation condition, a village farmer (coordinate: 119) in Cudrania tricuspidata, dendro, fujian province is taken ^。 51′59.7〞E，27 ^。 13'34.0 "" N) autotrophic adult porcine cecal mucosal segment, mucosal mucus was scraped from EP tubes using sterile slides, diluted 10% with sterile physiological saline ² Taking 0.1mL of the bacterial strain, coating the bacterial strain on an MRS agar plate containing a bromocresol purple indicator (0.01 percent, g/mL), placing the MRS agar plate in an anaerobic incubator after coating, culturing for 48 hours at 37 ℃, selecting bacterial colonies with a culture medium becoming yellow, streaking and purifying, selecting bacterial strains with gram-positive staining and no spores through microscopic examination, rescreening the bacterial strains on the MRS agar plate containing the bromocresol purple (0.01 percent, g/mL), tea polyphenol (0.01 percent, g/mL) and garlicin (0.01 percent, g/mL), and finally selecting bacterial strains with obvious yellow color, smooth and convex surfaces of the bacterial colonies, regular peripheries as target bacterial strains, wherein the bacterial strains are named as BD-1, and performing slant MRS preservation on the bacterial strains by using a slant culture medium.

2. Morphological observation

The morphological characteristics of the colony of the strain BD-1 were observed to show that: the surface of a bacterial colony on the MRS agar plate is smooth and convex, the periphery is neat, the bacterial colony is gray, the diameter of the bacterial colony is 0.5 to 1.2mm, the bacterial colony is G +, no spore exists, and the diameter of the bacterial colony is about 0.5 to 1 mu m.

3. Physiological and biochemical characteristics

The strain BD-1 is identified according to the biochemical identification test of the lactic acid bacteria, the biochemical characteristics such as motility and oxidase tests are respectively identified, and the test result is comprehensively judged according to Bergey's bacteria identification handbook. The results are shown in Table 1.

The result of the physiological series index measurement of the isolated strain is consistent with the result of the enterococcus lactis strain.

4. 16S rDNA sequencing alignment of BD-1 strains

Extracting the total DNA of the strain BD-1 by using a bacterial genome DNA extraction kit (DP 302-02, TIANGEN), and carrying out PCR amplification on the 16 rDNA sequence of the strain under the guide of primers P0 (5: 10 XBuffer 5.0 μ L, dNTP (10 mmol/L) 2.0 μ L, primer 1 (20 μmol/L) 1.0 μ L, primer2 (20 μmol/L) 1.0 μ L, template DNA 2.5 μ L, taq enzyme (5.0U/μ L) 0.5 μ L, ultrapure water 38 μ L, and total 50 μ L. The PCR reaction conditions are as follows: firstly, 94 ℃ for 5min; then, 30 cycles of 94 ℃ for 1min,55 ℃ for 1min and 72 ℃ for 1min are performed; finally, extension is carried out for 8min at 72 ℃. After the reaction is finished, carrying out 1% agarose gel electrophoresis detection on the PCR amplification product, and comparing with a marker to obtain a target band. The target band was recovered and purified using an agarose gel recovery kit from Axygen, and sent to Biotechnology engineering (Shanghai) Co., ltd for DNA sequencing, and the sequencing results are shown in SEQ ID No.1.

The determined sequence was compared with the 16S rDNA sequence in Genbank for Blastn similarity analysis, resulting in the strains BD-1 andEnterococcus faecium strain KCI1、Enterococcus lactisthe 16S rDNA sequence of strain K12419C is 100% homologous, so BD-1 was identified as enterococcus faecalis, and its phylogenetic tree is shown in FIG. 1.

The strain BD-1 of the invention is identified and classified and named as enterococcus faecalis by combining the morphological, physiological and biochemical characteristics and sequencing and evolutionary tree resultsEnterococcus faecalis）BD-1。

5. Influence of tea polyphenols on growth and pH of enterococcus faecalis BD-1

(1) Activation of enterococcus faecalis BD-1: washing slant strain of enterococcus faecalis BD-1 test tube with sterile normal saline in sterile environment, washing off thallus Porphyrae on the surface of culture medium, inoculating into MRS liquid culture medium, and static culturing at 37 deg.C for 48 hr;

(2) Inoculating the activated liquid of the enterococcus faecalis BD-1 obtained in the step (1) into an anaerobic serum bottle containing an MRS liquid culture medium in an anaerobic box according to the inoculation amount of 3% (volume ratio), wherein the gradient of tea polyphenol in the MRS liquid culture medium in the anaerobic serum bottle is sequentially set as (g/mL): 0%, 0.10%, 0.25%, 0.50%, 1.00%, and the results of measuring the pH value and viable count of the fermentation broth at the end point of the culture are shown in Table 2.

From the results of table 2, it can be seen that: the concentration of the tea polyphenol which can be tolerated by the enterococcus faecalis BD-1 is 1.0%, and the survival rate of thalli under the concentration is 70.26%.

6. Effect of allicin on growth and pH of enterococcus faecalis BD-1

(1) Activation of enterococcus faecalis BD-1: the same procedure as in step (1) of item 5 of this example;

(2) Inoculating the activated liquid of the enterococcus faecalis BD-1 obtained in the step (1) into an anaerobic serum bottle containing an MRS liquid culture medium in an anaerobic box according to the inoculation amount of 3% (volume ratio), wherein the gradient of allicin in the MRS liquid culture medium in the anaerobic serum bottle is sequentially set to be (g/mL): 0%, 0.010%, 0.025%, 0.050%, 0.10%, and the results of measuring the pH value and viable count of the fermentation broth at the end point of the culture are shown in Table 3;

from the results in Table 3, it can be seen that: the concentration of the tolerant allicin of the enterococcus faecalis BD-1 is 0.1 percent, and the survival rate of thalli under the concentration is 70.43 percent;

(3) Growth comparison of enterococcus faecalis resistant to tea polyphenols and allicin:

respectively inoculating the activated liquid of the enterococcus faecalis BD-1 (counted as A) obtained in the step (1) and the activated liquids of the control bacteria B, C and D into an anaerobic serum bottle containing an MRS liquid culture medium according to the inoculation amount of 3 percent (volume ratio), wherein the gradient of the tea polyphenol (1) in the MRS liquid culture medium in the anaerobic serum bottle is sequentially set as (g/mL): 0%, 1.00%, 2.00%, (2) gradient of allicin set in order (g/mL): 0%, 0.05%, 0.1%, continuously culturing for 48h, and using 0% +0% as control (viable count is defined as X) ₀ ) By the formula: cell loss rate = (X) ₀ -X）/ X ₀ * The loss rate of enterococcus faecalis cells was calculated at 100%, and the results are shown in Table 4.

From the results of table 4, it can be seen that: under the combined action of tea polyphenol (2%) and allicin (0.1%), the survival rate of enterococcus faecalis BD-1 can reach 53.7%, which exceeds 50%, and is far higher than that of similar strains in the market.

7. Enterococcus faecalis bacteriostasis experiment

(2) Anaerobic static fermentation of enterococcus faecalis BD-1 in MRS liquid culture medium:

inoculating the activating solution of the enterococcus faecalis BD-1 obtained in the step (1) into an anaerobic serum bottle containing an MRS liquid culture medium according to the inoculation amount of 3% (volume ratio), statically culturing at 37 ℃ for 48h, taking a fermentation liquid at the fermentation end point, detecting the bacteriostatic effect of the fermentation liquid on staphylococcus aureus, micrococcus luteus and Escherichia coli by using an Oxford cup method, and taking the non-inoculated MRS liquid culture medium as a reference;

(3) Aerobic fermentation of enterococcus faecalis BD-1 in MRS liquid medium:

and (2) inoculating the activating solution of the enterococcus faecalis BD-1 obtained in the step (1) into a 500 mL triangular flask filled with 100mL of MRS liquid culture medium according to the inoculation amount (volume ratio) of 3%, culturing at 37 ℃ and 150rpm for 48h, taking the fermentation liquid at the end of the culture, detecting the bacteriostatic effect on staphylococcus aureus, micrococcus luteus and Escherichia coli by using an Oxford cup method, and taking the non-inoculated MRS liquid culture medium as a control.

The diameter of the zone of inhibition is shown in Table 5.

From the results in Table 5, it can be seen that: the enterococcus faecalis BD-1 can obviously inhibit the growth of pathogenic bacteria staphylococcus aureus, micrococcus luteus and Escherichia coli, and the anaerobic culture is stronger than the aerobic culture.

Example 2 isolation and identification of Clostridium butyricum RCM-2

1. Separation, enrichment and purification of clostridium butyricum RCM-2

Under the condition of aseptic operation, a village farmer (coordinate: 119) in Zhende county, nidgei, fujian province ^。 52′04.1〞E，27 ^。 13'32.7 "" N) mucosal sections of the cecum of healthy adult roosters bred from circinate arena, mucosal mucus was scraped from EP tubes with sterilized slides, diluted 10 with sterile physiological saline ² Heating to 60 ℃ for 30min, taking 0.1mL of the bacterial colony coated with a bromocresol purple indicator (0.01 percent, g/mL) on an RCM agar plate, placing the RCM agar plate in an anaerobic incubator after coating, culturing for 48h at 37 ℃, selecting a bacterial colony with a yellowing culture medium, streaking and purifying, selecting a rod-shaped bacterial strain with gram-positive staining and spores for microscopic examination, re-screening on the RCM agar plate containing bromocresol purple (0.01 percent, g/mL), tea polyphenol (0.01 percent, g/mL) and garlicin (0.01 percent, g/mL), finally selecting a bacterial colony with obvious yellow color, smooth and convex surface, milky yellow and non-transparent and acid odor as a preparation bacterial strain, then coating the preparation bacterial strain on a starch culture medium, culturing for 48h at 37 ℃, selecting a strain with the largest transparent circle as a target bacterial strain, using the bacterial strain RCM-2 as a slant culture medium, and preserving the bacterial strain by using the RCM slant culture medium.

2. Morphological observations

The colony morphology characteristics of the strain RCM-2 are observed, and the following results can be obtained: the surface of a bacterial colony on the RCM agar plate is smooth and convex, the bacterial colony is milky, yellow and opaque, has slight acid odor, has a diameter of 0.5 to 1.5cm, is microscopically checked to be G +, has spores, a spore oval, and spore end growth or sub-end growth, and the thallus of the spore is spindle-shaped, and has a length of about 3.5 to 7.0 mu m.

3. Physiological and biochemical characteristics

The bacterial strains are subjected to physiological and biochemical identification according to Bergey's Manual of bacteria identification, and physiological and biochemical tests are respectively carried out by catalase, amylase, nitrate reduction, sugar and the like, and the results are shown in Table 6.

Comparing the physiological and biochemical characteristics of the clostridium butyricum in Bergey's Manual of bacteria identification and the manual of common bacteria system identification, the result shows that the strain RCM-2 basically conforms to the physiological and biochemical characteristics of the clostridium butyricum, so the strain RCM-2 is preliminarily identified to be the clostridium butyricum.

4. Sequencing and evolutionary trees

Extracting the total DNA of the strain RCM-2 by using a bacterial genome DNA extraction kit (DP 302-02, TIANGEN), and carrying out PCR amplification on the 16S rDNA sequence of the strain under the guidance of primers P0 (5': 10 XBuffer 5.0 μ L, dNTP (10 mmol/L) 2.0 μ L, primer 1 (20 μmol/L) 1.0 μ L, primer2 (20 μmol/L) 1.0 μ L, template DNA 2.5 μ L, taq enzyme (5.0U/μ L) 0.5 μ L, ultrapure water 38 μ L, and total 50 μ L. The PCR reaction conditions are as follows: firstly, 94 ℃ for 5min; then, 30 cycles of 94 ℃ for 1min,55 ℃ for 1min and 72 ℃ for 1min are performed; finally, extension is carried out for 8min at 72 ℃. After the reaction is finished, carrying out 1% agarose gel electrophoresis detection on the PCR amplification product, and comparing with a marker to obtain a target band. The target band was recovered and purified using an agarose gel recovery kit from Axygen, and sent to Biotechnology engineering (Shanghai) Co., ltd for DNA sequencing, and the sequencing results are shown in SEQ ID No.2.

The determined sequence is compared with the 16S rDNA sequence in Genbank by Blastn similarity analysis,result Strain RCM-2 andClostridium butyricumthe homology of 16S rDNA sequence of strain 1005-15098 reaches 98%, so RCM-2 is identified as Clostridium butyricum, and the phylogenetic tree is shown in FIG. 2.

The strain RCM-2 is identified and classified as clostridium butyricum by combining the morphological, physiological and biochemical characteristics and sequencing and evolutionary tree resultsClostridium butyricum）RCM-2。

5. Influence of tea polyphenols on growth and pH of Clostridium butyricum RCM-2

(1) Activation of Clostridium butyricum RCM-2: washing the clostridium butyricum RCM-2 test tube slant strains with sterile normal saline in a sterile environment, washing down the lawn on the surface of a culture medium, then inoculating the lawn into an MRS liquid culture medium, and statically culturing for 48h at 37 ℃;

(2) Inoculating the activated liquid of clostridium butyricum RCM-2 obtained in the step (1) into an anaerobic serum bottle containing MRS liquid culture medium in an anaerobic box according to the inoculation amount of 3 percent (volume ratio), wherein the gradient of tea polyphenol in the MRS liquid culture medium in the anaerobic serum bottle is sequentially set as (g/mL): 0%, 0.10%, 0.25%, 0.50%, 1.00%, and the results of measuring the pH value and viable count of the fermentation broth at the end point of the culture are shown in Table 7.

From the results of Table 7, it can be seen that: the concentration of tea polyphenol which can be tolerated by clostridium butyricum RCM-2 is 1.0%, and the survival rate of thalli at the concentration is 87.3%.

6. Effect of allicin on growth and pH of Clostridium butyricum RCM-2

(1) Activation of Clostridium butyricum RCM-2: the same as step (1) in item 5 of this embodiment;

(2) Inoculating the activated liquid of clostridium butyricum RCM-2 obtained in the step (1) into an anaerobic serum bottle containing MRS liquid culture medium in an anaerobic box according to the inoculation amount of 3 percent (volume ratio), wherein the gradient of allicin in the MRS liquid culture medium in the anaerobic serum bottle is sequentially set as (g/mL): 0%, 0.010%, 0.025%, 0.050%, 0.10%, and the results of measuring the pH value and viable count of the fermentation broth at the end point of the culture are shown in Table 8;

from the results in Table 8, it can be seen that: the concentration of allicin which can be tolerated by the clostridium butyricum RCM-2 is 0.1%, and the survival rate of thalli at the concentration is 85.5%;

(3) Clostridium butyricum tolerates tea polyphenols and allicin growth comparison:

inoculating the activation liquid of the clostridium butyricum RCM-2 (counted as A) obtained in the step (1) into an anaerobic serum bottle containing an MRS liquid culture medium in an anaerobic box according to the inoculation amount of 3 percent (volume ratio), wherein the gradient of (1) tea polyphenol in the MRS liquid culture medium in the anaerobic serum bottle is sequentially set to be (g/mL): 0%, 1.00%, 2.00%, (2) gradient of allicin set to (g/mL): 0%, 0.05%, 0.1%, continuously culturing for 48h, with 0% +0% as control (viable count is defined as X) ₀ ) By the formula: cell loss rate = (X) ₀ -X）/ X ₀ * The loss rate of Clostridium butyricum cells was calculated at 100%, and the results are shown in Table 9.

From the results of Table 9, it can be seen that: under the combined action of tea polyphenol (1%) and allicin (0.05%), the survival rate of the clostridium butyricum RCM-2 can reach 88.5%, which exceeds 50%, and is far higher than that of similar strains in the market.

7. Clostridium butyricum bacteriostatic test

(1) Activation of Clostridium butyricum RCM-2: the same procedure as in step (1) of item 5 of this example;

(2) Inoculating the activated liquid of clostridium butyricum RCM-2 obtained in the step (1) into an anaerobic serum bottle containing MRS liquid culture medium according to the inoculation amount of 3 percent (volume ratio), statically culturing at 37 ℃ for 48 hours, taking fermentation liquor at the fermentation end point, detecting the bacteriostatic effect of the fermentation liquor on staphylococcus aureus, micrococcus luteus and Escherichia coli by using an Oxford cup method, and taking the non-inoculated MRS liquid culture medium as a reference; the diameter of the zone is shown in Table 10.

From the results in Table 10, it can be seen that: the clostridium butyricum RCM-2 can obviously inhibit the growth of pathogenic bacteria staphylococcus aureus, micrococcus luteus and escherichia coli.

Example 3 growth promotion test of radix Pseudostellariae Leptoradix Pseudostellariae extract on fermentation Strain

(1) Activation of four fermentation strains:

activation of enterococcus faecalis BD-1: washing test tube slant strain of enterococcus faecalis BD-1 with sterile normal saline in sterile environment, washing off thallus Porphyrae on the surface of culture medium, inoculating into MRS liquid culture medium, and static culturing at 37 deg.C for 48 hr;

activation of Clostridium butyricum RCM-2: washing test tube slant strain of Clostridium butyricum RCM-2 with sterile normal saline in sterile environment, washing off thallus Porphyrae on the surface of culture medium, inoculating into MRS liquid culture medium, and static culturing at 37 deg.C for 48 hr;

activation of saccharomyces cerevisiae: inoculating commercial edible wine-making yeast powder into a liquid culture medium, and performing static culture at 32 ℃ for 32 hours; the viable count of the brewing yeast powder is more than or equal to 1.0 multiplied by 10 ⁸ CFU/g; the liquid culture medium comprises: wort 45%, glucose 6% and balance water, natural pH, wherein the percentage of wort is in volume percent and the percentage of glucose is in g/mL;

activation of young clostridium: inoculating the young clostridium liquid preserved in the frozen tube into an improved MRS liquid culture medium at one time, and performing static culture at 37 ℃ for 48 hours;

(2) Growth promotion test:

inoculating the activating solution of the enterococcus faecalis BD-1 obtained in the step (1) into an anaerobic serum bottle containing an MRS liquid culture medium in an anaerobic box according to the inoculation amount of 3% (volume ratio), wherein the gradient of the radix pseudostellariae fibrous extract in the MRS liquid culture medium in the anaerobic serum bottle is sequentially set as (g/mL): 0%, 0.10% and 0.25%, static culturing at 37 ℃ for 48h, and measuring the pH value and viable count of the fermentation liquor at the end point of the culture;

inoculating the activated liquid of the clostridium butyricum RCM-2 obtained in the step (1) into an anaerobic serum bottle (the top of the anaerobic serum bottle is externally connected with a sensitive gas pressure gauge device) containing an MRS liquid culture medium in an anaerobic box according to the inoculation amount of 3 percent (volume ratio), wherein the gradient of the radix pseudostellariae fibrous extract in the MRS liquid culture medium in the anaerobic serum bottle is sequentially set as (g/mL): 0%, 0.10% and 0.25%, static culturing at 37 ℃ for 48h, and measuring the pH value, viable count and end point pressure value of the fermentation liquor when the culture is at the end point;

inoculating the activated liquid of the saccharomyces cerevisiae obtained in the step (1) into an anaerobic serum bottle (the top of the anaerobic serum bottle is externally connected with a sensitive gas pressure gauge device) containing a liquid culture medium according to the inoculation amount of 5% (volume ratio), statically culturing for 32 hours at 32 ℃, and measuring the pH value, the viable count and the end point pressure value of the fermentation liquid when the culture is at the end point; the liquid culture medium consists of: 45% of wort, 6% of glucose, 6% of radix pseudostellariae fibrous extract and the balance of water, and the natural pH value is set, wherein the percentage of the wort is calculated by volume percentage, and the percentage of the glucose and the radix pseudostellariae fibrous extract is calculated by g/mL; the gradient of the radix pseudostellariae fibrous extract in the liquid culture medium in the anaerobic serum bottle is sequentially set as follows: 0%, 0.10%, 0.25%;

inoculating the activated liquid of the young clostridium obtained in the step (1) into an improved MRS liquid culture medium according to the inoculation amount of 5 percent (volume ratio), wherein the gradients of the radix pseudostellariae fibrous extracts in the improved MRS liquid culture medium are sequentially set as (g/mL): 0%, 0.10% and 0.25%, standing and fermenting at 37 deg.C for 32h, and measuring pH value and viable count of the fermentation liquid at the end point of the culture.

The viable cell count results are shown in Table 11, and the end point pressure increase rate results are shown in Table 12.

As can be seen from tables 11-12: the radix pseudostellariae fibrous extract can obviously promote the growth of enterococcus faecalis BD-1, clostridium butyricum RCM-2, saccharomyces cerevisiae and Clostridium youngyi; meanwhile, the radix pseudostellariae fibrous extract with the content of 0.1-0.25% not only obviously promotes the growth of RCM-2 and saccharomyces cerevisiae, but also obviously improves the fermentation gas production capacity of RCM-2 and saccharomyces cerevisiae, so that the addition of the radix pseudostellariae fibrous extract with the content of 0.1-0.25% provides reliable technical reference for promoting the growth of thalli and rapidly producing gas during the later-stage matrix solid fermentation so as to accelerate the exhaust rate of the sealed fermentation bag.

Example 4 symbiotic growth of enterococcus faecalis BD-1 and Clostridium butyricum RCM-2

(1) Activation of the Strain

(2) Then inoculating the activated liquid of enterococcus faecalis BD-1, the activated liquid of Clostridium butyricum RCM-2 and the activated liquid compounded by the two according to the volume ratio of 1 to 1 into a 200 mL anaerobic serum bottle of MRS liquid culture medium according to the inoculation amount of 3 percent (volume ratio), wherein the gradient of the radix pseudostellariae fibrous extract in the MRS liquid culture medium in the anaerobic serum bottle is sequentially set as (mass ratio): 0 percent and 0.25 percent, static culture is carried out for 48 hours at 37 ℃, the viable count of the fermentation liquor is measured when the culture end point is reached, and in addition, the spore rate of the clostridium butyricum RCM-2 is measured; the results are shown in Table 13.

As can be seen from table 13: the radix pseudostellariae fibrous extract can remarkably promote the symbiotic growth of enterococcus faecalis BD-1 and Clostridium butyricum RCM-2, and the symbiotic promotion degree of two strains is greater than the sum of the number of two single strains under the same concentration; in addition, the spore rate of RCM-2 can be promoted at a concentration of 0.25%, and the spore rate of symbiotic culture is further improved.

Example 5 safety test

(1) Activation of four fermentation strains:

activation of enterococcus faecalis BD-1: washing slant strain of enterococcus faecalis BD-1 with sterile normal saline in sterile environment, inoculating into MRS liquid culture medium containing radix Pseudostellariae fibrous extract 0.25%, and static culturing at 37 deg.C for 48 hr; wherein the percentage of the radix pseudostellariae fibrous extract is calculated by mass volume percentage g/mL;

activation of Clostridium butyricum RCM-2: washing test tube slant strain of Clostridium butyricum RCM-2 with sterile normal saline under sterile environment, inoculating into MRS liquid culture medium containing radix Pseudostellariae fibrous extract 0.25%, and static culturing at 37 deg.C for 48 hr; wherein the percentage of the radix pseudostellariae fibrous root extract is calculated by mass volume percentage g/mL;

activation of saccharomyces cerevisiae: inoculating commercial edible wine-making yeast powder into a liquid culture medium, and performing static culture at 32 ℃ for 32 hours; the viable count of the brewing yeast powder is more than or equal to 1.0 multiplied by 10 ⁸ CFU/g; the liquid culture medium comprises: 45% of wort, 6% of glucose, 0.25% of radix pseudostellariae fibrous extract and the balance of water, wherein the natural pH value is shown in the specification, the percentage of the wort is calculated by volume percentage, and the percentage of the glucose and the radix pseudostellariae fibrous extract is calculated by g/mL;

activation of young clostridium: inoculating the young clostridium bacterium liquid preserved in the frozen tube into an improved MRS liquid culture medium containing 0.25 percent of radix pseudostellariae fibrous extract at one time, and statically culturing for 48 hours at 37 ℃; wherein the percentage of the radix pseudostellariae fibrous root extract is calculated by mass volume percentage g/mL;

under an aseptic environment, the activating solution of enterococcus faecalis BD-1 obtained in the step (1) and the activating solution of Clostridium butyricum RCM-2 form a mixed activated seed solution according to the volume ratio of 1; wherein the percentage of the radix pseudostellariae fibrous extract is calculated by mass volume percentage g/mL;

(3) Preparing a saccharomyces cerevisiae seed solution, and calculating as a seed solution B:

under an aseptic environment, inoculating 8% volume percent of the activation solution of the saccharomyces cerevisiae obtained in the step (1) into a liquid culture medium, and performing static culture at 32 ℃ for 32 hours to obtain a seed solution B; the liquid culture medium consists of: 45% of wort, 6% of glucose, 0.25% of radix pseudostellariae fibrous extract and the balance of water, wherein the natural pH value is that the percentage of the wort is calculated by volume percentage, and the percentage of the glucose and the radix pseudostellariae fibrous extract is calculated by g/mL;

(4) Preparing the young clostridium seed liquid, wherein the seed liquid is the seed liquid C:

inoculating the activated solution of the young clostridium obtained in the step (1) into an improved MRS liquid culture medium containing 0.25% of radix pseudostellariae fibrous extract according to the volume percentage of 5% under the aseptic environment, and standing and fermenting at 37 ℃ for 32 hours to obtain a seed solution C; wherein the percentage of the radix pseudostellariae fibrous extract is calculated by mass volume percentage g/mL;

(5) Safety test

Test animals: KM mice, which are of SPF grade, have the weight of 18 to 22g and are female and male in half, are purchased from the institute of Chinese medicine in Chongqing.

Test bacterial liquid: and C = 1:1 and 5: 4: 3 in volume ratio.

And (3) testing: selecting 10 healthy mice, each half of the mice are male and female, and the average body weight is as follows: female (21.90 + -0.5) g, male (22.00 + -0.5) g. After the mice are fasted (without water) for 12 hours, the mice are subjected to intragastric administration by test bacterium liquid, and the test bacterium liquid is subjected to administration of equal dose for 3 times a day, wherein the total administration dose is 1.5mL. After administration, the mice are normally raised in a clean-grade raising room at room temperature (21 +/-0.5) DEG C and with the relative humidity of 45-60%, the animals are observed to have abnormal changes in activity, fur, diet and excrement, toxic symptoms and death are observed, and the results are shown in a table 14 after three weeks of continuous observation.

Therefore, the following steps are carried out: the fermentation strain composition is safe and nontoxic.

EXAMPLE 6 preparation of Microecological preparation

The preparation method of the microecological preparation comprises the following steps:

steps (1) - (4): the same procedures as in steps (1) to (4) of example 5, respectively;

(5) And preparing the fermented seed liquid by proportioning the seed liquid A, the seed liquid B and the seed liquid C:

mixing the seed liquids A, B and C obtained in the steps (2), (3) and (4) according to the volume ratio of A: B: C = 1:1 to obtain a fermented seed liquid;

(6) Preparing a fermentation substrate:

the fermentation substrate is prepared by crushing and mixing the following raw materials in parts by weight: 25 parts of radix pseudostellariae fibrous roots, 20 parts of dried orange peels, 20 parts of hawthorns, 20 parts of medicated leavens, 15 parts of green tea and 0.06 part of allicin;

(7) And (3) fermentation:

firstly, feeding the fermentation substrate prepared in the step (6) into stirring equipment (the equipment adopts utility model patent equipment, the patent number is 201620053478.5), premixing for 5min, the mixing speed is 100 rpm, adding the fermentation seed liquid obtained in the step (5) into the equipment, the flow rate is 30mL/s, stirring, the stirring speed is 100 rpm, and the retention time is 20min, wherein the fermentation seed liquid accounts for 35% of the fermentation substrate in terms of volume mass percent L/kg; after being uniformly stirred, the mixture is quickly weighed and bagged, the fermentation bag is arranged in a utility model patent bag (patent number: 201620018088.4), the bag surface is quickly paved and pressed to discharge redundant air after bagging and sealing, a one-way valve is upward, the bag surface is sequentially placed in a fermentation vehicle (utility model patent number: 201620981592.4), the fermentation vehicle is further propelled, the fermentation vehicle requires constant temperature (35 ℃) and light shielding, and fermentation is started after the fermentation bags are arranged and stacked in the fermentation vehicle.

Preparation of control microecologics: mixing commercially available mixed fermentation bacteria (purchased from Zhengzhou Oukebake Biotechnology limited, trade name: backer; mixed fermentation bacteria comprises Bacillus subtilis, lactobacillus, and yeast, and total viable count is not less than 1.0 × 10 ⁹ CFU/g) as a control strain, activating according to the method of the product specification to prepare a control fermentation seed solution to replace the fermentation seed solution of the embodiment, and fermenting under the same fermentation substrate and conditions as the invention.

Taking the time point when the fermentation bag starts to suck and shape as the end time point of fermentation, then sampling, respectively taking the total viable count, the end pH value, the plastic strength of the fermentation bag sucked bag and the contamination rate of mixed bacteria as test indexes, randomly mixing 5 bags, and sampling. Wherein the two types are fermented for 30 bags, and the bag amount is 10kg.

The results are shown in Table 15.

Example 7 preparation of a Microecological formulation

The difference from example 6 is that: in the step (5), A: B: C = 5: 4: 3; in the step (6), the fermentation substrate consists of: 20 parts of radix pseudostellariae, 20 parts of astragalus membranaceus, 10 parts of codonopsis pilosula, 10 parts of radix isatidis, 10 parts of folium isatidis, 5 parts of honeysuckle, 10 parts of pericarpium citri reticulatae, 10 parts of Chinese yam, 5 parts of green tea and 0.06 part of allicin.

The results are shown in Table 16.

As can be seen from tables 15-16: the microbial ecological agent has a shorter fermentation period, shortens about half of the time, and has higher total viable bacteria, lower substrate pH value, stronger fermentation bag absorption shape strength and lower mould pollution rate when reaching the fermentation end point.

Example 8 application example of the finished Microecological preparation

(1) Example 6 Effect of the Microecological preparation product of the present invention on broiler growth Performance

The experimental scheme is as follows: 120 1-day-old avium broiler chickens were randomly divided into 2 groups of 3 replicates each of which contained 20 replicates each. The control group was fed with a basal diet, and the test group added 0.2wt% (based on the mass percentage of the basal diet) of the microecological preparation of the present invention in example 6 to the basal diet, which was a corn-soybean meal type powdery material, prepared with reference to NRC (1994) broiler nutritional standard and chinese feed nutritional ingredient table. The experimental broiler chickens are raised in cages, each broiler chicken is fed independently and repeatedly, the broiler chickens are fed with food and water freely, and daily management and immunization procedures are carried out according to the conventional method. The cultivation time was set to 6 weeks. The weights were taken before the early feeding on the 4 and 6 weekends, and the daily gain and the feed-weight ratio were calculated, respectively, and the results are shown in Table 17.

(2) Example 7 Effect of the Microecological preparation product of the invention on the growth Performance of piglets

The experimental scheme is as follows: 60 growing, 21-day-old, triple-weaned piglets, which were 60 and 21 days old and had a body weight of about 20kg, were randomly divided into 2 groups of 3 replicates each, each 10 replicates. The control group was fed with a basal diet, and the test group was supplemented with 0.25wt% of the inventive probiotic product of example 7 on the basal diet, which had the following formula: 56.00 wt% of corn, 30.00wt% of soybean meal, 10.00wt% of bran and 4.00wt% of premix (each kilogram of premix contains 15000IU of vitamin A and vitamin D) ₃ 8000IU, vitamin E60 IU, VK 2.5 IU). The test pigs are fed in groups and are fully fed, 4 times a day, free drinking water is carried out, the test period is 40d, and the management is normal. Weight gain and feed conversion were determined at the end of the test and the results are shown in table 18.

As can be seen from tables 17-18: compared with a control group, the finished product microecological preparation group can obviously promote the growth of livestock and poultry and obviously reduce the feed conversion ratio.

SEQUENCE LISTING

<110> Fujian Beidi pharmaceutical Co Ltd

<120> a compound Chinese herbal medicine micro-ecological preparation and a rapid production method thereof based on solid state fermentation

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Claims

1. A quick production method of a compound Chinese herbal medicine microecological preparation based on solid state fermentation is characterized by comprising the following steps:

(1) Activation of four fermentation strains:

enterococcus faecalis: (Enterococcus faecalis) Activation of BD-1: flushing enterococcus faecalis BD-1 test tube slant strains with sterile normal saline in an aseptic environment, then inoculating the washed enterococcus faecalis BD-1 test tube slant strains into an MRS liquid culture medium containing 0.2 to 0.5 percent of radix pseudostellariae fibrous root extract, and statically culturing for 42 to 48h at 35 to 37 ℃; wherein, theThe percentage of the radix pseudostellariae fibrous extract is calculated by mass volume percentage g/mL; enterococcus faecalis BD-1 is a new strain, and the preservation number is CCTCC NO: m2017416;

clostridium butyricum (C)Clostridium butyricum) Activation of RCM-2: washing a clostridium butyricum RCM-2 test tube slant strain with sterile normal saline in an aseptic environment, then inoculating the clostridium butyricum RCM-2 test tube slant strain into an MRS liquid culture medium containing 0.2 to 0.5 percent of radix pseudostellariae fibrous extract, and statically culturing for 42 to 48h at 35 to 37 ℃; wherein the percentage of the radix pseudostellariae fibrous extract is calculated by mass volume percentage g/mL; the clostridium butyricum RCM-2 is a new strain, and the preservation number is CCTCC NO M2017396;

saccharomyces cerevisiae (Saccharomyces cerevisiae) Activation of (2): commercial edible brewing yeast powder is inoculated into a liquid culture medium, and static culture is carried out at 30 to 37 ℃ for 24 to 32h; the liquid culture medium consists of: 40 to 60 percent of wort, 5 to 10 percent of glucose, 0.2 to 0.5 percent of radix pseudostellariae fibrous extract and the balance of water, and the natural pH value is set; wherein the percentage of the wort is calculated by volume percentage, and the percentage of the glucose and the radix pseudostellariae fibrous extract is calculated by g/mL;

young's clostridium (Clostridium: (II)Clostridium ljungdahlii) Activation of (2): inoculating the young clostridium liquid preserved in a frozen storage tube into an improved MRS liquid culture medium containing 0.2 to 0.5 percent of radix pseudostellariae fibrous extract at one time, and statically culturing for 42 to 48h at 35 to 37 ℃; wherein the percentage of the radix pseudostellariae fibrous extract is calculated by mass volume percentage g/mL;

(2) And preparing mixed fermentation seed liquid of enterococcus faecalis BD-1 and clostridium butyricum RCM-2, wherein the mixed fermentation seed liquid is calculated as seed liquid A:

under an aseptic environment, mixing an activated liquid of the enterococcus faecalis BD-1 obtained in the step (1) and an activated liquid of the clostridium butyricum RCM-2 according to a volume ratio of 1:1 to 5: 1 to form a mixed activated seed liquid, inoculating the mixed activated seed liquid into an MRS liquid culture medium containing 0.2 to 0.5 percent of the radix pseudostellariae fibrous extract according to a volume percentage of 3 to 5 percent, and performing static fermentation at 35 to 37 ℃ for 36 to 48h to prepare a seed liquid A; wherein the percentage of the radix pseudostellariae fibrous extract is calculated by mass volume percentage g/mL;

under an aseptic environment, inoculating the activated solution of the saccharomyces cerevisiae obtained in the step (1) into a liquid culture medium according to the volume percentage of 5 to 10%, and statically culturing at 30 to 37 ℃ for 30 to 36h to obtain a seed solution B; the liquid culture medium consists of: 40 to 60 percent of wort, 5 to 10 percent of glucose, 0.2 to 0.5 percent of radix pseudostellariae fibrous extract and the balance of water, and the natural pH value; wherein the percentage of the wort is calculated by volume percentage, and the percentage of the glucose and the radix pseudostellariae fibrous extract is calculated by g/mL;

(4) Preparing a young clostridium seed solution, namely a seed solution C:

under an aseptic environment, inoculating 3-7% by volume of the activated solution of the young clostridium obtained in the step (1) into an improved MRS liquid culture medium containing 0.2-0.5% of a radix pseudostellariae fibrous extract, and performing static fermentation for 32-36h at the temperature of 35-37 ℃ to obtain a seed solution C; wherein the percentage of the radix pseudostellariae fibrous root extract is calculated by mass volume percentage g/mL;

mixing the seed liquids A, B and C obtained in the steps (2), (3) and (4) according to the volume ratio of A: B: C = 1:1 or 5: 4: 3 to obtain a fermentation seed liquid;

(6) Preparing a fermentation substrate:

(7) And (3) fermentation:

adding the fermentation seed liquid obtained in the step (5) into the fermentation substrate obtained in the step (6), stirring and mixing uniformly, filling into a fermentation bag, sealing, and fermenting the fermentation bag at the temperature of 32 to 37 ℃ for 4 to 7d under the condition of keeping out of the sun to obtain a compound Chinese herbal medicine microecological preparation; wherein the percentage of the fermentation seed liquid in the fermentation substrate matrix is 25 to 35 percent in terms of volume mass percent (L/kg).

2. A compound Chinese herbal medicine microecological preparation prepared by the production method of claim 1.