Method for preparing renshenhuang based on artificial synthetic flora fermentation of licorice and application thereof

Abstract

This invention discloses a method and application for preparing Renzhonghuang (a traditional Chinese medicine) from licorice using artificially synthesized bacterial cultures through fermentation. It belongs to the field of traditional Chinese medicine preparation technology. The method involves analyzing the dominant probiotic flora in healthy human fecal samples using metagenomic sequencing, screening out 15 representative intestinal probiotic strains, and constructing an artificially synthesized bacterial culture based on their relative abundance ratios in the natural gut. Licorice powder is mixed with a suspension of the artificially synthesized bacterial culture, and fermented at 37°C to prepare Renzhonghuang. The resulting Renzhonghuang is stable in quality and has high safety. Pharmacological experiments have confirmed that its anti-H1N1 influenza virus effect is superior to that of Renzhonghuang produced using traditional methods, significantly reducing viral load, inflammatory factor levels, and lung index in lung tissue, and improving lung pathological damage. This invention eliminates the unclean process of traditional cesspool fermentation, achieving standardized, controllable, and industrialized production of Renzhonghuang, and has significant clinical application value and market prospects.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine preparation technology, and more specifically to a method and application for preparing human-infused yellow licorice based on artificially synthesized microbial fermentation. Background Technology

[0002] Human placenta extract is a traditional Chinese medicine with the effects of clearing heat and cooling blood, purging fire and detoxifying. It is used for epidemic febrile diseases, rashes caused by febrile diseases, high fever and thirst, blood heat in smallpox, erysipelas, and sores. The Yuan Dynasty text *Danxi Xinfa* by Zhu Zhenheng records its preparation method as follows: "Human placenta extract is made by placing licorice powder into a bamboo tube, plugging both ends with bamboo or wood, soaking it in a manure pit during winter, and removing it in early spring to air-dry in a shady place. The bamboo is then split open to extract the licorice, which is then dried in the sun." The *Chinese Materia Medica Dictionary* records its preparation method as follows: Grind licorice into coarse powder, fill it into a bamboo tube with a node at one end, plug the other end with cloth, seal the opening with rosin, scrape off the outer layer of the bamboo tube, and soak it in a manure pit for 2-3 months. It is usually soaked in winter, and removed the following spring. It is then rinsed in clean water for 2-3 weeks, changing the water daily until it is odorless. After air-drying, the bamboo tube is split open, the licorice is extracted, and then dried in the sun.

[0003] However, the traditional preparation method of Ren Zhong Huang (a traditional Chinese medicine) poses serious hygiene and safety risks. The latrine pits contain various pathogenic microorganisms, such as bacteria, viruses, and parasite eggs, posing significant biosafety risks to users. Furthermore, the traditional production process relies entirely on the natural environment and individual experience, resulting in highly unstable active ingredient composition and efficacy of the final product, with significant batch-to-batch variations, making standardized quality evaluation and controllable. From an industrial production perspective, this method is strictly limited by season, region, and specific operating conditions, leading to low production efficiency and failing to meet the demands of modern industrial production and market distribution. Moreover, due to its unique raw material sources and preparation process, traditional Ren Zhong Huang is psychologically difficult for the general public to accept, severely impacting patient adherence and its application and promotion in the modern pharmaceutical market.

[0004] Currently, some studies have attempted to use a single microorganism to replace cesspool fermentation in the preparation of Renzhonghuang (CN106822227A). The preparation method involves fermenting licorice root in a solution of E. coli cultured from feces and a compound ammonified alkaline solution. Another method uses fermentation broth obtained from in vitro culture of intestinal microorganisms as a substitute for feces (CN113209157A). Specifically, feces are vortexed with sterile, anaerobic PBS solution, filtered through an 800-mesh filter to obtain a fecal inoculum. The intestinal microecological typing is confirmed to be Prevotella enterotype. The fecal inoculum is then inoculated into an in vitro fermentation tank for anaerobic culture, and the fermentation broth is collected as a substitute for the fecal inoculum in the preparation process of Renzhonghuang.

[0005] Methods relying solely on a single bacterial strain (such as E. coli) have significant limitations in metabolic function, failing to fully replicate the complex environment and metabolic network of multi-microbial synergy in traditional fermentation. Traditional fermentation of human licorice relies on the synergistic metabolism of various microorganisms (including bacteria and fungi) in the fecal environment. Different strains perform specific functions: some decompose macromolecules in licorice, while others utilize the metabolites of preceding strains to synthesize new active ingredients, forming a continuous and complex chain of component transformation. A single strain can only complete limited metabolic reactions, making it difficult to cover the entire chain of transformation involving multiple microbial communities. This not only easily leads to the loss of some key trace active ingredients but may also cause imbalances in the content of certain components due to the lack of metabolic regulation among microbial communities.

[0006] In vitro culture of gut microbiota and subsequent fermentation broth can replace septic tanks to simulate the synergistic fermentation of multiple strains. However, using only Prevotella enterotype as a substitute for fermentation broth cannot fully simulate the complex environment in septic tanks, and the microbiota will fluctuate during the fermentation process.

[0007] Therefore, there is an urgent need for a new preparation process for Renzhonghuang that can eliminate impure raw materials and ensure product safety, stability, and uniform quality under controllable conditions, thereby promoting the modernization of this traditional medicine. This is a technical problem that needs to be solved by those skilled in the art. Summary of the Invention

[0008] In view of this, the present invention provides a method and application for preparing human-derived yellow licorice by fermentation based on artificially synthesized microbial communities. The method uses synthetic microbial communities with well-defined microbial community structure and abundance, that is, the microbial community ratio and number are well-defined, so as to achieve precise control in the production process of human-derived yellow licorice. The resulting human-derived yellow licorice meets the requirements of drug safety and stable quality, and retains the core pharmacological material basis of traditional human-derived yellow licorice. It has important clinical application value and market prospects.

[0009] To solve the above-mentioned technical problems, this application adopts the following technical solution:

[0010] The primary objective of this application is to provide a method for preparing human-derived yellow licorice by fermentation of artificially synthesized microbial communities, comprising the following steps: (1) Strains screening: Dominant probiotics were analyzed from fecal samples of healthy individuals using metagenomic sequencing. Based on the sequencing results, an artificial synthetic flora consisting of 15 probiotic strains was designed and constructed, simulating the gut microbiota structure of healthy individuals. The 15 strains are as follows: Lactobacillus casei Lactobacillus paracasei CGMCC 1.2435; Lactobacillus gasseri Lactobacillus gasseri CICC 24878; Bifidobacterium adolescentis Bifidobacterium adolescentis CICC 6175; Pediococcus acidilactici Pediococcus acidilactici CICC 10344; Lactobacillus amyloliquefaciens Lactobacillus amylovorus CICC 6090; Lactobacillus johnsonii Lactobacillus johnsonii CICC 6084; Fermented Lactobacillus mucinus Limosilactobacillus fermentum CGMCC 1.1880; Bifidobacterium pseudosporidioides Bifidobacterium pseudocatenulatum CGMCC 1.2277; Lactobacillus rhamnosus Lactobacillus rhamnosus CICC 6141; Lactobacillus plantarum Lactiplantibacillus plantarum CICC 6240; Bifidobacterium bifidum Bifidobacterium bifidum CICC 6166; Pediococcus pentosaceus Pediococcus pentosaceus CGMCC 1.2695; short-lived lactobacillus Levilactobacillus brevis CICC 25354; Bifidobacterium longum Bifidobacterium longum CICC 6186; Mucosal lactobacilli Limosilactobacillus mucosae CGMCC 1.15986; (2) Construction of artificially synthesized microbial community: The 15 strains described in step (1) are expanded and cultured to prepare bacterial suspensions. The bacterial suspensions are mixed according to the optimized ratio to obtain artificially synthesized microbial community bacterial suspension. (3) Licorice processing: Licorice is washed, dried to constant weight, pulverized and sieved to obtain licorice powder; (4) Fermentation: The licorice powder obtained in step (3) is mixed with the artificially synthesized bacterial suspension obtained in step (2) and fermented, then dried to prepare the fermentation product; the fermentation product is boiled, extracted, concentrated and dried to prepare human yellow.

[0011] As a preferred technical solution, in step (2), according to the above-mentioned strain order, the ratio of viable bacteria counts of the 15 strains in the artificially synthesized bacterial suspension is: 3:7:23:3:10:9:4:17:1:3:2:1:2:13:2; the viable bacteria count in the bacterial suspension is 1×10⁻⁶. 8 CFU / mL.

[0012] As a preferred technical solution, the fermentation temperature in step (4) is 37°C and the fermentation time is 20 days; the drying temperature is 60°C.

[0013] As a preferred technical solution, the ratio of licorice powder to artificially synthesized bacterial suspension in step (4) is 1g:1ml.

[0014] Another object of this application is to provide: human placenta yellow prepared by the method.

[0015] Another object of this application is to provide the use of the human ginseng prepared by the method in the preparation of anti-influenza A virus drugs.

[0016] As a preferred technical solution, the influenza A virus is the H1N1 subtype.

[0017] Another object of this application is to provide: an artificially synthesized bacterial community composed of the following 15 strains: Lactobacillus casei Lactobacillus paracasei CGMCC 1.2435; Lactobacillus gasseri Lactobacillus gasseri CICC 24878; Bifidobacterium adolescentis Bifidobacterium adolescentis CICC 6175; Pediococcus acidilactici Pediococcus acidilactici CICC 10344; Lactobacillus amyloliquefaciens Lactobacillus amylovorus CICC 6090; Lactobacillus johnsonii Lactobacillus johnsonii CICC 6084; Fermented Lactobacillus mucinus Limosilactobacillus fermentum CGMCC 1.1880; Bifidobacterium pseudosporidioides Bifidobacterium pseudocatenulatum CGMCC 1.2277; Lactobacillus rhamnosus Lactobacillus rhamnosus CICC 6141; Lactobacillus plantarum Lactiplantibacillus plantarum CICC 6240; Bifidobacterium bifidum Bifidobacterium bifidum CICC 6166; Pediococcus pentosaceus Pediococcus pentosaceus CGMCC 1.2695; short-lived lactobacillus Levilactobacillus brevis CICC 25354; Bifidobacterium longum Bifidobacterium longum CICC 6186; Mucosal lactobacilli Limosilactobacillus mucosae CGMCC 1.15986; The viable cell count ratio of the above 15 strains is 3:7:23:3:10:9:4:17:1:3:2:1:2:13:2.

[0018] Another object of this application is to provide the application of the aforementioned synthetic microbial community in the preparation of human yellow.

[0019] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: (1) High safety: It abandons the unclean process of traditional cesspool fermentation and avoids the risk of contamination by pathogenic microorganisms; (2) Quality controllable: The use of artificially synthesized microbial communities with clear microbial community structure and abundance enables precise control of the fermentation process, ensuring stable and uniform product quality; (3) Full conversion of active ingredients: Through multi-strain synergistic fermentation, the conversion of active ingredients in licorice was promoted, the content of glycyrrhizin was significantly increased, and the content of glycyrrhizic acid and glycyrrhizin was moderately reduced, which is consistent with the pharmacological material characteristics of human yellow. (4) Excellent efficacy: The anti-H1N1 influenza virus effect is better than that of traditional process and cultured intestinal flora process. It has significant anti-inflammatory and antiviral effects. (5) Good prospects for industrialization: It is not limited by season or region, has high production efficiency, and can meet the needs of industrial production. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0021] Figure 1 Figure: Relative abundance of dominant bacterial groups in the feces of healthy individuals.

[0022] Figure 2 Here are the liquid phase diagrams of extracts of human yellow ginseng from different processes. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1 Dominant microbial community analysis Fecal sample collection: Healthy and qualified donors were selected according to the requirements of the "Chinese Expert Consensus on Standardized Technical Specifications for Intestinal Bacteria Transplantation Preparation and Quality Control (2023 Edition)". Fresh fecal samples from the donors were collected, mixed, and placed on an ice box for processing within 2 hours to obtain the same bacterial source as the traditional preparation process of human ginseng.

[0025] Fecal metagenomic sequencing: 1g of mixed feces was extracted, flash-frozen in liquid nitrogen, and subjected to metagenomic sequencing to analyze the composition of dominant probiotic communities in the fecal sample. Sequencing results are shown below. Figure 1 .

[0026] Sequencing results showed that mixed feces contained a variety of beneficial probiotics. Based on the sequencing analysis results, 15 representative intestinal probiotic strains were selected as candidate strains for subsequent experiments.

[0027] Source of candidate strains: The 15 selected strains were all purchased from the Culture Collection Center (CGMCC, CICC), and their internal numbers are HLA01-HLA15. The source and collection numbers of the strains are shown in Table 1. Table 1. Sources of candidate strains in this application

[0028] Example 2 Evaluation of the effect of single strain fermentation of licorice (1) Licorice processing: After washing the licorice, dry it in an oven at 37°C until constant weight, pulverize it and pass it through a 60-mesh sieve for later use.

[0029] (2) Preparation of single-strain suspensions: The 15 strains (HLA01-HLA15) described in Example 1 were inoculated into YCFA liquid medium (the components of each liter of YCFA medium are: 10 g casein peptone, 2.5 g yeast extract, 0.001 g resazurin, 0.45 g dipotassium hydrogen phosphate, 0.45 g potassium dihydrogen phosphate, 0.9 g sodium chloride, 4 g sodium bicarbonate, 0.044 g magnesium sulfate, 0.09 g calcium chloride, and 0.01 g heme chloride) and cultured to the logarithmic growth phase. The viable count in each bacterial suspension was adjusted to 1 × 10⁻⁶. 8 CFU / mL.

[0030] (3) Fermentation group setup: 15 single-strain fermentation groups were set up. 100g of licorice powder was mixed with 100mL of single-strain bacterial solution in each group and fermented in a 37℃ incubator for 30 days. After fermentation, the mixture was dried in a 60℃ oven to obtain the fermentation product.

[0031] (4) Preparation of fermentation product extract: The fermentation product is placed in a decoction bag, placed in a decoction pot, and soaked in 1 L of water for 1 h. It is decocted twice, each time for 1 h. The two filtrates are combined, and the filtrates are concentrated by rotary evaporation and spray-dried to obtain the extract.

[0032] Product detection: Modern pharmacology shows that during the fermentation of licorice into human yellow, the content of glycyrrhizin increases while the content of glycyrrhizic acid and glycyrrhizin decreases. Therefore, the content of glycyrrhizic acid and glycyrrhizin and the degradation rate of glycyrrhizin are used as evaluation indicators for comparison. The results are shown in Table 2.

[0033] Table 2. Detection results of fermentation products from different strains

[0034] Results Analysis: According to the detection results in Table 2, all 15 single bacterial strains fermented licorice could transform the active ingredients in licorice to varying degrees. After fermentation, the glycyrrhizin content ranged from 0.039% to 0.071%, the glycyrrhizic acid content ranged from 2.590% to 2.910%, and the glycyrrhizin degradation rate ranged from 78.90% to 82.10%. The results indicate that although single bacterial strains have a certain transformation capacity, their metabolic functions are significantly limited, and the transformation effects of different strains vary considerably, making it difficult to simultaneously achieve the dual goals of efficient glycyrrhizin degradation and sufficient glycyrrhizin accumulation. Based on this, further research on multi-strain combined fermentation will be conducted to improve the transformation efficiency of licorice's active ingredients through synergistic effects of the bacterial community.

[0035] Example 3 Progressive optimization of strain combinations (1) Preparation of mixed bacterial suspension: Fermentation was carried out under the same conditions (37℃, 30 days, material-to-liquid ratio 1:1) to prepare bacterial suspensions for each strain, so that the viable count of each strain was 1×10⁻⁶. 8 CFU / mL, and then a strategy of gradually increasing the number of strains was adopted, starting with a single strain, and successively constructing mixed bacterial suspensions composed of two, three, and up to fifteen strains in different volume ratios, as follows: Combination 1 (HLA01); Combination 2 (HLA01+HLA02): 24:76 (volume ratio); Combination 3 (HLA01+HLA02+HLA03): 7:21:72 (volume ratio); Combination 4 (HLA01+HLA02+HLA03+HLA04): 6:19:67:8 (volume ratio); Combination 5 (HLA01+HLA02+HLA03+HLA04+HLA05): 5:15:52:6:22 (volume ratio); Combination 6 (HLA01~HLA06): 4:13:44:5:19:15 (volume ratio); Combination 7 (HLA01~HLA07): 4:12:41:5:18:15:5 (volume ratio); Combination 8 (HLA01~HLA08): 3:10:32:3:13:11:4:24 (volume ratio); Combination 9 (HLA01~HLA09): 3:9:31:4:13:11:4:23:2 (volume ratio); Combination 10 (HLA01~HLA10): 3:9:29:4:13:10:3:22:2:5 (volume ratio); Combination 11 (HLA01~HLA11): 3:9:28:4:12:11:4:21:2:4:2 (volume ratio); Combination 12 (HLA01~HLA12): 3:8:28:4:12:11:3:21:2:4:2:2 (volume ratio); Combination 13 (HLA01~HLA13): 3:8:27:4:12:10:3:20:2:4:2:2:3 (volume ratio); Combination 14 (HLA01~HLA14): 3:7:23:3:10:9:4:17:1:4:2:1:2:14 (volume ratio); Combination 15 (HLA01~HLA15): 3:7:23:3:10:9:4:17:1:3:2:1:2:13:2 (volume ratio); (2) Licorice fermentation: Licorice was fermented according to the fermentation steps in Example 2, and the product was measured. The glycyrrhizin content, glycyrrhizic acid content and glycyrrhizin degradation rate were used as evaluation indicators to examine the combined effect.

[0036] (3) Traditional process for preparing human placenta extract: licorice powder is packed into bamboo tubes, packed tightly, and the end with the node removed is plugged with clean paper. The tubes are then sealed with rosin. The bamboo tubes are tied together in bundles of 10-15 using hemp rope or wire, immersed in a human excrement pit, and weighed down with heavy objects to prevent them from floating. After soaking for 30 days, the tubes are taken out, rinsed with running water, split open, and the human placenta extract is removed and dried. According to the extraction steps in Example 2, human placenta extract is prepared. The glycyrrhizin content, glycyrrhizic acid content, and glycyrrhizin degradation rate are used as evaluation indicators and compared with the fermentation combination.

[0037] (4) Preparation of human yellow by culturing microbial community: Referring to CN113209157A, the fermentation broth obtained after in vitro culture of intestinal microorganisms is used as a substitute for feces to prepare human yellow. The specific method is as follows: Feces are vortexed and mixed with sterile and anaerobic PBS solution, filtered through an 800-mesh filter membrane to obtain fecal inoculation solution. The intestinal microecological type is determined to be Prevotella enterotype. The fecal inoculation solution is inoculated into an in vitro fermentation tank for anaerobic culture. The fermentation broth is collected as a substitute for fecal inoculation in the preparation process of human yellow to prepare human yellow.

[0038] Table 3 Comparison of fermentation effects of different strain combinations on licorice

[0039] The results showed that with the increase of the number of bacterial strains, the glycyrrhizin content and glycyrrhizin degradation rate gradually increased, while the glycyrrhizic acid content gradually decreased, indicating that multi-strain synergistic fermentation is more conducive to the conversion of the effective components of Renzhonghuang (a traditional Chinese medicine formula). Combination 15 (a complete combination of 15 strains) showed the best results. Compared with the traditional process of fermenting licorice and the culture of intestinal flora to ferment Renzhonghuang, Combination 15 showed higher glycyrrhizin content and better degradation of glycyrrhizic acid and glycyrrhizin than the traditional process and the culture of intestinal flora. This indicates that the synthetic bacterial flora has a higher conversion efficiency, and the reason for this difference may be the strain differences between the bacteria in the cesspool and the synthetic bacterial flora.

[0040] Example 4 Effects of different strains on the fermentation efficiency of licorice To verify the irreplaceable role of the specific strains selected in this application in the synthetic microbial community, *Bifidobacterium adolescentis* from 15 combinations was used. Bifidobacterium adolescentis Taking strain CICC 6175 as an example, the model strain of Bifidobacterium adolescentis (strain number ATCC 15703) and Bifidobacterium adolescentis isolated from bovine stomach (strain number CICC6179) were selected to replace the original Bifidobacterium adolescentis strains, and a comparative experiment was set up. The experimental results are shown in Table 4.

[0041] Combination 1: HLA01~HLA15; Combination 2: The only difference from Combination 1 is that the HLA03 strain in HLA01 to HLA15 is replaced with the model strain of Bifidobacterium adolescentis (strain number ATCC15703). Combination 3: The only difference from Combination 1 is that Bifidobacterium adolescentis (strain number CICC 6179) replaces strain HLA03 in HLA01 to HLA15.

[0042] Table 4. Comparison of the effects of different *Bifidobacterium juvenilee* strains on fermented licorice.

[0043] The experimental results show that the HLA01-HLA15 combination fermentation effect is superior to that of Bifidobacterium adolescentis from other sources. Bifidobacterium adolescentis This indicates that the HLA01~HLA15 combination is irreplaceable. Example 5 Fermentation time optimization Using combination 15 (HLA01-HLA15 mixed bacteria), the fermentation method of Example 2 was followed, and the fermentation time was set to 0, 5, 10, 15, 20, 25 and 30 days respectively. The effects of different fermentation times on the content of glycyrrhizin and glycyrrhizic acid and the degradation rate of glycyrrhizin were investigated. The results are shown in Table 5.

[0044] Table 5. Effect of fermentation time on fermentation efficiency

[0045] The experimental results show that the content of glycyrrhizin and glycyrrhizic acid, as well as the degradation rate of glycyrrhizin, tend to stabilize after 20 days of fermentation. Therefore, 20 days is selected as the optimal fermentation time.

[0046] Example 6 Optimization of feed-liquid ratio Combination 15 (HLA01-HLA15 mixed bacteria) was used, and the fermentation time was 20 days. Following the fermentation method of Example 2, the material-to-liquid ratio was set to 1:0.8, 1:0.9, 1:1, 1:1.1, and 1:1.2 respectively to investigate the effect of different material-to-liquid ratios on the fermentation effect. The results are shown in Table 6.

[0047] Table 6. Effect of substrate-to-liquid ratio on fermentation efficiency

[0048] The experimental results show that when the material-to-liquid ratio is 1:1, the glycyrrhizin content is higher, the glycyrrhizic acid content is lower, and the glycyrrhizin degradation rate is higher. Therefore, 1:1 is selected as the optimal material-to-liquid ratio.

[0049] Example 7 Evaluation of the efficacy of anti-H1N1 virus drugs Traditional Craft Group: Based on the processing method recorded in "Danxi Xinfa" by Zhu Zhenheng of the Yuan Dynasty, it is made by: "Put licorice powder into a bamboo tube, plug both ends with bamboo or wood, soak it in a manure vat in winter, take it out in the wind and dry it in the shade in early spring, split the bamboo to get the grass, and dry it in the sun for use."

[0050] Intestinal flora cultivation process: Feces were vortexed and mixed with sterile and anaerobic PBS solution, and filtered through an 800-mesh filter to obtain fecal inoculum. The intestinal microecological type was determined to be Prevotella enterotype. The fecal inoculum was inoculated into an in vitro fermentation tank for anaerobic culture. The fermentation broth was collected as a substitute for fecal inoculum in the preparation process of Renzhonghuang.

[0051] Artificially synthesized microbial community process group: prepared using the optimal conditions of this invention (combination 15, fermentation time 20 days, material-liquid ratio 1:1).

[0052] SPF-grade Balb / c mice, weighing 12.7–19.5 g, with half males and half females, were randomly divided into four groups: normal control group, model control group, oseltamivir phosphate granule group, traditional processing group, intestinal flora culture processing group, and artificially synthesized flora processing group, with 20 mice per group. Two hours before the first administration, each group of animals was lightly anesthetized with ether and then instilled with 100 μL of H1N1 influenza A virus stock solution via nasal drip. The normal control group received an equal volume of 0.9% sodium chloride injection via nasal drip. Each group of animals was administered the corresponding concentration of the drug solution orally at a dose of 20 mL / kg once daily for 7 consecutive days. The normal control group and model control group received an equal volume of pure water.

[0053] The following indicators were tested in different groups: General clinical observation: After modeling, the animals in each group were weighed daily to compare changes in body weight during the experiment (Table 7). The physiological state of the animals before and after drug administration, such as activity level and mental state of the mice, was observed and recorded.

[0054] Table 7. Effects of different groups on mouse body weight ( (n=20)

[0055] Note: Compared with the normal control group, ++ P ≤0.01. Compared with the model control group, P ≤0.05, P ≤0.01.

[0056] Results analysis: After modeling, all mice showed reduced spontaneous activity, piloerection, and rapid breathing. The above symptoms were still present in the model control group, oseltamivir phosphate granule group, traditional process human ginseng group, cultured intestinal flora group, and artificially synthesized human ginseng group after drug administration. However, the symptoms were relieved to varying degrees in the later stages of drug administration.

[0057] Lung index detection: On the day after the last administration, the animals in each group were euthanized by cervical dislocation, the lungs were dissected, the blood was wiped off, and the lungs were weighed. The data were recorded and the lung index was calculated (Table 8).

[0058] Table 8. Effects of different groups on the lung index of mice ( (n=10)

[0059] Note: Compared with the normal control group, ++ P ≤0.01; compared with the model control group, P ≤0.05, P ≤0.01.

[0060] Results analysis: As shown in Table 8, compared with the normal control group, the lung index of rats in the model control group was significantly increased on the day after the last administration (P ≤ 0.01); compared with the model control group, the lung index of mice in the oseltamivir phosphate granule group, the traditional process human ginseng group, and the artificially synthesized intestinal flora process human ginseng group was significantly decreased (P ≤ 0.05 or P ≤ 0.01), and the effect of the artificially synthesized intestinal flora process human ginseng was better than that of the traditional process group and the intestinal flora culture process group.

[0061] Virus titer detection: On the day after the last administration, the animals in each group were euthanized by cervical dislocation, and the right lung of each group was dissected and the titer of H1N1 influenza A virus in the lung tissue was detected by hemagglutination inhibition method (Table 9).

[0062] Table 9. Effects of different groups on viral load in mouse lung tissue ( (n=10)

[0063] Note: Compared with the normal control group, ++ P ≤0.01. Compared with the model control group, P ≤0.05, P ≤0.01.

[0064] Results analysis: As shown in the table, compared with the normal control group, the viral titer in the lung tissue of rats in the model control group was significantly increased on the day after the last administration (P ≤ 0.01); compared with the model control group, the viral titer in the lung tissue of rats in the artificially synthesized bacterial culture group and the oseltamivir phosphate granule group was significantly decreased (P ≤ 0.05 or P ≤ 0.01), while the traditional process did not have a significant effect on reducing viral titer.

[0065] Inflammatory factor detection: On the day following the last administration, animals in each group were euthanized by cervical dislocation to expose the lungs, which were repeatedly irrigated with 0.9% sodium chloride injection. The irrigated fluid was collected, and the contents of IL-1β, TNF-α, and IL-6 in the bronchoalveolar lavage fluid were detected by ELISA (Table 10).

[0066] Table 10 Effects of different groups on inflammatory factors in mouse bronchoalveolar lavage fluid ( (n=10)

[0067] Note: Compared with the normal control group, ++P ≤0.01. Compared with the model control group, P ≤0.05, P ≤0.01.

[0068] Results Analysis: As shown in Table 10, compared with the normal control group, the levels of IL-6, IL-1β, and TNF-α in the bronchoalveolar lavage fluid of rats in the model control group were significantly increased on the day after the last administration (P ≤ 0.01). Compared with the model control group, the levels of IL-6, IL-1β, and TNF-α in the bronchoalveolar lavage fluid of rats in the artificially synthesized microbial culture group and the oseltamivir phosphate granule group were significantly decreased (P ≤ 0.05 or P ≤ 0.01). The levels of IL-6 and IL-1β in the traditionally processed human-derived ...

[0069] Histopathological examination: On the day following the last administration, animals in each group were euthanized by cervical dislocation. The left lung was dissected, fixed in 10% neutral formalin solution, sectioned, embedded, and stained with hematoxylin and eosin (HE) to observe the histopathological changes in the bronchi and lung tissue. The evaluation criteria for histopathological changes are shown in Table 11.

[0070] Table 11 Evaluation Criteria for Tissue Lesions

[0071] Table 12 Effects of different groups of mice on lung tissue lesion scores ( (n=10)

[0072] Note: Compared with the normal control group, ++ P ≤0.01; compared with the model control group. P ≤0.01.

[0073] Results analysis: As shown in the results, microscopic observation of the alveoli, bronchiolar mucosa and alveolar cavities of the normal control group animals on the day after the last administration showed that, compared with the normal group, the lung tissue of the model control group mice showed inflammatory and hemorrhagic exudation, thickening of alveolar septa, and inflammatory cell infiltration of bronchiolars and surrounding blood vessels; the lesions of yellow lung tissue in the oseltamivir phosphate granule group and the artificially synthesized microbial community group were reduced to varying degrees compared with the model control group.

[0074] Example 8 Comparison of component liquid phase diagrams High-performance liquid chromatography (HPLC) analysis was performed on extracts of human ginseng processed by traditional methods, intestinal flora culture methods, and artificially synthesized flora methods to compare the component differences among the different processes.

[0075] Preparation of test solution: Accurately weigh 20 mg of extract to prepare a 10 mg / mL test solution. Injection volume: 10 µL. Column type: Venusil C18 Plus (4.6 × 250 mm, 5 µm). Elution conditions: Acetonitrile as mobile phase B, 0.05% phosphoric acid as mobile phase A, gradient elution according to the table below, wavelength: 237 nm, column temperature: 30℃, flow rate: 1.0 mL / min.

[0076] Table 13 Liquid phase elution conditions

[0077] Results analysis: Liquid chromatography analysis showed that ( Figure 2 The chemical composition of human placenta obtained by different processes varies significantly, which may be an important factor in the differences in its pharmacodynamics.

[0078] In summary, under the experimental conditions, artificially synthesized human ginseng processed with gut microbiota significantly reduced viral load, lung index, and bronchoalveolar lavage fluid inflammatory factor levels in H1N1 influenza virus-infected mice, and improved the degree of lung lesions, suggesting that it has significant anti-H1N1 influenza virus efficacy. Moreover, the antiviral effect of artificially synthesized human ginseng processed with gut microbiota is superior to that of human ginseng processed with traditional methods and those processed with cultured gut microbiota.

[0079] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0080] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing human-derived yellow licorice by fermentation of artificially synthesized microbial communities, characterized in that, Includes the following steps: (1) Strains screening: Dominant probiotics were analyzed from fecal samples of healthy individuals using metagenomic sequencing. Based on the sequencing results, an artificial synthetic flora consisting of 15 probiotic strains was designed and constructed, simulating the gut microbiota structure of healthy individuals. The 15 strains are as follows: Lactobacillus casei Lacticaseibacillus paracasei CGMCC 1.2435; Lactobacillus gasseri Lactobacillus gasseri CICC 24878; Bifidobacterium adolescentis Bifidobacterium adolescentis CICC 6175; Lactobacillus Pediococcus acidilactici CICC 10344; Lactobacillus amyloliquefaciens Lactobacillus amylovorus CICC 6090; Lactobacillus johnsonii Lactobacillus johnsonii CICC 6084; Fermented Lactobacillus mucinus Limosilactobacillus fermentum CGMCC 1.1880; Bifidobacterium pseudosporidioides Bifidobacterium pseudocatenulatum CGMCC 1.2277; Lactobacillus rhamnosus Lacticaseibacillus rhamnosus CICC 6141; Lactobacillus plantarum Lactiplantibacillus plantarum CICC 6240; Bifidobacterium bifidum Bifidobacterium bifidum CICC 6166; Pediococcus pentosaceus Pediococcus pentosaceus CGMCC 1.2695; short-lived lactobacillus Levilactobacillus brevis CICC 25354; Bifidobacterium longum Bifidobacterium longum CICC 6186; Mucosal lactobacilli Limosilactobacillus mucosae CGMCC 1.15986; (2) Construction of artificially synthesized microbial community: The 15 strains described in step (1) are expanded and cultured to prepare bacterial suspensions. The bacterial suspensions are mixed according to the optimized ratio to obtain artificially synthesized microbial community bacterial suspension. (3) Licorice processing: Licorice is washed, dried to constant weight, pulverized and sieved to obtain licorice powder; (4) Fermentation: The licorice powder obtained in step (3) is mixed with the artificially synthesized bacterial suspension obtained in step (2) and fermented, then dried to prepare the fermentation product; the fermentation product is boiled, extracted, concentrated and dried to prepare human yellow.

2. The method according to claim 1, characterized in that, Step (2) The ratio of viable bacteria of the 15 strains in the artificially synthesized bacterial suspension is 3:7:23:3:10:9:4:17:1:3:2:1:2:13:2, in accordance with the order of the strains in claim 1. The viable cell count of the bacterial suspension was 1 x 10 8 CFU / mL.

3. The method according to claim 1, characterized in that, The fermentation temperature in step (4) is 37°C and the fermentation time is 20 days; the drying temperature is 60°C.

4. The method according to claim 1, characterized in that, In step (4), the ratio of licorice powder to artificially synthesized bacterial suspension is 1g:1ml.

5. The human placenta extract prepared by the method according to any one of claims 1-4.

6. The use of human ginseng prepared according to any one of claims 1-4 in the preparation of anti-influenza A virus drugs.

7. The application according to claim 6, characterized in that, The influenza A virus in question is the H1N1 subtype.

8. An artificially synthesized microbial community, characterized in that, It consists of the following 15 strains: Lactobacillus casei Lacticaseibacillus paracasei CGMCC 1.2435; Lactobacillus gasseri Lactobacillus gasseri CICC 24878; Bifidobacterium adolescentis Bifidobacterium adolescentis CICC 6175; Lactobacillus Pediococcus acidilactici CICC 10344; Lactobacillus amyloliquefaciens Lactobacillus amylovorus CICC 6090; Lactobacillus johnsonii Lactobacillus johnsonii CICC 6084; Fermented Lactobacillus mucinus Limosilactobacillus fermentum CGMCC 1.1880; Bifidobacterium pseudosporidioides Bifidobacterium pseudocatenulatum CGMCC 1.2277; Lactobacillus rhamnosus Lacticaseibacillus rhamnosus CICC 6141; Lactobacillus plantarum Lactiplantibacillus plantarum CICC 6240; Bifidobacterium bifidum Bifidobacterium bifidum CICC 6166; Pediococcus pentosaceus Pediococcus pentosaceus CGMCC 1.2695; short-lived lactobacillus Levilactobacillus brevis CICC 25354; Bifidobacterium longum Bifidobacterium longum CICC 6186; Mucosal lactobacilli Limosilactobacillus mucosae CGMCC 1.15986; The viable cell count ratio of the above 15 strains is 3:7:23:3:10:9:4:17:1:3:2:1:2:13:

2.

9. The application of the artificially synthesized microbial community according to claim 8 in the preparation of human yellow.

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