Solid beverage for improving intestinal flora and preparation method and application thereof

Abstract

This invention relates to the field of solid beverage preparation technology, and in particular to a solid beverage for improving intestinal flora and its preparation method and application. It includes the following steps: (1) Extraction of active substances from *Dendrobium nobile*: *Dendrobium nobile* is mixed with water, extracted by microwave constant temperature, concentrated, precipitated with alcohol, filtered through a membrane, and dried to obtain *Dendrobium nobile* extract; (2) Extraction of active substances from Pu-erh tea: Pu-erh tea is mixed with an extractant, extracted by reflux, concentrated, and dried to obtain Pu-erh tea extract; (3) The *Dendrobium nobile* extract and Pu-erh tea extract are mixed and sterilized to obtain a solid beverage for improving intestinal flora. This invention obtains its active substances through an optimized extraction process, significantly improving the extraction efficiency and bioavailability of the effective components. The two are scientifically combined and work synergistically on the intestinal microenvironment, promoting the growth of beneficial bacteria and inhibiting the reproduction of harmful bacteria, thereby effectively regulating the balance of intestinal flora and improving intestinal health.

Description

Technical Field

[0001] This invention relates to the field of solid beverage preparation technology, and in particular to a solid beverage that improves intestinal flora, its preparation method, and its application. Background Technology

[0002] With the fast pace of modern life and changes in dietary structure, gut microbiota imbalance has become a significant factor affecting public health. As an important "microbial organ" of the human body, the gut microbiota's homeostasis is closely related to digestion and absorption, immune regulation, metabolic function, and even the health of the nervous system. Studies have shown a significant association between gut microbiota dysbiosis and various chronic diseases such as obesity, diabetes, inflammatory bowel disease, autoimmune diseases, and mood disorders. Therefore, regulating gut microbiota balance through scientific intervention has become an important strategy for promoting overall health and preventing related diseases.

[0003] In existing technologies, products that improve gut microbiota mainly rely on core ingredients such as probiotics, prebiotics, or synbiotics. While traditional probiotic supplements can directly replenish beneficial bacteria, the survival rate of these strains faces significant challenges during storage, transportation, and passage through the digestive tract environment, including stomach acid and bile salts. Their activity is easily affected by factors such as temperature, humidity, and oxygen, resulting in an unstable number of effective live bacteria that actually reach and colonize the intestines. Furthermore, probiotic products containing single strains or simple combinations may have limited regulatory effects, making it difficult to comprehensively address complex gut microbiota imbalances.

[0004] Currently, the market offers a variety of products for improving gut microbiota, including capsules, tablets, powders, and liquid beverages. Among these, powders and solid beverages are favored due to their portability and flexible consumption. However, many existing solid beverage products still have some shortcomings: on the one hand, some products focus only on the addition of probiotics, neglecting the scientific formulation of synergistic components such as prebiotics and post-biotics, resulting in limited overall regulatory efficacy on the gut microbiota; on the other hand, in terms of product manufacturing processes, there may be a lack of effective protection technology for heat-sensitive strains, or the formulation may not be optimized during processing to improve taste, solubility, and stability, affecting user experience and long-term adherence.

[0005] Therefore, there is an urgent need in this field to develop a novel solid beverage for improving gut microbiota. The aim is to significantly improve the stability, survival rate, and bioavailability of the core functional components. Simultaneously, the product must also possess excellent reconstitution properties, palatability, and storage stability, thereby providing an effective, convenient, and superior gut microbiota regulation solution to meet the growing market demand. Summary of the Invention

[0006] The purpose of this invention is to provide a solid beverage that improves intestinal flora, its preparation method, and its application.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing a solid beverage that improves gut microbiota, comprising the following steps: (1) Extraction of active substances from Dendrobium nobile: The purple-skinned dendrobium was mixed with water at 55-75℃ and extracted by microwave constant temperature 2-3 times, each time for 16-20 minutes. The extracts were combined, concentrated, precipitated with alcohol, filtered through a membrane, and dried to obtain the purple-skinned dendrobium extract. The microwave frequency was 1500-1700MHz and the power was 1200-1400W. (2) Extraction of active substances from Pu-erh tea: Mix Pu-erh tea with the extractant and reflux at 85-90℃ for 1-2 times, each time for 1-3 hours. Combine the extracts, concentrate and dry to obtain Pu-erh tea extract. (3) The purple Dendrobium extract and Pu-erh tea extract are mixed and sterilized to obtain a solid beverage that improves intestinal flora.

[0008] Preferably, the water in step (1) contains maltose; the concentration of maltose in the water is 2.5~3.5 g / 100mL.

[0009] Preferably, step (1) involves concentrating the solution to a relative density of 1.15 to 1.25.

[0010] Preferably, the alcohol precipitation in step (1) involves adding 3 to 6 times the volume of concentrated ethanol and then letting it stand for 14 to 18 hours to obtain the precipitate.

[0011] Preferably, the membrane filtration in step (1) is as follows: the precipitate is dissolved in 25 to 30 times its mass of water, then passed through an ultrafiltration membrane with a molecular weight of 150 KD, the retentate is collected, and then passed through an ultrafiltration membrane with a molecular weight of 350 KD, the permeate is collected.

[0012] Preferably, the extractant in step (2) is an ethanol solution containing formic acid at a volume concentration of 0.1% to 1%.

[0013] Preferably, the volume concentration of the ethanol solution in step (2) is 85-90%.

[0014] Preferably, the relative density of the concentrate obtained in step (2) is 1.18 to 1.23.

[0015] The present invention provides a method for preparing the aforementioned solid beverage, which produces a solid beverage that improves intestinal flora, comprising 20-30 parts of Dendrobium officinale extract and 60-80 parts of Pu-erh tea extract.

[0016] This invention provides the application of the aforementioned solid beverage for improving gut microbiota in the preparation of beverages that have the effect of regulating gut microecological balance.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention relates to a solid beverage using natural plants, Dendrobium nobile and Pu-erh tea, as core ingredients. An optimized extraction process is employed to obtain their active substances, significantly improving the extraction efficiency and bioavailability of the effective components. Dendrobium nobile is rich in polysaccharides, flavonoids, and other active ingredients, possessing immunomodulatory and anti-inflammatory effects; Pu-erh tea contains tea polyphenols, tea pigments, and other substances with excellent antioxidant and metabolic regulatory functions. The scientific combination of these two ingredients works synergistically on the intestinal microenvironment, promoting the growth of beneficial bacteria and inhibiting the proliferation of harmful bacteria, thereby effectively regulating the balance of intestinal flora and improving intestinal health.

[0018] The extraction process employed in this invention is highly targeted, mild, and efficient. For Dendrobium nobile, microwave-assisted extraction is used to rapidly release active substances under low-temperature conditions, avoiding the damage to heat-sensitive components caused by high temperatures. For Pu-erh tea, reflux extraction with an ethanol solution containing formic acid is used, which can more fully extract fat-soluble and water-soluble active components such as tea polyphenols. Subsequent purification steps, such as alcohol precipitation and fractional ultrafiltration, further remove non-target impurities, improving the purity and stability of the extract and ensuring the reliability and consistency of the final product's efficacy.

[0019] This product is a solid beverage, offering advantages such as portability, ease of preparation, and a pleasant taste, making it easy for consumers to consume consistently over a long period. The preparation process does not rely on exogenous probiotics, avoiding the instability in survival rates that occurs with live bacteria preparations during storage, transportation, and consumption. This results in high product stability and a long shelf life. This solid beverage can be consumed directly or used as a functional base in other beverages or foods, expanding its application scenarios in regulating intestinal microecological balance and demonstrating promising market prospects and practical promotional value. Attached Figure Description

[0020] Figure 1 The stacked bar chart of the percentage of groups in the phylum-level microbial community composition analysis of HFD model mice shows the relative abundance and composition structure of gut microbiota at the phylum level for different experimental groups.

[0021] Figure 2 The stacked bar chart of the percentage of groups in the genus-level microbial community composition analysis of the HFD model mice shows the relative abundance and composition structure of the gut microbiota at the genus level for different experimental groups.

[0022] Figure 3 A stacked bar chart showing the percentage grouping of microbial community composition at the phylum level in HFD model mice (see attached). Figure 1(This will allow for further comparison of changes in phylum-level microbial community structure under different intervention conditions.)

[0023] Figure 4 A stacked bar chart showing the percentage grouping of microbial community composition at the phylum level in HFD model mice (see attached). Figure 3 (This section further illustrates the differences in bacterial flora at the phylum level under different drug dosages and in the positive control group.)

[0024] Figure 5 A stacked bar chart showing the percentage grouping of microbial community composition at the genus level in the HFD model mouse (continued) Figure 2 Further comparisons were made of changes in genus-level bacterial community structure under different intervention conditions.

[0025] Figure 6 A stacked bar chart showing the percentage grouping of microbial community composition at the genus level in the HFD model mouse (continued) Figure 5 This section further illustrates the differences in bacterial flora at the genus level under different drug dosages and in the positive control group.

[0026] Figure 7 Venn diagram showing the composition of microbial communities at the phylum and genus levels in HFD model mice.

[0027] Figure 8 HFD model mouse microbial community composition analysis at the phylum and genus levels, grouping and clustering heatmap.

[0028] Figure 9 Analysis of microbial community diversity in HFD model mice.

[0029] Figure 10 Diversity analysis of viral communities in three groups of samples: (A) ACE index (B) Shannon index (C) PCoA analysis (D) NMDS.

[0030] Figure 11 The composition of viral communities at the phylum (A) and family (B) levels in the three groups of samples.

[0031] Figure 12 The composition of viral communities in the three groups of samples (A) Venn (B) genus level.

[0032] Figure 13 Multiple comparisons of viral communities in the three groups of samples at the phylum level, family level, and genus level.

[0033] Figure 14 The viral community level LEfSe in the three groups of samples.

[0034] Figure 15 Functional annotations (A) and functional clustering heatmaps (B) of the three groups of viral communities from the UniProtKB database. Detailed Implementation

[0035] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1

[0037] A solid beverage for improving gut microbiota, composed of 20 parts of Dendrobium officinale extract and 60 parts of Pu-erh tea extract; its preparation method includes the following steps: (1) Extraction of active substances from Dendrobium nobile: The purple-skinned dendrobium was pulverized and mixed with 12 times its weight of water at 55°C. The mixture was then microwaved twice at a constant temperature for 16 minutes each time. The extracts were combined and concentrated to a relative density of 1.15. After alcohol precipitation and membrane filtration, the extract was dried into powder to obtain the purple-skinned dendrobium extract. The microwave frequency was 1500MHz and the power was 1200W. The water contains maltose; the concentration of maltose in the water is 2.5 g / 100 mL; The alcohol precipitation was achieved by adding 3 times the volume of concentrated ethanol, then letting it stand for 14 hours to obtain the precipitate. The membrane filtration process involves dissolving the precipitate in 25 times its mass of water, then passing it through an ultrafiltration membrane with a molecular weight of 150 KD, collecting the retentate, and then passing it through an ultrafiltration membrane with a molecular weight of 350 KD to collect the permeate.

[0038] (2) Extraction of active substances from Pu-erh tea: After pulverizing Pu-erh tea, mix it with 8 times its weight of the extractant, reflux at 85°C once for 1 hour each time, combine the extracts, concentrate until the relative density of the concentrate is 1.18, and then dry it into powder to obtain Pu-erh tea extract. The extractant is an ethanol solution containing 0.1% formic acid by volume; the volume concentration of the ethanol solution is 85%.

[0039] (3) The purple Dendrobium extract and Pu-erh tea extract are mixed and sterilized to obtain a solid beverage that improves intestinal flora.

[0040] Example 2

[0041] A solid beverage for improving gut microbiota, composed of 30 parts of Dendrobium officinale extract and 80 parts of Pu-erh tea extract; its preparation method includes the following steps: (1) Extraction of active substances from Dendrobium nobile: The purple-skinned dendrobium was pulverized and mixed with 10 times its weight of water at 75℃. The mixture was extracted three times at a constant temperature using a microwave, each time for 20 minutes. The extracts were combined and concentrated to a relative density of 1.25. After alcohol precipitation and membrane filtration, the extract was dried into powder to obtain the purple-skinned dendrobium extract. The microwave frequency was 1700MHz and the power was 1400W. The water contains maltose; the concentration of maltose in the water is 3.5 g / 100 mL; The alcohol precipitation was achieved by adding 6 times the volume of concentrated ethanol, then allowing it to stand for 18 hours to obtain the precipitate. The membrane filtration process involves dissolving the precipitate in 30 times its mass of water, then passing it through an ultrafiltration membrane with a molecular weight of 150 KD, collecting the retentate, and then passing it through an ultrafiltration membrane with a molecular weight of 350 KD to collect the permeate.

[0042] (2) Extraction of active substances from Pu-erh tea: After pulverizing Pu-erh tea, mix it with 9 times its weight of the extractant, reflux at 90℃ twice for 3 hours each time, combine the extracts, concentrate until the relative density of the concentrate is 1.23, and then dry it into powder to obtain Pu-erh tea extract; The extractant is an ethanol solution containing 1% formic acid by volume; the ethanol solution has a volume concentration of 90%.

[0043] (3) The purple Dendrobium extract and Pu-erh tea extract are mixed and sterilized to obtain a solid beverage that improves intestinal flora.

[0044] Example 3

[0045] A solid beverage for improving gut microbiota, composed of 23 parts of Dendrobium officinale extract and 65 parts of Pu-erh tea extract; its preparation method includes the following steps: (1) Extraction of active substances from Dendrobium nobile: The purple-skinned dendrobium was pulverized and mixed with 10 times its weight of water at 60°C. The mixture was then microwaved twice at a constant temperature for 17 minutes each time. The extracts were combined and concentrated to a relative density of 1.17. After alcohol precipitation and membrane filtration, the extract was dried into powder to obtain the purple-skinned dendrobium extract. The microwave frequency was 1550MHz and the power was 1250W. The water contains maltose; the concentration of maltose in the water is 2.7 g / 100 mL; The alcohol precipitation was achieved by adding 4 times the volume of concentrated ethanol, then letting it stand for 15 hours to obtain the precipitate. The membrane filtration process involves dissolving the precipitate in 26 times its mass of water, then passing it through an ultrafiltration membrane with a molecular weight of 150 KD, collecting the retentate, and then passing it through an ultrafiltration membrane with a molecular weight of 350 KD to collect the permeate.

[0046] (2) Extraction of active substances from Pu-erh tea: After pulverizing Pu-erh tea, mix it with 12 times its weight of the extractant, reflux at 87°C once for 1-3 hours each time, combine the extracts, concentrate until the relative density of the concentrate is 1.19, and then dry it into powder to obtain Pu-erh tea extract. The extractant is an ethanol solution containing 0.3% formic acid by volume; the volume concentration of the ethanol solution is 86%.

[0047] (3) The purple Dendrobium extract and Pu-erh tea extract are mixed and sterilized to obtain a solid beverage that improves intestinal flora.

[0048] Example 4

[0049] A solid beverage for improving gut microbiota, composed of 28 parts of Dendrobium officinale extract and 75 parts of Pu-erh tea extract; its preparation method includes the following steps: (1) Extraction of active substances from Dendrobium nobile: The purple-skinned dendrobium was pulverized and mixed with 9 times its weight of water at 70℃. It was extracted three times by microwave constant temperature, 18 min each time. The extracts were combined and concentrated to a relative density of 1.23. After alcohol precipitation, membrane filtration, and drying into powder, the purple-skinned dendrobium extract was obtained. The microwave frequency was 1650MHz and the power was 1350W. The water contains maltose; the concentration of maltose in the water is 3.3 g / 100 mL; The alcohol precipitation was achieved by adding 5 times the volume of concentrated ethanol and then letting it stand for 17 hours to obtain the precipitate. The membrane filtration process involves dissolving the precipitate in 28 times its mass of water, then passing it through an ultrafiltration membrane with a molecular weight of 150 KD, collecting the retentate, and then passing it through an ultrafiltration membrane with a molecular weight of 350 KD to collect the permeate.

[0050] (2) Extraction of active substances from Pu-erh tea: After pulverizing Pu-erh tea, mix it with 11 times its weight of the extractant, reflux extract twice at 88℃ for 3 hours each time, combine the extracts, concentrate until the relative density of the concentrate is 1.21, and then dry it into powder to obtain Pu-erh tea extract. The extractant is an ethanol solution containing formic acid at a volume concentration of 0.8%; the volume concentration of the ethanol solution is 88%.

[0051] (3) The purple Dendrobium extract and Pu-erh tea extract are mixed and sterilized to obtain a solid beverage that improves intestinal flora.

[0052] Example 5

[0053] A solid beverage for improving gut microbiota, composed of 25 parts of Dendrobium officinale extract and 70 parts of Pu-erh tea extract; its preparation method includes the following steps: (1) Extraction of active substances from Dendrobium nobile: The purple-skinned Dendrobium was pulverized and mixed with 8 times its weight of water at 65°C. The mixture was then microwaved at a constant temperature for 18 minutes each time. The extracts were combined and concentrated to a relative density of 1.20. After alcohol precipitation and membrane filtration, the extract was dried into powder to obtain the purple-skinned Dendrobium extract. The microwave frequency was 1600MHz and the power was 1300W. The water contains maltose; the concentration of maltose in the water is 3 g / 100 mL. The alcohol precipitation was achieved by adding 5 times the volume of concentrated ethanol and then letting it stand for 16 hours to obtain the precipitate. The membrane filtration process involves dissolving the precipitate in 28 times its mass of water, then passing it through an ultrafiltration membrane with a molecular weight of 150 KD, collecting the retentate, and then passing it through an ultrafiltration membrane with a molecular weight of 350 KD to collect the permeate.

[0054] (2) Extraction of active substances from Pu-erh tea: After pulverizing Pu-erh tea, mix it with 10 times its weight of the extractant, reflux at 88℃ twice, 2 hours each time, combine the extracts, concentrate until the relative density of the concentrate is 1.20, and then dry it into powder to obtain Pu-erh tea extract. The extractant is an ethanol solution containing 0.5% formic acid by volume; the volume concentration of the ethanol solution is 88%.

[0055] (3) The purple Dendrobium extract and Pu-erh tea extract are mixed and sterilized to obtain a solid beverage that improves intestinal flora.

[0056] (4) Raw material composition content: Dendrobium nobile: moisture 6.95%; polysaccharide 52.56%; mannose 44.26%; total polyphenols 0.42%; total flavonoids 0.51%; Dendrobium nobile extract yield: 57.4%; Pu-erh tea: Moisture 11.5%; Crude polysaccharide 3.7%; Mannose 0.05%; Total polyphenols 12.3%; Total flavonoids 0.7%; Theabrownins 13.1%; Gallic acid 1.3%; Protein 9.3%; Pu-erh tea extract yield: 37.5%.

[0057] Experimental Example 1

[0058] 1.1 Animal Experiments

[0059] Six-week-old SPF-grade male C57BL / 6J mice were housed in a standard barrier environment and underwent a one-week acclimatization period with free access to food and water before the experiment. Mice were randomly assigned to groups, and specific groupings and drug administration regimens are shown in Table 1. A long-term HFD (High-Frequency Hierarchical Discharge) metabolic load model was established in this experiment. Except for the NC and NDDYJ groups, all other groups were continuously fed under HFD until the end of the experiment. After the feeding change, mice were simultaneously administered different doses of DDYJ via gavage under continuous HFD for 12 weeks. After the last administration, mice were fasted but allowed free access to water for 12 hours. Blood samples were collected, and serum was separated for subsequent biochemical assays. Mice were then sacrificed, and liver and adipose tissue were quickly removed, rinsed with physiological saline, weighed, and fixed in 4% paraformaldehyde solution for histological analysis.

[0060] Table 1 Experimental Groups and Drug Administration Conditions

[0061] The preparation method of Dendrobium officinale pulp is as follows: 1 part of solid beverage is mixed with 50 parts of water to obtain DDYJ. The polysaccharide content of DDYJ used in the gavage experiment is greater than 2000 mg / 100 g, and the mannose content is greater than 1000 mg / 100 g.

[0062] 1.2 Collection of mouse fecal samples

[0063] Fecal samples were collected from mice at 0, 6, and 12 weeks for gut microbiome and metaviromic sequencing analysis. During sample collection, to minimize environmental matrix interference, mice were transferred to pre-sterilized, bedding-free individual cages and allowed to defecate naturally. Fresh fecal pellets were then quickly picked up with sterile forceps and placed in 1.5 mL sterile centrifuge tubes (EP tubes). The tube caps were immediately tightened to isolate the air, and the sample tubes were immediately placed in an ice bath to inhibit microbial metabolic activity and nucleic acid degradation. The forceps were disinfected with alcohol before the next sampling was performed until all mouse fecal samples were collected. Fecal samples from 5 mice per cage were pooled in the same centrifuge tube as one sample. The samples were immediately stored at -80°C.

[0064] 1.3 Metagenomic sequencing and results (Effects of DDYJ on gut microbiota in HFD mice)

[0065] 1.3.1 Data Quality Control and De-hosting Analysis

[0066] Metagenomic sequencing of mouse fecal samples was performed on the Illumina NovaSeq platform, using paired-end sequencing to obtain raw sequence data (FASTQ format). To ensure the accuracy and reliability of subsequent analyses, the raw sequencing data underwent quality control and host sequence removal.

[0067] This study obtained metagenomic sequencing data from three batches (A, B, and C) of mouse fecal samples at 0, 6, and 12 weeks. The number of raw reads for each sample ranged from 3.4 × 10⁻⁶. 7 -5.6×10 7 Between these values, the raw base was approximately 11-17 GB, and the proportions of Q20 and Q30 were both above 96%, indicating that the overall quality of the sequencing data was high and could meet the in-depth requirements of subsequent species annotation and functional analysis.

[0068] After quality control and host genome sequence filtering, most samples yielded a high proportion of CleanReads. The CleanReads proportion for 6-week-old samples mainly ranged from 60% to 93%; for 12-week-old samples, some individuals showed greater fluctuations, ranging from 22% to 93%. Overall, most samples still retained abundant and reliable microbial-derived sequences, with only a few samples showing an increased proportion of host-derived sequences.

[0069] 1.3.2 Species Composition Analysis

[0070] To reveal the compositional characteristics and trends of mouse gut microbiota under different intervention conditions, based on metagenomic sequencing results, we analyzed the gut microbiota at both the phylum and genus levels using group percentage stacked bar charts, Venn diagrams, and group clustering heat maps. Figure 3 This method allows for a comprehensive analysis of the species composition among samples.

[0071] Stacked bar charts at the phylum and genus levels can visually display the overall community composition of the gut microbiota in each group of mice. For example... Figure 1 As shown, at the phylum level, the gut microbiota of the NC group mice was mainly composed of Bacteroidetes and Bacillota, with other phyla (such as Verrucomicrobiota, Campylobacterota, Actinomycetota, etc.) having a lower proportion. In the early stages of the experiment (week 0), the proportion of Verrucomicrobiota was higher in some samples (such as A02), suggesting inter-individual differences. This difference may be related to the age of the mice, their dietary adaptation period, the gut microbiota adaptation process, or environmental stress responses at the initial stage of the experiment. As the experimental period progressed (to weeks 6 and 12), Bacteroidota and Bacillota became the dominant phyla, and the community composition gradually stabilized, reflecting the natural maturation trend of the gut microbiota over time. At the genus level ( Figure 2 In the initial stage of the experiment (week 0), *Rhizoctonia solani* was used. Muribaculum ), Duncanella ( Duncaniella ) and Bacteroides ( Bacteroides () is the dominant genus, but there are significant differences between individuals; as the experiment progresses, Muribaculum and Duncaniella Abundance increased, and Akkermansia Gradually replaced by other bacterial genera. This dynamic change reflects the natural maturation and stabilization of the gut microbiota in the NC group mice over time, gradually forming a mature and healthy ecological structure. Further comparison of the community structure of each experimental group showed that ( Figure 3 and Figure 4 Although the microbial communities in each group were dominated by Bacteroidota and had similar overall composition, some differences still existed. In the Dendrobium intervention groups, the proportion of Bacteroidota was slightly lower than in the NC group, and this proportion continued to decrease at 12 weeks. Meanwhile, Verrucomicrobiota was significantly more abundant in the NDDYJ and PC groups than in other groups. Actinomycetota accounted for a certain proportion in several groups, but its abundance increased in the DDYJ administration groups. This suggests that Dendrobium intervention may promote the growth of certain bacterial genera, thereby participating to some extent in the regulation of the mouse gut microbiota structure. In the genus-level composition analysis (… Figure 5 and Figure 6 Significant differences in microbial community structure were observed among different groups, with certain species exhibiting high enrichment in some groups. The main dominant genera included... Bacteroides , Muribaculum and Parabacterium spp. ( Parabacteroides These genera, such as […], dominate most groups, indicating they are core community members. It is noteworthy that... Akkermansia , Duncaniella and Microbacterium spp. ( Faecalibaculum The abundance of certain genera such as ( ) is higher in certain specific groups, which may be related to the specific environment or conditions of that group. Akkermansia Significantly enriched in NDDYJ and PC groups; Duncaniella The proportion is relatively high in NC and NDDYJ; Muribaculum Enriched in NC, NDDYJ, and PC groups; while Lactobacillus spp. ( Lactobacillus )and Faecalibaculum The proportions were higher in PC, DDYJ-H, and DDYJ-M, suggesting that Dendrobium intervention may improve gut homeostasis by promoting the expansion of certain genera with potential probiotic properties.

[0072] Further investigation was conducted using Venn diagrams at the phylum and genus levels to explore the overlap and differences in microbial composition among the groups. For example... Figure 7As shown in (AB), at 6 weeks, there were a total of 23 phyla across the groups, with a large overlap and high degree of intersection, indicating a high degree of similarity in the macroscopic structure of the gut microbiota. The phylum-level distribution was as follows: NC group: 28 species (1 endemic phylum); NDDYJ group: 29 species (0 endemic phyla); MG group: 33 species (2 endemic phyla); PC group: 31 species (2 endemic phyla); DDYJ group: 34 species (1 endemic phylum). This is consistent with the results of the group percentage stacked bar chart, showing that the composition at the phylum level was relatively stable, dominated by Bacteroidota and Bacillota, with only proportional differences. Genus-level analysis showed: NC group: 267 species (33 endemic genera); NDDYJ group: 261 species (21 endemic genera); MG group: 338 species (38 endemic genera); PC group: 298 species (15 endemic genera); DDYJ group: 418 species (65 endemic species), with a total of 159 genera. Compared to the phylum level, the differences at the genus level were more significant. Besides sharing a core community, the number of endemic genera increased significantly in each group, particularly in the DDYJ and MG groups, suggesting that the intervention led to the enrichment of new genera or the amplification of rare bacteria. At 12 weeks ( Figure 7 At the phylum level (CD), the total number of phyla decreased slightly to 19, but the community overlap remained high. The phylum level distribution was as follows: NC group 30 species (2 endemic phyla), NDDYJ group 30 species (1 endemic phylum), MG group 28 species (4 endemic phyla), PC group 25 species (0 endemic phyla), and DDYJ group 33 species (2 endemic phyla). At the genus level, there were 150 genera, with a slight increase in endemic genera compared to week 6: NC group 230 species (21 endemic genera), NDDYJ group 256 species (28 endemic genera), MG group 337 species (36 endemic genera), PC group 295 species (16 endemic genera), and DDYJ group 383 species (56 endemic species). Notably, at different time points, both the MG and DDYJ groups had more endemic operational taxonomic units (OTUs) than the NC group. Based on the stacked bar chart results, it can be inferred that dietary and drug interventions not only altered the proportions of the gut microbiota, but may also introduce or enrich some species that are not present in the healthy state. DDYJ intervention may enhance the functional diversity of the host gut ecosystem by promoting the amplification of specific metabolites, while the extended duration of intervention may promote the differentiation of the microbiota structure and ecological reconstruction.

[0073] Clustering heatmap analysis of each sample group was used to verify the above differences and identify the enrichment characteristics of key bacterial communities, thereby identifying the key microorganisms with different characteristics in each group. Results showed that at 6 weeks ( Figure 8At the phylum level, significant differences were observed in the microbial community structure among the different groups. The NC group was more homogeneous overall, with fewer extremely high-abundance clusters, indicating higher gut community stability. In contrast, the MG group showed multiple relatively high-abundance clusters, with significant enrichment of species such as Bacillota. Among the drug intervention groups, the community structures of the PC, NDDYJ, and DDYJ-M groups were similar to those of the NC group; the DDYJ-H group was enriched with phyla such as Actinomycetota. The enrichment of Actinomycetota is generally considered to be associated with the maturity and stability of the gut microbiota, suggesting that DDYJ-H may contribute to the improvement of health-related microbial community structure. At the genus level ( Figure 8 Analysis (B) further revealed this trend of "clustering towards health". The NC, NDDJ, PC, and DDYJ-M groups showed consistent behavior at the phylum level, exhibiting greater overall homogeneity and fewer extremely high-abundance clusters, with enrichment in some functional symbiotic or neutral bacteria (e.g., ...). Akkermansia Butymonas spp. Butyricimonas (etc.). Consistent with the enrichment of endemic genera shown in the Veen plot, MG and DDYJ-H have more endemic genera. The MG group samples show several distinct "red patches" (relatively high abundance) distributed in the genus *Broutella* (etc.). Blautia ), genus *Leptochrysis* ( Romboutsia In bacteria of the genera *[]*, DDYJ-H is enriched with [[...]]. Faecalibaculum , spp. of Osmotherium Dysosmobacter Butyric acid-producing bacteria such as *Bacillus*, but some individuals also contain *Enterococcus*. Enterococcus ) and Escherichia coli ( Escherichia The enrichment of conditionally pathogenic or stress-related genera, such as [list of genera], may be related to changes in the dynamic balance of the gut microbiota. By 12 weeks (…), Figure 8 At the phylum level, the overall high abundance blocks in each group decreased, the color bands became more balanced, fluctuations decreased, and the community structure became more balanced, still dominated by bacteria. A small number of archaea or fungi (such as Ascomycota) showed sporadic high-value blocks in individual samples, but these were not dominant in any group and were mostly due to individual differences. At the bacteria level, the differences between groups were more "converged" than at week 6, and more similar to the NC group. The distribution of core phyla such as Bacteroidota and Bacillota was closer, with fewer extreme values. The high imprint of MG at some phylum levels also weakened to some extent, indicating that long-term HFD intervention stabilized and reshaped the community structure at the macroscopic level. At the genus level ( Figure 8 In the MG group, sporadic high values ​​could still be seen in a few genera; while the difference in bacterial distribution between the DDYJ intervention group and the NC group was significantly reduced, with enrichment in genera with potential probiotic properties or metabolic regulatory effects, such as *Mediterranean*. Mediterraneibacter )and Faecalibaculum wait, Enterococcus The number of potentially harmful genera decreased compared to 6 weeks, suggesting that long-term DDYJ intervention may help promote gut microbiota stabilization and maintain intestinal ecological balance.

[0074] 3.2.6.3 Species diversity analysis

[0075] (1) α-diversity analysis

[0076] Species richness (Chao1 index) and species diversity (Shannon index) were used to reflect the species richness and community diversity of the bacterial community, respectively. The results showed that there were certain trends in the Chao1 and Shannon indices among the groups, but none reached a statistically significant level of difference. p > 0.05).

[0077] like Figure 9 (A) At 6 weeks, the Chao1 index was lowest in the NC group (approximately 550), indicating that the gut microbiota structure was relatively stable, with moderate richness and reasonable species composition under normal conditions. The Chao1 index in the MG group was at a higher level (approximately 850), indicating a higher species richness in the microbial community. This reflects the proliferation of low-abundance or opportunistic bacteria in the microbiota under HFD-induced growth, leading to an increase in overall richness but a decrease in stability, and a change in community structure. Among the drug intervention groups, the Chao1 index of the NDDYJ group was close to that of the NC group; the PC group and the DDYJ-M group were similar, both falling between the NC and MG groups (approximately 660-700); while the richness of the DDYJ-H group was higher, similar to the level of the MG group (approximately 850), suggesting that high-dose intervention may have caused strong stimulation to the microbiota in the short term, leading to an unstable state of species proliferation. Shannon index ( Figure 9 (B) Further analysis showed that although there were differences in species richness, the overall community diversity was not significantly different. The indices of the NDDYJ, PC, and DDYJ-M groups were similar to those of the NC group (approximately 4.1-4.5), while the indices of the MG and DDYJ-H groups were slightly higher (approximately 4.7). This indicates that although there were significant differences in species richness among the groups at 6 weeks, the overall community diversity was very similar. This suggests that although dietary and drug interventions altered species richness, they did not disrupt the species evenness within the community. While the number of microbial communities changed, the spatial competition and structure within the community remained relatively balanced.

[0078] By week 12, the Chao1 index in each group ( Figure 9The overall decline in the C) indicates that the gut microbiota gradually evolves towards a "stable core bacteria-dominated" structure during long-term feeding. Specifically, the Chao1 index in the NC group decreased to around 400; the NDDYJ group maintained a level close to that at 6 weeks (approximately 550), the closest to the NC group among all groups, showing a significant advantage in maintaining long-term microbiota stability; the indices in the PC and DDYJ-M groups were close to those at 6 weeks (approximately 660-700); while the indices in the MG and DDYJ-H groups both decreased to approximately 660-700, indicating that microbiota fluctuations gradually subsided during this stage. Shannon index ( Figure 9 Regarding the evenness index (D), the NC group showed a slight decrease compared to week 6 (approximately 3.8); the NDDYJ group was slightly higher than the NC group, and close to the level at week 6 (approximately 4.1); the remaining groups showed a slight increase compared to week 6, with the MG group having a higher index than the DDYJ-M and PC groups, showing a trend from "high abundance but unstable" to "low abundance but higher evenness". These results indicate that over time, the gut microbiota community structure of mice in each group gradually shifted from a "generalized" state containing many incidentally present species to a state dominated by core functional species, and the evenness of the community significantly improved. This characteristic is common in the chronic adaptation process of the gut microbiota ecosystem.

[0079] (2) β-diversity analysis

[0080] To further explore the impact of intervention on the overall differences in the microbial community structure, this study conducted principal coordinate analysis (PCoA) at the genus level based on Bray-Curtis distance.

[0081] The results showed that at 6 weeks ( Figure 9 The fecal samples from different groups showed significant separation on a two-dimensional coordinate system (Axis 1 and Axis 2). The first principal component (Axis 1) and the second principal component (Axis 2) explained 37.96% and 19.26% of the community differences, respectively, with a cumulative explained rate of 57.22%. The NC and NDDYJ groups clustered on the left, showing similar community structures, indicating a close similarity in their gut microbiota composition. The remaining groups clustered on the right, showing differences from the NC group. The MG group was significantly separated from the NC group, suggesting that HFD had caused significant structural changes. The drug intervention groups (DDYJ-H, DDYJ-M, and PC) were distributed between MG and NC, but there was some overlap between groups. The dispersion of sample points within the same group may be related to the differences in the effects of the drugs on different individuals during this period. This indicates that although the HFD-treated groups showed similarities in overall composition, the intervention could restore the gut microbiota structure to a certain extent, bringing it closer to a normal state, but individual differences still exist. The PERMANOVA test results further showed that the differences between groups were statistically significant. p < 0.05).

[0082] At 12 weeks ( Figure 9 The differences in community distribution further intensified, and the boundaries between clusters became clearer. The first principal component (Axis.1) and the second principal component (Axis.2) explained 47.55% and 19.83% of the community differences, respectively, increasing the cumulative explained value to 66.85%. The NC and MG groups remained clearly separated, and significant differences in gut microbiota persisted, with an overall trend similar to that at week 6. The NDDYJ group continued to cluster closely with the NC group, while the DDYJ-H and DDYJ-M groups maintained significant distance from the NC group, but with increased clustering within each group. Permanente analysis (R² = 0.649, F = 4.428, P = 0.001) demonstrated that group factors were one of the important determinants driving differences in gut microbiota structure.

[0083] 2.1 Metavirome sequencing and results

[0084] 2.1.1 Sample Collection

[0085] Fecal samples were collected from mice in the NC, MG, and DDYJ-H groups at week 12 for metaviromic sequencing. During sample collection, to minimize environmental matrix interference, mice were transferred to pre-sterilized, bedding-free individual cages and allowed to defecate naturally. Fresh fecal pellets were then quickly picked up with sterile forceps and placed in 1.5 mL sterile centrifuge tubes (EP tubes). The tube caps were immediately tightened to isolate the air, and the sample tubes were immediately placed in an ice bath to inhibit microbial metabolic activity and nucleic acid degradation. The forceps were disinfected with alcohol before the next sampling, until all mouse fecal samples were collected. Fecal samples from five mice per cage were pooled in the same centrifuge tube as one sample. The samples were immediately stored at -80°C.

[0086] 2.1.2 Metavirome Sequencing and Data Quality Control

[0087] Viral DNA extracted from nine samples was subjected to Illumina high-throughput sequencing, yielding approximately 91.54 Gb of clean reads with a Q30 base ratio >94%, meeting the requirements for downstream analysis. De novo assembly was performed using MEGAHIT, with an average N50 length of 3812.67 bp and a maximum contig of 153328 kb. BWA backfit evaluation showed an average read utilization rate of 95%, indicating good assembly integrity. Further BLAST alignment with the host genome to remove contaminating sequences revealed that non-host source contigs accounted for 99.99% of the total assembly. CD-HIT redundancy removal yielded 92,832 high-quality, non-redundant viral contigs. QUAST evaluation showed an average GC content of 47.96%, integrity >90%, and contamination rate <5%, meeting the standards for medium to high integrity viral genomes, suitable for subsequent functional annotation and taxonomic analysis.

[0088] The raw data was processed using Trimmomatic (v0.36) to remove low-quality data, resulting in high-quality data for subsequent analysis. BWA (v0.7.17, default parameters: mem–k 30) was used to align clean reads to both the ribosome database (Silva.132) and the host database, filtering out alignments shorter than 80% of the total read length and removing the corresponding host sequences. BWA was then used to align clean reads to the virus reference data (Virus-NT), again filtering out alignments shorter than 80% of the total read length for preliminary virus classification.

[0089] 2.1.3 Data Assembly and Bioinformatics Analysis

[0090] The clean data was assembled using Megahit (v1.1.2, default parameters: presets meta large min contig len 300) software. BWA software was used to compare the clean reads with the assembled results and calculate read utilization. Simultaneously, BLAST (v2.9.0+) software was used to align the assembled contigs with the host sequences and remove the host sequences. Additionally, CDHIT (v4.7, default parameters: c 0.95 aS 0.8) software was used to cluster the contigs of all samples to obtain unique.contig.

[0091] Viral sequence identification and annotation were performed on the assembled viral contigs using a combination of confirmed / suspected alignment with reference databases and denovo feature identification methods. Different classification labels were assigned based on the identification method and completeness. The annotation, host contamination removal, and false positive filtering of the assembled unique contigs were completed by Shenzhen MicroMed Technology Group Co., Ltd. This company employed a multi-dimensional joint identification strategy: first, integrating multi-source evidence such as nucleotide homology (BLASTn / BLASTx) and conserved amino acid domains (HMMsearch against VPFs / vFam) to screen candidate viral sequences; then, rigorous secondary alignment (NT / NR databases) and taxonomic consistency-based removal of sequences primarily belonging to non-viral groups eliminated false positives.

[0092] Alpha diversity analysis, Beta diversity analysis, distance matrix calculation, and PCA and PCoA analyses were all completed on the WeKeMeng cloud platform. Based on the Contig abundance matrix, this study adopted a stratified strategy for pairwise comparisons between groups: first, the normality of the data (Shapiro-Wilk test) and homogeneity of variance (Lvene test) were assessed; for data that met the parametric hypothesis, Student's t-test was used for independent samples and Paired t-test was used for paired samples; if the data violated the normality or homogeneity of variance assumptions, the nonparametric Wilcoxon rank-sum test (Mann-Whitney U test) or Wilcoxon signed-rank test was used, respectively. To control the risk of false positives from multiple hypothesis testing, all p-values ​​were corrected for the false discovery rate (FDR, Benjamini-Hochberg method).

[0093] 2.1.4 Phage Host Prediction

[0094] Viral host prediction was performed by identifying the CRISPR spacer region and tRNA sequence information in viral contigs. The CHERRY tool in PhaBOX (https: / / phage.ee.cityu.edu.hk / ) was used for host prediction. Prediction results were filtered using a score threshold (0-1). To obtain more accurate predictions, results with PhaMerScore and CHERRYScore greater than 0.9, and those with low confidence scores, were selected as host information for the viral groups. Subsequently, based on the filtered high-quality virus-host pairing data, Python was used to visualize and construct the interaction relationships, intuitively analyzing and displaying the specific interaction relationships and topological characteristics between viral groups and their hosts.

[0095] 2.2 Results and Analysis

[0096] 2.3 Diversity of DNA Viral Communities in HFD-Induced Groups

[0097] Viral sequence identification and annotation were performed using a combination of methods based on confirmed and suspected reference sequences and Denovo, with different classification tags assigned according to the identification method and completeness. A total of 3522 viral sequences with complete genome structures were identified from all samples, and subsequent analyses were based on these. Statistical results of the identified viral genome types showed that 76% of the contigs were identified as double-stranded DNA viruses, 15% as single-stranded DNA viruses, and 8% as unknown viruses. Phage identification results showed that 96% of all viral contigs were phage sequences, while non-phage viral contigs accounted for 4%. In summary, the viruses obtained from viral sequence identification and classification annotation were primarily phages.

[0098] The α and β diversity of DNA viral communities in mouse feces was analyzed based on contig levels. The α diversity results showed that the Shannon index of NC was higher than that of MG and DDYJ-H (…). Figure 10 A), the ACE index showed that the viral abundance in the DDYJ-H group was slightly higher ( Figure 10 B), but there were no statistically significant differences among the three groups in Shannon and ACE indices ( p >0.05). β-diversity analysis was performed using Bray-Curtis distance for NMDS (…). Figure 10 C) and PCoA sorting ( Figure 10 D), the results showed that the three groups of samples were spatially separated, especially the NC group and the MG and DDYJ-H groups had significant differences in viral community structure, although the PERMANOVA test did not reach a significant level ( p=0.085), but visualization results reveal that high-fat diets and interventions have a potential impact on the composition of the gut virus community.

[0099] 2.4 Composition of DNA Virus Community in HFD-Induced Group

[0100] All samples were annotated to 9 phyla, 10 classes, 10 orders, 16 families, 28 genera, and 163 species of viruses.

[0101] Door level ( Figure 11 A) Phixviricota, Uroviricota, and Cressdnaviricota were the main taxa in the different treated samples. Among them, the relative abundance of Phixviricota in MG samples (56.82%) was higher than that in NC samples (31.85%); the relative abundance of Uroviricota in NC samples (26.58%) and DDYJ-H group (13.17%) was higher than that in MG samples (9.6%).

[0102] At the scientific level ( Figure 11 B), Microviridae and Podoviridae were the main taxa in samples treated differently. Among them, the relative abundance of Microviridae in MG samples (56.83%) was higher than that in NC samples (31.85%); the relative abundance of Podoviridae in NC samples (19.85%) and DDYJ-H group (9.8%) was higher than that in MG samples (5.25%).

[0103] Venn analysis of the genus level in the three groups of samples showed that 24 genera were common to all three groups, and 4 genera ( Gammasphaerolipovirus, Unspecified_Autographiviridae , Punavirus , Pahexavirus This genus is unique to the DDYJ-H group samples; it is not unique to the NC and MG groups. Figure 12 A). At the horizontal level ( Figure 12 B), with a total of 28 genera annotated. Among viral taxa with known taxonomic positions, Lymphocryptovirus , Cytomegalovirus , Lentivirus , Scapunavirus , Gemykrogvirus , Tunavirus It is the main group among the three groups of samples.

[0104] Based on the results of the multiple comparison analysis, at the gate level ( Figure 13A), Cressdnaviricota and Hofneiviricota are the differentially expressed groups among the three sample groups. Cressdnaviricota showed significant differences from the other two groups in the DDYJ-H sample, while Hofneiviricota showed significant differences from the other two groups in the NC group. (At the scientific level) Figure 13 (B) Apart from the Unspecified group, Drexlerviridae and Inoviridae were the differentially expressed groups in the three sample groups. Drexlerviridae was significantly enriched in the DDYJ-H sample; Inoviridae was significantly enriched in the NC sample. At the genus level ( Figure 13 C), Tunavirus It is the differential group among the three groups of samples, with a higher abundance in the DDYJ-H sample and significant differences from the other two groups.

[0105] Analysis using LEfSe (LDA Effect Size) Figure 14This study systematically compared the differences in intestinal virus communities among the NC, MG, and DDYJ-H mouse groups at the species level, aiming to identify viral biomarkers with statistical and biological significance. Although the analysis included all three groups, the final results only showed the significantly different taxa between the NC and DDYJ-H groups, indicating that the MG group did not exhibit independent and strongly enriched characteristic viruses in terms of viral composition. This may be because its viral community was in a transitional state between NC and DDYJ-H, or because large individual variability prevented it from reaching the LDA threshold set by LEfSe (default LDA > 2). s_Dickeya_phage_phiDP23_1, Unspecified_Ackermannviridae, f_Ackermannviridae, s_crAssphage_cr113_1, o_Herpesviricetes, o_Herpesvirales, p_Peploviricota, f_Herpesviridae, and s_Siphoviridae_sp_ctJT77 were identified as significantly enriched viral markers in the DDYJ-H group in LEfSe analysis. These results indicate that intervention with Dendrobium officinale procrease specifically promotes the amplification of various phages, including members of the Ackermannviridae and Siphoviridae families, as well as the crAssphage group. In the healthy state (NC group), the gut contained a viral community dominated by Cressdnaviricota (a type of double-stranded DNA phage) and members of the Siphoviridae family. The enrichment of specific species-level viruses such as s_Chaetec_virus_UA24_2563 and s_Siphoviridae_sp_ctvok7 suggests that they may be related to normal gut microbiota homeostasis or immune regulation.

[0106] 2.5 Functional characteristics of viral communities in the HFD-induced group

[0107] As can be seen from the figure, the three groups differ in their distribution of functional categories. Figure 15 A) The reference protein had the highest proportion in the NC group, while its proportion decreased in the MG and DDYJ-H groups. Host cytoplasm, Capsid protein, and Virion-related functions had relatively high proportions in the MG group, suggesting that the model group may be associated with an enhanced viral life cycle and pathogenic process. The proportions of some functional categories in the DDYJ-H group converged with those in the NC group, indicating that intervention may have a certain moderating effect on functional abnormalities in the model group. Cluster heatmap ( Figure 15(B) Specific functional items such as Capsid protein, icosahedral capsid protein, Direct protein sequencing, and Host cytoplasm were highly expressed (red) in the MG group, while they were lowly expressed (blue) in the NC and DDYJ-H groups. The DDYJ-H group partially recovered to a low expression level, further supporting the possibility that the intervention suppressed the abnormally elevated virus-related or replication-related functions in the model group.

[0108] 2.7 HFD-induced phage host prediction

[0109] Based on host prediction analysis results, the core functional virus genus belongs to... Eponavirus , Brigitvirus , Oengusvirus , Carjivirus , Lagaffevirus , Toutatisvirus , Mushuvirus Mainly, it contains multiple specific phages. Tunavirus , Astrithrvirus , Prasinovirus . Eponavirus To maintain the absolute dominant genus in the basal microbial community turnover, Carjivirus , Oengusvirus These are closely related to homeostasis. Virus host range coverage Faecalibacterium , Butyricicoccus , Blautia , Ruminococcus Core bacteria that produce short-chain fatty acids Bacteroides , Prevotella Dominant symbiotic bacteria, Lactobacillus , Bifidobacterium Probiotics, and Salmonella , Enterococcus Conditionally pathogenic bacteria, and also include Lactococcus , Parabacteroides Other symbiotic bacteria, along with others, form a more complex virus-bacterial interaction regulatory network. Differences in intergroup interactions are evident in MG. Carjivirus , Brigitvirus , Oengusvirus Abundance decreased significantly, and targeted interaction links with butyric acid-producing bacteria and probiotics were significantly reduced. Enterococcus The interaction between viruses and opportunistic pathogens was enhanced. The intervention group DDYJ-H, however, recovered. Carjivirus Health markers and viruses Faecalibacterium , Ruminococcus Extensive interaction and enrichment of beneficial bacteria Tunavirus Targeted elimination of pathogens Shigella The bacteriophage, while enhancing the ability to target... Lactobacillus The specific phage flow demonstrates a precise regulatory and restorative effect on the imbalanced microecology.

[0110] In summary, fecal metagenomic sequencing data from different gavage cycles (0, 6, and 12 weeks) indicate that DDYJ can maintain overall gut microbiota stability while systematically adjusting its structure to approximate the characteristics of a normal diet group. Gavage administration of DDYJ can sustainably enrich bacterial genera closely related to short-chain fatty acid (SCFA) production, intestinal barrier function, and immune regulation (such as...). Akkermansia, Bifidobacterium, Faecalibaculum and Lactobacillus It also inhibits the abnormal proliferation of inflammation-related bacteria, indicating that DDYJ has a significant effect on regulating the gut microbiota.

[0111] Metavirome sequencing was used to evaluate the regulatory effects of DDYJ on enteroviruses in mice with metabolic abnormalities. DDYJ altered the intestinal DNA viral community structure, repaired viral functional imbalances, remodeled the phage-intestinal bacteria interaction network, and maintained intestinal microecological homeostasis. DDYJ intervention effectively alleviated physiological metabolic abnormalities and liver damage induced by HFD and demonstrated good safety in mice. Metavirome sequencing yielded high-quality viral sequences. Community analysis showed no significant difference in α-diversity among the three groups, but significant segregation in β-diversity, with double-stranded DNA phages being the predominant viral type. HFD altered the viral composition at the phylum, family, and genus levels, while DDYJ significantly enriched specific phage groups and restored differential viral abundance. Phage host prediction showed that HFD disrupted viral-beneficial bacteria interactions and enhanced associations with opportunistic pathogens, while DDYJ restored the healthy virus-core bacterial interaction network and precisely regulated intestinal microecological balance.

Claims

1. A method for preparing a solid beverage for improving intestinal flora, characterized by, Includes the following steps: (1) Extraction of active substances from Dendrobium nobile: The purple-skinned dendrobium was mixed with water at 55-75℃ and extracted by microwave constant temperature 2-3 times, each time for 16-20 minutes. The extracts were combined, concentrated, precipitated with alcohol, filtered through a membrane, and dried to obtain the purple-skinned dendrobium extract. The microwave frequency was 1500-1700MHz and the power was 1200-1400W. (2) Extraction of active substances from Pu-erh tea: Mix Pu-erh tea with the extractant and reflux at 85-90℃ for 1-2 times, each time for 1-3 hours. Combine the extracts, concentrate and dry to obtain Pu-erh tea extract. (3) The purple Dendrobium extract and Pu-erh tea extract are mixed and sterilized to obtain a solid beverage that improves intestinal flora.

2. The method for preparing a solid beverage according to claim 1, characterized by, The water in step (1) contains maltose; the concentration of maltose in the water is 2.5~3.5 g / 100 mL.

3. The method for preparing a solid beverage according to claim 1, characterized by, Step (1) involves concentrating the solution to a relative density of 1.15~1.

25.

4. The method for preparing a solid beverage according to claim 3, characterized by, The alcohol precipitation in step (1) involves adding 3 to 6 times the volume of concentrated ethanol and then letting it stand for 14 to 18 hours to obtain the precipitate.

5. The method for preparing a solid beverage according to claim 4, characterized by, The membrane filtration in step (1) is as follows: the precipitate is dissolved in 25 to 30 times its mass of water, then passed through an ultrafiltration membrane with a molecular weight of 150 KD, the retentate is collected, and then passed through an ultrafiltration membrane with a molecular weight of 350 KD, the permeate is collected.

6. The method of preparing a solid beverage according to claim 1, characterized in that, The extractant in step (2) is an ethanol solution containing formic acid at a volume concentration of 0.1% to 1%.

7. The method of preparing a solid beverage according to claim 1, characterized in that, The volume concentration of the ethanol solution in step (2) is 85-90%.

8. The method of preparing a solid beverage according to claim 1, characterized in that, The relative density of the concentrate in step (2) is 1.18~1.

23.

9. The method of claim 1-8 for the preparation of a solid beverage for the improvement of the intestinal flora, characterized by, It includes 20-30 parts of Dendrobium officinale extract and 60-80 parts of Pu-erh tea extract.

10. The application of the solid beverage for improving intestinal flora as described in claim 9 in the preparation of beverages with the function of regulating intestinal microecological balance.

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