A composition for enhancing immunity through intestinal flora and a preparation method and application thereof

Abstract

This invention discloses a composition for enhancing immunity through gut microbiota, its preparation method, and its application. The composition comprises the following raw materials: a complex prebiotic, polyphenols, inactivated *Saccharomyces boulardii*, and plant extracts. The complex prebiotic includes human milk oligosaccharides and arabinogalactan, the polyphenols include berry polyphenols and grape polyphenols, and the plant extracts include pomegranate seed extract and kale extract. This invention's composition, on the one hand, selectively enriches *A. AKK* bacteria through prebiotics; *A. AKK* can directly interact with various immune cells to enhance immunity. On the other hand, supplementing with yeast factors can also inhibit inflammation and enhance immune responses, thereby significantly increasing the number of *A. AKK* bacteria in the gut microbiota and simultaneously improving intestinal cellular immune function.

Description

Technical Field

[0001] This invention relates to the field of dietary supplement technology, specifically to a composition for enhancing immunity through gut microbiota, its preparation method, and its application. Background Technology

[0002] The interaction between the gut microbiota and the host immune system is a cutting-edge research topic in life sciences. The human gut is home to trillions of microorganisms, which together constitute a complex ecosystem. Through metabolic products, cell wall components, and immune regulatory signals, they significantly influence the development and homeostasis of the host's overall immune function. In recent years, numerous studies have confirmed that alterations in gut microbiota structure are closely related to susceptibility to infection, vaccine response efficacy, and the occurrence and development of autoimmune diseases. This provides a solid theoretical foundation for intervention strategies that enhance immunity by regulating the gut microbiota.

[0003] Among microbial species targeted for regulation, Akkermansia muciniphila (AKK) has attracted widespread attention due to its unique mucus layer localization ability and close interaction with the host's immune system. Studies have shown that the relative abundance of AKK is positively correlated with metabolic health and immune regulation, and it plays a key role in enhancing host immunity through mechanisms such as maintaining intestinal barrier integrity and regulating inflammatory responses. However, regulatory attitudes and progress regarding the direct use of AKK in the food sector vary across major global markets. Therefore, developing functional components that can specifically promote the proliferation of AKK has become an important direction in the field of prebiotic research.

[0004] The role of prebiotics in promoting the proliferation of AKK bacteria has been widely validated. Multiple studies have shown that specific dietary components can serve as growth substrates for AKK bacteria. For example, β-glucan (especially β-glucan derived from wheat) has a significant growth-promoting effect on AKK bacteria at a concentration of 20 mg / mL and can be utilized by AKK bacteria to produce short-chain fatty acids. Galacto-oligosaccharides (GOS), soybean oligosaccharides, and xylooligosaccharides have also been shown to significantly promote the growth of human AKK strains. Furthermore, prebiotics such as sodium alginate and xanthan gum, when used as microcapsule wall materials, can not only improve the survival rate of AKK bacteria during processing and storage but also enhance their tolerance in simulated gastrointestinal fluids.

[0005] Meanwhile, significant progress has been made in the application of yeast probiotics in immunomodulation. Traditional *Saccharomyces boulardii* has been widely used to maintain gut health, with its immunomodulatory mechanisms including enhancing the production of secretory immunoglobulin A (sIgA), regulating cytokine expression, and influencing antigen-presenting cell function. In recent years, the potential of non-*Saccharomyces* yeasts as novel probiotics has been increasingly recognized. Studies have shown that certain inactive yeasts and their derivatives also possess immunomodulatory activity, exhibiting good safety and processing adaptability. However, existing yeast immunomodulators mainly focus on live bacterial preparations and are mostly used alone; research on the combined use of inactive yeasts with other ingredients remains limited.

[0006] In summary, although prebiotics can selectively enrich AKK bacteria and inactive yeast has shown immunomodulatory potential, the exploration of their synergistic effects remains lacking. Therefore, developing a composition that enhances immunity by regulating the gut microbiota has significant theoretical value and application prospects. Summary of the Invention

[0007] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes a composition for enhancing immunity through gut microbiota, which significantly promotes the proliferation of AKK bacteria in the gut, thereby effectively solving the problem that AKK bacteria cannot be directly used in food formulations.

[0008] The present invention also provides a method for preparing the above composition.

[0009] The present invention also proposes applications of the above-described composition.

[0010] According to one aspect of the present invention, a composition for enhancing immunity through gut microbiota is provided, comprising the following ingredients: a complex prebiotic, polyphenolic substances, inactivated Saccharomyces boulardii, and plant extracts, wherein the complex prebiotic includes human milk oligosaccharides (HMOs) and arabinogalactan, the polyphenolic substances include berry polyphenols and grape polyphenols, and the plant extracts include pomegranate seed extract and kale extract.

[0011] The composition for enhancing immunity through gut microbiota according to embodiments of the present invention has at least the following beneficial effects: The composition of the present invention significantly proliferates AKK bacteria in the gut microbiota through a dual mechanism of "prebiotic-directed enrichment of key microbiota" and "synergistic enhancement of yeast immunomodulatory factors" (on the one hand, by direct enrichment of AKK bacteria through prebiotics, AKK can directly interact with various immune cells to enhance immunity; on the other hand, by supplementing yeast factors, inflammation can also be inhibited and immune responses enhanced), thereby enhancing the immune function of intestinal cells. Specifically, the compound prebiotic components in the composition can directionally proliferate AKK bacteria in the gut. AKK maintains the integrity of the intestinal barrier and regulates the tone of the immune system, while β-glucan in the yeast cell wall is a powerful immune activator. This combination has a complementary and synergistic effect on enhancing immunity. In addition, plant extracts have also been shown to have unique advantages in this regard: the polyphenols, alkaloids, capsaicin, and various plant-derived polysaccharides contained therein can all significantly increase the abundance of AKK bacteria; low-concentration decoctions of some medicinal and edible herbs have also shown the effect of increasing the number of AKK bacteria in in vitro and in vivo experiments, providing new ideas for the development of dietary supplements. In terms of immune regulation, yeast-derived β-glucan is the core active ingredient, which can significantly increase the levels of secretory antibodies (IgA) in the intestinal mucosa of animals. + The number of cells and specific immune T cells (CD4) + CD8 + This composition enhances the "precise" immune defense against specific pathogens by adjusting the ratio of prebiotics to various functional factors. Simultaneously, *Saccharomyces boulardii* has been shown to inhibit the secretion of pro-inflammatory signaling molecules (such as IL-8 and NF-κB) and activate aryl hydrocarbon receptors, helping to calm excessive and harmful inflammatory responses. In summary, this composition achieves the dual effects of *AKK* bacteria proliferation and intestinal immune regulation through the synergistic effect of prebiotics and multiple functional factors.

[0012] According to some embodiments of the present invention, the human milk oligosaccharide includes 2'-fucosylated lactose. 2'-Fucosylated lactose belongs to fucosylated HMOs, and studies have revealed novel mechanisms by which it repairs the intestinal barrier and works synergistically with specific beneficial bacteria.

[0013] According to some embodiments of the present invention, the mass fraction ratio of the compound prebiotic: polyphenolic substance: plant extract: inactivated yeast is 14~21:4~8:9~16:5~8.

[0014] According to some embodiments of the present invention, the composition comprises the following parts by weight of raw materials: 8-12 parts of human milk oligosaccharides; 6-9 parts of arabinogalactan; 2-4 parts of berry polyphenols; 2-4 parts grape polyphenols; 4-7 parts pomegranate seed extract; 5-9 parts of kale extract; Inactivate 5-8 portions of Saccharomyces boulardii.

[0015] According to some embodiments of the present invention, the mass fraction ratio of human milk oligosaccharide to arabinogalactan is 1~2: 1~1.5, the mass fraction ratio of berry polyphenols to grape polyphenols is 2~1: 1~2, and the mass fraction ratio of pomegranate seed extract to kale extract is 5~9: 5~8.

[0016] According to some embodiments of the present invention, the pomegranate seed extract and kale extract are commercially available alcohol extracts.

[0017] According to another aspect of the present invention, a method for preparing the above-described composition is provided, comprising the following steps: Mix the above ingredients to obtain the final product.

[0018] According to another aspect of the invention, the use of the above composition in the preparation of products having immunomodulatory functions is provided.

[0019] According to some embodiments of the present invention, the product includes at least one of health food, food composition or pharmaceutical.

[0020] According to some embodiments of the present invention, the immunomodulatory function includes intestinal immunomodulatory function. The compositions of the present invention are particularly suitable for enhancing intestinal immunity.

[0021] The composition of the present invention can significantly enhance immunity (especially intestinal immunity) and can be used in health foods / food compositions with immunomodulatory functions.

[0022] According to another aspect of the present invention, the use of the above composition in the preparation of products that increase intestinal flora is provided.

[0023] According to some embodiments of the present invention, the intestinal flora includes at least one of Akkermansia, Streptococcus, Odoribacter, Bacteroidota, or Verrucomicrobiota.

[0024] According to some embodiments of the present invention, the intestinal flora includes Akkermansia and / or Streptococcus. Akkermansia and Streptococcus are characteristic genera of the effects of this composition on the intestinal flora.

[0025] According to another aspect of the present invention, a dietary supplement or food is provided, the dietary supplement or food comprising the above-described composition.

[0026] According to some embodiments of the present invention, the dietary supplement or food also includes a thickener.

[0027] According to some embodiments of the present invention, the mass ratio of the thickener to the composition is 7~14:15~27.

[0028] According to some embodiments of the present invention, the thickener includes two of pectin-apple, maltodextrin, and aloe vera gel.

[0029] According to some embodiments of the present invention, the dietary supplement or food also includes a flavoring agent.

[0030] According to some embodiments of the present invention, the mass ratio of the flavoring agent to the composition is 5~8:15~27.

[0031] According to some embodiments of the present invention, the flavoring agent is selected from at least three of the following: citrus fruit powder, lychee fermentation powder, strawberry powder, banana powder, lemon fruit powder, pineapple powder, strawberry powder, papaya powder, and honey powder.

[0032] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0033] Figure 1 This is a graph showing the test results of the in vitro intestinal flora fermentation verification of the probiotic AKK proliferation effect in the test examples of this invention.

[0034] Figure 2 This is a graph showing the test results of improving the nutritional status of the thymus and spleen in mice in the test examples of this invention.

[0035] Figure 3 This is a graph showing the test results of the effect of the present invention on inhibiting intestinal inflammation and intestinal barrier damage.

[0036] Figure 4 This is a graph showing the test results of the indicators for improving liver damage in the test examples of this invention.

[0037] Figure 5 This is a graph showing the results of the immune factor regulation test in the test examples of this invention.

[0038] Figure 6 This is an α-diversity analysis diagram of the test results for improving gut microbiota diversity in the test examples of this invention.

[0039] Figure 7This is a β-diversity PCA analysis diagram of the test results for improving gut microbiota diversity in the test examples of this invention.

[0040] Figure 8 This is a β-diversity PCoA analysis diagram of the test results for improving gut microbiota diversity in the test examples of this invention.

[0041] Figure 9 This is a chart showing the top 30 phylum level analysis results of the test results for improving gut microbiota diversity in the test examples of this invention.

[0042] Figure 10 This is a graph showing the top 30 genera level analysis results of the test results for improving gut microbiota diversity in the test examples of this invention.

[0043] Figure 11 This is a linear discriminant analysis graph of the test results for improving gut microbiota diversity in the test examples of this invention.

[0044] Figure 12 This is a Spearman correlation analysis result graph showing the Top 30 bacterial genera in each group of samples and the corresponding detection indicators in the test examples of this invention. Detailed Implementation

[0045] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available. Unless otherwise specified, the same parameter value is the same in all embodiments. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0046] In the description of this invention, references to terms such as "some embodiments" indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0047] In the description of this invention, the use of terms such as "first," "second," etc., is for the purpose of distinguishing technical features only and should not be construed as indicating or implying relative importance, or implicitly indicating the number of technical features indicated, or implicitly indicating the order of the technical features indicated.

[0048] The raw material information for embodiments of the present invention is as follows: 2'-Fucose-based lactose CAS:41263-94-9.

[0049] Arabic galactomannan CAS: 9036-66-2.

[0050] Berry polyphenols CAS: 84082-34-8.

[0051] Grape polyphenols CAS: 501-36-0.

[0052] Pomegranate seed extract CAS: 476-66-4.

[0053] Kale extract from Mufan Biotechnology, product number: MF-005785.

[0054] Inactivated *Brasilaria sinensis* was obtained by isolating, propagating, and inactivating fresh litchi. Details are as follows: 1. Isolation and purification of *Bretschneidera sinensis*: Sample collection and pretreatment Select mature lychee peels with natural white bloom (fruit powder) on the surface and without damage. Cut off the lychee peels and put them into an Erlenmeyer flask containing sterile physiological saline. Shake and wash thoroughly to prepare a bacterial suspension.

[0055] Selective enrichment culture The bacterial suspension was inoculated into acidic YPD liquid medium (pH 4.5). After incubation at 30°C for 24 hours, the liquid medium became turbid, indicating that the yeast had proliferated rapidly.

[0056] Solid plate separation and purification The enrichment solution was serially diluted 10-fold, and 10... -6 The diluted solution was spread onto yeast isolation solid medium containing 0.1 g / L chloramphenicol (an antibacterial agent). Single colonies were isolated using the streak plate method. *Saccharomyces boulardii* typically appears creamy or slightly yellow on the medium, with a smooth, moist surface and regular edges. Colonies are slightly smaller and more raised than those of *Saccharomyces cerevisiae*. Under a microscope, the cells are oval or egg-shaped and reproduce by budding.

[0057] Molecular biological identification Suspected single colonies were selected for ITS sequence sequencing. The sequencing results were compared with the NCBI database. Only when the sequence showed >99% homology with Saccharomyces boulardii could it be confirmed as the target strain.

[0058] 2. Proliferation Culture After obtaining the pure strain, a large number of cells need to be obtained in a short time through fermentation.

[0059] 2.1 Seed liquid preparation Primary seed: The purified strain was inoculated into 5 mL of YPD liquid medium and cultured at 30°C and 200 rpm for 16 h with shaking.

[0060] Secondary expansion culture: Transfer the primary seed culture to a large Erlenmeyer flask or seed tank at an inoculation rate of 5% and culture until the logarithmic growth phase.

[0061] 2.2 High-density fermentation The culture medium was optimized, with 38 g / L glucose as the carbon source, supplemented with 2% litchi extract as a partial carbon source. The nitrogen sources were 36 g / L peptone, 5 g / L yeast extract, and 17 g / L corn steep liquor powder. Inorganic salts were added at 0.6 g / L magnesium sulfate and 0.8 g / L potassium dihydrogen phosphate. The fermenter temperature was set at 29.5℃, the pH was controlled at 5.6, and the dissolved oxygen level was maintained above 40%. A fed-batch culture strategy was employed, achieving a viable cell count exceeding 1 billion CFU / mL.

[0062] 3. Inactivation 3.1 Centrifugal Concentration After fermentation, the yeast slurry is separated using a continuous flow centrifuge. The centrifugation speed is typically 6800 rpm. The supernatant is discarded, and the milky white paste-like yeast slurry is collected.

[0063] 3.2 Inactivation treatment Prepare the yeast mixture into a suspension, rapidly heat it to 85°C, and maintain the temperature for 30 minutes.

[0064] 3.3 Enzymatic hydrolysis and autolysis After inactivation, adjust the pH to 6.0, control the temperature at 55℃, and add 2% papain. Incubate for autolysis for 24 hours.

[0065] 3.4 Drying and Grinding The inactivated yeast solution was instantly dried in a spray drying tower (inlet air temperature 210℃, outlet air temperature 90℃) to obtain a powdered product for subsequent experiments. Example 1

[0066] This example provides a composition for enhancing immunity through gut microbiota, prepared from the following components in parts by weight: 10 parts of human milk oligosaccharides (2'-fucosylated lactose); 8 parts of arabinogalactan; 4 parts berry polyphenols; 4 parts grape polyphenols; 5 parts pomegranate seed extract; 6 parts of kale extract; Eight portions of inactivated *Saccharomyces boulardii* were used.

[0067] Take the above components according to the specified weight parts, mix them, and you will get the product. Example 2

[0068] This example provides a formulation for enhancing immunity through gut microbiota, prepared from the following components in parts by weight: Example 1: Composition, thickener, and flavoring agent; wherein the thickener is pectin-apple and maltodextrin, and the flavoring agent is citrus fruit powder, banana powder, and honey powder.

[0069] The composition, thickener, and flavoring agent are in a mass ratio of 90:3:7. Specifically, the thickener's mass ratio is pectin-apple:maltodextrin = 1:5; the flavoring agent's mass ratio is citrus fruit powder: banana powder: honey powder = 1:2:4.

[0070] Test Case Performance Test 1. In vitro fermentation test to verify the effect of prebiotic AKK on proliferation. Fecal samples were collected from 10 individuals aged 40 and above. An in vitro simulated fermentation experiment was conducted according to the table above. After fermentation, the changes in AKK bacteria content were detected by qPCR. Fresh feces were collected from healthy volunteers, added to sterile PBS, and vortexed to prepare a 10% fecal suspension. This suspension was then filtered through an 80-mesh sterile sieve to remove large food debris particles, resulting in a mixed suspension of fecal microbiota. This mixed suspension was inoculated into YCFA medium (Qingdao Haibo, HB9212) containing different prebiotics and anaerobically cultured at 37°C for 48 hours. The AKK bacteria content in the fermentation broth was then detected. The test results are as follows: Figure 1 As shown.

[0071] from Figure 1 As can be seen, 2'-fucosylated lactose, arabinogalactan, xylooligosaccharides, and fructooligosaccharides significantly increased the number of AKK bacteria compared to the initial value at 0 hours. 6 After fermentation, the number of AKK bacteria in these four groups can reach 10. 7 The above results show a more than tenfold increase in quantity, while the growth effect of other substrates is not significant. Therefore, these four carbon sources can be used as prebiotic substrates for the growth of AKK bacteria. The compound prebiotics of this invention have a significant tendency to promote the proliferation of AKK bacteria, and their combination can achieve targeted proliferation of AKK bacteria in complex gut microbiota.

[0072] 2. Animal experiments This study involved 50 male BABL / c mice aged 6-8 weeks, provided by Jicui Pharmaceutical Co., Ltd. (Jiangsu, China). Mice were housed in a controlled environment with a stable temperature (23°C-25°C) and a consistent 12-hour light / dark cycle. After one week of acclimatization, the mice were weighed and randomly divided into five groups of 10 mice each. Mice in the control group received daily intraperitoneal injections of 0.9% saline, while the remaining 40 mice received intraperitoneal injections of cyclophosphamide (80 mg / kg, once daily) for three days to establish the BABL / c model.

[0073] After the model was established, mice in the normal group (NC) and the model group (M) were administered 0.9% saline by gavage daily; the treatment group (LH) was administered levamisole (40 mg / kg) by gavage daily; the low-dose composition group (L) was administered 200 mg / kg of the composition (prepared according to the formulation of Example 1) by gavage daily; and the high-dose composition group (H) was administered 400 mg / kg of the composition (prepared according to the formulation of Example 1). Cyclophosphamide and levamisole were provided by Shanghai Maclean Biochemical Technology Co., Ltd. (Shanghai, China).

[0074] The experiment lasted for one week.

[0075] After the experiment, fecal samples were collected, and the mice were euthanized by intraperitoneal injection of sodium pentobarbital (100 mg / kg) after anesthesia. Blood samples were collected from the ocular tracts of the anesthetized mice, and they were placed supine on a paraffin plate. The abdomen was immediately exposed by incision, and samples from the liver, colon, spleen, and thymus were collected and weighed.

[0076] detection indicators Blood chemistry assessment: Detect the levels of immunoglobulin A (IgA), immunoglobulin G (IgG), interleukin-2 (IL-2), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in serum.

[0077] Colonic histological analysis: Colonic samples were fixed in 4% paraformaldehyde, followed by trimming, dehydration, paraffin embedding, and paraffin embedding. Hematoxylin-eosin staining was performed to assess mucosal damage, inflammatory cell infiltration, epithelial repair, epithelial integrity, and villous structure.

[0078] Gut microbiota analysis: 16S rDNA sequencing was performed on fecal samples, focusing on the V3-V4 region, using primers 341F (5′CCTACGGGNGGCWGCAG-3′) and 805R (5′GACTACHVGGGTATCTAATCC-3′). Sequencing was performed using the Illumina platform.

[0079] Quantitative real-time reverse transcription-PCR analysis: A systematic molecular biology approach was used to analyze the expression of genes IFN-γ, IL-1β, TNF-α, IL-2, IL-4, and IL-12 in spleen samples. First, approximately 0.1 g of the sample was ground in liquid nitrogen, and total RNA was extracted using a TransGen Biotech kit. After purification using the chloroform-isopropanol method, RNA purity was detected spectrophotometer (A260 / A280 ratio 1.8–2.0), and its integrity was verified by agarose gel electrophoresis. Then, 1 μg of RNA was used to synthesize cDNA with oligonucleotide DT random primers and reverse transcriptase. The qPCR reaction was performed using the SYBR green fluorescent dye method in a 20 μL system, including 2×SYBR premix, 10 μm forward and reverse primers, and the cDNA template. The reaction procedure included 40-45 cycles of pre-denaturation at 94℃ for 30 seconds, amplification at 94℃ for 5 seconds, and amplification at 50-60℃ for 15 seconds (fluorescence was collected during this stage). Specificity was then verified by melting curve analysis. Gapdh mRNA was used as an internal reference gene, and all experiments were repeated three times to ensure data reliability.

[0080] Statistical analysis: Data are expressed as mean ± standard error of the mean. Analysis of variance (ANOVA) was performed using GraphPadPrism 9.0 (San Diego, California, USA), and Spearman correlation coefficients were used to determine the associations between variables. Significance levels are expressed as follows: *p<0.05 (*), **p<0.01 (**), ***p<0.001 (***), ****p<0.0001 (****).

[0081] The test results are as follows: Effects on improving the nutritional status of the thymus and spleen in mice Test results are as follows Figure 2 As shown in the figure, the body weight of mice in group M decreased by approximately 10%-20% compared to the normal group during the experiment, indicating that cyclophosphamide treatment had a significant impact on overall growth. Organ index analysis showed that the thymus and spleen indices of the model group mice were significantly lower than those of the normal control group (NC group), suggesting that cyclophosphamide-induced immunosuppression may lead to relative atrophy or functional decline of key organs. Notably, treatment in groups L and H significantly increased the thymus and spleen indices of mice, indicating that this treatment can alleviate cyclophosphamide-induced thymus and spleen damage to some extent and has the potential to improve the nutritional status of the thymus and spleen. Therefore, this composition can alleviate cyclophosphamide-induced thymus and spleen damage to some extent and has the potential to improve the nutritional status of the thymus and spleen.

[0082] 2) Effects of inhibiting intestinal inflammation and intestinal barrier damage Test results are as follows Figure 3 As shown in the figure, cyclophosphamide-induced immunosuppression significantly damaged the intestinal immune barrier in mice, manifested by a significantly increased intestinal macrophage infiltration compared to the normal control group. This suggests that the body activated the inflammatory response and damaged the mucosal barrier structure under immunosuppressive conditions. LH supplementation alleviated inflammation to some extent, with its effect mainly limited to the basal side of the intestine, indicating that LH helps to partially inhibit immune activation. However, its anti-inflammatory effect was relatively limited compared to the L and H treatment groups. The combined intervention significantly reduced inflammatory cell infiltration, indicating that it has a stronger effect in regulating the intestinal microenvironment. It may exert its immunomodulatory and anti-inflammatory effects synergistically from multiple aspects by regulating the intestinal flora structure, inhibiting the overgrowth of harmful bacteria, enhancing mucosal barrier function, and inhibiting pro-inflammatory pathways such as NF-κB. In summary, the combined intervention has significant advantages in improving cyclophosphamide-induced immunosuppression-related intestinal inflammation and barrier function damage, showing good intervention potential and application prospects. This indicates that the combined intervention significantly reduced inflammatory cell infiltration in mice and partially inhibited immune activation, demonstrating significant advantages in improving cyclophosphamide-induced immunosuppression-related intestinal inflammation and barrier function damage, showing good intervention potential and application prospects.

[0083] 3) Effects on improving liver damage indicators Test results are as follows Figure 4As shown in the figure, the levels of AST and ALT, liver damage indicators, in the model group (M) mice were significantly higher than those in the NC group, indicating that cyclophosphamide caused some liver damage while suppressing immunity. Furthermore, the IL-6 level in the M group mice was significantly elevated, indicating a significant inflammatory response in the model mice. Simultaneously, the IgA and IgG levels in the M group mice were significantly lower than those in the NC group, directly demonstrating that cyclophosphamide successfully suppressed humoral immunity, leading to reduced antibody synthesis. Notably, the level of the anti-inflammatory factor IL-10 was also significantly elevated in the M group mice, which may be a compensatory anti-inflammatory response to the aforementioned inflammation (high IL-6) and damage, but insufficient to completely reverse the immunosuppressive state. However, compared with the model group (M), mice treated with different doses of the composition showed an improving or recovering trend in multiple indicators, suggesting that the intervention may have a protective effect: AST and ALT levels in the L and H groups were lower than those in the model group, indicating that the composition intervention may help alleviate cyclophosphamide-induced hepatocellular damage; IL-6 levels in the L and H groups also showed a decreasing trend, indicating that the composition intervention may help alleviate excessive inflammatory response; IgA and IgG levels in the L and H groups rebounded to varying degrees compared with the model group, indicating that the composition intervention may, to some extent, counteract the immunosuppressive effect of cyclophosphamide and promote antibody production. These results show that the composition intervention has a positive effect on protecting liver function, inhibiting excessive inflammation, and promoting humoral immune recovery, and has potential nutritional intervention and therapeutic value. This indicates that the composition intervention significantly reduced AST and ALT levels in mice, and IL-6 levels also showed a decreasing trend. IgA and IgG levels rebounded to varying degrees compared with the model group, indicating that this composition intervention may, to some extent, counteract the immunosuppressive effect of cyclophosphamide and promote antibody production, showing a positive effect on protecting liver function, inhibiting excessive inflammation, and promoting humoral immune recovery, and has potential nutritional intervention and therapeutic value.

[0084] 4) Effects of regulating immune factors Test results are as follows Figure 5As shown in the figure, compared with the NC group, the mRNA levels of pro-inflammatory factors IL-1β, TNF-α, and IL-2 in the model group (M) were significantly reduced. This directly indicates that cyclophosphamide inhibits the activity and function of macrophages (producing IL-1β and TNF-α) and T lymphocytes (producing IL-2), and not just reduces cell number (as reflected by organ indices). Furthermore, the IL-4 mRNA level in the model group (M) was also significantly reduced, indicating that the inhibition by cyclophosphamide is broad, affecting not only Th1-related immunity (IL-2) but also Th2-related immunity (IL-4). In addition, the change in IFN-γ in the M group showed a downward trend, as IFN-γ is mainly produced by activated T cells and NK cells, and its suppressed expression also reflects a decline in cellular immune function. Conversely, compared to the model group (M), the composition treatment groups (L and H) exhibited a clear dose-dependent immune activation effect. The mRNA expression levels of IL-1β, TNF-α, IL-2, and IL-4, which were significantly inhibited by cyclophosphamide, showed a dose-dependent recovery in the composition treatment groups. The expression levels in the high-dose group (H) typically recovered to near or even reached the levels of the normal group (NC), with statistically significant differences. These results indicate that the composition can counteract the inhibitory effect of cyclophosphamide, reactivate macrophages and T lymphocytes, and promote the synthesis and release of key immunomodulatory factors, thereby initiating the recovery of immune function at the molecular level. This demonstrates that the composition intervention resulted in a dose-dependent recovery of IL-1β, TNF-α, IL-2, and IL-4 expression levels in mice, indicating that the composition can counteract the inhibitory effect of cyclophosphamide, reactivate macrophages and T lymphocytes, and promote the synthesis and release of key immunomodulatory factors, thereby initiating the recovery of immune function at the molecular level.

[0085] 5) Improves gut microbiota diversity To investigate the effects of the combined intervention on the diversity and abundance of gut microbiota in immunosuppressed mice, we performed an α-diversity analysis. The results are as follows: Figure 6 As shown in the figure, the ace and Chao1 indices showed significant statistical differences between the high-dose combination group (H) and the M group, indicating that the H group treatment significantly altered the richness of the gut microbiota. The Shannon index, which indicates changes in gut microbiota diversity, was significantly improved in the H group treatment, suggesting a significant improvement in gut microbiota diversity in immunosuppressed mice.

[0086] To investigate the changes in gut microbiota structure caused by treatment in immunosuppressed mice, a β-diversity analysis was further performed. The results are as follows: Figure 7 , Figure 8As shown in the figure, both PCA and PCoA analyses revealed significant differences in the gut microbiota structure between immunosuppressed mice and the NC group mice. Treatment in the H group significantly altered the overall structure of the gut microbiota in immunosuppressed mice.

[0087] Furthermore, the top 30 phyla and genera were analyzed, and the test results are as follows: Figure 9 , Figure 10 As shown in the figure, the phylum-level relative abundance stacking plot and cluster heatmap reveal that the relative abundance of Firmicutes in the group supplemented with the composition was significantly lower than that in the NC and M groups. Furthermore, Dethiobacteria were significantly enriched in the intestines of M mice, and their relative abundance decreased significantly after LH and composition treatment in immunosuppressed mice. Particularly noteworthy is that the H group treatment significantly increased the relative abundance of Verrucomicrobiota in the intestines of immunosuppressed mice. These results indicate that composition treatment has a certain impact on the intestinal flora of immunosuppressed mice. Genus-level relative abundance analysis shows that the reason why composition treatment significantly increased the relative abundance of Verrucomicrobiota in immunosuppressed mice is due to the effect of composition treatment on the relative abundance of Akkermansia. The genus-level relative abundance stacking plot and cluster heatmap show that the relative abundance of Akkermansia in the composition-treated group was significantly higher than in other groups. In addition, the abundance of beneficial bacteria genera such as *Lachnospiraceae* NK4A136 group was significantly increased in the composition-treated group, while the abundance of some potential opportunistic pathogens such as *Alistipes* was relatively decreased.

[0088] To identify the characteristic phylum and genus of each treatment group, linear discriminant analysis (LEfSe) was performed. The test results are as follows: Figure 11 As shown. The analysis results indicate that *Odoribacter* is the marker genus of the LH group. *Bacteroidota* and *Verrucomicrobiota* are the marker phyla of the high-dose combination treatment group, while *Akkermansia* and *Streptococcus* are the marker genera of this group.

[0089] The results above show that the intervention of this composition can significantly improve the diversity of gut microbiota in immunosuppressed mice, improve the overall structure of gut microbiota, significantly increase the abundance of beneficial bacteria, relatively decrease the abundance of some potential conditional pathogens, and increase the relative abundance of Akkermansia.

[0090] To explore the correlation between changes in gut microbiota and various indicators, Spearman correlation analysis was performed on the top 30 bacterial genera in each group of samples and their corresponding detection indicators. The test results are as follows: Figure 12 As shown in the figure. Analysis results indicate that at least one genus of bacteria, including *Colidextribacter*, *Desulfovibrio*, *Oscillibacter*, *Bilophila*, *Roseburia*, *Incertae_Sedis*, *Dorea*, and *Helicobacter*, was significantly negatively correlated with serum IgA, IgG, IFN-γ gene expression, and anti-inflammatory genes IL-2, IL-10, and IL-12 expression levels; and at least one genus was significantly positively correlated with serum AST, ALT, TGF-β1, IL-1β, and TNF-α expression levels. At least one genus of bacteria, including *Alistipes*, *Tyzzerella*, *Anaerotignum*, *Muribaculum*, *Eisenbergiella*, and *Odoribacter*, was significantly negatively correlated with serum AST, ALT, TGF-β1, IL-1β, and TNF-α expression levels; and at least one genus was significantly positively correlated with serum IgA, IgG, and IFN-γ, IL-2, IL-12, and IL-10 expression levels.

[0091] In summary, this invention provides a composition that can significantly proliferate Akkermansia (AKK) bacteria in the intestinal flora, increasing their abundance and quantity, and enhancing the immune function of intestinal cells. Specifically, supplementing with the prebiotic components described in this invention can directionally promote the proliferation of AKK bacteria in the intestine, thereby increasing their quantity. Simultaneously, the combination of compound prebiotics, inactivated Saccharomyces boulardii, and plant extracts can synergistically enhance the body's immune function: this combination has the potential to improve the nutritional status of the thymus and spleen; in a cyclophosphamide-induced immunosuppressed mouse model, the composition intervention significantly reduced inflammatory cell infiltration, partially inhibited immune activation, and showed significant advantages in improving intestinal inflammation and barrier function damage, demonstrating good intervention potential and application prospects. Furthermore, the composition intervention also significantly reduced serum AST and ALT levels in mice, with a decreasing trend in IL-6 levels, while IgA and IgG levels showed varying degrees of recovery compared to the model group, suggesting that this composition can partially antagonize the immunosuppressive effect of cyclophosphamide, promote antibody production, and has positive effects in protecting liver function, inhibiting excessive inflammation, and promoting humoral immune recovery, possessing potential nutritional intervention and therapeutic value. Furthermore, after intervention with the composition, the expression levels of IL-1β, TNF-α, IL-2, and IL-4 in mice showed a dose-dependent recovery, indicating that the composition can counteract the immunosuppressive effect of cyclophosphamide, reactivate macrophages and T lymphocytes, and promote the synthesis and release of key immunomodulatory factors, thereby initiating the recovery of immune function at the molecular level. Regarding the gut microbiota, the composition intervention significantly improved the diversity of gut microbiota in immunosuppressed mice and improved the overall structure of the microbiota: the abundance of beneficial bacteria significantly increased, while the abundance of some potential opportunistic pathogens relatively decreased, with Akkermansia showing a relatively increased abundance. The combined effect of Akkermansia and yeast was particularly significant—AKK bacteria help maintain the integrity of the intestinal barrier and regulate the tone of the immune system, while β-glucan in the yeast cell wall is a potent immune activator; the two have complementary and synergistic effects in enhancing immunity. This composition can effectively enhance immunity. Identification of specific functional bacterial groups revealed that supplementation with the composition increased the abundance of Akkermansia and Streptococcus in the gut. These two genera can serve as marker genera for the influence of the composition on the gut microbiota.

[0092] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A composition for enhancing immunity through gut microbiota, characterized in that: The product includes the following ingredients: compound prebiotics, polyphenols, inactivated Saccharomyces boulardii, and plant extracts. The compound prebiotics include human milk oligosaccharides and arabinogalactan, the polyphenols include berry polyphenols and grape polyphenols, and the plant extracts include pomegranate seed extract and kale extract.

2. The composition for enhancing immunity through gut microbiota according to claim 1, characterized in that: The human milk oligosaccharide includes 2'-fucosylated lactose.

3. The composition for enhancing immunity through gut microbiota according to claim 1, characterized in that: The mass fraction ratio of the compound prebiotic: polyphenolic substance: plant extract: inactivated yeast is 14~21:4~8:9~16:5~8.

4. The composition for enhancing immunity through gut microbiota according to claim 1, characterized in that: The composition comprises the following raw materials in parts by weight: 8-12 parts of human milk oligosaccharides; 6-9 parts of arabinogalactan; 2-4 parts of berry polyphenols; 2-4 parts grape polyphenols; 4-7 parts pomegranate seed extract; 5-9 parts of kale extract; Inactivate 5-8 portions of Saccharomyces boulardii.

5. A method for preparing the composition according to any one of claims 1 to 4, characterized in that: Includes the following steps: The raw materials are mixed to obtain the final product.

6. The use of the composition according to any one of claims 1 to 4 in the preparation of a product having immunomodulatory function.

7. The application according to claim 6, characterized in that: The product includes at least one of health food, food composition or pharmaceutical; and / or, the immunomodulatory function includes intestinal immunomodulatory function.

8. The use of the composition according to any one of claims 1 to 4 in the preparation of products that increase intestinal flora.

9. A dietary supplement or food, characterized in that: The dietary supplement or food includes the composition as described in any one of claims 1 to 4.

10. The dietary supplement or food according to claim 9, characterized in that: The dietary supplement or food also includes thickeners and flavorings. The thickeners include two of the following: pectin-apple, maltodextrin, and aloe vera gel. The flavorings are selected from at least three of the following: citrus fruit powder, lychee fermentation powder, strawberry powder, banana powder, lemon fruit powder, pineapple powder, strawberry powder, papaya powder, and honey powder.

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