Prebiotics for constructing a humanized microbiota animal (HMA) model and a preparation method thereof
By using a prebiotic combination of xylooligosaccharides, 2'-fucosylated lactose, resistant starch, and inulin in animal models, combined with fecal microbiota transplantation, the long-term stability problem of humanized gut microbiota in animal models was solved, achieving a high-fidelity and stable gut microecological environment, and improving the reproducibility and reliability of the model.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANGHAI RES CENT FOR MODEL ORGANISMS
- Filing Date
- 2026-03-23
- Publication Date
- 2026-06-05
AI Technical Summary
Existing technologies struggle to maintain stable humanized gut microbiota in experimental animal models over the long term, leading to reduced model reproducibility and reliability. Conventional prebiotics cannot effectively support the colonization and persistence of core symbiotic bacteria in the human gut.
A specific combination of prebiotics, including xylooligosaccharides, 2'-fucosylated lactose, resistant starch, and inulin, is used as a feed additive in conjunction with fecal microbiota transplantation from healthy adults to promote the colonization and persistence of human microbiota in animal models.
It significantly improved the colonization fidelity and persistence of human microbiota in animal models, maintained the stability of the gut microbiota ecosystem and the similarity of the metabolic profile, and enhanced the reliability and reproducibility of the model.
Smart Images
Figure CN122139860A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of animal model construction technology, and relates to the field of feed additive technology, and particularly to a prebiotic for constructing a humanized gut microbiota (HMA) animal model and its preparation method. Background Technology
[0002] Humanized gut microbiota (HMA) animal models are key tools for studying microbiota-host interactions, mechanisms of intestinal diseases, validating the efficacy of fecal microbiota transplantation (FMT), probiotics / prebiotics, and drugs, and exploring metabolic, immune, and neurological diseases (such as obesity, IBD, autism, and depression). Current research primarily uses SPF or germ-free mice to construct HMA animal models. Experimental miniature pigs (such as Bama miniature pigs and Göttingen pigs) have become important model animals in biomedical research due to their high similarity to humans in anatomical structure, physiological metabolism, and genetic background. Using miniature pigs to construct HMA models not only allows for a gut microbiota composition closer to humans but also enables precise control of donor microbiota types (healthy / disease-prone individuals), allowing for long-term observation of the effects of microbiota colonization, metabolism, and immunity, making them more valuable for translational studies than mouse models or conventional animal models. However, the uncontrollability of the microbial colonization environment remains a key factor affecting experimental reproducibility and the reliability of results. Establishing a humanized human microbiota transplantation (HMA) pig model and standardizing its microbial composition to significantly enhance its application value in disease mechanism research, drug evaluation, and translational medicine remains a challenge. A key challenge in constructing this model is that the transplanted human microbiota is difficult to maintain stably in the animal's gut for long periods, easily undergoing structural drift or being reverse-colonized by the animal's own microbiota. This leads to a decrease in the model's "fidelity"—that is, the similarity between the recipient's microbiota structure and the donor's experimental structure—and thus reduces reproducibility and reliability.
[0003] Currently, methods for maintaining stable human gut microbiota in pig models mainly focus on optimizing procedures during the transplantation phase (such as multiple transplantations and bacterial culture preparation) and pretreatment of recipient animals (such as antibiotic treatment and germ-free animals). However, these methods are costly, complex, and fail to fundamentally provide long-term, specific nutritional support for the transplanted human microbiota. While conventional porcine prebiotics (such as common oligosaccharides) can promote gut health, their effects are broad-spectrum and lack the selective growth-promoting ability for core human gut commensal bacteria, thus failing to effectively support the specific maintenance of human microbiota structure. Therefore, developing a targeted nutritional intervention strategy to enhance the colonization competitiveness and persistence of human gut microbiota in porcine models is crucial for improving the technical value of this model. Summary of the Invention
[0004] In order to address the problems existing in the prior art, the primary objective of this invention is to overcome the shortcomings of the prior art and provide a prebiotic that can significantly improve the colonization persistence and structural fidelity of human gut microbiota in HMA animal models.
[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:
[0006] The first aspect of this invention discloses a prebiotic for improving the colonization effect of human microbiota in the animal intestine, the prebiotic being composed of the following components in parts by weight: 15-30 parts xylooligosaccharide, 5-15 parts 2'-fucosylated lactose, 20-35 parts resistant starch, 10-20 parts inulin, and 8-15 parts carrier.
[0007] Preferably, the purity of the xylooligosaccharide is ≥95%.
[0008] Preferably, the resistant starch is RS3 type resistant starch.
[0009] Preferably, the carrier is maltodextrin.
[0010] Human milk oligosaccharides are unique components of human breast milk. 2'-Fucose-based oligosaccharide is one of the most abundant human milk oligosaccharides, and current research has found that it can effectively inhibit pathogen infection. This invention utilizes it as a substrate for gut microbiota to effectively promote the early colonization of Bifidobacteria. This invention also adds resistant starch and inulin as fermentation substrates, which can be utilized by gut microbiota in the colon to generate short-chain fatty acids such as butyrate. Butyrate is a major energy source for colonic epithelial cells, can inhibit inflammation, reduce oxidative stress, and create a healthy gut environment.
[0011] In a preferred embodiment of the present invention, the prebiotic is composed of the following components in parts by weight: 15-20 parts xylooligosaccharide, 10-15 parts 2'-fucosylated lactose, 20-30 parts resistant starch, 15-20 parts inulin, and 8-10 parts carrier.
[0012] The second aspect of this invention discloses a method for preparing the above-mentioned prebiotics, comprising the following steps: weighing each component according to the stated weight parts, placing them in a mixer, and physically mixing them uniformly under room temperature and dry conditions.
[0013] The third aspect of this invention discloses the application of the above-mentioned prebiotics in the preparation of products for improving the colonization effect of human microbiota in animal models.
[0014] The prebiotics are used as feed additives to colonize human microbiota in animal models, and the proportion of prebiotics added to the feed is 1%-5%.
[0015] The human gut microbiota animal model is a pig model constructed by transplanting fecal microbiota from healthy adults.
[0016] The fourth aspect of this invention discloses a method for constructing a humanized intestinal flora animal model, comprising feeding feed and the aforementioned prebiotics, wherein the proportion of the prebiotics added is 1%-5% of the total weight of the feed.
[0017] Compared with the prior art, the beneficial effects of this invention are as follows:
[0018] 1. Through the synergistic effect of specific prebiotic combinations, the colonization fidelity and persistence of human microbiota in animal models are significantly improved.
[0019] 2. The successful reconstruction of the gut microbiota ecosystem was demonstrated through multidimensional functional evidence (similarities in microbial community structure and metabolic profile). Attached Figure Description
[0020] Figure 1 PCA diagram analysis of gut microbiota in animal experiments;
[0021] Figure 2 PCA plot analysis of metabolites in animal experiments. Detailed Implementation
[0022] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but this does not limit the present invention to the scope of the described embodiments. Process parameters not specified in the embodiments of this application can be performed according to conventional methods, and all raw materials used can be obtained through commercial channels.
[0023] Example 1
[0024] This embodiment provides a prebiotic specifically for humanized intestinal flora animal models, which is made from the following components in parts by weight: 20 parts xylooligosaccharide, 10 parts 2'-fucosylated lactose, 30 parts RS3 resistant starch, 15 parts inulin, and 10 parts maltodextrin. All the above raw materials are added to a mixer and mixed at room temperature for 30 minutes until the color is uniform, thus obtaining feed additive premix A (denoted as Additive A).
[0025] Example 2
[0026] This embodiment provides a prebiotic specifically for humanized intestinal flora animal models, which is made from the following components: 15 parts xylooligosaccharide, 15 parts 2'-fucosylated lactose, 20 parts resistant starch, 20 parts inulin, and 8 parts maltodextrin. Mixed with the method of Example 1, feed additive premix B (denoted as Additive B) is obtained.
[0027] Comparative Example 1
[0028] This example provides a prebiotic specifically for a humanized gut microbiota animal model. Its formulation and preparation method are basically the same as those in Example 1, except that 2'-fucosylated lactose is not added.
[0029] Comparative Example 2
[0030] This example provides a prebiotic specifically for a humanized intestinal flora animal model. Its formulation and preparation method are basically the same as those in Example 1, except that resistant starch is not added.
[0031] Comparative Example 3
[0032] This example provides a conventional prebiotic for pigs, namely ordinary xylooligosaccharide, in a dosage of 75 parts.
[0033] To further illustrate the beneficial technical effects of the prebiotics in the various embodiments of this application, animal experiments were conducted on the samples obtained from Examples 1-2 and Comparative Examples 1-3 to verify the effect of the additives of the present invention on improving the colonization fidelity and persistence of fecal microbiota in healthy adults in piglets.
[0034] Animal Experiments 1. Laboratory Animals and Grouping
[0035] Twenty-four healthy 3-week-old SPF Bama miniature pigs were randomly divided into 6 groups of 4 pigs each. The specific procedures are as follows:
[0036] Control group: fed with a basal diet.
[0037] Experimental Group A: Feeding with basal diet + 2% (w / w) of additive A prepared in Example 1.
[0038] Experimental Group B: Feeded with basal diet + 2% (w / w) of additive B prepared in Example 2.
[0039] Experimental group C: fed with basal diet + 2% (w / w) additive prepared in Comparative Example 1.
[0040] Experimental group D: fed with basal diet + 2% (w / w) additive prepared in Comparative Example 2.
[0041] Experimental group E: fed with basal diet + 2% (w / w) additive prepared in Comparative Example 3.
[0042] 2. Feeding and transplantation
[0043] All groups were fed the appropriate feed from the start of the experiment (7 days before transplantation). On the 7th day, all piglets were transplanted by gavage with a suspension of fecal microbiota from healthy adults (1-3 ml each time, every other day, for one week).
[0044] 3. Clinical Tolerability Analysis
[0045] During the experiment, the weight and diarrhea status of miniature pigs were recorded, and the average daily weight gain and diarrhea incidence rate were calculated.
[0046] Average daily weight gain (g) = Average weight gain per piglet (kg) / Age in days × 1000.
[0047] Diarrhea incidence rate = (number of piglets with diarrhea ÷ total number of piglets) × 100.
[0048] 4. Sample collection and analysis:
[0049] Collection time points: Fecal samples were collected from piglets on day 1, day 7, and day 35 after transplantation.
[0050] Analytical methods: 16S rRNA gene high-throughput sequencing was used to analyze the microbial community composition, and the results were compared with donor fecal microbiota. Non-targeted metabolomics (LC-MS) was used to analyze the metabolite profile in feces, and the results were compared with donor fecal microbiota. Targeted metabolomics (GC-MS) was used to analyze the short-chain fatty acid profile in feces, and the results were compared with donor fecal microbiota.
[0051] The core indicator is the structural similarity of the microbial community and metabolites: Principal coordinate analysis (PCA) was used to calculate the similarity between the experimental group and the control group and the donor sample at each time point. After dimensionality reduction analysis, the samples have relative coordinate points on principal components p1 and p2. The distance between each coordinate point represents the degree of clustering and dispersion among the samples. The closer the distance, the higher the similarity between the samples, and the farther the distance, the greater the difference between the samples.
[0052] 5. Results Analysis
[0053] (1) Good clinical tolerance: During the trial, the diarrhea rate of experimental group A was 18.05% and that of experimental group B was 19.44%, which was significantly lower than that of the control group of 29.16%; there was no significant difference in the average daily weight gain of each group compared with the control group (P>0.05), indicating that the additive did not affect normal growth.
[0054] Table 1 Clinical Data
[0055] (2) Higher similarity in bacterial community structure: such as Figure 1 As shown, 16S rRNA gene sequencing analysis on days 1, 7, and 35 post-transplantation revealed that on days 7 and 35, compared with the control group and experimental groups C, D, and E, the microbial community structure of experimental groups A and B was more similar to that of the human microbial community (H_M group).
[0056] (3) Increased levels of propionic acid and butyric acid: Targeted metabolomics quantitative detection showed that the concentrations of butyric acid and propionic acid in the feces of experimental group A were significantly higher than those in the control group (P<0.01, Table 2).
[0057] Table 2. Concentration of short-chain fatty acids in feces (μg / g)
[0058] Grouping Acetic acid propionic acid butyric acid H_M group 3667.78±501.79 1242.71±377.40 1412.85±67.74 control group 1941.78±409.60 565.57±54.15 293.61±55.68 Experimental Group A 2562.06±521.67 973.17±18.96 493.16±150.67 Experimental Group B 2432.25±205.78 712.00±177.80 368.86±41.02 Experimental group C 2736.59±373.99 600.43±48.41 370.951±38.80 Experimental group D 2689.89±453.70 620.61±42.51 364.51±41.23 Experimental group E 2540.28±368.95 590.57±39.04 356.12±32.51 P-value (A vs. control) 0.11 0.01 0.04 P-value (B vs. control) 0.07 0.09 0.07 P-value (C vs. control) 0.02 0.04 0.07 P-value (D vs. control) 0.06 0.08 0.10 P-value (E vs. control) 0.37 0.16 0.48
[0059] (4) Higher structural similarity of metabolites: such as Figure 2 On day 35 post-transplantation, non-target metabolomics analysis revealed that, compared with the control group and the other experimental groups, the microbial community structure of experimental groups A and B was more similar to the metabolite profile structure of the human microbial community (H_M group).
[0060] 6. Discussion
[0061] The results showed that after gavage inoculation with fecal microbiota suspension from healthy adults, the rate of intestinal stress such as diarrhea in piglets was significantly lower than that in the control group, indicating that the additive helps to smoothly implant exogenous microbiota.
[0062] 16S rRNA gene sequencing results showed that, compared with the control group and the other experimental groups, the fecal microbiota of experimental groups A and B piglets were more similar to the microbiota of the donor adults at days 7 and 35, and remained relatively stable during the 35-day experimental period, indicating that the additive of the present invention can effectively maintain the high fidelity of human microbiota.
[0063] The fecal non-target metabolomics analysis of experimental groups A and B showed that the composition of fecal metabolites after transplantation was closer to the level of the donor adult microbiota, while the control group showed no significant effect. This indicates that the additive can promote the initial colonization of the human microbiota. Experimental group A (additive A), using the optimized ratio, significantly increased the concentrations of propionic acid and butyric acid, showing the best effect.
[0064] The above embodiments demonstrate that the prebiotics provided by this invention, when added to piglet feed in a specific manner and combined with fecal microbiota transplantation from healthy adults, can create and maintain an intestinal microenvironment conducive to the specific colonization and survival of human-derived microbiota. This successfully constructs a pig model of human-derived microbiota with high fidelity and stability, providing an effective solution to related technical challenges. This animal experiment is a preliminary verification; the results obtained can be used as a reference and provide a theoretical basis for subsequent implementation.
[0065] It is understood that the prebiotics of the present invention may also have beneficial effects when used in human microbiota transplantation models from other sources (such as infants).
[0066] It is foreseeable that the prebiotics of the present invention may also have similar beneficial effects when constructing human microbiota transplantation models in other mammals (such as mice and dogs).
[0067] This invention is not limited to the above-described embodiments. Any changes in shape or structure are within the scope of protection of this invention. The scope of protection of this invention is defined by the appended claims. Those skilled in the art can make various changes, modifications, substitutions, combinations, and simplifications to these embodiments without departing from the principles and essence of this invention. All such changes and simplifications should be considered equivalent substitutions and fall within the scope of protection of this invention.
Claims
1. A prebiotic for improving the colonization effect of human gut microbiota in animals, characterized in that, The prebiotic is composed of the following components in parts by weight: 15-30 parts xylooligosaccharide, 5-15 parts 2'-fucosylated lactose, 20-35 parts resistant starch, 10-20 parts inulin, and 8-15 parts carrier.
2. The prebiotic according to claim 1, characterized in that, The purity of the xylooligosaccharide is ≥95%.
3. The prebiotic according to claim 1, characterized in that, The resistant starch is RS3 type resistant starch.
4. The prebiotic according to claim 1, characterized in that, The carrier is maltodextrin.
5. The prebiotic according to claim 1, characterized in that, The prebiotic is composed of the following components in parts by weight: 15-20 parts xylooligosaccharide, 10-15 parts 2'-fucosylated lactose, 20-30 parts resistant starch, 15-20 parts inulin, and 8-10 parts carrier.
6. A method for preparing the prebiotic as described in any one of claims 1-5, characterized in that, Includes the following steps: Weigh each component according to the stated weight proportions, place them in a mixer, and physically mix them evenly under normal temperature and dry conditions to obtain the final product.
7. The use of the prebiotic as described in any one of claims 1-5 in the preparation of a prebiotic for improving the colonization effect of human microbiota in animal models.
8. The application according to claim 7, characterized in that, The prebiotic is used as a feed additive to colonize human microbiota in animal models, and the proportion of the prebiotic added to the feed is 1%-5%.
9. The application according to claim 7, characterized in that, The human gut microbiota animal model is a pig model constructed by transplanting fecal microbiota from healthy adults.
10. A method for constructing a humanized animal model of gut microbiota, characterized in that, It includes feed and prebiotics as described in any one of claims 1-5, wherein the proportion of the prebiotics added is 1%-5% of the total weight of the feed.