Stachyose composition and application thereof in treating diarrhea

Abstract

The invention discloses a stachyose composition and application thereof in treating diarrhea, and belongs to the technical field of biological medicine. The technical problem to be solved is that single prebiotics have limited effects in the aspects of promoting the generation of short-chain fatty acids (SCFAs), proliferating intestinal beneficial bacteria and repairing intestinal barrier functions. According to the technical scheme, the compound prebiotic composition is characterized in that stachyose (SS), xylooligosaccharide (XOS) and galactomannan (PHGG) are taken as core components, and the mass ratio of the stachyose (SS), the xylooligosaccharide (XOS) and the galactomannan (PHGG) is optimized, so that synergistic regulation and control on intestinal microecology are realized.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a stachyose composition and its application in the treatment of diarrhea. Background Technology

[0002] For understanding the technical content of this invention: Prebiotics refer to oligosaccharides, dietary fiber, or bioactive molecules that can be fermented by gut microbes in the colon and converted into SCFAs. Different types of prebiotics, such as oligosaccharides and dietary fiber, can affect the composition of the gut microbiota and the production of metabolites through different mechanisms.

[0003] SCFAs (such as acetic acid, propionic acid, and butyric acid) not only provide energy for intestinal cells but also regulate intestinal motility and inhibit inflammatory responses. Beneficial intestinal bacteria (such as Bifidobacteria, Akkermansia, and Lactobacillus) inhibit pathogenic bacteria and maintain intestinal homeostasis by competing for nutrients and secreting antimicrobial substances. The integrity of the intestinal barrier function depends on the normal expression of tight junction proteins (such as ZO-1, Occludin, and Claudin) and the mucus layer (such as MUC2), and their damage is closely related to intestinal diseases such as diarrhea. Among them, ZO-1, as a tight junction "bridge protein," can repair the intestinal barrier and reduce the diarrhea index when its expression is increased; Occludin and Claudin reduce intestinal permeability by enhancing the sealing of intercellular connections, and related studies have shown that their upregulation can significantly reduce the risk of diarrhea; MUC2, as a core component of the mucus layer, can strengthen the physical barrier when its expression is increased, and its upregulation can improve diarrhea symptoms.

[0004] Relevant patent documents retrieved: The document, published in China (CN104642870A) on May 27, 2015, discloses a composition for regulating the human gut microbiota, comprising stachyose and galactooligosaccharides as active ingredients, and may also contain mannose and / or xylooligosaccharides. It regulates the gut microbiota by promoting the growth of probiotics and inhibiting pathogenic bacteria.

[0005] Relevant non-patent literature retrieved: The journal is titled "The arabinofolia saccharides and polysaccharides of xylan and mannan in the health of monogastric animals", Volume 9, Publication Date: 2020. This article discloses that oligosaccharide forms of arabinoxylan (AX) and mannan (such as xylooligosaccharide XOS and mannan oligosaccharide MOS) have prebiotic functions and can affect gut microbiota, immune regulation, and intestinal barrier function.

[0006] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: Current research focuses on the regulatory effects of oligosaccharides (such as galactooligosaccharides and xylooligosaccharides) on gut microbiota abundance, but lacks verification of the direct causal relationship between "upregulation of intestinal barrier protein (ZO-1, Occludin, Claudin, MUC2) expression" and "improvement of diarrhea symptoms" in animal models or in vitro experiments. At the same time, the technical solutions do not disclose key dosages, process parameters, and experimental condition control standards, resulting in insufficient reproducibility. Summary of the Invention

[0007] The purpose of this invention is to provide: A stachyose composition and its application in the treatment of diarrhea, and related technologies, to address the technical problems of the limited effectiveness of single prebiotics in promoting the production of short-chain fatty acids (SCFAs), increasing beneficial intestinal bacteria, and repairing intestinal barrier function, or a combination thereof.

[0008] Terminology Explanation: Unless otherwise defined, all technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this subject matter pertains. Unless otherwise stated, all patents, patent inventions, and disclosures cited throughout this document are incorporated herein by reference in their entirety. Where multiple definitions exist for terms herein, the definitions provided in this chapter shall prevail.

[0009] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0010] The definition of the standard chemical term can be found in the reference "Prebiotic Science and Technology", China Light Industry Press, edited by Jiang Zhengqiang.

[0011] Unless otherwise stated, conventional methods within the scope of the art, such as qPCR for detecting gene expression levels and Western blot for detecting protein expression levels, shall be used.

[0012] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0013] Stachyose (SS): A naturally occurring oligosaccharide that, as a prebiotic, can be fermented and utilized by beneficial gut bacteria, promoting their proliferation and producing metabolites.

[0014] Xylooligosaccharides (XOS) are oligosaccharides composed of 2-7 xylose molecules linked by glycosidic bonds. They have good prebiotic properties and can selectively promote the growth of beneficial bacteria in the gut.

[0015] Galactomannan (PHGG): a water-soluble dietary fiber that can be fermented by gut microbes as a prebiotic and has a positive effect on gut health.

[0016] Short-chain fatty acids (SCFAs): Monocarboxylic acids containing 2-6 carbon atoms, are representative substances produced by bacterial fermentation in the gut, including acetic acid, propionic acid, butyric acid, etc. They provide a source of substances for cell metabolism and play an important role in maintaining gut health.

[0017] Intestinal barrier function: refers to the function of the intestinal mucosal epithelium in preventing harmful substances from entering the body. Its integrity depends on the normal expression and function of tight junction proteins (such as ZO-1, Occludin, Claudin, etc.) and the mucus layer (such as MUC2).

[0018] In a first aspect, the present invention provides: a stachyose composition comprising stachyose, xylooligosaccharide, and galactomannan in a mass ratio of 1-6:1-4:1-4.

[0019] The technical feature quality ratios are selected from: 4:4:2, 4:2:4, 4:3:3, 1:1:1, 2:1:1, and 6:2:2.

[0020] The preferred ratio of technical feature quality is 4:2-4:2-4. The preferred ratio of technical feature quality is 4:4:2, 4:2:4, or 4:3:3.

[0021] The preferred ratio of technical feature quality is 4:4:2.

[0022] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: The first preferred option uses a mass ratio of 4:4:2, 4:2:4, 4:3:3, 1:1:1, 2:1:1, and 6:2:2. This approach addresses the limited effectiveness of single prebiotics in promoting short-chain fatty acid (SCFA) production, increasing beneficial gut bacteria, and repairing the intestinal barrier, aiming to further achieve fundamental synergistic regulation of the gut microbiota.

[0023] The second preferred approach involves mass ratios of 4:4:2, 4:2:4, and 4:3:3. This approach further enhances the targetedness and effectiveness of synergistic regulation, addressing the shortcomings of single prebiotics in the generation of specific SCFAs and the proliferation of key bacterial communities.

[0024] The third preferred option is a mass ratio of 4:4:2. This technical solution further achieves optimal synergistic effects across multiple dimensions, addressing the balance issue of compound prebiotics in comprehensively improving the gut microbiota.

[0025] Secondly, the present invention provides the application of the above-mentioned stachyose composition in the preparation of functional foods or pharmaceuticals for promoting the production of short-chain fatty acids in the intestine.

[0026] Specifically, the short-chain fatty acids include acetic acid, propionic acid, and / or butyric acid.

[0027] Thirdly, the present invention provides the use of the above-mentioned stachyose composition in the preparation of functional foods or pharmaceuticals for selectively proliferating beneficial intestinal bacteria and inhibiting potentially pathogenic bacteria.

[0028] Specifically, the beneficial bacteria are Bifidobacterium, Lactobacillus, and / or Akkermania; the potential pathogens are Escherichia coli and Shigella.

[0029] Fourthly, the present invention provides the use of the above-described stachyose composition in the preparation of products for treating and / or preventing diarrhea.

[0030] The products mentioned include pharmaceuticals.

[0031] Fifthly, the present invention provides a pharmaceutical composition comprising the above-described stachyose composition.

[0032] The pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0033] More specifically, the pharmaceutically acceptable excipients include any one or more of the following: binders, fillers, disintegrants, lubricants, preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, or osmotic pressure regulators.

[0034] The dosage form of the pharmaceutical composition includes: liquid dosage form, gas dosage form, solid dosage form or semi-solid dosage form.

[0035] The liquid dosage forms include: solvent-based, aromatic aqueous solutions, tinctures, elixirs, colloidal solutions, pastes, suspensions, or emulsifiers.

[0036] The gaseous dosage form includes: aerosol or spray.

[0037] The solid dosage forms include: powders, pills, tablets, or films.

[0038] The semi-solid dosage forms include ointments, suppositories, or pastes.

[0039] The administration methods of the pharmaceutical composition include oral, injection, implantation, external application, spray, inhalation, or combinations thereof.

[0040] The injection administration methods include, but are not limited to: intraocular injection, intravenous injection, intramuscular injection, subcutaneous injection, intrathecal injection, intra-articular injection, or intratumoral injection.

[0041] The external application methods include, but are not limited to: mucosal administration or transdermal / local administration.

[0042] The mucosal administration includes, but is not limited to: nasal administration, oral buccal / sublingual administration, and ocular administration.

[0043] The transdermal / local drug delivery includes, but is not limited to, transdermal patch drug delivery.

[0044] The inhalation administration method includes, but is not limited to, nebulized inhalation or metered-dose inhaler inhalation.

[0045] Examples 1-6 and Detection Examples 1-6 of this invention at least support the protection of a stachyose composition, wherein the stachyose composition comprises stachyose, xylooligosaccharide, and galactomannan in a mass ratio of 1-6:1-4:1-4.

[0046] Regarding the above: The technical feature "combination of stachyose, xylooligosaccharides, and galactomannan" is summarized from the specific ratios (4:4:2, 4:3:3, 4:2:4, etc.) in the foregoing explanation and examples, and is based on the common feature of "synergistic effect of the three prebiotics." Those skilled in the art can reasonably infer that the subordinate concepts of this combination (such as stachyose, xylooligosaccharides, or galactomannan from different sources) and substantially equivalent technical means (such as the substitution of some components with structurally similar oligosaccharides) all fall within the scope of protection of this invention.

[0047] The technical feature "mass ratio of stachyose, xylooligosaccharide, and galactomannan 1-6:1-4:1-4" is derived from the specific ratios in the examples (4:4:2, 4:3:3, 4:2:4, 1:1:1, etc.) and summarized by the common feature "the ratio range covers the synergistic effect interval". For example, other ratios adjusted within this range (such as 3:3:2, 5:2:3, etc.) are still within the protection scope of this invention.

[0048] The technical feature of this invention, "use in the preparation of medicaments for treating and / or preventing diarrhea," and "pharmaceutical composition for protecting stachyose compositions," are supported by experiments in the examples of SCFAs generation (promoting intestinal motility), intestinal flora regulation (proliferation of beneficial bacteria), and intestinal barrier repair (upregulation of tight junction proteins). For example, the 4:4:2 ratio in the examples significantly increased acetic acid production (promoting defecation) and inhibited pathogenic bacteria (improving diarrhea), verifying its efficacy in treating diarrhea. Therefore, this application and equivalent applications (such as other products related to improving intestinal function) are within the scope of protection.

[0049] The present invention has at least the following beneficial effects: Compared to single prebiotics, the compositions of this invention exhibit superior performance in the production of total acid, acetic acid, propionic acid, and butyric acid. For example, the total acid production of the 4:4:2 ratio is comparable to that of the positive control inulin (approximately 6 μg / mL), significantly higher than that of the single prebiotic group (e.g., approximately 3 μg / mL when PHGG is used alone); the butyric acid production of the 4:2:4 ratio reaches 2.3 μg / mL, significantly higher than that of the single SS group (0.14 μg / mL), effectively enhancing intestinal metabolic function.

[0050] The composition can selectively proliferate beneficial bacteria (such as Akkermania, Bifidobacterium, and Lactobacillus) and inhibit pathogenic bacteria. For example, the 4:4:2 ratio resulted in Akkermania abundance being twice that of the control group, Bifidobacterium accounting for more than 70%, while reducing Escherichia Shigella (a potential pathogenic bacterium) abundance to below 20%, which is significantly better than the regulatory effect of a single prebiotic.

[0051] The fermentation broth of the composition significantly upregulated the expression of tight junction proteins (ZO-1, Occludin, Claudin-1) and mucin (MUC2). The expression level of MUC2 protein in the high-concentration group (100-fold dilution) was twice that of the control group, and the expression level of ZO-1 protein was nearly doubled, which can effectively repair the intestinal barrier and alleviate mucosal damage caused by diarrhea.

[0052] High safety: At a concentration of 100-200 times dilution, the survival rate of HT-29 cells is over 95%, with no cytotoxicity, providing a safety guarantee for clinical application. Attached Figure Description

[0053] Figure 1 To determine the concentration of SCFAs in the prebiotic combination, A represents the total acid concentration, B the acetic acid concentration, C the propionic acid concentration, and D the butyric acid concentration. In the figure, a, b, c, d, e, and f are considered to have no significant difference if they share a single letter, and significant difference if they have different letters.

[0054] Figure 2 The concentrations of SCFAs produced by a single prebiotic are shown in the figure. A represents the total acid concentration, B the acetic acid concentration, C the propionic acid concentration, and D the butyric acid concentration. In the figure, a, b, c, d, e, f, g, and h are considered to have no significant difference if they share a single letter, and significant difference if they have different letters.

[0055] Figure 3 To simulate the proliferation of intestinal flora after colonic fermentation with a combination of prebiotics, A represents total bacteria, B represents Akkermansia, C represents Bifidobacterium, and D represents Lactobacillus. In the figure, a, b, c, d, and e with the same letter are considered to have no significant difference, while those with different letters are considered to have significant difference.

[0056] Figure 4 To simulate the proliferation of gut microbiota after colonic fermentation with a single prebiotic in vitro, A represents total bacteria, B represents Akkermansia, C represents Bifidobacterium, and D represents Lactobacillus. In the figure, a, b, c, d, and e are considered to have no significant difference if they share a single letter, and significant difference if they have different letters.

[0057] Figure 5 The results of 16S rRNA sequencing at the gate level for the combination of prebiotics.

[0058] Figure 6 The results of 16S rRNA sequencing for the prebiotic combination are at the genus level.

[0059] Figure 7 To illustrate the effect of different concentrations of synbiotic fermentation dilutions on cell viability, the figures a, b, c, and d indicate no significant difference if they share a single letter, and significant difference if they have different letters.

[0060] Figure 8 The effect of different concentrations of synbiotic fermentation dilutions on MuC2 is shown. ns represents no significant difference compared to the control group, and ** represents p<0.01 compared to the control group.

[0061] Figure 9 The effect of different concentrations of synbiotic fermentation dilution on ZO-1 is shown in the figure. ns represents no significant difference compared with the control group, * represents p < 0.05 compared with the control group, and ** represents p < 0.01 compared with the control group.

[0062] Figure 10 The effect of different concentrations of synbiotic fermentation dilutions on Occludin is shown in the figure. ns represents no significant difference compared with the control group, * represents p < 0.05 compared with the control group, and ** represents p < 0.01 compared with the control group.

[0063] Figure 11 The effect of different concentrations of synbiotic fermentation dilutions on Claudin is shown in Figure 1. * indicates comparison with the control group, p<0.05, and ** indicates comparison with the control group, p<0.01.

[0064] Figure 12 The results are from a mouse colon strength test.

[0065] Figure 13 The results show the water content of mouse feces. Detailed Implementation

[0066] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0067] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0068] Examples 1-6 The formula is shown in Table 1 below.

[0069] Table 1

[0070] Preparation method: Weigh the above-mentioned stachyose, xylooligosaccharide and galactomannan according to Table 1, stir them thoroughly, and then package them into portions and store them at 4℃ for later use.

[0071] Comparative Example 1: Stachyose monosaccharide Formula composition: Weigh 30g of stachyose as a single prebiotic control group.

[0072] Comparative Example 2: Mono-xylooligosaccharides Formula composition: Weigh out 30g of xylooligosaccharide as a single prebiotic control group.

[0073] Comparative Example: 3 Monofructose Formula composition: Weigh out 30g of fructooligosaccharides as the control group with a single prebiotic.

[0074] Comparative Example 4: Monoisomaltooligosaccharide Formula composition: Weigh out 30g of isomaltooligosaccharide as a single prebiotic control group.

[0075] Comparative Example 5: Monogalacto-oligosaccharides Formula composition: Weigh 30g of galactooligosaccharides as a single prebiotic control group.

[0076] Comparative Example 6: Mono-hexagalactan Formula composition: Weigh 30g of galactomannan as a single prebiotic control group.

[0077] Comparative Example 7: Mono-polydextrose Formula composition: Weigh out 30g of oligosaccharides as the control group with a single prebiotic.

[0078] Comparative Example 8: Single resistant starch Formula composition: Weigh 30g of resistant starch as a single prebiotic control group.

[0079] Comparative Example 9: Mono-resistant dextrin Formula composition: Weigh 30g of resistant dextrin as a single prebiotic control group.

[0080] Detection Example 1: SCFA production status Stool samples were collected from healthy volunteers (3 women and 3 men) who had not taken antibiotics for at least 3 months. The stool samples were diluted with sterile PBS under anaerobic conditions to prepare a 10% (w / v) stool suspension. The stool suspension was centrifuged at 500 rpm for 5 minutes to remove food residues, and the supernatant was immediately stored in an anaerobic bottle for use. 1 mL of the mixed fecal suspension was added to 9.0 mL of basal nutrient medium containing 100 mg of intervention (0.1 g sodium chloride, 0.04 g each of dipotassium hydrogen phosphate and potassium dihydrogen phosphate, 0.01 g each of magnesium sulfate and calcium chloride, 2 g sodium bicarbonate, 2.5 g peptone, 4 g yeast extract, 0.4 g glucose, 2 g mucin, 0.5 g bile salts (Type III), 0.46 g cysteine ​​sulfate, and 2 mL Tween-80; after mixing, the pH was adjusted to 7.0, and the mixture was autoclaved at 121℃ for 15 min and cooled for later use). A sample group (2% intervention), a positive control (inulin), and a blank group were set up. The mixture was incubated in an anaerobic incubator at 37℃ (oxygen content ≤ 0.1%) for 24 h. The incubation was observed in the anaerobic incubator, and samples were collected at the specified time intervals.

[0081] After fermentation, the supernatant and bacterial sludge precipitate were separated by centrifugation at 12000 r / min for 10 min and placed in separate centrifuge tubes. 800 μL of the supernatant was added to 200 μL of 50% (w / v) concentrated sulfuric acid and vortexed. Immediately afterward, 1 mL of diethyl ether was added and vortexed for 1 min. The mixture was then incubated on ice for 10 min, centrifuged at 12000 r / min for 10 min, and dehydrated with anhydrous CaCl2. The supernatant was then filtered through a membrane for analysis. The mass concentration of each short-chain fatty acid was calculated using the external standard method. The detector was a flame ionization detector (FID) with N2 as the carrier gas. The detector temperature was 250 ℃, the injection volume was 1 μL, the injection port temperature was 240 ℃, the initial temperature was 80 ℃ (0.5 min), the temperature was increased to 150 ℃ (4 ℃ / min), and then increased to 230 ℃ (20 ℃ / min) and held for 10 min. The experimental results are shown in [Figure number missing]. Figure 1-2 .

[0082] like Figure 1 As shown in Figure A, inulin and Example 1 group had the highest total acid content (both labeled "a"), with no significant difference between them; Example 2 and Example 3 groups (both "b") followed closely behind, significantly higher than Example 4 group ("c"); Example 5 and Example 6 groups (both "d") were significantly better than the blank control group (Control, "e"), but showed poorer total acid production capacity compared to the other ratios. These results indicate that Example 1 group can achieve a level similar to the positive control inulin in terms of total SCFAs, while the other ratios (Example 2 and Example 3 groups) also have relatively considerable synergistic effects.

[0083] In the combination of prebiotics, the inulin group served as a positive control group, producing acetic acid ( ). Figure 1 The amount of B in the group was significantly higher than in all other groups; the overall performance of the Example 1 group was second only to the positive control group; the Example 2 and Example 3 groups were slightly lower than the Example 1 group, but were still significantly better than the Example 4, Example 5 and Example 6 groups. Therefore, from the perspective of acetic acid, the Example 1 combination showed a better synergistic effect under the complementary effects of multiple prebiotics, and was close to the acid production level of the positive group.

[0084] propionic acid ( Figure 1 The overall pattern of propionic acid production (C) in Example 1 was similar to that of the positive control group; Example 4 and Example 3 were in the middle tier, significantly higher than Example 2 and Example 5. Example 6 had the lowest propionic acid production among the groups, but it was still better than the blank control. This study shows that the formulation of Example 1 also has considerable potential in promoting propionic acid production, while Example 4 and Example 3 also maintained good increases, suggesting that a reasonable formulation design can take into account the synergistic effect of multiple bacterial communities, thereby producing considerable propionic acid levels.

[0085] Compared to the first two short-chain fatty acids, butyric acid ( Figure 1 The high values ​​of D in the samples were significantly different: Group 2 had the highest value, Groups 1 and 4 were in the middle, Group 3 was slightly lower, and Group 5 was even lower; while inulin and Group 6 were in the lower range, still significantly higher than the control group. Previous studies have shown that butyric acid plays an important role in maintaining intestinal epithelial integrity and regulating inflammation. These results indicate that certain specific ratios (such as increased PHGG content) can more effectively stimulate the growth and metabolism of butyric acid-producing bacteria. Different prebiotics often have different degradation rates and specific bacterial compatibility; reasonable formulation helps to balance rapid and long-lasting fermentation processes.

[0086] contrast Figure 1 and Figure 2 As can be seen, the total acid concentration of Examples 1-6 was 4.5-6.0 μg / mL, which was significantly higher than that of Comparative Examples 1-3 (2.0-3.5 μg / mL). This indicates that the combination of specific proportions can take into account the synergistic effect of multiple microbial communities, which helps to balance the fermentation process with both rapid and long-lasting effects.

[0087] Test Case 2: Proliferation of Beneficial Intestinal Bacteria After in vitro simulated colon fermentation, the bacterial sludge was settled, and the procedure was performed sequentially according to the method described in the fecal genomic DNA extraction kit (Beijing Jinbaite Biotechnology Co., Ltd.). Subsequently, real-time quantitative PCR was used to quantitatively analyze the target bacterial community. The PCR system and primer sequences are shown in Tables 2 and 3 below, and the experimental results are as follows: Figure 3-4 .

[0088] Table 2 PCR system

[0089] Table 3. Primer sequences for gut microbiota

[0090] like Figure 3 As shown in Figure A, the Control group had the highest bacterial count (labeled "a"), which was also the case for Example 1, Example 2, Example 3, and Example 5. This is consistent with previous results for single prebiotics (…). Figure 4 Although the total bacterial count of some compound formulations was on par with the control, the advantage of multiple prebiotics may lie in the selective promotion of specific beneficial bacteria genera, rather than simply increasing the overall bacterial count.

[0091] exist Figure 3As shown in Figure B, the effect of Example 1 was significantly higher than all other treatments, followed by Example 4 and Example 3, but all were higher than the positive control group. The effects of Example 2, Example 5, and Example 6 were further reduced; the control group showed the lowest effect. Compared with single prebiotics, different ratios of SS:XOS:PHGG significantly enhanced the upregulation effect on Akkermansia. Previous studies have shown that Akkermansia is closely related to the intestinal mucosal barrier and inflammation regulation; therefore, synergistic effects targeting this bacterium are of great significance for improving gut health.

[0092] like Figure 3 As shown in Figure C, the results for Example 1 group were higher than those for the positive control group, Example 4 group, and Example 3 group. This is slightly different from the strong proliferative effect of inulin on Bifidobacteria in previous single prebiotic experiments, indicating that the 4:4:2 ratio in the compound formulation has a better promoting effect on Bifidobacteria than inulin alone. Considering the close relationship between Bifidobacteria and host immune regulation and antagonism of pathogenic bacteria, this result also highlights the potential value of formulation design for the proliferation of key probiotic genera. Zhu et al. intervened by inoculating mice with Bifidobacterium bifidum and used RT-qPCR to examine changes in related markers. The results showed that the relative abundance of Lactobacillus and Bacteroides, specific gut microbiota, was increased.

[0093] like Figure 3 As shown in Figure D, Group 3 of Example 3 exhibited the highest abundance of lactic acid bacteria, while Groups 4 and 2 of Example 3 showed slightly lower levels. Compared to inulin alone, the compound formulations in Groups 3, 2, and 4 demonstrated a more pronounced promoting effect on lactic acid bacteria. Given the positive impact of lactic acid bacteria on maintaining the intestinal environment and host immunity, this result further confirms the potential advantages of synergistic use of multiple prebiotics.

[0094] In summary, different compound formulations each emphasized different aspects of the four indicators: total bacterial count, Akkermansia, Bifidobacteria, and Lactobacillus. The formulation in Example 1 significantly outperformed single prebiotics in specific beneficial bacteria genera, while other formulations (such as 4:3:3 and 1:1:1) also showed a synergistic effect on Lactobacillus. Compared with previous results using single prebiotics, it can be preliminarily inferred that the functional complementarity of different prebiotics can significantly enhance the abundance of specific key bacterial groups, laying the foundation for further optimization of compound prebiotic intervention strategies.

[0095] Example 3: Effects of different compound ratios of prebiotics on gut microbiota Based on a comprehensive evaluation of SCFA production and beneficial gut bacteria proliferation, Example 1 showed the greatest overall advantage: it demonstrated outstanding performance in five key indicators—total acid, acetic acid, propionic acid production, Akkermansia, and Bifidobacterium (P > 0.05). Example 2 and Example 3 showed excellent performance in butyric acid production and lactic acid bacteria production, respectively. Example 4 was at a moderate level in total bacterial count and propionic acid production, but its simple ratio design provided a reference for studying the basic mechanism of prebiotic synergistic effects and also served as a comparison with other formulations. Therefore, these four groups were selected for subsequent experiments.

[0096] Further investigation was conducted into the prebiotic compatibility ratio and its ability to regulate gut microbiota at different levels. The 16S rRNA sequencing results are as follows: The results of the door level test are shown below. Figure 5 .

[0097] like Figure 5 As shown, in the four formulation groups (Example 4, Example 2, Example 1, and Example 3), the Actinobacteria phylum was significantly higher than that in the control group, especially in Example 1 and Example 3, reaching 70-80% or even more. This phylum includes various Bifidobacteria, often considered probiotics, with functions such as synthesizing short-chain fatty acids and inhibiting the growth of pathogens. The significant increase suggests the selective promoting effect of the compound prebiotic on this type of probiotic group. Compared with the control group, this phylum was significantly increased in all four formulation combinations (P < 0.05).

[0098] The Proteobacteria phylum contains some pathogenic bacteria, and an excess of these bacteria may be associated with intestinal inflammation or metabolic disorders. Only the treatment in Example 1 showed a relative reduction in their abundance, presumably because the bacterial community structure gradually remodeled towards a healthy state.

[0099] In the control group, Bacteroidetes were significantly dominant, but this was greatly reduced in all compound treatment groups, replaced by an increase in Actinobacteria. Firmicutes were similarly reduced to a low level, indicating that in the utilization of competitive fermentation substrates, Bacteroidetes and Firmicutes were inferior to Actinobacteria after compounding with multiple prebiotics. While desulfurization bacteria, Fusobacteria, synergists, and verrucous microbes had a certain proportion in the control group, they were generally present in trace amounts or masked by the larger proportions of Actinobacteria, Verrucous Microbes, and Proteobacteria in the compound treatment groups. The significant reduction in Fusobacteria and some desulfurization bacteria with potential pathogenicity or inflammatory effects may reflect that the compound prebiotics help optimize the gut microbiota balance.

[0100] In summary, compared with the control, all four formulation ratios (Example 4, Example 2, Example 1, and Example 3) resulted in an increase in the abundance of Actinobacteria and a decrease in the abundance of Firmicutes and Bacteroidetes within the Verrucous microbiota. Different prebiotics exhibit selective promoting effects in regulating the gut microbiota structure. Specifically, Example 1 showed the highest abundance of Actinobacteria and Verrucous microbiota, reaching over 80% of the total flora, while Example 3 maintained around 70%. Combined with previous analyses of short-chain fatty acids and specific functional bacterial genera (such as Bifidobacteria, Lactobacillus, and Akkermansia), it can be inferred that polarity shifts at the phylum level have a potential positive impact on improving gut ecology and enhancing host health.

[0101] Horizontal test results as follows Figure 6 As shown.

[0102] In all four prebiotic formulations, Bifidobacterium was the dominant genus, accounting for over 50%, especially in Examples 1 and 3, where it reached approximately 70% or higher, significantly higher than the control group. This result echoes the previously observed significant enrichment of Actinobacteria at the phylum level, indicating a significant increase in the abundance of probiotics such as Bifidobacterium. This phenomenon verifies that the combination of multiple prebiotics can provide suitable fermentation substrates and growth environments for Bifidobacterium, thereby achieving selective proliferation. In the control group, Escherichia coli and Shigella dysenteriae accounted for a relatively high proportion, ranging from 25% to 30%. Only in Example 1 was the proportion suppressed to below 20%. This genus contains several potential opportunistic pathogens, and their excessive abundance may be associated with an increased risk of intestinal inflammation or infection. Therefore, the significant reduction in this genus suggests that the compound prebiotic in Example 1 has a positive effect on regulating the gut microbiota structure and inhibiting potential pathogens. In the control group, Bacteroides accounted for approximately 10-15%, and a certain proportion of Fusobacterium, Clostridium sensu-stricto, and Parabacterium were also observed. However, in the compound prebiotic group, Bacteroides generally decreased to below 5%, and in Example 3 and Example 1 groups, the proportion was almost undetectable, while Fusobacterium and other genera were significantly reduced or nearly disappeared. Excessive enrichment of these genera is often accompanied by an increased risk of inflammation or disease.

[0103] These results indicate that the compound prebiotics, to some extent, restructured the anaerobic bacterial community, shifting it in a direction more beneficial to host health. Compared to the control, these non-dominant bacterial genera were mostly replaced by beneficial bacteria such as Bifidobacteria.

[0104] The combination of various prebiotics provided by this invention allows different oligosaccharide structures to provide diverse carbon sources for Bifidobacteria, thereby competitively inhibiting other potential pathogens or conditional pathogens.

[0105] In clinical or functional assessments, an increase in the proportion of Bifidobacteria and a decrease in potential pathogens such as Escherichia coli and Shigella dysenteriae are often considered indicators of improved gut ecology. The results showed that the proportion in Example 1 group had multiple benefits, including anti-inflammatory effects and improved barrier function.

[0106] Example 4: Effect of Synbiotic Fermentation Broth on Cell Viability Using the group of Example 3 as the intervention, in vitro simulated colonic fermentation was carried out (method is the same as in Test Example 1). The 24-hour fermentation supernatant was used as raw material, and the efficacy and mechanism of action of the compound prebiotic product in helping to improve diarrhea were investigated using the HT-29 cell model.

[0107] This section uses the HT-29 cell model to investigate the efficacy and mechanism of action of Synbiotic products in helping to improve diarrhea.

[0108] This experiment used the CCK-8 assay (Shanghai Beyotime Biotechnology Co., Ltd., C0038) to analyze the effect of synbiotic fermentation broth on the growth of HT-29 cells. The cell viability test results are as follows: Figure 7 As shown in the figure. The results showed that, within the concentration range of 100 to 250 dilutions, the cell viability of the mixed fermentation broth pairs remained above 95%, with no significant difference compared to the control group (P<0.05).

[0109] This indicates that at these concentrations, SCFAs in the diluted fermentation broth neither promoted cell proliferation nor exhibited cytotoxicity, confirming their safety in cell experiments. Therefore, concentrations of 100 and 200 dilutions were selected as the high-dose group and low-dose group for subsequent experiments.

[0110] Example 5: Effects of stachyose-based colonic fermentation broth on the expression of intestinal barrier-related genes and proteins. HT-29 cells were cultured to passage 3, and the cell concentration was adjusted to 5 × 10⁶ cells / year. 5Cells were seeded at a density of 2 mL / well in 6-well plates. After complete cell adhesion, the culture medium was replaced with fresh medium. In the intervention group, 1 mL of the corresponding fermentation supernatant was added to each well, while the blank control group received an equal volume of cell culture medium. The plates were incubated at 37 °C for 24 h in a 5% CO2 incubator. Cells and culture supernatant were collected and stored at -80 °C for later use. Total RNA was extracted from co-cultured HT-29 cells using an RNA extraction kit and a reverse transcription kit (Beijing Jinbaite Biotechnology Co., Ltd.), and the RNA was reverse transcribed into cDNA. Real-time quantitative PCR was then used to detect the expression levels of cell mucin (MUC2) and tight junction proteins (ZO-1, Occludin, Claudin). The PCR reaction system was prepared according to the instruction manual, and the reaction conditions were: pre-denaturation 95 °C, 10 min; denaturation 95 °C, 15 s; annealing 60 °C, 60 s; 40 cycles. Final data are presented as follows. ΔΔ The analysis was performed using CT scans.

[0111] The effect of synbiotic in vitro simulated colon fermentation broth on the expression levels of cell-related signaling proteins was detected using Western blot. Logarithmic growth phase cells were collected and fermented in an in vitro culture medium of 5 × 10⁶ cells. 5Cells were seeded at a density of 2 mL cell suspension per well in 6-well plates. After 24 h of culture and cell attachment, the original culture medium was aspirated and the cells were gently washed with PBS. Subsequently, a blank control group, a high-dose fermentation broth group, and a low-dose fermentation broth group were set up, and corresponding treatment solutions were added to each group for intervention. After 24 h of treatment and culture, the culture medium was gently aspirated, and the cells were washed twice with PBS. Then, 200 μL of cell lysis buffer was added to each well, and the cells were thoroughly mixed by pipetting. The lysis buffer was collected in centrifuge tubes, centrifuged at 8000 r for 5 min, and the supernatant was collected for later use. After standardizing the concentration, 5× loading buffer was added to the protein sample, mixed, and boiled in a water bath for 10 min to denature. After cooling, the sample was mixed again and centrifuged. The protein sample was then added to a 12% SDS-PAGE gel at a concentration of 30 μg protein per well. After SDS-PAGE electrophoresis (80 V, 30 min; 120 V, 60 min), the protein in the gel was transferred to a methanol-activated PVDF (Bio-Rad) membrane using a constant current (200 mA, 60 min). After transfer, the PVDF membrane was blocked at room temperature for 2 h in a 5% skim milk solution prepared with TBST Buffer. Then, the band containing the target protein was cut and placed in an antibody incubation cassette, with the corresponding primary antibody solution added and incubated overnight at 4 °C. After incubation, the band was washed three times with TBST solution for 10 min each time. Subsequently, depending on the primary antibody used, each band was incubated in the corresponding secondary antibody solution at room temperature in the dark for 2 h. After incubation, the band was washed three times with TBST solution for 10 min each time. Finally, ECL chromogenic solution was prepared, and the membrane was incubated for color development, then photographed and observed using a gel imaging system.

[0112] MUC2 is an important secretory mucin in the intestinal epithelium that forms a mucus layer in the colon, playing a crucial role in maintaining the intestinal mucosal barrier and intestinal health. Diarrhea is associated with low levels of expression of the tight junction protein MUC2, and its upregulation typically enhances the mucosal surface's barrier function against harmful bacteria and toxins.

[0113] from Figure 8 The results show that with increasing concentrations of synbiotic intervention, the mRNA and protein expression levels of intestinal mucin MUC2 in HT-29 cells exhibited a positive correlation upward trend. The high-concentration group showed a significant difference compared to the control group (P < 0.01), while the low-concentration group showed no significant difference compared to the control group. Based on these results, it is hypothesized that synbiotic supplementation can enhance intestinal mucus barrier function by upregulating the transcription and translation levels of intestinal mucin MUC2, thus playing a positive role in improving diarrhea.

[0114] ZO-1 is a scaffold protein that maintains tight junction stability and is responsible for connecting transmembrane proteins to the cytoskeleton. As shown in the figure, with increasing concentrations of synbiotic intervention, the mRNA and protein expression levels of the tight junction protein ZO-1 in HT-29 cells generally increased. Figure 9 As can be seen, at the mRNA level, there was no significant difference between the low-concentration group and the control group, while there was a highly significant difference between the high-concentration group and the control group (P < 0.01). At the protein level, the low-concentration group showed a significant difference compared to the control group (P < 0.05), and the difference between the high-concentration group and the control group was even more significant (P < 0.01). These results indicate that synbiotic intervention can upregulate the transcription and translation levels of the tight junction protein ZO-1, especially at high concentrations. This demonstrates that the synbiotic fermentation broth provided by this invention can not only enhance intercellular tight junctions but also improve intestinal barrier function, thereby helping to improve diarrhea.

[0115] Occludin expression is typically associated with the tightness of intercellular spaces, forming, along with Claudin, "gated" structures of paracellular pathways. Increased ocludin levels help maintain proper fluid balance within the intestinal lumen, thereby improving diarrhea symptoms. Figure 10 As can be seen, with the increase of fermentation broth concentration, the mRNA and protein expression levels of the tight junction protein Occludin in HT-29 cells showed a significant upregulation trend. At the mRNA level, there was a significant difference between the control group and the low-concentration group (P < 0.05), while there was a highly significant difference between the control group and the high-concentration group (P < 0.01). At the protein expression level, there was no significant difference between the control group and the low-concentration group, but the difference between the control group and the high-concentration group was extremely significant (P < 0.01).

[0116] In conclusion, synbiotics can upregulate the transcription and translation levels of occludin, especially at high concentrations, indicating that they have a positive effect on enhancing cell tight junctions, improving intestinal barrier function, and alleviating diarrhea.

[0117] Claudin-1 is an important member of the tight junction protein family and plays a crucial role in epithelial barrier function. Its abnormal expression may be associated with altered intestinal permeability, inflammation, or motility disorders, indirectly affecting the occurrence of diarrhea. As shown in the figure, with increasing concentrations of synbiotic intervention, the overall mRNA and protein levels of the tight junction protein Claudin-1 in HT-29 cells showed a significant upregulation trend. Figure 11The results showed that at the mRNA level, compared with the control group, the low-concentration group showed a significant upregulation (P < 0.05), while the high-concentration group showed an even more significant increase (P < 0.01). At the protein level, compared with the control group, the protein expression levels in both the low-concentration and high-concentration groups were positively correlated with the dose-response relationship, showing highly significant differences (P < 0.01). These results indicate that synbiotic intervention can upregulate Claudin-1 expression at both the transcriptional and translational levels, especially at high concentrations, thereby helping to strengthen tight junctions and improve intestinal barrier function, potentially playing a positive role in alleviating diarrhea.

[0118] Example 6: Mouse Experiment In this study, we administered a prophylactic intervention to SPF-grade C57BL / 6 male mice using a stachyose composition at a daily dose of 500 mg / kg body weight. Subsequently, during the final week of the intervention, the mice were induced to develop a colitis mouse model using 3% sodium dextran sulfate (DSS). This design aimed to investigate the potential utility of stachyose in preventing or alleviating DSS-induced colitis diarrhea symptoms.

[0119] Thirty male SPF-grade C57BL / 6 mice, aged 6 weeks, were provided by Beijing SPF Biotechnology Co., Ltd. All mice were kept under SPF conditions with free access to food and water, at 21±2 ℃, and following a 12-hour light-dark cycle. Prior to the experiment, all mice underwent a one-week acclimatization period. The 30 mice were then randomly divided into three groups of 10 mice each, as follows: (1) Blank control group of C57BL / 6 mice (blank group) (2) C57BL / 6 mouse acute colitis model group (model group) (3) C57BL / 6 colitis mice + stachyose combination intervention group (stachyose combination group) The administration methods were as follows: the blank group drank water normally for 3 weeks, the model group and the stachyose composition group drank water normally for weeks 1 and 2, and 3% DSS was added to the water in week 3. The stachyose composition group was given the stachyose composition by gavage for 3 weeks at a dose of 500 mg / kg / d. The blank group and the model group were given an equal volume of distilled water by gavage for 3 weeks. The last week of the experiment was defined as the DSS period.

[0120] Seven aseptic stool collections were performed during the experiment. Throughout the DSS treatment period, all experimental animals underwent continuous and meticulous clinical observation to record their physiological state and behavioral changes. On day 22 of the experiment, all experimental animals were euthanized by cervical dislocation and their tissues were collected.

[0121] Colon length is generally considered a key macroscopic indicator for assessing colitis and is negatively correlated with the severity of colitis; that is, the shorter the colon, the more severe the inflammation. In addition to colonic shortening, mice also exhibited symptoms such as edema, hemorrhage, and even erosion of the colonic mucosa due to inflammation. Figure 12 It was found that after DSS treatment, the colon in the model group was shortened by 40% compared to the blank control group. The stachyose composition treatment significantly inhibited the shortening of the mouse colon compared to the model group, and the effect was significantly greater. Observing the colon morphology, the colon in the blank group was more intact, with more formed feces and a larger cecal volume; while in the model group, the feces were unformed, and some mice in the model group had blood clots in the cecum and more bloodstains in the colon, and the overall weight was also reduced. After intervention with the stachyose composition, the feces in the colon became formed, and the cecal volume increased, indicating that the stachyose composition intervention can significantly inhibit the colonic shortening and diarrhea symptoms caused by DSS-induced acute colitis, while also inhibiting bleeding and alleviating the inflammatory state in mice.

[0122] In addition, the characteristics of mouse feces were continuously monitored, and the fecal water content was measured. Figure 13 As shown, during the DSS induction phase, the fecal water content of the model group mice increased significantly, especially during days 19 to 21 of the experiment, when the mice exhibited watery stools and diarrhea with blood components, leading to a surge in fecal water content. This phenomenon also indicates the successful establishment of the DSS-induced acute colitis model. Meanwhile, the mice in the stachyose composition intervention group showed increased stool volume and significantly alleviated diarrhea symptoms.

[0123] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A stachyose composition, characterized in that, The stachyose composition consists of stachyose, xylooligosaccharide, and galactomannan in a mass ratio of 1-6:1-4:1-4.

2. The stachyose composition according to claim 1, characterized in that, The mass ratio of stachyose, xylooligosaccharide, and galactomannan is 4:2-4:2-4.

3. The stachyose composition according to claim 1, characterized in that, The mass ratio of stachyose, xylooligosaccharide, and galactomannan is 4:4:2, 4:2:4, 4:3:3, 1:1:1, 2:1:1, or 6:2:

2.

4. The stachyose composition according to claim 3, characterized in that, The mass ratio of stachyose, xylooligosaccharide, and galactomannan is 4:4:2, 4:2:4, or 4:3:

3.

5. The use of the stachyose composition according to any one of claims 1-4 in the preparation of functional foods or pharmaceuticals for promoting the production of short-chain fatty acids in the intestine.

6. The application according to claim 5, characterized in that, The short-chain fatty acids include acetic acid, propionic acid, and / or butyric acid.

7. The use of the stachyose composition according to any one of claims 1-4 in the preparation of functional foods or pharmaceuticals for selectively proliferating beneficial intestinal bacteria and inhibiting potentially pathogenic bacteria.

8. The application according to claim 7, characterized in that, The beneficial bacteria are Bifidobacterium, Lactobacillus, and / or Akkermania; the potential pathogens are Escherichia coli and Shigella.

9. The use of the stachyose composition according to any one of claims 1-4 in the preparation of products for treating and / or preventing diarrhea.

10. The application according to claim 9, characterized in that, The products mentioned include pharmaceuticals.

11. A pharmaceutical composition, characterized in that, The stachyose composition includes any one of claims 1-4.

12. The pharmaceutical composition according to claim 11, characterized in that, The dosage form of the pharmaceutical composition includes: liquid dosage form, gas dosage form, solid dosage form, or semi-solid dosage form.

13. The pharmaceutical composition according to claim 11, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

14. The pharmaceutical composition according to claim 13, characterized in that, The pharmaceutically acceptable excipients include any one or more of the following: binders, fillers, disintegrants, lubricants, preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, or osmotic pressure regulators.

15. The pharmaceutical composition according to claim 11, characterized in that, Including at least one of the following methods of administration: oral, injection, implantation, topical, spray, inhalation, or a combination thereof.

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