Composition for preventing and / or treating diarrhea and application thereof

Abstract

The invention relates to a composition for preventing and / or treating diarrhea and application thereof. The composition comprises three or more of 2 '-FL, LNT, 3'-SL and calcium citrate, and the composition contains calcium citrate. The composition provided by the invention can regulate intestinal flora balance and inhibit pathogenic bacteria, and has an excellent effect of improving diarrhea.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a composition for preventing and / or treating diarrhea and use thereof. Background Art

[0002] Diarrhea is a common health problem worldwide, with a particularly high incidence among infants and young children. Current treatments for diarrhea rely primarily on oral rehydration salts, antibiotics, and probiotics. While antibiotics can inhibit pathogens, they can disrupt the balance of intestinal flora, leading to drug resistance. The efficacy of probiotics is affected by strain, dosage, and individual variability, leading to limited stability. Antidiarrheal medications only alleviate symptoms without addressing the underlying cause.

[0003] Human milk oligosaccharides (HMOs), the third largest solid component in breast milk, have been shown in recent years to be one of the core active ingredients that benefit the health of breastfed infants. Their unique structural and functional properties enable them to affect intestinal health through intestinal microorganisms. For example, CN113519849B discloses that HMOs containing 3'-sialyllactose (3'-SL), 3-fucosyllactose (3'-FL) or 6'-sialyllactose (6'-SL) can significantly reduce the adhesion of diarrhea-causing Escherichia coli (such as ETEC and EPEC) to intestinal cells, repair intestinal barrier function, and reduce the release of the inflammatory factor IP-10. This patent document relies on in vitro experiments or animal studies and lacks clinical population verification. For example, some studies only draw conclusions through cell experiments or mouse models, and have not been systematically verified in humans (especially in different age groups or special populations), resulting in doubts about the practical application value of the results.

[0004] For example, CN114786501A discloses a nutritional composition for preventing or treating gastrointestinal infection / inflammation in infants and young children, the core ingredients of which are fucosylated oligosaccharides (such as 2'-fucosyllactose, 2'-FL) and N-acetylated oligosaccharides (such as lactose-N-neotetraose, LNnT) in a specific proportion. The application scenarios of these patents are narrow, the functions are single and the population coverage is insufficient. For example, some studies only target specific populations such as infants and young children, and have not been extended to adults, the elderly or other related health problems (such as immune regulation, metabolic health). In addition, the patents do not take into account the challenges of industrialization (such as ingredient stability and production costs), which affects the actual promotion potential of the technology. Future research needs to strengthen clinical verification, optimize compound formulas, and explore a wider range of applicable scenarios.

[0005] Therefore, there is an urgent clinical need to develop a safe and effective treatment for diarrhea. Summary of the Invention

[0006] In view of this, the object of the present invention is to provide a composition containing human milk oligosaccharides and calcium citrate, and use of the composition in preparing a composition for treating or preventing diarrhea.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0008] The present invention provides a composition for preventing and / or treating diarrhea. The composition comprises three or more of 2'-FL, LNT, 3'-SL and calcium citrate, wherein the composition comprises calcium citrate.

[0009] Preferably, the composition consists of 2'-FL, LNT, 3'-SL and calcium citrate.

[0010] Preferably, the mass ratio of the 2'-FL, LNT, 3'-SL and calcium citrate is (1-10):(0.1-5):1:(2-15).

[0011] Preferably, the mass ratio of the 2'-FL, LNT, 3'-SL and calcium citrate is (1-5):(0.5-3):1:(3-8).

[0012] Preferably, the mass ratio of the 2'-FL, LNT, 3'-SL and calcium citrate is (3.5-4.5):(1.5-2.5):1:(6.5-7.5).

[0013] Preferably, the mass ratio of the 2'-FL, LNT, 3'-SL and calcium citrate is 4:2:1:7.

[0014] In one aspect, the present invention provides a medicine, a nutritional product or a health product comprising any of the above compositions.

[0015] In one aspect, the present invention provides use of any of the above compositions or the above medicines, nutritional products or health products in the preparation of products for preventing and / or treating diarrhea.

[0016] Preferably, the diarrhea is bacterial infectious diarrhea, preferably E. coli infectious diarrhea.

[0017] Preferably, the bacteria are Escherichia coli ETEC and / or EPEC.

[0018] Preferably, the product is used to reduce small intestinal propulsion rate.

[0019] Preferably, the product is used to reduce the water content of feces.

[0020] Preferably, the product comprises a medicine, a nutritional product or a health product.

[0021] Due to the adoption of the above technical solution, the present invention has the following beneficial effects:

[0022] The present invention provides a composition containing human milk oligosaccharides and calcium citrate, specifically 2'-FL, LNT, and 3'-SL. The human milk oligosaccharides and calcium citrate in the present invention have significant synergistic advantages. HMOs act as prebiotics to promote the proliferation of beneficial bacteria such as Bifidobacterium, optimize the intestinal microenvironment, and enhance calcium bioavailability. At the same time, the complex formed by calcium ions and HMOs can delay carbohydrate fermentation and prolong the duration of prebiotic action. This combination not only synergistically enhances intestinal barrier function and reduces inflammatory responses, but also improves calcium absorption rate by regulating intestinal pH, thereby regulating intestinal flora balance and inhibiting pathogenic bacteria. It has an excellent effect on improving diarrhea, is safe, has no side effects, avoids drug resistance, and is suitable for long-term use (such as infant formula or functional foods). BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a bar graph showing the effects of each group on the small intestinal propulsion rate of mice in Example 2;

[0024] Figure 2 This is a bar graph of the moisture content of mouse feces in each group in Example 3;

[0025] Figure 3 The figure is a bar graph showing the loose stool rates of mice in each group in Example 3. DETAILED DESCRIPTION

[0026] To make the technical solutions and beneficial effects of the present invention more clearly understood, the following detailed description is given by way of specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly illustrate the details of the local features. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0027] Studies have shown that HMOs (such as 2'-FL, LNT, and 3'-SL) are exclusive nutrient sources for beneficial bacteria such as Bifidobacterium, which can significantly increase their abundance and optimize the intestinal microecology. HMOs competitively bind to the surface receptors of pathogens (such as ETEC and Salmonella), blocking their adhesion to the intestinal epithelium and reducing the risk of infectious diarrhea. Based on this, the inventors have developed a composition containing 2'-FL, LNT, 3'-SL and calcium citrate through a large number of experimental studies. The HMOs in the composition selectively promote the proliferation of probiotics and block the adhesion of pathogens. Calcium ions are involved in the regulation of smooth muscle contraction. Appropriate calcium supplementation helps maintain normal intestinal motility function, strengthen the intestinal barrier and neutralize inflammatory factors. The two work synergistically to enhance the efficacy of treating diarrhea. In particular, by adjusting the ratio of HMO types to calcium, it can adapt to various causes such as bacterial diarrhea, and subsequently provide precise intervention for different age groups (such as infants focusing on 2'-FL and adults focusing on LNT).

[0028] The composition provided by the present invention can significantly alleviate weight loss induced by Escherichia coli, particularly E. coli, reduce the rate of hyperactive small bowel movement caused by diarrhea, and lower stool water content, effectively improving diarrhea-related physiological indicators. The composition of the present invention can also avoid bacterial flora disturbances and drug resistance caused by antibiotics. Its natural ingredients make it particularly suitable for long-term use in sensitive populations such as infants and the elderly. Based on this, the present invention was completed.

[0029] The present invention provides a composition for preventing and / or treating diarrhea. The composition comprises three or more of 2'-FL, LNT, 3'-SL and calcium citrate, wherein the composition comprises calcium citrate.

[0030] In certain embodiments, the composition consists of 2'-FL, LNT, 3'-SL, and calcium citrate.

[0031] In certain embodiments, the mass ratio of 2'-FL, LNT, 3'-SL and calcium citrate is (1-10):(0.1-5):1:(2-15).

[0032] In certain embodiments, the mass ratio of 2'-FL, LNT, 3'-SL and calcium citrate is (1-5):(0.5-3):1:(3-8).

[0033] In certain embodiments, the mass ratio of 2'-FL, LNT, 3'-SL and calcium citrate is (3-5):(0.5-3):1:(6-8).

[0034] In certain embodiments, the mass ratio of 2'-FL, LNT, 3'-SL and calcium citrate is (3.5-4.5):(1.5-2.5):1:(6-8).

[0035] In certain embodiments, the mass ratio of 2'-FL, LNT, 3'-SL and calcium citrate is 4:2:1:7.

[0036] In one aspect, the present invention provides a medicine, a nutritional product or a health product comprising any of the above compositions.

[0037] In one aspect, the present invention provides use of any of the above compositions or the above medicines, nutritional products or health products in the preparation of products for preventing and / or treating diarrhea.

[0038] In certain embodiments, the diarrhea is bacterial infectious diarrhea.

[0039] In certain embodiments, the bacterium is E. coli.

[0040] In certain embodiments, the bacteria are Escherichia coli ETEC and / or EPEC.

[0041] In certain embodiments, the diarrhea is E. coli infectious diarrhea.

[0042] In certain embodiments, the product is used to reduce small intestinal propulsion rate;

[0043] In certain embodiments, the product is used to reduce fecal water content.

[0044] In certain embodiments, the product comprises a pharmaceutical, nutritional, or health product.

[0045] In the following examples, all ingredients used are conventional commercial products, which can be obtained from commercial products or synthesized according to known methods. For example, fucosyllactose and sialyllactose are produced by Suzhou Yixi Biotechnology Co., Ltd.

[0046] The present invention is described in more detail below by way of examples. Where specific experimental steps or conditions are not specified in the examples, they were performed according to conventional procedures or conditions in the art.

[0047] The materials and reagents used in the following examples are shown in Table 1, and the instruments and equipment are shown in Table 2.

[0048] Table 1 Main materials and reagents

[0049]

[0050] Table 2 Main instruments and equipment

[0051]

[0052] Example 1

[0053] 2'-FL, LNT, 3'-SL and calcium citrate were respectively taken and compounded into composition 1 in a mass ratio of 5:1:2:8.

[0054] 2'-FL, LNT, 3'-SL and calcium citrate were respectively taken and compounded into composition 2 in a mass ratio of 3:2:1:6.

[0055] 2'-FL, LNT, 3'-SL and calcium citrate were respectively taken and compounded into composition 3 in a mass ratio of 4:2:1:7.

[0056] Example 2

[0057] 1. 4-week-old BALB / C male mice (aged 6-8 weeks) were selected and housed in a controlled environment with free access to standard laboratory chow and water. The experimental environment was set up with a 12-h light-dark cycle, and the temperature and humidity were maintained between 22 ± 2°C and 45 ± 5%. After a week of acclimatization, the mice were randomly divided into 9 groups of 10 mice each.

[0058] 2. According to different groups, a diarrhea mouse model was constructed. The negative control group mice were gavaged with an equal volume of PBS, and the other groups were first disrupted with Escherichia coli once a day for one week. Subsequently, 2'-FL, LNT, 3'-SL, calcium citrate monomers and the combination were used for gavage treatment. The negative control group and the model group were gavaged with an equal volume of PBS once a day for one week (see Table 3 for experimental groups and gavage doses). The gavage dose was determined to be 1.2 g / L for infants and 250 mg / day for calcium requirement based on the dose of 2'-FL in breast milk and the added dose in infant products specified by domestic and international standards. The HMOs mouse dose was determined to be 1300 mg / Kg by body surface area conversion method (BSA method), and calcium citrate was approximately 1300 mg / Kg. The mixed group used a total amount equivalent to that of the other treatment groups, and the sugar ratio was based on breast milk, combined with domestic standards and EU regulations. The weight of the mice was recorded after the experiment.

[0059] Table 3 Experimental groups and dosages

[0060] Grouping Adaptive feeding / 7d Modeling / 7D Intervention / 7d NC - Equal volume of PBS Equal volume of PBS MC - E. coli Equal volume of PBS 2'-FL - E. coli 1300mg / Kg 2'-FL LNT - E. coli 1300mg / Kg LNT 3'-SL - E. coli 1300mg / Kg 3'-SL calcium citrate - E. coli 1300mg / Kg calcium citrate 2'-FL+LNT+3'-SL+calcium citrate - E. coli 1300mg / Kg Composition 1 2'-FL+LNT+3'-SL+calcium citrate - E. coli 1300mg / Kg Composition 2 2'-FL+LNT+3'-SL+calcium citrate - E. coli 1300mg / Kg Composition 3

[0061] 3. Small intestinal propulsion rate experiment: gavage at 0.1-0.2mL / 10g body weight (e.g. 0.2-0.4mL for a 20g mouse). Record the completion time of gavage (T0). Execute the animal 30 minutes after gavage. Execute the animal by cervical dislocation, quickly open the abdominal cavity, and separate the gastrointestinal tissue. Remove the small intestine, gently lift the pylorus (the junction of the stomach and duodenum) to the ileocecal region (the end of the small intestine), avoid pulling it too long, spread the small intestine flat on an ice tray, keep it in a naturally relaxed state, and measure the total length (the entire length from the pylorus to the ileocecal region). Find the distance from the front end of the charcoal powder (the farthest point marked by the black mark) to the pylorus. Calculate the propulsion rate.

[0062] Here are the results:

[0063] 1. Mouse weight growth curve

[0064] As shown in Table 4, during the modeling period, the body weight of the MC group and each treatment group decreased significantly, with significant differences compared to the NC group. Analysis showed that this change was mainly due to diarrhea caused by E. coli infection, resulting in impaired nutritional intake and weight loss. After entering the treatment period, both the MC group and the treatment group regained weight, with the growth trend in the treatment group being more significant. Compared with the MC group, the treatment group recovered weight faster after gavage with a combination of human milk oligosaccharides and calcium citrate, while the MC group only gavage with an equal amount of PBS, and the recovery effect was weaker. The results show that the combination of human milk oligosaccharides and calcium citrate has a positive effect in improving ETEC-induced weight loss. The experimental period consists of three stages: the first week is the adaptation period, the second week is the modeling period, and the third week is the treatment period.

[0065] Table 4 Mouse weight gain

[0066]

[0067]

[0068] 2. Effects of different combinations of human milk oligosaccharides on small intestinal propulsion rate in mice

[0069] The changes in small intestinal propulsion rate during diarrhea in mice can reflect the repair capacity of the intestinal barrier. The effects of different human milk oligosaccharides on small intestinal propulsion rate are as follows: Figure 1As shown, the MC group showed a significant increase in the small intestinal propulsion rate, suggesting that increased intestinal fluid after E. coli infection stimulates the peristaltic reflex. Inflammatory mediators directly excite smooth muscle, leading to accelerated peristalsis to expel harmful substances, resulting in hyperactive diarrhea. Compared with the MC group, intervention with human milk oligosaccharides and calcium citrate alone or in combination significantly reduced the elevated small intestinal propulsion rate associated with hyperactive diarrhea. 2'-FL, LNT, and 3'-SL, as important HMOs, regulate intestinal flora, improve intestinal barrier function, and reduce inflammation. Combined intervention with calcium citrate further enhanced these effects, synergistically promoting small intestinal repair. Combination 3 (2'-FL, LNT, 3'-SL, calcium citrate = 4:2:1:7) showed the greatest efficacy, likely due to the synergistic effect of the four components in this ratio, which comprehensively improved barrier integrity, promoted tissue regeneration, and reduced inflammation, bringing the intestinal propulsion rate closer to normal levels.

[0070] Example 3

[0071] 1. Treat mice according to steps 1 and 2 of Example 2;

[0072] 2. Determine fecal water content: Collect feces from each mouse before sacrifice and freeze-dry the feces. Fecal water content = (wet weight - dry weight) / wet weight;

[0073] 3. Determination of Diarrhea-Related Indicators: One day before the end of the experiment, all mice were housed in individual cages. Filter paper was placed on the cage floor. The total number of feces and loose stools produced by the mice over the 24-hour period were recorded. Diarrhea rate = total number of loose stools in a group / total number of feces in the group; loose stool rate = total number of loose stools in a group / total number of feces in the group.

[0074] Here are the results:

[0075] 1. Effects of different combinations of human milk oligosaccharides on fecal water content in mice

[0076] Fecal water content is an important indicator for measuring the severity of diarrhea symptoms. Bacterial diarrhea usually causes mice to have unformed feces. The higher the fecal water content, the more severe the diarrhea symptoms. Therefore, by monitoring the fecal water content of mice, the changes in diarrhea and the severity of symptoms can be effectively assessed. Figure 2As shown in the figure, the fecal water content of the MC group was significantly higher than that of the NC group, indicating that the diarrhea mouse model was successfully established. Human milk oligosaccharides (HMOs) and calcium citrate intervention significantly reduced the fecal water content and effectively alleviated the symptoms of diarrhea. The effect of combined intervention was more significant, indicating that different HMOs and calcium components may have complementary effects in improving intestinal function, regulating intestinal microecology, repairing barrier structure, and improving the intestinal ability to reabsorb water. The group with a composition ratio of 4:2:1:7 had the best effect, which may be because the synergistic effect of this ratio of composition further improved the expression of tight junction proteins, restored the integrity of the intestinal barrier, and reduced the level of inflammation, thereby effectively reducing the fecal water content and significantly alleviating diarrhea symptoms.

[0077] 2. Diarrhea-related indicators

[0078] The loose stool rate of mice in each group was Figure 3 ,Depend on Figure 3 The MC group showed a significant increase in the rate of loose stools and diarrhea, indicating that E. coli infection impairs intestinal barrier function, reduces water absorption capacity, and leads to abnormal intestinal motility, further exacerbating diarrheal symptoms. After the introduction of HMOs and calcium citrate, the rate of loose stools in all treatment groups was significantly reduced, demonstrating that both HMOs and calcium citrate are effective in alleviating diarrhea. The combination can reduce water loss in the intestinal lumen and lower the incidence of loose stools by regulating the intestinal microbiome, promoting the proliferation of beneficial bacteria, and improving intestinal barrier integrity.

[0079] The loose stool rates of mice in each group are shown in Table 5. Regarding diarrhea rates, the 3'-SL group showed relatively weaker improvement, possibly related to its mechanism of action and dependence on specific intestinal flora. However, the combined intervention of 3HMOs and calcium citrate significantly reduced diarrhea rates, likely due to the synergistic effect of these two substances in enhancing mucosal barrier function, reducing inflammatory responses, and maintaining intestinal homeostasis. Furthermore, the three HMOs may synergistically improve intestinal health through multi-target mechanisms, accelerating intestinal barrier repair and thus more effectively alleviating diarrheal symptoms.

[0080] Table 5 Effect of human milk oligosaccharide and calcium citrate combination on diarrhea rate in mice

[0081]

[0082] Example 4

[0083] 1. Experimental Methods

[0084] (1) Volunteer recruitment and grouping: 20 adult volunteers (aged 18-40 years) diagnosed with bacterial diarrhea were recruited and randomly divided into two groups:

[0085] Control group (Group A, n=10): took placebo (maltodextrin).

[0086] Experimental group (Group B, n=10): took a sample containing 3HMOs and calcium citrate composition (2'-FL+LNT+3'-SL+calcium citrate composition = 4:2:1:7, daily dose 1.2 g, orally administered twice).

[0087] (2) Experimental Design: Acclimation period (3 days): All volunteers stopped taking any anti-diarrhea medications, and baseline data (defecation frequency, stool characteristics, etc.) were recorded. Intervention period (10 days): Group A took a placebo daily, and Group B took a combination of 3HMOs and calcium citrate daily. Volunteers maintained a regular diet (avoiding high-fat and irritating foods) and recorded symptoms and medication use daily.

[0088] (3) Detection indicators: Diarrhea relief rate: the proportion of volunteers with ≤3 bowel movements per day and improved stool consistency (Bristol stool typing ≤5) after intervention. Stool consistency score: The Bristol stool typing scale (1-7, 1 for hard stool and 7 for watery stool) was used. Inflammatory markers: Fecal calprotectin levels were measured.

[0089] (4) Data collection and statistics: The number of bowel movements and stool characteristics were recorded daily; stool samples were collected before and after the intervention and stored at -80°C for inflammatory factor detection.

[0090] Here are the results:

[0091] 1. Diarrhea symptoms significantly improved:

[0092] Diarrhea Relief Rate (Table 6): After taking the combination of 3HMOs and calcium citrate, 80% of the volunteers in the experimental group (Group B) achieved diarrhea relief criteria (≤3 bowel movements per day and improved stool characteristics), which was significantly higher than the 30% in the control group (P < 0.05).

[0093] Stool characteristics (Table 6): According to the Bristol Stool Type Scale, Group B's stool improved from 5.7 ± 1.0 (nearly watery) before intervention to 3.4 ± 0.8 (nearly formed), while the control group showed only a slight improvement (5.8 ± 0.9 → 5.2 ± 1.1), indicating that the 3HMOs and calcium citrate combination effectively reduced intestinal water loss. Data in Tables 6 and 7 are mean ± SD before and after intervention.

[0094] Table 6 Effects of human milk oligosaccharides and calcium citrate combination on diarrhea relief rate and stool characteristics of volunteers

[0095]

[0096] 2. Reduced inflammatory response

[0097] The changes in fecal calprotectin expression in groups A and B before and after treatment are shown in Table 7. The level of fecal calprotectin (an inflammatory marker) in group B decreased from 125±20 μg / g to 65±15 μg / g (P<0.05), while the change in the control group was not significant (120±25→110±30 μg / g), indicating that the combination of 3HMOs and calcium citrate can alleviate intestinal inflammation.

[0098] Table 7 Changes in intestinal inflammation markers

[0099]

[0100] From the above results, it can be seen that the composition provided by the present application can effectively improve body weight (increase by 30%), reduce fecal water content (reduction by 35%), small intestinal propulsion rate (reduction by 30%), diarrhea rate (reduction by 80%) and loose stool rate (reduction by 86%), thereby effectively reducing intestinal water loss, quickly alleviating diarrhea symptoms, and repairing intestinal structural damage and enhancing nutrient absorption function.

[0101] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations of the claims. Various modifications and variations may be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments merely illustrate several implementations of the present invention and do not limit the scope of protection of the patent of this invention.

Claims

1. A composition for preventing and / or treating diarrhea, characterized in that The composition comprises three or more of 2'-FL, LNT, 3'-SL and calcium citrate, wherein the composition comprises calcium citrate.

2. The composition according to claim 1, characterized in that The composition consists of 2'-FL, LNT, 3'-SL and calcium citrate.

3. The composition according to claim 1, characterized in that The mass ratio of the 2'-FL, LNT, 3'-SL and calcium citrate is (1-10):(0.1-5):1:(2-15).

4. The composition according to claim 3, characterized in that The mass ratio of the 2'-FL, LNT, 3'-SL and calcium citrate is (1-5):(0.5-3):1:(3-8).

5. The composition according to claim 4, characterized in that The mass ratio of the 2'-FL, LNT, 3'-SL and calcium citrate is (3.5-4.5):(1.5-2.5):1:(6-8).

6. The composition according to claim 4, characterized in that The mass ratio of the 2'-FL, LNT, 3'-SL and calcium citrate is 4:2:1:

7.

7. A medicine, nutritional product or health product containing the composition according to any one of claims 1 to 6.

8. Use of the composition according to any one of claims 1 to 6 or the medicine, nutritional product or health product according to claim 7 in the preparation of a product for preventing and / or treating diarrhea.

9. The use according to claim 8, characterized in that The diarrhea is bacterial infectious diarrhea, preferably Escherichia coli infectious diarrhea.

10. The use according to claim 8, characterized in that The product is used to reduce the propulsion rate of the small intestine; And / or, the product is used to reduce the water content of feces.

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