Synbiotic composition for improving cognitive function and application thereof

Abstract

The invention relates to a synbiotics composition for improving a cognitive function and application of the synbiotics composition. The synbiotics composition comprises lactobacillus reuteri C501 and galactooligosaccharide. The synbiotics composition disclosed by the invention has potential application value in the aspects of preventing, repairing and improving the Alzheimer's disease and improving the cognitive function, and can be incorporated into foods, formula foods with special medical purposes, nutritional supplements, functional foods, health-care foods and medicines. Food acceptable auxiliary materials are added into the synbiotics composition to prepare the food. And adding a pharmaceutically acceptable medical non-toxic carrier to prepare the medicine. The screened synbiotics composition is relatively good in safety, can improve the expression level of brain inflammation genes, exert beneficial effects of the brain inflammation genes and generate exact healthy effects, and animal experiments show that the prepared synbiotics have the function of improving cognitive functions.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial additives, and in particular relates to a synbiotic composition for improving cognitive function and an application thereof. Background Art

[0002] Cognitive impairment refers to impairment in one or more cognitive domains, including memory, thinking, language, attention, executive function, reasoning, and visuospatial function, resulting from various causes. This impairment is highly prevalent in the elderly population and has complex etiologies, potentially caused by a variety of factors, including neurodegenerative diseases, cerebrovascular disease, and psychiatric illness. Cognitive impairment can be categorized by severity as mild cognitive impairment (MCI) and dementia. Alzheimer's disease (AD) is the most common type of dementia and the leading cause of cognitive impairment in the elderly. With the increasing aging population in my country, neurodegenerative diseases such as AD are becoming a major public health concern threatening the health of the population. It is estimated that there are currently over 16 million AD patients in my country, resulting in annual economic losses exceeding US$240 billion, a figure projected to double by 2030. Therefore, the prevention and treatment of geriatric diseases has become a research hotspot. Symptoms of AD include decreased cognitive memory, as well as certain language and behavioral impairments, which significantly impact patients' quality of life and place a heavy burden on their families and society. Synbiotics are a combination of probiotics and prebiotics. Probiotics are active microorganisms that are beneficial to the host. Prebiotics, on the other hand, are food ingredients or substances that are not easily absorbed by the human body but can selectively stimulate the growth of beneficial intestinal bacteria or enhance the activity of beneficial bacteria. As a microbial preparation, synbiotics can exert prebiotic effects by regulating the intestinal microenvironment and enhance host health. Synbiotics are now widely used in human and animal health care and disease prevention and treatment. Due to their many advantages, such as high efficiency and no residue, they are expected to become a new strategy for disease prevention and treatment in the future. As a microbial preparation, synbiotics primarily exert their prebiotic effects by regulating the production of metabolites by beneficial microorganisms. Therefore, increasing the levels of beneficial microbial metabolites in the body is the primary purpose of synbiotic supplementation. Research has shown a close link between the gut microbiome and AD. An imbalance in the gut microbiome can cause harmful substances to enter the circulatory system, triggering neuroinflammation and impairing cognitive function. Metabolites produced by gut microbes can influence the pathological progression of AD by regulating neurotransmitters and immune responses. Furthermore, the gut microbiome communicates directly with the brain through the "gut-brain axis," influencing mood and memory. Therefore, regulating the gut microbiome may provide a new strategy for the prevention and treatment of AD. Lactobacillus reuteri (L. reuteri), a common probiotic, has been found to improve behavioral manifestations similar to those in Autism Spectrum Disorder (ASD) in mouse models. Therefore, to address these issues, developing a synbiotic product based on L. reuteri to improve cognitive function is of great significance. Summary of the Invention

[0003] Technical problem to be solved: The purpose of the present invention is to provide a synbiotic composition for improving cognitive function, so as to effectively improve cognitive function damage and brain inflammation.

[0004] Technical solution: A synbiotic composition for improving cognitive function, comprising probiotics and prebiotics, wherein the probiotic is Lactobacillus reuteri C501; and the prebiotic comprises galacto-oligosaccharide. Furthermore, the Lactobacillus reuteri C501Lactobacillus reuteri was deposited in the Guangdong Provincial Microbiological Culture Collection Center on May 17, 2022, with the deposit number GDMCC No: 62470. The preparation process of the above-mentioned synbiotic composition for improving cognitive function based on Lactobacillus reuteri comprises the following steps: S1: Based on the complete genome information of Lactobacillus reuteri C501, all gene sequences were functionally predicted using bioinformatics methods to screen out all carbohydrate utilization-related genes; S2: Functional genomic analysis of carbohydrate utilization based on substrates to predict candidate prebiotics for the strain; S3: Validate the predicted information in vitro by using the predicted prebiotic as the sole carbon source and MRS medium without carbon source as the control. Measure the growth curve of the strain in the culture medium with the corresponding prebiotic as the substrate using a fully automatic microbial growth curve analyzer to determine the effect of the prebiotic. S4: Determine the composition and proportion of the synbiotics to obtain a synbiotic composition. Furthermore, the candidate prebiotics in S2 include galacto-oligosaccharides, fructo-oligosaccharides, chitosan, inulin, and highland barley β-glucan. Furthermore, the synbiotics in S4 are composed of Lactobacillus reuteri C501 and galacto-oligosaccharides. Furthermore, the polymerization degree of the galacto-oligosaccharide is 2-6. Furthermore, the dosage ratio of the above Lactobacillus reuteri C501 and galacto-oligosaccharide is (1×10 7 ~1×10 11 ) CFU / mL:(5~40)g / L. Furthermore, the dosage ratio of the above Lactobacillus reuteri C501 and galacto-oligosaccharide is (1×10 8 ~1×10 10 ) CFU / mL:(20~40)g / L. Furthermore, the concentration of the Lactobacillus reuteri C501 is 1×10 9 CFU / mL, and the concentration of galacto-oligosaccharide was 30 g / L. Furthermore, the above-mentioned synbiotic composition is used in foods for preventing and repairing Alzheimer's disease, cognitive function, special medical purpose formula foods, nutritional supplements, functional foods, health foods and medicines. Furthermore, a food-acceptable excipient is added to the synbiotic composition to prepare a food; or a pharmaceutically acceptable non-toxic carrier is added to the synbiotic composition to prepare a drug; the dosage form of the drug is selected from one of powder, tablet, granule, capsule, solution, suspension, emulsion, and lyophilized preparation. Beneficial effects: 1. The synbiotic composition of the present invention, namely the probiotic (Lactobacillus reuteri C501) and the prebiotic (galacto-oligosaccharide), is a newly discovered synbiotic combination. After research, it was found that both have a good effect on improving cognitive function. When the two are used together, the effect is significantly increased, and there is a clear synergistic effect, which is specifically manifested in significantly improving the structure of the intestinal flora, inhibiting the growth of harmful bacteria, and reducing the production of harmful metabolites, thereby improving cognitive function and inhibiting the level of brain inflammation. 2. The synbiotic composition of the present invention has a significant synergistic effect by combining probiotics and prebiotics and utilizing a reasonable ratio of the two. The prebiotics can effectively increase the number of probiotics in the composition and improve the survival rate of probiotics in the intestine. 3. The dosage of the method of administration of the present invention is reasonable and the efficacy is significant, which effectively avoids the ineffectiveness of the existing probiotic preparations due to unclear dosage or the adverse effects that may be caused by excessive intake. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 The effects of the intervention doses of specific synbiotics on the growth of probiotics in vitro in Example 1 were as follows: (A) 5 g / L, (B) 10 g / L, (C) 20 g / L, (D) 30 g / L, and (E) 40 g / L. Figure 2 The growth of Lactobacillus reuteri C501 under different concentrations of galacto-oligosaccharide in Example 2; Figure 3 Effects of specific synbiotics on cognitive impairment in humanized AD mice, including (A) open-arm to closed-arm time ratio in elevated plus maze test, (B) central-to-peripheral time ratio in open field test, and (C) preference index in novel object recognition test. Figure 4 The effects of specific synbiotics on neuroinflammatory response in AD humanized mice, including (A) IL-6 mRNA expression in the cortex, (B) IL-1β mRNA expression in the cortex; Figure 5 Effects of specific synbiotics on intestinal tissue morphology and intestinal barrier in AD humanized mice (A) H&E staining, (B) Claudin-1 immunofluorescence staining. DETAILED DESCRIPTION In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further described below in conjunction with specific examples, but the following examples are only preferred embodiments of the present invention, not all. Based on the examples in the embodiments, other embodiments obtained by those skilled in the art without making creative work all fall within the scope of protection of the present invention. Lactobacillus reuteri C501 in the present invention is deposited in Guangdong Province Microbial Culture Collection Center with a deposit number of GDMCC No: 62470; the experimental methods in the following examples, unless otherwise specified, are conventional methods, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels. In some specific implementation cases, the synbiotic composition includes probiotics and prebiotics, the probiotic is Lactobacillus reuteri C501, the prebiotic is galacto-oligosaccharide, and the synbiotic composition contains a dosage of 1×10 9 CFU / day of probiotics and 30g / L of galacto-oligosaccharides. The technical solution of the present invention is further described in detail below through examples and in conjunction with the accompanying drawings. However, the selected examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Example 1 Screening of prebiotics in synbiotic compositions 1. Materials and Methods 1.1 Experimental Materials Selected strain: Lactobacillus reuteri C501 Main reagents: MRS culture medium; galacto-oligosaccharide, purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; fructo-oligosaccharide, purchased from Shaanxi Baichuan Biotechnology Co., Ltd.; chitosan, purchased from Shaanxi Baichuan Biotechnology Co., Ltd.; inulin, purchased from Shanghai Yuanye Biotechnology Co., Ltd.; highland barley β-glucan, purchased from Shaanxi Baichuan Biotechnology Co., Ltd. 1.2 Experimental methods First, the whole genome information of Lactobacillus reuteri C501 was obtained through whole genome sequencing technology. The sequencing results of the strain were compared with the CAZy database to obtain the carbohydrate-active enzymes of the strain, and the glycoside hydrolases that can hydrolyze carbon sources were screened out, completing the prebiotic prediction of the strain. Secondly, the predicted information was verified in vitro: the strain was transferred to MRS liquid culture medium and cultured at 37°C for more than 24 hours; culture medium containing different carbon sources was prepared, and each carbon source was divided into a series of concentration gradients of 5g / L, 10g / L, 20g / L, 30g / L, and 40g / L. The predicted prebiotic was used as the only carbon source, MRS without glucose was used as the blank control, and glucose was used as the carbon source as the positive control. First, 300μL of carbon source was added to the well plate, and then 50μL of bacterial solution was added. It was then placed in an automatic growth curve analyzer and the OD value of 24h was measured. 600 The growth curve of microorganisms is drawn by changing the value. Finally, the effect of prebiotics is determined based on the growth curve results, the dominant prebiotics are screened out, and the composition of synbiotics is determined. Test results 1. Genome-wide functional analysis The gene information of Lactobacillus reuteri C501 was compared with the carbohydrate active enzyme database. The results are shown in Table 1. It was found that Lactobacillus reuteri C501 can metabolize five carbon sources. Table 1 Types of enzymes involved in the metabolism of five carbon sources possessed by Lactobacillus reuteri C501 2. Growth curve results Lactobacillus reuteri C501 was cultured in MRS medium (without glucose) supplemented with different carbon sources, and the growth of the strain was analyzed using an automatic growth curve analyzer. The results are as follows: Figure 1 As shown, it can be seen that except chitosan, the other four carbon sources can promote the growth of Lactobacillus reuteri, among which oligogalactose has the best growth promoting effect on Lactobacillus reuteri C501. Example 2 Determination of the ratio of synbiotics 1. Experimental materials: MRS culture medium, galacto-oligosaccharide 2. Experimental method: Lactobacillus reuteri C501 was inoculated into different concentrations of galacto-oligosaccharide culture medium and cultured for 24 hours to explore the optimal ratio of Lactobacillus reuteri C501 and galacto-oligosaccharide. The concentration of Lactobacillus reuteri C501 was 1×10 9 CFU / ml, and the concentration of galacto-oligosaccharide in MRS medium without glucose was 5g / L, 10g / L, 20g / L, 30g / L, and 40g / L. After adding 50μL of bacterial solution to the honeycomb plate, 300μL of carbon source solution was added. Each concentration was repeated three times. The growth of the strain in medium containing different concentrations of galacto-oligosaccharide was recorded using a growth curve analyzer to screen the optimal galacto-oligosaccharide concentration for the growth of Lactobacillus reuteri C501 and determine the appropriate composition of the synbiotic. Test results: The test results are as follows Figure 2 As shown in the figure, it can be seen that Lactobacillus reuteri C501 grows best at a galacto-oligosaccharide concentration of 30 g / L, so the synbiotic ratio is determined to be 1×10 9 CFU / mL, and the concentration of galacto-oligosaccharide was 30 g / L. A trial to improve cognitive impairment The present invention uses animal experiments to evaluate the efficacy of the synbiotic composition to demonstrate its effectiveness. Typically, a synbiotic composition is combined with probiotics and prebiotics and administered to mice via both drinking water and oral gavage. However, the present animal experiment used probiotics administered via oral gavage and prebiotics administered via drinking water, achieving the desired effect. 1. Grouping of experimental animals Sixty 4.5-month-old C57BL / 6 mice were selected for the experiment and divided into six groups, with 10 mice in each group. The mice were treated with probiotics, prebiotics, and synbiotics for two weeks. The specific groups are as follows: (1) Control group CON: intragastric administration of PBS + sterile water; (2) Healthy group FMT-HC+PBS: antibiotics + oral HC feces + oral PBS + sterile water; (3) AD patient group FMT-AD+PBS: antibiotics + feces of AD patients administered orally + PBS + sterile water administered orally; (4) AD patient probiotic group FMT-AD+Pro: antibiotics + feces of AD patients gavaged + Lactobacillus reuteri C501 gavaged + sterile water; (5) AD patient prebiotic group FMT-AD+Pre: antibiotics + oral administration of AD patient feces + oral administration of PBS + GOS drinking water; (6) AD patient synbiotic group FMT-AD+Syn: antibiotics + oral administration of AD patient feces + oral administration of Lactobacillus reuteri C501+GOS drinking water. 2. Processing methods for each group Control treatment: normal drinking water, and oral gavage supplemented with 100 μL of PBS every day. Prebiotic treatment: dissolve galacto-oligosaccharide in sterile water, adjust to a concentration of 30 g / L, and supplement with drinking water. Probiotic treatment: suspend the cultured Lactobacillus reuteri in an appropriate amount of PBS until the viable count reaches 10 9 CFU / mL, freshly prepared for use. 100 μL of bacterial suspension was supplemented daily by oral gavage. Synbiotic treatment: 10 live bacteria count per day 9 CFU / mL bacterial suspension was supplemented by gavage and 30g / L prebiotics were supplemented in drinking water. 3. Experimental Methods (1) Elevated plus maze test The elevated plus maze test is a behavioral test for measuring anxiety-like behavior in mice. A 1.5-meter-high cross-shaped platform is designed for mice. The cross-shaped arms of the platform are open horizontally (i.e., flat panels) and closed vertically (i.e., panels approximately 10 cm high on either side of the flat panels). This prevents the mouse from observing the ground in the closed arms. Mice are placed at the intersection, and the Supermaze animal behavior analysis system is used to record the time and distance the mouse spends in the open and closed arms over a 5-minute period. The time the mouse spends in the open arms is calculated. (2) Open field test The open field test can be used to assess the locomotor activity of experimental animals. First, test mice are randomly placed in the middle of the experimental apparatus. An automated data acquisition and processing system records the mouse's movement trajectory and total distance covered over a 5-minute period to assess the mouse's locomotor activity. It is important to note that before each mouse is tested, the experimental apparatus must be sprayed with alcohol and cleaned thoroughly to prevent interference from mouse odor. (3) New object recognition test The new object recognition experiment was conducted over three days: Acclimation Day: Remove the mouse from its cage and place it in an empty box (size: 40 cm × 40 cm × 40 cm). Allow the mouse to freely explore the box for 5 minutes. After exploration, temporarily place the mouse in an empty cage. Continue acclimating the mice until all mice in the cage have completed acclimation training. Thoroughly clean the box with 75% ethanol each time. On the training day, place two identical objects diagonally inside a box. Remove the test mouse from its cage and place it in the center of the box, equidistant from the two objects. Allow the mouse to explore freely for 5 minutes. At the end of the trial, temporarily place the mouse in an empty cage until all mice in that cage have completed the habituation training. Thoroughly clean the box and the objects with 75% ethanol between each trial. On the test day, one object used during the training day (the old object) and one new object were placed in the box at the same angles as on the training day. The mice were removed from their cages and placed in the center of the box, equidistant from the old and new objects. The mice were allowed to explore freely for 5 minutes. The time they spent exploring the new and old objects was recorded, and the entire exploration trajectory of the mice was recorded and stored in a computer. At the end of the test, the mice were removed. Test results The spatial memory and working memory abilities of mice were evaluated using the elevated plus maze test, open field test, and novel object recognition test. Figure 3 It can be seen that compared with HC mice, AD mice showed anxiety behavior and impaired spatial memory ability. After 2 weeks of synbiotic intervention, the results of the elevated plus maze test showed that the time AD mice stayed in the open arm was significantly improved, and was better than the prebiotic and probiotic groups. The open field test showed that the autonomous activity ability of AD mice was significantly improved after synbiotic intervention. The discrimination index of the new object recognition test showed that the mice's ability to recognize new objects was significantly better than that of AD mice after intervention. The above results show that synbiotics can improve the spatial memory and learning ability of Alzheimer's mice. Test for inhibiting the expression of pro-inflammatory factors in brain tissue 1. Experimental grouping and treatment are the same as in Example 2. 2. Experimental Methods The mice were killed after 2 weeks of intervention, and their brain tissues were collected to measure the levels of inflammatory factors in the cortex by qRT-PCR. Test results The level of brain tissue inflammation was detected by qRT-PCR. Figure 4 It can be seen that the transcription level of inflammatory factors in the brain of AD mice was significantly increased. After 2 weeks of synbiotic intervention, the expression of pro-inflammatory factors in their brain tissue was inhibited and the inflammation level was significantly reduced. Improvement of intestinal tissue morphology test 1. Experimental grouping and treatment are the same as in Example 2. 2. Experimental Methods The intestinal barrier function was evaluated by observing the intestinal tissue morphology by H&E staining and observing the expression of tight junction protein Claudin-1 by immunofluorescence staining. Test results The intestinal tissue morphology was observed by H&E staining, and the expression of tight junction protein Claudin-1 was observed by immunofluorescence staining. Figure 5 It can be seen that synbiotic intervention alleviated the crypt damage and inflammatory cell infiltration in AD humanized mice, significantly increased the expression level of tight junction proteins, and improved intestinal barrier damage. Example 3 The synbiotic composition is prepared into tablets A preparation of a synbiotic composition for improving cognitive impairment, wherein the preparation is prepared by adding a food-acceptable excipient to the synbiotic composition for improving cognitive impairment to prepare a food, or by adding a pharmaceutically acceptable excipient to prepare a tablet. Example 4 The synbiotic composition is prepared into granules A preparation of a synbiotic composition for improving cognitive impairment, wherein the preparation is prepared by adding a food-acceptable excipient to the synbiotic composition for improving cognitive impairment to prepare a food, or by adding a pharmaceutically acceptable excipient to prepare a granule.

Claims

1. A synbiotic composition for improving cognitive function, characterized in that: The invention comprises probiotics and prebiotics, wherein the probiotics are Lactobacillus reuteri C501 and the prebiotics include galacto-oligosaccharide.

2. The synbiotic composition for improving cognitive function according to claim 1, characterized in that The polymerization degree of the oligogalactose is 2-6.

3. The synbiotic composition for improving cognitive function according to claim 1, characterized in that: The Lactobacillus reuteri C501 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on May 17, 2022, with the deposit number GDMCCNo:62470.

4. The synbiotic composition for improving cognitive function according to claim 1, characterized in that The dosage ratio of Lactobacillus reuteri C501 and galacto-oligosaccharide is (1×10 7 ~1×10 11 )CFU / mL:(5~40)g / L.

5. The synbiotic composition for improving cognitive function according to claim 1, characterized in that The dosage ratio of Lactobacillus reuteri C501 and galacto-oligosaccharide is (1×10 8 ~1×10 10 )CFU / mL:(20~40)g / L.

6. The synbiotic composition for improving cognitive function according to claim 1, characterized in that: The concentration of Lactobacillus reuteri C501 is 1×10 9 CFU / mL, and the concentration of galacto-oligosaccharide was 30 g / L.

7. Use of the synbiotic composition for improving cognitive function according to any one of claims 1 to 6 in preventing, repairing and improving Alzheimer's disease, in foods for cognitive function, special medical purpose formula foods, nutritional supplements, functional foods, health foods and medicines.

8. The use according to claim 7, characterized in that The synbiotic composition is prepared by adding excipients acceptable to food to the composition; or, the synbiotic composition is prepared by adding a pharmaceutically acceptable non-toxic carrier to the composition.

9. The use according to claim 7, characterized in that The dosage form of the drug is selected from one of powder, tablet, granule, capsule, solution, suspension, emulsion and freeze-dried preparation.

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