Compound composition as well as application and product thereof

Abstract

The invention relates to a compound composition. The compound composition is prepared from bifidobacterium breve i1088, choline and phosphatidylserine. The bifidobacterium breve i1088, choline and phosphatidylserine are proportionally combined, so that the components are compounded, and the spatial learning and memory ability of a mouse with APP / PS1 congenital cognitive impairment is remarkably improved. Due to the synergistic effect, the effect of the composition in improving cognition is remarkably improved, and side effects possibly occurring when the components are independently used can be reduced.

Description

Technical Field

[0001] The present invention relates to the field of microbial engineering technology, and relates to a composition and application that can promote intellectual development and alleviate Alzheimer's disease. Specifically, it relates to a composition containing Bifidobacterium breve i1088, choline and phosphatidylserine that can improve cell resistance to oxidative stress and spatial learning and memory ability of APP / PS1 mice, and its application in products. Background Art

[0002] In the application of microbial engineering technology, Bifidobacterium breve, as an important intestinal probiotic, has been widely studied and applied to improve intestinal health and enhance immunity. Bifidobacterium breve is not only sensitive to most antibiotics, but also has good resistance to gastric acid and bile salts, which makes it have broad application prospects in the pharmaceutical and food industries.

[0003] Choline is a key component of cell membrane phospholipids. It is involved in the synthesis of neurotransmitters and is essential for cell structure and function. Phosphatidylserine is a naturally occurring phospholipid that plays an important role in maintaining cell membrane fluidity and function and is believed to enhance nerve signaling. The combination of these two substances can enhance the cell's ability to resist oxidative stress, thereby protecting cells from damage by free radicals.

[0004] Studies have shown that oxidative stress plays a key role in the pathogenesis of Alzheimer's disease (a type of senile dementia). It not only causes irreversible damage to cells and interferes with neurological function, but also triggers pro-inflammatory signaling, cell apoptosis and necrosis (see Bhatt S, Puli L, Patil CR. Role of reactive oxygen species in the progression of Alzheimer's disease [J]. Drug discovery today, 2021, 26 (3): 794-803). Nrf2 (Nuclear factor erythroid 2-related factor 2) is a transcription factor that plays a key role in maintaining cellular redox homeostasis and regulating inflammatory responses. In addition, in the brains of patients with Alzheimer's disease, the expression of Nrf2 and its driver genes (NQO1, HO-1 and GCLC) is decreased. Activating Nrf2 gene expression can provide cellular protection for AD and resist a variety of pathological changes including neurodegenerative lesions (see Osama A, Zhang J, Yao J, et al. Nrf2: a dark horse in Alzheimer's disease treatment [J]. Ageing research reviews, 2020, 64: 101206). This provides a potential therapeutic strategy for alleviating Alzheimer's disease. Summary of the invention

[0005] The following is a summary of the subject matter described in detail herein. This summary is not intended to limit the scope of the claims.

[0006] In order to solve the above deficiencies in the prior art, the present application improves the spatial learning and memory ability of APP / PS1 mice by combining Bifidobacterium breve i1088 with choline and phosphatidylserine in a ratio, and by improving the anti-oxidative stress ability of cells. Among them, APP / PS1 mice are a commonly used Alzheimer's disease model, and the effect of the composition can be verified by evaluating the behavioral and physiological changes of mice. In addition, the composition of the present invention can also be applied to various products, such as food, medicine or health products, to improve cell anti-oxidative stress, promote intellectual development and improve Alzheimer's function, and provide a new raw material combination for the prevention of Alzheimer's disease and the promotion of intellectual development.

[0007] The innovation of this application is that by combining Bifidobacterium breve i1088 with choline and phosphatidylserine, the compounding of these ingredients can better improve the function of improving cell resistance to oxidative stress and improving the spatial learning and memory ability of APP / PS1 mice. This synergistic effect not only improves the effect of the composition, but also may reduce the side effects that may occur when these ingredients are used alone.

[0008] The present application also provides a composition for improving cognition for use in preparing middle-aged and elderly formula milk powder and probiotic-containing health food.

[0009] To achieve the above objectives, the technical solutions adopted in this application are as follows:

[0010] The first aspect of the present application provides a composite composition comprising Bifidobacterium breve i1088. Wherein, the Bifidobacterium breve i1088 has been deposited in the General Microbiological Culture Collection Center of the China Microbiological Culture Collection Administration on May 8, 2023, with a deposit number of CGMCC NO.27281, and its Latin name is Bifidobacterium breve.

[0011] In an exemplary embodiment, the composite composition comprises Bifidobacterium breve i1088, choline and phosphatidylserine.

[0012] In an exemplary embodiment, the usage ratio of Bifidobacterium breve i1088, choline and phosphatidylserine is 0.4 mg to 500 mg: 51 mg to 594.5 mg: 60 mg to 699.4 mg.

[0013] In an exemplary embodiment, the viable count of Bifidobacterium breve i1088 is 1×10 5 CFU / g~5×10 11 CFU / g or 1×10 5 CFU / ml~5×10 11 CFU / ml; for example, 1×10 5 CFU / g or CFU / ml, 5×10 5 CFU / g or CFU / ml, 1×10 6 CFU / g or CFU / ml, 5×10 6 CFU / g or CFU / ml, 1×10 7 CFU / g or CFU / ml, 5×10 7 CFU / g or CFU / ml, 1×10 8 CFU / g or CFU / ml, 5×10 8 CFU / g or CFU / ml, 1×10 9 CFU / g or CFU / ml, 5×109 CFU / g or CFU / ml, 1×10 10 CFU / g or CFU / ml, 5×10 10 CFU / g or CFU / ml, 1×10 11 CFU / g or CFU / ml, 5×10 11 CFU / g or CFU / ml or any two of them are used as range values ​​between endpoint values, but are not limited to the listed values. Other unlisted values ​​within the numerical range are also applicable.

[0014] In an exemplary embodiment, the viable count of Bifidobacterium breve i1088 is 2×10 6 CFU / g~5×10 10 CFU / g or 2×10 6 CFU / ml~5×10 10 CFU / ml.

[0015] In an exemplary embodiment, the compound composition comprises the bacterial liquid or bacterial powder of Bifidobacterium breve i1088.

[0016] In an exemplary embodiment, the concentration of the bacterial solution or powder of Bifidobacterium breve i1088 is 3×10 10 CFU / g or CFU / ml.

[0017] In an exemplary embodiment, the daily intake of Bifidobacterium breve i1088, choline and phosphatidylserine are: 1×10 9 CFU, 94.5mg and 99.4mg.

[0018] The second aspect of the present application provides a product, which comprises the compound composition.

[0019] In an exemplary embodiment, the product is a food, a health product, a medicine or a pharmaceutical composition.

[0020] In an exemplary embodiment, the product is formula milk powder or a probiotic health product.

[0021] In an exemplary embodiment, the formula milk powder is a middle-aged and elderly formula milk powder.

[0022] In an exemplary embodiment, the compounded composition is added to the product in an amount of 0.2% to 13% by weight.

[0023] The third aspect of the present application provides an application of the above-mentioned compound composition or the above-mentioned product, which includes the application of the compound composition or the product in the preparation of a product that enhances the oxidative stress capacity of mammalian cells and improves the expression level of antioxidant-related genes in mammalian cells.

[0024] In an exemplary embodiment, the mammal comprises a human.

[0025] In an exemplary embodiment, the cellular antioxidant-related genes include Nrf2 gene and SODmRNA gene.

[0026] In an exemplary embodiment, the application includes the use of the compound composition or the product in preparing a product having at least one of improving cognitive function and promoting intellectual development.

[0027] In an exemplary embodiment, improving cognitive function includes preventing, alleviating and / or improving cognitive function decline in an individual.

[0028] In an exemplary embodiment, the individual is diagnosed with Alzheimer's disease or early related symptoms or childhood intellectual disability.

[0029] Compared with the related art, this application has the following technical effects:

[0030] By combining Bifidobacterium breve i1088 with choline and phosphatidylserine, it was achieved that the combination of these ingredients showed better improvement in spatial learning and memory ability of APP / PS1 congenital cognitive impairment mice. This synergistic effect not only improves the effect of the combination, but also may reduce the side effects that may occur when these ingredients are used alone.

[0031] Other features and advantages of the present application will be described in the following description, and partly become apparent from the description, or be understood by implementing the present application. Other advantages of the present application can be realized and obtained by the schemes described in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings are used to provide an understanding of the technical solution of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solution of the present application and do not constitute a limitation on the technical solution of the present application.

[0033] Figure 1This is a comparison of the effects of Bifidobacterium breve i1088, Bifidobacterium breve JMCC0794 and Bifidobacterium breve JMCC1106 and their compositions in Example 1 on improving the anti-oxidative stress capacity of cells, wherein ###: compared with the model group, P<0.001; **: compared with the experimental group 1, P<0.05; ***: compared with the experimental group 1, P<0.01;

[0034] Figure 2 The comparison of the effects of Bifidobacterium breve i1088, Bifidobacterium breve JMCC0794 and Bifidobacterium breve JMCC1106 and the combination thereof on the SOD mRNA gene expression of Caco-2 cells under LPS stress in Example 2;

[0035] Figure 3 The comparison of the effects of Bifidobacterium breve i1088, Bifidobacterium breve JMCC0794 and Bifidobacterium breve JMCC1106 and their combination on the expression of NRF2 mRNA gene in Caco-2 cells under LPS stress in Example 2;

[0036] Figure 4 is the percentage of the time each group of mice stayed in the original platform position area in the water maze test in Example 3, where, compared with the blank control group, **p<0.01; compared with the model control group, #p<0.05, ##p<0.001;

[0037] Figure 5 It is the number of times each group of mice crossed the original platform position area in the water maze test in Example 3; compared with the blank control group, **p<0.01; compared with the model control group, #p<0.05, ##p<0.001. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below through specific embodiments and drawings. It should be understood that the described embodiments are only used to explain the present invention, but not to limit the present invention.

[0039] Unless otherwise specified, the materials and reagents used in the examples of the present invention can be obtained from commercial sources. Experimental methods without specific conditions in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0040] The raw materials used in this application are all conventional products on the market.

[0041] In the embodiment:

[0042] Bifidobacterium breve i1088, the strain was deposited in the China General Microbiological Culture Collection Center on May 8, 2023, with the deposit number CGMCC NO.27281, and the Latin name is Bifidobacterium breve;

[0043] The commercial Bifidobacterium breve strains JMCC0794 and JMCC1106 were provided by Junlebao Microbiologiccal Culture Collection (JMCC) and were derived from the intestines of healthy infants.

[0044] Example 1 Bifidobacterium breve i1088, choline and phosphatidylserine synergistically enhance cellular oxidative stress capacity

[0045] Experimental cells: Human colorectal adenocarcinoma cells (Caco-2)

[0046] (1) Experimental reagents, instruments and consumables:

[0047] DMEM cell culture medium, penicillin-streptomycin mixture (100X), cell culture plate (6-well plate), cell scraper, CCK-8 kit (Cell Counting Kit-8), cell workbench, constant temperature incubator (5% CO2), microplate reader (450nm filter), 96-well plate, 3% hydrogen peroxide, pipette (10μL\100μL\1000μL), Bifidobacterium breve i1088, Bifidobacterium breve JMCC0794, Bifidobacterium breve JMCC1106.

[0048] (2) Experimental methods:

[0049] 1. Culture the cells to a confluence of more than 95% and plate them evenly in a 96-well plate, with a fixed cell volume of 5×10 4 Around, culture overnight until adherent;

[0050] 2. Add 8mM hydrogen peroxide and different components of intervention substances to each well and intervene for 6 hours;

[0051] 3. After intervention, add 10 μL of CCK-8 reagent to each well, mix well, incubate at 37°C in the dark for 0.5 h, and then immediately measure the absorbance at 450 nm;

[0052] 4. Experimental groups: blank group (containing cells and culture medium), model group (containing cells, culture medium and hydrogen peroxide), experimental group 1 (containing choline, phosphatidylserine, cells, culture medium and hydrogen peroxide), experimental group 2 (containing i1088, cells, culture medium and hydrogen peroxide), experimental group 3 (containing i1088, choline, phosphatidylserine, cells, culture medium and hydrogen peroxide), experimental group 4 (containing JMCC0794, cells, culture medium and hydrogen peroxide), experimental group 5 (containing JMCC0794, choline, phosphatidylserine, cells, culture medium and hydrogen peroxide), experimental group 6 (containing JMCC1106, cells, culture medium and hydrogen peroxide), experimental group 7 (containing JMCC1106, choline, phosphatidylserine, cells, culture medium and hydrogen peroxide);

[0053] 5. Analysis method: The absorbance at 450 nm is used to indirectly reflect the cell activity index for evaluation. The higher the cell activity, the better the effect of the cells after intervention in resisting oxidative stress.

[0054] 3) Result analysis:

[0055] Compared with the model group, experimental groups 1, 2, 3, 5, 6 and 7 were able to improve the anti-oxidative stress capacity of cells, which was manifested in that the cell activity was significantly different from that of the model group, while the improvement effect of experimental group 4 was not obvious. Intra-group comparison showed that experimental group 3 had a better effect in improving the anti-oxidative stress of cells (see Figure 1 ).

[0056] Example 2 Combination of i1088, choline and phosphatidylserine upregulates the expression level of antioxidant-related genes such as Nrf2 in CaCo-2 cells

[0057] Nrf2 (Nuclear factor erythroid 2-related factor 2) is an important intracellular anti-oxidative stress transcription factor that plays a key role in maintaining cellular redox balance, metabolism and protein homeostasis.

[0058] (1) Experimental method: CaCo-2 cells were seeded in 6-well plates, with approximately 2×10 cells per well. 6cell, the experimental group and the positive control group were treated with 10μg / mL lipopolysaccharide modeling (LPS) for 24 hours to complete the modeling. Except for the blank group and the positive control group, the other groups added different intervention substances (experimental groups 1 to 7 as described in Example 1), intervened for 12 hours, eluted bacteria, extracted total cell RNA and reverse transcribed cDNA using a kit. After the synthesized cDNA was diluted a certain multiple, 1μL of cDNA, 0.5uL of upstream and downstream primers, 3uL of ddH2O, and 5uL of SYBR Green were added to the total system of 10μL, and real-time PCR analysis was performed using a CFX 96TMReal-timeSystem instrument. 2 -△△Ct The relative expression of mRNA was calculated by the method. Figures 2 to 3 , among which, compared with the blank group, *P<0.05, **P<0.01, ***P<0.001. The primer sequences used in this study are shown in Table 1 below.

[0059] Table 1

[0060]

[0061] Intra-group comparison showed that the combination containing Bifidobacterium breve i1088, choline and phosphatidylserine had a better effect on improving the expression levels of the antioxidant gene Nrf2 and SODmRNA gene in CaCo-2 cells.

[0062] Example 3 Combination of i1088, choline and phosphatidylserine improves spatial learning and memory ability in mice

[0063] The preparation method of the composition in this embodiment is: choline + PS (phosphatidylserine) + i1088 bacteria, which is equivalent to the daily intake of 94.5 mg + 99.4 mg + 1×10 9 CFU

[0064] (1) Experimental animals

[0065] C57BL / 6 mice, male, 14 weeks old

[0066] APP / PS1 mice, male, 14 weeks old

[0067] (2) Experimental reagents, instruments and consumables

[0068] Disposable 5 ml syringe (Shanghai Kangdeli, China), electronic analytical balance (Precisa12A, Precisa, Switzerland), depyrogenation ultrapure water system (Millipo3150, Millipore, USA), mouse gavage needle (Zhongke Life Science, China), electric constant temperature water bath (HW-SY11-K P2, Beijing Changfeng Instrument Co., Ltd., China), centrifuge tube (1.5 ml, Wuxi Nice Life Science Co., Ltd., China), water maze apparatus (ACT-200A, Beijing Youcheng Biotechnology Co., Ltd., China).

[0069] Positive control drug: Edaravone (M70800-5G, Sigma-Aldrich, USA).

[0070] (3) Experimental methods

[0071] Animal grouping and oral administration

[0072] After 7 days of adaptive feeding, 50 mice were divided into 10 groups: blank group, model group, positive drug group, experimental group 1 to experimental group 7 as described in Example 1. The blank group used C57BL / 6 male mice without any treatment, and the other groups used APP / PS1 congenital cognitive impairment male mice.

[0073] The mice in each group were gavaged daily, and the mice in the blank group and the model group were given an equal volume of saline daily. The mice in the positive drug group were given 3 mg / kg of edaravone daily; the same gavage dose was controlled in experimental groups 1 to 7: choline equivalent to 94.5 mg per day for the human body, phosphatidylserine equivalent to 99.4 mg per day for the human body, and Bifidobacterium breve equivalent to 1×10 9 CFU. Gavage once a day for 42 consecutive days.

[0074] Water maze experimental training and testing

[0075] 24 hours after the last gavage, each group began water maze training: 4 training sessions per day, each time starting from a different quadrant of the water maze and entering the swimming pool, and one training session ended when the mouse found the underwater platform or lasted for 60 seconds, with an interval of 15 minutes between each training session, and training continued for 4 days.

[0076] After 4 days of training, the water maze test began on the 5th day: each mouse entered the swimming pool (without platform) 4 times, starting from a different quadrant of the water maze each time, and recorded the movement trajectory of the mice within 120 seconds. The proportion of the time each group of mice stayed in the original platform position area during the 120-second test time, as well as the number of times they crossed the original platform position area, were analyzed.

[0077] (4) Results analysis

[0078] The composition containing Bifidobacterium breve i1088, choline and phosphatidylserine has the effect of improving the spatial learning and memory ability of mice. Specifically, it can increase the proportion of APP / PS1 mice's residence time in the original platform position area in the water maze test p<0.01, and increase the number of crossing the original platform position area p<0.01. In addition, the improvement effect of the composition is better than the combination of Bifidobacterium breve A (No. JMCC0794) and Bifidobacterium breve B (No. JMCC1106) of the same genus and choline, phosphatidylserine, indicating that the combination of Bifidobacterium breve i1088 with choline and phosphatidylserine has a certain synergistic effect. See details Figures 4 to 5 .

[0079] Example 4 Application of the composition for improving cognitive efficacy in middle-aged and elderly formula powder

[0080] The choline + PS (phosphatidylserine) + i1088 bacteria (equivalent to the daily intake of 94.5mg + 99.4mg + 1×10 9 CFU) was added to the middle-aged and elderly formula milk powder, wherein the viable count of Bifidobacterium breve i1088 was 2×10 6 ~5×10 7 CFU / g; the added amount of the composition is 0.3-3g / 100g.

[0081] Example 5 Application of the composition for improving cognitive function in health products containing probiotics

[0082] The choline + PS (phosphatidylserine) + i1088 bacteria (equivalent to the daily intake of 94.5mg + 99.4mg + 1×10 9 CFU) is added to a probiotic health product, wherein the viable count of Bifidobacterium breve i1088 is 5×10 7 ~8×10 10 CFU / g, the added amount of the composition is 0.2-20g / 100g.

[0083] Conclusion: In this application, the composition comprising Bifidobacterium breve i1088, choline and PS (phosphatidylserine) can enhance the oxidative stress capacity of mammalian cells, increase the expression level of antioxidant-related genes in mammalian cells, and significantly increase the proportion of the residence time of APP / PS1 congenital cognitive impairment mice in the original platform position area in the water maze experiment test, and increase the number of crossing the original platform position area. Therefore, this application applies a composition with improved cognitive function to middle-aged and elderly formula milk powder, yogurt and health food, in order to provide support for the cognitive health of the middle-aged and elderly population through the adjustment of daily diet, and prevent, alleviate or improve Alzheimer's disease or mixed cognitive impairment.

[0084] It should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the scope of protection of the claims of the present invention.

Claims

1. A composite composition, characterized in that: The composite composition comprises Bifidobacterium breve i1088, choline and phosphatidylserine. The Bifidobacterium breve i1088 has been deposited in the China General Microbiological Culture Collection Center of the China Microbiological Culture Collection Administration on May 8, 2023, with a collection number of CGMCC NO.27281, and its Latin name is Bifidobacteriumbreve.

2. The composite composition according to claim 1, characterized in that The dosage ratio of the Bifidobacterium breve i1088, choline and phosphatidylserine is 0.4mg-500mg: 51mg-594.5mg: 60mg-699.4mg.

3. The composite composition according to claim 1 or 2, characterized in that: The viable count of Bifidobacterium breve i1088 is 1×10 5 CFU / g~5×10 11 CFU / g or 1×10 5 CFU / ml~5×10 11 CFU / ml; Optionally, the viable count of Bifidobacterium breve i1088 is 2×10 6 CFU / g~5×10 10 CFU / g or 2×10 6 CFU / ml~5×10 10 CFU / ml.

4. The composite composition according to claim 1 or 2, characterized in that: The composite composition comprises the bacterial liquid or bacterial powder of Bifidobacterium breve i1088; and / or The concentration of the bacterial solution or powder of Bifidobacterium breve i1088 is 3×10 10 CFU / g or CFU / ml.

5. A product comprising the composite composition according to any one of claims 1 to 4, characterized in that: The product comprises the compound composition according to any one of claims 1 to 4, and optionally, the product is a food, a health product, a medicine or a pharmaceutical composition.

6. The product according to claim 5, characterized in that The product is formula milk powder or probiotic health product; Optionally, the formula milk powder is formula milk powder for middle-aged and elderly people.

7. The product according to claim 5 or 6, characterized in that Calculated by weight percentage, the compound composition is added in an amount of 0.2% to 13% in the product.

8. Use of the composite composition according to any one of claims 1 to 4 or the product according to any one of claims 5 to 7, characterized in that: The application shown includes the use of the compound composition or the product in preparing a product that enhances the oxidative stress capacity of mammalian cells and improves the expression level of antioxidant-related genes in mammalian cells.

9. The use according to claim 8, characterized in that: The mammal includes a human; and / or The cell antioxidant related genes include Nrf2 gene and SODmRNA gene.

10. The use according to claim 8 or 9, characterized in that: The application includes the application of the compound composition or the product in preparing a product having at least one of improving cognitive function and promoting intelligence development; Optionally, the improving cognitive function includes preventing, alleviating and / or improving cognitive function decline of an individual; Optionally, the individual is diagnosed with Alzheimer's disease or early related symptoms or childhood intellectual disability.

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