Application of probiotic composition in improvement of intestinal flora and preparation of product containing probiotic composition

Abstract

The invention discloses application of a probiotic composition to improvement of intestinal flora and preparation of a product containing the intestinal flora. The probiotic composition comprises (1) lactobacillus plantarum; the preservation number is CCTCC (China Center For Type Culture Collection) NO: M2022699 (2) bifidobacterium breve; the preservation number of the strain is CCTCC NO: M 20221846. The probiotic composition and the composition containing the probiotic composition can effectively regulate intestinal flora disorder caused by high-fat diet, improve the abundance of beneficial bacteria in intestinal tracts, reduce the abundance of harmful bacteria, enhance the richness and diversity of intestinal flora and relieve intestinal inflammation.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an application of a probiotic combination in improving intestinal flora and preparing a product containing the probiotic combination. Background Art

[0002] Intestinal flora refers to the microbial community in the human intestine, including bacteria, fungi, viruses and other microorganisms. Beneficial and harmful bacteria in the intestinal flora work together in the intestine and participate in various physiological functions such as human digestion, nutrient absorption, and immune regulation. At the same time, the intestinal flora can also synthesize a variety of beneficial substances, such as vitamins, amino acids, short-chain fatty acids, etc., which play an important regulatory role in human health. The intestinal barrier refers to the immune barrier composed of intestinal mucosal cells, mucus layer, immune cells, etc., which is easily affected by external factors such as different dietary structures. Studies have confirmed that the balance of intestinal flora is closely related to the maintenance of intestinal barrier function. Excessive consumption of fat can lead to intestinal flora disorder and intestinal barrier dysfunction. The interaction between the latter two will further increase the risk of metabolic diseases. Some studies have shown that a high-fat diet can lead to an imbalance of intestinal flora, including a decrease in flora diversity, a decrease in the number of beneficial flora, and an increase in the number of harmful flora. This disorder is often accompanied by the occurrence and development of intestinal inflammation, which further aggravates metabolic dysfunction and induces intestinal inflammation, inflammatory bowel disease, obesity and metabolic diseases. Restoring intestinal flora homeostasis through flora intervention and regulation can help reduce intestinal inflammation caused by a high-fat diet, restore intestinal barrier function, and reduce the risk of other diseases.

[0003] At present, the methods for regulating intestinal flora mainly include the following: (1) Using antibiotics for regulation: Antibiotics are widely used drugs that can quickly and effectively kill intestinal pathogens and achieve the purpose of regulating flora; (2) Regulation through diet: The main principle of dietary regulation is that by eating foods rich in dietary fiber and other ingredients, this type of dietary fiber is fermented by intestinal microorganisms in the intestine to produce a variety of short-chain fatty acids, which can promote the growth and reproduction of beneficial bacteria and inhibit the growth of harmful bacteria; (3) Regulating intestinal flora through the supplementation of probiotics and prebiotics: Probiotics and prebiotics are effective methods for regulating intestinal flora. They can promote the growth and reproduction of beneficial bacteria and inhibit the growth of harmful bacteria; (4) Regulating intestinal flora through the use of plant extracts, etc.: Some components in plants can affect intestinal flora. For example, scutellaria baicalensis extract, wolfberry extract, etc. can inhibit the growth of harmful bacteria, while dandelion extract, etc. can promote intestinal peristalsis and reduce the growth of harmful bacteria.

[0004] Although the above methods of regulating intestinal flora are good, they all have certain disadvantages. Using antibiotics to regulate harmful intestinal flora is a quick and effective method, but it will also destroy beneficial flora and more easily lead to dysbacteriosis. In addition, antibiotics can also cause damage to the intestinal mucosal barrier, increase the invasion of foreign harmful substances, aggravate intestinal inflammation, and thus cause a variety of intestinal diseases; dietary regulation of intestinal flora is a safe and effective method, but it requires long-term persistence and adjustment of eating habits. For some people, dietary changes may not be easy to achieve, and different people may have different effects on the same diet. The supplementation of probiotics and prebiotics is a very popular method of regulating intestinal flora, and its safety and effectiveness have been widely recognized. However, there are many types of probiotics and prebiotics, and different types of probiotics and prebiotics have different effects on different flora, requiring individualized regulation; plant extracts, as natural ingredients, may have the disadvantages of slow onset and unclear effects when used alone, and need to be used for a long time. In addition, there are certain differences in the types and abundance of microorganisms between the intestinal flora disorders caused by a high-fat diet and those caused by other factors, and the ways of regulating the flora also show certain differences. Therefore, the development of safer and more effective methods, especially for regulating the intestinal flora disorders caused by a high-fat diet, is still a hot topic and difficulty in current research. Summary of the invention

[0005] In view of the deficiencies of the prior art and actual needs, the present invention provides an application of a probiotic combination in improving intestinal flora and preparing products containing the probiotic combination, wherein the probiotic combination comprises: (1) Lactobacillus plantarum; the preservation number is CCTCC NO: M2022699; and (2) Bifidobacterium breve; the preservation number is CCTCC NO: M 20221846. The probiotic combination composition is a composition composed of spinach extract and probiotics. The innovative combination of probiotics and spinach extract can effectively regulate the intestinal flora disorder caused by a high-fat diet, increase the abundance of beneficial intestinal bacteria, reduce the abundance of harmful bacteria, enhance the richness and diversity of intestinal flora, and reduce intestinal inflammation.

[0006] In order to solve the above technical problems, the first aspect of the present invention provides an application of a probiotic combination in improving intestinal flora or preparing a product containing the probiotic combination, wherein the probiotic composition comprises:

[0007] (1) Lactobacillus plantarum; the deposit number is CCTCC NO: M 2022699;

[0008] (2) Bifidobacterium breve; the deposit number is CCTCC NO:M 20221846.

[0009] In order to solve the above technical problems, the second aspect of the present invention provides an application of a composition in improving intestinal flora or preparing a product containing the composition, wherein the composition comprises the probiotic combination as described in the first aspect of the present invention.

[0010] The probiotic composition of the present invention can increase the relative abundance of beneficial intestinal flora (Oscillibacter, Akkermansia, Rikenellaceae RC9, Muribaculaceae, Bacteroides) under a high-fat diet, reduce the relative abundance of harmful intestinal bacteria (Lachnoclostridium, Enterorhabdus), and improve the diversity and richness of intestinal flora. The combination of the two can effectively regulate the changes in intestinal flora and systemic inflammatory response (reducing the levels of serum proinflammatory factors IL-1β, IL-6 and TNF-α, and increasing the level of serum anti-inflammatory factor IL-10) caused by a high-fat diet.

[0011] In some preferred embodiments of the present invention, the composition further comprises a plant extract.

[0012] In some more preferred embodiments of the present invention, the chlorophyll content of the plant extract is 1-5%, such as 3%.

[0013] In some embodiments of the present invention, the plant extract is spinach extract. Spinach extract can enhance the efficacy of probiotics in inhibiting intestinal pathogens by promoting the growth of probiotics. At the same time, the effect of spinach extract is not interfered by the gastrointestinal environment, which solves the shortcoming that probiotics take effect slowly when used alone.

[0014] In some preferred embodiments of the present invention, the mass ratio of the probiotics to the spinach extract is 1:1-1:5.5.

[0015] In some embodiments of the present invention, the preparation of the spinach extract comprises the following steps: fresh spinach is crushed, a mixture of ethanol and isopropanol is added at a solid-liquid ratio of 1:10, the mixture is centrifuged to obtain the supernatant, the supernatant is concentrated to 1 / 10-1 / 30 of the original volume, and the lower precipitate is obtained.

[0016] In some embodiments of the present invention, the preparation of the spinach extract satisfies one or more of the following conditions:

[0017] (1) In the mixed solution, ethanol:isopropanol=2:1;

[0018] (2) the centrifugation is performed at 8000-15000 rpm for 10-30 min, for example, at 10000 rpm for 15 min;

[0019] (3) The precipitate is obtained by standing or centrifuging; for example, standing overnight at 4° C.;

[0020] (4) The spinach extract is obtained by freeze-drying the precipitate.

[0021] In some embodiments of the present invention, the composition comprises, by mass fraction:

[0022] 8.00%~25.00% Lactobacillus plantarum;

[0023] 8.00%~25.00% Bifidobacterium breve;

[0024] 50.00%~84.00% spinach extract.

[0025] In some embodiments of the present invention, the composition comprises, by mass fraction, 8% Lactobacillus plantarum, 8% Bifidobacterium breve and 84% spinach extract; or, 12.50% Lactobacillus plantarum, 12.50% Bifidobacterium breve and 75.00% spinach extract; or, 25% Lactobacillus plantarum, 25% Bifidobacterium breve and 50% spinach extract.

[0026] In some embodiments of the present invention, the probiotic composition is prepared by the following method: weigh the probiotic combination and the plant extract according to a proportion, and mix them evenly to obtain the probiotic composition.

[0027] In some preferred embodiments of the present invention, the probiotics and the plant extracts in the probiotic combination are freeze-dried products.

[0028] In some embodiments of the present invention, the application is for non-therapeutic purposes; the performance of improving the function of intestinal flora is selected from one or more of the following:

[0029] (1) Promote human digestion and nutrient absorption;

[0030] (2) Maintaining the balance of intestinal flora, including maintaining or increasing the number of beneficial flora and maintaining or reducing the number of harmful flora;

[0031] (3) Maintain or restore intestinal barrier function and reduce intestinal inflammation.

[0032] In some embodiments of the present invention, the product is used in food, health products or medicines for improving intestinal flora disorders, improving intestinal barrier dysfunction and treating metabolic diseases.

[0033] In some preferred embodiments of the present invention, the intestinal flora disorder, intestinal barrier dysfunction and metabolic diseases are caused by a high-fat diet, including one or more of the following:

[0034] (1) Imbalance of intestinal flora, including decreased diversity of intestinal flora, decreased number of beneficial flora, and increased number of harmful flora;

[0035] (2) Metabolic dysfunction;

[0036] (3) intestinal barrier dysfunction;

[0037] (4) intestinal inflammation, inflammatory bowel disease, obesity, and metabolic diseases;

[0038] (5) Systemic inflammatory response.

[0039] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0040] The reagents and raw materials used in the present invention are commercially available.

[0041] The positive and progressive effects of the present invention are:

[0042] The probiotic combination of the present invention and the composition containing the probiotic combination can increase the relative abundance of intestinal native beneficial bacteria (Oscillibacter, Akkermansia, Rikenellaceae RC9, Muribaculaceae, Bacteroides) under a high-fat diet, reduce the relative abundance of intestinal pathogens (Lachnoclostridium, Enterorhabdus), and improve the diversity and richness of intestinal flora. The combination of the two can effectively regulate the changes in intestinal flora and systemic inflammatory response (reducing the levels of serum proinflammatory factors IL-1β, IL-6 and TNF-α, and increasing the level of serum anti-inflammatory factor IL-10) caused by a high-fat diet. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The species composition at the phylum level of the intestinal flora sequencing of each group of mice in Example 2 of the present application;

[0044] Figure 2 This is the species composition at the genus level of the intestinal flora sequencing of each group of mice in Example 2 of the present application.

[0045] Description of biological material deposit

[0046] Lactobacillus plantarum BGI-N1 ​​(Lactobacillus plantarum BGI-N1) was deposited in the China Center for Type Culture Collection (CCTCC) on May 23, 2022, with the address being No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code 430072. The culture name is Lactobacillus plantarum BGI-N1, the classification name is Lactobacillus plantarum (Lactobacillus plantarum), and the preservation number is CCTCC NO: M 2022699.

[0047] Bifidobacterium breve BGI-N5 (Bifidobacterium breve BGI-N5) was deposited in the China Center for Type Culture Collection (CCTCC) on November 30, 2022, with the address being No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code 430072. The culture name is Bifidobacterium breve BGI-N5, the classification name is Bifidobacterium breve (Bifidobacterium breve), and the preservation number is CCTCCNO: M 20221846. DETAILED DESCRIPTION

[0048] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0049] Example 1 Preparation of composition

[0050] Lactobacillus plantarum BGI-N1 ​​and Bifidobacterium breve BGI-N5 were isolated from human intestinal samples and preserved in the China Center for Type Culture Collection. After the strains were isolated and identified, they were prepared into freeze-dried bacterial powder by high-density fermentation, centrifugation and vacuum freeze-drying using conventional processes. The live bacterial content of Lactobacillus plantarum BGI-N1 ​​was 3.0×10 11 CFU / g, the content of live bacteria of Bifidobacterium breve BGI-N5 is 5.0×10 11 CFU / g. Spinach extract is extracted from fresh spinach. After the fresh spinach is crushed by a blender, a mixture of ethanol and isopropanol (ethanol: isopropanol = 2:1) is added according to a solid-liquid ratio of 1:10. The crushed mixture is centrifuged at 10000r / min for 15 minutes, and the supernatant is taken. The supernatant is concentrated to 1 / 20 of the original volume using a rotary evaporator, placed in a 4°C refrigerator for 12 hours, and the dark green precipitate is taken and freeze-dried to obtain the spinach extract. The chlorophyll concentration in the obtained extract is 3%.

[0051] The preparation method of the composition of experimental group 1 for regulating intestinal flora disorder caused by high-fat diet is to prepare the composition according to the following mass ratio: freeze-dried bacterial powder of Lactobacillus plantarum BGI-N1 ​​(8.00%), freeze-dried bacterial powder of Bifidobacterium breve BGI-N5 (8.00%), spinach extract (84.00%), weigh the above ingredients according to the proportion, mix them well to obtain the composition with the effect of regulating intestinal flora disorder caused by high-fat diet.

[0052] The preparation method of the composition of experimental group 2 for regulating intestinal flora disorder caused by high-fat diet is prepared according to the following mass ratio: freeze-dried bacterial powder of Lactobacillus plantarum BGI-N1 ​​(12.50%), freeze-dried bacterial powder of Bifidobacterium breve BGI-N5 (12.50%), spinach extract (75.00%), weigh the above ingredients according to the proportion, mix them well to obtain the composition with the effect of regulating intestinal flora disorder caused by high-fat diet.

[0053] The preparation method of the composition of experimental group 3 for regulating intestinal flora disorder caused by high-fat diet is prepared according to the following mass ratio: freeze-dried bacterial powder of Lactobacillus plantarum BGI-N1 ​​(25.00%), freeze-dried bacterial powder of Bifidobacterium breve BGI-N5 (25.00%), spinach extract (50.00%), weigh the above ingredients according to the proportion, mix them well to obtain the composition with the effect of regulating intestinal flora disorder caused by high-fat diet.

[0054] Example 2 Effect verification of the composition: Animal test on mice fed a high-fat diet

[0055] To explore the regulatory effect of probiotics and spinach extract composition on intestinal flora disorder and systemic inflammatory response caused by high-fat diet, the present embodiment uses animal experiments to verify the effect. C57BL / 6N mice (3 weeks old, male, 32 in total, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were selected for experiment. The feeding of experimental animals was carried out according to standard guidelines, 12h light and dark alternation, free access to food and water. The experimental animals were randomly divided into 2 groups according to body weight after one week of adaptive feeding, and experimental grouping and sample preparation were carried out according to Table 1. The experiment was a total of 10 weeks, and intestinal flora was measured in the 10th week.

[0056] Table 1: Animal experiment grouping and intervention measures

[0057]

[0058] Index detection:

[0059] After 10 weeks of intervention, mice were anesthetized with ether and blood was collected from the ocular vein. The blood was centrifuged at 4000 r / min for 10 min at 4°C to obtain the upper serum and stored at -80°C for later use. The mice were then killed and the cecal contents were obtained. The ELISA kit was used to measure the serum tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-10 (IL-10). The PowerMax fecal genomic DNA extraction kit was used to extract total bacterial genomic DNA from the cecal contents of mice.

[0060] The V4 hypervariable region of the 16S rRNA gene was amplified using universal primers 515F (5'-GTGCCAGCMGCCGCGGTAA-3'; SEQ ID NO: 1) and 806R (5'-GGACTACHVGGGTWTCTAAT-3'; SEQ ID NO: 2). The amplification procedure was as follows: 98°C pre-denaturation for 30s, 95°C denaturation for 15s, 58°C annealing for 15s, 72°C extension for 15s, for a total of 25 cycles, and a final extension at 72°C for 1min. The amplified products were sequenced by the second generation and quality controlled using FastQC software. The sequences at the 97% similarity level were divided into an operational taxonomic unit (OTU) using USEARCH (Version 10.0), and the SILVA138 database was used for species annotation. Alpha diversity was used to reflect species diversity and abundance, and Beta diversity was used to reflect group composition and differences.

[0061] Experimental results:

[0062] The regulatory effect of the probiotic spinach extract composition on the disorder of microbiota and systemic inflammatory response induced by high-fat diet in mice was evaluated by sequencing the 16S rRNA gene of the intestinal flora of mice and measuring the cytokines in the whole blood serum of mice. The data were statistically analyzed by SPSS V26.0, and the details are shown in Table 2 (in the same row of data, different lowercase letters with shoulders indicate significant differences between the data (p < 0.05), and the same lowercase letters indicate no significant differences between the data).

[0063] The Shannon index is often used to evaluate the richness and diversity of intestinal flora. The higher the Shannon index, the higher the species richness and diversity of intestinal flora. Compared with the high-fat diet control group, the Shannon index of the experimental groups fed with different proportions of probiotic spinach extract compositions increased to varying degrees (p < 0.05), indicating that the combination of probiotics and spinach extract can effectively improve the reduction in intestinal flora diversity caused by a high-fat diet. The relative abundance of intestinal flora at the phylum level and genus level of each group of mice is shown in Figure 2. Figure 1 and Figure 2 . Studies have shown that obesity caused by a high-fat diet is significantly associated with an increase in the ratio of intestinal Firmicutes to Bacteroidetes (F / B). Some strains of Firmicutes can absorb more energy from the intestine and are clearly related to weight gain. Some strains of Firmicutes can produce harmful substances through metabolism, affect intestinal permeability and induce inflammatory responses in the body, further affecting metabolic health and systemic immune health; while some strains of Bacteroidetes have better metabolic functions, can improve sugar and lipid metabolism, and reduce the body's absorption of energy, thereby reducing fat accumulation and improving the body's health. From the experimental results, compared with the high-fat diet control group, the Firmicutes / Bacteroidetes ratio of mice in each experimental group showed an extremely significant decrease, among which the decrease in experimental group 2 was the most obvious.

[0064] At the genus level, the combined intervention of probiotics and spinach extract changed the microbiota structure at the genus level in mice fed a high-fat diet. Previous studies have shown that the relative abundance of Oscillibacter in the intestine is causally related to the reduction of triglyceride concentration. In the results of this experiment, compared with the high-fat diet control group, the relative abundance of Oscillibacter in the three experimental groups fed with probiotics and spinach extract increased significantly (p<0.05). At the same time, beneficial bacterial genera such as Akkermansia, Rikenellaceae RC9, Muribaculaceae, and Bacteroides can use succinate, lactate, or acetate as substrates to produce a variety of beneficial metabolites such as propionate and butyrate. Short-chain fatty acids (SCFAs). The higher abundance of these beneficial bacterial genera helps maintain intestinal health, maintain microbiota balance, and promote fat metabolism and reduce fat storage, thereby improving metabolic health. The relative abundance of the above bacterial genera in the three experimental groups showed a significant increase (p<0.05); in addition, the relative abundance of Lachnolostridium, a marker of obesity, and Enterobacter, a harmful intestinal bacterium, showed a significant downward trend in the three experimental groups (p<0.05). In summary: The combination of probiotics and spinach extract can effectively regulate the imbalance of the mouse flora induced by a high-fat diet, increase beneficial bacteria and reduce harmful bacteria, thereby maintaining intestinal health.

[0065] From the results of this experiment, mice consuming a high-fat diet had an imbalance of the microbiota and were accompanied by obvious systemic inflammation, which was manifested in the increase of inflammatory factors (IL-1β, IL-6, TNF-α) and the decrease of anti-inflammatory factors (IL-10); feeding the probiotics and spinach extract combination significantly reduced the levels of proinflammatory factors IL-1β, IL-6 and TNF-α in mice fed a high-fat diet (p < 0.05), and significantly increased the level of anti-inflammatory factor IL-10 (p < 0.05), which indicates that the probiotics and spinach extract combination can improve the systemic inflammatory response caused by a high-fat diet.

[0066] Table 2: Intestinal flora structure and cytokine levels of mice in each group

[0067]

Claims

1. Use of a probiotic combination in improving intestinal flora or preparing a product containing the probiotic combination, characterized in that: The probiotic combination comprises: (1) Lactobacillus plantarum; the deposit number is CCTCC NO: M 2022699; (2) Bifidobacterium breve; the deposit number is CCTCC NO:M 20221846.

2. Use of a composition for improving intestinal flora or preparing a product containing the composition, characterized in that: The composition comprises the probiotic combination as claimed in claim 1; Preferably, the probiotic composition further comprises a plant extract; More preferably, the chlorophyll content of the plant extract is 1-5%, such as 3%.

3. The use according to claim 2, characterized in that The plant extract is spinach extract; Preferably, the mass ratio of the probiotic combination to the spinach extract is 1:1-1:5.

5.

4. The use according to claim 3, characterized in that The preparation of the spinach extract comprises the following steps: fresh spinach is crushed, a mixed solution of ethanol and isopropanol is added at a solid-liquid ratio of 1:10, the mixture is centrifuged to obtain a supernatant, the supernatant is concentrated to 1 / 10-1 / 30 of the original volume, and a lower layer of precipitate is obtained.

5. The use according to claim 4, characterized in that The preparation of the spinach extract meets one or more of the following conditions: (1) In the mixed solution, ethanol:isopropanol=2:1; (2) the centrifugation is performed at 8000-15000 rpm for 10-30 min, for example, at 10000 rpm for 15 min; (3) The precipitate is obtained by standing or centrifuging; for example, standing overnight at 4° C.; (4) The spinach extract is obtained by freeze-drying the precipitate.

6. The use according to any one of claims 2 to 5, characterized in that: The composition comprises, by mass fraction: 8.00%~25.00% Lactobacillus plantarum; 8.00%~25.00% Bifidobacterium breve; 50.00%~84.00% spinach extract.

7. The use according to claim 6, characterized in that The composition comprises, by mass fraction, 8% of lactobacillus plantarum, 8% of bifidobacterium breve and 84% of spinach extract; or, 12.50% of lactobacillus plantarum, 12.50% of bifidobacterium breve and 75.00% of spinach extract; or, 25% of lactobacillus plantarum, 25% of bifidobacterium breve and 50% of spinach extract.

8. The use according to any one of claims 2 to 7, characterized in that: The composition is prepared by the following method: weighing the probiotic combination and the plant extract according to a proportion, and mixing them evenly to obtain the composition; Preferably, the probiotics and plant extracts in the probiotic combination are freeze-dried products.

9. The use according to any one of claims 1 to 8, characterized in that The application is for non-therapeutic purposes; the performance of improving the function of intestinal flora is selected from one or more of the following: (1) Promote human digestion and nutrient absorption; (2) Maintaining the balance of intestinal flora, including maintaining or increasing the number of beneficial flora and maintaining or reducing the number of harmful flora; (3) Maintain or restore intestinal barrier function and reduce intestinal inflammation.

10. The use according to any one of claims 1 to 9, characterized in that The products include foods, health products or drugs that improve intestinal flora disorders, improve intestinal barrier dysfunction and treat metabolic diseases; Preferably, the intestinal flora disorder, intestinal barrier dysfunction and metabolic diseases are caused by a high-fat diet.

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