Functional probiotic composition and application thereof

Abstract

The invention discloses a functional probiotic composition and application thereof, and relates to the technical field of microbial engineering. Bifidobacterium longum, lactobacillus acidophilus and enterococcus faecalis are combined to serve as a microbial agent, the microbial agent and active substances of the tea leaves form a composition, then the composition is combined with prebiotics, it is ensured that viable bacteria directly reach the intestinal tract through a microencapsulation technology, and the effect of improving the immune function is achieved; tea polyphenol in the active substances of the tea leaves can serve as a carbon source of prebiotics to stimulate proliferation of the bifidobacterium longum and the lactobacillus acidophilus, so that the acid resistance and intestinal colonization capacity of the microbial inoculum are enhanced, and meanwhile, theanine in the active substances of the tea leaves, the bifidobacterium longum and the lactobacillus acidophilus synergistically promote secretion of gamma-aminobutyric acid, so that the effect of improving the intestinal colonization is achieved. And the tea polyphenol can inhibit the large intestine rod from adhering to the intestinal mucosa, and the probiotic agent secretes antibacterial peptide, so that the integrity of the intestinal barrier is maintained together.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial engineering, and in particular to a functional probiotic composition and application thereof. Background Art

[0002] The intestinal flora is the largest and most diverse group of microorganisms. They have a crucial impact on human digestion, metabolism, immunity, mood, and other physiological and psychological processes, and are closely related to human health. The intestinal flora is called a "superorganism" because its total bacterial cell count is approximately 10 times that of the human body. Numerous studies have confirmed that the metabolites of the intestinal flora can produce or alter neurotransmitters, activate immune signaling pathways, and act on the vagus nerve and immune system to influence brain function and structure. Therefore, the interactive pathway between the intestinal flora and the brain is called the "gut-brain axis."

[0003] There's an old Chinese saying in Traditional Chinese Medicine: "If the stomach isn't in harmony, sleep won't be easy." This also highlights the relationship between the gut and mental health. An imbalance in the gut flora can trigger various intestinal disorders, such as irritable bowel syndrome and inflammatory bowel disease. These conditions not only present with obvious intestinal symptoms like abdominal pain, bloating, and abnormal bowel movements, but can also lead to psychological and psychiatric conditions, such as anxiety, depression, insomnia, and cognitive impairment. This is precisely due to the central nervous system damage caused by an imbalance in the gut flora. Therefore, therapeutic interventions to regulate and restore the gut flora are crucial, and probiotics are microbial agents that improve the balance of intestinal microorganisms. However, probiotics require high nutritional requirements, proliferate slowly, and must survive the harsh gastric environment and remain active until they reach the large intestine to be effective. This requires yogurt to contain a significant probiotic content before consumption to ensure its predominance upon reaching the large intestine. Summary of the Invention

[0004] The purpose of the present invention is to provide a functional probiotic composition and application thereof, so as to solve the problems existing in the prior art.

[0005] In order to solve the above technical problems, the present invention provides the following technical solution: a functional probiotic composition, comprising a probiotic agent, tea active substances and prebiotics.

[0006] Furthermore, the probiotic agent consists of Bifidobacterium longum, Lactobacillus acidophilus, and Enterococcus faecalis.

[0007] Furthermore, the longum bifidobacterium is Bifidobacterium longum CCFM760, which was deposited on December 7, 2017 in the General Microbiology Center of China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCCNO.15032.

[0008] Furthermore, the Lactobacillus acidophilus is the Lactobacillus acidophilus (LactobaciLLusacidophiLus) strain HLYJ001, which was deposited in the General Microbiology Center of the China Culture Collection Administration on April 26, 2022, with the address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCCNO.24782.

[0009] Furthermore, the Enterococcus faecae is Enterococcus faeca Lis.

[0010] Furthermore, the tea active substance consists of theanine and tea polyphenols.

[0011] Furthermore, the prebiotic is at least one of fructooligosaccharide, galacto-oligosaccharide and xylo-oligosaccharide.

[0012] The present invention also provides a functional bacterial agent, the above-mentioned functional probiotic composition.

[0013] The present invention also provides the use of the functional bacterial agent in enhancing immunity.

[0014] The present invention also provides the use of the functional probiotic composition in enhancing immunity.

[0015] Compared with the prior art, the beneficial effects achieved by the present invention are as follows: the present invention utilizes a combination of Bifidobacterium longum, Lactobacillus acidophilus, and Enterococcus faecalis as a bacterial agent, forms a composition with tea active substances, and then combines prebiotics through microencapsulation technology to ensure that the live bacteria reach the intestines directly and achieve the effect of improving immune function; the tea polyphenols in the tea active substances can serve as a carbon source for prebiotics, stimulate the proliferation of Bifidobacterium longum and Lactobacillus acidophilus, and thereby enhance the acid resistance and intestinal colonization ability of the bacterial agent; at the same time, theanine in the tea active substances synergistically promotes the secretion of γ-aminobutyric acid with Bifidobacterium longum and Lactobacillus acidophilus, thereby regulating the stress response through the gut-brain axis and enhancing the body's immune system; and the tea polyphenols can inhibit the adhesion of Escherichia coli to the intestinal mucosa, while the probiotics secrete antimicrobial peptides, which improve immunity by jointly maintaining the integrity of the intestinal barrier. DETAILED DESCRIPTION

[0016] Various exemplary embodiments of the present invention are now described in detail. Unless otherwise specified, the methods in the examples are conventional methods. Unless otherwise specified, the reagents used are conventional commercially available reagents or reagents prepared using conventional methods. This detailed description should not be considered a limitation of the present invention, but rather should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0017] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0018] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0019] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0020] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0021] Example 1 Activity Test

[0022] 1. Bifidobacterium longum, Lactobacillus acidophilus, and Enterococcus faecalis were spread on MRS solid culture medium according to the three-zone method and cultured at 37°C for 15 hours. Then, single colonies of Bifidobacterium longum, Lactobacillus acidophilus, and Enterococcus faecalis were selected and inoculated into MRS liquid culture medium and cultured at 37°C for 15 hours. Finally, the mixture was centrifuged at 12,000 rpm for 5 minutes, and the bacterial sludge was collected and mixed to obtain a probiotic agent; the mass ratio of Bifidobacterium longum, Lactobacillus acidophilus, and Enterococcus faecalis was 2:1:1;

[0023] 2. The probiotic agent, tea polyphenols, theanine, and galacto-oligosaccharide were mixed, and then a 5 wt % sodium alginate aqueous solution was added, and the mixture was stirred at 100 rpm for 1 hour. A 6 wt % calcium chloride aqueous solution was added, and the mixture was stirred at 8000 rpm for 30 minutes. The mixture was then pre-frozen at -80°C for 2 hours, and then dried at -50°C with a vacuum degree of 10 Pa for 38 hours to obtain a functional probiotic composition; the mass ratio of the probiotic agent, tea polyphenols, theanine, galacto-oligosaccharide, 5 wt % sodium alginate aqueous solution, and 6 wt % calcium chloride aqueous solution was 1:0.5:0.5:1:10:5;

[0024] 3. The functional probiotic composition was placed in a pepsin solution with a pH of 2.5 and containing 0.3% pepsin, and cultured at 37° C. for 2 h with shaking. The viable bacterial count was determined to be 90%.

[0025] Example 2 Immune function test (determination of serum hemolysin half-value hemolysis)

[0026] 1. Bifidobacterium longum, Lactobacillus acidophilus, and Enterococcus faecalis were spread on MRS solid culture medium according to the three-zone method and cultured at 37°C for 15 hours. Then, single colonies of Bifidobacterium longum, Lactobacillus acidophilus, and Enterococcus faecalis were selected and inoculated into MRS liquid culture medium and cultured at 37°C for 15 hours. Finally, the mixture was centrifuged at 12,000 rpm for 5 minutes, and the bacterial sludge was collected and mixed to obtain a probiotic agent; the mass ratio of Bifidobacterium longum, Lactobacillus acidophilus, and Enterococcus faecalis was 2:1:1;

[0027] 2. The probiotic agent, tea polyphenols, theanine, and galacto-oligosaccharide were mixed, and then a 5 wt % sodium alginate aqueous solution was added, and the mixture was stirred at 100 rpm for 1 hour. A 6 wt % calcium chloride aqueous solution was added, and the mixture was stirred at 8000 rpm for 30 minutes. The mixture was then pre-frozen at -80°C for 2 hours, and then dried at -50°C with a vacuum degree of 10 Pa for 38 hours to obtain a functional probiotic composition; the mass ratio of the probiotic agent, tea polyphenols, theanine, galacto-oligosaccharide, 5 wt % sodium alginate aqueous solution, and 6 wt % calcium chloride aqueous solution was 1:0.5:0.5:1:10:5;

[0028] 3. A functional probiotic composition with a concentration of 0.42 mg / (kg·d) was prepared with purified water and administered to mice by gavage once a day for 30 consecutive days. On the 31st day, 0.2 L of 2% (V / V) sheep red blood cells were injected intraperitoneally into the mice, and blood was collected by eye removal on the 4th day after injection. The blood was placed in a centrifuge tube and centrifuged for 1 hour to collect the upper serum; the serum was diluted 300 times and 50 μL was placed in a 96-well plate, and 25 μL of 10% (V / V) sheep red blood cells and 50 μL of the functional probiotic composition were added in sequence. The reaction was terminated after 30 minutes in a constant temperature incubator at 37°C and centrifuged again to obtain 0.05 mL of the supernatant, and 0.15 mL of Vinci's reagent was added as a sample. At the same time, a half-hemolysis well was set, 12.5 μL of 10% (V / V) sheep red blood cells were taken, and Vinci's reagent was added to 0.2 mL. The reagents of each group were fully mixed. After standing for 10 minutes, the half-hemolysis value was measured at 540 nm and was 83.65.

[0029] It can be seen that the present invention reduces Th2 type cytokines and increases Th1 type by combining tea polyphenols with probiotics, corrects immune deviation, and promotes antibody diversification; Enterococcus faecalis enhances the ability of macrophages to present antigens through the TLR4 / NF-κB pathway, indirectly activates B cells, and thus reduces the interference of immunosuppressive factors, ensuring efficient antibody production, and prolongs the continuous release of sheep red cells by combining prebiotics with microencapsulation, thereby stably improving the half hemolytic value.

[0030] Example 3 Immune function test (antibody-producing cell detection)

[0031] 1. Bifidobacterium longum, Lactobacillus acidophilus, and Enterococcus faecalis were spread on MRS solid culture medium according to the three-zone method and cultured at 37°C for 15 hours. Then, single colonies of Bifidobacterium longum, Lactobacillus acidophilus, and Enterococcus faecalis were selected and inoculated into MRS liquid culture medium and cultured at 37°C for 15 hours. Finally, the mixture was centrifuged at 12,000 rpm for 5 minutes, and the bacterial sludge was collected and mixed to obtain a probiotic agent; the mass ratio of Bifidobacterium longum, Lactobacillus acidophilus, and Enterococcus faecalis was 2:1:1;

[0032] 2. The probiotic agent, tea polyphenols, theanine, and galacto-oligosaccharide were mixed, and then a 5 wt % sodium alginate aqueous solution was added, and the mixture was stirred at 100 rpm for 1 hour. A 6 wt % calcium chloride aqueous solution was added, and the mixture was stirred at 8000 rpm for 30 minutes. The mixture was then pre-frozen at -80°C for 2 hours, and then dried at -50°C with a vacuum degree of 10 Pa for 38 hours to obtain a functional probiotic composition; the mass ratio of the probiotic agent, tea polyphenols, theanine, galacto-oligosaccharide, 5 wt % sodium alginate aqueous solution, and 6 wt % calcium chloride aqueous solution was 1:0.5:0.5:1:10:5;

[0033] 3. A functional probiotic composition with a concentration of 0.42 mg / (kg·d) was prepared with purified water and administered to mice by gavage once daily for 30 consecutive days. 1 mL of packed sheep red blood cells was added to 5 mL of blank mouse serum and placed at 4°C for 30 minutes. The supernatant was centrifuged and stored at -70°C for use as complement. On the 31st day, 0.2 mL of 2% (v / v) sheep red blood cells was injected intraperitoneally into the mice. Four days after the injection, the mice in each group were sacrificed and their spleens were removed and stored in Hank's solution. The spleens were ground, filtered through a mesh, and centrifuged to prepare a cell suspension. The cell concentration was adjusted to 5×10 6 Dissolve the surface culture medium by heating and place in a 45°C water bath for preservation. Mix with an equal volume of 2-fold concentrated Hank's solution and dispense into test tubes at a volume of 0.5 mL per tube. Add 50 μL of 10% (V / V) sheep red blood cells and 20 μL of spleen cell suspension, mix quickly, and pour onto a glass slide brushed with a thin layer of agarose. After the agarose solidifies, place the slide horizontally on a slide rack and place in a CO2 incubator for a further 1.5 hours of incubation. Add the prepared complement and place it in the groove of the slide. After incubation for 1.5 hours, count the number of hemolytic plaques to 275 per whole spleen cell.

[0034] It can be seen that the butyrate and other sheep red sheep cells produced by the combined metabolism of Bifidobacterium longum, Lactobacillus acidophilus and Enterococcus faecalis in the present invention can stimulate the differentiation of B cells in the intestinal lymphoid follicles, promote the production of antibodies by plasma cells, and provide sustained energy through the sustained release of prebiotics, prolong the metabolic activity of the flora, and indirectly support immune enhancement.

[0035] From the experimental data in the examples, it can be found that the present invention utilizes a combination of Bifidobacterium longum, Lactobacillus acidophilus, and Enterococcus faecalis as a bacterial agent to form a composition with tea active substances, and then combines prebiotics through microencapsulation technology to ensure that the live bacteria reach the intestines directly and achieve the effect of improving immune function; the tea polyphenols in the tea active substances can serve as a carbon source for prebiotics, stimulate the proliferation of Bifidobacterium longum and Lactobacillus acidophilus, and thus enhance the acid resistance and intestinal colonization ability of the bacterial agent, and the tea polyphenols can inhibit the adhesion of Escherichia coli to the intestinal mucosa, while the probiotics secrete antimicrobial peptides, which jointly maintain the integrity of the intestinal barrier, thereby maintaining the activity of probiotics and improving immunity.

[0036] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be encompassed therein. Any reference in a claim should not be construed as limiting the claim to which it relates.

Claims

1. A functional probiotic composition, characterized in that: Including probiotics, tea active substances and prebiotics.

2. A functional probiotic composition according to claim 1, characterized in that The probiotic agent consists of Bifidobacterium longum, Lactobacillus acidophilus and Enterococcus faecalis.

3. A functional probiotic composition according to claim 2, characterized in that: The long form Bifidobacterium is Bifidobacterium longum CCFM760, which was deposited on December 7, 2017 at the General Microbiology Center of China Culture Collection Administration Committee, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCCNO.15032.

4. A functional probiotic composition according to claim 2, characterized in that, The Lactobacillus acidophilus is the Lactobacillus acidophilus (LactobaciLLusacidophiLus) strain HLYJ001, which was deposited in the General Microbiology Center of the China Culture Collection Administration on April 26, 2022, with the address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCCNO.24782.

5. A functional probiotic composition according to claim 2, characterized in that, The Enterococcus faecae is Enterococcus faeca Lis.

6. A functional probiotic composition according to claim 1, characterized in that: The tea active substance consists of theanine and tea polyphenols.

7. A functional probiotic composition according to claim 1, characterized in that: The prebiotic is at least one of fructooligosaccharide, galacto-oligosaccharide and xylo-oligosaccharide.

8. A functional bacterial agent, characterized in that The invention comprises the functional probiotic composition according to any one of claims 1 to 7.

9. Use of the functional bacterial agent according to claim 8 in enhancing immunity.

10. Use of the functional probiotic composition according to any one of claims 1 to 7 in enhancing immunity.