Probiotic and prebiotic composition for improving intestinal microecology and assisting in resisting food allergy and application of probiotic and prebiotic composition

Abstract

The invention belongs to the technical field of biological medicines, and discloses a probiotic and prebiotic composition for improving intestinal microecology and assisting in resisting food allergy, and the composition comprises bifidobacterium longum and oligosaccharide in a mass ratio of (1-3): (1-3). The probiotic and oligosaccharide composition provided by the invention is clear in mechanism and wide in application range, and the effect of relieving food anaphylactic reaction is achieved by enhancing intestinal barriers and regulating intestinal flora. And under the participation of the oligosaccharide, the anti-allergic effect of the probiotics is obviously enhanced. The invention can solve the problem that the existing anti-allergic medicine treats both symptoms and root causes, simplifies the composition, defines the action mechanism of the composition, and expands the range of applicable people.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular to a probiotic and prebiotic composition for improving intestinal microecology and assisting in preventing food allergies, and application thereof. Background Art

[0002] Food allergy is a common disease worldwide. Currently, about 30-40% of the world's population is affected by allergic diseases. For susceptible organisms, incompletely digested or abnormally digested food antigens will pass through the intestinal epithelial barrier, enter the lamina propria, and then trigger an immune response. As an important line of host defense, the intestinal barrier (intestinal microorganisms, intestinal mechanical barriers, etc.) significantly affects the occurrence and development of food allergies. Studies have shown that people with food allergies are usually accompanied by intestinal microbial disorders, and damage to the intestinal mechanical barrier is the final result of repeated exposure of susceptible organisms to food antigens.

[0003] Current methods for controlling the occurrence and development of food allergies include dietary avoidance and anti-allergic drugs used clinically. Although they can effectively reduce the incidence of allergies, they cannot fundamentally change the body's susceptibility.

[0004] Probiotics are defined as live microbial supplements that favorably affect the host by improving the balance of intestinal microorganisms. Studies have shown that probiotics can maintain intestinal and body immune homeostasis by regulating the composition of intestinal flora, maintaining or enhancing the expression of tight junction proteins, and affecting the release of intestinal epithelial cytokines. Probiotics can fundamentally change the body's susceptibility state and have important application prospects in controlling the occurrence and development of food allergies. However, there are differences in the in vivo colonization and effects of strains, and a technology that can enhance the effects of probiotics is urgently needed.

[0005] At present, probiotic compositions are mostly used to enhance the anti-allergic effect of strains. Various methods for enhancing the prevention and treatment of allergic diseases with probiotics are known, see CN108065400 and CN113908176A, which use a multi-bacteria mixture method, but the mixed bacteria method cannot confirm the rationality of its compatibility and cannot be verified; CN107114794 uses a compound of bacteria with polysaccharides and vitamin C, but the glycemic index of the product is high, limiting the scope of applicable population; CN110710659A uses a compound of strains with polysaccharides and mixed fruit and vegetable powders, on the one hand, the product has a high glycemic index, and on the other hand, the addition of mixed fruit and vegetable powders introduces more potential allergens into the product, thereby limiting the scope of use of the product.

[0006] In summary, there is an urgent need for a probiotic composition with a clear mechanism of action and a wide range of applications for the prevention and treatment of food allergies. Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a probiotic and prebiotic composition and its application for improving intestinal microecology and assisting in preventing food allergies.

[0008] The technical solution adopted by the present invention to solve its technical problem is:

[0009] A probiotic and prebiotic composition for improving intestinal microecology and assisting in preventing food allergies, the composition comprising Bifidobacterium longum and oligosaccharides in a mass ratio of 1-3:1-3.

[0010] Furthermore, the oligosaccharide is selected from one or two of 2'-fucosyllactose and 3'-fucosyllactose, which have been widely proven to protect the intestinal barrier.

[0011] Furthermore, the probiotic is Bifidobacterium longum subsp. infantis, with a preservation number of CGMCC No. 25762 and a preservation date of September 21, 2022, at the General Microbiology Center of the China Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The probiotic has strong oligosaccharide utilization ability, strong gastric acid tolerance, and strong intestinal adhesion ability.

[0012] Furthermore, the anti-allergic effects of the composition include: significantly reducing sIgE levels, inhibiting the differentiation of Th0 cells into Th2 cells, maintaining intestinal flora homeostasis, enhancing intestinal tight junction protein expression, reducing intestinal inflammatory factor secretion, and improving intestinal barrier damage.

[0013] Furthermore, the dosage form of the composition is one of tablets, granules, powders, capsules, solutions, suspensions, and freeze-dried preparations.

[0014] Furthermore, the composition comprises 25-50 parts by weight of oligosaccharides and 50-75 parts by weight of Bifidobacterium longum, wherein the content of Bifidobacterium longum is 10 8 -10 10 Colony forming units.

[0015] The preparation method of the composition as described above is characterized in that: a powder of a certain mass fraction is redissolved in sterile distilled water, and then consumed after homogenization.

[0016] Use of the composition as described above in preparing food and / or medicine for inflammatory intestinal barrier damage.

[0017] Furthermore, the inflammatory intestinal barrier damage includes inflammatory intestinal barrier damage associated with aging, obesity, diabetes, and irritable bowel syndrome.

[0018] The advantages and positive effects achieved by the present invention are:

[0019] 1. The Bifidobacterium longum CGMCC.25762 and oligosaccharide composition of the present invention can significantly improve the intestinal homeostasis of sensitized organisms and alleviate the occurrence of allergic reactions, and the effect is significantly better than the effect of Bifidobacterium longum or oligosaccharides alone.

[0020] 2. The composition of the present invention has no risk of introducing new food allergens. The oligosaccharides in the composition of the present invention are mainly metabolized and utilized by the intestinal flora of the body, and do not burden the body's blood sugar metabolism. The composition of the present invention can be used to prepare anti-allergic products and products that improve inflammatory intestinal barrier damage related to aging, obesity, diabetes, irritable bowel syndrome, etc., and has broad application prospects.

[0021] 3. The present invention provides a probiotic and oligosaccharide composition with a clear mechanism and a wide range of applications, which can alleviate food allergic reactions by strengthening the intestinal barrier and regulating the intestinal flora. With the participation of oligosaccharides, the anti-allergic effect of probiotics is significantly enhanced. The present invention can solve the problem that current anti-allergic drugs only treat the symptoms but not the root cause, and at the same time simplify the composition of the composition, clarify the mechanism of action of its components, and expand the scope of its applicable population.

[0022] 4. The composition of the present invention can significantly improve intestinal inflammatory damage; can effectively inhibit the differentiation of intestinal lymphocytes Th0 to Th2, and the level of specific IgE antibodies in sensitized organisms; thereby achieving the effect of improving intestinal homeostasis and resisting food allergies. Compared with the effects of probiotics or oligosaccharides alone, the composition containing probiotics and oligosaccharides obtained by the present invention has the best efficacy. The composition of the present invention can also be widely used to improve inflammatory intestinal barrier damage related to aging, obesity, diabetes, irritable bowel syndrome, etc., and has high market application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the apparent index diagram of sensitized mice in the present invention;

[0024] Figure 2 This is a diagram showing that Bi / FL / SY intervention alleviates allergic reactions in sensitized mice in the present invention;

[0025] Figure 3 This is a diagram of the imbalance of small intestine homeostasis in sensitized mice in the present invention;

[0026] Figure 4 This is a diagram showing that Bi / FL / SY intervention improves small intestinal homeostasis in sensitized mice in the present invention;

[0027] Figure 5 This is a diagram showing the improvement of the small intestinal flora composition of sensitized mice by Bi / FL / SY intervention in the present invention;

[0028] Figure 6This is a diagram showing the changes in the short-chain fatty acid composition of the small intestine of sensitized mice after Bi / FL / SY intervention in the present invention. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0030] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0031] A probiotic and prebiotic composition for improving intestinal microecology and assisting in preventing food allergies, the composition comprising Bifidobacterium longum and oligosaccharides in a mass ratio of 1-3:1-3.

[0032] Preferably, the oligosaccharide is selected from one or two of 2'-fucosyllactose and 3'-fucosyllactose, which have been widely proven to protect the intestinal barrier.

[0033] Preferably, the probiotic is Bifidobacterium longum subsp. infantis, with a preservation number of CGMCC No. 25762 and a preservation date of September 21, 2022 at the General Microbiology Center of China Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The probiotic has strong oligosaccharide utilization ability, strong gastric acid tolerance, and strong intestinal adhesion ability.

[0034] Preferably, the anti-allergic effects of the composition include: significantly reducing sIgE levels, inhibiting the differentiation of Th0 cells into Th2 cells, maintaining intestinal flora homeostasis, enhancing intestinal tight junction protein expression, reducing intestinal inflammatory factor secretion, and improving intestinal barrier damage.

[0035] Preferably, the dosage form of the composition is one of tablets, granules, powders, capsules, solutions, suspensions, and freeze-dried preparations.

[0036] Preferably, the composition comprises 25-50 parts by weight of oligosaccharides, 50-75 parts by weight of Bifidobacterium longum, and the content of Bifidobacterium longum is 10 8 -10 10 Colony forming units.

[0037] The preparation method of the composition as described above is characterized in that: a powder having a certain weight portion is redissolved in sterile distilled water and then consumed after homogenization.

[0038] Use of the composition as described above in preparing food and / or medicine for inflammatory intestinal barrier damage.

[0039] Preferably, the inflammatory intestinal barrier damage includes inflammatory intestinal barrier damage associated with aging, obesity, diabetes, and irritable bowel syndrome.

[0040] Specifically, the relevant preparation and detection are as follows:

[0041] The 2'-fucosyllactose involved in the examples of the present invention was purchased from Chemical Sugar Research Institute Co., Ltd.

[0042] The culture medium involved in the embodiments of the present invention is as follows:

[0043] Bifidobacterium longum uses mMRS medium (pH: 6.2-6.4), and its formula is (1L): 10g peptone, 5g beef extract powder, 1ml Tween 80, 2g dipotassium hydrogen phosphate, 4g yeast extract powder, 0.2g magnesium sulfate, 2g triammonium citrate, 20g glucose, 0.05g manganese sulfate, 5g sodium acetate, and 50g L-cysteine.

[0044] The detection method involved in the embodiments of the present invention is as follows:

[0045] The levels of serum sIgE and mcpt-1 in the mice in the modeling group were significantly increased, their activity level was reduced, and their eyes were red and swollen, indicating that the conditions for the allergic model had been met.

[0046] Blood biochemical index detection method:

[0047] The levels of sIgE and mcpt-1 in serum were determined by indirect ELISA.

[0048] Intestinal inflammatory factor detection method:

[0049] 20 mg of mouse intestinal tissue was homogenized in 0.5 mL of tissue lysis buffer for 90 seconds, centrifuged at 1000 g for 5 minutes, and the supernatant was taken. The contents of IL-13, IL-4, IL-2, and IFN-γ in the tissue were determined using a commercial kit.

[0050] Intestinal tight junction protein expression detection method:

[0051] 20 mg of mouse intestinal tissue was taken and added to 1 mL of TriQuick Reagent to extract RNA. A reverse transcription kit was used to obtain cDNA. The designed primers were used to detect the expression levels of tight junction proteins (ZO-1, Muc-2, Occludin, Claudin1) in intestinal tissue.

[0052] Intestinal flora composition detection method:

[0053] The intestinal contents of mice were taken, DNA was extracted using the CTAB method, and then PCR was performed on the V3-V4 fragment of the bacteria using it as a template. The target gene was purified using agarose gel electrophoresis, and a small fragment library was constructed based on the characteristics of the amplified region. The library was sequenced at both ends based on the Illumina NovaSeq sequencing platform, and effective data was screened for species annotation and analysis.

[0054] Method for determination of intestinal flora metabolites:

[0055] The intestinal contents of mice were thoroughly ground in a tissue homogenizer, and the content of short-chain fatty acids was determined by high performance liquid chromatography with an injection volume of 10 μL, a flow rate of 0.8 mL / min, a column temperature of 60°C, a mobile phase of 5 mM dilute sulfuric acid, and a UV detector (210 nm).

[0056] Example 1

[0057] A probiotic and prebiotic composition for improving intestinal microecology and assisting in preventing food allergies, the composition consisting of equal weight parts of Bifidobacterium longum CGMCC No.25762 and 2'-fucosyllactose.

[0058] The preparation method of the probiotic composition is as follows: by weight, the weight ratio of Bifidobacterium longum and 2'-fucosyllactose is 1:1, and 10 8 The colony forming units of Bifidobacterium longum and 100 mg of 2'-fucosyllactose were redissolved in 400 μL of sterile distilled water and homogenized to obtain the composition prepared in Example 1.

[0059] The composition prepared in Example 1 was used to carry out relevant tests, as follows:

[0060] 1. Bifidobacterium longum / 2-fucosyllactose combination preparation alleviates food allergy in mice

[0061] The specific steps are as follows:

[0062] Experimental groups and treatment methods:

[0063] Five groups were set up: blank control group (Ct), allergic model group (OA), Bifidobacterium longum intervention group (Bi), 2'-fucosyllactose intervention group (FL), and Bifidobacterium longum / 2'-fucosyllactose combination (SY) intervention group.

[0064] The preparation method of Bifidobacterium longum CGMCC:25762 is as follows: placing Bifidobacterium longum in mMRS culture medium and culturing it to the logarithmic growth phase, centrifuging to obtain bacterial mud, and resuspending it with physiological saline to obtain a resuspended liquid.

[0065] The preparation method of the probiotic composition is as follows: by weight, the weight ratio of Bifidobacterium longum and 2'-fucosyllactose is 1:1, and 10 8 The colony forming units of Bifidobacterium longum and 100 mg of 2'-fucosyllactose were redissolved in 400 μL of sterile distilled water and homogenized to obtain the composition prepared in Example 1.

[0066] The specific experiments are as follows:

[0067] Female Balb / c mice, 6 weeks old, were raised in the Animal Experiment Center of Nankai University (25±2°C, 12 / 12 h light / dark period). The mice were given free water and food under laboratory conditions and adapted to feeding for one week. Then, the mice were divided into 5 groups (n=7 per group). The animal experiment design is shown in Table 1.

[0068] Table 1 Animal experiment design

[0069]

[0070] At week 7, all mice were killed by cervical dislocation, blood was collected, and spleen tissue was weighed.

[0071] The results show:

[0072] (1) Figure 1 As shown, after subcutaneous injection of the sensitizing protein, compared with the control group (Ct), the serum sIgE and mcpt-1 levels of the sensitized group mice (OA) were significantly increased, the clinical symptoms were significant, the spleen was enlarged, and the modeling conditions were met.

[0073] (2) Figure 2 As shown, intervention with Bifidobacterium longum and / or 2'-fucosyllactose significantly reduced the secretion of mcpt-1 in serum and alleviated the clinical symptoms of mice. The reduction rates of serum sIgE were 47.5% (Bifidobacterium longum group-Bi), 24.4% (combination group-SY), and 29% (2'-fucosyllactose group-FL), and the spleen weight reduction rates were 20.7% (Bi), 27.6% (SY), and 16.9% (FL). Overall, the Bifidobacterium longum / 2-fucosyllactose combination intervention group (SY) had the most significant effect in alleviating allergic reactions.

[0074] In summary, the probiotic composition provided by the present invention can significantly alleviate the allergic reaction of sensitized mice and play an anti-allergic role. It can also be seen that the combined use of Bifidobacterium longum and 2'fucosyllactose in the composition of the present invention has a synergistic effect between Bifidobacterium longum and 2'fucosyllactose, which can synergistically improve the relevant properties of the prepared composition.

[0075] 2. Bifidobacterium longum / 2'-fucosyllactose combination preparation improves intestinal inflammatory damage in food allergic mice

[0076] Five groups were set up: blank control group (Ctrl), allergic model group (OA), Bifidobacterium longum intervention group (Bi), 2'-fucosyllactose intervention group (FL), and Bifidobacterium longum / 2'-fucosyllactose combination (SY) intervention group.

[0077] The specific implementation method is the same as that in the experiment (i.e., the related operations of the Bifidobacterium longum / 2-fucosyllactose composition preparation to alleviate food allergy in mice), and the intestines and intestinal contents of the mice are collected after the experiment.

[0078] The results show:

[0079] (1) Figure 3 As shown, after subcutaneous injection of the sensitizing protein, the expression of intestinal tight proteins (ZO-1, Occludin, Claudin, Muc-2) in the sensitized group of mice was downregulated, and the secretion of intestinal inflammatory factors (IL-2, IFN-γ, IL-4, IL-13) increased (this is partly to confirm that the disease model was successfully established.).

[0080] (2) Figure 4 As shown, intervention with Bifidobacterium longum and / or 2'-fucosyllactose significantly upregulated the expression of tight junction proteins and enhanced the intestinal barrier. The reduction rates of intestinal proinflammatory cytokine IL-4 were 70.6% (Bi), 97.7% (SY), and 94.8% (FL), respectively; the reduction rates of intestinal proinflammatory cytokine IL-13 were 21% (Bi), 68.8% (SY), and 36.9% (FL), respectively. Overall, the Bifidobacterium longum / 2'-fucosyllactose combination intervention group had the most significant effect in alleviating allergic reactions.

[0081] (3) Figure 5 As shown in the figure, after subcutaneous injection of allergenic proteins, the intestinal flora structure of mice in the sensitized group deviated from that of the control group, and the abundance of intestinal pro-inflammatory bacteria increased.

[0082] (4) Figure 5 As shown, the intervention group significantly maintained the homeostasis of intestinal flora, and the effect of the Bifidobacterium longum / 2'-fucosyllactose combination intervention group (SY) was the most significant.

[0083] (5) Figure 6 As shown, acetate was the main SCFA measured in the small intestine; the levels of lactic acid and acetate were significantly reduced after sensitization.

[0084] (6) Figure 6As shown, intervention treatment with Bifidobacterium longum and / or 2'-fucosyllactose composition can significantly increase the content of isobutyric acid and isovaleric acid (isobutyric acid and isovaleric acid play an important role in maintaining intestinal mucosa and immune homeostasis), and the Bifidobacterium longum / 2'-fucosyllactose composition has the best effect on improving the small intestine.

[0085] In summary, the composition provided by the present invention can significantly improve inflammatory intestinal barrier damage and is widely used in products for improving intestinal barrier function.

[0086] It can also be seen that the combined use of Bifidobacterium longum and 2'-fucosyllactose in the composition of the present invention has a synergistic effect, and can synergistically improve the relevant properties of the prepared composition.

[0087] In the prior art, probiotic compositions are often used to enhance the anti-allergic effect of strains. Various methods for enhancing the prevention and treatment of allergic diseases with probiotics are known, see CN108065400 and CN113908176A, which use a multi-bacteria mixture method, but the rationality of the combination cannot be confirmed and verified; CN107114794 uses a compound of bacteria with polysaccharides and vitamin C, but the product has a high glycemic index, which limits the scope of applicable population; CN110710659A uses a compound of strains with polysaccharides and mixed fruit and vegetable powders, which, on the one hand, has a high glycemic index, and on the other hand, the addition of mixed fruit and vegetable powders introduces more potential allergens into the product, thereby limiting the scope of use of the product.

[0088] The composition of the present invention has no risk of introducing new food allergens. The present invention provides a probiotic and oligosaccharide composition with a clear mechanism and a wide range of applications. The 2'-fucosyllactose used in the present invention is a typical representative of human milk oligosaccharides. The lack of 2'-fucosyllactose in breast milk can easily lead to a higher risk of infant food allergies. The present invention applies it to dietary intervention for food allergies for the first time and confirms its effectiveness. The present invention uses human bifidobacteria, which have excellent colonization ability in the body, which is superior to the use of strains derived from fermented foods in existing patents.

[0089] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A probiotic and prebiotic composition for improving intestinal microecology and assisting in preventing food allergies, characterized in that: The composition comprises Bifidobacterium longum and oligosaccharide in a mass ratio of 1-3:1-3.

2. The composition according to claim 1, characterized in that: The oligosaccharide is selected from one or two of 2'fucosyllactose and 3'fucosyllactose, which have been widely proven to be able to protect the intestinal barrier.

3. The composition according to claim 1, characterized in that: The probiotic is Bifidobacterium longum subsp. infantis, with a preservation number of CGMCC No. 25762 and a preservation date of September 21, 2022, at the General Microbiology Center of the China Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The probiotic has strong oligosaccharide utilization ability, strong gastric acid tolerance, and strong intestinal adhesion ability.

4. The composition according to claim 1, characterized in that: The anti-allergic effects of the composition include: significantly reducing sIgE levels, inhibiting the differentiation of Th0 cells into Th2 cells, maintaining the homeostasis of intestinal flora, enhancing the expression of intestinal tight junction proteins, reducing the secretion of intestinal inflammatory factors, and improving intestinal barrier damage.

5. The composition according to claim 1, characterized in that: The dosage form of the composition is one of tablets, granules, powders, capsules, solutions, suspensions and freeze-dried preparations.

6. The composition according to any one of claims 1 to 5, characterized in that: The composition comprises 25-50 parts by weight of oligosaccharides and 50-75 parts by weight of Bifidobacterium longum, wherein the content of Bifidobacterium longum is 10 8 -10 10 Colony forming units.

7. The method for preparing the composition according to any one of claims 1 to 6, characterized in that: Dissolve the powder in a determined amount by mass in sterile distilled water and homogenize before consumption.

8. Use of the composition according to any one of claims 1 to 6 in the preparation of food and / or medicine for inflammatory intestinal barrier damage.

9. The use according to claim 8, characterized in that: The inflammatory intestinal barrier damage includes inflammatory intestinal barrier damage associated with aging, obesity, diabetes, and irritable bowel syndrome.

Images