L-malic acid and bifidobacterium breve composition and application thereof in preparation of product for preventing colitis

Abstract

The invention discloses an L-malic acid and bifidobacterium breve composition and application thereof in preparation of a product for preventing colitis, and belongs to the technical field of enteric microorganisms. According to the invention, the L-malic acid and the bifidobacterium breve are combined for use, so that the colitis symptom can be more effectively improved. L-malic acid promotes field planting and reproduction of bifidobacterium breve in intestinal tracts by enhancing the viability of bifidobacterium breve in the intestinal tracts, so that the probiotic effect of bifidobacterium breve is better played, and colitis is improved.

Description

Technical Field

[0001] The invention relates to an L-malic acid and Bifidobacterium breve composition and application thereof in preparing a product for preventing colitis, belonging to the technical field of intestinal microorganisms. Background Art

[0002] As a widespread intestinal inflammatory disease, colitis has a complex pathogenesis, involving multiple aspects such as excessive activation of intestinal inflammatory response, severe damage to intestinal barrier function, and abnormal regulation of the immune system. These pathological changes not only lead to a significant decline in the patient's quality of life, but may also further induce more serious intestinal and even systemic diseases. Among the many influencing factors, the imbalance of intestinal microbiota is recognized as one of the key factors in the onset of colitis. In particular, the significant reduction in the number of beneficial bacteria in the intestine (such as Bifidobacterium breve, etc.) and the excessive proliferation of harmful bacteria (such as certain Clostridium species, Escherichia coli, etc.) have jointly aggravated the deterioration of the intestinal environment.

[0003] Bifidobacterium breve, as an important member of the probiotic family, has attracted much attention for its outstanding intestinal health-promoting effects. It can not only reduce inflammatory symptoms by regulating intestinal immune responses, but also effectively enhance intestinal barrier function and reduce the invasion of harmful substances into the intestine. However, a major challenge faced by Bifidobacterium breve in practical applications is its poor tolerance to bile salts. Bile salts are an important component in the intestine and play an indispensable role in maintaining the normal physiological function of the intestine, but they also pose a survival threat to many probiotics. The survival ability of Bifidobacterium breve in the intestine is limited, especially glycodeoxycholic acid, which can severely damage its cell membrane and reduce its growth activity, resulting in a significant reduction in its survival rate and colonization efficiency in the intestine, thereby limiting its potential in the treatment of colitis.

[0004] L-malic acid, as an organic acid naturally present in many fruits, has gradually been favored by researchers in recent years due to its wide range of biological activities. It not only has antioxidant and anti-inflammatory effects, can effectively combat oxidative stress and inflammatory responses in the intestine, but may also play an important role in regulating the balance of intestinal flora. However, L-malic acid needs to be consumed for a long time to have a certain therapeutic effect on inflammation.

[0005] Therefore, how to overcome the survival barriers of Bifidobacterium breve in the intestine, improve its intestinal survival rate and colonization ability, and thus more effectively exert its potential in the treatment of colitis has become a key issue that needs to be urgently addressed. Summary of the invention

[0006] In order to solve the deficiencies of the prior art, the present invention provides a composition of L-malic acid and Bifidobacterium breve. The combination of L-malic acid and Bifidobacterium breve can more effectively improve the symptoms of colitis.

[0007] The present invention is achieved through the following technical solutions:

[0008] The first object of the present invention is to provide an application of a composition of L-malic acid and Bifidobacterium breve in preparing a product for preventing colitis.

[0009] In one embodiment of the present invention, the Bifidobacterium breve is Bifidobacterium breve Bb18.

[0010] In one embodiment of the present invention, the content of L-malic acid in the composition is 3 to 5 g / L.

[0011] In one embodiment of the present invention, the content of Bifidobacterium breve in the composition is 1×10 7～10 CFU / mL.

[0012] In one embodiment of the present invention, the composition is an oral preparation.

[0013] In one embodiment of the present invention, the composition is a tablet or a liquid preparation.

[0014] In one embodiment of the present invention, the composition further comprises a pharmaceutically acceptable excipient.

[0015] The second object of the present invention is to provide a composition of L-malic acid and Bifidobacterium breve.

[0016] In one embodiment of the present invention, the Bifidobacterium breve is Bifidobacterium breve Bb18.

[0017] In one embodiment of the present invention, the content of L-malic acid is 3-5 g / L, and the content of Bifidobacterium breve is 1×10 7～10 CFU / mL.

[0018] Beneficial effects of the present invention:

[0019] The present invention uses L-malic acid in combination with Bifidobacterium breve, which can more effectively improve the symptoms of colitis. L-malic acid enhances the survival ability of Bifidobacterium breve in the intestine, promotes its colonization and reproduction in the intestine, thereby better exerting its prebiotic effect and improving colitis. The method not only provides a new idea for intestinal health maintenance, but also opens up a new way for the prevention of related diseases, showing broad application prospects and far-reaching significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 This is a graph showing the experimental results of using L-malic acid to improve the bile salt tolerance of Bifidobacterium breve.

[0022] Figure 2 This is a graph showing the experimental results of using different acids to improve the bile salt tolerance of Bifidobacterium breve.

[0023] Figure 3 This is the experimental result of the intestinal colonization rate of Bifidobacterium breve after 7 and 14 days of oral administration.

[0024] Figure 4 This is a graph showing the experimental results of L-malic acid combined with Bifidobacterium breve alleviating the disease activity index (DAI) of mice.

[0025] Figure 5 This is a graph showing the experimental results of L-malic acid combined with Bifidobacterium breve alleviating colon shortening in mice.

[0026] Figure 6 This is the experimental result of L-malic acid combined with Bifidobacterium breve alleviating colon damage in mice.

[0027] Figure 7 This is a graph showing changes in levels of colon inflammatory markers in mice.

[0028] Among them, *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with specific examples. These implementation cases are only used to illustrate the present invention and are not used to limit the scope of the present invention. In addition, after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the claims attached to the application.

[0030] The culture medium involved in the following examples is:

[0031] MRS medium: tryptone 10g / L, beef extract 10g / L, diammonium citrate 2g / L, glucose 20g / L, Tween 80 1g / L, sodium acetate 5g / L, magnesium sulfate heptahydrate 0.1g / L, manganese sulfate 0.05g / L, potassium dihydrogen phosphate 2g / L, L-cysteine ​​0.5g / L, autoclaved at 115℃ for 30min. For solid medium, add 20g / L agar.

[0032] Bile salt stress simulation: 1 g / L of sodium glycodeoxycholate (GDCA) was additionally added to the MRS medium, and the pH was adjusted to 6.2±0.2 with 1 M NaOH solution before sterilization, and then sterilized by high-pressure steam at 115°C for 30 min.

[0033] L-malic acid modification: MRS medium (containing 1 g / L GDCA) was supplemented with 4 g / L L-malic acid, and the pH was adjusted to 6.2 ± 0.2 with 1 M NaOH solution before sterilization, and sterilized by high-pressure steam at 115 °C for 30 min.

[0034] The biochemical materials involved in the following embodiments are as follows:

[0035] Bifidobacterium breve Bb18 was from DuPont Nutrition & Biosciences, batch number 1103787639.

[0036] The methods involved in the following embodiments are:

[0037] 1. Strain activation and cultivation

[0038] Take a tube of Bifidobacterium breve Bb18 stored at -80℃ and streak it on MRS solid medium, then invert the plate and culture it in a 37℃ anaerobic incubator for 24-36h until white colonies grow. Then pick the colonies and inoculate them into MRS liquid medium, culture them in a 37℃ anaerobic incubator for 16h, and then inoculate them into new medium at a ratio of 2% (v / v) for anaerobic culture at 37℃.

[0039] 2. Animal Experiment Grouping

[0040] Thirty-two clean-grade healthy male C57 / 6J mice were randomly divided into 4 groups, namely blank control group, model control group, experimental group 1 (intervention with Bifidobacterium breve), and experimental group 2 (combination intervention with L-malic acid and Bifidobacterium breve), with 8 mice in each group. After adaptive feeding for 1 week, the blank control group and the model control group were gavaged with normal saline, and the experimental group 1 was gavaged with 0.2 mL of Bifidobacterium breve Bb18 solution (1×10 9 CFU / mL) once a day, and experimental group 1 was gavaged with 0.2 mL of Bifidobacterium breve Bb18 solution (1×10 9CFU / mL, compounded with 4g / L L-malic acid) 10mL / kg each time, for a total of 21 days. In the last week, except for the blank control group, mice in other groups drank 2.5% dextran sulfate (DSS) solution freely to construct a mouse ulcerative colitis model. After the experiment, all mice were killed, and serum, colon tissue and intestinal content samples of all mice were collected. Specific experimental grouping and treatment methods are shown in Table 1.

[0041] Table 1 Grouping of experimental animals

[0042]

[0043]

[0044] 3. Testing and recording of body weight and disease activity index (DAI):

[0045] The body weight of mice was measured once a week and once a day during the modeling stage. The mental state of mice was observed and the disease activity index (DAI) of mice was evaluated. The judgment criteria of the disease activity index (DAI) are shown in Table 2.

[0046] Table 2 Disease Activity Index (DAI) score

[0047]

[0048] Note: Normal stool: formed stool; Loose stool: Pasty, semi-formed stool that does not stick to the anus; Loose stool: Loose watery stool that sticks to the anus.

[0049] 4. Analysis of colonization of Bifidobacterium breve:

[0050] The DNA of the fecal flora of mice in each treatment group was collected and extracted once a week and frozen at -80°C. The colonization of Bifidobacterium breve was determined by real-time quantitative fluorescence PCR. The Bifidobacterium-specific primers fell within the full length of the 16S rRNA gene, and the clone of the 16S rRNA-specific gene fragment of the Bifidobacterium strain was constructed as a quantitative standard. Using Bifidobacterium breve Bb18 as a DNA template, specific primers were used to amplify the 16S rRNA-specific gene fragment. The target fragment was 169bp. After ligation and transformation, the 16S rRNA-specific gene fragment cloning template of Bifidobacterium breve Bb18 was obtained. Randomly pick one positive clone after enrichment in LB liquid medium and use a kit to extract plasmid DNA for use as a standard. Determine the concentration of plasmid DNA, and calculate the plasmid copy number using the following formula: N = mR / Mw, where N represents the copy number in copies / μL; m represents the DNA concentration in g / μL; R represents Avogadro's constant (R = 6.022×10 23); Mw represents the molecular weight of the plasmid, Mw = number of bases in the plasmid × 660, and the unit is g / mol.

[0051] The plasmid DNA was diluted in different gradients to a copy number of 1×10 3 to 1×10 9 (copies / μL), 2 μL was taken as amplification template to draw the standard curve. Similarly, 2 μL (20 ng) of environmental sample DNA was taken as amplification template for the corresponding quantitative PCR reaction.

[0052] The specific amplification primers are: Bb18-F (5'-TTTCTACGGAGAATACGGC) (SEQ ID NO. 1) and Bb18-R (5'-CGATCCGGGAGCCCAAAAG) (SEQ ID NO. 2), and the target fragment length is about 169 bp.

[0053] The 25 μL quantitative PCR amplification reaction system includes: 20 ng DNA (2 μL 10 ng / μL DNA), 1×Mix (containing Taq enzyme), and 0.5 μM of each primer.

[0054] The amplification reaction program was as follows: pre-denaturation at 95°C for 30 s; 40 cycles of 95°C for 5 s and 60°C for 10 s.

[0055] 5. Determination of serum inflammatory factor related indicators:

[0056] After blood was collected, serum was collected by centrifugation (3000r, 4°C), and the levels of inflammatory factors (TNF-α, IL-1β, IL-6 and IL-10) in serum were determined using ELISA kits.

[0057] 6. Colon histopathology sections:

[0058] The colon tissue was soaked in 4% paraformaldehyde solution, eluted with different concentrations of ethanol, and soaked in xylene solution for 2.5 hours. After embedding in paraffin, the sections were sliced ​​(3-5 μm), dewaxed with xylene, and then eluted with different concentrations of ethanol until colorless. The colon sections of each mouse in each group were stained with HE staining, and the morphological changes of the small intestinal tissue of each group of mice were observed and compared by scanning.

[0059] 7. Determination of related indicators of inflammatory factors in colon tissue:

[0060] A certain amount of colon tissue was weighed, and pre-cooled (4°C) physiological saline was added in a certain proportion. The tissue was homogenized several times using a high-throughput tissue grinder (70 Hz, 30 s) until no colon tissue was visible to the naked eye. The tissue homogenate was centrifuged at high speed to collect the supernatant. The levels of inflammatory factors (TNF-α, IL-1β, IL-6, and IL-10) in the colon tissue of mice with ulcerative colitis were determined using ELISA kits.

[0061] The technical solution of the present invention is described in detail below in conjunction with specific embodiments. In the following embodiments, unless otherwise specified, the reagents, materials and equipment used can be purchased from commercial sources, or prepared by conventional methods, or are commonly used in the industry.

[0062] Example 1: L-malic acid combined with Bifidobacterium breve culture in a bile salt-containing medium

[0063] Through in vitro culture experiments, according to the strain activation and culture methods in the above experimental methods, the Bifidobacterium breve strain was activated and cultured, and its growth in the culture medium containing 1g / L bile salt was observed (respectively cultured in MRS medium under bile salt stress and MRS medium containing bile salt modified by L-malic acid, and the OD600 after 16h of culture was tested). The experimental results are shown in Figure 1 The experimental results showed that after adding L-malic acid, Bifidobacterium breve could still maintain good growth activity in an environment with high bile salt concentration. Compared with the untreated control group, the bacterial growth of the L-malic acid treated group was significantly improved.

[0064] In addition, according to the above method, L-malic acid was replaced with other acids. The inventors also studied the effects of D-malic acid (D-MA), citric acid (CA), α-ketoglutaric acid (α-KG), fumaric acid (FMA) and oxaloacetic acid (OAA) in improving the bile salt tolerance of Bifidobacterium breve. The experimental results are shown in Figure 2 The results showed that only L-malic acid had a good effect on improving the bile salt tolerance of Bifidobacterium breve, while other acids could not significantly improve the growth activity of the bacteria.

[0065] Example 2: L-malic acid synergistically improves the intestinal colonization rate of Bifidobacterium breve

[0066] According to the animal grouping in Table 1, in the mouse colitis model, oral administration of an aqueous solution containing L-malic acid (concentration of 4 g / L) and Bifidobacterium breve Bb18 (1*10 9CFU / mL) mixture, administered once a day for 21 days, and quantitative determination of Bifidobacterium breve Bb18 in feces was performed. The experimental results showed that the bile salt tolerance of Bifidobacterium breve was significantly enhanced, and the colonization ability of Bifidobacterium breve in the intestine was significantly improved. The specific experimental results are shown in Figure 3 .

[0067] Example 3: L-malic acid combined with Bifidobacterium breve alleviates colitis in mice

[0068] After 21 days of intervention with L-malic acid combined with Bifidobacterium breve, the symptoms of DSS-induced colitis were significantly alleviated, the weight loss of mice was alleviated, the length of the colon was shortened, the degree of damage to the colon tissue was reduced, and the intestinal barrier function was restored. The effect of relieving colitis in mice was significantly better than that of Bifidobacterium breve alone. The experimental results are shown in Figure 4-6 .

[0069] Example 4: L-malic acid combined with Bifidobacterium breve reduces the level of inflammatory factors in mice

[0070] By measuring the levels of inflammatory factors in the intestine of the mouse colitis model, it was found that the levels of TNF-α, IL-1β, IL-6 and IL-10 in the mice treated with L-malic acid combined with Bifidobacterium breve were significantly lower than those in the control group, indicating that this method can effectively inhibit the immune inflammatory response associated with colitis. The results of colon inflammatory factor detection are shown in Figure 7 .

[0071] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of a composition of L-malic acid and Bifidobacterium breve in the preparation of a product for preventing colitis.

2. The use according to claim 1, characterized in that: The Bifidobacterium breve is Bifidobacterium breve Bb18.

3. The use according to claim 1, characterized in that: In the composition, the content of L-malic acid is 3-5 g / L.

4. The use according to any one of claims 1 or 3, characterized in that: In the composition, the content of Bifidobacterium breve is 1×10 7～10 CFU / mL.

5. The use according to claim 1, characterized in that: The composition is an oral preparation.

6. The use according to claim 5, characterized in that: The composition is in the form of a tablet or a liquid preparation.

7. The use according to any one of claims 5 or 6, characterized in that: The composition also includes pharmaceutically acceptable excipients.

8. A composition of L-malic acid and Bifidobacterium breve.

9. The composition according to claim 8, characterized in that The Bifidobacterium breve is Bifidobacterium breve Bb18.

10. The composition according to claim 8, characterized in that The content of L-malic acid is 3-5 g / L, and the content of Bifidobacterium breve is 1×10 7～10 CFU / mL.

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