Use of oleanolic acid in promoting the colonization of the gastrointestinal tract by probiotic ecn strain

Abstract

The application discloses application of oleanolic acid in promoting colonization of probiotic EcN strain in a gastrointestinal tract, and the oleanolic acid can promote formation of a biofilm of the probiotic EcN, enhance hydrophobicity of a surface of the probiotic EcN strain, and inhibit movement ability of the EcN strain, thereby benefiting adhesion and aggregation of the EcN strain, and further stabilizing colonization and survival in the intestinal tract, and having potential to be developed into probiotic food and preparations.

Description

TECHNICAL FIELD

[0001] The application relates to the medical and pharmaceutical technical field and in particular to application of oleanolic acid in promoting colonization of a probiotic EcN strain in a gastrointestinal tract. BACKGROUND

[0002] Biofilm (BF) can be defined as a microbial community embedded in self-produced extracellular polymeric substance (EPS) matrix, attached to biological or non-biological surfaces, and is a complex, highly hydrated three-dimensional structure. At present, the research on biofilm mainly focuses on biofilm-associated infections (BAI) caused by pathogenic bacteria, and the research on probiotic biofilm is still in its infancy. Escherichia coli Nissle 1917 (EcN) is an intestinal probiotic with good colonization characteristics, and the formation of EcN biofilm will be more conducive to stable colonization in the intestine, and the main mechanism is to adhere to the intestinal epithelium, secrete antibiotics, and then inhibit the colonization of pathogenic bacteria on the intestinal epithelium. However, most probiotics cannot effectively colonize in the intestine after oral administration, resulting in poor clinical use effect, so the proliferation culture of probiotics is particularly important, and biofilm as a new strategy for probiotic colonization is attracting widespread attention. Reports indicate that the addition of exogenous active substances can promote the growth and activity of probiotics, Ding Ting et al. found that the addition of whey protein hydrolysate can promote the growth of Lactobacillus acidophilus, and the biofilm and extracellular polysaccharide content are significantly increased, and the probiotic effect may be to increase the quorum sensing effect of Lactobacillus acidophilus, improve the ability to resist the external environment and promote bacterial reproduction.

[0003] At present, the use of natural compounds from plant sources to promote the formation of probiotic biofilm has been proved to be a promising strategy for the prevention and treatment of various BAI. Oleanolic acid studied in this paper is a pentacyclic triterpenoid compound widely existing in the plant kingdom, and is widely used due to its potential anticancer, hepatoprotective, hypolipidemic, antioxidant, antibacterial and other effects. However, there is no report on the effect of oleanolic acid on the formation of probiotic biofilm. Therefore, this paper mainly investigates the promotion of oleanolic acid on the stable colonization of ECN in the intestinal environment, and lays a theoretical foundation for the development of new probiotic products. SUMMARY

[0004] Therefore, the purpose of the present application is to provide application of oleanolic acid in promoting colonization of a probiotic EcN strain in a gastrointestinal tract.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical scheme:

[0006] 1. Application of oleanolic acid in promoting colonization of a probiotic EcN strain in a gastrointestinal tract.

[0007] In some embodiments of the present application, the concentration of the oleanolic acid is 8-64 μg / mL.

[0008] In some embodiments of the present application, the use of oleanolic acid in promoting the biofilm formation of probiotic EcN strain.

[0009] In some embodiments of the present application, the use of oleanolic acid in inhibiting the biofilm formation of pathogenic Escherichia coli standard strain ATCC 25922 and Staphylococcus aureus standard strain ATCC 29213.

[0010] In some embodiments of the present application, oleanolic acid enhances the hydrophobicity of the surface of probiotic EcN strain, thereby facilitating the adhesion and aggregation of EcN strain, and further promoting the formation of biofilm.

[0011] In some embodiments of the present application, the use of oleanolic acid in inhibiting the motility of EcN strain.

[0012] In some embodiments of the present application, the use of oleanolic acid in enhancing the adhesion ability of EcN strain.

[0013] The present application has the following advantages: oleanolic acid has antibacterial activity on probiotic EcN, with a minimum inhibitory concentration of 128 μg / mL. Oleanolic acid at concentrations of 8, 16, 32 and 64 μg / mL can promote the formation of EcN biofilm. When the drug intervention is at a concentration of 64 μg / mL, the growth rate of EcN biofilm can be as high as 178.08% (p<0.001). At the same concentration, the highest inhibition rate of oleanolic acid on the biofilm of Staphylococcus aureus standard strain (ATCC 29213) and Escherichia coli standard strain (ATCC 25922) can be as high as 52.77% and 48.25%, respectively. Therefore, subsequent experiments are carried out within this concentration range. MTT method is used to evaluate the metabolic activity of the biofilm bacteria, and the metabolic activity of the bacteria is 152.83% (p<0.001). The surface hydrophobicity of EcN treated with oleanolic acid is 55.06% (p<0.01), which is 2.92 times that of the control group. Compared with the control group, the bacterial swimming movement diameter is reduced by 51.13% after drug intervention.

[0014] In the acid resistance test, it was found that EcN had no bacterial growth at pH = 1.0, and at pH = 2.0, it had no growth in the first 12 hours, and then slowly grew after 24 hours. At pH = 3.0, the growth of EcN was stable. After culturing EcN in different concentrations of bile salt for 24 hours, the number of viable bacteria showed no significant difference compared with the control group. EcN was cultured in simulated artificial gastric juice for 0, 2, 4, and 6 hours, respectively, and the number of viable bacteria showed no statistical significance in the first 4 hours, but significantly increased at 6 hours (p<0.001). In simulated artificial intestinal fluid, the number of viable bacteria showed no significant difference after 0, 2, 4, 6, and 8 hours of culture (p>0.05). Conclusion: Oleanolic acid can promote the formation of biofilm of probiotic bacteria EcN, and can stabilize the colonization and survival in the intestinal tract, and has the potential to be developed as probiotic food and preparation. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to make the purpose, technical scheme and beneficial effects of the present application clearer, the present application provides the following drawings for illustration:

[0016] Figure 1 The growth curve of EcN treated with oleanolic acid;

[0017] Figure 2 The effect of oleanolic acid on the formation of different bacterial biofilms of probiotic bacteria EcN strain (A), Staphylococcus aureus strain (B) and Escherichia coli standard strain (C);

[0018] Figure 3 The effect of oleanolic acid on the metabolic activity of EcN biofilm;

[0019] Figure 4 The representative graph of CLSM observation of EcN biofilm formation intervened by oleanolic acid and the Biofilm Q biofilm data quantification analysis graph; A (Control) B (oleanolic acid) represents the orthogonal graph; C (Control) D (oleanolic acid) represents the 3D graph; E: biofilm quantity; F: biofilm volume; G: biofilm surface area; H: biofilm base area; I: total biofilm fluorescence intensity;

[0020] Figure 5 The effect of oleanolic acid on the hydrophobicity of EcN cell surface;

[0021] Figure 6 The effect of oleanolic acid on the motility of EcN;

[0022] Figure 7 The growth curve of EcN and its acid resistance;

[0023] Figure 8 The effect of oleanolic acid on the bile salt resistance of EcN;

[0024] Figure 9 Effect of artificial gastric juice and intestinal juice on growth of EcN. DETAILED DESCRIPTION

[0025] The present application will be further described below in conjunction with the drawings and specific examples so that those skilled in the art can better understand the present application and implement it. The examples are not intended to limit the present application.

[0026] Test strains: Escherichia coli Nissle 1917 (EcN), Staphylococcus aureus standard strain (ATCC 29213), and Escherichia coli standard strain (ATCC 25922).

[0027] Oleuropein (purity > 97%) and dimethyl sulfoxide were purchased from Shanghai Maikelin Biotechnology Co., Ltd.

[0028] Example 1, Preliminary exploration of the mechanism of oleanolic acid promoting EcN biofilm formation

[0029] I. Preparation of bacterial suspension

[0030] A single colony of EcN was inoculated into 7 mL of MH medium and cultured at 37°C in a constant temperature shaking incubator for 6-8 h. The bacterial suspension was adjusted to OD 600 = 0.1 (about 1 x 10 8 CFU / mL) using a UV-visible spectrophotometer.

[0031] II. Determination of minimum inhibition concentration (MIC)

[0032] The concentration of the bacterial solution was adjusted to 1 x 10 6 CFU / mL, and the minimum inhibition concentration of oleanolic acid on EcN was determined by micro-broth dilution method. In a 96-well plate, 40 μL of MH broth was added, and 40 μL of drug solution at 16384 μg / mL was added for successive dilution by a factor of two, and finally 40 μL of working bacterial solution was added. At the same time, bacterial solution positive control, drug negative control, and broth negative control were set. After incubation at 37°C for 16-18 h, the results were observed.

[0033] III. Effect on dynamic growth curve of EcN

[0034] The overnight culture of EcN was diluted to OD 600=0.1, dispensed into 5 EP tubes, and added an equal volume of oleanolic acid solution to achieve final concentrations of 8, 16, 32, and 64 μg / mL, respectively. The blank group consisted of an equal volume of 2% DMSO solution. The tubes were incubated in a 37℃ air bath with shaking. OD values ​​were measured at 600 nm using a spectrophotometer at 0, 2, 4, 6, 8, 10, 12, and 24 h. Within a certain wavelength range, the absorbance value was directly proportional to the bacterial concentration. Growth curves were constructed by plotting the relationship between absorbance and incubation time.

[0035] IV. Effects of Biofilm Mass

[0036] Add OD to polyethylene 96-well cell culture plates 600 50 μL of EcN (or pathogenic bacteria) working culture at 0.1 g / mL was added simultaneously with oleanolic acid solution to achieve final oleanolic acid concentrations of 8, 16, 32, and 64 μg / mL in the 96-well plates. The blank control consisted of an equal volume of 2% DMSO. After static incubation at 37°C for 24 h, the drug solution and bacterial culture were aspirated, and the plates were washed three times with PBS buffer. Methanol solution was added for fixation for 10 min. After washing away the methanol and allowing it to evaporate, crystal violet solution was added for staining for 20 min. The staining solution was discarded, and the biofilm was washed. Acetic acid solution was added to dissolve the biofilm, and the OD value was measured at 590 nm to determine the biofilm amount.

[0037] V. Effects on the metabolic activity of biological membranes

[0038] The experimental method of Martinez et al. was referenced and partially adjusted. The MTT stock solution was prepared at 5 mg / ml and dissolved in PBS solution (pH = 7.4). The 5 mg / ml MTT stock solution was diluted 10 times to prepare the working solution. After EcN and oleanolic acid formed a biofilm for 24 hours, the 96-well cell culture plate was removed from the 37°C incubator. The drug in the test wells was removed, and 100 μL of PBS solution was added. The plate was gently shaken, and the PBS solution was aspirated. This process was repeated 2-3 times. Then, 100 μL of MTT working solution was added to each well, and the plate was incubated in the dark (37°C, 3 hours). The MTT solution was aspirated, dissolved in 100 μL of DMSO, and the absorbance was measured at 590 nm.

[0039] VI. Observation of biofilm morphology using laser confocal scanning microscopy (CLSM)

[0040] At Lab-Tek TM Add 250 μL of EcN working bacterial culture and 250 μL of oleanolic acid to the coverslip in chamber II. Use 250 μL of 2% DMSO solution as a control. Incubate at 37°C for 24 h. After 24 h of incubation, remove the coverslip, discard the supernatant, wash twice with 0.9% NaCl solution, and use a filmtracer. TM IVE / DEADTM Biofilm Viability Kit staining for 20 min, and then washed with sterile water to remove residual dye. The morphology of the biofilm was observed under CLSM. The excitation wavelengths were 561 nm (PI) and 488 nm (SYTO), respectively. Three independent experiments were performed, and random fields were photographed. At least 12 images were selected for biofilm-related parameter analysis, including biofilm number, fluorescence intensity, substrate area, volume, etc. Biofilm Q software was used for data analysis.

[0041] Seven, determination of bacterial surface hydrophobicity

[0042] According to the affinity of hydrocarbons, the surface hydrophobicity of EcN treated with oleanolic acid was determined. The method of Fonseca et al. was used as a reference and was modified. The bacterial suspension was centrifuged at 8000 rmp / min for 10 min, the supernatant was discarded, and the bacteria were resuspended in PBS solution. The OD 600nm The absorbance value (A0) was measured at 600 nm. Oleanolic acid was added to the EcN bacterial suspension and mixed with 1 ml of xylene. The blank group was treated with an equal amount of PBS buffer. The mixture was shaken in a vortex oscillator for 3 minutes, and then incubated at 37°C for 1 hour. The aqueous phase was collected, and the absorbance (A1) was measured at 600 nm. The surface hydrophobicity calculation formula is as follows:

[0043] Hydrophobicity (%) = (A0-A1) / A0 x 100%

[0044] Where A0 is the initial absorbance of the bacteria, and A1 is the final absorbance after xylene treatment.

[0045] Eight, determination of bacterial motility

[0046] 0.5 g of tryptone, 0.25 g of yeast extract, 0.25 g of sodium chloride, and 0.15 g of bacterial agar powder were weighed. 50 mL of purified water was measured, and the mixture was thoroughly stirred and dissolved. High-pressure steam sterilization was performed for 15 min. Oleanolic acid solution was added as the drug group, and the plate was prepared. 1.5 μL of working bacterial solution was dropped onto the center of the control group and the drug group plates, respectively. After the bacterial solution was dried, the plates were incubated in a 37°C constant temperature incubator for 48 h.

[0047] Results and discussion

[0048] One, different MICs of oleanolic acid have no effect on the growth of EcN

[0049] The MIC of oleanolic acid for EcN was 128 μg / mL, and the MIC of DMSO for EcN was 12.5%. In this paper, the concentration of DMSO used was 2%, which had no effect on the test results.

[0050] The growth curve of EcN treated with oleanolic acid was measured by spectrophotometryFigure 1 ), and the results showed that:

[0051] The selected concentrations of oleanolic acid did not affect the growth of EcN, and subsequent experiments could be conducted.

[0052] II. Oleanolic acid promotes the formation of EcN biofilm

[0053] The OD value measured by crystal violet staining method represents the amount of EcN biofilm, and compared with the control group, the results are as follows Figure 2 , A shows that when the concentration of oleanolic acid is 8, 16, 32 and 64 μg / mL, the growth rate of EcN biofilm is 138.22% (p<0.05), 142.66% (p<0.01), 159.94% (p<0.001) and 178.08% (p<0.001) respectively. The quantitative test results show that oleanolic acid significantly promotes the production of EcN biofilm. It is worth noting that when screening active substances for promoting the formation of biofilm of probiotics, the inhibitory effect of the active substance on pathogenic bacteria should also be investigated. In view of this, the crystal violet staining method was used to screen oleanolic acid, which can inhibit the formation of biofilm of Staphylococcus aureus standard strain (ATCC 29213) and Escherichia coli standard strain (ATCC 25922) Figure 2 , B and 2, C). The results showed that when oleanolic acid was intervened, the highest inhibition rate of Staphylococcus aureus standard strain (ATCC 29213) and Escherichia coli standard strain (ATCC 25922) biofilm could reach 52.77% and 48.25% respectively.

[0054] III. Effect of oleanolic acid on metabolic activity of EcN biofilm bacteria

[0055] MTT method can preliminarily evaluate the effect of different concentrations of oleanolic acid on the metabolic activity of EcN biofilm bacteria Figure 3 ). The results showed that when the concentration of oleanolic acid was 64 μg / mL, the metabolic activity increased by 152.83% (p<0.001).

[0056] IV. Morphological observation of biofilm

[0057] The representative orthogonal graph and 3D graph of oleanolic acid intervention EcN biofilm were observed by confocal laser scanning microscopy (CLSM) Figure 4), green (SYTO) represents live bacteria, and red (PI) represents dead bacteria. The results showed that after oleanolic acid intervention, the bacteria formed "mushroom-like" accumulation, the biofilm was highly dense, the field of view was clear, the total fluorescence intensity, number, volume and surface area of EcN were significantly different (p<0.0001), which increased by 3.01 times, 0.91 times, 4.39 times and 0.93 times, respectively, and the biofilm basal area had no significant difference.

[0058] V. Determination of the Hydrophobicity of Bacteria by Oleanolic Acid

[0059] The results of the influence of oleanolic acid on the hydrophobicity of EcN are shown in Figure 5 Compared with the control group, the hydrophobicity of EcN strain after oleanolic acid intervention was 55.06% (P<0.01), which was 2.92 times that of the control group. It is reported that when the hydrophobicity is less than 40%, it is called a hydrophilic strain; when the hydrophobicity of the strain is 40%-60%, it can be considered as a moderately hydrophobic strain. The adhesion ability of probiotics is the key to its effective colonization in the intestinal tract, and the higher surface hydrophobicity of bacteria helps the adhesion and aggregation of bacterial bodies, which is more conducive to the formation of biofilm. Therefore, the intervention of oleanolic acid enhances the surface hydrophobicity of EcN, which is more conducive to the adhesion and aggregation of EcN, and further promotes the formation of biofilm.

[0060] VI. Oleanolic Acid Promotes the Motility of EcN

[0061] The influence of oleanolic acid on the motility of EcN is shown in Figure 6 After the action of oleanolic acid, the movement diameter of bacteria was reduced by 51.13% (p<0.001) compared with the control group. The weakening of the movement ability of bacteria will lead to the enhancement of its adhesion ability, which is conducive to the initial adhesion of biofilm, and thus can promote the formation of biofilm.

[0062] Example 2, EcN Tolerance Determination

[0063] I. Preparation of Artificial Digestive Juice

[0064] Artificial intestinal juice: weigh 0.68 g of potassium dihydrogen phosphate and dissolve in 50 mL of distilled water, add 1 g of trypsin, make up to 100 mL, filter sterilization with 0.22 μm microporous filter membrane.

[0065] Artificial gastric juice: weigh 0.2 g of sodium chloride, 1 g of pepsin, take 0.7 mL of concentrated hydrochloric acid, add water to make up to 100 mL, adjust the pH to 3 with 1 mol / L hydrochloric acid, filter sterilization with 0.22 μm microporous filter membrane.

[0066] II. Acid Tolerance Test

[0067] The pH of sterile water was adjusted to 1.0, 2.0 and 3.0 with 0.1 mol / L hydrochloric acid, respectively. The medium was prepared with water of different pH values and sterilized for use. 5% of EcN working solution in the logarithmic growth phase was inoculated into the medium of different pH values and cultured at 37°C on a shaking table. The bacterial solution was collected every 2h, and the medium without 0.1 mol / L hydrochloric acid was used as a control. The absorbance value of the bacterial solution was measured at a wavelength of 600 nm, and the growth curve of EcN acidity tolerance was drawn.

[0068] III. Bile salt tolerance test

[0069] 0.03g of bovine bile salt was weighed and dissolved in the medium, and then filtered to remove bacteria for standby use. 0.1%, 0.2% and 0.3% bile salt solutions were prepared. 200μL of EcN working solution was inoculated into 4.8mL of bile salt medium of different concentrations, and the medium without bile salt was used as a control. After 24h of culture at 37°C on a shaking table, the bacterial colony count was performed.

[0070] IV. Simulated artificial gastrointestinal fluid test

[0071] 5% EcN working solution was inoculated into the above artificial gastric and intestinal fluids, respectively, and cultured at 37°C on a shaking table. Samples were taken every 2h, diluted with sterile water in a 10-fold gradient, and plated for colony count.

[0072] V. Data processing and statistical analysis

[0073] All experimental data were statistically processed using Excel and Prism 8.0. Single factor analysis of variance was used and multiple tests were performed to calculate the statistical significance of the differences. * represents p<0.05 for significant difference, **p<0.01, ***p<0.001, ****p<0.0001 for extremely significant difference.

[0074] Results and discussion

[0075] I. EcN growth curve

[0076] Figure 7 As shown in Fig. 1A, EcN strain entered the logarithmic growth phase after 2h of culture at 37°C, entered the stationary phase after 12h of culture, and remained stable until 24h of culture. This growth curve conforms to the growth rule of most bacteria. Whether other culture temperatures will affect the growth of ECN strain remains to be explored.

[0077] II. Acid tolerance test

[0078] As shown in Fig. 2A, EcN strain entered the logarithmic growth phase after 2h of culture at 37°C, entered the stationary phase after 12h of culture, and remained stable until 24h of culture. This growth curve conforms to the growth rule of most bacteria. Whether other culture temperatures will affect the growth of ECN strain remains to be explored. Figure 7, B can be known that EcN has no bacterial growth at pH 1.0 in the culture medium, indicating that the extremely low pH acidic environment has a bactericidal effect; at pH 2.0, there is no growth in the first 12 hours, and some bacteria grow slowly at 24 hours; at pH 3.0, the bacterial growth stable period is delayed. The results show that the acidic environment has an inhibitory effect on the growth of EcN, and the bacteria can grow at pH 2.0 and 3.0, and can tolerate higher pH acidic environment.

[0079] III. Bile salt tolerance determination

[0080] From Figure 8 It can be seen that after culturing EcN in different concentrations of bile salt for 24 hours, the number of viable bacteria has no statistical significance compared with the control group with the increase of bile salt concentration. It shows that EcN can still survive in high bile salt environment and has tolerance to higher bile salt environment.

[0081] IV. EcN tolerance determination to artificial gastric juice

[0082] From Figure 9 , A can be known that EcN is cultured in simulated artificial gastric juice with pH 3.0 for 0, 2, 4, 6 and 8 hours, respectively, and the number of viable bacteria has no statistical significance in the first 4 hours, and the number of viable bacteria increases significantly at 6 hours (p<0.001). The results show that the number of bacteria does not decrease in the simulated artificial gastric juice environment, but gradually increases with the extension of the treatment time, that is, EcN has strong tolerance to artificial gastric juice and can adapt to the acidic environment.

[0083] V. Tolerance determination to artificial intestinal juice

[0084] The primary condition for probiotics to play a probiotic role is to be stably colonized in the intestinal tract. For example Figure 9 , B, EcN is cultured in simulated artificial intestinal juice for 0, 2, 4, 6 and 8 hours, respectively, and the number of viable bacteria has no significant difference (p>0.05) in the first 8 hours. It shows that EcN has good tolerance to simulated artificial intestinal juice and can stably survive in the intestinal tract. Some literature points out that the number of viable bacteria of probiotics should be more than Log 106 when they play a probiotic role, and the number of viable bacteria of EcN used in this test is far more than this, which shows that EcN has good tolerance to artificial intestinal juice and can be stably colonized and survive in the intestinal tract, which lays a good foundation for its probiotic role.

[0085] Analysis and discussion

[0086] Currently, there are a large number of reports on biofilm bacterial infection, but mainly focus on most pathogenic bacteria, and the potential bacterial population of drug-resistant bacteria in clinical infection is increasing. Under the environment of limiting antibiotics in feed and breeding, it is urgent to find alternative drugs. At present, the research on biofilm of probiotics is still in the initial stage, but the growth advantage of biofilm of probiotics is obvious. It is reported that the formation of non-pathogenic bacterial biofilm plays a crucial role in maintaining the balance of the body and can inhibit the growth and adhesion of pathogenic microorganisms.

[0087] EcN as an intestinal probiotic has excellent colonization characteristics, and the formation of EcN biofilm will be more conducive to stable colonization in the intestine, improve its anti-bacterial characteristics, and better prevent certain microorganisms from long-term proliferation in the intestine. It is reported that endogenous active substances such as heparin and hydrolyzed milk protein can promote the formation of probiotic biofilm, and plant-derived active substances are the main source of newly developed drugs, second only to other natural sources and synthetic compounds. It is found that trans-resveratrol can promote the formation of biofilm of Lactobacillus paracasei ATCC334 by enhancing its adhesion ability.

[0088] In the previous study, oleanolic acid was screened to promote the formation of EcN biofilm. With the increase of drug concentration, the effect of promoting biofilm formation was more obvious. Probiotic EcN is a good biofilm former, which adheres to the intestinal epithelial cells through pili and flagella, and secretes antibacterial substances to inhibit the adhesion of pathogenic bacteria to the intestinal epithelium. In view of this, two strains of gram-negative and positive bacteria with strong biofilm formation ability were successfully screened. The results showed that the biofilm formation of Escherichia coli standard strain ATCC 25922 and Staphylococcus aureus was inhibited by oleanolic acid intervention. Lu Tingting et al. found that sub-inhibitory concentration of rhein could significantly inhibit the biofilm formation of Staphylococcus xylosus. With the increase of rhein concentration, the biofilm formation was significantly reduced in a concentration-dependent manner, with an average inhibition rate of 40.73%, which was similar to the results of this study. Zhang Wenyang et al. found that tea polyphenols, lemon aldehyde and cinnamyl aldehyde had different degrees of biofilm inhibition on Staphylococcus aureus and Salmonella enteritidis at sub-inhibitory concentration. In short, oleanolic acid can be used as a plant metabolite to inhibit the aggregation of pathogenic microorganisms and enhance the biofilm formation of probiotics. Natural small molecules from plants have potential use in dietary supplements for the food industry. Therefore, this study lays a theoretical foundation for the development of oleanolic acid as a probiotic preparation. MTT method was used to determine the effect of biofilm bacterial metabolic activity. The results showed that with the increase of drug concentration, the bacterial activity gradually increased. Laser confocal scanning microscopy (CLSM) was used to observe the effect of oleanolic acid intervention on the number and volume of biofilm. The results showed that the number and volume of biofilm increased significantly with the intervention of oleanolic acid. This study evaluated the effect of oleanolic acid on EcN biofilm by combining the above three methods. The results showed that oleanolic acid could promote the formation of EcN biofilm.

[0089] The adhesion ability of probiotics is the key to its effective colonization in the intestinal tract, and most studies have shown that higher surface hydrophobicity of bacteria helps bacterial adhesion and aggregation, and is more conducive to biofilm formation. Therefore, the intervention of oleanolic acid enhances the surface hydrophobicity of EcN strain, which is more conducive to the adhesion and aggregation of EcN strain, and further promotes the formation of its biofilm. Zhang Wenxiao et al. found that the potential respiratory probiotic D-19 has a hydrophobicity of 51.34%, which is a moderately hydrophobic strain. The authors pointed out that the surface hydrophobicity of the bacterial body is positively correlated with its adhesion ability, but there are many factors affecting the surface hydrophobicity, such as culture time, temperature, polysaccharides and proteins, etc. In addition, the weakening of bacterial motility will lead to the enhancement of adhesion ability, which is more conducive to the initial adhesion of biofilm, and further promotes the formation of biofilm. Gong Bingxue et al. found that the motility of EcN and EcN△csgA is basically the same, which proves that curli pili does not affect the motility of bacteria, and there is no significant difference in the amount of biofilm formed by EcN and EcN△csgA, which shows that EcN△csgA has no effect on the formation of EcN biofilm. However, EcN has type1 and F1C pili in addition to curli pili, so it can be indirectly speculated that oleanolic acid acts on the type1 and F1C pili of EcN, which weakens its motility, enhances its adhesion, and is more conducive to the initial adhesion and formation of biofilm. However, the specific target of action needs to be further explored.

[0090] It is well known that oral probiotics have a wide range of applications, and are safer and more convenient. However, before reaching the gastrointestinal tract to exert its effective role, it will be interfered by factors such as gastric acid, degrading enzymes, intestinal fluid, which is also the main reason for the low bioavailability of most probiotics. Xiao Xinyun et al. found that Debaryomyces hansenii did not survive at pH 1.0, and the viable count decreased significantly at pH 1.5, 2.0 and 2.5 compared with the control group. It can be seen that Debaryomyces hansenii can also tolerate a higher acidic environment, similar to the results of this study. EcN did not inhibit bacterial activity in a strong acidic environment, and could adapt to growth and exert a probiotic effect, with the potential to be developed as a probiotic food and preparation. The tolerance of bacteria to the gastrointestinal tract is an important indicator for judging the effectiveness of probiotics. Rao Zebin et al. found that Lactobacillus amylovorus TZR-PI001 was resistant to artificial gastric juice and could adapt to pH 2.0 artificial gastric juice within 3h and start to proliferate. EcN in simulated artificial gastric juice showed that the viable count did not decrease, but instead increased continuously with the extension of treatment time, i.e. it had strong resistance to artificial gastric juice and could adapt to proliferation. In the simulated artificial intestinal fluid environment, compared with 0h, the viable count increased continuously after 2, 4, 6 and 8h treatment, i.e. EcN had good resistance to artificial intestinal fluid and could stably colonize and survive in the intestinal tract, which laid a good foundation for exerting its probiotic effect. In view of the overall evaluation of EcN in simulated gastrointestinal environment, it was found that EcN had good stress resistance and adaptability, and had the development value of probiotics, providing a basis for its use as a probiotic preparation. However, the safety of EcN, including its toxicity test, side effects and other issues still need to be verified in the next step.

[0091] The above-described embodiments are only preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. Any equivalent substitution or transformation made by those skilled in the art based on the present application is within the protection scope of the present application. The protection scope of the present application is subject to the claims.

Claims

1. The application of oleanolic acid in the preparation of products that promote the colonization of probiotic EcN strains in the gastrointestinal tract, characterized in that, The application of oleanolic acid in promoting biofilm formation of probiotic EcN strain, wherein the concentration of oleanolic acid is 8~64 μg / mL.

2. The application according to claim 1, characterized in that, Oleanolic acid enhances the hydrophobicity of the surface of the probiotic EcN strain, thereby facilitating the adhesion and aggregation of the EcN strain and promoting the formation of biofilm.

3. The application according to claim 1, characterized in that, Application of oleanolic acid in inhibiting the motility of EcN strains.

4. The application according to claim 1, characterized in that, Application of oleanolic acid in enhancing the adhesion ability of EcN strains.

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