<i>Bifidobacterium longum </i>subsp. <i>infantis </i>with fimbriae and applications thereof

Abstract

A strain of Bifidobacterium longum subsp. infantis Y46 with fimbriae (BI_Y46) includes a unique flagella structure, exhibits strong abilities in utilizing 2-fucosyllactose (2-FL), galacto-oligosaccharides and short-chain galacto-oligosaccharides, possesses excellent probiotic characteristics with higher levels of extracellular polysaccharides and surface proteins than a strain of Bifidobacterium longum subsp. infantis M63 (BI_M63) and a strain of Bifidobacterium longum subsp. infantis ATCC 15697 (BI_15697), and further displays stronger ability to tolerate gastric acid and inhibitory ability of the cell-free supernatant to Escherichia coli ATCC 15922 than the BI_15697. Therefore, the BI_Y46 has excellent probiotic potential and can be used to develop probiotics, infant food, prebiotic products, and functional foods.

Claims

1. A method for inhibiting Escherichia coli, comprising: administering to a subject a product comprising a strain of Bifidobacterium longum subsp. infantis Y46 with fimbriae (BI_Y46) in an effective amount, wherein the BI_Y46 is deposited in China Typical Culture Preservation Center, Wuhan, China, with an accession number of CCTCC NO: M 20221253, dated Aug. 9, 2022.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows morphology of strains.

(2) FIG. 2 shows adhesion ability of strains of Bifidobacterium longum subsp. infantis Y46 with fimbriae (BI_Y46) to HT-29 cells.

SPECIFIC EMBODIMENTS

(3) The present invention is further elaborated in combination with specific embodiments and attached drawings.

(4) Bifidobacterium longum subsp. infantis M63 (BI_M63) is a strain preserved in the laboratory. Bifidobacterium longum subsp. infantis ATCC 15697 (BI_15697) and Escherichia coli ATCC 25922 (EC_25922) are purchased from China Industrial Microbiology Culture Collection Management Center (CICC).

(5) Reducing Solution comprises: 10 g/L of bacterial peptone, 5 g/L of sodium chloride, 0.5 g/L of L-Cysteine H Cl, 1 mL/L of Tween 80 which are sterilized at 121? C. for 15 minutes. RB Agar Medium is prepared according to a method described by Hartemink[19] and sterilized at 118? C. for 15 minutes. MRSC Medium is prepared as follows: supplementing MRS broth medium with 0.5 g/L of L-Cysteine HCl, adding 15 g/L of agar for obtaining solid MRSC medium, and then sterilizing at 118? C. for 15 minutes.

(6) No Carbon Source MRSC Medium comprises: 10 g/L of bacterial peptone, 5 g/L of yeast extract, 0.5 g/L of L-Cysteine HCl, 2 g/L of potassium dihydrogen phosphate, 5 g/L of anhydrous sodium acetate, 2 g/L of ammonium citrate, 0.2 g/L of magnesium sulfate heptahydrate, 0.05 g/L of manganese(II) sulfate monohydrate, 1 mL/L of Tween 80, which are sterilized at 118? C. for 15 minutes.

(7) Different Carbon Source Media are prepared by: separately adding 2-fucosyllactose, short-chain galacto-oligosaccharides, galacto-oligosaccharides, glucose, and lactose to each of the Carbon Source MRSC Medium mentioned above, adjusting each final concentration to 10 g/L, and obtaining 2-fucosyllactose medium, short-chain galacto-oligosaccharides medium, galacto-oligosaccharides medium, glucose medium, and lactose medium, which are sterilized at 118? C. for 15 minutes.

Embodiment 1: Isolation and Purification of Bifidobacterium longum

(8) Use a sterile fecal collection device to collect fecal samples from breast-fed healthy infants in Harbin, Heilongjiang Province, China who are exclusively breastfed, full-term, delivered vaginally, aged within 6 months, and have not been introduced to solid foods or probiotic products within the past two weeks, immediately transfer collected fecal samples to a cooler with ice packs and transport the same to the laboratory, upon arrival at the laboratory, store the collected fecal samples in a ?80? C. freezer, and perform isolation and purification experiment of bifidobacteria promptly as follows:

(9) Take 1?2 grams of the collected fecal samples and perform a ten-fold serial dilution via a reducing solution, then prepare dilutions of 10.sup.5, 10.sup.6, and 10.sup.7, spread each dilution onto RB agar plates, place the RB agar plates in an anaerobic incubator set at 37? C., and incubate for 24?72 hours under anaerobic conditions.

(10) After a designated incubation period, carefully examine the plates for the growth of distinct colonies. Select colonies that exhibit typical morphological characteristics for further analysis and purification.

(11) After Obtaining Single Bacterial Colonies:

(12) Pick a single bacterial colony and transfer the same to MRSC liquid medium, incubate the culture anaerobically at 37? C. for 24 hours, perform Gram straining and evaluate the strain for Gram-positive characteristics and negative catalase reaction, additionally, observe if the strain appears as rod-shaped or bifurcated, if a strain meets the criteria of being Gram-positive, catalase-negative, rod-shaped or bifurcated, streak the strain onto an agar plate in order to obtain isolated colonies, then repeat the above steps including picking single colonies, streaking, and incubating, until microscopic examination confirms the presence of pure cultures, assign each strain a unique identification number, add an equal volume of sterile glycerol (13% v/v) to the pure cultures, and store the strains at ?20? C. in a freezer.

(13) Inoculate a loopful of the strains from a preservation tube into MRSC liquid medium and incubate anaerobically at 37? C. for 24 hours, then, inoculate 1% (v/v) of the culture into MRSC liquid medium and continue anaerobic incubation for 18 hours as a reserve.

(14) Use a scanning electron microscope (SEM) S-3400N equipped with a tungsten filament to observe bacterial morphology.

(15) Generally, bifidobacterium observed under scanning electron microscope (SEM) exhibits a branching or rod-shaped morphology with irregular arrangement. Control strains BI_15697 and BI_M63 display smooth surfaces and appear as short rods or curved rods. However, strains BI_Y46 are more unique, as SEM images reveal a Y-shaped morphology, with the presence of flagella on surfaces of cells (see FIG. 1).

(16) Use the Omega D4015-01 Bacterial Genomic DNA Extraction Kit from Omega Bio-Tek to extract bacterial genomic DNA, amplify extracted DNA by using specific primers for Bifidobacterium (Bif164-F:5-GGGTGGTAATGCCGGATG-3; PbiR2:5-GACCATGCACCACCTGTGAA-3), perform 1% agarose gel electrophoresis to verify amplification products and send the same to Shanghai Sangon Biotech Co., Ltd. for sequencing. After sequencing, obtain 16S rDNA sequence and compare the same on the BLAST website, revealing that Bifidobacterium BI_Y46 belongs to Bifidobacterium longum subsp. infantis (abbreviated as BI_Y46), which has been preserved at the China Center for Type Culture Collection (CCTCC) in Wuhan, with a preservation number of CCTCC NO: M 20221253, on Aug. 9, 2022.

Embodiment 2: Capability Evaluation of Bifidobacterium longum in Utilizing 2-Fucosyllactose (2-FL) and Short-Chain Galacto-Oligosaccharides

(17) Centrifuge 1 mL of BI_Y46, BI_15697, and BI_M63 bacterial solution cultured in MRSC medium at 10,000?g for 5 minutes at 4? C., discard the supernatant, wash the precipitate with 1 mL of sterile water, and re-suspend bacterial cells in 1 mL of sterile water.

(18) Add 200 ?L of 2-fucosyllactose (2-FL) medium, short-chain galacto-oligosaccharides medium, lactulose medium, glucose medium, and lactose medium into separate wells of a 96-well plate, inoculate each well with re-suspended bacterial cells at a 1% (v/v) inoculum size, make sure to record well numbers and set up following groups: control group. MRSC medium without a carbon source group, 2-fucosyllactose medium group, short-chain galacto-oligosaccharides medium group, lactulose medium group, glucose medium group, lactose medium group, and MRSC medium without a carbon source with added bacteria group, incubate all groups anaerobically at 37? C., and measure absorbance values at 595 nm at 0 h, 24 h, and 48 h to evaluate capabilities in utilizing 2-fucosyllactose and short-chain galacto-oligosaccharides.

(19) The results show that Bifidobacterium longum subsp. infantis BI_Y46, along with the control strains BI_15697 and BI_M63, exhibited different metabolic capabilities towards 2-fucosyllactose, short-chain galacto-oligosaccharides, galacto-oligosaccharides, glucose, and lactose, as shown in Table 1. Both BI_15697 and BI_Y46 demonstrate good utilization of glucose and lactose. The utilization ability of BI_Y46 for oligo-galactose is comparable to BI_M63 and superior to BI_15697. Moreover, BI_Y46 shows significantly stronger utilization abilities for short-chain oligo-galactose and 2-fucosyllactose compared to BI_15697 and BI_M63. The results indicate that BI_Y46 has a stronger ability to utilize oligo-galactose compared to BI_15697, and additionally, BI_Y46 exhibits excellent utilization capabilities for human milk oligosaccharides such as 2-fucosyllactose and galacto-oligosaccharides.

(20) TABLE-US-00001 TABLE 1 Highest absorbance values (OD595 nm) of experimental strains within 48 hours of growth by separately using 2-fucosyllactose, galacto- oligosaccharides, and short-chain galacto-oligosaccharides Strain 2-FL SC-GOS GOS Glucose Lactose Bl_M63 0.65 ? 0.12b 0.96 ? 0.05b 1.27 ? 0.17a 1.09 ? 0.17b 1.27 ? 0.12b Bl_15697 0.11 ? 0.03c 0.25 ? 0.01c 0.63 ? 0.03b 1.36 ? 0.12ab 1.36 ? 0.09ab Y46 0.92 ? 0.11a 1.05 ? 0.03a 1.32 ? 0.11a 1.54 ? 0.23a 1.54 ? 0.16a Note: different lower-case letters in the above form indicate a significant difference among different strains.

Embodiment 3: Probiotic Properties of Bifidobacteria

(21) Extracellular Polysaccharide (EPS) Extraction and Determination:

(22) Centrifuge the above bacterial solution at 6000?g for 10 minutes at 4? C., mix the supernatant with three times the volume of ethanol, precipitate at 4? C. for 16 hours, centrifuge the precipitate at 10000?g for 10 minutes at 4? C., after air-drying the precipitate, add 5 mL of water for re-suspending, dialyze the suspension with a 3.5 KD dialysis bag for 48 hours, change the water every 8 hours, and measure the extracellular polysaccharide (EPS) production by using the Alcian Blue method.

(23) Add 50 ?L of different concentrations of xanthan gum solutions to separate 1.5 mL EP tubes, then add 100 ?L of 7% (v/v) ice-cold acetic acid and 100 ?L of 0.5 mg/mL Alcian Blue, allow the reaction to proceed at room temperature for 30 minutes, then centrifuge at 6800?g for 5 minutes, take 200 ?L of the supernatant, transfer to a 96-well plate, and measure absorbance values of the supernatant at 595 nm. Establish a standard curve equation and a cubic function equation based on the concentrations of xanthan gum solutions and the absorbance values.

(24) Add 50 ?L of the sample to a 1.5 mL EP tube, followed by adding 100 ?L of 7% (v/v) ice-cold acetic acid and 100 ?L of 0.5 mg/mL Alcian Blue. Allow the reaction to proceed at room temperature for 30 minutes, then centrifuge at 10,000?g for 5 minutes. Measure absorbance values of the supernatant at 595 nm. Use the method of standard curve analysis and perform a univariate solution to convert the absorbance values at 595 nm to milligrams of xanthan gum equivalent per milliliter.

(25) Determination of Protein Content in Fermentation Broth and Surface Protein Content of Bacterial Cells:

(26) Centrifuge 10 mL of the above bacterial solution at 6000?g for 10 minutes at 4? C., wash the precipitate twice with PBS, add 2 mL of a 5 mol/L solution of LiCl to the precipitate and incubate by constant shaking (200 r/min) for 1 hour at 37? C., centrifuge the mixture again at 6000?g for 10 minutes at 4? C. mix the supernatant with twice the volume of ice-cold acetone and place overnight at ?20? C. and measure surface protein content of the bacterial cells by using the Bradford method.

(27) Determination of Self-Aggregation Ability:

(28) Centrifuge 5 mL of the above bacterial solution at 4500?g for 15 minutes at 4? C., wash the precipitate with PBS buffer and re-suspend, measure absorbance values of the bacterial suspension at 595 nm at 0 hour, 2 hours, 4 hours, and 6 hours. The aggregation rate (A %) can be calculated using the following formula:
A %=(1?At/A0)?100%,

(29) Where A0 is an initial absorbance value at 0 hours and At is a absorbance value at a specified time point.

(30) Determination of Biofilm Formation Ability:

(31) Centrifuge 1 mL of the above bacterial solution at 10,000?g for 5 minutes at 4? C., discard the supernatant, wash with 1 mL of sterile water, vortex thoroughly and centrifuge again at 10,000?g for 5 minutes at 4? C., and re-suspend the precipitate in 1 mL of sterile water. Add 200 uL of culture medium to each well of a 96-well plate, incubate at 37? C. under anaerobic conditions for 96 hours, remove the culture medium and wash each well with 200 uL of sterile water. Add 200 uL of 0.1% (w/v) crystal violet dye and incubate for 30 minutes, wash the wells again and allow them to air dry for 30 minutes, dissolve the cell-bound crystal violet by adding 200 uL of ethanol-acetone solution (80:20) and incubating for 10 minutes. Transfer 135 uL from each well to a new 96-well plate and measure absorbance values at 595 nm.

(32) Simulated Gastric Fluid Tolerance Analysis

(33) Inoculate the above bacterial solution into artificial gastric fluid at a volume ratio of 1:5 (gastric pepsin 10 g/L, adjusted to pH 2.0 with hydrochloric acid). Incubate at 37? C. without agitation. At 0 h, 0.5 h, and 3 h, perform serial dilutions of the bacterial suspension and plate the same on MRSC solid agar plates for colony counting.

(34) Bile Salt Tolerance Analysis

(35) Centrifuge 1 mL of the above bacterial solution at 10,000?g for 5 minutes at 4? C. Wash bacterial cells twice and re-suspend in MRSC culture medium containing 0.2% porcine bile salts. Incubate the suspension at 37? C. under anaerobic conditions. Perform colony counting respectively at 0 hour and 0.5 hours.

(36) Determination of Bacteriostatic Ability of Cell-Free Supernatant:

(37) Centrifuge 5 mL of the above bacterial culture at 10,000?g for 5 minutes at 4? C., collect the supernatant and filter the same through a 0.22 ?m microporous membrane, and use EC_25922 (Escherichia coli strain) as an indicator strain and apply Oxford cup assay to assess the antibacterial activity of each bacterial strain.

(38) All experiments were conducted independently 3 times, and the results are presented as the means?standard deviation (x?SD). The experimental data were analyzed for statistical significance using the SPSS software with the Wald-Duncan test, with a significance level set at P<0.05, indicating a significant difference.

(39) The present invention measured the extracellular polysaccharide content, bacterial surface protein content, biofilm formation, bile salt tolerance, inhibitory ability of cell-free supernatant against EC_25922 (as shown in Table 2), self-aggregation capacity of the BI_Y46 strain (as shown in Table 3), and simulated gastric fluid tolerance (as shown in Table 4) in comparison to control strains BI_15697 and BI_M63.

(40) The results showed that the extracellular polysaccharide content of BI_Y46 was higher than that of the two control strains, but the difference was not significant. The bacterial surface protein content of BI_Y46 was significantly higher than that of the two control strains. Results from Table 1 indicated that BI_Y46 exhibited significantly higher tolerance to 0.2% pig bile salts compared to the control strains. Table 3 revealed that BI_Y46 demonstrated intermediate tolerance to gastric acid between BI_M63 and BI_15697. When using sterile MRSC medium as a control, the inhibitory ability of BI_Y46's cell-free supernatant against EC_25922 was comparable to BI_M63 but significantly higher than BI_15697. Furthermore, BI_Y46 exhibited significantly higher self-aggregation ability than the control strains,

(41) TABLE-US-00002 TABLE 2 Comparison of different properties of experimental strains (x ? SD, n = 3) Item Bl_M63 Bl_15697 Bl_Y46 Exopolysaccharide 0.36 ? 0.06a 0.36 ? 0.03a 0.44 ? 0.02a content (mg/mL) Surface protein content 0.27 ? 0.01b 0.22 ? 0.02c 0.31 ? 0.02a of bacterial cells(mg/mL) Biofilm-forming capacity 0.11 ? 0.02a 0.12 ? 0.03a 0.11 ? 0.02a (OD595) 0.2% Porcine bile salt 7.68 ? 0.1b 8.08 ? 0.11a 7.25 ? 0.04c tolerance (Log reduction of bacterial colonies (CFU/mL)) Diameter of antibacterial 13.50 ? 0.50a 8.00 ? 0.00b 12.83 ? 0.29a ring (mm) Note: Different lower-case letters in the above form indicate a significant difference among different strains.

(42) TABLE-US-00003 TABLE 3 Self-aggregation ability of each experimental strain (x ? SD, n = 3) Strain 2 h (%) 4 h (%) 6 h (%) Bl_M63 17.67 ? 2.74Cb 27.37 ? 1.89Bb 38.98 ? 0.96Ac Bl_15697 18.92 ? 1.49Cb 32.80 ? 3.82Bb 48.43 ? 0.90Ab Y46 30.56 ? 2.51Ca 48.83 ? 5.76Ba 68.56 ? 3.16Aa Note: in the above form, capital letters indicate significant differences in growth conditions for a same strain at different time points, and lowercase letters indicate significant differences in growth conditions among different strains at a same time point.

(43) TABLE-US-00004 TABLE 4 Simulated Gastric Tolerance at pH 2.0 (x ? SD, n = 3) 0 h 0.5 h 3 h Strain (Log (CFU/mL)) (Log (CFU/mL)) (Log (CFU/mL)) Bl_M63 7.08 ? 0.20Bb 7.95 ? 0.02Aa 8.23 ? 0.15Aa Bl_15697 7.88 ? 0.02Aa 6.09 ? 0.08Bc 5.46 ? 0.28Cc Bl_Y46 7.97 ? 0.02Aa 7.76 ? 0.01Bb 7.81 ? 0.03Bb Note: in the above form, capital letters indicate significant differences in growth conditions for a same strain at different time points, and lowercase letters indicate significant differences in growth conditions among different strains at a same time point.

Embodiment 4: Determination of Cell Adhesion Capability

(44) Colon cancer cells (HT-29) stored in the laboratory of the present invention are routinely cultured in a complete RPMI medium while changing medium every 2 days until a confluent monolayer is formed through cell fusion. To conduct adhesion experiments, an HT-29 monolayer is digested with trypsin and transferred to a 24-well tissue plate (10.sup.5 cells per well). The cells are then cultured in a complete RPMI medium with 5% of CO.sub.2 for 24 hours. Subsequently, the culture media is removed, and adherent HT-29 cells are washed twice with PBS. Then, 1 mL of antibiotic-free RPMI is added to each well. Overnight cultured bifidobacteria are diluted to obtain a bacterial suspension having a concentration of 10.sup.7 CFU/mL in the complete RPMI medium, the bacterial suspension is then added to the wells to achieve an initial infection ratio of 100:1 (bacteria: cells). The cells are incubated at 37? C. with 5% CO.sub.2 for 0.5 hours. Afterward, the cells are washed twice with PBS buffer to remove non-adherent bacteria. To determine the number of adhered bacteria, the cells are treated with trypsin for 10 minutes. After centrifugation, the cell pellet is re-suspended in 1 mL of RPMI medium. Serial dilutions are performed, and the bacteria are counted by using plate counting method. The percentage of cell adhesion ability is defined as the number of adhered bacteria divided by the total number of inoculated bacteria, multiplied by 100%.

(45) Adhesion to the gastrointestinal tract is considered a crucial prerequisite for bifidobacteria to colonize the human gut and exert its probiotic effects. In the present experiment, HT-29 colon cancer cells are used as a model to investigate the adhesion capacity of BI_Y46 strain compared to control strains on epithelial cells. The results (see FIG. 2) show that control strains BI_M63, BI_15697, and BI_Y46 all exhibited adhesion to HT-29 cells. However, BI_Y46 exhibited significantly higher cell adhesion capability compared to the two control strains. BI_Y46 had higher concentrations of surface proteins and self-aggregation ability than the control strains, and a structure called bacterial fimbriae is formed on surface of BI_Y46. The findings suggest that BI_Y46 possesses more adhesive proteins, which enhances its ability to adhere to intestinal epithelial cells. As a result, BI_Y46 can tightly bind to host epithelial cells, compete with pathogens for the same receptors, and replace pathogenic bacteria in host cells, thus playing a beneficial role in the host's gut health.

(46) The above description are merely some embodiments of the present invention and do not limit the scope of the patent. Any equivalent modifications or applications in the relevant technical field, whether made directly or indirectly based on the content of this specification, are also included within the scope of patent protection of the present invention.