Method for screening strain having high HMO utilization rate and strain having high HMO utilization rate

Abstract

Disclosed in the present invention are a method for screening a strain having a high HMO utilization rate and a strain having a high HMO utilization rate.

Description

A screening method for HMO high utilization rate strains and high HMO utilization rate strains Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and in particular relates to a method for screening a strain with high HMO utilization rate and a strain with high HMO utilization rate. Background Art

[0002] Human milk oligosaccharides (HMOs) are important prebiotics with unique benefits for infant health. 2'-Falcalacyl lactose (2'-FL) and lacto-N-neotetraose (LNnT), two abundant HMOs, are gaining increasing attention due to their unique benefits for infants and significant commercial value. They possess superior prebiotic properties for infants, and other health effects have also been demonstrated, including immunomodulation, anti-inflammatory properties, prevention of necrotizing enterocolitis, anti-adhesion antimicrobial activity, antiviral activity, and promotion of intestinal epithelial cell maturation. Safety assessments and clinical trials have demonstrated that 2'-FL and LNnT are safe and well-tolerated in infants, and they are now commercially added as functional ingredients to infant formula.

[0003] Infants' gut microbiota are typically rich in Bifidobacterium spp. Among the diverse gut microbiota, infant-associated Bifidobacteria are known to efficiently assimilate HMOs. Studies have shown that HMO assimilation capacity varies significantly between different Bifidobacterium species and strains. Therefore, to truly maximize the benefits of prebiotic and probiotic supplementation in commercial formulations, a more detailed understanding of the HMO assimilation behavior of probiotics is needed. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a high-throughput and quantitative screening method that can utilize HMO strains. Based on a probiotic bacterial library, a Bifidobacterium longum subsp. infantis that can simultaneously and efficiently utilize 2'-salinasyllactose and lactose-N-neotetraose is screened and obtained, named dipro-F, dipro-15 or dipro-105.

[0005] The strain dipro-F obtained by screening in the present invention has a higher utilization rate of human milk oligosaccharides than the listed strains edible for infants on the market.

[0006] In order to solve the above technical problems, the present invention provides a method for screening strains with high HMO utilization rate, the screening method comprising the following steps:

[0007] 1) Activating multiple strains of Bifidobacterium to be tested to obtain seed liquids, centrifuging the seed liquids, washing with PBS, and resuspending the resulting suspensions to obtain bacterial suspensions. 1%-10% by volume of the bacterial suspensions are then added to a carbon-free MRS+ medium containing bromocresol purple and cultured, with 2'-FL and LNnT added as sole carbon sources, respectively. Glucose is used as a positive control, and water is used as a negative control. The color of the culture medium is observed. If the strain grows, the culture medium color turns yellow; otherwise, the culture medium color remains purple. The strain corresponding to the yellow culture medium color is preliminarily determined to be a strain capable of simultaneously utilizing 2'-FL and LNnT.

[0008] 2) Using the method of step 1), bacterial suspensions were prepared from the strains obtained in step 1), and the bacterial suspensions were inoculated into MRS+ medium containing no carbon source, 2'-FL and LNnT were added as the sole carbon source, and glucose was used as a positive control and water was used as a negative control. Based on the number of sampling times, multiple parallel cultures of bacteria were prepared, and the OD600 was measured every few hours. Growth curves were plotted, and the area under the curve (AUC) was calculated based on the growth curves. The utilization rate of different carbon sources by different tested strains was determined based on the AUC; the formula for the AUC is as follows:

[0009] Wherein, t is time, 0<i<48h, OD represents OD600 value, and when the AUC value of the tested strain is more than 10 times greater than that of the standard strain (M16V strain), it is a strain with high HMO utilization rate.

[0010] The bifidobacterium comprises one or more of Bifidobacterium breve, Bifidobacterium longum subsp. infantis, Bifidobacterium bifidum, Bifidobacterium pseudocatenulatum, Bifidobacterium breve and Bifidobacterium animalis.

[0011] In step 1), the carbon-free MRS+ medium is prepared by adding cysteine ​​to the carbon-free MRS medium at a final concentration of 0.5 g / L.

[0012] The carbon-free MRS medium comprises the following components: 10.0 g of peptone, 5.0 g of yeast extract powder, 10.0 g of beef extract, 5.0 g of anhydrous sodium acetate, 2.0 g of anhydrous dipotassium hydrogen phosphate, 2.0 g of diammonium hydrogen citrate, 0.58 g of magnesium sulfate heptahydrate, 0.25 g of manganese sulfate tetrahydrate, and 0.1 mL of Tween-80. Distilled water is added to 1000 mL, the pH value is adjusted to 6.2-6.4, and sterilized at 121°C for 15 minutes.

[0013] The working concentrations of glucose, 2'-FL and LNnT in step 1) and step 2) are all 2-20 g / L. Preferably, the working concentration is 10 g / L.

[0014] Wherein, the concentration of the bacterial suspension in step 1) is OD600=0.5-5.

[0015] Wherein, the concentration of the bacterial suspension in step 2) is OD600=0.5-5.

[0016] The present invention also includes a strain with high HMO utilization rate obtained based on the screening method.

[0017] Wherein, the strains with high HMO utilization rate include dipro-F, dipro-15 or dipro-105.

[0018] The strain dipro-F was deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms, with the deposit number CGMCC No. 29343, the deposit date being December 18, 2023, and the deposit address being Beijing, China.

[0019] The strain dipro-15 was deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with the deposit number CGMCC No. 29341, the deposit date was December 18, 2023, and the deposit address was Beijing, China.

[0020] The strain dipro-105 was deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms, with the deposit number CGMCC No. 29342, the deposit date being December 18, 2023, and the deposit address being Beijing, China.

[0021] The present invention also includes a probiotic preparation, which includes the strain.

[0022] Bifidobacterium dipro-F and its application

[0023] The present invention also provides Bifidobacterium dipro-F, a composition or product containing the Bifidobacterium dipro-F, and applications thereof, especially applications in enhancing the intestinal mucosal barrier and / or improving enteritis.

[0024] The Bifidobacterium dipro-F of the present invention is Bifidobacterium longum infantis, which may also be called Bifidobacterium infantis.

[0025] The Bifidobacterium dipro-F of the present invention can efficiently utilize human milk oligosaccharides 2'-FL and LNnT. In addition, Bifidobacterium dipro-F is a probiotic that can be widely distributed in the intestine and has no adverse effects on the host.

[0026] Preferably, the present invention further provides a composition or product containing Bifidobacterium dipro-F and optionally HMO (such as LNnT and / or 2'-FL). The composition or product of the present invention can be used to improve intestinal diseases.

[0027] Experiments have shown that a composition containing LNnT and Bifidobacterium infantis dipro-F can effectively alleviate inflammatory bowel disease. In one embodiment, in a mouse model of acute colitis induced by dextran sulfate sodium (DSS), the dipro-F-containing composition of the present invention significantly alleviated acute colitis without significant toxic side effects.

[0028] The compositions of the present invention may contain one or more different HMOs (e.g., human milk HMOs). Representative examples include (but are not limited to) LNnT, 2'-FL, or a combination thereof. Preferred HMOs include LNnT, which is an important prebiotic in human milk and has unique benefits for infant health.

[0029] Preferably, the dipro-F-containing product of the present invention comprises a fermentation product, and representative fermentation products include (but are not limited to): fermentation supernatant, fermentation precipitate, bacterial suspension, or a combination thereof.

[0030] In the present invention, the dosage form of the product of the present invention is not particularly limited, and representative examples include (but are not limited to): powder, capsule, tablet, pill, film-coated agent, aerosol, granule, liquid, liposome, transdermal agent, suppository or lyophilized powder injection.

[0031] Typically, the product further comprises a protective agent, a functional aid / or an auxiliary additive, a drug carrier and / or a pharmaceutical excipient. Beneficial effects:

[0032] Compared with the prior art, the main advantages of the present invention include: the present invention has developed a high-throughput screening method for strains with high HMO utilization rates. This method is simple, rapid, and the screening process is clear at a glance. It can be used to screen the utilization of HMO as the sole carbon source, as well as the utilization of other carbon sources as the sole carbon source. The present invention has developed a method for quantitative screening of HMO utilization, which avoids the impact of repeated introduction of oxygen on the growth of anaerobic bacteria during the growth curve determination process, providing a feasible experimental scheme for the growth curve determination of strict anaerobic bacteria. The Bifidobacterium longum subspecies infantis dipro-F, dipro-15, and dipro-105 screened by the present invention can efficiently utilize HMO and have better HMO utilization efficiency than the listed strains for infants and young children sold on the market. The HMO utilization mechanism has been determined through whole genome sequencing, and the strains have been experimentally verified to have no safety risks. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 shows the high-throughput chromogenic screening of strains that can utilize HMOs in a bacterial library;

[0034] Figure 2 shows the growth curves of 8 bacterial strains under three carbon sources;

[0035] Figure 3 is a heat map of the AUCs of the eight strains;

[0036] Figure 4 shows the growth curves of three HMO-utilizing strains when glucose was used as the carbon source;

[0037] Figure 5 shows the growth curves of three HMO-utilizing strains when 2′-FL was used as the carbon source;

[0038] Figure 6 shows the growth curves of three HMO-utilizing strains when LNnt was used as the carbon source;

[0039] Figure 7 shows the animal experiment flow chart;

[0040] Figure 8 shows the measurement of body weight changes in experimental mice in different groups;

[0041] Figure 9 shows the colon length measurements of different groups of experimental mice; A is a colon diagram of different groups of experimental mice; B is a statistical diagram of colon length of different groups of experimental mice

[0042] Figure 10 shows the levels of lipopolysaccharide (LPS) in the blood of experimental mice in different groups;

[0043] Figure 11 shows the H&E staining results of colon tissues of experimental mice in different groups;

[0044] Figure 12 shows the mRNA expression levels of IL-10 in colon tissues of mice in different groups;

[0045] FIG13 shows the mRNA expression levels of LGR5 in colon tissues of mice in different groups. DETAILED DESCRIPTION

[0046] Example 1

[0047] 1. Culture medium configuration:

[0048] MRS medium containing glucose was prepared as the growth medium for probiotics.

[0049] Prepare MRS liquid medium: 10.0 g peptone, 5.0 g yeast extract powder, 10.0 g beef extract, 20.0 g glucose, 5.0 g anhydrous sodium acetate, 2.0 g anhydrous dipotassium phosphate, 2.0 g diammonium hydrogen citrate, 0.58 g magnesium sulfate heptahydrate, 0.25 g manganese sulfate tetrahydrate, and 0.1 mL Tween-80. Add distilled water to 1000 mL and adjust the pH to 6.2-6.4. Aliquot 10 mL of the culture medium into anaerobic tubes, aerate with sufficient nitrogen, cap and seal to isolate the air, and sterilize at 121°C for 15 minutes. For MRS solid medium, add 15 g agar powder to the liquid medium.

[0050] Prepare carbon-free MRS medium as the screening medium for probiotics with a pH of 7, add bromocresol purple with a final concentration of 0.17 g / L as a color indicator, and sterilize at 121°C and high temperature and high pressure for 15 minutes.

[0051] Carbon-free MRS medium preparation: peptone 10.0 g, yeast extract 5.0 g, beef extract 10.0 g, anhydrous sodium acetate 5.0 g, anhydrous dipotassium hydrogen phosphate 2.0 g, diammonium hydrogen citrate 2.0 g, magnesium sulfate heptahydrate 0.58 g, manganese sulfate tetrahydrate 0.25 g, Tween-80 0.1 mL, add distilled water to 1000 mL, adjust the pH to 6.2-6.4, and sterilize at 121°C for 15 min.

[0052] Carbon-free MRS+ medium is prepared by adding 0.5 g / L cysteine ​​to carbon-free MRS medium. Prepare a 250 g / L cysteine ​​stock solution, filter it through 0.22 μm, and add it to carbon-free MRS medium at a final concentration of 0.5 g / L before use. The medium supplemented with 0.5 g / L cysteine ​​is designated MRS+. To prepare MRS+ plates, add 0.5 g / L cysteine ​​to MRS solid medium.

[0053] Carbohydrates were prepared at a concentration of 100 g / L. After preparation, they were filter-sterilized and added to MRS+ medium at a ratio of 1:10 as the sole carbon source. The carbohydrates and their concentrations were as follows: 100 g / L glucose, 100 g / L 2'-salvinyllactose, and 100 g / L lactose-N-neotetraose.

[0054] 2. Strain activation

[0055] From the strain library of Difuluo Company, glycerol bacteria of Bifidobacterium (including Bifidobacterium breve, Bifidobacterium longum infantis subsp., Bifidobacterium bifidum, and Bifidobacterium pseudoselaginosa) were selected in a -80°C refrigerator. The infant edible list strains M16V (Bifidobacterium breve) and Bb12 (Bifidobacterium animalis) were used as reference strains. MRS solid plates (Bifidobacterium streaked MRS+ plates) were streaked. After culturing at 37°C for 48 hours, single clones on the plates were picked and cultured in MRS or MRS+ liquid culture medium for 24 hours. Then, they were transferred to fresh culture medium at a ratio of 10% and continued to be cultured for 24 hours.

[0056] 3. Seed solution transfer and high-throughput screening

[0057] The activated seed liquid was centrifuged at 4000rpm and 4℃ for 3 minutes, washed once with PBS at pH 6.5, and resuspended in equal volume to obtain a bacterial suspension. Bacteria were cultured in 96-well plates, and 20μL of the bacterial suspension was added to each experimental group and control group containing 180μL of carbon-free MRS+ culture medium (bromocresol purple was added to the culture medium of each group, and glucose was added to the culture medium as a positive control; water was added to the culture medium as a negative control; 1% carbon source was added to the culture medium as the experimental group, 2'-FL was added as the only carbon source as experimental group 1, and LNnT was added as the only carbon source as experimental group 2). After culturing at 37℃ for 48h, the color of the culture medium was observed. The screening results are shown in Figure 1. If the strain grows, the color of the culture medium turns yellow, otherwise the color of the culture medium remains purple. At the same time, a blank control group was set up: carbon-free MRS+ culture medium+water; a positive control group: carbon-free MRS+ culture medium+glucose;

[0058] The specific groups are as follows:

[0059] In Figure 1, yellow represents strains capable of utilizing carbon sources, while purple represents strains unable to utilize carbon sources. As shown in Figure 1, among the strains screened, seven were able to utilize both 2'-FL and LNnT, five were able to utilize 2'-FL alone, and one was able to utilize LNnT alone.

[0060] The specific strains screened are as follows:

[0061] According to the above screening results, the high-throughput colorimetric screening method of the present invention can quickly and intuitively screen out strains that can utilize HMOs. Multiple experimental repetitions have demonstrated that this method has good repeatability.

[0062] 4. Quantitative screening method

[0063] Seven strains capable of utilizing both 2'-FL and LNnT were identified using a high-throughput screening method. Subsequently, OD600 values ​​of the eight strains were measured using the above method, using M16V as a reference strain, and growth curves were plotted. The results are shown in Figure 2.

[0064] The area under the curve (AUC) of the growth curves of the eight strains on three carbon sources (glucose, 2'-FL, and LNnT) was calculated. The area under the curve is a commonly used indicator to evaluate the efficiency of microorganisms in utilizing a specific carbon source. A larger area indicates that the microorganism is utilizing the carbon source more efficiently, resulting in a higher utilization rate. The results are shown in Table 1:

[0065] Table 1 Area under the curve of 8 bacterial strains under three different carbon sources

[0066] A heat map based on the area under the curve is shown in Figure 3. The darker the color in the heat map, the larger the area under the curve and the higher the carbon source utilization rate. As shown in Figure 3, after high-throughput colorimetric screening and quantitative screening, the present invention obtained three strains with high HMO utilization rates:

[0067] 2-bifidobacterium infantis (specific strain name: Bifidobacterium longum infantis subspecies DSM 20088T DSM_2, designated dipro-F). Its 2'-FL utilization rate was 20.5 times that of the standard strain (M16V), and its LNnt utilization rate was 41.6 times that of the standard strain (M16V).

[0068] 4-Bifidobacterium bifidum, the specific strain name is dipro-15, the 2'-FL utilization rate is 13.2 times that of the standard strain (M16V), and the LNnt utilization rate is 34.2 times that of the standard strain (M16V);

[0069] 5-Bifidobacterium bifidum, the specific strain name is: dipro-105, the 2'-FL utilization rate is 18.5 times that of the standard strain (M16V), and the LNnt utilization rate is 37.2 times that of the standard strain (M16V).

[0070] 5. Whole-genome analysis of strains with high HMO utilization

[0071] HMO use of genetic analysis

[0072] Based on the amino acid sequences of the HMO metabolic genes of dipro-F, dipro-15, and dipro-105 (the reference amino acid sequences encoding the genes are Blon_0343, Blon_2202, FL transporter; Bbr_1554, LNnT transporter; Blon_2334, β-1,4-Galactosidase; Blon_2335, 1,2-α-l-Fucosidase; and Blon_2336, 1,3 / 4-l-Fucosidase), a BLAST analysis was performed on the sequence results obtained from whole-genome sequencing. It was found that the Dipro F strains screened in the present invention all contained sequences with a similarity of greater than 93% to the above genes, and possessed a full set of metabolic genes capable of degrading HMOs.

[0073] Example 2 Growth curve determination of HMO high utilization rate strain

[0074] 1. Experimental methods:

[0075] 1. Culture medium configuration

[0076] Prepare MRS liquid medium: 10.0 g peptone, 5.0 g yeast extract powder, 10.0 g beef extract, 20.0 g glucose, 5.0 g anhydrous sodium acetate, 2.0 g anhydrous dipotassium phosphate, 2.0 g diammonium hydrogen citrate, 0.58 g magnesium sulfate heptahydrate, 0.25 g manganese sulfate tetrahydrate, and 0.1 mL Tween-80. Add distilled water to 1000 mL and adjust the pH to 6.2-6.4. Aliquot 10 mL of the culture medium into anaerobic tubes, aerate with sufficient nitrogen, cap and seal to isolate the air, and sterilize at 121°C for 15 minutes. For MRS solid medium, add 15 g agar powder to the liquid medium.

[0077] Preparation of chemically defined medium: The formula is shown in Table 2. The carbon sources in the medium are glucose, 2'-FL, and LNnT, respectively. To minimize nutrient loss, the mineral salts and amino acids in the medium were sterilized at 121°C for 15 minutes. The carbon source, vitamins, and nucleic acid bases were sterilized by filtration through a 0.22 μm microporous membrane and mixed before use.

[0078] Table 2 Chemically defined medium formulation

[0079] 2. Strain activation

[0080] Dipro-F, dipro-15, and dipro-105 glycerol strains stored at -80°C were inoculated into anaerobic tubes containing MRS liquid culture medium and activated. Then, they were transferred sequentially to anaerobic tubes containing MRS liquid culture medium at a 1% inoculum volume. Using the activated strain from the third passage, 9 ml of the bacterial solution was placed in a 10 ml sterile centrifuge tube in a clean bench and centrifuged at 4500 rpm and 4°C for 15 minutes. The supernatant was discarded, and 9 ml of sterile saline was added and mixed to serve as the inoculum. The inoculum was transferred to chemically defined medium at a 1% inoculum volume. Under a sterile environment, the inoculated culture was transferred to 96-well cell culture plates, with 100 μl per well. Three wells were replicated per plate for each time point and incubated at 37°C under anaerobic conditions.

[0081] 3. Strain growth

[0082] The growth curve of bifidobacteria was determined using a multifunctional microplate reader, and the area under the curve (AUC) was calculated.

[0083] 2. Growth curve of high HMO-producing strains and calculation of area under the curve

[0084] 1. When glucose was used as the carbon source, the growth curves of the three HMO-utilizing strains were shown in Figure 4;

[0085] 2. When 2'-FL was used as the carbon source, the growth curves of the three HMO-utilizing strains were shown in Figure 5;

[0086] 3. When LNnt was used as the carbon source, the growth curves of the three HMO-utilizing strains were shown in Figure 6;

[0087] Based on the above growth curves, the area under the curve (AUC) of the three HMO-utilizing strains under different carbon sources was calculated. The results are shown in Table 3:

[0088] Table 3 Area under the curve (AUC) of three HMO-utilizing strains under different carbon sources

[0089] According to the data in Table 3, under the experimental conditions, dipro-F had the highest HMO utilization rate under different carbon source conditions. In addition, the other two strains also had high HMO utilization rates. Among them, dipro-F's utilization rate of 2'-FL was 20.5 times that of the standard strain (M16V), and its utilization rate of LNnt was 41.6 times that of the standard strain (M16V).

[0090] The utilization rate of 2'-FL by dipro-15 was 13.2 times that of the standard strain (M16V), and the utilization rate of LNnt was 34.2 times that of the standard strain (M16V); the utilization rate of 2'-FL by dipro-105 was 18.5 times that of the standard strain (M16V), and the utilization rate of LNnt was 37.2 times that of the standard strain (M16V).

[0091] Example 3 Dipro-F Safety Determination

[0092] 1. Test Preparation

[0093] Preparation of intraperitoneal injection solution: Dipro-F bacteria were inoculated on MRS plates containing cysteine ​​hydrochloride. After incubation, the culture on the second generation plate was scraped and suspended in sterile saline. The concentration was adjusted with an appropriate amount of sterile saline to make a bacterial concentration of 5.0×10 7 CFU / mL bacterial suspension for intraperitoneal injection.

[0094] Preparation of oral gavage bacterial solution: Dipro-F bacteria were inoculated on MRS plates containing cysteine ​​hydrochloride. After incubation, the culture on the second generation plate was scraped and suspended in sterile saline. The concentration was adjusted with an appropriate amount of sterile saline to make a bacterial concentration of 2.5×10 8 CFU / mL and 1.25×10 9 CFU / mL bacterial suspension for oral gavage.

[0095] 2. Mouse Experiment

[0096] Mouse experiments were performed by oral gavage: at least 80 mice were randomly divided into 8 groups (10 mice per group) for each bacterial strain, including a male mouse culture medium control group, a male mouse bacterial suspension group, a male mouse 5-fold concentrated culture medium control group, and a male mouse 5-fold concentrated bacterial suspension group; and a female mouse culture medium control group, a female mouse bacterial suspension group, a female mouse 5-fold concentrated culture medium control group, and a female mouse 5-fold concentrated bacterial suspension group. Each group was gavaged with 0.2 mL of the test substance per mouse, so that the amount of bacteria per mouse in each test group was 1.0 × 10 7 CFU (stock solution group) and 5.0×10 7 CFU (5-fold concentrated solution group) were administered orally for 3 consecutive days. Animals were fasted overnight (16 hours) before the first oral administration and fed 3-4 hours after oral administration.

[0097] Mouse experiments: Intraperitoneal injection: 40 mice were used for each bacterial strain, half male and half female, and randomly divided into 4 groups (10 mice each), including a male mouse sterile saline control group, a male mouse bacterial suspension group, a female mouse sterile saline control group, and a female mouse bacterial suspension group. The injection volume was 0.2 mL per mouse, meaning that each mouse in the test group received at least 1.0 × 10 7CFU.

[0098] 3. Experimental Results

[0099] Observe and record any abnormalities in the mouse's skin and fur, eyes and mucous membranes, breathing, limb movements, and behavior. Pay special attention to any signs of tremors, convulsions, diarrhea, lethargy, salivation, or coma.

[0100] The experimental results are shown in Table 4. No adverse reactions or deaths were observed in the tested mice. This indicates that the dipro-F strain of the present invention is non-pathogenic and has extremely high safety.

[0101] Table 4 Safety test results of dipro-F

[0102] Example 4: Dipro-F and its combined use with LNnT improve colitis

[0103] In this example, a composition containing dipro-F and LNnT was prepared, and its effect on improving colitis was verified.

[0104] 4.1 Preparation of Bifidobacterium infantis dipro-F and LNnT

[0105] Remove a culture of Bifidobacterium infantis dipro-F from a -80°C freezer, store in 30% glycerol, and streak onto solid MRS medium to isolate individual colonies. Place the streaked plate in an anaerobic incubator and add an anaerobic gas-producing bag to simulate the anaerobic growth environment of the intestine. Seal the bag and place the plate in a 37°C incubator for 48 hours. Pick a single colony from the plate and culture it in MRS liquid medium containing 0.1% L-cysteine ​​for 48 hours. Transfer the colony to a 50ml centrifuge tube containing fresh medium at a 10% transfer ratio. Incubate the tube in a 37°C incubator until the logarithmic growth phase.

[0106] Take out the infant Bifidobacterium dipro-F that has grown to the logarithmic phase, centrifuge at 8000 rpm for 5 minutes, discard the supernatant, wash the bacterial pellet with physiological saline, centrifuge again, and wash three times. After discarding the supernatant, resuspend the bacterial pellet with 100 g / L LNnT to make the bacterial solution concentration reach 5×10 9 Each mouse was gavaged with 200 μl of bacterial solution containing 20 mg of LNnT and 1×10 dipro-F. 9 CFU.

[0107] Effects of Bifidobacterium infantis dipro-F and its combination with LNnT on body weight in colitis mice

[0108] Six-week-old SPF C57BL / 6J male mice were divided into five groups: blank group (NC), model group (DSS) and experimental group (LNnT, dipro-F, LNnT+dipro-F). The LNnT group was orally administered with 20 mg of LNnT; the dipro-F group was orally administered with 1×10 9 CFU of Bifidobacterium infantis dipro-F; LNnT+dipro-F group was gavaged with 1×10 9 Six animals per group were housed at Shanghai Pengli Biopharmaceutical Technology Co., Ltd. at a constant temperature of 21-26°C, humidity of 40-70%, noise level of 60dB or less, and illumination of 15-20LX (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Committee). The specific animal experimental process is shown in Figure 7:

[0109] The experimental period lasted for 20 days.

[0110] Days -11 to -7 were the adaptation period: during the adaptation period, mice in each cage had free access to water and were given normal growth and reproduction feed.

[0111] Days -6 to 0: dipro-F and LNnT groups (1×10 9 CFU of Bifidobacterium infantis dipro-F);

[0112] The other groups were given normal drinking water every day.

[0113] Days 1 to 5,

[0114] DSS group: given 3% DSS drinking water every day;

[0115] LNnT group: 3% DSS drinking water + 20 mg LNnT gavage daily;

[0116] dipro-F group: 3% DSS drinking water + oral gavage 1×10 9 CFU of Bifidobacterium infantis dipro-F;

[0117] LNnT+dipro-F group: 3% DSS drinking water + 1×10 9 CFU of Bifidobacterium infantis dipro-F and 20 mg LNnT;

[0118] NC group: normal drinking water was given every day.

[0119] Days 6 to 9:

[0120] dipro-F group: 1×10 9CFU of Bifidobacterium infantis dipro-F;

[0121] LNnT+dipro-F group: 1×10 9 CFU of Bifidobacterium infantis dipro-F and 20 mg LNnT;

[0122] Each cage of mice was given normal drinking water every day.

[0123] Statistical analysis was performed on the body weight monitored for 9 consecutive days from day 1 to day 9.

[0124] The results are shown in Figure 8. Compared with the DSS group, the infant B. infantis dipro-F and LNnT groups significantly slowed the percentage of weight loss in mice. On the last day of intervention, the weight change in the DSS group was 80.43%, while that in the infant B. infantis dipro-F and LNnT combination group was 91.93%, an increase of 11.5% compared to the DSS group. After intervention with LNnT alone and infant B. infantis dipro-F alone, the weight change values ​​were 81.18% and 86.43%, respectively, which were only 0.75% and 6.00% higher than those in the DSS group. Therefore, the combined use of infant B. infantis dipro-F and LNnT can more effectively slow the percentage of weight loss in mice.

[0125] Effects of Bifidobacterium infantis dipro-F and its combination with LNnT on colon length in colitis mice

[0126] After the experiment, the mice were dissected according to ethical requirements, and the colon was removed and measured in length.

[0127] As shown in Figure 9, compared to the NC group's colon length of 73.2 mm, the DSS group's colon length was significantly reduced to 46.79 mm. Under the intervention of the combination of Bifidobacterium infantis dipro-F and LNnT, the colon length significantly increased to 68.12 mm, a 45.58% increase compared to the DSS group's colon length. After intervention with LNnT alone and Bifidobacterium infantis dipro-F alone, the colon lengths were 47 mm and 58.67 mm, respectively, which were only 0.44% and 25.39% higher than those in the DSS group. Therefore, the combination of Bifidobacterium infantis dipro-F and LNnT can more effectively slow the shortening of the mouse colon length.

[0128] Effects of Bifidobacterium infantis dipro-F and its combination with LNnT on serum inflammatory cytokine levels in colitis mice

[0129] After the animal experiment, according to ethical requirements, the mouse blood was collected into an anticoagulant tube and centrifuged at 3000×g for 15 minutes. The supernatant was aspirated and the LPS concentration in the blood was determined according to the instructions of the ELISA kit.

[0130] Figure 10 shows the LPS concentration in the blood. Quantitative results in Figure 10 indicate that the LPS concentration in the DSS group was 4.59 μg / mL. The combination of Bifidobacterium infantis dipro-F and LNnT significantly reduced the LPS concentration to 2.65 μg / mL, a 42.26% decrease compared to the DSS group. LNnT alone reduced the LPS concentration to 4.15 μg / mL, a 9.5% decrease compared to the DSS group. Therefore, the combination of Bifidobacterium infantis dipro-F and LNnT can more effectively suppress LPS levels in colonic tissue and alleviate colonic inflammation.

[0131] 4.5 Effects of Bifidobacterium infantis dipro-F and its combination with LNnT on inflammatory cell infiltration in colitis mice

[0132] After the animal experiment, the entire colon was removed and measured in length according to ethical requirements. The colon tissue approximately 1 cm long near the anus was cut and fixed in formalin for at least 5 h for tissue sectioning and HE staining.

[0133] (1) Paraffin tissue embedding and sectioning

[0134] a. Fix the colon tissue in 4% formaldehyde solution for at least 5 hours and then remove the tissue;

[0135] b. Dehydrate the tissue and immerse it in wax in the following solutions: 70% ethanol for 60 minutes → 85% ethanol for 40 minutes → 95% ethanol for 60 minutes → 95% ethanol for 40 minutes → anhydrous ethanol for 60 minutes → anhydrous ethanol for 40 minutes → xylene for 50 minutes → xylene for 50 minutes → paraffin for 30 minutes → paraffin for 60 minutes, and then embed the tissue.

[0136] c. Use a paraffin tissue slicer to slice the tissue to a thickness of 5 μm. After spreading, use tissue-resistant slides to mount the slices. Bake the slices at 70°C for at least 1 hour and then store them at room temperature for long-term storage.

[0137] (2) HE staining

[0138] a. Dewax paraffin sections in xylene and a gradient of ethanol: xylene for 10 min → xylene for 8 min → anhydrous ethanol for 2 min → anhydrous ethanol for 1 min → 95% ethanol for 1 min → 85% ethanol for 1 min → 70% ethanol for 1 min → tap water for 1 min.

[0139] b. Stain with hematoxylin for 5 minutes, rinse with tap water for 30 seconds, differentiate with 0.5% hydrochloric acid ethanol solution for 10 seconds, rinse with tap water for 30 seconds, stain with eosin solution for 1 minute, and rinse with tap water for 30 seconds.

[0140] c. After removal, place the slides in the following order: 70% ethanol for 2 seconds → 85% ethanol for 4 seconds → 95% ethanol for 20 seconds → anhydrous ethanol for 1 minute → anhydrous ethanol for 1 minute → xylene for 2 minutes → xylene for 2 minutes, then seal the slides with neutral gum and store them in the air.

[0141] (3) Microscope observation

[0142] An ordinary upright microscope was used to observe the pathological damage of colon tissue and collect image information.

[0143] The results of HE staining of colonic tissue are shown in Figure 11. As shown in Figure 11, compared with the NC group, the colonic tissue in the DSS group showed significant inflammation, with significant infiltration of neutrophils and lymphocytes. At the same time, the colonic mucosal structure was damaged to a certain extent, including a decrease in goblet cells and structural disorder of the glands. However, the intervention of the Bifidobacterium infantis dipro-F and LNnT combination significantly alleviated the occurrence of these pathological changes in colonic tissue. Therefore, the Bifidobacterium infantis dipro-F and LNnT combination can more effectively inhibit the occurrence of colonic tissue inflammation and alleviate the pathological damage caused by colonic inflammation.

[0144] 4.6 Effects of Bifidobacterium infantis dipro-F and its combination with LNnT on colonic tissue in colitis mice

[0145] After the animal experiment, mice were dissected in accordance with ethical guidelines. Colon tissue was weighed and extracted. Total RNA was extracted from the colon using TriZol reagent and reverse transcribed. Relative cDNA content was analyzed using a SYBR qPCR Master Mix real-time PCR thermocycler. GAPDH-normalized IL-10 and LGR5 levels were calculated using 2-ΔΔCT.

[0146] The mRNA expression of IL-10 in colon tissue is shown in Figure 12. As shown in the quantitative results of Figure 12, the mRNA expression of IL-10 in the DSS group was 1.06, while the mRNA expression of IL-10 under the intervention of the combination of Bifidobacterium infantis dipro-F and LNnT was 0.56, which was 47.17% lower than that in the DSS group and had no significant difference with the NC group. After intervention with LNnT alone and Bifidobacterium infantis dipro-F alone, the mRNA expression of IL-10 was 0.91 and 0.76, respectively, which was only 14.15% and 28.30% lower than that in the DSS group. Therefore, the combination of Bifidobacterium infantis dipro-F and LNnT can more effectively alleviate the occurrence of colon tissue inflammation.

[0147] The mRNA expression of LGR5 in colon tissue is shown in Figure 13. The quantitative results in Figure 13 show that the mRNA expression of LGR5 in the DSS group was 4.22. The mRNA expression of LGR5 was significantly increased to 8.45 after intervention with the combination of Bifidobacterium infantis dipro-F and LNnT, which was 100.23% higher than that in the DSS group. After intervention with LNnT alone and Bifidobacterium infantis dipro-F alone, the mRNA expression of LGR5 was 2.67 and 6.31, respectively, which were only -36.72% and 49.52% higher than those in the DSS group. Therefore, the combination of Bifidobacterium infantis dipro-F and LNnT can more effectively promote the mRNA expression of LGR5 in colon tissue.

Claims

1. A strain with high HMO utilization rate, characterized in that: The high HMO utilization strain is strain dipro-F, which is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No. 29343, a deposit date of December 18, 2023, and a deposit address of Beijing, China.

2. The strain with high HMO utilization rate according to claim 1, characterized in that The dipro-F can simultaneously and efficiently utilize the human milk oligosaccharides 2'-FL and LNnT.

3. A probiotic preparation, characterized in that The probiotic preparation comprises the high HMO utilization strain according to claim 1.

4. The probiotic preparation according to claim 3, wherein The preparation is a fermentation product.

5. The probiotic preparation according to claim 4, wherein The fermentation product includes fermentation supernatant, fermentation precipitate or bacterial suspension of dipro-F.

6. The probiotic preparation according to claim 3, wherein Also contains human milk oligosaccharides (HMOs).

7. The probiotic preparation according to claim 6, wherein The human milk oligosaccharide is selected from the following group: LNnT, 2'-FL, or a combination thereof.

8. The probiotic preparation according to claim 6, wherein The human milk oligosaccharide is LNnT.

9. A probiotic product, characterized in that The probiotic product is a fermented product containing the high HMO utilization strain according to claim 1.

10. The product according to claim 9, wherein the fermented product comprises a fermentation supernatant, a fermentation precipitate or a bacterial suspension.

11. The product according to claim 9, characterized in that The dosage forms of the product include powder, capsule, tablet, pill, film-coated agent, aerosol, granule, liquid, liposome, transdermal agent, suppository or lyophilized powder injection.

12. The product according to claim 9, wherein The product also includes: a protective agent, a functional auxiliary agent / or an auxiliary additive, a drug carrier and / or a pharmaceutical excipient.

13. The product according to claim 9, characterized in that The product is a medicine or a food.

14. The product according to claim 9, wherein Also contains human milk oligosaccharides (HMOs).

15. The product according to claim 9, wherein The human milk oligosaccharide is selected from the following group: LNnT, 2'-FL, or a combination thereof.

16. Use of the high HMO utilization strain or its fermentation product according to claim 1 or the probiotic preparation according to claim 3, characterized in that: Used to prepare products that enhance intestinal mucosal barrier and / or improve enteritis.

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