Screening method of strain with high HMO utilization rate and strain with high HMO utilization rate
Through high-throughput and quantitative screening methods, the Bifidobacterium longan infant subspecies that can efficiently utilize 2’-FL and LNnT were screened, solving the problem of low HMO utilization in the prior art and achieving higher HMO utilization efficiency and safety.
Patent Information
- Application Number
- CN202411768318.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-01-29
- Publication Date
- 2025-02-28
AI Technical Summary
The prior art is difficult to effectively screen out the Bifidobacterium longan infant subspecies that can efficiently utilize 2’-Saltol-Black Lactose and Lactose-N-Neotetrasaccharide, which limits the maximization of HMO in infant formula milk powder.
Using high-throughput and quantitative screening methods, the HMO utilization rate of the strain was judged by adding Bifidobacterium seed solution to the carbon-free source MRS+ culture medium containing bromophenol, using 2’-FL and LNnT as the only carbon sources.
The Bifidobacterium longan infant subspecies dipro-F, dipro-15 and dipro-105 with higher HMO utilization were successfully screened. Compared with the edible list of infants and young children sold on the market, its HMO utilization efficiency was better, and its safety was verified by whole-genome sequencing.
Smart Images
Figure 00000000_0000_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of microbial technology, and specifically relates to a method for screening a strain with high HMO utilization rate and a strain with high HMO utilization rate. This patent application is a divisional application of application number 2024101203609. Background Art
[0002] Human milk oligosaccharides (HMOs) are important prebiotics with unique effects on infant health. 2'-Fluorinated lactose (2'-FL) and lacto-N-neotetraose (LNnT), two abundant HMOs, have attracted increasing attention due to their unique benefits for infants and great commercial value. They have superior prebiotic properties for infants, and other health effects have also been confirmed, including immunomodulation, anti-inflammatory, prevention of necrotizing enterocolitis, anti-adhesion antibacterial, antiviral activity, and promotion of intestinal epithelial cell maturation. Safety assessments and clinical trials have shown that 2'-FL and LNnT are safe and well tolerated for infants, and have been commercially added as functional ingredients to infant formula.
[0003] The intestinal microbiota of infants is usually rich in Bifidobacterium spp., and among the different intestinal microbiota, infant gut-associated Bifidobacteria can effectively assimilate HMOs. Studies have shown that HMO assimilation capacity varies significantly between different Bifidobacterium species and strains. Therefore, to truly maximize the role 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] Purpose of the invention: 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, to screen and obtain Bifidobacterium longum infantis subspecies that can simultaneously and efficiently utilize 2'-salinasyllactose and lactose-N-neotetraose, named dipro-F, dipro-15 or dipro-105.
[0005] The strain Dipro F screened and obtained by the present invention has a higher utilization rate of human milk oligosaccharides than the listed strains edible to infants on the market.
[0006] Technical solution: In order to solve the above technical problems, the present invention provides a method for screening HMO high utilization rate strains, the screening method comprising the following steps:
[0007] 1) Activating multiple strains of bifidobacteria to be tested to obtain seed liquids, then centrifuging the seed liquids, washing with PBS, and resuspending them to obtain bacterial suspensions, then adding 1%-10% of the bacterial suspensions by volume to a carbon-free MRS+ medium containing bromophenol methyl violet for culture, adding 2'-FL and LNnT as the sole carbon sources, respectively, and then using glucose as a positive control and water as a negative control to observe the color of the culture medium. If the strain grows, the color of the culture medium turns yellow, otherwise the color of the culture medium remains purple, and the strain corresponding to the yellow culture medium color is preliminarily judged as a strain that can simultaneously utilize 2'-FL and LNnT;
[0008] 2) Using the method of step 1), the strains obtained in step 1) are respectively used to prepare bacterial suspensions, and the bacterial suspensions are inoculated into MRS+ medium containing no carbon source, and 2'-FL and LNnT are respectively added as the sole carbon source, and then glucose is used as a positive control and water is used as a negative control. According to the sampling time and number, multiple parallel cultured bacteria are configured, and OD600 is measured every few hours, and a growth curve is drawn. According to the growth curve, the area under the curve AUC is calculated, and the utilization rate of different carbon sources by different strains tested is judged according to the AUC; the formula of 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] Wherein, in step 1), the carbon source-free MRS+ medium is prepared by adding cysteine with a final concentration of 0.5 g / L to the carbon source-free MRS medium.
[0012] The carbon source-free MRS medium includes 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. Add distilled water to 1000 mL, adjust the pH value to 6.2-6.4, and sterilize at 121° C. for 15 minutes.
[0013] Wherein, 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 is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration. The strain dipro-F is classified and named Bifidobacterium longum subsp. infantis, the deposit number is CGMCC No. 29343, the deposit date is December 18, 2023, and the deposit address is Beijing, China.
[0019] The strain dipro-15 is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration. The strain dipro-15 is classified and named Bifidobacterium bifidum, the deposit number is CGMCC No. 29341, the deposit date is December 18, 2023, and the deposit address is Beijing, China.
[0020] The strain dipro-105 is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration. The strain dipro-105 is classified and named Bifidobacterium bifidum, the deposit number is CGMCC No. 29342, the deposit date is December 18, 2023, and the deposit address is Beijing, China.
[0021] The present invention also includes a probiotic preparation, which includes the strain.
[0022] Beneficial effects: Compared with the prior art, the advantages of the present invention are: the present invention has developed a high-throughput screening method for strains with high HMO utilization, which is simple and fast, and the screening is clear at a glance. It can be used for screening the utilization of HMO as the sole carbon source, and can also be used for screening 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 influence of oxygen on strain growth caused by repeated removal and placement of anaerobic bacteria during the growth curve determination process, and provides a feasible experimental scheme for the growth curve determination of strict anaerobic bacteria. The infant subspecies of Bifidobacterium longum dipro-F, dipro-15 and dipro-105 screened by the present invention can efficiently utilize HMO, and have better HMO utilization efficiency than the edible list 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 hazards. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 To screen strains that can utilize HMO in bacterial libraries by high-throughput colorimetric screening;
[0024] Figure 2 The growth curves of 8 bacterial strains under three carbon sources;
[0025] Figure 3 This is the AUC heat map of 8 strains;
[0026] Figure 4 Movable element analysis;
[0027] Figure 5 Growth curves of three HMO-utilizing strains when glucose was used as the carbon source;
[0028] Figure 6 Growth curves of three HMO-utilizing strains when 2'-FL was used as the carbon source;
[0029] Figure 7 Growth curves of three HMO-utilizing strains using LNnt as the carbon source. DETAILED DESCRIPTION
[0030] Example 1
[0031] 1. Culture medium configuration:
[0032] MRS medium containing glucose was prepared as the growth medium for probiotics.
[0033] Preparation of MRS liquid culture medium: 10.0g peptone, 5.0g yeast extract powder, 10.0g beef extract, 20.0g glucose, 5.0g anhydrous sodium acetate, 2.0g anhydrous potassium hydrogen phosphate, 2.0g diammonium hydrogen citrate, 0.58g magnesium sulfate heptahydrate, 0.25g manganese sulfate tetrahydrate, 0.1mL Tween-80, add distilled water to 1000mL, adjust the pH value to 6.2-6.4, dispense 10mL of culture solution into anaerobic tubes, fill with sufficient nitrogen, cover with plug to isolate air, and sterilize at 121℃ for 15 minutes. MRS solid culture medium is added with 15g agar powder on the basis of liquid culture medium.
[0034] MRS medium without carbon source was prepared as the screening medium for probiotics, with a pH value of 7, and bromophenol violet with a final concentration of 0.17 g / L was added as a color indicator, and sterilized at 121° C. for 15 minutes.
[0035] The carbon-free MRS medium was prepared as follows: 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. Add distilled water to 1000 mL, adjust the pH to 6.2-6.4, and sterilize at 121°C for 15 minutes.
[0036] MRS+ medium without carbon source is to add 0.5g / L cysteine to MRS medium without carbon source. Prepare 250g / L cysteine stock solution, filter it through 0.22μm, and add it to MRS medium without carbon source at a final concentration of 0.5g / L before use. After adding 0.5g / L cysteine to the medium, it is named MRS+. If you need to prepare MRS+ plates, add 0.5g / L cysteine to MRS solid medium.
[0037] The carbohydrate concentration was 100 g / L. After preparation, it was filtered and sterilized, and added to the MRS+ medium at a ratio of 1:10 before use as the sole carbon source. The carbohydrates and their concentrations were as follows: glucose 100 g / L, 2'-salinalgyl lactose 100 g / L, lactose-N-neotetraose 100 g / L.
[0038] 2. Strain activation
[0039] 46 strains of bifidobacterium glycerol bacteria (including Bifidobacterium breve, Bifidobacterium longum infantis subspecies, Bifidobacterium bifidum, and Bifidobacterium pseudoselaginosa) were selected from the strain library of Difuluo Company in a -80°C refrigerator, and the infant edible list strains M16V (Bifidobacterium breve) and Bb12 (Animal Bifidobacterium) were used as reference strains. MRS solid plates (Bifidobacterium streaked MRS+ plates) were streaked, and after culturing at 37°C for 48 hours, single clones on the plates were picked, and after culturing in MRS or MRS+ liquid culture medium for 24 hours, they were transferred to fresh culture medium at a ratio of 10% and continued to be cultured for 24 hours.
[0040] 3. Seed solution transfer and high-throughput screening
[0041] The activated seed solution 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. Use a 96-well plate to culture bacteria, and add 20μL of the bacterial suspension to each experimental group and control group containing 180μL of MRS+ medium without carbon source (bromophenol violet 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% of the carbon source was added to the culture medium as the experimental group, and 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), and after culturing at 37℃ for 48h, the color of the culture medium was observed, and the screening results were as follows: Figure 1 , if the strain grows, the culture medium will turn yellow, otherwise the culture medium will remain purple. At the same time, a blank control group was set up: MRS without carbon source + culture medium + water; a negative control group was set up: MRS without carbon source + culture medium + glucose;
[0042] The specific groups are as follows:
[0043]
[0044] Figure 1 Yellow represents strains that can utilize carbon sources, and purple represents strains that cannot utilize carbon sources. Figure 1 It can be seen that among the 46 strains screened, 7 strains can utilize 2'-FL and LNnT simultaneously, 5 strains can utilize 2'-FL alone, and 1 strain can utilize LNnT alone.
[0045] The specific strains screened are as follows:
[0046] serial number Accession number Strain name dipro-F CGMCC No.29343 dipro-F 1 CGMCC No.29341 dipro-15 2 CGMCC No.29342 dipro-105
[0047] 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 HMO. Multiple experimental repetitions have proven that this method has good repeatability.
[0048] 4. Quantitative screening method
[0049] Seven strains that can utilize 2'-FL and LNnT simultaneously were identified by high-throughput screening. Subsequently, OD600 of the eight strains was measured using M16V as a reference strain, and growth curves were drawn. The results are shown in Figure 2. Figure 2 .
[0050] The area under the curve (AUC) of the growth curves of the eight strains under 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. The larger the area, the more effectively the microorganism utilizes the carbon source and the higher the utilization rate. The results are shown in Table 1:
[0051] Table 1 Area under the curve of 8 strains under three different carbon sources
[0052]
[0053] Draw a heat map based on the area under the curve 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. Figure 3 As shown, after high-throughput colorimetric screening and quantitative screening, the present invention obtained three strains with high HMO utilization rates:
[0054] 2-bifidobacteriuminfantis, the specific strain name is Bifidobacteriumlonguminfantis subspecies DSM 20088T DSM_2, named dipro-F. The utilization rate of 2'-FL is 20.5 times that of the standard strain (M16V), and the utilization rate of LNnt is 41.6 times that of the standard strain (M16V);
[0055] 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);
[0056] 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).
[0057] 5. Whole genome analysis of strains with high HMO utilization
[0058] 5.1 Security Analysis
[0059] According to the VFDB and CARD databases, the BLAST tool was used to analyze the drug resistance genes and virulence-related genes of dipro-F, dipro-15 and dipro-105, respectively. The analysis results showed that there were no drug resistance genes and virulence-related genes in the genome of this strain. The transferable elements in the genome were further analyzed using Island viewer software. Figure 4 The results showed that there were no virulence genes, antibiotic genes, or pathogen-related genes near the transferable elements of the strain.
[0060] 5.2 HMO Utilization Genetic Analysis
[0061] According to the amino acid sequences of HMO metabolic genes of dipro-F, dipro-15 and dipro-105 (the reference amino acid sequence encoding 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; Blon_2336, 1,3 / 4--l-Fucosidase), BLAST analysis was performed on the sequence results obtained by whole genome sequencing, and it was found that the Dipro F strains screened by the present invention all contained sequences with a similarity of greater than 93% with the above genes, and had a full set of metabolic genes capable of degrading HMO.
[0062] Example 2 Growth curve determination of HMO high utilization rate strain
[0063] 1. Experimental methods:
[0064] 1. Culture medium configuration
[0065] Preparation of MRS liquid culture medium: 10.0g peptone, 5.0g yeast extract powder, 10.0g beef extract, 20.0g glucose, 5.0g anhydrous sodium acetate, 2.0g anhydrous potassium hydrogen phosphate, 2.0g diammonium hydrogen citrate, 0.58g magnesium sulfate heptahydrate, 0.25g manganese sulfate tetrahydrate, 0.1mL Tween-80, add distilled water to 1000mL, adjust the pH value to 6.2-6.4, dispense 10mL of culture solution into anaerobic tubes, fill with sufficient nitrogen, cover with plug to isolate air, and sterilize at 121℃ for 15 minutes. MRS solid culture medium is added with 15g agar powder on the basis of liquid culture medium.
[0066] Preparation of chemically defined medium: The formula is shown in Table 2, where the carbon sources in the medium are glucose, 2'-FL, and LNnT. To avoid the loss of nutrient elements, the mineral salts and amino acids in the medium are sterilized at 121°C for 15 min, and the carbon source, vitamins, and nucleic acid bases are sterilized by filtration using a 0.22 μm microporous membrane and mixed before use.
[0067] Table 2 Chemically defined medium formulation
[0068]
[0069] 2. Strain activation
[0070] Dipro-F, dipro-15 and dipro-105 glycerol bacteria stored in -80℃ environment were inoculated into MRS liquid culture medium anaerobic tubes for activation, and then transferred to MRS liquid culture medium anaerobic tubes in sequence with 1% inoculation volume. Using the activated strain of the third passage, 9 ml of bacterial solution was taken in a 10 ml sterile centrifuge tube in a clean bench, centrifuged at 4500rpm and 4℃ for 15 minutes, the supernatant was poured out, and 9 ml of sterile physiological saline was added and mixed as inoculation liquid. The inoculation liquid was transferred to the chemical defined medium at 1% inoculation volume, and the inoculated culture liquid was transferred into a 96-well cell culture plate in a sterile environment, with each well being 100 microliters system, one plate at each time point, and 3 wells were made in parallel at each time point, and cultured at 37℃ in an anaerobic environment.
[0071] 3. Strain growth
[0072] The growth curve of bifidobacterium was determined using a multifunctional microplate reader, and the area under the curve (AUC) was calculated.
[0073] 2. Growth curve of high HMO-utilizing strains and calculation of area under the curve
[0074] 1. When glucose was used as the carbon source, the growth curves of the three HMO utilizing strains were as follows: Figure 5 ;
[0075] 2. When 2'-FL was used as the carbon source, the growth curves of the three HMO utilizing strains were as follows: Figure 6 ;
[0076] 3. When LNnt was used as the carbon source, the growth curves of the three HMO utilizing strains were as follows: Figure 7 ;
[0077] According to 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:
[0078] Table 3
[0079]
[0080] According to the data in Table 3, under the experimental conditions, the utilization rate of HMO of Dipro F was the highest under different carbon source conditions. In addition, the other two strains also had high HMO utilization rates. Among them, the utilization rate of dipro-F for 2'-FL was 20.5 times that of the standard strain (M16V), and the utilization rate of LNnt was 41.6 times that of the standard strain (M16V); the utilization rate of dipro-15 for 2'-FL 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 dipro-105 for 2'-FL 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).
Claims
1. A method for screening HMO high utilization rate strains, characterized in that: The screening method comprises the following steps: 1) Activating multiple strains of bifidobacteria to be tested to obtain seed liquids, then centrifuging the seed liquids, washing with PBS, and resuspending them to obtain bacterial suspensions, then adding the bacterial suspensions to a carbon source-free MRS+ medium containing bromophenol methyl violet at a volume percentage of 1%-10% for culture, adding 2'-FL and LNnT as the sole carbon sources, respectively, and then using glucose as a positive control and water as a negative control to observe the color of the culture medium. If the strain grows, the color of the culture medium turns yellow, otherwise the color of the culture medium remains purple, and the strain corresponding to the yellow culture medium color is preliminarily judged as a strain that can simultaneously utilize 2'-FL and LNnT; 2) Using the method of step 1), the strains obtained in step 1) are respectively used to prepare bacterial suspensions, and the bacterial suspensions are inoculated into MRS+ medium containing no carbon source, 2'-FL and LNnT are respectively added as the sole carbon source, and then glucose is used as a positive control and water is used as a negative control. According to the sampling time and number, multiple parallel cultured bacteria are configured, and OD600 is measured every few hours, and a growth curve is drawn. According to the growth curve, the area under the curve AUC is calculated, and the utilization rate of different carbon sources by different strains tested is judged according to the AUC; the formula of the AUC is as follows: 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), it is a strain with high HMO utilization rate.
2. The method for screening HMO high utilization rate strains according to claim 1, characterized in that: The bifidobacterium comprises one or more of Bifidobacterium breve, Bifidobacterium longum subsp. infantis, Bifidobacterium bifidum, Bifidobacterium pseudocatenulatum, Bifidobacterium breve and Bifidobacterium animalis.
3. The method for screening HMO high utilization rate strains according to claim 1, characterized in that: Step 1) Carbon-free MRS+ medium is prepared by adding cysteine to the carbon-free MRS medium at a final concentration of 0.5 g / L.
4. The method for screening HMO high utilization rate strains according to claim 3, characterized in that: The carbon source-free MRS culture 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.
5. The method for screening HMO high utilization rate strains according to claim 3, characterized in that: The working concentrations of glucose, 2'-FL and LNnT in step 1) and step 2) are all 2-20 g / L.
6. The method for screening HMO high utilization rate strains according to claim 3, characterized in that: The concentration of the bacterial suspension in step 1) is OD600=0.5-5.
7. The method for screening HMO high utilization rate strains according to claim 3, characterized in that: Step 2) The concentration of the bacterial suspension is OD600=0.5-5.
8. A strain with high HMO utilization rate, characterized in that: The high HMO utilization strain is strain dipro-105, which is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration. The classification name of the strain dipro-105 is Bifidobacterium bifidum, the deposit number is CGMCC No. 29342, the deposit date is December 18, 2023, and the deposit address is Beijing, China.
9. A probiotic preparation, characterized in that: The probiotic preparation comprises the strain dipro-105 according to claim 8.