Breast milk oligosaccharide for reducing escherichia coli virulence gene and application of breast milk oligosaccharide
By using 2'-fucosyl lactose, lactose-N-neotetrasaccharide and 6'-sialic acid lactose at a concentration of 10 mg/mL, the problem of poor anti-adhesion in the prior art was solved, and a more effective anti-E. coli adhesion and virility reduction effect was achieved.
Patent Information
- Application Number
- CN202510617544.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-13
- Publication Date
- 2025-08-19
AI Technical Summary
In the prior art, breast milk oligosaccharide products that resist E. coli adhesion cannot effectively inhibit the expression of E. coli virulence genes, and the anti-adhesion mechanism is unclear.
The 2'-fucosyl lactose, lactose-N-neotetrasaccharide and 6'-sialic acid lactose were incubated with E. coli at a concentration of 10 mg/mL to inhibit the expression of adhesion-related genes gfcE, etk, espZ, espL2, nleB, espP, toxB and iha, and the effect was verified by transcriptomic experiments.
It significantly inhibits E. coli's adhesion and colonization in the intestine, reduces the expression of virulence genes, and provides better anti-E. coli adhesion effect.
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Figure CN120504711A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of functional oligosaccharides, in particular to human milk oligosaccharides capable of reducing virulence genes of Escherichia coli and applications thereof. Background Art
[0002] Human milk oligosaccharides (HMOs) have multiple biological activities: first, they competitively block the binding of pathogen surface adhesins to host glycoconjugates by acting as glycosylated molecular ligand mimics, thereby forming a biological barrier against intestinal pathogens (as described in Reference 1); second, they act as regulators of intestinal flora homeostasis, indirectly enhancing immune responses and neurodevelopmental functions by promoting the proliferation of beneficial bacteria such as Bifidobacterium.
[0003] Jiang Yikang et al. introduced in the 2024 issue of "Food and Machinery" Volume 40, Issue 2 (Total Issue 268) that enterotoxigenic Escherichia coli (ETEC) and uropathogenic Escherichia coli (UPEC) rely on the hemagglutinin-mediated glycan binding mechanism to achieve colonization, and sialylated HMOs can reduce their hemagglutination titer; Table 1 of the document further quantified the type distribution characteristics and mass concentration (g / L) of HMOs components in breast milk, among which 2'-fucosyllactose (2'-FL), lactose-N-neotetraose (LNnT) and 6'-sialyllactose (6'-SL) were 2.74, 0.79 and 0.64, respectively.
[0004] Existing technologies use breast milk or sialylated HMOs to inhibit E. coli adhesion to intestinal epithelial cells, but the anti-adhesion mechanism has not been clearly elucidated. There is an urgent need to develop human milk oligosaccharide products that are more effective in inhibiting E. coli adhesion than breast milk or sialylated HMOs and to determine their effectiveness in inhibiting E. coli virulence genes. Summary of the Invention
[0005] The object of the present invention is to provide a use of human milk oligosaccharides in the preparation of medicines or foods for resisting Escherichia coli adhesion.
[0006] The purpose of the present invention is achieved through the following technical solutions:
[0007] Use of a human milk oligosaccharide group that reduces the virulence gene of Escherichia coli in the preparation of medicines or foods that resist Escherichia coli adhesion.
[0008] Human milk oligosaccharides that reduce the virulence genes of Escherichia coli include 2'-fucosyllactose (2'-FL), lactose-N-neotetraose (LNnT) and 6'-sialyllactose (6'-SL) at a concentration of 10 mg / mL; the purity of 2'-fucosyllactose is 97.2%, the purity of lactose-N-neotetraose is 97.9%, and the purity of 6'-sialyllactose is 96.5%.
[0009] Furthermore, the human milk oligosaccharide group reduced the expression of adhesion-related genes gfcE, etk, espZ, espL2, nleB, espP, toxB and iha in Escherichia coli.
[0010] Furthermore, the Escherichia coli is E. coli O157:H7.
[0011] The beneficial effects of the present invention are:
[0012] The present invention has confirmed through relative adhesion rate experiments that when 2'-fucosyllactose, lactose-N-neotetraose and 6'-sialyllactose at a concentration of 10 mg / mL are incubated with Escherichia coli, they have better anti-Escherichia coli adhesion effects than the blank treatment group.
[0013] In addition, the relative adhesion rate experiment was verified by transcriptomics experiments, and the working principle of the present invention was revealed at the molecular biology level: after pre-incubation with Escherichia coli, 2'-fucosyllactose, lactose-N-neotetraose and 6'-sialyllactose inhibited the expression process of Escherichia coli's gfcE, etk, espZ, espL2, nleB, espP, toxB and iha genes in terms of virulence, reduced the virulence effect, and affected the host cell interaction; ultimately, it made it difficult for Escherichia coli to adhere to and colonize the intestine. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 Graph showing the regulatory effects of human milk oligosaccharides in Examples 1, 2, and 3 on virulence-related genes of Escherichia coli O157:H7. DETAILED DESCRIPTION
[0015] The following is further explained in conjunction with specific implementation methods:
[0016] The concentrations of the three human milk oligosaccharides in Examples 1-3 and Comparative Example 1 are:
[0017]
[0018] Chemical reagents and materials:
[0019]
[0020]
[0021] 1. Relative Adhesion Rate Experiment
[0022] 1. Experimental Methods
[0023] The cultured E. coli O157:H7 was washed and resuspended in a medium containing 2% (v / v) FBS to a final concentration of 1×10 6CFU / mL. 10 mg / mL of sample (Examples 1-3 respectively) was added to the bacterial suspension. The untreated bacterial solution was used as the control group (Comparative Example 1). These mixed solutions were incubated at 37°C and 5% CO2 for 1 hour, and then 1 mL was added to the HT-29 cell model and incubated for 1 hour. After incubation, the cells were rinsed 4 times with PBS to remove unattached bacteria. In order to determine the number of bacteria adhering to the cells, HT-29 cells were lysed using 0.1% (v / v) Triton X-100 solution at 37°C, and the resulting lysate was diluted 10 times in a maximum rejuvenation dilution (MRD), then inoculated into MHA medium and cultured overnight at 37°C. The bacterial adhesion was quantified by counting colony forming units (CFU) and expressed as a relative adhesion rate (%) compared to the control group.
[0024] 2. Experimental Results
[0025] Example 1: 16.87%;
[0026] Example 2: 12.53%;
[0027] Example 3: 11.06%;
[0028] Comparative Example 1: 100.00%.
[0029] 3. Conclusion
[0030] By comparing the adhesion of Escherichia coli treated with Examples 1-3 and Comparative Example 1, it was found that 2'-fucosyllactose, lactose-N-neotetraose and 6'-sialyllactose at a concentration of 10 mg / mL could significantly inhibit the adhesion rate of Escherichia coli. It can be inferred without a doubt that 2'-fucosyllactose, lactose-N-neotetraose and 6'-sialyllactose have a positive effect on the adhesion of Escherichia coli.
[0031] 2. Transcriptomics Experiment
[0032] 1. Test Methods
[0033] 1-1. RNA extraction and detection
[0034] 1-1-1, RNA extraction
[0035] The concentration of the overnight cultured bacterial suspension was adjusted to 2 × 10 6CFU / mL (4 groups in total), add equal volumes of 10 mg / mL of sterile examples 1-3 and comparative example 1 that have been membrane-passed. 1 mL of the treated bacterial suspension was added to an enzyme-free centrifuge tube, and 3 parallels were set up for each treatment. The total RNA of each sample was extracted using a column-type bacterial total RNA extraction and purification kit. According to the operating instructions of the kit instructions, bacteria in the logarithmic growth phase were collected by centrifugation at 4 ° C and 8,000 rpm for 1 min, the supernatant was discarded, and the bacterial pellet was resuspended in 100 μL of lysozyme solution (400 μg / mL) and incubated at room temperature for 5 min. Subsequently, the bacterial solution was lysed using Rlysis-BG lysis buffer, and after treatment with anhydrous ethanol, it was allowed to stand for 1 min, centrifuged at 12,000 rpm at room temperature for 1 min, the waste liquid in the collection tube was discarded, and the RNA was bound to the centrifuge column. Subsequently, the tube was washed with GT and NT buffers in sequence and allowed to stand for 1 min, centrifuged at 10,000 rpm for 1 min at room temperature, the waste liquid in the collection tube was discarded, and finally the total RNA was eluted with 50 μL of diethyl pyrocarbonate-treated water.
[0036] 1-1-2. RNA quality and concentration determination
[0037] The extracted total RNA was immediately placed on ice, and its concentration and purity were determined using a NanoDrop 2000C spectrophotometer. The integrity of the RNA was analyzed by agarose gel electrophoresis and further verified using an Agilent 2100 Bioanalyzer.
[0038] 1-2, Library construction and quality control
[0039] The Zymo-Seq RiboFree Total RNA Library Kit was used to remove rRNA from extracted bacterial total RNA for Illumina sequencing. Divalent cations were then used to randomly fragment the RNA. The RNA was then fragmented and used as a template for reverse transcription to synthesize complementary DNA (cDNA). The resulting double-stranded cDNA was purified and screened for 400-500 bp fragments using the AMPure XP Nucleic Acid Purification Kit. Subsequent steps included double-end repair, insertion of an A base at the 3' end, and ligation of sequencing adapters. Polymerase chain reaction (PCR) amplification was then performed, and the PCR product was purified again using AMPure XP beads to generate the final library.
[0040] Library quality was assessed using a bioanalyzer and a high-sensitivity DNA kit. The Quant-iT PicoGreends DNA Quantification Kit was used to measure total library concentration, and qPCR was used to quantify effective library concentration. Equal volumes of normalized DNA libraries from multiple samples were mixed, serially diluted, and quantified before sequencing on an Illumina sequencer in PE150 mode.
[0041] 1-3. Transcriptome Analysis
[0042] 1-3-1, Data Quality Control
[0043] The image files generated by sequencing were converted into FASTQ raw data using the software provided by the sequencing platform. Fastp (0.22.0) was used to remove 3'-end forked sequences and reads with an average quality score lower than Q20. Sequences with adapters and low quality were further filtered to avoid significant interference with subsequent information analysis. All subsequent analyses were high-quality analyses based on clean data.
[0044] 1-3-2, Alignment and gene expression analysis
[0045] E. coli O157:H7 is a prokaryotic organism, and its transcriptome data was used to construct a reference genome index using Bowtie2 (v2.4.1) and the filtered sequences were compared with the reference genome.
[0046] HTSeq (v0.9.1) was used to compare the read counts of each E. coli gene and used as the raw gene expression level. To ensure comparability of gene expression levels between different genes and samples, expression levels were normalized using fragments per kilobase per million.
[0047] 1-3-3, screening of differentially expressed genes.
[0048] Differentially expressed genes (DEGs) were screened using DESeq2 (v1.38.3), with a fold change of |log2FoldChange| > 1 and a significance of p < 0.05. GO enrichment analysis was further performed using topGO, and p values were calculated using the hypergeometric distribution method. GO terms significantly enriched in upregulated, downregulated, and all differentially expressed genes were screened according to the significant enrichment criterion of p < 0.05. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using clusterProfiler (v4.6.0), also focusing on pathways significantly enriched with p < 0.05, to determine the primary biological functions of the differentially expressed genes in virulence.
[0049] 2. Experimental Results
[0050] See Gene Expression Fold Difference Table, Gene Expression Difference Table and Figure 1 .
[0051] Gene expression fold difference table:
[0052]
[0053]
[0054] 3. Conclusion
[0055] Transcriptomics results further confirmed the accuracy of the "relative adhesion rate experiment" results. By treating E. coli with Examples 1-3 and Comparative Example 1, it was found that 2'-fucosyllactose, lactose-N-neotetraose, and 6'-sialyllactose all exhibited strong inhibitory effects on the expression of gfcE, etk, espZ, espL2, nleB, espP, toxB, and iha genes. This reveals the application principle of the present invention at the molecular biological level.
[0056] The above embodiments and descriptions are only for explaining the principles and best embodiments of the present invention. Without departing from the spirit and scope of the present invention, the present invention may be subject to various changes and improvements, which shall fall within the scope of the invention to be protected.
Claims
1. A human milk oligosaccharide that reduces the virulence gene of Escherichia coli, characterized in that: The present invention includes any one or more of 2'-fucosyllactose, lactose-N-neotetraose and 6'-sialyllactose.
2. The human milk oligosaccharide for reducing Escherichia coli virulence genes according to claim 1, characterized in that: The concentration of any one or more of the 2'-fucosyllactose, lactose-N-neotetraose and 6'-sialyllactose monomers is 10 mg / mL.
3. Use of the human milk oligosaccharide for reducing the virulence gene of Escherichia coli as claimed in claim 1 in the preparation of medicines, foods or health products for resisting Escherichia coli adhesion.
4. The human milk oligosaccharide for reducing the virulence gene of Escherichia coli according to claim 1, characterized in that Human milk oligosaccharide monomers or compositions are used to inhibit the expression of adhesion-related genes gfcE, etk, espZ, espL2, nleB, espP, toxB and iha in Escherichia coli.
5. The human milk oligosaccharide for reducing the virulence gene of Escherichia coli according to claim 1, characterized in that: The Escherichia coli is E. coli O157:H7.
6. Use of 2'-fucosyllactose, lactose-N-neotetraose and 6'-sialyllactose monomers or a combination thereof in the preparation of substances against genes gfcE and / or etk related to O-antigen capsule synthesis.
7. Use of 2'-fucosyllactose, lactose-N-neotetraose, and 6'-sialyllactose monomers or combinations thereof in the preparation of substances that resist espZ, espL2, nleB, espP, toxB, and iha, genes associated with the type III secretion system (T3SS), that affect host cell interactions. 。 8. Use of 2'-fucosyllactose, lactose-N-neotetraose and 6'-sialyllactose monomers or their combination in the preparation of drugs for treating various diseases caused by Escherichia coli.
9. A method for determining whether 2'-fucosyllactose, lactose-N-neotetraose and 6'-sialyllactose monomers or a combination thereof reduce virulence genes in Escherichia coli, characterized in that: The concentrations of the three human milk oligosaccharides in Examples 1-3 and Comparative Example 1 are: Chemical reagents and materials: Relative adhesion rate experiment 1. Experimental Methods The cultured E. coli O157:H7 was washed and resuspended in a medium containing 2% (v / v) FBS to a final concentration of 1×10 6 CFU / mL; 10 mg / mL of sample (Examples 1-3, respectively) was added to the bacterial suspension; an untreated bacterial suspension served as a control group (Comparative Example 1); these mixtures were incubated at 37°C, 5% CO2 for 1 hour, and then 1 mL of each was added to the HT-29 cell model and incubated for 1 hour; after incubation, the cells were rinsed four times with PBS to remove unattached bacteria; to determine the number of bacteria adhering to the cells, HT-29 cells were lysed at 37°C using a 0.1% (v / v) Triton X-100 solution. The resulting lysate was serially diluted 10-fold in maximum rejuvenation dilution (MRD) and then inoculated into MHA medium and incubated overnight at 37°C. Bacterial adhesion was quantified by counting colony-forming units (CFU) and expressed as a relative adhesion rate (%) compared to the control group.
10. The method for determining whether 2'-fucosyllactose, lactose-N-neotetraose and 6'-sialyllactose reduce the virulence gene of Escherichia coli according to claim 9, characterized in that: Test method: 1-1, RNA extraction and detection; 1-1-1, RNA extraction; The concentration of the overnight cultured bacterial suspension was adjusted to 2 × 10 6 CFU / mL (a total of 4 groups), were added with equal volumes of 10 mg / mL of sterile Examples 1-3 and Comparative Example 1 that had been membrane-passed; 1 mL of the treated bacterial suspension was added to an enzyme-free centrifuge tube, with 3 parallels set for each treatment; total RNA of each sample was extracted using a column-type bacterial total RNA extraction and purification kit; according to the operating instructions of the kit instructions, bacteria in the logarithmic growth phase were collected by centrifugation at 4°C and 8,000 rpm for 1 min, the supernatant was discarded, the bacterial pellet was resuspended in 100 μL of lysozyme solution (400 μg / mL), and incubated at room temperature for 5 min; subsequently, the bacterial solution was lysed using Rlysis-BG lysis buffer, treated with anhydrous ethanol, and allowed to stand for 1 min, centrifuged at 12,000 rpm at room temperature for 1 min, the waste liquid in the collection tube was discarded, and the RNA was bound to the centrifuge column; subsequently, the cells were washed with GT and NT buffers in sequence and allowed to stand for 1 min, centrifuged at 10,000 rpm at room temperature for 1 min, the waste liquid in the collection tube was discarded, and finally the total RNA was eluted using 50 μL of diethyl pyrocarbonate-treated water; 1-1-2, RNA quality and concentration determination; The extracted total RNA was then immediately placed on ice, and its concentration and purity were determined using a NanoDrop 2000C spectrophotometer. The integrity of the RNA was analyzed by agarose gel electrophoresis and further verified using an Agilent 2100 bioanalyzer. 1-2, library construction and quality control; The Zymo-Seq RiboFree Total RNA Library Kit was used to remove rRNA from extracted bacterial total RNA for Illumina sequencing. Divalent cations were then used to randomly fragment the RNA. The RNA was then fragmented and reverse transcribed to synthesize complementary DNA (cDNA) using the RNA as a template. The resulting double-stranded cDNA was purified and screened for 400-500 bp fragments using the AMPure XP Nucleic Acid Purification Kit. Subsequent steps included double-end repair, insertion of an A base at the 3' end, and ligation of sequencing adapters. The resulting cDNA was then amplified by polymerase chain reaction (PCR), and the PCR product was purified again using AMPure XP beads to generate the final library. Library quality was tested using a bioanalyzer and a high-sensitivity DNA kit. The Quant-iT PicoGreends DNA quantification kit was used to measure the total library concentration, and qPCR quantitative detection was used to quantify the effective library concentration. Equal volumes of normalized multi-sample DNA libraries were mixed, gradually diluted and quantified, and then sequenced on an Illumina sequencer in PE150 mode. 1-3, transcriptome analysis; 1-3-1, data quality control; The image files generated by sequencing were converted into FASTQ raw data using the software provided by the sequencing platform. Fastp (0.22.0) was used to remove 3'-end forked sequences and reads with an average quality score lower than Q20. Sequences with adapters and low quality were further filtered to avoid significant interference with subsequent information analysis. This ensured that all subsequent analyses were high-quality analyses based on clean data. 1-3-2, alignment and gene expression analysis; As a prokaryotic organism, the transcriptome data of E. coli O157:H7 were used to construct a reference genome index using Bowtie2 (v2.4.1) and the filtered sequences were compared with the reference genome; HTSeq (v0.9.1) was used to statistically compare the read counts of each E. coli gene as the raw expression level of the gene. To make the gene expression levels comparable between different genes and different samples, the expression levels were normalized using fragments per kilobase per million. 1-3-3, screening of differentially expressed genes; DESeq2 (v1.38.3) was used to screen differentially expressed genes (DEGs), and DEGs with expression difference fold |log2FoldChange|>1 and significance p<0.05 were screened; GO enrichment analysis was further performed using topGO, and the p value was calculated using the hypergeometric distribution method. According to the significant enrichment standard p<0.05, GO terms with significant enrichment of upregulated, downregulated, and all differential genes were screened; KEGG pathway enrichment analysis was performed using clusterProfiler (v4.6.0), also focusing on the significantly enriched pathways with p<0.05, to determine the main biological functions of differentially expressed genes in virulence.