Composition comprising human milk oligosaccharide and probiotic, and use of composition in improving intestinal development

Abstract

The present invention relates to a composition comprising a human milk oligosaccharide and a probiotic. The human milk oligosaccharide is a neutral fucosylated human milk oligosaccharide, and the probiotic is bifidobacterium animalis. Specifically, the composition comprises 2'-fucosyllactose (2'-FL) and bifidobacterium animalis CP-9. In addition, the present invention also relates to a use of the composition in the preparation of a food or a healthcare supplement, and a use of the composition in the preparation of a product for improving intestinal development.

Description

Composition comprising human milk oligosaccharides and probiotics and its use for improving gut development

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese patent application No. 2024113122316, filed on September 20, 2024, entitled “Composition comprising human milk oligosaccharides and probiotics and its use for improving gut development”, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present document relates to a composition of human milk oligosaccharides and probiotics and the use of the composition in the manufacture of a preparation for improving the development of the glycocalyx layer of intestinal epithelial cells. In particular, the present document relates to a composition of 2’-fucosyllactose (2’-FL) and Bifidobacterium animalis CP-9, the use of the composition in the manufacture of a preparation for improving the development of the glycocalyx layer of intestinal epithelial cells. BACKGROUND

[0004] The intestinal barrier is considered as the gatekeeper of human health. The intestinal barrier is responsible for the passage and absorption of nutrients, but also plays a role in preventing the entry of pathogens into the human body. In addition, it also has the function of regulating the crosstalk between the luminal microbiota and macromolecules, and maintaining tolerance and immune response. After birth, the gatekeeping function of the intestinal barrier has not yet fully developed in newborns, and any disruption of the intestinal barrier function in newborns will have long-term effects and can play a role in the development of gastrointestinal infections and inflammatory bowel disease (IBD). In addition, the intestinal barrier function also affects the development of other diseases in childhood and adulthood, such as obesity and allergy, etc.

[0005] Human milk is the gold standard for infant nutrition, and human milk oligosaccharides (HMOs) are the main components that distinguish human milk from the milk of other mammals. In infant formula, one important function of HMOs is to stimulate the colonization of the gastrointestinal tract by the microbiota, improving the development of the intestinal barrier function. One possible way to achieve this is to stimulate the development of the epithelial glycocalyx on the intestinal epithelium. The glycocalyx on the intestinal epithelium of newborns provides binding sites for commensal microorganisms, and a well-developed glycocalyx can prevent the adhesion of pathogens and act as a barrier to luminal toxins and enzymes, thereby promoting intestinal development. The ability of probiotics to adhere to the intestinal epithelium plays a key role in influencing the composition of the intestinal flora, and the colonization of the intestinal epithelium by strains competes with other species through greater adhesion. In recent years, many studies have shown that HMOs can enhance the adhesion ability of probiotics by affecting the expression of bacterial adhesins, but few studies have shown that HMOs can promote the adhesion ability of probiotics by improving the development of the glycocalyx layer of intestinal epithelial cells.

[0006] Currently, there are few clinical intervention studies on the direct regulation of HMOs on intestinal cells and structures. However, preclinical research data show that HMOs have a positive impact on the maturation of the intestine. In vitro, intestinal cell line cultures such as Caco-2 are often used to study the mechanisms by which nutrients and microbial metabolites affect epithelial integrity. Studies have shown that N-lacto-neotetraose (LnNT), 2'-fucosyllactose (2'-FL) and 6'-sialyllactose (6'-SL) can promote tight junction protein expression and increase cell differentiation along the crypt-villus axis; 2'-fucosyllactose and 3-fucosyllactose have a positive effect on the expression of mucin glycoproteins. In vivo animal experiments have found that supplementation with 2'-FL can significantly increase the integrity of the intestinal epithelium and reduce the incidence of colitis. Currently, there are few studies on the effect of neutral fucosylated human milk oligosaccharides and animal bifidobacterium compositions on the development of the glycosyl layer of intestinal epithelial cells, especially whether the combination of the two has a synergistic effect on the development of the glycosyl layer of intestinal epithelial cells.

[0007] Therefore, this paper explores the effect of 2'-FL and animal bifidobacterium CP-9 composition on the development of the glycosyl layer of intestinal epithelial cells at the cellular level, and lays a theoretical foundation for the combination of 2'-FL and animal bifidobacterium CP-9. SUMMARY

[0008] In a first aspect, the purpose of this paper is to provide a composition comprising human milk oligosaccharides and probiotics, the human milk oligosaccharides being neutral fucosylated human milk oligosaccharides, and the probiotics being animal bifidobacterium.

[0009] Specifically, the neutral fucosylated human milk oligosaccharides are 2'-fucosyllactose (2'-FL), and the animal bifidobacterium is animal bifidobacterium CP-9. The composition is an efficacy component of a food, health product or drug.

[0010] In an embodiment, the final concentration of 2'-fucosyllactose (2'-FL) in the composition is 1.0 mg / mL to 2.6 mg / mL and / or the addition concentration of animal bifidobacterium is 10 6 cfu / mL to 10 8 cfu / mL.

[0011] For example, the final concentration of 2'-fucosyllactose (2'-FL) is 1.0 mg / mL, 1.2 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, 2.0 mg / mL, 2.1 mg / mL, 2.2 mg / mL, 2.3 mg / mL, 2.4 mg / mL or 2.6 mg / mL.

[0012] In an embodiment, the concentration of 2'-fucosyllactose (2'-FL) is 1.2 mg / mL to 2.4 mg / mL.

[0013] In a second aspect, another object of the present disclosure is to provide use of a composition of human milk oligosaccharide and probiotic in preparation of a food or health product.

[0014] In a third aspect, another object of the present disclosure is to provide use of a composition of human milk oligosaccharide and probiotic in preparation of a medicament for improving intestinal development.

[0015] In a fourth aspect, another object of the present disclosure is to provide use of a composition of human milk oligosaccharide and probiotic in improving intestinal development.

[0016] In an embodiment, the composition described above is used for improving development of the glycocalyx layer of intestinal epithelial cells. Specifically, the composition of 2'-fucosyllactose (2'-FL) and Bifidobacterium animalis CP-9 can synergistically improve the development of the glycocalyx layer on intestinal epithelial cells, thereby improving intestinal development.

[0017] In an embodiment, the manifestation of improving development of the glycocalyx layer of intestinal epithelial cells is selected from at least one of the following:

[0018] Protecting the glycocalyx protein skeleton, promoting the synthesis of glycocalyx hyaluronic acid, and promoting the synthesis of glycocalyx heparan sulfate.

[0019] In an embodiment, the manifestation of improving development of the glycocalyx layer of intestinal epithelial cells is selected from at least one of the following: upregulating the transcription level of the glycocalyx development-related gene glypican 1, upregulating the transcription level of the hyaluronic acid synthase-encoding gene, and upregulating the transcription level of the exostin glycosyltransferase-encoding gene.

[0020] In an embodiment, the manifestation of improving development of the glycocalyx layer of intestinal epithelial cells is selected from at least one of the following: upregulating the transcription level of gpc1, the transcription level of has1, the transcription level of has2, the transcription level of has3, the transcription level of ext1, and the transcription level of ext2.

[0021] In an embodiment, the composition has at least one of the following uses:

[0022] (1) 2'-fucosyllactose (2'-FL) improves the adhesion of Bifidobacterium animalis to intestinal epithelial cells;

[0023] (2) Bifidobacterium animalis and 2'-FL synergistically promote the development of the intestinal glycocalyx layer.

[0024] In an embodiment, 2'-fucosyllactose (2'-FL) improves the adhesion rate of intestinal epithelial cells to Bifidobacterium animalis.

[0025] In an embodiment, the composition is used to improve the transcription level of the glypican 1 (GPC1) proteoglycan-encoding gene gpc1, thereby improving the development of the protein skeleton of the glycocalyx layer; improve the transcription level of hyaluronic acid synthase 1-encoding genes has1, has2 and has3, thereby promoting the synthesis of hyaluronic acid synthase of the glycocalyx layer; and / or improve the transcription level of exostin glycosyltransferase 1-encoding genes ext1, ext2, thereby promoting the synthesis of heparan sulfate of the glycocalyx layer.

[0026] In an embodiment, 2'-fucosyllactose (2'-FL) further synergizes with the animal bifidobacteria adhering to the intestinal epithelial cells to promote the development of the intestinal glycocalyx layer while improving the adhesion of the animal bifidobacteria to the intestinal epithelial cells. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 shows the effect of 2'-FL on the adhesion rate of animal bifidobacteria CP-9 on Caco-2 cells.

[0028] Figure 2 shows the effect of the composition of 2'-FL and animal bifidobacteria CP-9 on the gpc1 transcription level of Caco-2 cells.

[0029] Figure 3 shows the effect of the composition of 2'-FL and animal bifidobacteria CP-9 on the has1 transcription level of Caco-2 cells.

[0030] Figure 4 shows the effect of the composition of 2'-FL and animal bifidobacteria CP-9 on the has2 transcription level of Caco-2 cells.

[0031] Figure 5 shows the effect of the composition of 2'-FL and animal bifidobacteria CP-9 on the has3 transcription level of Caco-2 cells.

[0032] Figure 6 shows the effect of the composition of 2'-FL and animal bifidobacteria CP-9 on the ext1 transcription level of Caco-2 cells.

[0033] Figure 7 shows the effect of the composition of 2'-FL and animal bifidobacteria CP-9 on the ext2 transcription level of Caco-2 cells. DETAILED DESCRIPTION

[0034] In order to make the purposes, technical solutions and advantages of the embodiments herein clearer, the technical solutions herein will be described clearly and completely below in conjunction with the following specific embodiments. Obviously, the described specific embodiments are part of the embodiments herein. All other embodiments obtained by a person of ordinary skill in the art without creative effort based on the described embodiments herein belong to the scope of protection of the present disclosure.

[0035] The "glycocalyx" described herein is composed of glycans and proteins, and the adhesion of probiotics to host cells is also closely related to extracellular polysaccharides. Modulating the structure of the glycocalyx on the surface of intestinal epithelial cells or improving the development of the glycocalyx can improve the mutual adhesion of probiotics to intestinal epithelial cells.

[0036] The "human milk oligosaccharide (HMO)" described herein can be a neutral fucosylated human milk oligosaccharide, preferably 2'-fucosyllactose (2'-FL).

[0037] The glycocalyx is composed of glycans and proteins. Proteoglycans are generally considered to be the most important component of the glycocalyx and form the skeleton of the glycocalyx. In addition, the glycocalyx contains glycosaminoglycan chains connected to the core proteins of the proteoglycans, among which heparan sulfate (HS) and hyaluronic acid (HA) are the main glycosaminoglycan components in the glycocalyx.

[0038] Therefore, the evaluation of the development of the glycocalyx layer of intestinal epithelial cells herein is mainly determined by measuring the transcriptional level of the glycocalyx development-related genes glypican 1 (gpc1), hyaluronic acid synthase 1 (has1, has2, has3), and exostin glycosyltransferase 1 (ext1, ext2) in Caco-2 cells. Specifically, gpc1 is the coding gene of Glypican 1 (GPC1) proteoglycan, and GPC1 is the protein skeleton of the glycocalyx layer and an important carrier of glycosaminoglycan chains including hyaluronic acid (HA) and heparan sulfate (HS). has1, has2, and has3 are the coding genes of hyaluronic acid synthase (HAS), which is essential for the synthesis of HA, and HA is a highly viscous component of the intestinal mucus layer, which is responsible for tissue repair, stability, and anti-inflammatory effects. Compared with HAS1 and HAS2, HAS3 synthesizes low molecular weight HA, which is necessary for the development of intestinal stem cells. In addition, the ext1 and ext2 genes are involved in the extension of heparan sulfate (HS) chains and are responsible for integrating the HS chains with nucleotide sugars in the Golgi apparatus, and HS synthesis supports organogenesis, growth factor signaling, and bacterial adhesion.

[0039] In a first aspect, the purpose of the present application is to provide a composition comprising a human milk oligosaccharide and a probiotic, wherein the human milk oligosaccharide is a neutral fucosylated human milk oligosaccharide, and the probiotic is an animal Bifidobacterium.

[0040] Specifically, the neutral fucosylated human milk oligosaccharide is 2'-fucosyllactose (2'-FL), and the animal Bifidobacterium is animal Bifidobacterium CP-9. The composition is an efficacy component of a food, health product, or drug.

[0041] In an embodiment, the final concentration of 2'-fucosyllactose (2'-FL) in the composition is 1.0 mg / mL to 2.6 mg / mL and / or the addition concentration of the animal Bifidobacterium is 106 cfu / mL ~ 10 8 cfu / mL.

[0042] For example, the final concentration of 2'-fucosyllactose (2'-FL) is 1.0 mg / mL, 1.2 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, 2.0 mg / mL, 2.1 mg / mL, 2.2 mg / mL, 2.3 mg / mL, 2.4 mg / mL, or 2.6 mg / mL.

[0043] In one embodiment, the concentration of 2'-fucosyllactose (2'-FL) is 1.2 mg / mL ~ 2.4 mg / mL.

[0044] In a second aspect, another object of the present disclosure is to provide use of a composition of human milk oligosaccharide and probiotic in preparation of a food or health product.

[0045] In a third aspect, another object of the present disclosure is to provide use of a composition of human milk oligosaccharide and probiotic in preparation of a medicament for improving intestinal development.

[0046] In a fourth aspect, another object of the present disclosure is to provide use of a composition of human milk oligosaccharide and probiotic in improving intestinal development.

[0047] In one embodiment, the above-mentioned composition is used for improving development of the glycocalyx layer of intestinal epithelial cells. Specifically, the composition of 2'-fucosyllactose (2'-FL) and Bifidobacterium animalis ssp. lactis CP-9 can synergistically improve the development of the glycocalyx layer on the intestinal epithelial cells, thereby improving intestinal development.

[0048] In one embodiment, the manifestation of improving the development of the glycocalyx layer of intestinal epithelial cells is selected from at least one of the following:

[0049] Protecting the glycocalyx protein skeleton, promoting the synthesis of glycocalyx hyaluronic acid, and promoting the synthesis of glycocalyx heparan sulfate.

[0050] In one embodiment, the manifestation of improving the development of the glycocalyx layer of intestinal epithelial cells is selected from at least one of the following: up-regulating the transcription level of the glycocalyx development-related gene glypican 1, up-regulating the transcription level of the hyaluronic acid synthase-encoding gene, and up-regulating the transcription level of the exostin glycosyltransferase-encoding gene.

[0051] In one embodiment, the manifestation of improving the development of the glycocalyx layer of intestinal epithelial cells is selected from at least one of the following: up-regulating the transcription level of gpc1, the transcription level of has1, the transcription level of has2, the transcription level of has3, the transcription level of ext1, and the transcription level of ext2.

[0052] Within a certain concentration range, 2’-FL can significantly up-regulate the transcription level of gpc1, and the increase of animal bifidobacterium CP-9 helps to up-regulate the transcription level of gpc1. When 2’-FL and animal bifidobacterium CP-9 are used in combination, the relative transcription level of gpc1 is significantly higher than that of animal bifidobacterium CP-9 alone after 1.2 mg / mL-2.4 mg / mL 2’-FL is added to culture the host cells and then animal bifidobacterium CP-9 adhering to the host cells is used for intervention; therefore, 2’-FL and animal bifidobacterium CP-9 adhering to the host cells have a synergistic promotion effect on improving the relative transcription level of gpc1, and can protect the protein skeleton of glycocalyx.

[0053] Higher concentration of 2’-FL and animal bifidobacterium CP-9 adhering to the host cells have a synergistic promotion effect on improving the relative transcription level of has1, has2 and has3, and can promote the synthesis of hyaluronic acid in the glycocalyx.

[0054] Combination of specific concentration of 2’-FL and animal bifidobacterium CP-9 adhering to the host cells has a synergistic promotion effect on improving the transcription level of ext1, and can promote the synthesis of heparan sulfate in the glycocalyx.

[0055] In an embodiment, the composition has at least one of the following uses:

[0056] (1) 2’-fucosyllactose (2’-FL) improves the adhesion of animal bifidobacterium to intestinal epithelial cells;

[0057] (2) Animal bifidobacterium and 2’-FL synergistically promote the development of intestinal glycocalyx layer.

[0058] In an embodiment, 2’-fucosyllactose (2’-FL) improves the adhesion rate of intestinal epithelial cells to animal bifidobacterium.

[0059] In an embodiment, the composition is used to improve the transcription level of gpc1, the coding gene of Glypican 1 (GPC1) proteoglycan, thereby improving the development of protein skeleton of glycocalyx layer; improve the transcription level of has1, has2 and has3, the coding genes of hyaluronic acid synthase 1, thereby promoting the synthesis of hyaluronic acid synthase in the glycocalyx layer; and / or improve the transcription level of ext1, ext2, the coding genes of exostosin glycosyltransferase 1, thereby promoting the synthesis of heparan sulfate in the glycocalyx layer.

[0060] In an embodiment, 2’-fucosyllactose (2’-FL) improves the adhesion of animal bifidobacterium to intestinal epithelial cells, and animal bifidobacterium adhering to intestinal epithelial cells and 2’-FL further synergistically promote the development of intestinal glycocalyx layer.

[0061] Embodiments

[0062] The embodiments herein are only used for exemplarily describing the technical solutions herein, but do not mean that the technical solutions herein are limited to these specific embodiments.

[0063] The inventors of the present case have proved through specific experiments that the combination of 2'-fucosyllactose (2'-FL) and animal bifidobacterium CP-9 herein has a synergistic promoting effect on improving the development of the glycocalyx layer of intestinal epithelial cells, and 2'-fucosyllactose (2'-FL) improves the adhesion of animal bifidobacterium in intestinal epithelial cells.

[0064] The experimental methods and subject conditions used in each embodiment and control are as follows:

[0065] The culture methods of Caco-2 cells and animal bifidobacterium (CP-9) used in the embodiments are as follows:

[0066] 1. Culture of Caco-2 cells

[0067] Human colon adenocarcinoma cells (Caco-2, purchased from Nanjing Sunbeiqia Biotechnology Co., Ltd.) were cultured in MEM complete medium (purchased from Nanjing Sunbeiqia Biotechnology Co., Ltd.) containing 20% high-quality fetal bovine serum, and grown in 25cm 2 cell culture bottles in a carbon dioxide incubator at 37°C, 5% CO2, and a relative humidity of 95%. The culture medium was replaced every other day or every two days. When the cells grew to 80%-90% confluence, they were digested with 0.25% trypsin digestion solution and subcultured at a ratio of 1:3.

[0068] 2. Culture of animal bifidobacterium (CP-9)

[0069] The freeze-dried and preserved animal bifidobacterium CP-9 (purchased from Anhui Jinqiao Biotechnology Co., Ltd.) was inoculated in modified MRS medium (purchased from Hibeier Biotechnology Co., Ltd.). After 24h of culture at 37°C, it was inoculated in fresh modified MRS medium (purchased from Hibeier Biotechnology Co., Ltd.) at a ratio of 1%. After 24h of culture at 37°C, plate viable count was performed using the pouring method, and the viable bacteria count of the animal bifidobacterium CP-9 after activation for the second time was recorded.

[0070] Embodiment 1

[0071] This embodiment provides an experiment on the effect of 2'-FL on the adhesion of animal bifidobacterium CP-9.

[0072] (1) Adjust the density of Caco-2 cells to 1×10 5 cells / cm 2, inoculated in 24-well plates, and incubated for 3 days in MEM medium with a final concentration of 0.6 mg / mL of 2’-FL. The cultured Bifidobacterium animalis CP-9 was centrifuged to remove the supernatant, washed twice with sterile PBS, and resuspended with MEM complete medium to adjust the viable concentration of CP-9 to 1 x 10 8 CFU / mL of bacterial suspension.

[0073] (2) The supernatant of the incubated Caco-2 cell culture medium was removed, and 2’-FL was added at a concentration of 0.6 mg / mL and MEM complete medium (without double antibiotics) containing 1 x 10 8 CFU / mL of Bifidobacterium animalis CP-9 was added, and incubated for 2 h at 37°C in a 5% CO2 incubator. The liquid in the wells was discarded, and then washed twice with sterile PBS solution to remove unadhered bacteria. 150 μL of trypsin cell digestion solution was added, and after the cells were completely detached, 350 μL of complete cell culture solution was added to terminate the digestion, and diluted by 10-fold gradient.

[0074] The number of adhered bacteria was determined by pour plate colony counting method, and the adhesion rate was calculated according to formula (1), with blank group 1 without the addition of 2’-FL as a control.

[0075] Example 2

[0076] This example provides an experiment on the effect of 2’-FL on the adhesion of Bifidobacterium animalis CP-9. (1) The final concentration of 2’-FL in MEM was 1.2 mg / mL; (2) the supernatant of the incubated Caco-2 cell culture medium was removed, and 2’-FL was added at a concentration of 1.2 mg / mL and MEM complete medium (without double antibiotics) containing 1 x 10 8 CFU / mL of Bifidobacterium animalis CP-9 was added, and incubated for 2 h at 37°C in a 5% CO2 incubator. Other steps were the same as in Example 1.

[0077] Example 3

[0078] This example provides an experiment on the effect of 2’-FL on the adhesion of Bifidobacterium animalis CP-9. (1) The final concentration of 2’-FL in MEM was 2.4 mg / mL; (2) the supernatant of the incubated Caco-2 cell culture medium was removed, and 2’-FL was added at a concentration of 2.4 mg / mL and MEM complete medium (without double antibiotics) containing 1 x 10 8 CFU / mL of Bifidobacterium animalis CP-9 was added, and incubated for 2 h at 37°C in a 5% CO2 incubator. Other steps were the same as in Example 1.

[0079] For the specific concentrations of 2'-FL and CP-9 strains in blank group 1 and Examples 1-3, please refer to Table 1 below.

[0080] Table 1

[0081] As shown in Figure 1, compared with the control group 1, the adhesion rate of Caco-2 cells to Bifidobacterium animalis CP-9 was increased after co-culturing with different concentrations of 2'-FL. In particular, the adhesion rate of Caco-2 cells to Bifidobacterium animalis CP-9 was significantly increased by 1.89 times and 2.12 times, respectively, under the intervention of 1.2 mg / mL and 2.4 mg / mL of 2'-FL (p < 0.05).

[0082] The following examples test the effects of 2'-FL and CP-9 on the development of glycocalyx in intestinal epithelial cells.

[0083] Examples 4-5

[0084] Adjust Caco-2 cell density to 1×10⁻⁶ 5 pcs / cm 2 Caco-2 cells were seeded in 24-well plates and incubated with MEM intact medium containing a specific concentration of 2'-FL for 48 hours. In Example 4, 1.2 mg / mL of 2'-FL was added, and in Example 5, 2.4 mg / mL of 2'-FL was added. After incubation, total RNA was extracted from the cells and analyzed by reverse transcription and qPCR.

[0085] Examples 6-7

[0086] Adjust Caco-2 cell density to 1×10⁻⁶ 5 pcs / cm 2 Cells were inoculated into 24-well plates and cultured in MEM intact medium for 36 hours. When the confluence exceeded 70%, specific numbers of viable Bifidobacterium animalis CP-9 were added, with 10 in Example 6. 7 cfu / mL Bifidobacterium animalis CP-9, added in Example 7 10 8 CFU / mL Bifidobacterium animalis CP-9. After incubation for 2 hours, the cells were washed twice with sterile PBS solution, retaining only Bifidobacterium animalis CP-9 adhering to Caco-2 cells. Following the method in section 2.1, the adhesion of Bifidobacterium animalis CP-9 to Caco-2 cells in three replicates was measured (see Table 2 below). The remaining three replicates were then incubated for another 10 hours with intact MEM medium. Total RNA was then extracted and subjected to reverse transcription and qPCR assays.

[0087] Examples 8-11

[0088] Adjust Caco-2 cell density to 1×10⁻⁶ 5 pcs / cm 2 The culture medium was inoculated into 24-well plates, and MEM containing a specific concentration of 2'-FL was added. The plates were incubated for 36 hours. When the fusion rate exceeded 70%, a specific number of viable Bifidobacterium animalis CP-9 was added. After incubation for 2 hours, the plates were washed twice with sterile PBS solution, retaining only the Bifidobacterium animalis CP-9 adhering to Caco-2 cells. Following the method in Example 1, the adhesion of Bifidobacterium animalis CP-9 to Caco-2 cells in three replicates was measured (see Table 2 below). The remaining three replicates were then supplemented with MEM containing a final concentration of 1.2 mg / mL of 2'-FL. After incubation for another 10 hours, total RNA was extracted and subjected to reverse transcription and qPCR.

[0089] Specifically, in Example 8, MEM complete medium containing 1.2 mg / mL of 2'-FL was used for co-incubation for 36 hours. When the fusion rate exceeded 70%, 10 mg / mL of MEM was added. 7 The concentration of Bifidobacterium animalis CP-9 at cfu / mL was determined to be 10 CFU / mL in Caco-2 cells. 6 cfu / mL; In Example 9, MEM complete medium containing 2'-FL was used for co-incubation for 36 h. When the fusion rate exceeded 70%, 10 cfu / mL was added. 7 The concentration of Bifidobacterium animalis CP-9 at cfu / mL was determined to be 10 CFU / mL in Caco-2 cells. 6 cfu / mL; In Example 10, MEM complete medium containing 1.2 mg / mL 2'-FL was used for co-incubation for 36 h. When the fusion rate exceeded 70%, 10 cfu / mL was added. 8 The concentration of Bifidobacterium animalis CP-9 at cfu / mL was determined to be 10 CFU / mL in Caco-2 cells. 7 cfu / mL; and, in Example 11, MEM complete medium containing 2.4 mg / mL 2'-FL was used for co-incubation for 36 h. When the fusion rate exceeded 70%, 10 cfu / mL was added. 8 The concentration of Bifidobacterium animalis CP-9 at cfu / mL was determined to be 10 CFU / mL in Caco-2 cells. 7 cfu / mL.

[0090] The specific concentrations of the added and interventional substances 2'-FL and CP-9 in each embodiment are shown in Table 2 below. Meanwhile, MEM culture medium was set up as a blank control group 2.

[0091] qPCR was performed using the qPCR primer sequences shown in Table 3 below to determine the transcription level of the glypican 1 (gpc1), hyaluronic acid synthase 1 (has1, has2, has3), and exostin glycosyltransferase 1 (ext1, ext2) genes related to the development of the glycocalyx in Caco-2 cells.

[0092] Table 2

[0093] Table 3

[0094] (I) Evaluation of the effects of 2’-FL and CP-9 on the protein skeleton of the glycocalyx layer in different embodiments

[0095] According to the above experimental preparation steps and specific experimental methods, the results of the relative transcription level of gpc1 mRNA are shown in FIG. 2.

[0096] As can be seen, the different concentrations of 2’-FL in Examples 4-5 can significantly up-regulate the relative transcription level of gpc1 (p<0.05), but as the concentration of 2’-FL increases, the relative transcription level of gpc1 shows a downward trend.

[0097] According to the relative transcription level of gpc1 mRNA in Examples 6-7, it can be found that when only the animal bifidobacterium CP-9 adhering to Caco-2 is used as an intervention substance, the relative transcription level of gpc1 of Caco-2 has no significant change compared with the blank group 2 (p>0.05), but as the adherent strain increases, the relative transcription level of gpc1 shows an upward trend.

[0098] According to the relative transcription level of gpc1 mRNA in Examples 8-11, it can be seen that after 1.2 mg / mL-2.4 mg / mL of 2’-FL is first added to culture Caco-2 cells, and then the animal bifidobacterium CP-9 adhering to Caco-2 is used for intervention, the relative transcription level of gpc1 is significantly higher than that in the intervention with only the animal bifidobacterium CP-9 adhering to Caco-2 (Examples 6-7) (p<0.05). At the same time, as the concentration of 2’-FL increases, the relative transcription level of gpc1 (p<0.05) shows an upward trend, and when 2.4 mg / mL of 2’-FL is first added, i.e., in Examples 10-11, the relative transcription level of gpc1 is significantly higher than that in the remaining examples (p<0.05).

[0099] In summary, 2’-FL and the animal bifidobacterium CP-9 adhering to Caco-2 cells have a synergistic promoting effect on improving the relative transcription level of gpc1, which can protect the protein skeleton of the glycocalyx.

[0100] (ii) Evaluation of the effect of 2’-FL and CP-9 on the synthesis of hyaluronic acid in the glycan layer in different embodiments

[0101] According to the above experimental preparation steps and specific experimental methods, the obtained mRNA relative transcription levels of hasl, has2 and has3 are shown in Figures 3-5.

[0102] The different concentrations of 2’-FL in Examples 4-5 can significantly up-regulate the transcription level of hasl (p<0.05), and 1.2 mg / mL of 2’-FL can significantly up-regulate the relative transcription levels of has2 and has3, and with the increase of the concentration of 2’-FL, the relative transcription levels of hasl, has2 and has3 all show a downward trend.

[0103] According to Examples 6-7, it can be found that when only the adherent animal bifidobacterium CP-9 is used as an intervention substance, it can only significantly up-regulate the relative transcription level of hasl in Caco-2, and has a smaller improvement degree on the mRNA relative transcription levels of has2 and has3 in Caco-2.

[0104] According to the mRNA relative transcription levels of hasl, has2 and has3 in Examples 8-11, it can be found that after 1.2 mg / mL-2.4 mg / mL of 2’-FL is first added to culture Caco-2 cells, and then the animal bifidobacterium CP-9 adhered to Caco-2 is intervened, the mRNA relative transcription level of hasl is significantly higher than that of hasl mRNA relative transcription level in Examples 4-7 (p<0.05). At the same time, the mRNA relative transcription levels of has2 and has3 in Examples 10 and 11 are significantly higher than those of has2 and has3 in Examples 5-7 (2.4 mg / mL of 2’-FL is added alone or CP-9 with an adhering concentration of 10 6 cfu / mL-10 7 cfu / mL is used as an intervention substance) (p<0.05).

[0105] In summary, higher concentrations of 2’-FL and animal bifidobacterium CP-9 adhered to Caco-2 cells have a synergistic promotion effect on improving the relative transcription levels of hasl, has2 and has3, and can promote the synthesis of hyaluronic acid in the glycan layer.

[0106] (iii) Evaluation of the effect of 2’-FL and CP-9 on the synthesis of heparan sulfate in the glycan layer in different embodiments

[0107] According to the above experimental preparation steps and specific experimental methods, the obtained mRNA relative transcription levels of extl and ext2 are shown in Figures 6-7.

[0108] It can be seen that only 2’-FL at a concentration of 1.2 mg / mL in Examples 4-5 can significantly up-regulate the transcription levels of ext1 and ext2, and the transcription levels of ext1 and ext2 show a downward trend with the increase of the concentration of 2’-FL.

[0109] According to the relative transcription levels of ext1 and ext2 mRNA in Examples 6-7, it can be found that when only the animal bifidobacterium CP-9 adhering to Caco-2 is retained as an intervention substance, the relative transcription levels of ext1 and ext2 in Caco-2 have no significant difference (p>0.05) compared with the control of blank group 2.

[0110] According to the relative transcription levels of ext1 mRNA in Examples 8-11, it can be found that after Caco-2 cells are cultured by adding 1.2 mg / mL-2.4 mg / mL 2’-FL first and then intervened by animal bifidobacterium CP-9 adhering to Caco-2, the transcription levels of ext1 in Examples 8-9 are significantly higher than those in the remaining examples (p<0.05).

[0111] According to the relative transcription levels of ext2 mRNA in Examples 8-11, it can be found that after Caco-2 cells are cultured by adding 1.2 mg / mL-2.4 mg / mL 2’-FL first and then intervened by animal bifidobacterium CP-9 adhering to Caco-2, the transcription levels of ext2 have no significant improvement compared with the transcription levels of ext2 in Examples 4-7, but the transcription levels of ext2 are lower than those of using different concentrations of 2’-FL alone.

[0112] According to Examples 6-7, when CP-9 is used as an intervention substance alone, CP-9 has no effect on promoting the gene transcription levels of ext1 and ext2 in Caco-2, which may be due to the strain itself or the metabolic products have no up-regulation effect or have certain inhibition effect on the gene transcription levels of ext1 and ext2. Therefore, after the adhesion of animal bifidobacterium CP-9 is promoted by adding 2’-FL first, the effect of animal bifidobacterium CP-9 on the gene transcription levels of ext1 and ext2 (no up-regulation or inhibition) is amplified, so that no synergistic promotion effect on the transcription level of ext2 is shown.

[0113] It is confirmed that 2’-FL and animal bifidobacterium CP-9 adhering to Caco-2 cells need to be used at a specific concentration to show a synergistic promotion effect on the gene transcription levels of ext1 and ext2, and it is not a simple superposition of effects.

[0114] In summary, the combination of 2’-FL at a specific concentration and animal bifidobacterium CP-9 adhered to Caco-2 cells has a synergistic effect on improving the transcription level of ext1, which can promote the synthesis of glycosylphosphatidylinositol (GPI)-anchored heparan sulfate. Industrial applicability

[0115] The use of a composition containing 2’-fucosyllactose (2’-FL) at a specific concentration range and animal bifidobacterium CP-9 can synergistically improve the development of the intestinal epithelial cell glycocalyx layer, prevent the adhesion of pathogens through good development of the intestinal epithelial cell glycocalyx layer, provide a good binding site for symbiotic microorganisms, and further promote the development of intestinal barrier function and intestinal development.

[0116] The use of 2’-fucosyllactose (2’-FL) at a specific concentration range can improve the adhesion rate of intestinal epithelial cells to animal bifidobacterium, thereby affecting the composition of intestinal flora and promoting the development of intestinal barrier function and intestinal development.

Claims

1. A composition comprising a human milk oligosaccharide and a probiotic, characterized in that, The human milk oligosaccharide is neutral fucosylated human milk oligosaccharide, and the probiotic is Bifidobacterium animalis.

2. The composition of claim 1, wherein, The neutral fucosylated human milk oligosaccharide is 2'-fucosyllactose (2'-FL).

3. The composition of claim 1, wherein, The Bifidobacterium animalis is Bifidobacterium animalis CP-9.

4. The composition according to any one of claims 1 to 3, characterized in that, The final concentration of the 2'-fucosyllactose (2'-FL) in the composition is 1.0 mg / mL to 2.6 mg / mL and / or the added concentration of the animal bifidobacterium is 10 6 cfu / mL to 10 8 cfu / mL.

5. The composition according to any one of claims 1 to 3, characterized in that, The concentration of the 2'-fucosyllactose (2'-FL) is 1.2 mg / mL to 2.4 mg / mL.

6. Use of the composition according to any one of claims 1 to 5 in the manufacture of a food or health product.

7. Use of the composition according to any one of claims 1 to 5 in the manufacture of a medicament for improving intestinal development.

8. Use of the composition according to any one of claims 1 to 5 for improving intestinal development.

9. Use according to claim 7 or 8, characterized in that, The composition is used for improving the development of the glycocalyx layer of intestinal epithelial cells.

10. Use according to claim 9, characterized in that, The manifestation of the improved development of the glycocalyx layer of intestinal epithelial cells is selected from at least one of the following: protecting the glycocalyx protein skeleton, promoting the synthesis of glycocalyx hyaluronic acid, and promoting the synthesis of glycocalyx heparan sulfate.

11. Use according to claim 10, characterized in that, The manifestation of the improved development of the glycocalyx layer of intestinal epithelial cells is selected from at least one of the following: up-regulating the transcription level of the glycocalyx development-related gene glypican 1, up-regulating the transcription level of the hyaluronic acid synthase-encoding gene, and up-regulating the transcription level of the exostosin glycosyltransferase-encoding gene.

12. Use according to claim 11, characterized in that, The manifestation of the improved development of the glycocalyx layer of intestinal epithelial cells is selected from at least one of the following: up-regulating the transcription level of gpc1, the transcription level of has1, the transcription level of has2, the transcription level of has3, the transcription level of ext1, and the transcription level of ext2.

13. Use according to any one of claims 7 to 12, characterized in that, The composition has at least one of the following uses: (1) 2'-fucosyllactose (2'-FL) improves the adhesion of Bifidobacterium animalis to intestinal epithelial cells; (2) Bifidobacterium animalis and 2'-FL synergistically promote the development of the intestinal glycocalyx layer.

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