Composition for relieving intestinal inflammation and application thereof

Abstract

The invention relates to the field of infant foods, in particular to the field of nutritional compositions containing breast milk oligosaccharides, and particularly relates to a composition for relieving intestinal inflammation and application thereof. The composition is prepared from breast milk oligosaccharide and lactobacillus rhamnosus. The composition provided by the invention can relieve neonatal necrotizing enterocolitis, and solves the problems of insufficient solutions, unknown mechanisms and the like for enteritis such as neonatal necrotizing enterocolitis in the prior art. According to the application disclosed by the invention, the combination of breast milk oligosaccharide and lactobacillus rhamnosus can be used for relieving neonatal necrotizing enterocolitis by inhibiting a TLR4 / NF-kappa B inflammation pathway, improving cell oxidative stress, protecting a cell barrier and reducing cell apoptosis, and the application has an important application value in the field of functional foods.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of infant food, in particular to the field of nutritional compositions comprising human milk oligosaccharides, and in particular to a composition for alleviating intestinal inflammation and uses thereof. BACKGROUND

[0002] Necrotizing enterocolitis (NEC) is one of the main diseases affecting the life and health of premature infants. About 5%-10% of premature infants will suffer from necrotizing enterocolitis, and the mortality rate of NEC patients is as high as 25%-50%. Surgical treatment of NEC can produce complications such as short bowel syndrome, intestinal failure, parenteral nutrition-related liver disease, and neurodevelopmental delay. Therefore, it is crucial to prevent the occurrence of NEC. The intestinal barrier is a natural barrier to prevent bacteria and their products from entering the blood, and can also enhance the host's sensitivity to endotoxins, regulate immune responses, and its functional integrity is of great significance to maintain the ecological balance inside the human intestine. NEC is a multifactorial disease, and newborns are prone to NEC under the condition of immature intestinal development. The intestinal barrier blocks the invasion of pathogens with the help of physical and immune defense layers, and the damage of intestinal mucosal integrity and insufficient barrier function leads to the combination of microbial toxins and epithelial toll-like receptor 4 (TLR4), which activates the TLR4 / nuclear factor-kappa-B (NF-κB) signaling pathway, transmits signals to the downstream, and destroys the balance of anti-inflammatory and pro-inflammatory, releases a large amount of inflammatory cytokines. This pathway can induce cell oxidative stress, reduce antioxidant enzymes, and destroy the oxidation / antioxidant balance. Inflammatory cytokines induce cell apoptosis and reduce tight junction proteins and mucins, and multiple pathways strengthen each other to increase the risk of NEC.

[0003] HMOs are the most abundant prebiotics in breast milk, with over 200 different structures. Due to its composition and complex structure, it is particularly difficult to supplement HMOs in formula to prevent NEC. Studies have reported that 2'-fucosyllactose (2'-FL) and 6'-sialyllactose (6'-SL) can alleviate NEC by inhibiting TLR4 signaling, reducing cell apoptosis and protecting the intestinal barrier. Jantscher-Krenn et al. also found that disialyllacto-N-tetraose (DSLNT) can alleviate the severity of NEC and reduce the incidence of NEC. Wu et al. proposed the protective effect of HMOs on Caco-2 cells and elucidated the relationship between HMO structure and function. In addition, probiotics have also been proposed to treat NEC. Probiotics can maintain the integrity of the intestinal mucosal barrier, regulate the colonization of bacteria, assist the intestinal immune defense system and improve intestinal inflammation. A meta-analysis also showed that the supplementation of bifidobacteria and lactobacilli can reduce the incidence and mortality of NEC, and lactobacilli and bifidobacteria are more effective than other strains. It is known that HMOs are important metabolic substrates, but the use of related probiotics has specificity. For example, in a model of NEC piglets, the combination of 3'-SL and BL. infantis did not significantly reduce the incidence of NEC compared with single use. Therefore, the combination of HMOs and probiotics to alleviate NEC needs further research. SUMMARY

[0004] The purpose of the present application is to provide a composition for alleviating necrotizing enterocolitis in newborns, so as to solve the problems of insufficient solutions and unknown mechanisms in the prior art for necrotizing enterocolitis in newborns.

[0005] In a first aspect of the present application, a composition for improving necrotizing enterocolitis in newborns is provided, which comprises breast milk oligosaccharides and Lactobacillus rhamnosus.

[0006] The breast milk oligosaccharides comprise at least one neutral oligosaccharide and at least one sialylated oligosaccharide.

[0007] Further, the neutral oligosaccharide is selected from fucosyllactose, lacto-N-neotetraose and lacto-N-tetraose.

[0008] Further, the neutral oligosaccharide comprises 3-fucosyllactose.

[0009] Further, the sialylated oligosaccharide comprises 3'-sialyllactose.

[0010] In a preferred embodiment, the weight ratio of 3-fucosyllactose to 3'-sialyllactose is (0.5-50):1. Preferably, the weight ratio of 3-fucosyllactose to 3'-sialyllactose is (1-20):1. Further preferably, the weight ratio of 3-fucosyllactose:3'-sialyllactose is (1-7):1. More preferably, the 3-fucosyllactose is 0.1-3 mg / mL.

[0011] Further, the concentration of the Lactobacillus rhamnosus is 10 6 -10 14 CFU / g. In a preferred embodiment, the Lactobacillus rhamnosus is Lactobacillus rhamnosus 1.0320, with the accession number of CGMCC No. 15557.

[0012] Further, the composition can be a liquid or a solid, and preferably is a powdered solid in terms of production, transportation, storage and use convenience.

[0013] The second aspect of the present application is to provide a formula food comprising the above-mentioned composition.

[0014] Further, the formula food, the added amount of the composition is 0.1-10 g / 100 g.

[0015] The formula food includes pasta, beverage, instant food, baked cakes, sauce or functional nutritional supplement food.

[0016] More preferably, the formula food is an infant formula food or an infant complementary food, and the added amount of the composition is: 3-fucosyllactose content 0.3-1.75 g / 100 g, 3'-sialyllactose content 0.1-0.28 g / 100 g, and Lactobacillus rhamnosus content 10 6 -10 14 CFU / 100 g.

[0017] The third aspect of the present application is to provide the use of the above-mentioned composition in the preparation of a product for relieving intestinal inflammation; the product is a food, a feed additive or a health product; preferably, the intestinal inflammation is necrotizing enterocolitis of newborns.

[0018] Further, the improvement of necrotizing enterocolitis of newborns includes the following: (1) relieving or recovering one or more of intestinal edema, pneumatosis cystoides intestinalis, intestinal villus shedding, intestinal barrier damage, and tissue necrosis; (2) inhibiting the release of inflammatory factors promoted by TLR4 / NF-κB pathway; (3) improving the degradation ability of lipid peroxides; (4) inhibiting the increase of Enterobacter abundance of NEC pathogenic bacteria; (5) restoring the short-chain fatty acid content in the intestinal tract.

[0019] Further, the lipid peroxide can be MDA.

[0020] The difference between neonatal necrotizing enterocolitis and ordinary enteritis is that the pathological target of neonatal necrotizing enterocolitis (NEC) is more, and NEC is caused by the imperfect development of the intestinal tract of a newborn, so that the pathological process presents a "disaster level" reaction, a large number of pathogens invade the intestinal wall to produce cytotoxins and produce gas, causing intestinal gas accumulation, abnormal structure of the intestinal wall due to oxidative ischemia, and finally leading to intestinal necrosis in a short period of time, and even causing more serious intestinal perforation and portal vein gas accumulation. Ordinary enteritis is only a local immune and inflammatory reaction caused by pathogens, and has a higher reversibility.

[0021] In addition, the pathogenic bacteria Enterobacter is the main pathogenic bacteria of NEC, and the composition has a significant effect on inhibiting the abundance of the pathogenic bacteria Enterobacter, which shows that it has a particularly unique effect on NEC.

[0022] The beneficial effects of the present application are as follows: The present application finds that the combination of human milk oligosaccharides and Lactobacillus rhamnosus can synergistically play a role in preventing, improving and relieving neonatal necrotizing enterocolitis (NEC) through a multi-target and integrated mechanism of inhibiting the TLR4 / NF-κB inflammatory pathway, improving cell oxidative stress, protecting cell barriers, regulating intestinal flora and reducing cell apoptosis. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a diagram of the effect of 3-FL, 3'-SL alone or in combination with Lactobacillus rhamnosus 1.0320 on cell viability induced by LPS.

[0024] Figure 2 is a diagram of the effect of 3-FL, 3'-SL alone or in combination with 1.0320 on the release amount of cell LDH induced by LPS.

[0025] Figure 3 is a diagram of the effect of 3-FL, 3'-SL alone or in combination with 1.0320 on cell inflammation factors induced by LPS.

[0026] Figure 4 is a diagram of the effect of 3-FL, 3'-SL alone or in combination with 1.0320 on cell oxidative stress induced by LPS.

[0027] Figure 5 is a diagram of the effect of 3-FL + 3'-SL and 1.0320 alone or in combination on cell apoptosis induced by LPS.

[0028] Figure 6 Figure is the impact of 3-FL + 3'-SL and 1.0320 alone or in combination on the TLR4 / NF-κB inflammatory pathway in LPS-induced cell injury.

[0029] Figure 7 Figure is the impact of 3-FL + 3'-SL and 1.0320 alone or in combination on the Nrf2 / NQO1 pathway in LPS-induced cell injury.

[0030] Figure 8 Figure is the impact of 3-FL + 3'-SL and 1.0320 alone or in combination on the apoptosis pathway in LPS-induced cell injury.

[0031] Figure 9 Figure is the impact of 3-FL + 3'-SL and 1.0320 alone or in combination on tight junction proteins and MUC2 in LPS-induced cell injury.

[0032] Figure 10 Figure is the impact of 3-FL and 3'-SL combined with Lactobacillus rhamnosus 1.0320 on necrotic enterocolitis in suckling rats.

[0033] Figure 11 Figure is the impact of 1.0320 group, FS group and 1.0320 FS group on oxidative stress indicators in the intestinal tissue of rats with necrotic enterocolitis.

[0034] Figure 12 Figure is the impact of 1.0320 group, FS group and 1.0320 FS group on TLR4 / NF-κB pathway protein expression in the intestine of rats with necrotic enterocolitis.

[0035] Figure 13 Figure is the impact of 1.0320 group, FS group and 1.0320 FS group on Nrf2 pathway-related protein expression in the intestine of rats with necrotic enterocolitis.

[0036] Figure 14 Figure is the impact of 1.0320 group, FS group and 1.0320 FS group on the intestinal barrier of rats with necrotic enterocolitis.

[0037] Figure 15 Figure is the impact of 1.0320 group, FS group and 1.0320 FS group on Beta diversity.

[0038] Figure 16 Figure is the impact of 1.0320 group, FS group and 1.0320 FS group on the composition of bacterial genera at the level.

[0039] Figure 17 Figure is the impact of 1.0320 group, FS group and 1.0320 FS group on short-chain fatty acids. DETAILED DESCRIPTION

[0040] In order to make the objectives, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0041] The present application finds that the combined effect of human milk oligosaccharides and probiotics can improve intestinal inflammation, especially for neonatal necrotizing enterocolitis.

[0042] Among them, neutral oligosaccharides can be divided into neutral fucosyl lactose (which contains fucose at the terminal position) and neutral non-fucosylated HMOs (which contains N-acetylglucosamine at the terminal position) based on their core structure.

[0043] Among them, fucosyl lactose is an oligosaccharide with a fucose residue. The following illustrates the types of fucosyl lactose: 2'-fucosyllactose (2'-FL), which is a neutral trisaccharide composed of L-fucose, D-galactose and D-glucose units, wherein the monosaccharide L-fucose is connected to the disaccharide D-lactose by an α(1→2) bond. 3-fucosyllactose (3-FL), which is a neutral trisaccharide composed of L-fucose, D-galactose and D-glucose units, wherein the monosaccharide L-fucose is connected to D-glucose by an α(1→3) bond. Lactose-N-fucopentaose I (LNFP I), which is a neutral pentasaccharide composed of L-fucose, D-glucose, 2 molecules of D-galactose, N-acetylglucosamine units, wherein the monosaccharide L-fucose is connected to D-glucose by an α(1→3) bond. Lactose-N-difucosylhexose I (LNDFH I), which is a neutral hexose composed of 2 molecules of L-fucose, D-glucose, D-galactose, N-acetylglucosamine units, wherein the monosaccharide L-fucose is connected to D-galactose and N-acetylglucosamine by an α(1→2) bond and an α(1→4) bond, respectively. Lactose-N-difucosylhexose II (LNDFH II), which is a neutral hexose composed of 2 molecules of L-fucose, D-glucose, D-galactose, N-acetylglucosamine units, wherein the monosaccharide L-fucose is connected to D-glucose and N-acetylglucosamine by an α(1→3) bond and an α(1→4) bond, respectively. The human milk oligosaccharides HMOs described in the present application at least include the above-mentioned neutral fucosyl lactose, and in some preferred embodiments, the fucosyl lactose is preferably 3-fucosyllactose (3-FL).

[0044] The sialylated oligosaccharide according to the present application includes any one or more of 3'-sialylated lactose, 6'-sialylated lactose, sialylated-lactose-N-tetraose a, sialylated-lactose-N-tetraose b, sialylated-lactose-N-tetraose c, and disialyl-lactose-N-tetraose. In some preferred embodiments, the sialylated oligosaccharide according to the present application is 3'-sialylated lactose.

[0045] The source of 3-FL and 3'-SL according to the present application is not particularly limited, and typically, it can be obtained by means of ordinary chemical synthesis method, microbial fermentation method, etc. in the art. In addition, 3-FL and 3'-SL can also be derived from animal milk such as cow milk, etc.

[0046] The probiotic according to the present application is Lactobacillus rhamnosus, and the source of Lactobacillus probiotic according to the present application is not particularly limited, and typically, it can be obtained by microbial fermentation or purchased as a commercial product. In some specific embodiments, the Lactobacillus rhamnosus according to the present application is Lactobacillus rhamnosus 1.0320 disclosed in patent CN201810944744.7, with the accession number of CGMCC No.15557, which is isolated from a traditional kumiss sample, has high production of bacteriocin, high tolerance to cholate, high intestinal adhesion capacity, and the bacteriocin produced has significant bacteriostatic capacity. Although the probiotics including Lactobacillus rhamnosus in the prior art can alleviate intestinal inflammation by restoring intestinal barrier and reducing inflammatory response, etc., the colonization, growth and functional expression of probiotics in practical application are limited by nutritional competition of pathogenic bacteria and complex intestinal environment, and it is difficult to exert the probiotic efficacy.

[0047] In one specific embodiment of the present application, especially the composition of 3-FL, 3'-SL and Lactobacillus rhamnosus, it is found that the combined action of the three can alleviate LPS-induced IEC-6 cell inflammation by inhibiting TLR4 / NF-κB inflammatory pathway, improving cell oxidative stress, maintaining cell barrier and promoting NO production.

[0048] The formula food according to the present application is not particularly limited, and typically, it can be noodles, beverages, instant foods, baked cakes, sauces or functional nutritional supplement foods. For the instant foods, typically, it can be instant milk powder products such as infant milk powder, adult milk powder, middle-aged and elderly milk powder, etc. Especially, the composition of the present application is very suitable for the preparation of infant milk powder. In addition, for the functional nutritional supplement foods, there is no particular limitation, and it can be used as a nutritional supplement or a meal replacement. In such foods, in addition to the two functional components according to the present application, one or more of the following components can also be included: protein, fat, essential carbohydrate, dietary fiber, supplemental element, vitamin, plant or dietary ingredient, etc.

[0049] Unless otherwise specified in the examples, all techniques or conditions are conventional or described in the literature in the field or according to the manufacturer's instructions. Unless otherwise specified, reagents and instruments used are conventional products that can be purchased through regular channels.

[0050] Example 1 Analysis method: The raw data was analyzed by SPSS 27.0.1, and Tukey method was used for multiple comparisons. There was a significant difference when P<0.05. The experimental data was expressed as mean ± standard deviation, and each group was repeated at least 3 times.

[0051] 1. Determination of the administration concentration of 3-FL, 3'-SL and Lactobacillus rhamnosus 1.0320 Through the sample toxicity pre-experiment, it was shown that when the pre-treatment concentrations of 3-FL and 3'-SL were 1 mg / mL and 0.01 mg / mL, respectively, the cell viability was the highest. When the concentration of 1.0320 was 10 7 CFU / mL, the cell survival rate increased significantly to 82.48±3.44% (P<0.05), so the concentration of 10 7 CFU / mL of Lactobacillus rhamnosus was selected for cell pretreatment.

[0052] 2. Effect of 3-FL, 3'-SL and Lactobacillus rhamnosus 1.0320 alone or in combination on cell viability IEC-6 cells were seeded in 96-well plates at 6000 cells / well and incubated for 24 h until the cells were completely adherent. They were divided into Control group (no LPS induction treatment), LPS group (only LPS induction treatment, without the addition of 3-FL, 3'-SL and Lactobacillus rhamnosus 1.0320), 1.0320 group (Lactobacillus rhamnosus 1.0320 added at 10 7 cfu / mL), 3-FL group (3-FL added at 1 mg / mL), 3'-SL group (3'-SL added at 0.01 mg / mL), 3-FL+3'-SL group (3-FL added at 0.5 mg / mL and 3'-SL added at 0.005 mg / mL), 1.0320+3-FL group (3-FL added at 0.5 mg / mL and Lactobacillus rhamnosus 1.0320 added at 0.5×10 7 cfu / mL), 1.0320+3'-SL group (3'-SL added at 0.005 mg / mL and Lactobacillus rhamnosus 1.0320 added at 0.5×10 7cfu / mL), 1.0320+3-FL+3'-SL group (3-FL added amount was 0.33 mg / mL, 3'-SL added amount was 0.003 mg / mL, Lactobacillus rhamnosus 1.0320 added amount was 0.33 x 10 7 cfu / mL), 1.0320+3-FL+3'-SL group (3-FL added amount was 0.33 mg / mL, 3'-SL added amount was 0.003 mg / mL, Lactobacillus rhamnosus 1.0320 added amount was 0.33 x 10

[0053] Results are shown in the following table: Figure 1 The results showed that 1.0320, 3-FL and 3'-SL alone or in combination with pretreatment had a mitigating effect on LPS-induced decrease in cell survival rate, which increased to 78.69±4.57%, 79.79±2.68%, 77.09±2.29%, 81.76±2.13%, 84.62±2.78%, 81.89±2.76%, 88.04±2.25%, respectively, which were significantly different from the LPS treatment group (65.52±2.23%) (P<0.05). In addition, the data showed that the 3-FL+3'-SL+1.0320 group had the best effect on restoring cell survival rate, followed by the 3-FL+3'-SL group. The cell survival rate of the 3-FL+3'-SL group was higher than that of the two single HMOs, and the effect of the two HMOs combined with 1.0320 was better than that of single HMO or 1.0320.

[0054] 3. Measurement of lactate dehydrogenase (LDH) release IEC-6 cells were seeded in 96-well plates at 6000 cells / well and incubated for 24 h until the cells were completely adherent. The cells were treated according to the same grouping method in step 2 for 24 h, and the original culture medium was discarded. After centrifugation, the supernatant was collected after treatment with LPS-containing medium (10 μg / mL) for 24 h. According to the instructions of the LDH kit (70920, BIOBASE), the release of LDH in the supernatant was measured.

[0055] Results are shown in the following table: Figure 2As shown, LPS-induced IEC-6 cell release significantly increased after 24 h (P < 0.05). However, LDH release significantly decreased when treated with 3-FL, 3'-SL, and 1.0320 alone or in combination (P < 0.05). These results indicate that the combined or single-treatment effects of 3-FL, 3'-SL, and 1.0320 can reduce LDH leakage by inhibiting LPS-induced cell membrane damage in IEC-6 cells. The 3-FL + 3'-SL + 1.0320 group showed the best effect, followed by the 3-FL + 3'-SL group. Furthermore, the combined treatment groups were all more effective than their respective single-treatment groups (P < 0.05), indicating that the combined treatment groups were superior to the single-treatment groups in terms of LDH release.

[0056] 4. Measurement of IEC-6 cellular inflammatory factors IEC-6 cells were seeded at 6000 cells / well in 96-well plates and incubated for 24 h until complete cell adhesion. Cells were pretreated for 24 h using the same grouping method as in step 2, then cultured in LPS-containing medium for 24 h before centrifugation, and the supernatant was collected. The levels of tumor necrosis factor-α, interleukin-1β, IL-10, and IL-6 in the supernatant were measured using an enzyme-linked immunosorbent assay (ELISA) kit, with all measurements performed according to the kit instructions.

[0057] The production of LPS-induced inflammatory factors in IEC-6 cells was detected using an ELISA kit to investigate the effects of 3-FL, 3'-SL, and 1.0320 on intestinal inflammation and immune function. Figure 3 As shown, after LPS treatment, the levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6 in cells significantly increased (P < 0.05), reaching 569.92 ± 12.03 ng / L, 65.52 ± 1.53 ng / L, and 271.11 ± 5.66 pg / mL, respectively, while the level of anti-inflammatory factor IL-10 was significantly decreased (P < 0.05), reaching 59.33 ± 2.94 ng / L. This indicates that 10 μg / mL LPS treatment leads to an increase in the levels of pro-inflammatory factors and a decrease in the levels of anti-inflammatory factors in IEC-6. Pretreatment with 3-FL, 3'-SL, and 1.0320, alone or in combination, significantly inhibited the increase in TNF-α, IL-1β, and IL-6 levels and the decrease in IL-10 levels in cells (P < 0.05). The best effect was observed in the 3-FL + 3'-SL + 1.0320 group, which reduced TNF-α, IL-1β, and IL-6 by 59.56%, 59.04%, and 60.34% respectively compared to the LPS group, and increased IL-10 by 225.77% compared to the M group. This indicates that the effects of 3-FL, 3'-SL, and 1.0320, alone or in combination, can alleviate LPS-induced cellular inflammation from the perspectives of inflammatory factors and immunity.

[0058] 5. IEC-6 cell antioxidant level analysis IEC-6 cells were seeded in 96-well plates at 6000 cells per well and incubated for 24 h to allow the cells to adhere completely. The cells were pretreated for 24 h according to the same grouping method as in step 2, and then treated with LPS-containing medium for 24 h. After washing twice with PBS, the cells were trypsinized and centrifuged, and then collected. The cells were added to cell lysis buffer and lysed completely, and then centrifuged (2000 x g, 15 min) to obtain the supernatant for subsequent detection. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), peroxidase (CAT), and trace reduced glutathione (GSH) were determined using a Nanjing Jiancheng kit. All determinations were performed according to the kit instructions.

[0059] As shown in Figure 4 , LPS induction had a significant impact on the antioxidant enzyme system defense system (P < 0.05), and the GSH, CAT, and SOD contents of the LPS group were reduced to one-fourth of the control group. However, 3-FL, 3'-SL, and 1.0320 alone or in combination significantly restored the activities of GSH, CAT, and SOD (P < 0.05), especially in the LFS group, which were restored to 68.63 ± 1.75 umol / L, 7.28 ± 0.19 U / mL, and 19.02 ± 0.71 U / mL, respectively, followed by the LF treatment group. At the same time, MDA, as the end product of lipid peroxidation, was significantly increased in the model M group (15.57 ± 0.25 nmol / mL), while 3-FL, 3'-SL, and 1.0320 alone or in combination significantly reduced the MDA content (P < 0.05). Similarly, the 3-FL + 3'-SL + 1.0320 group had the best effect in reducing MDA, followed by the 3-FL + 3'-SL treatment group, which were reduced to 5.56 ± 0.38 nmol / mL and 6.96 ± 0.58 nmol / mL, respectively. The results showed that 3-FL, 3'-SL, and 1.0320 alone or in combination could inhibit LPS-induced oxidative stress by reducing MDA and activating the intracellular antioxidant system, thereby alleviating cell damage caused by lipid peroxide accumulation in IEC-6 cells.

[0060] 6. Morphological observation of cell damage The 1.0320+3-FL+3'-SL group showed the best alleviating effect, so the following five groups (based on the grouping in step 2) were selected for further mechanism research and result comparison: the Control group, the LPS group, the 1.0320 group, the 3-FL+3'-SL group, and the 1.0320+3-FL+3'-SL group.

[0061] The IEC-6 cells were incubated for 24 h after plating, and then treated with the above grouping for 24 h, and the original culture medium was discarded. The cells were treated with LPS-containing medium for 24 h. The cells in each group were observed by inverted fluorescence microscope, and the entire situation was observed at 100x magnification during the photographing process, and the image clarity was 1920x1200.

[0062] The cells were observed by inverted fluorescence microscope. The cells in the Control group showed polygonal or angular shape and were closely arranged. The cells in the LPS group became rounder or showed aggregation phenomenon, and the density decreased. The cell density treated with 1.0320 and 3-FL+3'-SL alone or in combination was close to the normal level, the round cells decreased, and most of them were normal cells, indicating that 3-FL+3'-SL and 1.0320 could alleviate the cell morphological changes induced by LPS.

[0063] 7. Flow cytometry analysis Flow cytometry was determined by Annexin V-FITC / PI double staining method. Figure 5 The results showed that the apoptosis rate increased from 5.79±0.39% to 33.35±2.63% after LPS induction, and it could be seen that LPS could significantly increase cell apoptosis (P<0.05). After treatment with 1.0320, 3-FL+3'-SL group, the cell apoptosis was reduced, and the 1.0320 combined with 3-FL+3'-SL group showed the best effect. Therefore, 1.0320 and 3-FL+3'-SL alone or in combination could reduce LPS-induced cell apoptosis.

[0064] 8. Effect on TLR4 / NF-κB inflammatory pathway TLR4 / NF-κB inflammatory pathway is closely related to the occurrence of NEC. In the TLR4 signaling pathway, LPS can activate TLR4 receptor to activate the TLR4 / MyD88 dependent signaling pathway, so that the expression of TLR4 and MyD88 increases significantly. Then through a series of complex reactions, NF-κB inhibitory protein kinase is activated, so that NF-κB inhibitory protein is phosphorylated and degraded, and free NF-κB enters the nucleus, NF-Κb p65 is phosphorylated, the transcription of related inflammatory factor target genes is started, and the transcription and expression of pro-inflammatory factors are promoted at the gene level. To study the effects of 3-FL, 3'-SL and Lactobacillus rhamnosus 1.0320 on the TLR4 / NF-κB inflammatory pathway of IEC-6 cells, the expression levels of related proteins in the cells were determined by immunoblotting.

[0065] As shown in Figure 6 , the protein expression levels of TLR4 and MyD88 in IEC-6 cells were only 0.006±0.0004 and 0.106±0.016 under normal conditions. After LPS treatment, the protein expression levels increased significantly (P<0.05). After pretreatment with 3-FL, 3'-SL and 1.0320 alone or in combination, the protein expression levels of TLR4 and MyD88 decreased significantly, and the effect of the 3-FL+3'-SL+1.0320 group was the best, with a decrease to 0.069±0.022 and 0.194±0.016. After immunoblotting detection of NF-κB p65 and P-NF-κB p65, it was found that the protein expression level of phosphorylated NF-κB p65 (P-NF-κB p65) increased significantly after LPS induction (P<0.05), and decreased significantly after pretreatment with 3-FL, 3'-SL and 1.0320 alone or in combination. The results showed that LPS can activate TLR4 and increase the expression of TLR4 and MyD88, so that NF-κB enters the nucleus, thereby activating the TLR4 / NF-κB inflammatory pathway. Pretreatment with 3-FL, 3'-SL and 1.0320 alone or in combination can effectively reduce the expression of related inflammatory pathway proteins, indicating that 3-FL, 3'-SL and 1.0320 can inhibit LPS-induced cell damage from the TLR4 / NF-κB inflammatory pathway.

[0066] 9. Effect on Nrf2 / NQO1 pathway The expression levels of key proteins such as Nrf2, NQO1 and HO-1 were analyzed by immunoblotting. As shown in Figure 7Compared with the Control group, the Nrf2 / NQOl pathway was inhibited after LPS induced IEC-6 cells for 24 h, and the expression levels of Nrf2, NQOl and HO-1 were significantly reduced (P<0.05), which were 0.07±0.02, 0.15±0.13, 0.09±0.18, respectively, only 11.82%, 19.65%, 10.63% of the Control group. However, the protein expression levels of Nrf2, NQOl and HO-1 in the Nrf2 / NQOl pathway were significantly restored after 3-FL, 3'-SL and 1.0320 were treated alone or in combination. The above results show that 3-FL, 3'-SL and 1.0320 alone or in combination can restore the protein expression levels of Nrf2, NQOl and HO-1, which may be one of the potential mechanisms of 3-FL, 3'-SL and 1.0320 to eliminate ROS and improve the oxidative stress state of cells, and also provides an important pathway for 3-FL, 3'-SL and 1.0320 to improve NEC.

[0067] 10、Effects on the apoptosis pathway of IEC-6 cells The expression levels of three key proteins involved in cell apoptosis, Caspase3, Bax and Bcl-2, were determined, and the results are shown in Figure 8 Compared with the apoptosis pathway-related proteins in the Control group, the expression levels of Caspase-3 and Bax in the LPS group were significantly increased (P<0.05), and the expression level of Bcl-2 was significantly decreased (P<0.05), which indicated that LPS induced severe mitochondrial-mediated apoptosis in IEC-6. Compared with the LPS group, the expression levels of Caspase-3 and Bax in cells were significantly reduced, and the expression level of Bcl-2 was significantly increased (P<0.05) after 3-FL, 3'-SL and 1.0320 were treated alone or in combination. In addition, the protein expression levels of Caspase3, Bax and Bcl-2 in the 3-FL+3'-SL+1.0320 group were similar to those in the Control group, which were 0.33±0.024, 0.18±0.023, 0.58±0.024, respectively, which indicated that 3-FL, 3'-SL and 1.0320 alone or in combination had an effective inhibitory effect on mitochondrial-mediated apoptosis. These results show that regulating the expression of key proteins in the mitochondrial apoptosis pathway to reduce intestinal tissue cell apoptosis may also be one of the potential mechanisms of 3-FL, 3'-SL and 1.0320 to alleviate NEC.

[0068] 11、Analysis of the protein expression of tight junction and MUC2 in the barrier of IEC-6 cells In neonates and preterm infants, more and more studies have shown the importance of its intestinal epithelial barrier function, the intestinal barrier is composed of important physical barrier formed by the tight arrangement of intestinal epithelial cells, mucus layer and intestinal immune system. Among them, tight junction proteins are considered to be important structures for maintaining the tight connection between cells and ensuring the integrity of the intestinal barrier. The representative tight junction proteins are mainly ZO-1, Occludin and Claudin-1. And mucin (MUC2) is also an important component of maintaining the integrity of the intestinal barrier. In the study of et al, it was observed that MUC2 expression was reduced in the NEC mouse model, and HMO treatment could promote MUC2 expression. Therefore, in this study, the protein expression levels of ZO-1, Occludin, Claudin-1 and MUC2 were analyzed by immunoblotting to explore the mechanism of 3-FL, 3'-SL and 1.0320 on the intestinal epithelial barrier.

[0069] As shown in Figure 9 , after LPS treatment, the cell tight junction proteins (ZO-1, Occludin and Claudin-1) were significantly reduced compared with the control group (P<0.05), and LPS could lead to a decrease in tight junction proteins in IEC-6 cells, thereby destroying the cell barrier. Then the protein expression of tight junction proteins in IEC-6 cells pretreated with 3-FL, 3'-SL and 1.0320 was determined. The results showed that the expression levels of ZO-1, Occludin and Claudin-1 were significantly increased compared with the LPS group (P<0.05), and the expression levels of 3-FL+3'-SL+1.0320 group were close to normal levels, and the expression levels of tight junction proteins were restored to 0.58±0.015, 0.51±0.021 and 0.74±0.015.

[0070] The expression level of MUC2 protein, another defense line of intestinal epithelial barrier, was also determined. The results showed that the expression of MUC2 protein was significantly reduced after LPS treatment (P<0.05), and the decrease of MUC2 was alleviated after pretreatment with 3-FL, 3'-SL and 1.0320. These results suggest that LPS can destroy the intestinal barrier by reducing the expression of tight junction proteins and MUC2 in IEC-6 cells, which further contributes to the occurrence of NEC. Compared with the LPS group, the pretreatment with 3-FL, 3'-SL and 1.0320 significantly restored the expression of tight junction proteins and MUC2, indicating that 3-FL, 3'-SL and 1.0320 can inhibit LPS-induced cell damage through the intestinal barrier, providing a mechanism for 3-FL, 3'-SL and 1.0320 to alleviate NEC.

[0071] This study provides an important theoretical basis for the use of HMOs in combination with probiotics to alleviate necrotizing enterocolitis in animal models, and demonstrates that the synergistic effect of prebiotics and probiotics is better than that of single components, providing a new method for preventing necrotizing enterocolitis in newborns.

[0072] Example 2 The present application further verifies the effect of breast milk oligosaccharides 3-FL and 3'-SL and their combination with Lactobacillus rhamnosus 1.0320 on the alleviation of intestinal inflammation in mice, as follows: (I) Experimental procedure 1. Animal experiments Con group: no additional treatment, normal feeding treatment, fed with high osmotic formula milk (15 g PreNAN milk powder dissolved in 75 mL Esbilac dog milk replacer), 4 times / day, increasing by 0.1 mL every 24 h.

[0073] NEC group: on the basis of the Con group, NEC procedure induction: placed in a low oxygen environment (95% N2+5% O2) for 10 minutes, then placed in a 4 ℃ environment for 10 minutes, 3 times / day, for 3 consecutive days.

[0074] FS group: on the basis of the NEC group, additional feeding of 3-FL and 3'-SL every day, with an addition of 1500 mg / kg and 300 mg / kg of the two in the feed.

[0075] 1.0320 group: on the basis of the NEC group, additional feeding of 2x10 8 CFU of Lactobacillus rhamnosus 1.0320 every day.

[0076] 1.0320FS group: on the basis of the NEC group, additional feeding of 3-FL, 3'-SL and Lactobacillus rhamnosus 1.0320 every day, with an addition of 750 mg / kg and 150 mg / kg of 3-FL and 3'-SL in the feed, and a feeding amount of 10 8 CFU of Lactobacillus rhamnosus 1.0320.

[0077] 2. NEC severity assessment Before sacrificing the rats, observe the rat condition from aspects such as appearance, tactile response, natural activity and body color. According to the scoring standard of the clinical disease index (Table 1), evaluate the overall activity of the rats. After euthanizing the rats, observe the intestinal appearance, and score the macroscopic condition of the intestine from aspects such as intestinal continuity, intestinal color and intestinal distension according to the scoring standard (Table 2).

[0078] Table 1 Scoring of clinical disease index

[0079] Table 2 Macroscopic intestinal condition score

[0080] 3. Histopathological analysis Spleen and ileum terminal segments in paraformaldehyde fixative were dehydrated and embedded. Embedded tissue samples were cut into 4 pm and deparaffinated, then mounted after hematoxylin and eosin (H&E) staining. Sections were observed using an inverted fluorescence microscope and images were scored. NEC assessment was performed on ileum terminal tissue sections according to the following criteria: 0, intact structure without damage; 1, upper half of villi destroyed and submucosa slightly separated; 2, mild villus separation, submucosa slightly edematous; 3, villi sloughed, submucosa edematous; 4, most of the villi sloughed or entire structure completely lost. Mice with a score of 2 and above were considered to have developed NEC disease.

[0081] 4. Inflammatory cytokine determination Tissue homogenates were prepared by mixing 0.1 g of rat ileum tissue samples with physiological saline, and after centrifugation at 4°C (3000 g, 10 min), the supernatant was collected and stored at -80°C for testing. According to the instructions, the enzyme-linked immunosorbent assay (ELISA) kit was used to determine the content of inflammatory cytokines (TNF-a), (IL-1β), (IL-6), (IL-10) and immunoglobulins (IgA), (IgG) in the tissue supernatant.

[0082] The contents of malondialdehyde (MDA), (GSH), (SOD), (CAT) in the supernatant were determined using commercially available detection kits, and the operation steps were carried out according to the instructions.

[0083] 5. Immunofluorescence staining After the ileum terminal wax block was cut into 4 pm sections and deparaffinated and rehydrated, 5% BSA was used for blocking (room temperature, 20 min). After PBS washing to remove BSA, the sections were incubated with ZO-1, Occludin, Claudin-1 antibodies in a wet box at 4°C overnight, and then washed with PBS for 3 times. Then the secondary antibody was added to cover the tissue sections and incubated at room temperature for 50 min. Then PBS was used to wash 3 times in the dark. Finally, 4,6-diamino-2-phenylindole (DAPI) was added to stain the nuclei at room temperature for 10 min, and then PBS was used for washing. The images were observed using a microscope and quantitative analysis was performed.

[0084] 6. Real-time quantitative polymerase chain reaction (RT-qPCR) Total RNA was extracted from small intestinal tissue samples using the Trizol kit, and cDNA was obtained according to the operating instructions of the reverse transcription kit. Finally, the expression level of the target gene was determined using the fluorescent quantitative PCR kit. The data were normalized by GAPDH as an internal reference. -ΔΔCt Method calculation analysis, and normalization by GAPDH as an internal reference.

[0085] 7. Western blot The small intestinal tissue sample was homogenized and centrifuged (10000 g, 5 min) after adding a lysis solution containing protease and phosphatase inhibitors. The protein content was determined using the BCA kit. The protein sample was separated by 10% polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene fluoride (PVDF) membrane, and blocked with 5% BSA at room temperature for 1 h. Then the PVDF membrane was incubated with antibodies (TLP4, MyD88, NF-kB p65, p-NF-kB p65) at 4°C overnight, and then incubated with secondary antibodies at room temperature for 1 h, washed 3 times, developed with an ECL chemiluminescence detection kit and exposed. Finally, the gray value of the target band was analyzed using software.

[0086] 8. Intestinal flora analysis based on 16S rRNA sequencing The 16S rRNA sequencing process was carried out according to the established method: genomic DNA was extracted from fresh fecal samples using the OMEGA Soil DNA kit, the DNA concentration was quantified using a Nanodrop spectrophotometer, and the DNA integrity was detected by 0.8% agarose gel electrophoresis. Subsequently, the V3-V4 hypervariable region of the 16S rRNA gene was amplified using primers 338F (5'-ACTCCTACGGAGGCAGCA-3') and 806R (5'-GGACTACCAGGGTATCTAAT-3') using a PCR amplifier (ABI2720), the PCR product concentration was determined using a Quant-iTPicoGreen dsDNA detection kit in a microplate reader, a TruSeq Nano DNA LT library preparation kit was selected to construct a sequencing library, and finally sequencing was completed through the Illumina NovaSeq platform.

[0087] 9. Quantification of short-chain fatty acids The small intestinal tissue was homogenized in PBS, the supernatant was mixed with phosphoric acid, an internal standard and diethyl ether. After centrifugation for 2 minutes, the supernatant was measured after passing through a 0.22 μm organic membrane. Detection was performed using a Thermo Trace 1310 gas phase system.

[0088] 10. Statistical analysis.

[0089] The experimental data were statistically analyzed by one-way ANOVA, supplemented by Tukey's multiple comparison test. The results were expressed as mean ± standard deviation (SD). P<0.05 was considered statistically significant.

[0090] (II) Experimental results (1) NEC severity assessment There was no difference in the initial body weight of the pups in each group, but the body weight of the Con group gradually increased over time, while the body weight of the NEC group and other treatment groups remained unchanged or decreased slightly. During the modeling process, there were deaths in each group, and after autopsy of the dead pups, no formula milk was found in the chest cavity, indicating that it was not artificial death caused by gavage.

[0091] As Figure 10 , the results of clinical disease score showed that the pups in the NEC group were sluggish, had loose skin, and moved slowly and the whole body was pale. The scores of the 1.0320 group, the FS group, and the 1.0320FS group were significantly lower than that of the NEC group, and the score of the 1.0320FS group was the lowest, which decreased by 55.37% compared with the NEC group ( Figure 10 upper left).

[0092] The macroscopic condition of the intestinal tract of the pups was scored, and it was found that the intestinal tract of the Con group was pink and healthy. The pups in the NEC group had extensive edema and bubbles ( Figure 10 upper right red arrow, from top to bottom, con, nec, 1.0320, FS, 1.0320FS group), the intestinal tract was swollen and fragile (which is a hallmark of NEC), and the intestinal tract damage of the other treatment groups was lighter. Among them, the intestinal tract of the 1.0320 group and the FS group was still swollen and had bubbles, while the intestinal tract of the 1.0320FS group had no obvious damage.

[0093] H&E staining showed that the intestinal tract structure of the Con group was normal, while the intestinal villi of the NEC group were destroyed and shed, and necrosis occurred. The intestinal tract structure of the other treatment groups was improved accordingly, and the villi were not found to be obviously shed and arranged in order. The histological score of the intestinal tract showed that the score of the NEC group was 2.66±0.15, and the modeling was successful. The scores of the 1.0320 group, the FS group, and the 1.0320FS group were 1.98±0.13, 1.34±0.15, and 0.89±0.16, respectively, which decreased by 25.56%, 49.62%, and 66.54% compared with the NEC group, respectively, indicating that the intestinal tract damage induced by NEC was relieved in all treatment groups, and the combined group had better effect than the single treatment group.

[0094] (2) Inflammatory factor analysis The contents of TNF-a, IL-1β and IL-6 in the intestinal tissue of the NEC group were significantly higher than those of the Con group (P < 0.05), indicating that the intestinal tract of the neonatal rats was seriously inflamed after the NEC induction. Compared with the NEC group, the contents of the corresponding inflammatory factors in the intestinal tissue of the 1.0320 group, the FS group and the 1.0320FS group were significantly reduced (P < 0.05), and the effect of the 1.0320FS group was the most significant, with the contents of TNF-a, IL-1β and IL-6 being reduced by 50.39%, 41.37% and 59.45% compared with the NEC group. In addition, the content of the anti-inflammatory cytokine IL-10 in the NEC group was significantly lower than that in the Con group, and the 1.0320 group, the FS group and the 1.0320FS group reversed the negative effects induced by the NEC program. The experiment showed that the effect of the combined treatment group was better than that of the single treatment group, indicating that the two groups synergistically enhanced the ability to relieve NEC.

[0095] (3) Analysis of oxidative stress parameters The results are shown in Table 2: Figure 11 The NEC induction had a significant effect on the antioxidant enzyme system defense system (P < 0.05), and the contents of GSH, CAT and SOD in the NEC group were significantly lower than those in the Con group, indicating that the NEC program treatment caused serious oxidative stress in the intestinal tract of the neonatal rats. The FS and 1.0320 alone or in combination significantly restored the antioxidant enzyme activity in the intestinal tract (P < 0.05), and the effect of the 1.0320FS group was the most significant. At the same time, the MDA level in the NEC group was significantly increased, and the MDA content was significantly reduced after the FS combined with 1.032 pretreatment (P < 0.05). The results showed that the FS combined with 1.032 treatment could relieve the intestinal injury caused by NEC by reducing MDA and restoring the antioxidant enzyme content in the intestinal tract, and the 1.0320FS group had the most significant pretreatment effect, followed by the FS group, and then the 1.0320 group.

[0096] (4) Effect on the TLR4 / NF-κB inflammatory pathway The results are shown in Table 2: Figure 12FS and 1.0320 on TLR4 / NF-κB pathway were further confirmed by Western blotting detection of corresponding proteins, and the expression levels of TLR4 and MyD88 were significantly higher than those in the Con group. After TLR4 activation, the downstream gene NF-κBp65 was phosphorylated and entered the nucleus to promote the release of inflammatory factors. In addition, the NF-κBp65 and p-NF-κBp65 in the NEC group were significantly increased, and the phosphorylation ratio of NF-κBp65 was the highest (P < 0.05), indicating that the activation of TLR4 / NF-κB inflammatory pathway after NEC program induction promoted the phosphorylation of NF-κBp65, thereby inducing inflammation in the intestinal tract. Compared with the NEC group, the phosphorylation ratios of TLR4, MyD88 and NF-κBp65 in each treatment group were significantly decreased (P < 0.05), indicating that 1.0320 and FS could alleviate intestinal inflammation by inhibiting the TLR4 pathway and reducing the phosphorylation of NF-κBp65. Among them, the effect of the 1.0320FS group was the best, followed by the FS group, and finally the 1.0320 group, proving that the combination of 1.0320 and FS could have a synergistic effect to enhance the inhibition of inflammation and have a better remission effect on NEC mice.

[0097] (5) Effect on Nrf2 / NQO1 pathway The results of Western blotting detection showed Figure 13 Compared with the Con group, the protein expression of Nrf2 and NQO1 in the NEC group was significantly reduced (P < 0.05). The 1.0320 group, the FS group and the 1.0320FS group significantly reduced the protein expression of Nrf2 and NQO1, and the NQO1 protein expression in the 1.0320FS group was significantly lower than that in the 1.0320 group and the FS group alone, indicating that the combination of the two promoted the remission effect on NEC.

[0098] (6) Effect on intestinal barrier integrity The results of immunofluorescence analysis of the intestinal barrier damage of the NEC program induced in the mouse and the intestinal barrier protection effect of the synergistic effect of 1.0320 and FS on the NEC mouse are as follows Figure 14The average fluorescence intensity analysis results showed that the TJ protein expression in the ileum tissue of the Con group was higher (ZO-1, Claudin-1 and Occludin), and the TJ protein expression in the ileum tissue of the NEC group was significantly reduced (P < 0.05), indicating that the intestinal barrier of the neonatal rats was severely damaged after the NEC procedure induction, and the bacterial toxins entered to exacerbate intestinal inflammation. After the treatment of 1.0320 and FS alone or in combination, the expression of TJ protein was significantly enhanced (P < 0.05), indicating that 1.0320 and FS can enhance the integrity of the intestinal barrier. Among them, the average fluorescence intensity of ZO-1 and Claudin1 in the 1.0320FS group had no significant difference with the Con group (P > 0.05), and was significantly better than the 1.0320 group and the FS group alone. The above results show that the intestinal barrier integrity of the NEC neonatal rats is damaged, and the recovery of the intestinal barrier after the treatment of 1.0320 and FS relieves the severity of NEC, and the synergistic effect of the two is better than that of the single treatment group.

[0099] (7) Intestinal microbial beta diversity analysis Figure 15 The genus level heat map shows the changes in the intestinal flora of the neonatal rats in each group. The intestinal flora of the NEC group of neonatal rats was significantly changed compared with the Con group, and the 1.0320 group and the 1.0320FS group had a higher degree of similarity in composition compared with the Con group. It is indicated that after the combined treatment of 1.0320 and FS, the intestinal flora gradually changes to the composition of the Con group, which relieves the damage induced by the NEC procedure.

[0100] (8) Changes in intestinal flora composition at genus level The composition analysis of the intestinal flora of the neonatal rats in each group at the genus level is as follows: Figure 16As shown, at the genus level, the main bacteria are *Proteus*, *Enterococcus*, *Lactobacillus*, and *Staphylococcus*. Compared with the Con group, the relative abundance of the harmful bacteria genus *Proteus* was significantly increased in the NEC group, while there was no significant difference between the 1.0320 group and the NEC group. However, the relative abundance of *Proteus* was significantly reduced after combined treatment with 1.0320 and FS, indicating that FS and 1.0320 have a synergistic effect in inhibiting the growth of harmful bacteria. *Enterococcus* is widely distributed in the human digestive tract. As shown in the figure, the relative abundance of *Enterococcus* was significantly reduced in the NEC group, indicating that NEC induction may lead to a decrease in *Enterococcus* abundance, while there was no significant difference in *Enterococcus* abundance between the treatment group and the NEC group. Notably, the relative abundance of Lactobacillus in the 1.0320FS group was significantly higher than in other groups. This may be due to the interaction between FS and 1.0320, which increases the abundance of Lactobacillus in the intestine and significantly reduces the relative abundance of Staphylococcus. Furthermore, Enterobacter (Enterobacter sakazakii) was found in the intestines of NEC group suckling mice. Enterobacter sakazakii is considered a major pathogenic bacterium for NEC. No large amounts of Enterobacter were found in the Con group and the treatment group, indicating that the treatment group inhibited the colonization of this bacterium and reduced the occurrence of NEC.

[0101] (9) Short-chain fatty acid analysis The results are as follows Figure 17 As shown, the levels of acetic acid, propionic acid, and butyric acid in the intestines of NEC suckling rats were significantly lower than those in the Con group. However, after combined treatment with 1.0320 and FS, the levels of short acids in the intestines of suckling rats were restored. There was no significant difference in butyric acid levels between the 1.0320 and FS groups and the NEC group. However, the butyric acid level in the 1.0320FS group was significantly higher than that in the NEC group, indicating that the two treatments had a synergistic effect in promoting the production of short acids in the intestines.

Claims

1. A composition for ameliorating necrotizing enterocolitis in a neonate, wherein, The composition comprises human milk oligosaccharides and Lactobacillus rhamnosus.

2. The composition of claim 1, wherein, The human milk oligosaccharides comprise at least one neutral oligosaccharide and at least one sialylated oligosaccharide.

3. The composition of claim 2, wherein, The neutral oligosaccharide is selected from fucosyllactose; preferably, the fucosyllactose comprises 3-fucosyllactose.

4. The saccharide composition according to claim 2, wherein, The sialylated oligosaccharide comprises 3'-sialyllactose.

5. The composition according to claim 3 or 4, wherein, The weight ratio of 3-fucosyllactose: 3'-sialyllactose is (0.5-50): 1; Preferably, the weight ratio of 3-fucosyllactose: 3'-sialyllactose is (1-20): 1; Further preferably, the weight ratio of 3-fucosyllactose: 3'-sialyllactose is (1-7):

1.

6. The composition according to claims 1-5, wherein, the amount of the lactobacillus rhamnosus is 10 6 -10 14 CFU; Preferably, the concentration of Lactobacillus rhamnosus is 10 6 -10 14 CFU / g; Further preferably, the Lactobacillus rhamnosus is Lactobacillus rhamnosus 1.0320, with the preservation number of CGMCC No. 15557.

7. A formula food comprising the composition of any one of claims 1-6.

8. The formula of claim 7, wherein, The formula is an infant or baby formula or an infant or baby complementary food, the composition is added in an amount of 3-fucosyl lactose content 0.3-1.75 g / 100 g, 3'-sialyllactose content 0.1-0.28 g / 100 g, Lactobacillus rhamnosus content 10 6 -10 14 CFU / 100 g, calculated as a solid.

9. Use of the composition of any one of claims 1-6 in the preparation of a product for improving necrotizing enterocolitis in a newborn. The product is a food, a feed additive or a health product.

10. Use according to claim 9, characterized in that, The improvement of necrotizing enterocolitis in a newborn comprises the following: (1) relieving or recovering one or more of intestinal edema, pneumatosis cystoides intestinalis, intestinal villus shedding, intestinal barrier damage, and tissue necrosis; (2) inhibiting the release of inflammatory factors promoted by TLR4 / NF-κB pathway; (3) improving the degradation ability of lipid peroxides; (4) inhibiting the increase of Enterobacter abundance, a pathogenic bacterium of NEC; (5) recovering the content of short-chain fatty acids in the intestine.

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