CN117158588A

CN117158588A - Infant preparation containing 3' SL and two lactobacillus and application thereof

Info

Publication number: CN117158588A
Application number: CN202311104758.5A
Authority: CN
Inventors: 李明; 袁杰力; 芮滨琪; 宁茜茜
Original assignee: Dalian Medical University
Current assignee: Dalian Medical University
Priority date: 2023-08-29
Filing date: 2023-08-29
Publication date: 2023-12-05

Abstract

The application belongs to the related technical field of infant nutrition, and particularly relates to an infant preparation containing 3' SL and two kinds of lactobacillus and application thereof. The application provides a preparation for improving infant immune imbalance, which comprises the following components: the preparation can effectively improve related diseases caused by infant immune development deficiency due to gestational diabetes and other diseases of infants.

Description

Infant preparation containing 3' SL and two lactobacillus and application thereof

Technical Field

The application belongs to the related technical field of infant nutrition, and particularly relates to an infant preparation containing 3' SL and two kinds of lactobacillus and application thereof.

Background

Gestational diabetes mellitus (Gestational diabetes mellitus, GDM) refers to temporary diabetes induced by pregnancy. The prevalence of GDM is increasing in our country and becomes a major public health problem. The early allergic diseases of the GDM infants are highly developed and can be closely related to the intestinal flora disturbance and the immune imbalance of the infants. According to studies, the content of 3 '-sialyllactose (3' SL), an oligosaccharide in breast milk, has a significant effect on regulating the intestinal flora of infants, which is an important factor in the immune development of infants. Compared with a healthy mother, the 3' SL content in the GDM mother breast milk is changed, so that the intestinal flora and the immune balance of the offspring are affected, but the research on oligosaccharides in the GDM breast milk in the prior art is relatively lacking, and particularly, the influence of the GDM breast milk oligosaccharide change on the colonisation of the intestinal flora and the formation of immune tolerance of the offspring is to be revealed.

Disclosure of Invention

In view of the above, the present application provides a preparation for improving immune imbalance in infants and young children, which comprises 3' -sialyllactose, lactobacillus johnsonii and lactobacillus reuteri, to improve immune development and other related disorders in infants and young children caused by gestational diabetes.

The application provides a preparation for improving infant immune imbalance and/or preventing and treating allergic diseases, which comprises the following components: 3' -sialyllactose, lactobacillus johnsonii and lactobacillus reuteri.

In the application, the preparation comprises the following components in parts by mass: 3' -sialyllactose 0.01-0.70 g/L, lactobacillus johnsonii 5×10 ⁷ ～1.5×10 ⁹ cfu/g and Lactobacillus reuteri (Limosilactobacillus reuteri) 1X 10 ⁸ ～1.1×10 ¹⁰ cfu/g. Further, in the present application, the lactobacillus johnsonii is preferably lactobacillus johnsonii AS1.3221, and the lactobacillus reuteri is preferably lactobacillus reuteri DSM 17938.

In the present application, the formulation is a liquid and/or a powder and/or a solid.

The application also provides application of the preparation in preparing a medicament for treating infant immune imbalance.

The application also provides application of the preparation in preparing a medicament for treating infant allergic symptoms.

The application also provides application of the preparation in preparing a medicament for promoting expression of Treg cells RORγt in infants suffering from immune imbalance.

The application also provides application of the preparation in preparing foods and/or supplements and/or complementary foods and/or drinks for improving the immune imbalance of infants.

The application also provides application of 3' -sialyllactose in preparing medicines and/or foods for promoting expression of Treg cells RORγt in infants suffering from immune imbalance.

Experiments show that the 3' SL content of oligosaccharide in gestational diabetes mellitus (Gestational diabetes mellitus, hereinafter referred to as GDM) murine milk is obviously reduced, so that the abundance of lactobacillus and bifidobacterium in intestinal tracts of offspring is reduced, and the immune balance is further influenced. The abundance of lactobacillus johnsonii and lactobacillus reuteri in the intestinal tract of the offspring model of food allergy (hereinafter referred to as FA) fed by the GDM female mice is lower, so that the expression of Treg cells RORγt in the Pi's knots is inhibited, and the establishment of immune tolerance is further influenced.

According to in vitro cell culture experiments, the combined use of lactobacillus reuteri, lactobacillus johnsonii and 3' -SL can effectively improve the proportion of Treg cells in the spleen of a newborn mouse, and simultaneously remarkably promote the expression level of RORgamma tmRNA in the intestinal tissue of the newborn mouse, which indicates that the combination can promote the development of RORgamma t+ cells of the newborn mouse; furthermore, the application discovers that the combination of the lysate of the lactobacillus reuteri, the supernatant of the lactobacillus johnsonii and the 3' -SL can significantly increase the number of Tregs, in particular RORγt+ Treg cells.

Drawings

FIG. 1 is a schematic diagram of a mouse FA model establishment;

FIG. 2 shows inflammatory factor levels in GDM mice after FA model establishment;

FIG. 3 is an analysis of intestinal flora alpha diversity (Shannon and Simpson index representation);

FIG. 4 shows the beta diversity of PCoA analysis of intestinal flora;

FIG. 5 is a graph showing the relative abundance of the intestinal primary flora level in mice;

FIG. 6 is the relative abundance of the intestinal primary flora level of mice;

FIG. 7 shows the dominant flora (p < 0.05) of LEfSe analyzed with significant differences between CON, GDM, CON+OVA and GDM+OVA groups; four sets of total LEfSe analysis comparisons;

FIG. 8 is a graph showing the dominant bacterial population (p < 0.05) of LEfSe analyzed with significant differences between CON, GDM, CON+OVA and GDM+OVA groups; comparing the CON group with the GDM group LEfSe analysis;

FIG. 9 shows the dominant flora (p < 0.05) of LEfSe analyzed with significant differences between CON, GDM, CON+OVA and GDM+OVA groups; comparison of CON+OVA group with GDM+OVA group LEfSe analysis;

FIG. 10 is a graph showing the ratio of mouse lymphocytes; red box is spleen Treg cell fraction map, blue box is peyer's patch rorγt ⁺ Treg cell fraction map;

FIG. 11 is a statistical chart of the proportion of lymphocytes in mice; wherein Spleen: spleen, PPs: a peyer's patch; * p <0.05, < p <0.01, < p <0.001;

FIG. 12 is a flow cytometer used to detect neonatal mouse spleen Treg cells for prognosis of a dry condition;

FIG. 13 is the effect of combined intervention of 3' -SL and L.spp on the expression level of RORγt mRNA in neonatal mouse intestinal tissue;

FIG. 14 is a flow cytometer analysis results of fecal metabolites co-cultured with T lymphocytes obtained from mouse Pp in vitro;

FIG. 15 shows the results of in vitro incubation of mouse lymphocytes with 3' -SL and supernatant/cell lysates of Lactobacillus reuteri and Lactobacillus johnsonii.

Detailed Description

TABLE 1 Experimental reagents

Table 2 laboratory apparatus

Statistical analysis method

All data herein are expressed as x+ -SEM (n.gtoreq.3). Statistical analysis and differential comparison of experimental data were performed with GraphPad Prism 8.1 software, a non-parametric t-test was performed between the two groups, and a one-way anova and Tukey test was used between the groups to evaluate if there was a statistical difference between the groups. Spearman correlation coefficient evaluates the relationship between breast milk oligosaccharides and intestinal flora. p-values <0.05 are considered statistically significant. * p <0.05, < p <0.01, < p <0.001, < p <0.0001.

1. Construction of a mother mouse GDM model

20 healthy 8-week-old female ICR mice were randomly divided into a healthy control group (CON group) and a gestational diabetes group (GDM group). The GDM mice were subjected to high-fat diet intervention for the first four weeks, and were mated with male mice of the same condition (age and strain) from the fifth week, and the female mice with the pregnant bolts were considered successful in conception by checking whether the pregnant bolts are present around the vaginal orifice of the female mice in the early morning. streptozotocin-STZ (30 mg/kg) was injected intraperitoneally for 3 consecutive days in the GDM group of pregnant mice, and the CON group of pregnant mice was injected with the same amount of physiological saline. The tail vein blood is taken after 3 days after the last injection to measure the blood sugar of the female mice, and the random blood sugar level is detected to be more than 16.1mmol/L for 3 continuous days, so that the molding is successful.

After delivery, on day 12 (middle and 28 (late lactation), respectively, mother-mouse faeces, breast milk, son-mouse faeces, pie-knot and the like were collected and relevant indexes were detected, and the effects of GDM mother-mouse breast milk oligosaccharide and intestinal flora on the establishment of sub-intestinal flora and development of the immune system at different stages were analyzed. CON mice were given a normal diet during lactation and GDM group mice continued to be given a high fat diet. Based on the experimental results, the GDM group offspring were subsequently modeled for Food Allergy (FA) and a series of experiments were performed.

2. Mouse FA model establishment

To better investigate whether GDM offspring were more allergic or the degree of allergy was more severe, GDM master mice and 4 week old mice of healthy control master mice were randomly divided into four groups and FA models were established for their OVA sensitization, i.e. CON group, con+ova group, GDM group and gdm+ova group. Sensitization of mice: 100 μg of OVA protein and 2mg of aluminum hydroxide adjuvant were weighed, dissolved in 100 μl of physiological saline, and sensitized to mice by subcutaneous injection 1 time every 2 days for 2 times. After an interval of 7 days, the excitation was performed: mice were perfused with 50mg of OVA 1 time every other day for 4 times. Eyeball blood was taken 1 hour after the last excitation. The modeling flow is schematically shown in FIG. 1 (in the figure, each mark is NS, physiological saline, i.p, intraperitoneal injection, s.c, subcutaneous injection, i.g, gastric lavage, prime, challlenge, challenge, and sacrifice, sacrifice).

The mice of two groups of CON and GDM mice are divided into two groups respectively, one group of mice is used as a blank control, and the other group of mice is used for FA model establishment, and four groups are the CON group, the CON+OVA group, the GDM group and the GDM+OVA group. Neither the CON group of mice nor the mice were modeled; the CON+OVA group master mice are not modeled, and the mice establish an FA model; the GDM group of mice establishes a GDM model, and the mice are not modeled; GDM+OVA group of mice established GDM model, mice established FA model. And (3) establishing a GDM model: four weeks of high fat diet intervention was performed on 8 week ICR female mice, mated with the same strain of male mice, and three consecutive days after pregnancy, STZ (30 mg/kg) was intraperitoneally injected. And (3) FA model establishment: on day 1 of week 4 of birth, mice were sensitized on day 4: OVA protein (100. Mu.g/dose) and aluminum hydroxide (2 mg/dose) were injected subcutaneously. Excitation was performed on day 3, day 5, day 7 and day 2 of week 6 of week 5: the mice were perfused with stomach OVA (50 mg/mouse). Mice were sacrificed 1 hour after the last challenge.

3. Statistical analysis of related data such as milk oligosaccharide content of female mice and offspring intestinal flora

(1) Detecting the oligosaccharide content of the female mouse milk: a500. Mu.L sample of the mother mouse's milk was taken, defatted, deproteinized, and oligosaccharide detected on the machine. The method comprises the following steps: the supernatant was removed by centrifugation at 8,000rpm for 15min at 4℃and 2mL of ethanol was added and the protein was removed by centrifugation at 8,000rpm for 10min at 4℃in a centrifuge.

(2) Extraction of genomic DNA from intestinal flora

Taking 150mg of mouse faeces sample according toThe Stool DNA kit operation shows that DNA extraction is carried out on the feces of the mice, the concentration (ng/mu L) and the purity (A260/A280) of the extracted DNA sample are detected, and the involutory sample is subpackaged and stored and put into a refrigerator at the temperature of minus 80 ℃ for standby.

(3) High throughput sequencing analysis of intestinal flora 16S rRNA gene

The mouse fecal DNA was sequenced using Illuminate Hiseq PE25016S rRNA gene sequencing analysis technique. And carrying out OTUs clustering, species classification and other relevant analysis by using the effective data.

(4) Spleen lymphocyte subpopulation proportion detection

Fresh spleen tissue of each group of mice is taken in PBS buffer solution, the spleen is lightly ground by a glass slide to prepare single cell suspension, the single cell suspension is filtered by a filter screen, centrifuged at 2500rpm of a centrifuge at 4 ℃ for 5min, the supernatant is discarded, and 3mL of erythrocyte lysate is added for standing for 5min. Cells were washed and resuspended in PBS buffer. 1X 10 cells were counted ⁶ The individual cells were resuspended in 100. Mu.L PBS buffer and blocked with 1. Mu.L anti-mouse CD16/CD32 mAb at room temperature for 1h. After adding 1. Mu.L of anti-mouse CD4 mAb, incubating for 30min on ice in the dark, washing cells with PBS buffer, adding 500. Mu.L of membrane rupture solution, incubating for 1h at room temperature in the dark, washing cells with PBS buffer, and re-suspending. mu.L of anti-mouse FoxP3 mAb was added and incubated on ice for 40min in the dark. The cells were washed with PBS buffer and resuspended, and after filtration with a filter screen, the spleen Treg cell fraction was detected by a machine flow cytometer and the data was analyzed using Flowjo software.

(5) Proportion detection of peyer's patch lymphocyte subpopulations

Grinding 8-10 fresh mouse intestinal Petri tissue in PBS buffer solution to obtain single cell suspension, filtering with filter screen, centrifuging to remove supernatant, and re-suspending cells, counting cells, and collecting about 1×10 ⁶ Individual cells, blocked with blocking antibodies at room temperature. Incubating anti-mouse CD4 mAb on ice in a dark place, incubating anti-mouse FoxP3 mAb on ice in a dark place after cell rupture, and detecting the proportion of intestinal tract Treg cells on the machine, wherein the method comprises the specific operationThe procedure was as described above for spleen lymphocyte detection.

4. Conclusion of early stage experiments

(1) GDM mice had higher inflammatory levels than the control group after FA model establishment

The prior studies reported that food allergy disease was high in children of GDM mother, while immunological imbalance was found in the GDM female nursing offspring. To further investigate the effect of GDM on the occurrence of sub-allergic diseases, the offspring of CON and GDM mice were subjected to food allergy model (FA) establishment and divided into four groups. IL-4, MMCP-1, OVA-sIgE, INF-gamma and TGF-beta levels in the sera of each group of mice after modeling were first examined. The results are shown in figure 2 (ELISA method for detecting cytokines IL-4, MMCP-1, OVA-sIgE as inflammatory factors, INF-gamma, TGF-beta as anti-inflammatory factors; p <0.05; p < 0.01) in mice after modelling, IL-4 levels were significantly higher in CON+OVA and GDM+OVA groups than in control mice, MMCP-1 levels were significantly higher in CON+OVA and INF-gamma levels were significantly lower in CON+OVA and GDM groups. The TGF-beta levels were significantly lower in the CON+OVA group than in the CON group. Prompting the establishment of the FA allergy model of the mice to be successful.

(2) GDM mice have significantly reduced intestinal lactobacillus abundance after FA model establishment

To further observe the changes in intestinal flora of FA mice, a diversity analysis of the simpson index and shannon index was first performed, and the results showed no significant differences between the four groups (fig. 3), with the biodiversity remaining stable in individuals. Beta diversity analysis found that there was a significant difference between the con+ova group, gdm+ova group and the CON group, GDM group, and no significant difference between the con+ova group and the gdm+ova group (fig. 4). At the genus level, the relative abundance of the mouse Lachnoclostrichum fed to the GDM master was higher, with the relative abundance of the GDM+OVA group being significantly higher than that of the other three groups (FIG. 6). Lachnoclostrichum is reported to be enriched in faeces of obese patients and highly associated with colon cancer. Of the offspring fed from CON mice, the relative abundance of Lactobacillus and bifidobacteria was higher (fig. 5, 6), with gdm+ova group Lactobacillus being significantly lower relative abundance than con+ova group (< 0.001, fig. 5). LEfSe analysis showed that the CON group Lactobacillusjohnsonii was more abundant than the GDM group (fig. 7). The relative abundance of con+ova groups Lactobacillus reuteri and Lactobacillus johnsonii is significantly higher than that of gdm+ova groups (fig. 8, 9).

3.3GDM mice ROR gamma t after setting up FA model ⁺ Treg cells were significantly reduced

Previous studies have shown that roryt ⁺ Treg cells mediate gut symbiont-induced tolerance. To determine whether alterations in intestinal microbiota of FA progeny affected their immune tolerance, progeny rorγt was tested ⁺ The proportion of Treg cells and other related immune cells (figures 10 and 11). Flow cytometry analysis shows that the ratio of spleen Treg cells in the CON+OVA group and the GDM+OVA group is reduced compared with the ratio of spleen Treg cells in the CON+OVA group and the GDM group, and the ratio of spleen Treg cells in the CON+OVA group is not obviously different from the ratio of spleen Treg cells in the GDM+OVA group. The proportion of progeny pekinetosome Treg cells fed by the GDM master is significantly lower than that of the progeny fed by the CON master, i.e., the GDM and gdm+ova groups have significantly lower proportion of pekinetosome Treg cells than the CON and con+ova groups (p<0.01). Gdm+ova group pekine rorγt ⁺ The Treg cell fraction was significantly lower than in the con+ova group (p<0.05 GATA 3) ⁺ The Treg cell fraction was significantly higher than in the con+ova group (p<0.05)。

The above test results found that the relative abundance of lactobacillus in the intestinal tract of FA mice fed by GDM mice was significantly lower than that of FA mice fed by CON mice, particularly lactobacillus johnsonii and lactobacillus reuteri. Thus, a decrease in the abundance of both lactobacillus species, FA progeny fed by the GDM master, aggravates the autoimmune response. The results show that the intestinal peter junction Treg cell ratio of the FA mice fed by the GDM mice is significantly lower than that of the FA mice fed by the CON mice, wherein the roryt+ Treg cell ratio is lower than that of the FA mice fed by the CON mice, and the gata3+ Treg cell ratio is higher than that of the FA mice fed by the CON mice. The results indicate that the food allergy symptoms of the FA mice fed to the GDM female mice are more severe.

Taken together, the results of this study demonstrate that GDM affects breast milk oligosaccharides and that the content of GDM in the mother's breast milk oligosaccharides results in delayed colonization of the intestinal beneficial bacteria of the offspring. Low levels of 3' sl in GDM murine milk lead to reduced bifidobacteria and lactobacillus abundance in the intestinal tract of the mice, leading to an immune imbalance in the offspring. The abundance of lactobacillus johnsonii and lactobacillus reuteri and the proportion of roryt+ Treg cells in the FA progeny intestinal tract of the GDM master mice are lower, and the degree of allergy is more severe. The data provides a certain reference basis for GDM breast milk oligosaccharide research and the influence of the GDM breast milk oligosaccharide on intestinal flora and immune tolerance of offspring, and provides a new direction for preventing and treating neonatal allergic diseases.

5. Verification experiment

1) In vitro culture and stimulation of mouse T cells

In order to analyze the effect of intestinal flora changes on proliferation of Treg cells in the mouse peyer's patch lymph nodes (PPs)/spleen, the present application extracts fecal metabolites from GDM mice fed with milk oligosaccharides and co-cultures T lymphocytes obtained from PPs in vitro.

0.25g of mouse feces was mixed with 1ml of PBS and centrifuged at 13000rpm for 30min. The supernatant obtained by centrifugation was filtered through a 0.45 μm filter and a 0.22 μm filter in this order. The liquid obtained after filtration can be regarded as feces dilution, diluted 10 times, and 20. Mu.L is added to 1ml containing 1X 10 ⁶ Cells in RPMI1640 medium. All cells were cultured in a cell incubator at 37℃with 5% CO2 for 6 hours.

In order to examine the effect of lactobacillus reuteri, lactobacillus johnsonii and 3'-SL on proliferation of Tregs cells in mouse PPs, the present application isolated total lymphocytes from neonatal mouse PPs and co-cultured with 3' -SL, lactobacillus reuteri and lactobacillus johnsonii supernatant and cell lysate, respectively.

The specific operation is as follows: 1mL of Lactobacillus reuteri and Lactobacillus johnsonii culture (OD 600 = 1.5) were centrifuged at 3000rpm for 15min to obtain supernatant and pellet, respectively, and the supernatants obtained herein were considered as bacterial secretions. The pellet was resuspended in 1ml PBS and sonicated to release intracellular components, and then centrifuged at 3000rpm for 15 minutes to give a supernatant, which was filtered with a 0.22 μm filter as lysate.

Diluting the supernatant and lysate of Lactobacillus reuteri and Lactobacillus johnsonii obtained by the above extraction 10 times, and collecting 20 μl to 1ml of the supernatant and lysate containing 1×10 ⁶ Cells in RPMI1640 medium. Equal amounts of 3' -SL were added to the lacto-oligosaccharide group at a final concentration of 1mg/ml.

All cells were cultured in a cell incubator at 37℃with 5% CO2 for 6 hours.

2) Experiments for intervention of FIG7A-F

Offspring of GDM master mice were divided into 4 groups, and treated with physiological saline, lactobacillus reuteri and Lactobacillus johnsonii (10) ⁷ cfu/15. Mu.L) or 3' -SL (0.1 mg/15. Mu.L) were lavaged, and each mouse in the combined intervention group received Lactobacillus reuteri (10) ⁷ cfu/5. Mu.L), lactobacillus johnsonii (10) ⁷ cfu/5. Mu.L) and 3' -SL (0.1 mg/5. Mu.L). Supplementation was started on day 10 post-natal mice, once a day, until weaning on day 21.

6. Results

1) Flow cytometry detects post-intervention neonatal mouse spleen Treg cells. As shown in fig. 12.

Although the total lymphocyte counts were similar between groups, the proportion of Treg (cd4+foxp3+) cells in spleen of neonatal mice supplemented with lactobacillus reuteri, lactobacillus johnsonii and 3' -SL was significantly higher than that of the control group (p=0.0012), indicating that the combined intervention of lactobacillus reuteri, lactobacillus johnsonii and 3' -SL was more effective in increasing Treg cell numbers than 3' -SL alone or lactobacillus reuteri+lactobacillus johnsonii alone.

2) The remarkable promoting effect of the combined intervention of 3' -SL and l.spp on roryt mRNA expression levels in neonatal mice intestinal tissue (fig. 13, p=0.0205), suggests that supplementation with this combination can promote the development of roryt+ cells in neonatal mice, which has been shown to be essential for the development of intestinal mucosal immune tolerance.

3) The interaction between intestinal microbiome changes and the immune response of GDM neonatal mice was further analyzed, fecal metabolites were extracted from neonatal mice supplemented with lactobacillus or 3' -SL and co-cultured with T lymphocytes obtained from mouse Pp in vitro (fig. 14). Flow cytometry analysis showed that the fecal metabolites stimulated Treg cell percentage up-regulation after GDM mice supplementation compared to saline group. The percentage of Treg cells was significantly higher in the lactobacillus reuteri, lactobacillus johnsonii and 3' -SL combined intervention group than in the other control group (p=0.0464). It was demonstrated that changes in the intestinal microbiome directly affect neonatal mouse immune responses.

4) To determine if lactobacillus reuteri and 3'-SL had a direct effect on roryt+ Treg cell development, lymphocytes were isolated from the Pp of mice and incubated in vitro with 3' -SL and supernatant/cell lysates of lactobacillus reuteri and lactobacillus johnsonii (fig. 15). After incubation, it was found that the number of Tregs, in particular roryt+ Treg cells, was significantly increased after stimulation with lactobacillus reuteri lysate, lactobacillus johnsonii supernatant and 3' -SL (p=0.0029, p=0.0080). The lactobacillus reuteri lysate, lactobacillus johnsonii supernatant and 3' -SL were shown to be effective in enhancing Treg cell development.

While the application has been described in terms of preferred embodiments, it is not intended to be limited thereto, but rather to enable any person skilled in the art to make various changes and modifications without departing from the spirit and scope of the present application, which is therefore to be limited only by the appended claims.

Claims

1. A formulation for improving an immune imbalance and/or preventing and treating allergic diseases in infants, the formulation comprising the following components: 3' -sialyllactose, lactobacillus johnsonii and lactobacillus reuteri.

2. The preparation according to claim 1, characterized in that the preparation comprises the following components in parts by mass: 3' -sialyllactose 0.01-0.70 g/L, lactobacillus johnsonii 5X 10 ⁷ ～1.5×10 ⁹ cfu/g and Lactobacillus reuteri 1X 10 ⁸ ～1.1×10 ¹⁰ cfu/g。

3. Formulation according to claim 1, characterized in that it is a liquid and/or a powder and/or a solid.

4. Use of the formulation of claim 1 for the manufacture of a medicament for the treatment of an immune imbalance in infants.

5. Use of the formulation of claim 1 for the manufacture of a medicament for the treatment of allergy symptoms in infants.

6. Use of the formulation of claim 1 for the manufacture of a medicament for promoting expression of Treg cells rorγt in infants suffering from immune imbalance.

7. Use of the formulation of claim 1 for the preparation of a food and/or supplement and/or complementary food and/or beverage for improving an immune imbalance in infants.

Use of 3' -sialyllactose for the preparation of a medicament and/or food for promoting the expression of Treg cells rorγt in infants suffering from an immune imbalance.