CN118020946A - Nutritional composition for protecting intestinal barrier and/or preventing central nervous system diseases and application thereof - Google Patents

Abstract

The present invention discloses a nutritional composition for protecting the intestinal barrier and/or preventing central nervous system diseases and its application, wherein the nutritional composition comprises TGF-β1 and lactoferrin. The nutritional composition provided by the present invention can promote the expression of intestinal epithelial tight junction proteins, increase the transmembrane resistance of intestinal epithelial cells, prevent and/or repair the damage of epithelial barrier function caused by enterohemorrhagic Escherichia coli, thereby protecting the intestinal barrier function and preventing central nervous system diseases.

Description

Nutritional composition for protecting intestinal barrier and/or preventing central nervous system diseases and its application

Technical Field

The present invention relates to the field of food technology, and in particular to a nutritional composition for protecting intestinal barriers and/or preventing central nervous system diseases and an application thereof.

Background technique

Breast milk is the best food for infants, providing them with essential nutrients for their growth and development. When breast milk is not available, infant formula becomes the only energy source for young infants. Among them, hydrolyzed (partially or extensively hydrolyzed) cow's milk protein formula is usually the choice for infants with weak digestive or immune functions. However, during the processing of proteolytic enzymes, some proteins with important physiological functions but low content, such as transforming growth factor, are decomposed and not supplemented.

Breast milk contains more than 21,000 different proteins. In addition to the main nutrients and immune components, it also contains trace amounts of growth factors and other components. Transforming growth factor beta (TGF-β) is a multifunctional protein that can affect the growth, differentiation, apoptosis and immune regulation of a variety of cells. Transforming growth factor-β includes three subtypes: transforming growth factor-β1 (TGF-β1), transforming growth factor-β2 (TGF-β2) and transforming growth factor-β3 (TGF-β3). The concentration of TGF-β1 in breast milk varies greatly between different races and individuals. During normal lactation, the concentration of TGF-β1 decreases slightly from colostrum to mature milk. Breast milk research usually focuses on the association between TGF-β1 and a reduced incidence of allergic diseases, asthma, atopic dermatitis, etc. in infants (Khaleva, 2019).

Among the proteins with immune activity in breast milk, lactoferrin is an immune-related protein that is abundant in colostrum. In the body of infants, lactoferrin receptors are more abundant in the small intestine. Orally ingested lactoferrin has direct and indirect antibacterial activity in the intestine. The direct antibacterial activity is through binding to lipopolysaccharide or phosphatidylic acid on the surface of pathogens. The indirect antibacterial activity may be through lactoferrin cells entering intestinal cells, regulating the gene transcription of intestinal cells, thereby affecting the mucosal immune function and ultimately killing bacteria (Lönnerdal, 2009).

However, the current hydrolyzed (partially or extensively) cow's milk protein formula contains almost no TGF-β1 and lactoferrin due to the processing and formula design of proteolytic enzymes, and has a significant difference in the types of protein composition in breast milk. At the same time, most of the infant formula milk powders with hydrolyzed cow's milk protein on the market do not consider the importance of growth factors such as TGF-β1 for the normal growth and development of infants and young children.

The applicant unexpectedly discovered that lactoferrin can regulate the cytokines of intestinal epithelial cells, increase the transcription of the TGF-β1 gene of intestinal cells, and thus increase endogenous TGF-β1. At the same time, the applicant discovered that TGF-β1 also plays an important role in maintaining the integrity of the intestinal physical barrier.

The intestinal barrier is mainly composed of the mucus layer, epithelial barrier and intestinal vascular barrier. It plays a vital role in health and disease by promoting nutrient absorption and preventing pathogens from entering, while participating in the regulation of the microbiota-gut-brain axis. When the intestinal epithelial tight junction protein is tightly connected with a hole or dysfunction, the gap between epithelial cells becomes larger and the intestinal permeability increases. The Lancet sub-journal reported that impaired intestinal barrier function and increased intestinal permeability are associated with central nervous system diseases such as cognitive impairment; clinical and animal evidence shows that impaired intestinal barrier causes microbiota, microbiota pathogenic metabolites or central nervous system-related pathological proteins to enter the blood ectopically, or activate inflammatory responses, or spread to the brain, causing central nervous system lesions; improving the integrity of the intestinal barrier through probiotics/prebiotics, microbiota metabolites, etc. is beneficial to the prevention and treatment of related central nervous system diseases (Pellegrini, C. 2023). Therefore, the integrity of the intestinal barrier can not only prevent the invasion of pathogens and ensure the efficient absorption and utilization of nutrients, but also prevent central nervous system lesions caused by increased intestinal permeability.

Summary of the invention

In order to overcome the deficiencies of the prior art, one of the objectives of the present invention is to provide a nutritional composition for protecting the intestinal barrier and/or preventing central nervous system diseases.

The second object of the present invention is to provide the application of the nutritional composition for protecting the intestinal barrier and/or preventing central nervous system diseases.

One of the purposes of the present invention is achieved by the following technical solution:

A nutritional composition for protecting intestinal barrier and/or preventing central nervous system diseases, comprising TGF-β1 and lactoferrin.

As a preferred embodiment of the present invention, in the nutritional composition, the content of TGF-β1 is 0.5-100 ng/mL. Preferably, the content of TGF-β1 is 0.5-50 ng/mL, the content of TGF-β1 is 0.5-10 ng/mL, the content of TGF-β1 is 1-10 ng/mL, and more preferably, the content of TGF-β1 is 10 ng/mL.

More specifically, the content of TGF-β1 in the nutritional composition is 0.5 ng/mL, 1 ng/mL, 1.2 ng/mL, 1.8 ng/mL, 3.35 ng/mL, 9.78 ng/mL, 10 ng/mL, 20 ng/mL, 30 ng/mL, 40 ng/mL, 50ng/mL, 60 ng/mL, 70 ng/mL, 80 ng/mL, 90 ng/mL, and 100 ng/mL.

As a preferred embodiment of the present invention, in the nutritional composition, the content of TGF-β1 is 0.5-100 ng/g. Preferably, the content of TGF-β1 is 0.5-50 ng/g, the content of TGF-β1 is 0.5-10 ng/g, the content of TGF-β1 is 1-10 ng/g. More preferably, the content of TGF-β1 is 10 ng/g.

More specifically, the content of TGF-β1 in the nutritional composition is 0.5 ng/g, 1 ng/g, 1.2 ng/g, 1.8 ng/g, 3.35 ng/g, 9.78 ng/g, 10 ng/g, 20 ng/g, 30 ng/g, 40 ng/g, 50 ng/g, 60 ng/g, 70 ng/g, 80 ng/g, 90 ng/g, and 100 ng/g.

As a preferred embodiment of the present invention, in the nutritional composition, the content of lactoferrin is 10-1200 ug/mL. Preferably, the content of lactoferrin is 10-800 ug/mL, and more preferably, the content of lactoferrin is 100 ug/mL.

More specifically, the content of lactoferrin in the nutritional composition is 10 ug/mL, 15 ug/mL, 20 ug/mL, 30 ug/mL, 40 ug/mL, 50 ug/mL, 60 ug/mL, 70 ug/mL, 80 ug/mL, 90 ug/mL, 100 ug/mL, 150 ug/mL, 200 ug/mL, 250 ug/mL, 300 ug/mL, 350 ug/mL, 400 ug/mL, 440 ug/mL, 500 ug/mL, 550 ug/mL, 600 ug/mL, 700 ug/mL, 800 ug/mL, 900 ug/mL, 1000 ug/mL, 1100 ug/mL, and 1200 ug/mL.

As a preferred embodiment of the present invention, in the nutritional composition, the content of lactoferrin is 10-1200 ug/g. Preferably, the content of lactoferrin is 100-800 ug/g, and more preferably, the content of lactoferrin is 100 ug/g.

More specifically, the content of lactoferrin in the nutritional composition is 10 ug/g, 15 ug/g, 20 ug/g, 30 ug/g, 40 ug/g, 50 ug/g, 60 ug/g, 70 ug/g, 80 ug/g, 90 ug/g, 100 ug/g, 150 ug/g, 200 ug/g, 250 ug/g, 300 ug/g, 350 ug/g, 400 ug/g, 440 ug/g, 500 ug/g, 550 ug/g, 600 ug/g, 700 ug/g, 800 ug/g, 900 ug/g, 1000 ug/g, 1100 ug/g, and 1200 ug/g.

As a preferred embodiment of the present invention, the TGF-β1 is derived from one or any combination of the following: whey protein fragments, polypeptide growth factors separated from milk, and recombinant TGF-β1 produced by bioengineering.

More specifically, the TGF-β1 source may be in the form of biologically active peptides such as whey protein fragments of TM0301 or XP-828L from Armor Proteines (France), or in the form of polypeptide growth factors isolated from milk, or in the form of recombinant TGF-β1 produced by bioengineering.

As a preferred embodiment of the present invention, the TGF-β1 is derived from cow's milk, goat's milk, horse's milk or camel's milk. More preferably, the TGF-β1 is derived from cow's milk.

As a preferred embodiment of the present invention, the lactoferrin is derived from cow's milk, goat's milk, horse's milk or camel's milk. More preferably, the lactoferrin is derived from cow's milk.

As a preferred embodiment of the present invention, the lactoferrin is used to stimulate the expression of transforming growth factor in intestinal epithelial cells, thereby inducing the production of endogenous TGF-β1 in the intestine.

As a preferred embodiment of the present invention, the TGF-β1 and/or lactoferrin protects the intestinal barrier and/or prevents central nervous system diseases through one or more of the following (I) to (III):

(I) Promote the expression of intestinal epithelial tight junction proteins;

(II) Increase the transmembrane resistance of intestinal epithelial cells;

(III) Prevent and/or repair damage to epithelial barrier function caused by enterohemorrhagic Escherichia coli.

As a preferred embodiment of the present invention, the tight junction protein includes Claudin-1.

As a preferred embodiment of the present invention, the increasing the transmembrane resistance of intestinal epithelial cells includes increasing: a decrease in the transmembrane resistance of intestinal epithelial cells caused by chronic or acute effects.

As a preferred embodiment of the present invention, the enterohemorrhagic Escherichia coli includes enterohemorrhagic Escherichia coli O157:H7.

The second object of the present invention is achieved by adopting the following technical solution:

The present invention also provides the use of the TGF-β1 and lactoferrin complex in preparing products for protecting the intestinal barrier and/or preventing central nervous system diseases.

As a preferred embodiment of the present invention, the products include foods, health products, nutritional supplements and medicines.

As a preferred embodiment of the present invention, the product includes dairy products, and the dairy products include milk powder, liquid milk, cheese, cream, condensed milk or other dairy products.

The present invention also provides a milk-based hydrolyzed formula, comprising TGF-β1 and lactoferrin.

As a preferred embodiment of the present invention, the milk-based hydrolyzed formula may further include one or any combination of the following components: carbohydrates, protein sources, lipid sources, probiotics, prebiotics, and the like.

As a preferred embodiment of the present invention, the prebiotics include human milk oligosaccharides.

The invention provides a milk-based hydrolyzed formula, including TGF-β1 and lactoferrin, based on the growth and development needs of infants and young children, the composition of breast milk and colostrum, and the latest breast milk research.

The milk-based hydrolyzed formula provided by the present invention, when administered to a target subject such as an infant, can promote the integrity of the intestinal barrier of the infant and/or prevent central nervous system diseases.

As a preferred embodiment of the present invention, the milk-based hydrolyzed formula is a partially hydrolyzed formula or an extensively hydrolyzed formula. In the partially hydrolyzed formula, 35% to 60% of the protein/peptide group has a molecular weight of less than 1000 Dalton. In the extensively hydrolyzed formula, at least 95% of the protein/peptide group has a molecular weight of less than 1000 Dalton.

As a preferred embodiment of the present invention, the milk-based hydrolyzed formula is a partially hydrolyzed formula. More preferably, the partially hydrolyzed protein with a whey protein:casein ratio of 6:4 is used.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The nutritional composition provided by the present invention is a protein composition that supports the integrity of the intestinal mucosal mechanical barrier, can reduce intestinal permeability, and effectively prevent bacteria from penetrating the mucosa and entering deep tissues.

(2) The nutritional composition provided by the present invention is a protein composition that can prevent barrier damage caused by enterohemorrhagic Escherichia coli (EHEC), prevent the reduction of tight protein secretion caused by EHEC, and prevent diarrhea and enterohemorrhagic Escherichia coli enteritis.

(3) The nutritional composition provided by the present invention is a protein composition that can supplement the content of TGF-β1 in the hydrolyzed formula and promote the production of endogenous TGF-β1. It can narrow the difference in the types and levels of growth factors between the current hydrolyzed infant formula and breast milk, and meet the needs of young infants and young children who consume hydrolyzed milk-based formula to maintain the integrity of the intestinal barrier and prevent central nervous system diseases.

(4) The nutritional composition provided by the present invention can be used to prepare hydrolyzed milk-based formula milk powder to make up for the growth factors lost during the hydrolysis of protein processing. The addition of lactoferrin with immune function can narrow the differences between the current hydrolyzed infant formula and breast milk in terms of protein types and immune efficacy, making the product closer to breast milk.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a diagram showing the results of partially hydrolyzed protein and lactoferrin promoting the transcription of transforming growth factor TGF-β1 gene in Caco-2 in experimental system 1 of the present invention;

FIG2 is a result diagram of the contents of tight junction proteins Claudin-1, Claudin-2 and Claudin-4 in intestinal epithelial cells in experimental system 2 of the present invention;

FIG3 is a graph showing the 72-hour change in transepithelial electrical resistance (TER) of human colon epithelial cells after pretreatment with different proteins in experimental system 3 of the present invention;

FIG4 is a graph showing the 72-hour change in transepithelial electrical resistance (TER) of human colon epithelial cells after treatment with different concentrations of protein compositions under experimental system 3;

FIG5 is a graph showing the results of cell transmembrane potential of intestinal epithelial cells in system 4 of Example 1 of the present invention after being treated with different proteins for 16 hours;

6 is a graph showing the results of cell transmembrane potential of intestinal epithelial cells in system 4 of Example of the present invention after being treated with protein compositions of different concentrations for 16 hours.

Detailed ways

The present invention is further described below in conjunction with the accompanying drawings and specific embodiments. It should be noted that, under the premise of no conflict, the embodiments or technical features described below can be arbitrarily combined to form new embodiments. The raw materials, equipment, etc. used in the following embodiments can be obtained by purchase unless otherwise specified.

In the experimental system provided in the embodiment of the present invention, lactoferrin (LF) is lactoferrin derived from bovine milk, and transforming growth factor (TGF-β1) is TGF-β1 derived from bovine milk.

Experimental system 1: The protein combination can upregulate the transcription of the TGF-β1 gene in intestinal epithelial cells and promote the production of endogenous TGF-β1

Human colon adenocarcinoma cells Caco-2 (American Type Tissue Culture Collection, Rockville, MD), which express a variety of cytokines, are commonly used as a model system to study the interaction of nutrients with intestinal epithelial cells. For this experiment, Caco-2 cells will be used between passage 20 and 30 and cultured in minimal essential medium containing fetal bovine serum (FBS, 10%), penicillin and streptomycin (10 units/mL and 1 mg/mL, respectively) in a humidified cell culture incubator at 37°C and 5% CO ₂ .

To evaluate the effect of lactoferrin on TGF-β1 gene transcription, Caco-2 cells were cultured in 6-well plates and treated with partially hydrolyzed protein (PHP) and lactoferrin (LF) (both at 100 µg/mL) for 72 h at 37 °C on day 8, with serum-free medium as blank control, and then total RNA was extracted for qRT-PCR. RNA integrity was assessed by electrophoresis on agarose gel (1%) stained with ethidium bromide (Sigma). cDNA was generated from RNA (1 µg) using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA).

Gene-specific primers for TGF-β1 will be used using Primer Express software (AppliedBiosystems). Real-time PCR will then be performed on the cDNA reaction mixture (2 µL) and SYBR Green (Applied Biosystems).

Table 1 TGF-β1 gene transcription experiment group record table

The results are shown in Figure 1, and the results are shown as mean fold change ± SD, n = 6. There are statistical differences between groups without common letter marks (P < 0.05). As can be seen from the figure, compared with the control group and PHP group, the level of TGF-β1 in the LF group was significantly increased (p < 0.05), and there was no statistically significant difference between the PHP group and the control group. This shows that lactoferrin (LF) can stimulate the expression of transforming growth factor beta 1 (TGF-β1) in human intestinal epithelial cells, thereby inducing the production of endogenous TGF-β1 in the intestine of infants and young children.

Experimental system 2: Effect of protein composition on the expression of tight junction proteins in intestinal epithelial cells

Tight junctions (TJs) are structures that bring adjacent cell membranes together to form a physical barrier around the cell. Tight junctions can form a continuous seal around the cell and act as a physical barrier to prevent solutes and water from freely passing through the paracellular space. The family of tight junction proteins (Claudins) is a key component of tight junctions between epithelial cells, including Claudin-1, Claudin-2, Claudin-4 proteins, etc. The more tight junction proteins there are between intestinal epithelial cells, the smaller the paracellular gap, and the stronger the integrity of the intestinal epithelial barrier. This experiment was used to explore the effects of lactoferrin (endogenous TGF-β1) and (exogenous) TGF-β1 on the expression of tight junction proteins in the intestinal epithelium.

Human colon epithelial cells T84 were cultured in medium containing 10% fetal bovine serum. T84 cells were seeded onto filter supports (106 cells/well, surface area ^{1 cm2} ) and grown to confluence (≥6 days). On day 7, they were treated with partially hydrolyzed protein (PHP) (100 µg/mL), lactoferrin (LF) (100 µg/mL), TGF-β1 (10 ng/mL), and lactoferrin + TGF-β1 (100 µg/mL + 10 ng/mL) at 37 °C for 72 hours. The partially hydrolyzed protein was used as the control group. The expression of tight junction proteins in the three groups after 72 hours of treatment was compared by quantitative western blot. After immunoblotting of eluted tight junction proteins, β-actin protein was re-detected to assess total protein loading. Claudin-4 expression was used as an internal control in this experiment because it is a blocking protein and is not altered by infection or transforming growth factor.

The experimental results showed (Figure 2) that in the protein extracts of monolayer T84 cells 72 hours after treatment with LF, TGF-β1 and LF+TGF-β1, the expression of Claudin-1 increased compared with the control group, while Claudin-2 and Claudin-4 did not increase. The experiment showed that the endogenous TGF-β1 produced by LF can increase the expression of Claudin-1 protein (indicated by the arrow), and the exogenous TGF-β1 group and both endogenous and exogenous TGF-β1 can increase the expression of Claudin-1 (indicated by the arrow), and the expression-promoting effect of LF+TGF-β1 is more obvious (indicated by the arrow), thereby enhancing intercellular connections and contributing to the integrity of the epithelial cell barrier.

Experimental system 3: Effect of protein composition on transmembrane potential of intestinal epithelial cells

The third part verifies the protective effect of the protein composition (lactoferrin + TGF-β1) provided in the embodiment of the present invention on the intestinal barrier of infants and young children, which can enhance the transepithelial electrical resistance (TER) and maintain the integrity of the intestinal epithelial cell barrier.

Human colon epithelial cells T84 were cultured in a medium containing 10% fetal bovine serum. T84 cells were inoculated onto filter carriers (106 cells/well, surface area ^{1 cm2} ) and grown to confluence (≥6 days), at which time TER (Millipore-ERS; Millipore) was monitored by a voltmeter and chopstick electrode. TER can reflect paracellular permeability, that is, the integrity of the intestinal epithelial cell barrier. When the holes or dysfunction of the tight junctions of the intestinal epithelial tight junction proteins are tightly connected, the gaps between cells become larger, and the intestinal permeability increases, then TER decreases. The TER measurement value at 0 hours was used as the baseline data for each group. When the concentration of tight junction proteins is higher, the TER value is higher, and the integrity of the intestinal epithelial barrier is stronger. In order to evaluate the chronic effect of the protein composition on the secretion of tight junction proteins by intestinal epithelial cells, five groups of protein combinations were added to the basolateral surface of the T84 monolayer as shown in Table 2, and TER was recorded 4, 8, 16, 24, 48 and 72 hours after addition to detect the chronic effect of each group of proteins. Table 2 shows the experimental grouping of the effect of protein combinations on the TER of intestinal epithelial cells.

The experimental results are shown in Figure 3, where the ordinate (TER % pretreatment) is the percentage of TER relative to TER before pretreatment. Compared with the control group, the TER of the other five groups increased within 72 hours. The TER of the LF+TGF-β1 group (dark blue line) was significantly higher than that of the other groups at 8h, 48h and 72h (one-way ANOVA, F=3.22, p<0.05, n=6 per group; Bonferroni method was used for pairwise comparison, p<0.05). The average TER of the control group (green line) was approximately 768 ± 186 Ω/cm².

The TER of the LF+TGF-β1 protein combination was the highest within 72 hours, higher than the TER of other groups. Compared with the control group, the TER of the other five groups also increased within 72 hours. At 16 hours and 24 hours, the results of one-way ANOVA indicated that there was no difference in the TER values of the six experimental groups (p>0.05);

Throughout the experiment, there was no difference in TER values between the PHP+TGF-β1 group and the OPN+TGF-β1 group and the group with TGF-β1 protein added alone (p>0.05); this also proves that partially hydrolyzed protein (PHP) and OPN have no effect on the growth of intestinal epithelial cells and cannot produce endogenous TGF-β1. At 8h, 48h and 72h, the TER of the LF+TGF-β1 protein combination was significantly higher than that of the LF group and the TGF-β1 protein group (one-way ANOVA, p<0.05, n=6 in each group; Bonferroni method was used for pairwise comparison, p<0.05), indicating that the LF+TGF-β1 protein combination has higher transepithelial resistance and better epithelial physical barrier integrity than when used alone.

In addition, the same experiment was carried out according to the experimental groups in Table 3, and the results are shown in Figure 4. The following conclusions can be drawn from Figure 4.

Group A: Similar to the results of the LF + TGF-β1 group of the same color (dark blue) in Figure 3, the expression of endogenous TGF-β1 continued to increase in the first half of 72 hours due to the appropriate concentration of TGF-β1 (10 ng/ml) and the promotion of lactoferrin in the later stage.

Group B: Since the levels of LF and TGF-β1 in the protein combination were very low, the effect was slightly better than that of the control group, but there was no significant effect.

Group C: Due to the addition of 100 ng/ml TGF-β1, TER increased rapidly in the early stage, but due to the limited lactoferrin receptors and the fact that the concentration of 1200 µg/mL lactoferrin had a certain inhibitory effect on the growth of intestinal epithelial cells, the growth rate did not reach the fastest before 24 hours. At the same time, lactoferrin will produce endogenous TGF-β1 from 24 to 72 hours, and some lactoferrin will bind to epithelial cells and be transported into the cells, so the inhibitory effect will be weakened, and the growth rate of TER will be accelerated, which has a better effect.

Group D: With the addition of 50 ng/ml of TGF-β1, there was an initial rapid increase, and the amount of lactoferrin added was within the effective range and did not reach the level of inhibition, so the effect was better than that of Group C.

Group E: The level of TGF-β1 added was low, and the increase was slow in the early stage. As lactoferrin produced endogenous TGF-β1 in the later stage, it promoted tight junctions between intestinal epithelial cells and maintained the integrity of the intestinal barrier. Therefore, the protein combination took effect mainly in the second half, so it showed a slow growth in the first half and an improvement in the second half.

At the same time, the acute effect of the LF+TGF-β1 protein combination was evaluated by a washout experiment. The monolayer cells were exposed to the protein combination [LF (100ug/ml) + TGF-β1 (10ng/mL)] for 1 hour, and then washed twice with culture medium. After being maintained in fresh culture medium every day, the TER value recorded 72 hours after addition was 1344±256 Ω/cm², which was 173% of the TER value before pretreatment, slightly lower than the TER (210%) at 72 hours under the chronic action of the target protein combination. Compared with the control group, acute treatment with the LF+TGF-β1 protein combination for only 1 hour can also increase the transepithelial resistance of intestinal epithelial cells (independent sample T test, n=6, p<0.05), improving the integrity of the physical barrier.

Table 2 Experimental groups for the effects of protein combinations on TER of intestinal epithelial cells

Table 3 Experimental groups for the effects of different concentrations of protein combinations on TER of intestinal epithelial cells

Experimental system 4: Preventive effect of protein combination (LF+TGF-β1) on intestinal barrier damage caused by pathogenic bacteria

Experimental system 4 is used to verify the preventive effect of the protein composition (LF+TGF-β1) provided in the embodiment of the present invention on the damage of the intestinal barrier caused by pathogenic bacteria. By detecting the changes in the transepithelial electrical resistance (TER) of the human intestinal cell barrier after infection with pathogenic bacteria (hemorrhagic Escherichia coli (EHEC) O157:H7), the function of the intestinal barrier is reflected, and the protein composition is simulated to prevent the damage of pathogenic bacteria to the intestinal barrier of infants and young children, and reduce the symptoms of diarrhea and other pathogenic bacteria infection in infants and young children due to damage to the intestinal barrier caused by pathogenic bacteria.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a group of E. coli that has the ability to produce Shiga-like toxins, which can cause acute hemorrhagic enteritis and, in some cases, secondary hemolytic uremic syndrome (HUS). Intestinal epithelial cells are the main site of EHEC colonization.

Enterohemorrhagic Escherichia coli (strain CL-56) was grown for 6 to 84 hours (to mid-logarithmic growth phase) prior to infection of T84 monolayers. The bacterial suspension was pelleted (21,000 rpm, 20 min), resuspended in antibiotic-free T84 cell culture medium, and 10 ⁸ CFU were added to the apical surface of the epithelial monolayer at an MOI of 100 (multiplicity of infection; 100 infectious units/epithelial cell).

T84 monolayers were treated with the protein combination 45 minutes before infection, the protein combination was maintained during infection, and TER was recorded after 16 hours (a time point determined based on the proliferation characteristics of EHEC). Table 4 shows the groups and doses of protein combination pretreatment.

As shown in Figure 5, the ordinate (TER % pretreatment) is the percentage of TER relative to TER before pretreatment. Groups without the same letters have statistical differences (after one-way ANOVA, Bonferroni method was used for pairwise comparison, n=6 for each group).

As can be seen in the figure, after EHEC infection, the TER of T84 cells in the EHEC group was significantly reduced compared with that in the control group (one-way ANOVA, n=6, p<0.05), indicating that EHEC infection destroys the integrity of the intestinal epithelial physical barrier.

The TER of T84 cells in the EHEC + PHP and EHEC + LF groups increased slightly, but there was no statistical difference among the three groups compared with the EHEC group infected with EHEC (one-way ANOVA, n=6, p>0.05).

After adding TGF-β1, PHP + TGF-β1 or LF + TGF-β1 protein combination, TER of T84 cells was increased (one-way ANOVA, n=6, p<0.05).

After adding the LF + TGF-β1 protein combination, the TER of T84 cells increased, and there was no statistical difference compared with the control group that was not infected with EHEC (one-way analysis of variance, n=6, p<0.05), indicating that under the condition of viral infection, the LF + TGF-β1 protein combination still maintains the transepithelial electrical resistance of intestinal epithelial cells under normal physiological conditions and maintains the integrity of the epithelial physical barrier.

In addition, the experimental groups were grouped according to different concentrations of protein combinations to verify the protective effect of the protein combination on the mucosal barrier. The experimental method was consistent with the experimental grouping method in Table 4. The results are shown in Figure 6. The following conclusions can be drawn from Figure 6.

Control group: Because EHEC was not added, the intercellular barrier was intact and maintained at more than 100% of the TER value at 0 h, which was higher than all other groups and had statistical significance.

EHEC group: Because of EHEC infection and the absence of any protein combination, the cell barrier damage was the most serious among all groups.

Group A: The results were similar to those of the EHEC + LF + TGF-β1 group in Figure 5, ranging from 85% to 90%.

Group B: The level of protein combination was lower, so the results were slightly better than the control group, but there was no statistical difference.

Group C: Due to the effects of TGF-β1 and lactoferrin receptor, the intercellular barrier was not seriously damaged, but the relatively high lactoferrin would provide iron ions to bacteria. Therefore, the effect of this group was between the groups marked with numbers b and c, but there was no statistical difference with groups b and c.

Group D: Similar to Group C, but the concentration range of lactoferrin did not reach the level of inhibitory effect on epithelial cells, so the effect was better than Group C, but there was no statistical difference with Groups B and C.

Group E: The level of TGF-β1 was low. Lactoferrin directly bound to bacteria, which slightly increased TER. However, since it takes time for lactoferrin to promote the production of endogenous TGF-β1 in intestinal epithelial cells, there was a certain improvement effect when tested after 16 hours, which was not statistically different from the EHEC group and group B.

Table 4 Experimental groups of protein combination pretreatment on mucosal barrier protection

Table 5 Experimental groups of different concentrations of protein combinations on mucosal barrier protection

The protein nutritional composition (lactoferrin + TGF-β1) provided in the embodiment of the present invention can promote the expression of intestinal epithelial tight junction proteins, increase the transmembrane resistance of intestinal epithelial cells, prevent and/or repair the damage of epithelial barrier function caused by enterohemorrhagic Escherichia coli, thereby protecting the intestinal barrier function and preventing central nervous system diseases.

The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims (10)

1. A nutritional composition for protecting the intestinal barrier and/or preventing disorders of the central nervous system, comprising TGF- β1 and lactoferrin.

2. A nutritional composition according to claim 1, wherein the TGF- β1 is present in the nutritional composition in an amount of 0.5 to 100 ng/mL or 0.5 to 100 ng/g and the lactoferrin is present in an amount of 10 to 1200 ug/mL or 10 to 1200 ug/g;

Preferably, the content of the TGF-beta 1 is 0.5-50 ng/mL or 0.5-50 ng/g, and the content of the lactoferrin is 100-800 ug/mL or 100-800 ug/g;

Preferably, the TGF-beta 1 content is 10 ng/mL or 10 ng/g and the lactoferrin content is 100 ug/mL or 100 ug/g.

3. A nutritional composition according to claim 1, wherein the TGF- β1 is derived from one or any combination of the following: whey protein fragments, polypeptide growth factors isolated from milk, recombinant TGF-beta 1 produced bioengineered.

4. A nutritional composition according to claim 1, wherein the TGF- β1 is derived from cow's milk, sheep's milk, horse's milk or camel's milk; the lactoferrin is derived from cow milk, sheep milk, horse milk or camel milk;

Preferably, the TGF- β1 is derived from cow's milk; the lactoferrin is derived from cow milk.

5. A nutritional composition according to claim 1 wherein lactoferrin is used to stimulate the expression of transforming growth factors in the intestinal epithelial cells, thereby inducing endogenous TGF- β1 production in the intestinal tract.

6. A nutritional composition according to claim 1, wherein the TGF- β1 and/or lactoferrin effects protection of the intestinal barrier and/or prevention of central nervous system diseases by one or more of the following (i) - (iii):

promoting expression of intestinal epithelial tight junction protein;

(II) increasing the transmembrane resistance of intestinal epithelial cells;

(III) prevention and/or repair of impaired epithelial barrier function caused by enterohemorrhagic Escherichia coli.

7. The nutritional composition of claim 6, wherein the tight junction protein comprises Claudin-1; the enterohemorrhagic escherichia coli comprises enterohemorrhagic escherichia coli O157: H7.

8. Use of a nutritional composition for the preparation of a product for protecting the intestinal barrier and/or preventing a central nervous system disorder, characterized in that the nutritional composition comprises TGF- β1 and lactoferrin.

9. The use according to claim 8, wherein the products include food products, health products, nutritional supplements and pharmaceuticals.

10. A milk-based hydrolysis formulation comprising TGF- β1 and lactoferrin.