Human Milk Oligosaccharides for Improving Resistance of Organism Against Staphylococcus Aureus Infection

Abstract

The present invention relates to the field of food and pharmaceutical products, and specifically to human milk oligosaccharide for improving resistance of organism against Staphylococcus aureus infection. In particular, the present invention relates to use of human milk oligosaccharide in preparation of a nutritional composition or medicament for prevention and/or treatment of diseases associated with Staphylococcus aureus infection in individuals, or for alleviation of discomfort associated with Staphylococcus aureus infection, or for improvement of an individual's resistance against Staphylococcus aureus infection, or for improvement of an individual's innate immunity and/or anti-aging.

Claims

1. Use of a human milk oligosaccharide in the preparation of a nutritional composition or medicament, wherein the nutritional composition or medicament is used for preventing and/or treating a disease associated with Staphylococcus aureus infection in an individual, or for alleviating discomfort associated with Staphylococcus aureus infection, or for improving an individual's resistance against Staphylococcus aureus infection, or for improving an individual's innate immunity and/or anti-aging; the human milk oligosaccharide being selected from the group consisting of: 2′-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, 6′-sialyllactose and any combination thereof, and a combination formed by 3′-sialyllactose together with one or more selected from 2′-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, and 6′-sialyllactose.

2. The use according to claim 1, which is characterized by one or more of the following: (1) the human milk oligosaccharide is administered at a dose of 0.012 to 12 g/day; (2) the nutritional composition is selected from a nutritional supplement, a functional food, a beverage product, a diet replacement or a food additive; (3) the disease or discomfort associated with Staphylococcus aureus infection is selected from food poisoning, enteritis, pneumonia, skin infection, wound ulceration, and meningitis; and (4) the individual is an infant, a toddler, a child, or an adult.

3. The use according to claim 1, wherein the nutritional composition is an infant formula food, such as an infant formula milk; preferably, the infant formula food further comprises the following ingredients: proteins, fat, carbohydrates, vitamins, minerals or any combination thereof; preferably, the infant formula food further comprises dietary fibers, nucleotides, inositol, taurine, L-carnitine, docosahexaenoic acid, eicosatetraenoic acid or any combination thereof.

4. The use according to claim 1, wherein the nutritional composition is a supplementary food or nutritional supplement for infants and toddlers.

5. The use according to any one of claims 1 to 4, wherein the human milk oligosaccharide is selected from the group consisting of: (1) a combination consisting of 2′-fucosyllactose and 3-fucosyllactose; (2) a combination consisting of 2′-fucosyllactose and lacto-N-tetraose; (3) a combination consisting of 2′-fucosyllactose and 6′-sialyllactose; (4) a combination consisting of 2′-fucosyllactose, 3-fucosyllactose and lacto-N-tetraose; (5) a combination consisting of 2′-fucosyllactose, 3-fucosyllactose and 6′-sialyllactose; (6) a combination consisting of 2′-fucosyllactose, lacto-N-tetraose and 6′-sialyllactose; (7) a combination consisting of 3-fucosyllactose and lacto-N-tetraose; (8) a combination consisting of 3-fucosyllactose and 6′-sialyllactose; (9) a combination consisting of 3-fucosyllactose, lacto-N-tetraose and 6′-sialyllactose; (10) a combination consisting of lacto-N-tetraose and 6′-sialyllactose; (11) a combination consisting of 2′-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose and 6′-sialyllactose; and a combination of any one of the above (1) to (11) together with 3′-sialyllactose.

6. The use according to claim 1, wherein when the nutritional composition or medicament is used for preventing and/or treating a disease associated with Staphylococcus aureus infection in an individual, or for alleviating discomfort associated with Staphylococcus aureus infection, or for improving an individual's resistance against Staphylococcus aureus infection, or for improving an individual's innate immunity and/or anti-aging, an effective amount of the human milk oligosaccharide or the nutritional composition or medicament is administered to an individual in need thereof; preferably, the human milk oligosaccharide is provided in a form of the nutritional composition or medicament suitable for oral or gavage feeding.

7. A nutritional composition or medicament comprising a human milk oligosaccharide, wherein the human milk oligosaccharide is selected from the group consisting of: 2′-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, 6′-sialyllactose and any combination thereof, and a combination formed by 3′-sialyllactose together with one or more selected from 2′-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, and 6′-sialyllactose; when the nutritional composition or medicament is liquid, the content of the human milk oligosaccharide in the nutritional composition or medicament is 0.03 to 12 g/L, preferably 0.03 to 6 g/L; when the nutritional composition or medicament is a solid (e.g., powder), the content of the human milk oligosaccharide in the nutritional composition or medicament is 0.02 to 9.09 g/100 g, preferably 0.02 to 4.55 g/100 g; preferably, the nutritional composition is selected from the group consisting of a nutritional supplement, a functional food, a beverage product, a diet replacement or a food additive.

8. The nutritional composition or medicament according to claim 7, wherein the nutritional composition is an infant formula food, such as an infant formula milk; preferably, the infant formula food further comprises the following ingredients: proteins, fat, carbohydrates, vitamins, minerals or any combination thereof; preferably, the infant formula food further comprises dietary fibers, nucleotides, inositol, taurine, L-carnitine, docosahexaenoic acid, eicosatetraenoic acid or any combination thereof.

9. The nutritional composition or medicament according to claim 7, wherein the nutritional composition is a supplementary food or nutritional supplement for infants and toddlers.

10. The nutritional composition or medicament according to any one of claims 7 to 9, wherein the human milk oligosaccharide is selected from: (1) a combination consisting of 2′-fucosyllactose and 3-fucosyllactose; (2) a combination consisting of 2′-fucosyllactose and lacto-N-tetraose; (3) a combination consisting of 2′-fucosyllactose and 6′-sialyllactose; (4) a combination consisting of 2′-fucosyllactose, 3-fucosyllactose and lacto-N-tetraose; (5) a combination consisting of 2′-fucosyllactose, 3-fucosyllactose and 6′-sialyllactose; (6) a combination consisting of 2′-fucosyllactose, lacto-N-tetraose and 6′-sialyllactose; (7) a combination consisting of 3-fucosyllactose and lacto-N-tetraose; (8) a combination consisting of 3-fucosyllactose and 6′-sialyllactose; (9) a combination consisting of 3-fucosyllactose, lacto-N-tetraose and 6′-sialyllactose; (10) a combination consisting of lacto-N-tetraose and 6′-sialyllactose; (11) a combination consisting of 2′-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose and 6′-sialyllactose; and a combination of any one of the above (1) to (11) together with 3′-sialyllactose.

11. A method for reducing infectivity of Staphylococcus aureus, comprising contacting Staphylococcus aureus with an effective amount of a human milk oligosaccharide selected from the group consisting of: 2′-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, 6′-sialyllactose and any combination thereof, and a combination formed by 3′-sialyllactose together with one or more selected from 2′-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, and 6′-sialyllactose; preferably, the effective amount is at least 1.5 mg/mL (e.g., 1.5 mg/mL to 10 mg/mL, 10 mg/mL to 30 mg/mL, or at least 30 mg/mL).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0081] FIG. 1 shows the effect of different doses of the human milk oligosaccharide 2′-FL on the survival rate of C. elegans when infected by Staphylococcus aureus. The interventions (HMOs) added during the infection phase were the same for each group in the figure as for the culture phase, respectively.

[0082] FIG. 2 shows the effect of a certain dose of the human milk oligosaccharide 2′-FL on the survival rate of C. elegans when infected by Staphylococcus aureus. The interventions (HMOs) added during the infection phase were the same for each group in the figure as for the culture phase, respectively.

[0083] FIG. 3 shows the effect of multiple different doses of the human milk oligosaccharide 2′-FL on the survival rate of C. elegans on the third day of infection by Staphylococcus aureus.

[0084] FIG. 4: pathogenic bacteria survival test using Staphylococcus aureus in the nematode model with 2′-FL tested at a concentration of 15 mg/mL. The interventions (HMOs) added during the infection phase were the same for each group in the figure as for the culture phase, respectively.

[0085] FIG. 5 shows the effect of different doses of the human milk oligosaccharide 3-FL on the survival rate of C. elegans when infected by Staphylococcus aureus. The interventions (HMOs) added during the infection phase were the same for each group in the figure as for the culture phase, respectively.

[0086] FIG. 6 shows a comparison of the different effects of different doses of the human milk oligosaccharides 2′-FL and 3-FL on the survival rate of C. elegans on the third day of infection by Staphylococcus aureus.

[0087] FIG. 7 shows the effect of different doses of the human milk oligosaccharide LNT on the survival rate of C. elegans when infected by Staphylococcus aureus. The interventions (HMOs) added during the infection phase were the same for each group in the figure as for the culture phase, respectively.

[0088] FIG. 8 shows the effect of several different doses of the human milk oligosaccharide LNT on the survival rate of C. elegans on the third day of infection by Staphylococcus aureus.

[0089] FIG. 9 shows the effect of different doses of the human milk oligosaccharide 6′-SL on the survival rate of C. elegans when infected by Staphylococcus aureus. The interventions (HMOs) added during the infection phase were the same for each group in the figure as for the culture phase, respectively.

[0090] FIG. 10 shows the effect of several different doses of the human milk oligosaccharide 6′-SL on the survival rate of C. elegans on the third day of infection by Staphylococcus aureus.

[0091] FIG. 11 shows the effect of different doses of the human milk oligosaccharide 3′-SL on the survival rate of C. elegans when infected by Staphylococcus aureus. The interventions (HMOs) added during the infection phase were the same for each group in the figure as for the culture phase, respectively.

[0092] FIG. 12 shows the effect of different doses of the human milk oligosaccharide 3′-SL on the survival rate of C. elegans on the third day of infection by Staphylococcus aureus.

[0093] FIG. 13 shows the effect of different doses of the human milk oligosaccharide compositions on the survival rate of C. elegans when infected by Staphylococcus aureus. The interventions (HMOs) added during the infection phase were the same for each group in the figure as for the culture phase, respectively.

[0094] FIG. 14 shows the effect of different doses of the human milk oligosaccharide compositions on the survival rate of C. elegans on the third day of infection by Staphylococcus aureus.

[0095] FIG. 15 shows a comparison of the different effects of 10 mg/mL of the human milk oligosaccharide composition and each of the five human milk oligosaccharides alone on the survival rate of C. elegans on the third day of infection by Staphylococcus aureus.

[0096] FIG. 16 shows a comparison of the different effects of 30 mg/mL of the human milk oligosaccharide composition and each of the five human milk oligosaccharides alone on the survival rate of C. elegans on the third day of infection by Staphylococcus aureus.

[0097] FIG. 17 shows a comparison of the different effects of 30 mg/mL of the human milk oligosaccharide composition and each of the five human milk oligosaccharides alone on the survival rate of C. elegans on the fifth day of infection by Staphylococcus aureus.

[0098] FIG. 18: pathogenic bacteria survival test using Staphylococcus aureus in the nematode model with composition A tested at concentrations of 8 and 15 mg/mL. The interventions (HMOs) added during the infection phase were the same for each group in the figure as for the culture phase, respectively.

[0099] FIG. 19: pathogenic bacteria survival test using Staphylococcus aureus in the nematode model with the composition G tested at concentrations of 8 and 15 mg/mL. The interventions (HMOs) added during the infection phase were the same for each group in the figure as for the culture phase, respectively.

[0100] FIG. 20: pathogenic bacteria survival test using Staphylococcus aureus in the nematode model with the composition H tested at concentrations of 8 and 15 mg/mL. The interventions (HMOs) added during the infection phase were the same for each group in the figure as for the culture phase, respectively.

[0101] FIG. 21: pathogenic bacteria survival test using Staphylococcus aureus in the nematode model with the composition J tested at concentrations of 8 and 15 mg/mL. The interventions (HMOs) added during the infection phase were the same for each group in the figure as for the culture phase, respectively.

[0102] FIG. 22: percentage of nematode survival after 5 days of infection. Nematodes were co-cultured with the HMO compositions A, G, H, J at different doses (8 and 15 mg/mL). The dotted line shows nematode survival in the absence of any interventions upon Staphylococcus aureus infection. The asterisks represent the statistical differences between the test groups and the S. aureus control group (no intervention).

[0103] FIG. 23 shows the observation of the presence or absence of a direct bacteria inhibition of the various human milk oligosaccharides alone co-cultured with Staphylococcus aureus in a Petri dish disc diffusion test and the comparison with the positive control gentamicin.

DETAILED DESCRIPTION OF THE INVENTION

[0104] The technical solutions in the embodiments of the present invention will be clearly and fully described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of, but not all of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtainable by those of ordinary skill in the art fall within the scope of protection of the present invention.

[0105] Unless otherwise specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the relevant field.

[0106] In addition, to avoid repetition, the common procedures that are necessary for the experiments in each example, such as culture of strains, are listed below.

[0107] Materials and Methods

TABLE-US-00001 TABLE 1 Tested uman milk oligosaccharide samples English Chinese Name English Name Abbreviations 2′-  2′-fucosyllactose 2′-FL 3-  3-fucosyllactose 3-FL Lacto-N-Tetraose LNT 3′-  3′-sialyllactose 3′-SL 6′-  6′-sialyllactose 6′-SL Human Milk HMO mix Oligosaccharide mix

TABLE-US-00002 TABLE 2 Formulation of the tested human milk oligosaccharide compositions Ratios (%) 2′-FL 3-FL LNT 3′-SL 6′-SL Composition A 53 21 16 5 5 Composition G 5 41 32 22 0 Composition H 30 30 25 12 3 Composition J 2 0 10 80 8

[0108] Human milk oligosaccharide solutions were prepared with distilled water and incubated in Petri dishes containing a nematode growth medium at different final concentrations (1, 10, and 30 mg/mL, respectively). To better determine the range of the ideal concentration, a preliminary screening on the dose-effect relationship of 2′-FL was first performed, and the 2′-FL concentrations used were 0.001, 0.01, 0.1, 1 and 10 mg/mL. For the HMO compositions, included were a test concentration of 8 mg/mL and a 15 mg/mL, respectively, after the addition into the nematode growth medium.

[0109] Bifidobacterium animalis BB-12 was grown in an MRS medium supplemented with 1% cysteine and incubated overnight at 37° C. in an anaerobic environment. Cells were harvested and rinsed with a saline solution, adjusted for bacterial concentration, and cultivited in plates containing nematode growth medium at a final concentration of 1×10.sup.8CFU.

[0110] Nematode Infection Model and Lifespan Assay

[0111] Age-consistent nematodes were obtained and cultured in Petri dishes containing nematode agar media (nematode media containing E. coli OP50 as food) with different doses of HMO (1, 8, 10, 15, 30 mg/mL) for co-culture. After the nematodes came into adulthood, they were transferred to petri dishes inoculated with Staphylococcus aureus ATCC25923 (supplemented at approximately 10.sup.8 to 10.sup.9CFU/mL) to simulate the condition of infection by Staphylococcus aureus. The intervening substances (HMO) added in the infection stage of each group were the same as those in the culture stage. Two controls were used, one without the pathogen (C. elegans dish containing E. coli OP50) and one with Staphylococcus aureus infection without any intervention (dish with Staphylococcus aureus only). In addition, a group in which an intervening substance was added in the culture phase and no intervention in the infection phase, and a group in which the intervention substance was not added in the cultivation phase but added in the infection phase were set up.

[0112] Several days into the culture of the nematodes, their survival rate was counted on a daily basis. If the nematodes do not respond to the platinum wire, they were considered dead. Two independent assays were carried out for each condition (that is, unless otherwise specified, the experimental data in the examples were the results of two independent experiments).

[0113] Statistical comparative analysis of survival curves was performed, and log rank survival significance analysis was performed using the GraphPad Prism 4 or GraphPad Prism 9 statistical software package. One-way ANOVA was used to analyze for significant differences in survival rates among the groups on the third or fifth day of infection. For the comparison of the groups, an asterisk*represents the degree of the significant difference, with ****indicating P<0.0001, and ***indicating P<0.001. NS means not significant.

EXPERIMENTAL RESULTS

Example 1. Effects of Different Concentrations of the Human Milk Oligosaccharide 2′-FL on the Survival Rate of Nematodes after Staphylococcus aureus Infection

[0114] First, to determine the dose range of the HMO tested in the experiments, six different doses of 2′-FL were tested. As shown in FIG. 1, doses at 1 mg/mL or less did not show any effects, and 10 mg/mL caused a considerable increase in nematode survival rate which was significant (P<0.0001). Therefore, as shown in FIG. 2, higher doses were tested, and when the dose of 2′-FL reached 30 mg/mL, the protective effect was significantly increased, and the effect was significant (P<0.0001). Seen as such, 2′-FL had a significant protective effect against Staphylococcus aureus, which was dose-dependent and optimal at 30 mg/mL. The histogram in FIG. 3 shows the different effects of different doses of 2′-FL on the survival rate of nematodes after being infected by Staphylococcus aureus for 3 days, showing that the protective effect is more pronounced at higher concentrations.

[0115] In another batch of experiments of the present invention, the improvement of the sole HMO 2′-FL at a concentration of 15 mg/mL on nematode survival rate after Staphylococcus aureus infection was also evaluated, as in the results shown in FIG. 4. It can be seen that at this concentration, 2′-FL alone significantly improves the survival rate of nematodes and significantly prolongs the lifespan of nematodes, demonstrating the regulation and enhancement of 2′-FL on the immune function of biological organisms, especially the innate immunity, and also showing a great potential of 2′-FL in improving the anti-aging of biological organisms.

Example 2. Effect of Different Concentrations of the Human Milk Oligosaccharide 3-FL on Nematode Survival Rate after Staphylococcus aureus Infection

[0116] As shown in FIG. 5, a dose-effect relationship was also observed for 3-FL, with greater protection shown at higher doses. At the concentration of 10 and 30 mg/mL, 3-FL had a better protective function (P<0.0001), with the effect maximized at 30 mg/mL. There was no effect at 1 mg/mL. As shown in FIG. 6, comparison of the data on the effect of 3-FL and 2′-FL as well as Bifidobacterium animalis BB-12 on the survival rate of nematodes showed that upon infection by Staphylococcus aureus for 3 days, the same effect was achieved with 3-FL at 10 mg/mL as that with 2′-FL at 30 mg/mL. As such, 3-FL was more effective in protecting nematodes from Staphylococcus aureus infection than 2′-FL at the same dose. It was deducted that 3-FL at less than 10 mg/mL had the potential to have a notably better protection on nematode survival in the presence of Staphylococcus aureus infection than that of the negative control group.

Example 3. Effects of Different Concentrations of the Human Milk Oligosaccharide LNT on Nematode Survival Rate after Staphylococcus aureus Infection

[0117] As shown in FIG. 7, similarly, when LNT was added at a concentration of 1 mg/mL, the survival rate of Caenorhabditis elegans (in short, nematodes) after Staphylococcus aureus infection was not greatly improved, but when LNT was added at a concentration of 10 mg/mL mL or 30 mg/mL, the survival rate of nematodes after Staphylococcus aureus infection was significantly improved. Seen as such, LNT also has a dose-effect relationship in the regulation of nematode growth after Staphylococcus aureus infection. The histogram in FIG. 8 shows the different effects of different doses of LNT on the survival rate of nematodes after being infected by Staphylococcus aureus for 3 days, showing that the protective effect is more pronounced at higher concentrations.

Example 4. Effects of Different Concentrations of the Human Milk Oligosaccharide 6′-SL on the Survival Rate of Nematodes after Staphylococcus aureus Infection

[0118] As shown in FIG. 9, a similar effect was also observed in 6′-SL:when 6′-SL was added at a concentration of 1 mg/mL, the survival rate of Caenorhabditis elegans (in short, nematodes) after Staphylococcus aureus (in short, S. aureus) infection was not greatly improved, but when 6′-SL was added at a concentration of 10 mg/mL or 30 mg/mL, the survival rate of nematodes after infection with S. aureus was significantly improved. Seen as such, 6′-SL also has a dose-effect relationship in the regulation of nematode growth after Staphylococcus aureus infection. The histogram in FIG. 10 shows the different effects of different doses of 6′-SL on the survival rate of nematodes after being infected by Staphylococcus aureus for 3 days, showing that the protective effect is more pronounced at higher concentrations. Also, the survival rate with 6′-SL at 10 mg/mL was similar to that at 30 mg/mL, and compared with other tested HMOs alone, it was found that the survival rate of nematodes under the protection of 6′-SL was higher, up to 75%. It was deducted that 6′-SL at lower than 10 mg/mL had a potential to have a notably better protection for nematode survival in the presence of Staphylococcus aureus infection than that of the negative control group.

[0119] In addition, three concentrations (1 mg/mL, 10 mg/mL and 30 mg/mL) were also tested in the same way for the 6′-SL isomer, 3′-SL, but no significant effect was observed (FIG. 11, FIG. 12). Thus, the response to Staphylococcus aureus infection is affected by the specific structure of the material, and not all oligosaccharide materials can be effective, while different materials may also have different effects.

Example 5. Effects of Different Concentrations of Human Milk Oligosaccharide Compositions on the Survival Rate of Caenorhabditis elegans after Staphylococcus aureus

Infection, and Comparison with the Same Dose of the .SUB.individual .HMOs

[0120] First, to determine the dose range of the HMO composition tested in the experiment, six different doses of the HMO Composition A were tested. As shown in FIG. 13, doses at 1 mg/mL or less did not show any effect, while 10 mg/mL caused a considerable increase in nematode survival rate with a significant effect (P<0.0001). Therefore, as shown in FIG. 13, higher doses were tested, and when the dose of HMO Composition A reached 30 mg/mL, the protective effect was significantly increased, and the effect was significant (P<0.0001). Seen as such, the HMO Composition A had a significant protective effect against Staphylococcus aureus, which was dose-dependent and more desirable at 10 mg/mL and 30 mg/mL. The histogram in FIG. 14 shows the different effects of different doses of the HMO Composition A on the survival rate of nematodes after being infected by Staphylococcus aureus for 3 days, showing that the protective effect is more pronounced at higher concentrations.

[0121] In addition, on the third day of culture with Staphylococcus aureus, the human milk oligosaccharides composition A was compared with each HMO at doses of 10 mg/mL (FIG. 15) and 30 mg/mL (FIG. 16), and it was found that at 10 mg/mL, the effect of the human milk oligosaccharides composition A was better than that of the HMOs such as 2′-FL, LNT and 3′-SL alone at the same dose, which was significant. At 30 mg/mL, the effect of the human milk oligosaccharide composition A was better than that of the HMOs such as 2′-FL, LNT and 3′-SL alone at the same dose, which was significant, and had a tendency to be better than that of 3-FL and 6′-SL alone, showing a synergistic effect to a certain extent.

[0122] On the fifth day of culture with Staphylococcus aureus, the human milk oligosaccharide composition A was compared with each HMO again at a dose of 30 mg/mL (FIG. 17), and the effect of the human milk oligosaccharide composition was better than that of all the tested HMOs, including 2′-FL, 3-FL, LNT, 3′-SL and 6′-SL, alone at the same dose, which was significant and demonstrated a synergistic effect and advantage of the composition.

[0123] In another batch of experiments of the present invention, the HMO composition A was also evaluated for the improvement of the survival rate of nematodes after infection by Staphylococcus aureus at a concentration of 8 mg/mL and 15 mg/mL, with the results shown in FIG. 18. It could be seen that at the concentration of 8 mg/mL (P<0.001) and 15 mg/mL (P<0.0001), the composition A could improve the survival rate of nematodes, showing a protective effect against Staphylococcus aureus infection. Comparison of the protective effects of both concentrations indicated that the protective effect was higher at the concentration of 15 mg/mL (P<0.001). Seen as such, a dose-effect response of the composition was observed at this ratio.

[0124] Likewise, the HMO composition G was compared at two different concentrations, with the results shown in FIG. 19. It was found that at a concentration of 15 mg/mL, the HMO composition had a stronger protective effect on nematode survival under Staphylococcus aureus infection (P<0.0001). At a concentration of 8 mg/mL, the composition G also showed a significant protective effect (P<0.0001) compared to the control without any test substance added. Seen as such, a dose-effect response of the composition was observed at this ratio.

[0125] FIG. 20 presents nematode survival curves of the HMO composition H at two different concentrations. It can be seen that both concentrations of 8 mg/mL (P<0.001) and 15 mg/mL (P<0.0001) have significantly higher survival rates than the control group without the test substance, in which the protective effect of the HMO composition H at 15 mg/mL is slightly higher than that at 8 mg/mL (P<0.05). Seen as such, a dose-effect response of the composition was observed at this ratio.

[0126] Also evaluated in the present invention was the effect of the HMO Composition J. As shown in FIG. 21, both concentrations of 8 mg/mL (P<0.01) and 15 mg/mL (P<0.05) slightly improve the survival rate compared to the control group without the test substance, and there is no difference in the survival-improving effect between the two concentrations (P>0.05). Seen as such, no dose-effect response of the composition was observed at this ratio.

[0127] The protective effects of the tested HMO compositions at various ratios on the survival rate of nematodes under Staphylococcus aureus infection were compared, with the results shown in FIG. 22. It was found that on the fifth day post-infection, a protective effect on the nematode survival was shown at all ratios. Among them, the HMO composition A and composition G, both under the condition of 15 mg/mL, showed the highest protective effect (P<0.0001). In addition, the HMO composition G at 15 mg/mL also resulted in higher nematode survival on the fifth day after infection (P<0.0001). Almost all the groups tested had lower protection at 8 mg/mL than that at 15 mg/mL, except for the HMO Composition J. The protective effect of HMO composition J on nematode survival was the lowest at both doses, and there was no difference between the doses (P>0.05).

[0128] As such, the HMO composition A, the HMO composition G, and the HMO composition H all showed a good dose-effect response, that is, the protective effect at a higher concentration was significantly better than that of a lower concentration. However, the HMO composition J did not show a dose-effect response, that is, there was no difference in the protective effect between higher and lower concentrations. Also, compared with the negative control, the effect of the HMO composition J was inferior on improving the survival rate than those of the HMO composition A, the HMO composition G and the HMO composition H at different ratios.

Example 6. Disc Diffusion Bacteriostatic Zone Experiment

[0129] In order to determine whether the tested materials themselves have an antibacterial effect, a bacteriostatic zone experiment for disk diffusion was carried out. Discs were prepared with sterile filter paper impregnated with the tested HMO materials (10 mg/mL, 30 mg/mL), and the discs were dried overnight under sterile conditions. Staphylococcus aureus ATCC25923 (1.0×10.sup.6CFU/mL) was spread evenly on the surface of a NGM agar plate, and then the discs were placed on the surface of the inoculated agar plate petri dish. Gentamicin (200 μg/mL) was used as a positive control. All dishes were incubated at 37° C. for 18 hours. Whether the test materials can directly inhibit bacterial growth or has antibacterial activity was confirmed by observing whether a transparent annular ring was formed around the disc.

[0130] FIG. 23 shows the observation of the presence or absence of a direct bacteria inhibition of the various human milk oligosaccharides alone co-cultured with Staphylococcus aureus in a Petri dish disc diffusion test and the comparison with the positive control gentamicin.

[0131] In order to investigate whether the protective effects of individual HMOs or their compositions against Staphylococcus aureus infection, as presented in previous tests, were derived from direct bactericidal or bacteriostatic function, a test was carried out in which the test material was incubated with the bacteria population and observed whether an bacteriostatic zone of was formed. As shown in the figure, at two different concentration (left: 10 mg/mL, right: 30 mg/mL), none of the human milk oligosaccharides alone formed a transparent annular bacteriostatic zone around the inoculation site. The positive control, gentamicin, showed a bacteriostatic effect, which was marked by the formation of a transparent annular bacteriostatic zone. Seen as such, the protective effects of each individual human milk oligosaccharide and the compositions thereof in this study on nematode against Staphylococcus aureus infection are not resulted from the direct bactericidal function of the materials, but a possible underlying mechanism by improving the resistance of the organism against S. aureus infection, such as activating the innate immune system of the organism, which in turn plays a role in the resistance and protection against bacteria.

[0132] Described above are only preferred embodiments of the present invention, which are not intended to limit the present invention. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.