Composition Containing Bifidobacterium Lactis and Human Milk Oligosaccharides and Application Thereof

Abstract

The present disclosure provides a composition comprising Bifidobacterium lactis and human milk oligosaccharides (HMOs) and use thereof. The composition is capable of effectively enhancing resistance of an organism to Staphylococcus aureus infection, improving innate immunity of an organism, and/or anti-aging, and can be added to various health foods and healthcare foods.

Claims

1. A composition comprising Bifidobacterium lactis and human milk oligosaccharides (HMOs).

2. The composition according to claim 1, wherein the Bifidobacterium lactis includes the strain Bifidobacterium lactis HN019.

3. The composition according to claim 1 or 2, wherein the HMOs include one or more of 2-fucosyllactose, 3-fucosyllactose, lactose-N-tetraose, 3-sialyllactose, and 6-sialyllactose; preferably, the HMOs include at least three or four of 2-fucosyllactose, 3-fucosyllactose, lactose-N-tetraose, 3-sialyllactose, and 6-sialyllactose in a weight ratio of (0% to 73%):(0% to 61%):(0% to 52%):(0% to 42%):(0% to 25%); more preferably, the HMOs include 2-fucosyllactose, 3-fucosyllactose, lactose-N-tetraose, 3-sialyllactose, and 6-sialyllactose in a weight ratio of (0% to 53%):(21% to 41%):(16% to 32%):(5% to 22%):(0% to 5%).

4. The composition according to claim 1 or 2, wherein the ratio of the Bifidobacterium lactis to the HMOs is (110.sup.3 to 110.sup.12 CFU):(0.1 to 10 g), preferably (110.sup.6 to 110.sup.10 CFU):(1 to 10 g).

5. Use of the composition according to any one of claims 1 to 4 in the manufacture of a food product or medicament for effectively enhancing resistance of an organism to Staphylococcus aureus infection, improving innate immunity of an organism, and/or anti-aging.

6. The use according to claim 5, wherein the enhancing resistance of an organism to Staphylococcus aureus infection includes: enhancing an individual's ability to prevent Staphylococcus aureus infection, reducing the ability of Staphylococcus aureus to infect an individual, and/or alleviating food poisoning, enteritis, pneumonia, skin infection, wound ulceration, and/or meningitis caused by Staphylococcus aureus infection in an individual.

7. The use according to claim 5, wherein the food product is a liquid beverage, a solid beverage, an oral liquid, a diary product, a tablet, or a capsule, such as milk powder, particularly infant formula powder.

8. The use according to claim 5 or 7, wherein the Bifidobacterium lactis in the composition is used in an amount of 110.sup.3 to 110.sup.12 CFU per day, preferably 110.sup.6 to 110.sup.11 CFU per day.

9. The use according to claim 5 or 7, wherein the HMOs in the composition are used in an amount of 0.015 to 15 g per day, further preferably 0.1 to 12 g per day, and more preferably 1.5 to 10 g per day.

10. A food product comprising the composition according to any one of claims 1 to 4; preferably, the Bifidobacterium lactis in the food product is used in an amount of 110.sup.3 to 110.sup.12 CFU per day, and more preferably 110.sup.6 to 110.sup.11 CFU per day; preferably, the HMOs in the food product are used in an amount of 0.015 to 15 g per day, further preferably 0.1 to 12 g per day, and more preferably 1.5 to 10 g per day.

11. Use of HMOs in improving the potency of Bifidobacterium lactis HN019 in enhancing resistance of an organism to Staphylococcus aureus infection, improving innate immunity of an organism, and/or anti-aging.

12. A method for enhancing resistance of an organism to Staphylococcus aureus infection, improving innate immunity of an organism, and/or anti-aging, comprising: administering to the organism an effective amount of the composition according to any one of claims 1 to 4.

13. The method according to claim 12, wherein the enhancing resistance of an organism to Staphylococcus aureus infection includes: enhancing an individual's ability to prevent Staphylococcus aureus infection, reducing the ability of Staphylococcus aureus to infect an individual, and/or alleviating food poisoning, enteritis, pneumonia, skin infection, wound ulceration, and/or meningitis caused by Staphylococcus aureus infection in an individual.

14. The method according to claim 12 or 13, wherein the composition according to any one of claims 1 to 4 is administered to the organism in the form of a food product, wherein the food product is a liquid beverage, a solid beverage, an oral liquid, a diary product, a tablet, or a capsule, such as milk powder, particularly infant formula powder; preferably, the Bifidobacterium lactis in the food product is used in an amount of 110.sup.3 to 110.sup.12 CFU per day, and more preferably 110.sup.6 to 110.sup.11 CFU per day; preferably, the HMOs in the food product are used in an amount of 0.015 to 15 g per day, further preferably 0.1 to 12 g per day, and more preferably 1.5 to 10 g per day.

Description

DESCRIPTION OF THE DRAWINGS

[0045] FIG. 1 shows the effect of a composition of 10 mg/mL of HMO mix A and Bifidobacterium lactis HN019 on the survival rate of Caenorhabditis elegans infected by Staphylococcus aureus. For each group indicated in the figure, the addition of intervening substance (probiotic and/or HMOs) in the infection stage is the same as that in the culture stage.

[0046] FIG. 2 shows the effect of a composition of 30 mg/mL of HMO mix A and Bifidobacterium lactis HN019 on the survival rate of Caenorhabditis elegans infected by Staphylococcus aureus. For each group indicated in the figure, the addition of intervening substance (probiotic and/or HMOs) in the infection stage is the same as that in the culture stage.

[0047] FIG. 3 shows the effect of a composition of HMO mix A and Bifidobacterium lactis HN019 on the survival rate of Caenorhabditis elegans on Day 3 after infection by Staphylococcus aureus. For each group indicated in the figure, the addition of intervening substance (probiotic and/or HMOs) in the infection stage is the same as that in the culture stage.

[0048] FIG. 4 shows the effect of a composition of HMO mix A and Bifidobacterium lactis HN019 on the survival rate of Caenorhabditis elegans on Day 5 after infection by Staphylococcus aureus. For each group indicated in the figure, the addition of intervening substance (probiotic and/or HMOs) in the infection stage is the same as that in the culture stage.

[0049] FIG. 5 is a picture of the results of the disc diffusion test of inhibition zone with gentamicin.

[0050] FIG. 6 is a picture of the results of the disc diffusion test of inhibition zone with Bifidobacterium lactis HN019.

[0051] FIG. 7 shows the different effects of the co-culture of HN019 (110.sup.8 CFU) and HMO mix A (10 mg/mL) in improving the survival rate of C. elegans before and/or during S. aureus infection.

[0052] FIG. 8 shows the effects of HMO mix A (8 mg/mL) and Bifidobacterium lactis HN019 (110.sup.8 CFU), alone and in combination, on the survival rate of C. elegans infected by Staphylococcus aureus.

[0053] FIG. 9 shows the effects of HMO mix G (8 mg/mL) and Bifidobacterium lactis HN019 (110.sup.8 CFU), alone and in combination, on the survival rate of C. elegans infected by Staphylococcus aureus.

[0054] FIG. 10 shows the effects of HMO mix H (8 mg/mL) and Bifidobacterium lactis HN019 (110.sup.8 CFU), alone and in combination, on the survival rate of C. elegans infected by Staphylococcus aureus.

[0055] In the figures: * represents p<0.05, ** represents p<0.01, *** represents p<0.001, **** represents p<0.0001, NS represents no significant difference (not significant).

DETAILED DESCRIPTION OF THE INVENTION

[0056] In order to provide a better understanding of the technical features, purposes and advantageous effects of the present disclosure, the technical solutions of the present disclosure are described in details hereinafter, which should not be construed as limitation to the implementable scope of the present disclosure.

Example 1: Effects of a Composition of an HMO Mix and Bifidobacterium lactis HN019 on the Survival Rate of Caenorhabditis elegans Infected by Staphylococcus aureus

[0057] 1. Experimental Materials and Methods

[0058] 1.1 The Tested HMO Samples are Shown in Table 1.

TABLE-US-00001 TABLE 1 English Name abbreviation Source 2-fucosyllactose 2-FL Jennewein 3-fucosyllactose 3-FL Jennewein Lacto-N-Tetraose LNT Jennewein 3-sialyllactose 3-SL Jennewein 6-sialyllactose 6-SL Jennewein HMO mix A of 5 HMOs HMO mix Jennewein

[0059] In HMO mix A, the five HMOs were in a ratio of: 53% 2-FL, 21% 3-FL, 16% LNT, 5% 3-SL, and 5% 6-SL.

[0060] A solution of HMOs was prepared with distilled water and incubated in dishes containing a nematode growth medium at different final concentrations (1, 10, 30 mg/mL, respectively).

[0061] The probiotic to be tested was grown in an MRS medium supplemented with 1% cysteine, and incubated overnight at 37 C. in an anaerobic environment. The cells were harvested and rinsed with physiological saline, the concentration of the bacteria was adjusted, and the bacteria were then inoculated on a plate containing a nematode growth medium at a final concentration of 110.sup.8 CFU.

[0062] 1.2 Nematode Infection Model

[0063] Nematodes of the same age were obtained and cultured in a dish containing a nematode agar medium (NGM medium, a nematode medium containing E. coli OP50 as food), and a composition of an HMO mix at varying dosages (8 mg/mL, 10 mg/mL, 30 mg/mL) and the probiotic (110.sup.8 CFU) was added for co-culturing (the total volume of liquid added to each nematode culture dish was 10 mL; for the test group having HMOs at a concentration of 10 mg/mL, the final amount of HMOs in each dish was 100 mg; for the test groups having HMOs at a concentration of 8 mg/mL and 30 mg/mL, the final amount of HMOs in each dish was 80 mg and 300 mg, respectively; thus, in each plate, the ratio of probiotic to HMOs was 110.sup.8 CFU:80-300 mg). After the nematodes grew into an adult form, they were transferred to dishes inoculated with Staphylococcus aureus ATCC25923 at an amount of 10.sup.8-10.sup.9 CFU/mL to simulate the infection by Staphylococcus aureus. For each group, the addition of the intervening substance (probiotic and/or HMOs) in the infection stage was the same as that in the culture stage. Two controls were set to respectively create the condition without pathogenic bacteria (nematode dishes containing E. coli OP50, i.e., the OP50 group), and the condition with pathogenic S. aureus infection but without any intervening material (dishes containing only S. aureus). Further groups wherein the intervening substance was added during the culture stage but not during the infection stage, and groups wherein the intervening substance was not added during the culturing stage but was added during the infection stage, were established.

[0064] After the nematodes were cultured for a few days, their survival rate was counted on a daily basis. If the nematodes did not respond to a platinum wire, they were considered dead. Two independent assays were carried out for each condition.

[0065] Statistical comparative analysis of the survival curves was carried out, and a log rank survival analysis of significance was carried out by using the GraphPad Prism 4 statistics software package. Differences in the effect on nematode survival between groups shown on each day were first analyzed by Two-way ANOVA, and then the comparison of the groups was done by a Tukey's post hoc test. Differences between the group of HMO mix alone or the probiotic alone and the group of the prebiotic-probiotic combination were marked with asterisks in the figures. One-way ANOVA and Dunnett's post hoc test were used to analyze the significant difference in the survival rates of each group on Day 3 or 5 after infection.

[0066] 1.3 Disc Diffusion Test of Inhibition Zone

[0067] In order to determine whether the test substances per se had an antibacterial effect, a disc diffusion test of inhibition zone was carried out. Discs were prepared with sterile filter paper, the test substances HMO (10 mg/mL, 30 mg/mL) or probiotic (110.sup.8 CFU) were introduced thereto, and the discs were dried overnight under sterile conditions. Staphylococcus aureus ATCC25923 (1.010.sup.6 CFU/mL) was uniformly applied on the surface of an NGM agar plate, and then the discs were placed on the surface of the inoculated agar plate. Gentamicin (200 g/mL) was used as a positive control. All plates were incubated at 37 C. for 18 hours. Presence or absence of a transparent ring formed around the discs was observed to determine whether the test substances were capable of directly inhibiting the growth of the bacterium or had antibacterial activity.

[0068] 2. Experimental Results

[0069] The effects of Bifidobacterium lactis HN019 and HMO mix A of five HMOs at two different doses on the survival rate of C. elegans infected by Staphylococcus aureus are shown in FIGS. 1 and 2. Throughout the entire experiment course, with the HMO mix at 10 mg/mL, the effect of the prebiotic-probiotic combination was significantly better than that of the HMO mix alone or the probiotic alone (P<0.0001). However, with the HMO mix A at 30 mg/mL, the combination of HMOs and probiotic did not show an advantage throughout the entire experiment course.

[0070] As shown in FIG. 1, with the HMO mix A at 10 mg/mL, the effects of the combination of the prebiotic HMOs and the probiotic HN019 on Days 4 and 5 were significantly better than those of the probiotic HN019 alone (p<0.05), and on Days 2, 4 and 5 were significantly better than those of the HMO mix alone (Day 2: p<0.0001; Days 4, 5: p<0.05). Regarding a synergistic effect, the survival rates with the combination of HMOs and probiotic were compared with those with the HMO mix alone or with the probiotic alone, and it was found that the survival rate with the combination of HMOs and probiotic was significantly higher than the sum of the survival rate with the HMO mix alone and the survival rate with the probiotic alone on both Days 4 and 5, indicating a synergistic effect of the combination of HMOs and probiotic (Table 2).

TABLE-US-00002 TABLE 2 Days of S. aureus + S. aureus + Syner- experi- HMO mix A S. aureus + HN019 + HMO gistic ment (10 mg/mL) HN019 mix A (10 mg/mL) effect 1 100 100 100 No 2 67 76 73 No 3 48 58 61 No 4 25 25 56 Yes 5 20 11 51 Yes

[0071] FIGS. 3 and 4 show a comparison of the effect of the combination of HMO mix A and the probiotic on the survival rate of Caenorhabditis elegans on Day 3 and Day 5 of infection. On Day 5 of infection, the combination of the prebiotic and the NH019 showed a better effect than the other test groups.

[0072] In addition, further experiments of the present disclosure demonstrate that the survival rate of Caenorhabditis elegans in the group with the intervention of the combination of the HMOs and HN019 in the culture stage but without any intervention in the infection stage was significantly higher than that of the groups without intervention either in the culture stage or in the infection stage.

[0073] In order to find out whether the protective effect of the combination of HMOs and probiotic against Staphylococcus aureus infection shown in the previous tests was due to a direct bactericidal or antibacterial effect, an inhibition zone test was carried out by culturing the test substances together with a bacterial population. As shown in FIGS. 5 and 6, the Bifidobacterium lactis HN019 strain did not form a transparent inhibition zone around the inoculation site (FIG. 6). The positive control gentamicin showed an antibacterial effect, as indicated by the formation of a transparent inhibition zone (FIG. 5). In addition, the inhibition zone test for each individual HMO at two different concentrations (10 mg/mL, 30 mg/mL) also showed a similar result, and none of the individual HMOs could directly inhibit the growth of S. aureus. Seen as such, the protective effect of the combination of HMOs and probiotic for C. elegans against Staphylococcus aureus infection shown in the present study was not due to a direct antibacterial action of the substances.

Example 2: Experiment of Efficacy in Improving Innate Immunity and Anti-Aging with a Combination of Different HMO Mixes and Bifidobacterium lactis HN019

[0074] In this example, the components and contents of the tested HMO mixes are shown in Table 3.

TABLE-US-00003 TABLE 3 2-FL 3-FL LNT 3-SL 6-SL percent- percent- percent- percent- percent- HMO mix age age age age age HMO mix A 53 21 16 5 5 HMO mix G 5 41 32 22 0 HMO mix H 30 30 25 12 3 [0075] Experiment 1 for efficacy of the tested compositions (investigation of the preventive effect of the tested substances in terms of enhancing resistance of an organism to Staphylococcus aureus infection, improving innate immunity of an organism, and/or anti-aging): nematodes of the same age were taken and cultured in a dish containing a nematode agar medium, and a composition of HMO mix A (10 mg/mL) and the probiotic HN019 (110.sup.8 CFU) in a certain ratio was added for co-culturing until the nematodes grew into an adult form. Then the nematodes were transferred to an infection plate, and were infected by the pathogenic bacterium Staphylococcus aureus ATCC25923, without addition of the composition of HMOs and probiotic. [0076] Experiment 2 for efficacy of the tested compositions (investigation of the therapeutic effect of the tested substances in terms of enhancing resistance of an organism to Staphylococcus aureus infection, improving innate immunity of an organism, and/or anti-aging): nematodes of the same age were taken and cultured in a dish containing a nematode agar medium. When the nematodes grew into an adult form, the nematodes were transferred to a plate supplemented with a composition of HMO mix A (10 mg/mL) and the probiotic HN019 (110.sup.8 CFU) in a certain ratio, and were infected by the pathogenic bacterium Staphylococcus aureus ATCC25923.

[0077] In addition, another group in which the composition of HMOs and probiotic was added in both the stages before and after the nematodes were infected was compared with the above two cases in Experiments 1 and 2 as a control group.

[0078] After the nematodes were cultured for a few days, their survival rate was counted on a daily basis. If the nematodes did not respond to a platinum wire, they were considered dead. Two control groups were further set, one without the pathogenic bacterium (a normal culturing plate supplemented with E. coli OP50 as food), and the other one as the infection control group (a plate with only S. aureus added). Two independent tests were performed for each condition. Statistical comparative analysis of the survival curves was carried out, and a log rank survival analysis of significance was carried out by using the GraphPad Prism 4 statistics software package.

[0079] Other operations during the experiments were done according to Example 1.

[0080] Experimental Results

[0081] 1. Effect of Test Substances Co-Cultured with Nematodes Before and/or During Infection

[0082] As mentioned above, in order to determine whether the test substances have a preventive effect, nematodes were co-cultured with HMO mix A and HN019 only before S. aureus infection. In order to determine whether the test substances have a therapeutic effect, nematodes were co-cultured with HMO mix A and HN019 only during S. aureus infection. Also included was a case where the test substances were co-cultured with the nematodes both before and during the infection.

[0083] The results are shown in FIG. 7. The survival curve shows that from Day 1 to Day 5 after culturing, no matter whether the test substances were added to the co-culture before the infection or during the infection, there was an improvement in the survival rate and protection for nematodes under an infecting condition with Staphylococcus aureus. Both the case where the test substances were added to the nematodes during Staphylococcus aureus infection, and the case where the test substances were present in the whole process, had similar effects in terms of the improvement in survival rate and protection against Staphylococcus aureus. Furthermore, as compared with the case where the test substances were added only before the infection, stronger protection for the survival of nematodes was seen in both the case where the test substances were added during the infection and the case where the test substances were present in the whole process.

[0084] 2. Effect of HMO Mix a (8 mg/mL) and Bifidobacterium lactis HN019, Alone or in Combination, on the Survival Rate of Nematodes Under Staphylococcus aureus Infection

[0085] The results are shown in FIG. 8. It can be seen that from Day 1 to Day 5 after culturing, both the HMO mix A alone at 8 mg/mL and the probiotic HN019 (10.sup.8 cfu) alone can significantly improve the survival rate of nematodes under an infecting condition with Staphylococcus aureus, and when they were combined, the improvement was even more significant (P<0.0001). This suggests there is an excellent synergistic effect between the probiotic HN019 and HMO mix A (8 mg/mL).

[0086] 3. Effect of HMO Mix G (8 mg/mL) and Bifidobacterium lactis HN019, Alone or in Combination, on the Survival Rate of Nematodes Under Staphylococcus aureus Infection

[0087] The results are shown in FIG. 9. It can be seen that from Day 1 to Day 5 after culturing, both HMO mix G alone at 8 mg/mL and the probiotic HN019 (10.sup.8 cfu) alone can significantly improve the survival rate of nematodes under an infecting condition with Staphylococcus aureus, and when they were combined, the improvement was even more significant. This suggests there is an excellent combinational effect between the probiotic HN019 and HMO mix G (8 mg/mL).

[0088] 4. Effect of HMO Mix H (8 mg/mL) and Bifidobacterium lactis HN019, Alone or in Combination, on the Survival Rate of Nematodes Under Staphylococcus aureus Infection

[0089] The results are shown in FIG. 10. It can be seen that from Day 1 to Day 5 after culturing, both HMO mix H alone at 8 mg/mL and the probiotic HN019 (10.sup.8 cfu) alone can significantly improve the survival rate of nematodes under an infecting condition with Staphylococcus aureus, and when they were combined, the improvement was even more significant. This suggests there is an excellent combinational effect between the probiotic HN019 and HMO mix H (8 mg/mL).

Example 3: Infant Formula Powder with Addition of a Composition of a HMO Mix and Bifidobacterium lactis HN019

[0090] This example provided formula powder for infant 0-6 months old. In this formula powder, the total protein content was 11.1 g/100 g powder, the fat content was 28.3 g/100 g powder, the carbohydrate content was 52.9 g/100 g powder, the content of the HMO mix was 2.5 g/100 g powder, and the Bifidobacterium lactis was 410.sup.8 CFU/100 g. The components and their ratio in the HMO mix were: 2-fucosyllactose (2-FL):3-fucosyllactose (3-FL):lactose-N-tetraose (LNT):3-sialyllactose (3-SL):6-sialyllactose (6-SL)=53:21:16:5:5.