Composition containing human milk oligosaccharide and probiotics, application thereof, and milk powder and microbial preparation containing the same

Abstract

The present invention relates to a composition comprising human milk oligosaccharides and probiotics, its application, and milk powder and microbial preparations comprising the same. The human milk oligosaccharide is lactose-N-trisaccharide LNT-II, and the probiotic is animal bifidobacterium. The combined use of LNT II with animal bifidobacterium at a specific concentration of the present invention can improve the competitive adhesion ability of animal bifidobacterium lactis subspecies relative to pathogenic bacteria, thereby achieving the effect of competitive inhibition of pathogenic bacteria.

Description

Technical Field

[0001] The present invention relates to the field of food technology, and in particular to a composition comprising human milk oligosaccharides and probiotics, an application thereof, and milk powder and a microbial preparation comprising the same. Background Art

[0002] Intestinal flora is an important component of the human intestinal microecological system and plays an important role in human health. Early studies have found that intestinal flora is related to digestion, absorption, metabolism, immunity and other functions, and is also involved in the occurrence of diseases such as tumors, diarrhea, obesity, and hyperlipidemia. As research deepens, a large amount of evidence shows that changes, instability, and diversity of intestinal flora are characteristics of a series of intestinal dysfunction, metabolic and immune diseases.

[0003] Currently, 14 strains have been used to improve the health of infants and young children. Both prebiotics and probiotics are regarded as microecological management tools to improve the health of the body, which can change, regulate and reorganize the existing intestinal flora. After passing through the gastrointestinal tract, probiotics reach the intestine in a certain number. Adherence to the intestinal mucosa is an important prerequisite for them to exert their physiological effects. In addition, this adhesion is also an important way for probiotics to inhibit pathogenic bacteria. Probiotics and human milk oligosaccharides have a synergistic effect, so my country's infant formula foods often use the method of adding prebiotics and probiotics at the same time to enhance the probiotic effect.

[0004] With the continuous advancement of chemical and biosynthetic technologies, lactose-N-trisaccharide (LNT-Ⅱ) can be obtained by enzymatic synthesis or acid hydrolysis, and has been proven to have excellent safety characteristics and no genotoxicity. Animal Bifidobacterium has multiple probiotic functions and is also called Bifidobacterium lactis. However, the synergistic antibacterial effect of animal Bifidobacterium and LNT-Ⅱ has not been studied. Summary of the invention

[0005] In view of this, the present invention provides a composition comprising human milk oligosaccharides and probiotics, wherein the human milk oligosaccharides are LNT II, ​​and the probiotics are animal Bifidobacterium.

[0006] Furthermore, another object of the present invention is to provide a composition comprising human milk oligosaccharides and probiotics for use in preparing a product for regulating intestinal flora. The composition achieves the effect of inhibiting pathogenic bacteria by improving the competitive adhesion ability of animal Bifidobacterium lactis subspecies with pathogenic bacteria. The product can be a food or a health product.

[0007] Furthermore, another object of the present invention is to provide an application of the composition in preparing food or health care products.

[0008] Furthermore, another object of the present invention is to provide a milk powder comprising the composition.

[0009] Furthermore, another object of the present invention is to provide a microbial preparation comprising the composition.

[0010] The above-mentioned object of the present invention is achieved through the following technical solutions.

[0011] In one aspect, the present invention provides a composition comprising human milk oligosaccharides and probiotics, wherein the human milk oligosaccharides are lactose-N-trisaccharide (LNT-Ⅱ), and the probiotics are animal Bifidobacterium ( Bifidobacterium animalis ).

[0012] Optionally, the animal Bifidobacterium is animal Bifidobacterium lactis subsp. Bifidobacterium animalis subsp . lactis ).

[0013] Optionally, the Bifidobacterium animalis subspecies lactis is selected from the group consisting of Bifidobacterium animalis subspecies lactis CP-9 and HN019, preferably Bifidobacterium animalis subspecies lactis CP-9.

[0014] Optionally, the concentration of animal Bifidobacterium is 10 7 CFU / mL~10 9 CFU / mL, preferably 10 8 CFU / mL~10 9 CFU / mL, more preferably 10 9 CFU / mL; and / or the content of LNT-Ⅱ is 0.25w / v%~0.50w / v%, preferably 0.30w / v%~0.50w / v%, preferably 0.40w / v%~0.50w / v%, more preferably 0.50w / v%.

[0015] In another aspect, the present invention provides use of the composition in preparing food or health care products.

[0016] In yet another aspect, the present invention provides use of the composition in preparing a product for regulating intestinal flora.

[0017] Optionally, the composition is used to improve the competitive adhesion ability of animal Bifidobacterium lactis subspecies with pathogenic bacteria.

[0018] Optionally, the pathogenic bacteria is Salmonella typhimurium ( Staphylococcus aureus ) or Staphylococcus aureus ( Staphylococcus aureus ).

[0019] In yet another aspect, the present invention provides a milk powder comprising the composition.

[0020] In yet another aspect, the present invention provides a microbial preparation comprising the composition.

[0021] The beneficial effects achieved by the present invention include but are not limited to:

[0022] The invention discloses that the combined use of LNT II with a specific concentration and animal bifidobacterium can improve the competitive adhesion ability of animal bifidobacterium lactis subspecies relative to pathogenic bacteria, so as to achieve the effect of competitively inhibiting pathogenic bacteria. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution of the present invention will be clearly and completely described in combination with the following specific implementation methods. Obviously, the specific implementation methods described are part of the implementation methods of the present invention. Based on the described implementation methods of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] The terms "competitive inhibition", "competitive adhesion inhibition" and the like used in the present invention mean that bifidobacteria can reduce the adhesion of pathogenic bacteria through competitive occupation, colonization and reproduction.

[0025] The terms "repulsion and antibacterial effect", "repulsion and adhesion and antibacterial effect" and the like used in the present invention mean that bifidobacteria can reduce the adhesion of pathogenic bacteria by repelling pathogenic bacteria.

[0026] Example

[0027] The present invention is further described below in conjunction with the embodiments. The embodiments of the present invention are only used to exemplarily describe the technical solutions of the present invention, but do not mean that the technical solutions of the present invention are limited to these specific embodiments.

[0028] It should be noted that the experimental methods used in the present invention are all conventional methods unless otherwise specified.

[0029] Unless otherwise specified, the reagents and materials used in the present invention are prepared by conventional methods or obtained from commercial channels.

[0030] The experimental methods and test substances used in each embodiment and control are as follows:

[0031] 1. Experimental preparation steps

[0032] 1.1 Strain activation

[0033] Animal Bifidobacterium lactis subsp. lactis CP-9 and HN019 (CP-9 was purchased from Jinqiao Biotechnology Co., Ltd., and HN019 was purchased from Danisco USA Inc.) were stored in the state of freeze-dried bacterial powder before the experiment. During the experiment, they were inoculated in modified Bifidobacterium MRS liquid culture medium for activation, and then separated and purified on modified MRS agar plates (0.05% cysteine ​​hydrochloride solution, 0.005% mupirocin lithium salt, filtered through a 0.22 μm microporous filter membrane and added to MRS culture medium).

[0034] Lactobacillus rhamnosus HN001 and MP108 (HN001 was purchased from International flavors and fragrances inc, and MP108 was purchased from Jinqiao Biotechnology Co., Ltd.) were stored in the state of freeze-dried powder before the experiment, inoculated in MRS liquid culture medium for activation during the experiment, and then isolated and purified on MRS agar plates.

[0035] Salmonella typhimurium ATCC 13311 and Staphylococcus aureus ATCC 6538 (both purchased from Shanghai Biotechnology Collection Center (SHBCC)) were stored in glycerol tubes, inoculated into LB liquid medium for activation during the experiment, and then isolated and purified on LB agar plates.

[0036] 1.2 Preparation of suspension solution

[0037] The suspension solution used in the present invention comprises:

[0038] (1) PBS solution;

[0039] (2) LNT II solution: PBS solution containing LNT II (purchased from Shandong Henglu Biotechnology Co., Ltd.), where the final concentration of LNT-II is 5.0 mg / mL (0.5%, w / v);

[0040] (3) 3'-FL solution: PBS solution containing 3'-fucosyllactose (3'-FL), where the final concentration of 3'-FL is 5.0 mg / mL (0.5%, w / v);

[0041] (4) 3'-SL solution: PBS solution containing 3'-sialyllactose (3'-SL), wherein the final concentration of 3'-SL is 5.0 mg / mL (0.5%, w / v);

[0042] (6) Combination solution 1: PBS solution containing LNT II and 3'-FL oligosaccharide combination, with the final concentration of LNT II being 2.5 mg / mL (0.25%, w / v) and the final concentration of 3'-FL being 2.5 mg / mL (0.25%, w / v);

[0043] (7) Combination solution 2: PBS solution containing LNT II and 3'-SL oligosaccharide combination, with the final concentration of LNT II being 2.5 mg / mL (0.25%, w / v) and the final concentration of 3'-SL being 2.5 mg / mL (0.25%, w / v);

[0044] (8) Combination solution 3: PBS solution containing a combination of LNT II, ​​galacto-oligosaccharides (GOS) and fructo-oligosaccharides (FOS), with the final concentration of LNT II being 2.5 mg / mL (0.25%, w / v), the final concentration of GOS being 1.25 mg / mL (0.125%, w / v), and the final concentration of FOS being 0.25 mg / mL (0.25%, w / v).

[0045] 1.3 Preparation of strain suspension

[0046] Animal Bifidobacterium lactis subsp. CP-9 and HN019 were inoculated into modified MRS medium at 3% inoculum. After 24 h of culture, the cells were collected by centrifugation (10,000 r / min, 2 min) and washed three times with PBS. Finally, the viable cell count was adjusted to 10 with a suspension solution. 9 CFU / mL, for future use.

[0047] Lactobacillus rhamnosus HN001 and MP108 were inoculated into MRS medium at a 3% inoculum. After culturing for 24 h, the cells were collected by centrifugation (10,000 r / min, 2 min) and washed three times with PBS. Finally, the number of viable cells was adjusted to 10 with a suspension solution. 9 CFU / mL, for future use.

[0048] Salmonella typhimurium ATCC 13311 and Staphylococcus aureus ATCC 6538 were inoculated into LB medium at a 3% inoculum. After culturing for 12 h, the cells were collected by centrifugation (10,000 r / min, 2 min) and washed three times with PBS. Finally, the number of viable cells was adjusted to the order of 10 using a suspension solution. 9 CFU / mL, for future use.

[0049] 1.4 Caco-2 cell culture

[0050] Human colorectal adenocarcinoma cells (Caco-2, purchased from Hunan Fenghui Biotechnology Co., Ltd.) were stored in liquid nitrogen and passaged after activation. When Caco-2 cells were stable, they were cultured until they formed a dense monolayer with good morphology and clear boundaries. Subsequently, Caco-2 cells were cultured at 2×10 5The cells were seeded at a concentration of 10 cells / well in a 24-well cell culture plate and cultured at 5% CO2, 95% relative humidity and 37°C until a monolayer of cells was formed for subsequent adhesion assays.

[0051] 2. Examples and Comparative Examples

[0052] Example 1. Effect of oligosaccharides on the adhesion of pathogenic bacteria

[0053] Wash the Caco-2 monolayer cells twice with PBS solution (pH 7.2), add 200 μL of pathogenic bacteria suspension (the content of pathogenic bacteria was determined to be about 2×10 9 CFU / mL). The suspension solutions used in the strain suspensions were PBS solution, LNT II solution, 3'-FL solution, 3'-SL solution, combination solution 1, combination solution 2 and combination solution 3.

[0054] The pathogens were incubated with cells for 2 h at 37 °C, 5% CO2, and 95% relative humidity. The Caco-2 monolayer cells were washed three times with PBS solution (pH 7.2) to remove unbound pathogens. Subsequently, 150 μL of 0.25% trypsin digestion solution was added, and after digestion at 37 °C for 15 min, 350 μL of MEM culture medium supplemented with 10% fetal bovine serum was added to each well to terminate the digestion. The solution in the well was serially diluted 10-fold, and the number of viable bacteria was tested by plate colony counting method. The number of viable bacteria counted was the number of bacteria adhering to the cells.

[0055] Table 1 Effects of different oligosaccharides on the adhesion of pathogenic bacteria

[0056]

[0057] Note: Different superscript letters in the same column indicate significant differences ( p <0.05).

[0058] According to the results shown in Table 1, the addition of different oligosaccharides or oligosaccharide combinations promoted the adhesion of pathogenic bacteria to Caco-2 cells to varying degrees. For Salmonella typhimurium and Staphylococcus aureus, the addition of different oligosaccharides or oligosaccharide combinations can promote the adhesion of pathogenic bacteria on Caco-2 cells, among which LNT II solution has no inhibitory effect on the adhesion of pathogenic bacteria.

[0059] Example 2. Effect of the combination of oligosaccharides and animal Bifidobacterium on the adhesion ability of pathogenic bacteria

[0060] Wash the Caco-2 monolayer cells twice with PBS (pH 7.2), add 200 μL of pathogenic bacteria (about 10 9CFU / mL) and Bifidobacterium animalis (approximately 10 9 CFU / mL). The pathogenic bacteria is Salmonella typhimurium ATCC 13311 or Staphylococcus aureus ATCC 6538; the animal Bifidobacterium is animal Bifidobacterium lactis subspecies CP-9 or HN019; the suspension solution is PBS solution, LNT II solution, 3'-FL solution, 3'-SL solution, combination solution 1, combination solution 2 or combination solution 3.

[0061] The mixed bacterial suspension was incubated for 2 h at 37 ° C, 5% CO2 and 95% relative humidity. The Caco-2 monolayer cells were washed three times with PBS solution (pH 7.2) to remove unbound pathogenic bacteria and animal Bifidobacterium. Subsequently, 150 μL of 0.25% trypsin digestion solution was added, and after digestion at 37 ° C for 15 min, 350 μL of MEM culture medium supplemented with 10% fetal bovine serum was added to each well to terminate the digestion. The solution in the well was diluted 10 times in a continuous gradient, and the number of viable bacteria was tested by the plate colony counting method. The number of viable bacteria counted was the number of bacteria attached to the cells. The results are shown in Tables 2 to 4:

[0062] Table 2. Effects of oligosaccharides combined with CP-9 on the adhesion ability of pathogenic bacteria

[0063]

[0064] Note: Different superscript letters in the same column indicate significant differences ( p <0.05).

[0065] According to the results in Table 2, Bifidobacterium animalis subsp. lactis CP-9 has competitive inhibition on both Staphylococcus aureus and Salmonella. When CP-9 is added alone, the adhesion amount of Staphylococcus aureus ATCC 6538 and Salmonella typhimurium ATCC 13311 to Caco-2 cells increases from 2.46×10 9 CFU / mL and 1.18×10 8 The CFU / mL decreased to 1.16×10 9 CFU / mL and 0.61×10 8 CFU / mL.

[0066] At the same time, the combined use of CP-9 and LNT-Ⅱ solution further enhanced the competitive inhibition effect of CP-9 on Staphylococcus aureus ATCC 6538 and Salmonella typhimurium ATCC 13311. That is to say, although LNT II solution has no inhibitory effect on the adhesion of pathogenic bacteria, the combination of CP-9 and LNT-Ⅱ solution can further reduce the adhesion of pathogenic bacteria. However, when CP-9 is used in combination with other oligosaccharides or multiple oligosaccharides (3'-FL solution, 3'-SL solution, combination solution 1, combination solution 2 or combination solution 3), the adhesion of pathogenic bacteria is increased compared with the addition of CP-9 alone.

[0067] It can be seen that animal Bifidobacterium lactis subspecies CP-9 and LNT II have a synergistic antibacterial effect.

[0068] Table 3. Effects of oligosaccharides combined with HN019 on the adhesion ability of pathogenic bacteria

[0069]

[0070] Note: Different superscript letters in the same column indicate significant differences ( p <0.05).

[0071] According to the results in Table 3, Bifidobacterium animalis subspecies lactis HN019 has competitive inhibition on both Staphylococcus aureus and Salmonella. When HN019 was added alone, the adhesion amount of Staphylococcus aureus ATCC 6538 and Salmonella typhimurium ATCC 13311 to Caco-2 cells increased from 24.2×10 8 CFU / mL and 1.08×10 8 The CFU / mL decreased to 4.05×10 8 CFU / mL and 0.9×10 8 CFU / mL.

[0072] At the same time, the combined use of HN019 and LNT-Ⅱ solution further enhanced the competitive inhibition effect of HN019 on Staphylococcus aureus ATCC 6538 and Salmonella typhimurium ATCC 13311. That is to say, although LNT II solution has no inhibitory effect on the adhesion of pathogenic bacteria, the combination of HN019 and LNT-Ⅱ solution can further reduce the adhesion of pathogenic bacteria. However, when HN019 is used in combination with other oligosaccharides or multiple oligosaccharides (3'-FL solution, 3'-SL solution, combination solution 1, combination solution 2 or combination solution 3), the adhesion of pathogenic bacteria is increased compared with the addition of HN019 alone.

[0073] It can be seen that animal Bifidobacterium lactis subspecies HN019 and LNT II have a synergistic antibacterial effect.

[0074] Comparative Example 1. Effect of the combination of oligosaccharides and other probiotics on the adhesion ability of pathogenic bacteria

[0075] Wash the Caco-2 monolayer cells twice with PBS (pH 7.2), add 200 μL of pathogenic bacteria (about 10 9 CFU / mL) and Lactobacillus rhamnosus (about 10 9 CFU / mL). The pathogenic bacteria is Salmonella typhimurium ATCC 13311 or Staphylococcus aureus ATCC 6538; the Lactobacillus rhamnosus is Lactobacillus rhamnosus HN001 or MP108; the suspension solution is PBS solution, LNT II solution, 3'-FL solution, 3'-SL solution, combination solution 1, combination solution 2 or combination solution 3.

[0076] The mixed bacterial suspension was incubated for 2 h at 37 °C, 5% CO2, and 95% relative humidity. The Caco-2 monolayer cells were washed three times with PBS solution (pH 7.2) to remove unbound pathogenic bacteria and Lactobacillus rhamnosus. Subsequently, 150 μL of 0.25% trypsin digestion solution was added, and after digestion at 37 °C for 15 min, 350 μL of MEM culture medium supplemented with 10% fetal bovine serum was added to each well to terminate the digestion. The solution in the well was diluted 10 times in a series, and the number of viable bacteria was tested by the plate colony counting method. The number of viable bacteria counted was the number of bacteria attached to the cells. The results are shown in Table 5.

[0077] (1) Oligosaccharides and Lactobacillus rhamnosus HN001

[0078] Table 4. Effects of oligosaccharides combined with HN001 on the adhesion of pathogenic bacteria

[0079]

[0080] Note: Different superscript letters in the same column indicate significant differences (P<0.05)

[0081] According to the results in Table 4, Lactobacillus rhamnosus HN001 has competitive inhibition on both Staphylococcus aureus and Salmonella. However, the combination of Lactobacillus rhamnosus HN001 and one or more oligosaccharide solutions did not show a synergistic effect of the combination of oligosaccharides and HN001 on the competitive adhesion and antibacterial ability of Staphylococcus aureus and Salmonella pathogens.

[0082] (2) Oligosaccharides and Lactobacillus rhamnosus MP108

[0083] Table 5. Effect of oligosaccharide combined with MP108 on the adhesion ability of pathogenic bacteria

[0084]

[0085] Note: Different superscript letters in the same column indicate significant differences (P<0.05)

[0086] According to the results in Table 5, Lactobacillus rhamnosus MP108 has competitive inhibition on both Staphylococcus aureus and Salmonella. However, when Lactobacillus rhamnosus MP108 was used in combination with each oligosaccharide solution, no synergistic effect of the combination of oligosaccharides and HN001 on the competitive adhesion and antibacterial ability of Staphylococcus aureus and Salmonella pathogens was shown.

[0087] In summary, the combination of LNT-Ⅱ at a specific concentration and animal Bifidobacterium can produce a synergistic effect of competitive adhesion inhibition on intestinal pathogens, thereby achieving the effect of inhibiting intestinal pathogens.

[0088] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composition comprising human milk oligosaccharides and probiotics, characterized in that: The human milk oligosaccharide is lactose-N-trisaccharide, the probiotic is animal Bifidobacterium lactis subspecies, and the animal Bifidobacterium lactis subspecies is selected from animal Bifidobacterium lactis subspecies CP-9 or HN019, Wherein, the concentration of the subspecies of Bifidobacterium animalis is 10 7 CFU / mL~10 9 CFU / mL, and / or the content of lactose-N-trisaccharide is 0.25w / v%~0.50w / v%.

2. Use of the composition comprising human milk oligosaccharides and probiotics according to claim 1 in preparing food.

3. Use of the composition comprising human milk oligosaccharides and probiotics according to claim 1 in the preparation of health products.

4. Use of the composition comprising human milk oligosaccharides and probiotics according to claim 1 in the preparation of a product for regulating intestinal flora.

5. The use according to claim 4, characterized in that The composition comprising human milk oligosaccharides and probiotics is used to improve the competitive adhesion ability of animal Bifidobacterium lactis subspecies with pathogenic bacteria.

6. The use according to claim 5, characterized in that The pathogenic bacteria is Salmonella typhimurium ( Staphylococcus aureus ) or Staphylococcus aureus ( Staphylococcus aureus ).

7. A milk powder, characterized in that: The composition comprising human milk oligosaccharides and probiotics as claimed in claim 1.

8. A microbial preparation, characterized in that: The composition comprising human milk oligosaccharides and probiotics as claimed in claim 1.

Images