A composition and a food containing the same

Abstract

The present application relates to the field of food, in particular to a composition and food and medicine containing the same. The present application finds that human milk-derived Bifidobacterium lactis can effectively utilize LNB, and accordingly provides a corresponding composition, which can improve the colonization, growth and proliferation of Bifidobacterium lactis in the intestinal tract, thereby effectively improving the efficacy of human milk-derived Bifidobacterium lactis, and enabling it to act together with other infant-derived Bifidobacterium to efficiently improve the intestinal health of infants and young children. At the same time, with the help of LNB and Bifidobacterium lactis, the abundance of Bifidobacterium in the intestinal tract of adults (especially the elderly) can be improved, and the intestinal health can be promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of food, in particular to a composition and a food containing the composition. BACKGROUND

[0002] Research has found that human milk is the best food for infants and young children, and is essential for the health of infants and young children. Currently, potential probiotic substances isolated from human milk include bifidobacteria (B. longum, B. breve, B. bifidum, etc.), lactobacillus (S. salivarius, L. gasseri, L. rhamnosus, L. fermentum, etc.), and human milk oligosaccharides (HMOs) with specific proliferation probiotics. These bacteria and human milk oligosaccharides (HMOs) are extremely important for the establishment and healthy development of the intestinal flora of infants and young children.

[0003] Existing B. longum, B. breve and B. bifidum generally have the ability to utilize part of human milk oligosaccharides (HMOs), including the ability to utilize lacto-N-tetraose (LNT) and LNT metabolites lacto-N-biose (LNB, Lacto-N-Biose I). However, the commonly used B. lactis (mostly non-human or adult sources) has not been found to have the ability to utilize human milk oligosaccharides (HMOs). Therefore, it is generally believed that B. lactis cannot interact with human milk oligosaccharides (HMOs) and has a relatively low effect on the intestinal health of infants and young children. SUMMARY

[0004] The present application accidentally found that human milk-derived B. lactis cannot utilize LNT, but can effectively utilize LNB, a metabolite of LNT, while B. lactis from fermented milk and adult sources does not have this ability. Therefore, the combination of human milk-derived B. lactis and LNB can effectively exert the function of B. lactis and also prove that human milk-derived B. lactis can help the health of infants and young children. The combination of B. lactis and LNB can also be used for adults, with the help of the specificity of LNB and the more effective function of B. lactis, to regulate intestinal health.

[0005] LNB can be used as a new type of human milk-derived prebiotic. Although it is not usually isolated from human milk and lacks the structural-functional potential of human milk oligosaccharides (HMOs), LNB is derived from type I human milk oligosaccharides (HMOs) such as LNT and LNFPI (lacto-N-fucopentaose I), which are present in the intestines of infants and young children after consuming breast milk. It is also known that LNB has specific proliferation properties for infant-derived bifidobacteria (such as Bifidobacterium longum, Bifidobacterium bifidum, etc.) and some lactobacilli. Therefore, it plays an important role in regulating intestinal flora, the health of the intestinal mucosa, and thus the health of the entire gastrointestinal tract.

[0006] Based on the above findings, the present invention first provides the use of lactose-N-disaccharide in any of the following aspects:

[0007] (1) In promoting the growth of human milk-derived Bifidobacterium lactis ( Bifidobacterium lactis ) for the colonization, growth or proliferation of plants;

[0008] (2) In the preparation of Bifidobacterium lactis derived from human milk ( Bifidobacterium lactis ) colonization, growth or proliferation of a composition.

[0009] Lactose-N-disaccharide can be used to promote the growth of human milk-derived Bifidobacterium lactis in the intestine ( Bifidobacterium lactis ) colonization, growth and proliferation to improve intestinal health and / or regulate immune levels; it can also be applied to the production of human milk-derived Bifidobacterium lactis ( Bifidobacterium lactis ) cultivation, screening and fermentation, etc.

[0010] The present invention further provides a composition comprising:

[0011] (1) Bifidobacterium lactis from human milk ( Bifidobacterium lactis );as well as

[0012] (2) At least one of the following:

[0013] (1) Bifidobacterium lactis from human milk ( Bifidobacterium lactis );as well as

[0014] (2) At least one of the following:

[0015] a) Lactose-N-disaccharide;

[0016] b) a precursor of lactose-N-disaccharide; the precursor can react with one or more digestive tract contents and / or can be decomposed by one or more digestive tract contents to obtain lactose-N-disaccharide;

[0017] c) a precursor of lactose-N-biose and a bacterium that hydrolyzes the precursor into lactose-N-biose.

[0018] By using human milk-derived Bifidobacterium lactis ( Bifidobacterium lactis The combined use of lactose-N-disaccharide (Lactose-N-disaccharide) and lactose-N-disaccharide can improve the colonization, growth, and proliferation of Bifidobacterium lactis in the intestines, thereby promoting its beneficial effects. Simultaneously, this combination also helps increase the abundance of other beneficial bacteria in the intestines (such as Bifidobacterium longum and Bifidobacterium breve), thereby improving the regulatory effect on the intestinal flora.

[0019] Although human milk-derived Bifidobacterium lactis cannot directly utilize the precursor of lactose-N-disaccharide, if its precursor can react with one or more contents of the digestive tract and / or can be decomposed by one or more contents of the digestive tract to obtain lactose-N-disaccharide, it can still be used in combination with human milk-derived Bifidobacterium lactis and achieve the effects of the present invention. In some embodiments, a precursor of lactose-N-disaccharide and a bacterium that hydrolyzes the precursor into lactose-N-disaccharide can also be added to the composition to further ensure that the precursor of lactose-N-disaccharide in the composition or the intestine can be effectively converted into lactose-N-disaccharide, thereby further facilitating its compounding effect with human milk-derived Bifidobacterium lactis.

[0020] Similarly, the effects of the present invention can also be achieved by combining all the components mentioned in (1) and (2) above.

[0021] Preferably, the human milk-derived Bifidobacterium lactis ( Bifidobacterium lactis ) is separated from breast milk. Specific separation methods include:

[0022] The breast milk was diluted and inoculated into the modified MRS medium, and then cultured at 37±2℃ under anaerobic conditions. The results showed that the lactobacillus ( Bifidobacterium lactis );

[0023] The modified MRS medium contains 0.4-0.6 g / L of L-cysteine ​​(L-cys) and 40-60 mg / L of mupirocin lithium salt (MUP), and has a pH value of 7.0±0.2.

[0024] In a preferred embodiment, the modified MRS medium contains 0.5 g / L L-cysteine ​​and 50 mg / L mupirocin lithium salt.

[0025] As preferred, the diluent for diluting the breast milk contains potassium dihydrogen phosphate 4.3-4.7 g / L, disodium hydrogen phosphate 5.8-6.2 g / L, L-cysteine hydrochloride 0.4-0.6 g / L, agar 0.9-1.1 g / L, Tween-80 0.8-1.2 mL / L; the pH value is 6.8±0.2.

[0026] In a preferred embodiment, the diluent for diluting the breast milk contains potassium dihydrogen phosphate 4.5 g / L, disodium hydrogen phosphate 6.0 g / L, L-cysteine hydrochloride 0.5 g / L, agar 1.0 g / L, Tween-80 1.0 mL / L.

[0027] In the specific implementation, the Bifidobacterium lactis from human milk source (Bifidobacterium lactis from human milk source) Bifidobacterium ) can be obtained by the following isolation method:

[0028] (1) Gradient dilution is performed on the breast milk sample in the diluent;

[0029] The formula of the diluent is as follows: potassium dihydrogen phosphate 4.5 g / L, disodium hydrogen phosphate 6.0 g / L, L-cysteine hydrochloride 0.5 g / L, agar 1.0 g / L, Tween-80 1.0 mL / L, and the solvent is water; the pH value is 6.8±0.2, and high-pressure sterilization at 115℃ for 20 min is performed;

[0030] (2) The sample diluent with a dilution degree of 10 0 ~10 -3 is coated into the modified MRS culture medium, after complete absorption of the plate, culture is performed at 37℃±2℃ in an anaerobic condition for 48 h, so as to obtain the Bifidobacterium lactis; and then the Bifidobacterium lactis is obtained through 16S determination;

[0031] The formula of the modified MRS culture medium is as follows: peptone 10.0 g / L, meat extract 8.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, sodium acetate 5.0 g / L, potassium hydrogen phosphate 2.0 g / L, ammonium citrate 2.0 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate tetrahydrate (MnSO4•4H2O) 0.05 g / L, Tween 80 1 mL, L-cysteine 0.5 g / L, mupirocin lithium salt 50 mg / L, and the solvent is water; the pH value is 7.0±0.2, and high-pressure sterilization at 115℃ for 20 min is performed.

[0032] Through the above isolation method, the Bifidobacterium lactis from human milk source (Bifidobacterium lactis from human milk source) lactis Bifidobacterium lactis) include Bifidobacterium lactis Probio-M8, Bifidobacterium lactis MN-B137, Bifidobacterium lactis MN-B138, Bifidobacterium lactis MN-B157, Bifidobacterium lactis MN-B275, Bifidobacterium lactis MN-B277 and Bifidobacterium lactis MN-B279.

[0033] Among them, Bifidobacterium lactis Probio-M8 has been disclosed in CN113018318A. It was isolated from the breast milk of healthy women in 2017 and was deposited on September 20, 2019 at the General Microbiology Center of the Chinese Culture Collection Administration of Microorganisms, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, and was classified and named Bifidobacterium lactis ( Bifidobacterium lactis ), the deposit number is CGMCC No.18610, and the Genbank sequence number is: MN372119.

[0034] Bifidobacterium lactis MN-B137, Bifidobacterium lactis MN-B138, Bifidobacterium lactis MN-B157, Bifidobacterium lactis MN-B275, Bifidobacterium lactis MN-B277 and Bifidobacterium lactis MN-B279 are deposited in the culture collection of Mengniu Beijing Company.

[0035] In a specific implementation, the human milk-derived Bifidobacterium lactis ( Bifidobacterium longum ) all have the effects mentioned in the present invention and are not limited to the specific strains mentioned above.

[0036] Preferably, the precursor substance is lactose-N-tetraose (LNT) and / or lactose-N-fucopentaose I (LNFPI). More preferably, the precursor substance is lactose-N-tetraose (LNT).

[0037] Preferably, the composition further contains Bifidobacterium longum ( Bifidobacterium bifidum ) and Bifidobacterium bifidum ( Bifidobacterium lactis ) one or both.

[0038] As a preferred embodiment, the composition comprises:

[0039] (1) Bifidobacterium lactis from human milk ( Bifidobacterium );as well as

[0040] (2) At least one of the following:

[0041] a) Lactose-N-disaccharide;

[0042] b) lactose-N-tetraose and / or lactose-N-fucopentaose I, and selected from Bifidobacterium longum ( longum Bifidobacterium bifidum ) and Bifidobacterium bifidum ( Bifidobacterium) one or both.

[0043] LNT can be hydrolyzed into LNB by bacteria secreting LnbX or LnbB, such as Bifidobacterium longum or Bifidobacterium bifidum, in the intestines (including infants and adults) and / or in the composition. This LNB is then shared with the intestines, allowing human milk-derived Bifidobacterium lactis to utilize it. LNFPI can be hydrolyzed and utilized by bacteria in the intestines (including infants and adults) and / or in the composition, such as Bifidobacterium bifidum or individual Bifidobacterium longum (such as Bifidobacterium longum BBMN68). For example, LNT is first hydrolyzed by afca in Bifidobacterium bifidum to form LNT, which is then hydrolyzed by LnbB to form LNB. This LNB is then shared with the intestines, allowing human milk-derived Bifidobacterium lactis to utilize it. Furthermore, LNT and LNFPI themselves promote the proliferation of beneficial microorganisms in the intestine. Therefore, when LNT and LNFPI are used in the composition of the present invention, the components can achieve synergistic effects, further contributing to human intestinal health.

[0044] Preferably, the composition further contains bifidobacteria ( Bifidobacterium ).

[0045] The present invention also found that, regardless of whether it is from infants or adults, bifidobacteria ( Bifidobacterium ) not only breaks down LNT into LNB but also utilizes LNB. When used in the compositions of the present invention, their use further optimizes the intestinal flora. They can be used in the compositions of the present invention as bacteria that break down LNT into LNB, or as additional probiotics when adult-derived bacteria that break down LNT into LNB are already present.

[0046] Preferably, the Bifidobacterium ( Bifidobacterium ) is selected from Bifidobacterium breve ( breve Bifidobacterium longum ), Bifidobacterium longum ( Bifidobacterium ) and Bifidobacterium bifidum ( bifidum Bifidobacterium ) one or more.

[0047] More preferably, the Bifidobacterium ( Bifidobacterium ) including Bifidobacterium bifidum ( bifidum Bifidobacterium breve )Bb-06, Bifidobacterium breve ( Bifidobacterium longum ) MN-B280, Bifidobacterium longum ( Bifidobacterium breve )BBMN68. The above strains have more prominent effects when used in the present invention.

[0048] Bifidobacterium breve BBMN68 has been disclosed in CN101649303A and preserved in the China General Microbiological Culture Collection Center, No. 3, Yuanlin, Beichen West Road, Chaoyang District, Beijing, China on November 26, 2007, and classified and named as Bifidobacterium breve (Bifidobacterium breve) Bifidobacterium longum ), with a preservation number of CGMCC No. 2265.

[0049] Bifidobacterium breve BBMN68 has been disclosed in CN101649303A and preserved in the China General Microbiological Culture Collection Center, No. 3, Yuanlin, Beichen West Road, Chaoyang District, Beijing, China on November 26, 2007, and classified and named as Bifidobacterium breve (Bifidobacterium breve) Bifidobacterium lactis ), with a preservation number of CGMCC No. 2265.

[0050] The person skilled in the art can adjust the amount of each component in the composition according to common sense, so that the amount of LNB utilized by Bifidobacterium lactis (Bifidobacterium lactis) Bifidobacterium lactis ) is basically the same as the content of LNB (including the content of LNB obtained after the reaction and / or decomposition of the precursor substance), but when there is surplus Bifidobacterium lactis (Bifidobacterium lactis) Bifidobacterium lactis ), the LNB contained in the intestinal tract can be utilized; when there is surplus LNB, the proliferation of other beneficial bacteria in the intestinal tract can be promoted, which can achieve the above-mentioned effects, and the amount of each component is not limited further.

[0051] The person skilled in the art can combine the above-mentioned schemes according to common sense to obtain a more preferred embodiment of the composition of the present application.

[0052] The present application further provides the use of the composition in the preparation of food.

[0053] The present application further provides a food containing the composition.

[0054] The food mentioned in the present application includes ordinary food and health food.

[0055] Based on the above technical scheme, the present application has the following beneficial effects:

[0056] The present invention discovered that human milk-derived Bifidobacterium lactis can effectively utilize LNB. Based on this discovery, a corresponding composition is provided, which can improve the colonization, growth, and proliferation of Bifidobacterium lactis in the intestine, thereby effectively enhancing the efficacy of human milk-derived Bifidobacterium lactis and enabling it to work synergistically with other infant-derived Bifidobacteria to effectively improve intestinal health in infants and young children. Furthermore, the combination of LNB and Bifidobacterium lactis can also increase the abundance of Bifidobacteria in the intestines of adults, particularly the elderly, promoting intestinal health. Furthermore, the components of the present composition are clearly derived, making it safer for use in humans. DETAILED DESCRIPTION

[0057] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0058] The strains used in the following examples are shown in Table 1-2.

[0059] Table 1 Bifidobacterium lactis (human milk, adult, and non-human sources)

[0060]

[0061] Table 2 Other Bifidobacteria

[0062]

[0063] Among them, Bifidobacterium lactis Probio-M8 has been disclosed in CN113018318A. It was isolated from the breast milk of healthy women in 2017 and deposited on September 20, 2019 at the General Microbiology Center of the Chinese Culture Collection Administration of Microorganisms, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, and was classified and named Bifidobacterium lactis ( Bifidobacterium animalis ), with a preservation number of CGMCC No.18610. Bifidobacterium lactis MN-B137, Bifidobacterium lactis MN-B138, Bifidobacterium lactis MN-B157, Bifidobacterium lactis MN-B275, Bifidobacterium lactis MN-B277, and Bifidobacterium lactis MN-B279 are obtained by the aforementioned separation method. In specific implementation, those skilled in the art can obtain specific human milk-derived Bifidobacterium lactis strains by the aforementioned separation method, all of which have the effects mentioned in the present invention.

[0064] Bifidobacterium animalis subsp. lactis Mn-Gup has been disclosed in CN111826299A and was deposited on April 10, 2018, at the General Microbiology Center of China Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, and was classified as Bifidobacterium animalis (Bifidobacterium longum ), and its preservation number is CGMCC No. 15578, and the preservation date is April 10, 2018. In the present application, the Bifidobacterium animalis subsp. lactis MN-Gup, referred to as lactobifidobacterium MN-Gup.

[0065] Bifidobacterium longum BBMN68 has been disclosed in CN101649303A and preserved in the General Microbiological Center of China Microorganism Culture Collection Committee, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, China, on November 26, 2007, with a postal code of 100101, and is classified and named as Bifidobacterium longum (Bifidobacterium longum). Bifidobacterium breve ), and the preservation number is CGMCC No. 2265.

[0066] Bifidobacterium breve MN-B51 was preserved in the General Microbiological Center of China Microorganism Culture Collection Committee, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, China, on September 18, 2020, with a postal code of 100101, and is classified and named as Bifidobacterium breve (Bifidobacterium breve). Bifidobacterium longum subsp. Longum ), and the preservation number is CGMCC No. 20679.

[0067] Bifidobacterium longum MN-B111 was preserved in the General Microbiological Center of China Microorganism Culture Collection Committee, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, China, on September 16, 2020, with a postal code of 100101, and is classified and named as Bifidobacterium longum subsp. longum (Bifidobacterium longum subsp. longum). Bifidobacterium breve ), and the preservation number is CGMCC No. 20660.

[0068] Bifidobacterium breve MN-B280 was preserved in the General Microbiological Center of China Microorganism Culture Collection Committee, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, China, on September 18, 2020, with a postal code of 100101, and is classified and named as Bifidobacterium breve (Bifidobacterium breve). Bifidobacterium lactis ), and the preservation number is CGMCC No. 20678.

[0069] Bifidobacterium adolescentis BBMN23 has been disclosed in Songling Liu et al., "Complete genome sequence of Bifidobacterium adolesentis BBMN23, a probiotic strain from healthy centenarian" in 2015, and deposited in China General Microbiological Culture Collection Center, No. 1 Xibeierxilu, Jiaoyuan, Chaoyang District, Beijing, China, Postcode: 100101, classified as Bifidobacterium adolescentis, and the accession number is CGMCC No. 2264.

[0070] Other strains are obtained through commercial channels.

[0071] The formula of the modified PTYG medium used in the examples is shown in Table 3, wherein the formula of the salt solution is as follows: anhydrous CaCl20.2 g, MgSO4•7H2O 0.48 g, K2HPO41.0 g, KH2PO41.0 g, NaHCO310.0 g, NaCl 2.0 g; the preparation method is as follows: first, dissolve anhydrous CaCl2and MgSO4•7H2O in 300 mL distilled water, measure 500 mL distilled water, and slowly add other salts while stirring. Continue stirring until completely dissolved, add 200 mL distilled water, and mix and store at 4°C for standby use.

[0072] Table 3 Modified PTYG medium

[0073]

[0074] Unless otherwise specified, the techniques or conditions in the examples are carried out according to the techniques or conditions described in the literature in the field, or according to the product instructions. Unless otherwise specified, the reagents or instruments used are conventional products that can be purchased through regular channels.

[0075] Example 1

[0076] 1. Test method:

[0077] 1) Activate the strains: the above-mentioned strains are activated for three generations at 37°C with a 4% inoculation amount under anaerobic conditions in PTYG liquid medium, and each culture is 24 h.

[0078] 2) Preparation of sugar-free PTYG liquid medium: mix and stir the reagents in Table 3 except glucose until dissolved, then adjust the pH to 6.8-7.0 with sodium hydroxide, sterilize at 115°C for 20 minutes, and cool after completion for standby use.

[0079] 3) Preparation of PTYG+LNB liquid medium: LNB 2g was weighed and dissolved in sterilized and cooled sugar-free PTYG medium to prepare PTYG+LNB medium containing LNB at a concentration of 2%. After membrane filtration and sterilization, it was ready for use.

[0080] 2. Test results:

[0081] 1) Bifidobacterium lactis Probio-M8 (4% inoculum) and Bifidobacterium bifidum Bb-06 (4% inoculum) and mixed Bifidobacterium lactis Probio-M8 (Probio-M8 at 2% inoculum and Bb-06 at 2% inoculum) in Table 1 and Table 2 were inoculated into sugar-free PYTG medium and PTYG+LNB medium respectively, and cultured anaerobically at 37°C for 16h. The OD600 increase value was measured, and the absorbance difference between the two media was the increase value of LNB utilization. The results are shown in Table 4.

[0082] Table 4 OD600 increase value of LNB utilization

[0083]

[0084] It can be seen that Bifidobacterium lactis Probio-M8 can effectively utilize LNB as a carbon source.

[0085] 2) Bifidobacterium lactis Probio-M8 and other bifidobacteria in Table 1 and Table 2 were inoculated into sugar-free PYTG medium and PTYG+LNB medium at an inoculum of 4%, and cultured anaerobically at 37°C for 16h. The OD600 increase value was measured, and the absorbance difference between the two media was the increase value of LNB utilization. The results are shown in Table 5.

[0086] Table 5 OD600 increase value of LNB utilization

[0087]

[0088] The results show that the adult source of B. adolescentis cannot use LNB, and BB12 of non-human origin cannot use it, other infantile mainly existing Bifidobacterium species (B. bifidum, B. breve, B. longum can use LNB (which is consistent with the literature description), B. bifidum Bb-06, B. breve MN-B280, B. longum BBMN68, B. breve MN-B51 and B. longum MN-B111 all have significant proliferation), although Lactobacillus bifidus Probio-M8 has a close ability to use LNB, but the effect of using LNB is not as good as the main Bifidobacterium species in the infant intestine, which is different from the current understanding in the art that Lactobacillus bifidus cannot use LNB (the source of Lactobacillus bifidus in existing literature is non-human source and adult source), and is significantly better than the Bifidobacterium species of adult source and non-human source (almost no use).

[0089] 3) Lactobacillus bifidus in Table 1 was inoculated into sugar-free PYTG medium and PTYG+LNB medium at an inoculation amount of 4%, and cultured anaerobically at 37°C for 16h, and the OD600 increase value was measured. The absorbance difference under the two culture media is the increase value of using LNB. The results are shown in Table 6.

[0090] Table 6 OD600 under LNB increase value

[0091]

[0092] The results show that adult-derived Lactobacillus bifidus MN-Gup and fermentation milk-derived BB12 cannot use LNB, and human milk-derived Lactobacillus bifidus Probio-M8 and other 9 strains of Lactobacillus bifidus have different degrees of LNB utilization ability, therefore, human milk-derived Lactobacillus bifidus combined with LNB can synergistically act to exert better efficacy.

[0093] 4) Lactobacillus bifidus Probio-M8 and other Bifidobacterium in Table 1 and Table 2 were inoculated into sugar-free PYTG medium and PTYG+LNB medium at an inoculation amount of 4%, and cultured anaerobically at 37°C for 8h, and the OD600 increase value was measured. The absorbance difference under the two culture media is the increase value of using LNB. The results are shown in Table 7.

[0094] Table 7 OD600 under LNB increase value

[0095]

[0096] The results reaffirmed that human milk-derived Bifidobacterium lactis Probio-M8 and MN-B137 can utilize LNB. Adult-derived Bifidobacterium lactis MN-Gup and fermented milk-derived BB12 cannot utilize LNB. However, Bifidobacterium breve, Bifidobacterium longum, and Bifidobacterium bifidum, species present in infancy, can utilize LNB to varying degrees.

[0097] As can be seen, compared to the carbon-free medium without LNB, all Bifidobacterium lactis except BB12 and MNGUP significantly proliferated in the presence of LNB, and all other Bifidobacterium lactis were derived from human milk. Therefore, it can be assumed that human milk-derived Bifidobacterium lactis has the ability to utilize LNB, and its utilization is comparable to that of major infant gut bacteria such as Bifidobacterium longum and Bifidobacterium breve, while non-human milk-derived Bifidobacterium lactis essentially does not utilize LNB. Since LNB is exclusively found in breast milk and persists in the infant gut due to its utilization of HMOs, it can be assumed that human milk-derived Bifidobacterium lactis is more adapted to the intestinal tract of breastfed infants than other Bifidobacterium lactis, potentially benefiting infant intestinal health. Due to the specificity of LNB, the combination of LNB and human milk-derived Bifidobacterium lactis can further enhance the health benefits of human milk-derived Bifidobacterium lactis and promote intestinal health.

[0098] Example 2

[0099] This embodiment provides a composition that can be used in foods such as fermented milk, milk powder, and solid beverages, which contains Bifidobacterium lactis ( Bifidobacterium lactis )Probio-M8 2.0×10 10 CFU, corresponding to 30 mg of LNB.

[0100] Example 3

[0101] This embodiment provides a composition that can be used in foods such as fermented milk, milk powder, and solid beverages, which contains Bifidobacterium lactis ( Bifidobacterium bifidum )Probio-M8 1.5×10 10 CFU, corresponding to LNT 40 mg and Bifidobacterium bifidum ( Bifidobacterium lactis )Bb-06 5.0×10 9 CFU.

[0102] Example 4

[0103] This embodiment provides a composition that can be used in foods such as fermented milk, milk powder, and solid beverages, which contains Bifidobacterium lactis ( Bifidobacterium bifidum )Probio-M8 2.0×10 10 CFU, corresponding to LNB 20 mg, LNT20 mg, and Bifidobacterium bifidum ( Bifidobacterium lactisBb-06 5.0 x 10 9 CFU.

[0104] Example 5

[0105] This example provides a composition that can be applied in fermented milk, milk powder, solid beverage, and the like, which contains Bifidobacterium lactis (Bifidobacterium animalis lactis) Bifidobacterium longum Probio-M8 1.5 x 10 10 CFU, corresponding to 40 mg of LNT, and Bifidobacterium longum (Bifidobacterium longum) Bifidobacterium lactis BBMN68 5.0 x 10 9 CFU.

[0106] Example 6

[0107] This example provides a composition that can be applied in fermented milk, milk powder, solid beverage, and the like, which contains Bifidobacterium lactis (Bifidobacterium animalis lactis) ​ Probio-M8 2.0 x 10 10 CFU, corresponding to 20 mg of LNB, 20 mg of LNFPI, and Bifidobacterium longum (Bifidobacterium longum) BBMN68 5.0 x 10 9 CFU.

[0108] Test Example 1

[0109] I. Test Design:

[0110] Detection basis: "Regulation of Intestinal Flora Function Test Method" in "Health Food Inspection and Evaluation Technical Specifications (2003 Edition)".

[0111] II. Test Animals:

[0112] BALB / c mice, 18~22g, male. After the animals were purchased, they were adapted to the environment for 5 days, and the animal experiment was performed.

[0113] III. Test Products:

[0114] The compositions of Examples 2, 3, 4, 5, and 6.

[0115] The compositions of Examples 2-6 were respectively made into the form of solid beverage, and in the formulation of each solid beverage, except for the composition of the example, the rest was filled with malt dextrin to 2g. The administration volume was 0.2mL / 20g, the administration medium was deionized water (2g dissolved in 60ml water), and the administration amount was adjusted regularly as the weight of the mice increased.

[0116] IV. Grouping and Dose Levels (Oral Administration, Grouping is Shown in Table 8)

[0117] Table 8 Test Grouping

[0118]

[0119] 5. Test steps

[0120] Before administering the test samples, 0.1 g of mouse feces was aseptically collected and serially diluted 10-fold. The appropriate dilution was selected and inoculated onto each culture medium. Following incubation, colonies were identified and counted using colony morphology, Gram staining, and biochemical analysis. The number of bacteria per gram of wet feces was calculated and the logarithm was used for statistical analysis. Twenty-four hours after the last administration of the test sample, the mice were sacrificed by cervical dislocation, and rectal feces were collected for intestinal microbial analysis.

[0121] VI. Indicators and Testing

[0122] 1. Clinical observation, observation indicators are shown in Table 9

[0123] Table 9 Observation indicators

[0124]

[0125] a. Box-side observations include general health and possible toxicity symptoms.

[0126] b. Detailed clinical observations include skin and coat condition, eyes and mucous membranes, respiratory system, circulatory system, autonomic movement, central nervous system, and behavioral characteristics.

[0127] c. Calculate the number of bacteria per gram of wet feces, take the logarithm, and perform statistical analysis. Pay attention to the method and time of fecal collection.

[0128] 2.Weight

[0129] Body weight was measured every three days and the drug was administered according to body weight until the end of the experiment. The mice were weighed after the experiment.

[0130] VII. Data Processing and Result Determination

[0131] SPSS software was used to perform variance analysis and all data were expressed as mean ± standard deviation.

[0132] 1. If any of the following items are met, the animal test results of the test sample can be determined to be positive.

[0133] 1.1 Judgment Criteria 1

[0134] The number of bifidobacteria and / or lactobacilli in feces increased significantly, while there was no significant change in enterobacteria and enterococci.

[0135] 1.2 Judgment Criteria 2

[0136] The bifidobacteria and / or lactobacilli in the feces are significantly increased, and the enterobacteria and / or enterococci are significantly increased, but the increase is lower than that of the bifidobacteria / lactobacilli.

[0137] VIII. Test results

[0138] 1) Lactobacilli:

[0139] As shown in Table 10 below, the G2-G6 groups can significantly increase the level of lactobacilli in the intestines (P<0.05) after 14 days compared with the initial 0 days, and are significantly better than the blank control group G1 (P<0.05). The G4 and G6 groups have a very significant increase (P<0.01) compared with 0 days. Therefore, the five combinations all have the ability to increase the level of lactobacilli. The combination of the G4 and G6 groups is a relatively optimal group.

[0140] Table 10 Change in lactobacilli

[0141]

[0142] Compared with G1 (P<0.05)

[0143] # Compared with 0 days (P<0.05)

[0144] ## Compared with 0 days (P<0.01)

[0145] 2) Bifidobacteria:

[0146] As shown in Table 11 below, the G2-G6 groups can very significantly increase the level of bifidobacteria in the intestines (P<0.01) after 14 days compared with the initial 0 days, and are significantly better than the blank control group G1 (P<0.05). The G4 group has the most bifidobacteria proliferation. Therefore, the five combinations all have the ability to increase the level of bifidobacteria. The combination of the G4 group is a relatively optimal group.

[0147] Table 11 Change in bifidobacteria

[0148]

[0149] Compared with G1 (P<0.05)

[0150] # Compared with 0 days (P<0.05)

[0151] ## Compared with 0 days (P<0.01)

[0152] 3) Enterococci:

[0153] As shown in Table 12, the enterococci levels in the intestines of the groups G2-G6 were significantly reduced (P<0.05) after 14 days compared with the initial 0 day, and were also significantly lower than the blank control group G1 (P<0.05). The enterococci levels in the groups G4 and G5 were extremely significantly reduced (P<0.01) after 14 days compared with the initial 0 day. Thus, the five combinations all have the ability to reduce the enterococci levels. The combinations of the groups G4 and G5 are the better combinations.

[0154] Table 12 Changes in enterococci

[0155]

[0156] Compared with G1 (P<0.05)

[0157] # Compared with 0 day (P<0.05)

[0158] ## Compared with 0 day (P<0.01)

[0159] 4) Enterobacteriaceae:

[0160] As shown in Table 13, the enterobacteriaceae levels in the intestines of the groups G2-G6 were extremely significantly reduced (P<0.01) after 14 days compared with the initial 0 day, and were also significantly lower than the blank control group G1 (P<0.05). The enterobacteriaceae levels in the groups G2, G3 and G4 were the most reduced after 14 days compared with the initial 0 day. Thus, the five combinations all have the ability to reduce the enterobacteriaceae levels. The combinations of the groups G2, G3 and G4 are the better combinations.

[0161] Table 13 Changes in enterobacteriaceae

[0162]

[0163] Compared with G1 (P<0.05)

[0164] # Compared with 0 day (P<0.05)

[0165] ## Compared with 0 day (P<0.01)

[0166] From the above results, according to the determination method of the "Regulation of Intestinal Flora Function Test Method" in the Health Food Inspection and Evaluation Technical Specifications (2003 Edition), the five combinations G2-G6 (Examples 2, 3, 4, 5 and 6) are positive, and all have the function of regulating intestinal flora.

[0167] While the application has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various modifications or changes can be made therein without departing from the spirit and scope thereof. Accordingly, it is intended that all such modifications and changes be included within the scope of the application as claimed.

Claims

1. Lactose-N-disaccharide is used to prepare Bifidobacterium lactis from human milk ( Bifidobacterium lactis ) use in compositions for colonization, growth or proliferation; The human milk-derived Bifidobacterium lactis includes Bifidobacterium lactis Probio-M8.

2. The use according to claim 1, characterized in that The composition contains: (1) Bifidobacterium lactis from human milk ( Bifidobacterium lactis );as well as (2) At least one of the following: a) Lactose-N-disaccharide; b) a precursor of lactose-N-disaccharide; the precursor being capable of reacting with one or more digestive tract contents and / or being capable of being decomposed by one or more digestive tract contents to produce lactose-N-disaccharide; c) a precursor of lactose-N-biose and a bacterium that hydrolyzes the precursor into lactose-N-biose.

3. The use according to claim 1 or 2, characterized in that The human milk-derived Bifidobacterium lactis ( Bifidobacterium lactis ) is separated from breast milk. Specific separation methods include: The breast milk was diluted and inoculated into the modified MRS medium, and then cultured at 37±2℃ under anaerobic conditions. The results showed that the lactobacillus ( Bifidobacterium lactis ); The modified MRS medium contains 0.4-0.6 g / L of L-cysteine ​​(L-cys) and 40-60 mg / L of mupirocin lithium salt (MUP), and has a pH value of 7.0±0.

2.

4. The use according to claim 3, characterized in that The diluent used to dilute breast milk contains 4.3-4.7 g / L of potassium dihydrogen phosphate, 5.8-6.2 g / L of disodium hydrogen phosphate, 0.4-0.6 g / L of L-cysteine ​​hydrochloride, 0.9-1.1 g / L of agar, and 0.8-1.2 mL / L of Tween-80; the pH value is 6.8±0.

2.

5. The use according to claim 2, characterized in that The precursor substance is lactose-N-tetraose and / or lactose-N-fucopentaose I.

6. The use according to claim 2 or 5, characterized in that The bacteria that hydrolyzes the precursor of lactose-N-disaccharide into lactose-N-disaccharide are specifically selected from Bifidobacterium longum ( Bifidobacterium longum ) and Bifidobacterium bifidum ( Bifidobacterium bifidum ) one or both.

7. The use according to any one of claims 2, 4 and 5, characterized in that: The composition contains: (1) Bifidobacterium lactis from human milk ( Bifidobacterium lactis );as well as (2) At least one of the following: a) Lactose-N-disaccharide; b) lactose-N-tetraose and / or lactose-N-fucopentaose I, and selected from Bifidobacterium longum ( Bifidobacterium longum ) and Bifidobacterium bifidum ( Bifidobacterium bifidum ) one or both.

8. The use according to any one of claims 2, 4 and 5, characterized in that: The composition also contains bifidobacteria ( Bifidobacterium ).

9. The use according to claim 8, characterized in that The Bifidobacterium ( Bifidobacterium ) is selected from Bifidobacterium breve ( Bifidobacterium breve ), Bifidobacterium longum ( Bifidobacterium longum ) and Bifidobacterium bifidum ( Bifidobacterium bifidum ) one or more.

10. The use according to claim 8, characterized in that The Bifidobacterium ( Bifidobacterium ) including Bifidobacterium bifidum ( Bifidobacterium bifidum )Bb-06, Bifidobacterium breve ( Bifidobacterium breve )MN-B280, Bifidobacterium longum ( Bifidobacterium longum )BBMN68; wherein, Bifidobacterium breve ( Bifidobacterium breve )MN-B280 has a preservation number of CGMCC No. 20678, Bifidobacterium longum ( Bifidobacterium longum )BBMN68 has a deposit number of CGMCC No. 2265.

11. Use of lactose-N-disaccharide for promoting the colonization, growth or proliferation of Bifidobacterium lactis derived from human milk for non-therapeutic purposes; The human milk-derived Bifidobacterium lactis includes Bifidobacterium lactis Probio-M8.

Images