Bifidobacterium longum subsp.infantis Imu-01 and application thereof

Abstract

The invention relates to a bifidobacterium longum subsp. Infantis Imu-01 and an application thereof. Specifically, the invention provides bifidobacterium longum subsp.infantis Imu-01, a culture, a preparation and application of the bifidobacterium longum subsp.infantis Imu-01 in preparation of a product for improving the immune function. The bifidobacterium longum subsp. Infantis provided by the invention can regulate and control the immune level of an object, improve the content of butyric acid in intestinal tracts and improve the utilization of oligosaccharide in breast milk.

Description

Technical Field

[0001] The present invention relates to the fields of microorganisms and food, and particularly relates to a Bifidobacterium longum subsp. infantis and its application. Background Art

[0002] According to the definition by the Food and Agriculture Organization of the United Nations and the World Health Organization (FAO / WHO) in 2001, probiotics are a class of live microorganisms that, when ingested in sufficient amounts, can produce beneficial effects on the host. Probiotics regulate the immune system through multiple mechanisms: 1) activating immune cells: stimulating the activities of natural killer cells (NK cells), macrophages, and T / B lymphocytes; 2) enhancing barrier function: strengthening the intestinal barrier by increasing mucin secretion and the expression of tight junction proteins in intestinal epithelial cells (Khalighi et al.); 3) regulating inflammatory responses: balancing the secretion of Th1 / Th2 cytokines, reducing the levels of pro-inflammatory factors (such as IL-8, TNF-α), and alleviating excessive immune responses; 4) secreting antibacterial substances: producing antibacterial peptides such as lactobacillin to directly inhibit pathogens. Existing studies have shown that specific probiotic strains (such as HN001, HN019, BB-12, etc.) can effectively enhance immunity under standardized use, but the principles of strain specificity, dosage requirements, and individual adaptation need to be strictly followed.

[0003] Studies have shown that indole-3-lactic acid (ILA) has a variety of biological functions and plays an important role in antioxidant, anti-inflammatory and regulation of intestinal microecology. First of all, ILA scavenges reactive oxygen species (ROS) and activates the Nrf2 (nuclear factor erythroid 2-related factor 2) signaling pathway, increasing the expression of antioxidant enzymes (such as glutathione peroxidase and superoxide dismutase), thus effectively reducing the damage of oxidative stress to cells and protecting the health of the body. Secondly, ILA has significant anti-inflammatory functions. It can inhibit the excessive release of pro-inflammatory factors (such as TNF-α, IL-1β) and promote the expression of anti-inflammatory factors by activating the aryl hydrocarbon receptor (AHR) signaling pathway, thereby regulating intestinal and systemic inflammatory responses. In addition, ILA also plays a key role in maintaining the balance of intestinal microecology. It can promote the proliferation of probiotics (such as Bifidobacterium and Lactobacillus), while inhibiting the growth of pathogenic bacteria, and by enhancing the expression of tight junction proteins (such as ZO-1, Occludin), improving the intestinal barrier function, reducing intestinal permeability and the risk of toxin leakage. Indole-3-lactic acid (ILA) is an aromatic lactic acid derivative produced by tryptophan metabolism. It is an important indole compound produced by intestinal microorganisms. It is derived from the catabolic pathway of tryptophan and is mainly produced by probiotics such as Lactobacillus and Bifidobacterium in the intestine.

[0004] Due to the significant differences in the metabolic capabilities of different microbial strains for ILA, screening for high-yield ILA strains is of great value for in-depth study of its functional mechanisms and the development of ILA-based functional foods and biological agents. Through efficient screening techniques (liquid chromatography-tandem mass spectrometry analysis, cell model evaluation) and animal experiment verification, not only can the metabolite production capabilities of different strains be rapidly evaluated, providing high-quality strain resources and theoretical basis for future clinical trials, but also new strategies and ideas can be provided for the development of functional foods or health products. Therefore, screening and applying high-quality ILA-producing probiotics has important research value and application prospects.

[0005] In addition, affected by geographical environment, genetic genes and eating habits, there are significant differences in the intestinal flora composition of humans in different countries around the world. Studies have found that the diversity of the intestinal flora in the Chinese population is significantly higher than that in the Western population, and there are also significant differences in the composition of the dominant intestinal flora between the two. This makes it possible that imported probiotic strains may not fully adapt to the intestinal environment of the Chinese people, and there may be situations such as low colonization efficiency or even no beneficial effects.

[0006] Therefore, it is of great practical significance to develop a strain of probiotics from breastfed infants in China that can produce high yields of ILA and effectively enhance immunity. Summary of the Invention

[0007] The object of the present invention is to provide a strain of Bifidobacterium longum subsp. infantis that can produce high yields of ILA and effectively enhance the body's immunity, and apply it to related immune products.

[0008] The present invention provides a Bifidobacterium longum subsp. infantis Imu-01, and its 16S RNA sequence is shown in SEQ NO ID: 1.

[0009] In one or more embodiments, the Bifidobacterium longum subsp. infantis is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, and the taxonomic name is Bifidobacterium longum subsp. infantis, and the deposit number is CGMCC 33792.

[0010] In one or more embodiments, the Bifidobacterium longum subsp. infantis Imu-01 is from the infant intestine and is an infant-derived probiotic.

[0011] In one or more embodiments, the characteristics of the Bifidobacterium longum subsp. infantis Imu-01 are: the colonies are round, convex, and have smooth edges; the colonies are small, with a size of 1-2 mm, the color is milky white, slightly yellowish; the colony surface is smooth and moist, and the texture is relatively viscous.

[0012] In one or more embodiments, the Bifidobacterium longum subsp. infantis Imu-01 produces high yields of indole-3-lactic acid (ILA).

[0013] In one or more embodiments, the Bifidobacterium longum subsp. infantis Imu-01 can enhance immune function.

[0014] In one or more embodiments, the Bifidobacterium longum subsp. infantis Imu-01 can use human milk oligosaccharides and / or their derivatives as a carbon source or the sole carbon source for growth.

[0015] In one or more embodiments, the human milk oligosaccharides are selected from one or more of the following: 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 3'-sialyllactose (3'-SL), difucosyllactose (DFL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), and / or 6'-sialyllactose (6'-SL).

[0016] In one or more embodiments, the human milk oligosaccharide is DFL.

[0017] In one or more embodiments, Bifidobacterium infantis subsp. Imu-01 can grow using any one of the following groups of human milk oligosaccharides: (1) 2'-FL; (2) 3'-SL; (3) LNT; (4) DFL; (5) LNnT; (6) 5HMO Mix. In one or more embodiments, Bifidobacterium infantis subsp. Imu-01 has good fermentation ability, preferably the fermentation ability within 48 h.

[0018] In one or more embodiments, when Bifidobacterium infantis subsp. Imu-01 uses LNnT and LNT as substrates, the fermentation rate is significantly higher than that when using glucose as a substrate.

[0019] In one or more embodiments, Bifidobacterium infantis subsp. Imu-01 has good acid resistance, bile salt resistance and adhesion ability.

[0020] In one or more embodiments, Bifidobacterium infantis subsp. Imu-01 has good safety.

[0021] In one or more embodiments, Bifidobacterium infantis subsp. Imu-01 grows and metabolizes to produce butyric acid, preferably in the intestine.

[0022] The present invention also provides a medium for culturing Bifidobacterium infantis subsp. described herein. The medium comprises a basal medium suitable for Bifidobacterium infantis subsp. and one or more human milk oligosaccharides selected from the following as a carbon source: 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 3'-sialyllactose (3'-SL), difucosyllactose (DFL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT) and / or 6'-sialyllactose (6'-SL).

[0023] In one or more embodiments, the basal medium is MRS medium.

[0024] In one or more embodiments, the medium comprises any one of the following groups of human milk oligosaccharides: (1) 2'-FL; (2) 3'-SL; (3) LNT; (4) DFL; (5) LNnT; (6) 5HMO Mix.

[0025] In one or more embodiments, the content of oligosaccharide in the medium is 0.1-10%, preferably 1-3%.

[0026] The present invention also provides a method for promoting the proliferation of Bifidobacterium longum subsp. infantis as described herein, which includes adding one or more of the following human milk oligosaccharides selected as a carbon source to a culture medium: 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 3'-sialyllactose (3'-SL), difucosyllactose (DFL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT) and / or 6'-sialyllactose (6'-SL).

[0027] The present invention also provides a culture of Bifidobacterium longum subsp. infantis Imu-01 with the preservation number of CGMCC No: 33792.

[0028] In one or more embodiments, the culture further contains a culture medium, such as MRS medium.

[0029] In one or more embodiments, the culture medium is a modified MRS medium.

[0030] In one or more embodiments, the modified MRS liquid medium contains: MRS medium, lithium mupirocin and cysteine hydrochloride.

[0031] In one or more embodiments, the conventional culture medium contains tryptone, beef extract, yeast extract, glucose, dipotassium hydrogen phosphate, ammonium citrate, sodium acetate, magnesium sulfate, manganese sulfate, Tween 80 and agar.

[0032] In one or more embodiments, the conventional culture medium contains 10 g / L of peptone, 8 g / L of beef extract, 4 g / L of yeast extract, 20 g / L of glucose, 2 g / L of dipotassium hydrogen phosphate, 2 g / L of ammonium citrate, 5 g / L of sodium acetate, 0.2 g / L of magnesium sulfate, 0.04 g / L of manganese sulfate, 1 g / L of Tween 80 and 15 g / L of agar.

[0033] In one or more embodiments, the pH value of the culture medium is 6.5 - 7.0, preferably 7.0.

[0034] The present invention also provides a preparation containing Bifidobacterium longum subsp. infantis Imu-01 with the preservation number of CGMCC No: 33792, its culture, lysate or extract.

[0035] In one or more embodiments, the preparation is a microbial preparation.

[0036] In one or more embodiments, the preparation is selected from one or more of the following: powder, pill, capsule, granule, tablet, liquid preparation or gel preparation.

[0037] In one or more embodiments, the preparation further comprises at least one excipient suitable for microbial preparations.

[0038] In one or more embodiments, the preparation is a bacterial suspension, comprising Bifidobacterium longum subsp. infantis and a buffer. The buffer may be a phosphate buffer, preferably PBS.

[0039] In one or more embodiments, the preparation is a bacterial powder, comprising Bifidobacterium longum subsp. infantis as described herein. The method for preparing the bacterial powder comprises: centrifuging the fermentation broth of Bifidobacterium longum subsp. infantis, collecting the bacterial cells, adding a freeze-drying protectant to the obtained bacterial cells, and performing vacuum freeze-drying to obtain a freeze-dried powder.

[0040] The present invention also provides a product comprising Bifidobacterium longum subsp. infantis Imu-01 according to any one of the embodiments herein, the culture according to any one of the embodiments herein, and / or the preparation according to any one of the embodiments herein.

[0041] In one or more embodiments, the Bifidobacterium longum subsp. infantis in the product is a live bacterial suspension, with a dose of 1×10 6 ~1×10 12 cfu / d / animal / day (1E6~1E12 cfu / d / animal / day), preferably, the dose is 1×10 8 ~1×10 10 cfu / d / animal / day (1E8~1E10 cfu / d / animal / day), and more preferably 1×10 9 cfu / animal / day (1E9 cfu / d / animal / day).

[0042] In one or more embodiments, the product is a food, a health product, or a pharmaceutical composition.

[0043] In one or more embodiments, the food further comprises raw and auxiliary materials, and the auxiliary materials in the raw and auxiliary materials include but are not limited to additives and / or nutrient fortifiers.

[0044] In one or more embodiments, the additives include but are not limited to flavoring agents, stabilizers, thickeners, preservatives, antioxidants, emulsifiers.

[0045] In one or more embodiments, the nutrient fortifiers include but are not limited to vitamins, minerals, amino acids, fatty acids, dietary fiber.

[0046] In one or more embodiments, the types of the food include but are not limited to: plant-based foods, animal-based foods, microbial fermentation foods, processed foods, food additives.

[0047] In one or more embodiments, the food includes, but is not limited to, dairy products, soy products, probiotic powders, probiotic oil droplets, dietary fiber supplements, nutrition bars, rice flour, purees, fruit and vegetable juices, food solid beverages, fruit juices, ice cream, candies, and biscuits.

[0048] In one or more embodiments, the dosage forms of the health products include, but are not limited to, powders, tablets, granules, capsules, solutions, emulsions, and suspensions.

[0049] In one or more embodiments, the dosage forms of the pharmaceutical composition include, but are not limited to, powders, tablets, granules, capsules, solutions, emulsions, and suspensions.

[0050] In one or more embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable excipient.

[0051] In one or more embodiments, the product may further contain prebiotics, such as human milk oligosaccharides. In one or more embodiments, human milk oligosaccharides include one or more selected from the following: 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 3'-sialyllactose (3'-SL), difucosyllactose (DFL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-sialyllactose (6'-SL).

[0052] In one or more embodiments, the product is a composition containing Bifidobacterium longum subsp. infantis and LNnT.

[0053] In one or more embodiments, the ratio of Bifidobacterium longum subsp. infantis to LNnT is 1×10 6 -1×10 12 cfu / d / animal / day: 10 - 10000 mg / kg / d (1E6~1E12 cfu / d / animal / day: 10 - 10000 mg / kg / d), preferably, the ratio is 1×10 8 -1×10 10 cfu / d / animal / day: 100 - 1000mg / kg / d (1E8~1E10 cfu / d / animal / day: 100 - 1000mg / kg / d), more preferably, the ratio is 1×10 9 cfu / animal / day: 500 mg / kg / d (1E9 cfu / d / animal / day: 500 mg / kg / d).

[0054] In one or more embodiments, the product has the use of enhancing immunity.

[0055] In one or more embodiments, the subject of the immunization is a mammal, preferably a human.

[0056] The present invention also provides the use of Bifidobacterium longum subsp. infantis, the culture, and the preparation described in any embodiment herein in the preparation of products having the functions of regulating the immune level of a subject, increasing the content of butyric acid in the intestine, and improving the utilization of human milk oligosaccharides.

[0057] In one or more embodiments, the regulation of the immune level of the subject includes one or more selected from the following: increasing the organ index, increasing the proliferation level of immune cells, upregulating inflammatory factors, and increasing the proportion, number, or activity of immune cells.

[0058] In one or more embodiments, the organ index is the organ index of the thymus and / or spleen.

[0059] In one or more embodiments, the proliferation level of immune cells is the proliferation level of splenic lymphocytes.

[0060] In one or more embodiments, the regulation is: upregulating the content of Imu-01 in the subject, thereby (i) increasing the organ index of the thymus and / or spleen, (ii) proliferating splenic lymphocytes, (iii) enhancing the activity of NK cells, and (iv) upregulating inflammatory factors.

[0061] In one or more embodiments, the subject is an immunosuppressed individual.

[0062] In one or more embodiments, immune cells include one or more selected from the following: white blood cells, lymphocytes, neutrophils, monocytes.

[0063] In one or more embodiments, the immune cells are blood immune cells.

[0064] In one or more embodiments, increasing the activity of immune cells includes increasing the activity of NK cells.

[0065] In one or more embodiments, increasing the proportion of immune cells includes increasing the proportion of neutrophils.

[0066] In one or more embodiments, increasing the number of immune cells includes increasing the number of one or more immune cells selected from the following: white blood cells, lymphocytes, neutrophils, monocytes.

[0067] In one or more embodiments, the product is a food, a health product, or a pharmaceutical composition.

[0068] In one or more embodiments, the food further comprises raw and auxiliary materials, and the auxiliary materials in the raw and auxiliary materials include, but are not limited to, additives and / or nutritional fortifiers.

[0069] In one or more embodiments, the additives include, but are not limited to, flavors and fragrances, stabilizers, thickeners, preservatives, antioxidants, emulsifiers.

[0070] In one or more embodiments, the nutritional fortifiers include, but are not limited to, vitamins, minerals, amino acids, fatty acids, dietary fiber.

[0071] In one or more embodiments, the types of the food include, but are not limited to: plant-based food, animal-based food, microbial fermentation food, processed food, food additives.

[0072] In one or more embodiments, the food includes, but is not limited to, dairy products, soy products, probiotic powder, probiotic oil droplets, dietary fiber supplements, nutrition bars, rice flour, puree, fruit and vegetable juice, food solid beverages, fruit juice, ice cream, candies, biscuits.

[0073] In one or more embodiments, the dosage forms of the health products include, but are not limited to, powders, tablets, granules, capsules, solutions, emulsions or suspensions.

[0074] In one or more embodiments, the dosage forms of the pharmaceutical compositions include, but are not limited to, powders, tablets, granules, capsules, solutions, emulsions or suspensions.

[0075] In one or more embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable auxiliary materials.

[0076] In one or more embodiments, the product may further contain prebiotics, such as human milk oligosaccharides. In one or more embodiments, the human milk oligosaccharides include one or more selected from the following: 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 3'-sialyllactose (3'-SL), difucosyllactose (DFL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-sialyllactose (6'-SL).

[0077] In one or more embodiments, the product is a solid beverage, probiotic oil droplets, chewable candies, dairy products, formulated milk powder, infant milk powder.

[0078] The present invention also provides the use of human milk oligosaccharides and one or more selected from the following in the preparation of a product having a regulated immune level of a target: Bifidobacterium longum subsp. infantis described in any embodiment herein, the culture described in any embodiment herein, the preparation described in any embodiment herein.

[0079] In one or more embodiments, the human milk oligosaccharides include one or more selected from the following: 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 3'-sialyllactose (3'-SL), difucosyllactose (DFL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-sialyllactose (6'-SL); preferably LNnT.

[0080] In one or more embodiments, the regulated immune level of the subject includes increasing the proportion, quantity or activity of immune cells in the blood.

[0081] In one or more embodiments, increasing the activity of immune cells includes increasing the activity of NK cells.

[0082] In one or more embodiments, increasing the proportion of immune cells includes increasing the proportion of neutrophils.

[0083] In one or more embodiments, increasing the quantity of immune cells includes increasing the quantity of neutrophils.

[0084] In one or more embodiments, the product is a food, a health product or a pharmaceutical composition.

[0085] In one or more embodiments, the product is a solid beverage, probiotic oil droplets, chewable tablets, dairy products, formulated milk powder, infant milk powder.

[0086] The present invention also provides a kit, comprising (1) a prebiotic and (2) Bifidobacterium longum subsp. infantis as described in any one of the embodiments herein, the culture as described in any one of the embodiments herein, and / or the preparation as described in any one of the embodiments herein.

[0087] In one or more embodiments, the prebiotic is a human milk oligosaccharide. In one or more embodiments, the human milk oligosaccharides include one or more selected from the following: 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 3'-sialyllactose (3'-SL), difucosyllactose (DFL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-sialyllactose (6'-SL). Preferably, the human milk oligosaccharide is LNnT.

[0088] On the other hand, the present invention also provides a method for screening the high ILA-producing strain, comprising the steps of: (1) Collecting a sample and performing spread plate isolation on the sample; (2) Separating and purifying after culturing and observing the colony morphology; (3) Preserving the strain; (4)ELISA primary screening and liquid chromatography-tandem mass spectrometry (LC-MS / MS) were used to qualitatively and quantitatively screen the ILA production capacity of the strains to be screened, respectively.

[0089] In one or more embodiments, the culture medium used for the coating and separation in step (1) is modified MRS agar medium; in one or more embodiments, the culture conditions in step (2) are culturing at 37 °C under anaerobic conditions for 48 - 72 h; in one or more embodiments, the preservation in step (3) is to prepare the purified strain using MRS liquid medium mixed with 10% glycerol and store it in an environment of -80 °C for long-term preservation; in one or more embodiments, the mobile phase used in the liquid chromatography in step (4) includes mobile phase A (0.1% formic acid aqueous solution) and mobile phase B (0.1% formic acid acetonitrile solution).

[0090] In one or more embodiments, in the screening method, it is also necessary to perform tests after gradient dilution of the standard solution of ILA and tryptophan (Trp), make a standard curve, and then compare the test results obtained in the above steps with the standard curve to calculate the ILA concentration in the sample.

[0091] The beneficial effects of the present invention are as follows: The present invention aims to screen a pure-breastfed infant-derived probiotic strain with high ILA production and significantly improved immunity.

[0092] The Bifidobacterium longum subsp. infantis Imu-01 of the present invention has an enhanced immune function, and the relevant subjects can be mammals, or even humans.

[0093] The Bifidobacterium longum subsp. infantis Imu-01 of the present invention is from the healthy infant intestine and has good safety.

[0094] The Bifidobacterium longum subsp. infantis Imu-01 of the present invention can utilize a variety of human milk oligosaccharides, and the compound of Imu-01 and LNnT has the function of enhancing immunity.

[0095] In the implementation of the present invention, the related products of Imu-01 can be live bacteria drugs, medicines, health products, ordinary foods, etc.; the form can be powder, liquid or oil drops. Description of the Drawings

[0096] Figure 1 It is the growth curve of Bifidobacterium longum subsp. infantis Imu-01.

[0097] Figure 2 It is the acid tolerance of Bifidobacterium longum subsp. infantis Imu-01.

[0098] Figure 3It is the 0.3% bile salt tolerance ability of Bifidobacterium longum subsp. infantis Imu-01.

[0099] Figure 4 It is the adhesion ability of Bifidobacterium longum subsp. infantis Imu-01.

[0100] Figure 5 It is the hemolytic plate test result of Bifidobacterium longum subsp. infantis Imu-01. 1. Negative control bacterium: Listeria innocua CICC 10417; 2. Positive control bacterium: Staphylococcus aurecus CICC 10473; 3. Sample: Bifidobacterium longum subsp. infantis 1mu-01. Note: Test report number: CICC 25-0284-00499.04-00914

[0101] Figure 6 It is the effect of Bifidobacterium longum subsp. infantis Imu-01 on white blood cell level.

[0102] Figure 7 It is the effect of Bifidobacterium longum subsp. infantis Imu-01 on lymphocyte level.

[0103] Figure 8 It is the effect of Bifidobacterium longum subsp. infantis Imu-01 on neutrophil level.

[0104] Figure 9 It is the effect of Bifidobacterium longum subsp. infantis Imu-01 on monocyte level.

[0105] Figure 10 It is the effect of Bifidobacterium longum subsp. infantis Imu-01 on fecal butyric acid content.

[0106] Figure 11 It is the utilization of 2'-FL by Bifidobacterium longum subsp. infantis Imu-01.

[0107] Figure 12 It is the utilization of 3'-SL by Bifidobacterium longum subsp. infantis Imu-01.

[0108] Figure 13 It is the utilization of LNT by Bifidobacterium longum subsp. infantis Imu-01.

[0109] Figure 14 It is the utilization of DFL by Bifidobacterium longum subsp. infantis Imu-01.

[0110] Figure 15 It is the utilization of LNnT by Bifidobacterium longum subsp. infantis Imu-01.

[0111] Figure 16 Utilization of Bifidobacterium longum subsp. infantis Imu-01 on 5HMOs.

[0112] Figure 17 Effect of Bifidobacterium longum subsp. infantis Imu-01 + LNnT on NK cell viability.

[0113] Figure 18 Effect of Bifidobacterium longum subsp. infantis Imu-01 + LNnT on the proportion of neutrophils.

[0114] Figure 19 Effect of Bifidobacterium longum subsp. infantis Imu-01 + LNnT on the number of neutrophils.

[0115] Figure 20 Colony morphology of Bifidobacterium longum Imu-01. Specific implementation manners

[0116] It should be understood that within the scope of the present invention, the above various technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form preferred technical solutions.

[0117] The present invention provides a Bifidobacterium longum subsp. infantis, named Imu-01, which was deposited at the China General Microbiological Culture Collection Center (No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Zip Code 100101) on March 11, 2025. Its taxonomic name is Bifidobacterium longum subsp. infantis, and the deposit number is CGMCC No: 33792.

[0118] The colony of Bifidobacterium longum subsp. infantis Imu-01 is round, convex, with a smooth edge; the colony is small, with a size of 1 - 2 mm, and the color is milky white with a slightly yellowish tint; the colony surface is smooth and moist, and the texture is relatively viscous. It was isolated from the intestines of healthy breastfed infants in Beijing, China, and is an infant-derived probiotic. This strain has good acid tolerance, bile salt tolerance, and adhesion ability, and can produce high levels of indole-3-lactic acid (ILA).

[0119] Generally, strains are preserved in the form of bacterial agents (such as bacterial powder, bacterial liquid), and usually need to be activated before use. According to the type of strain and the preservation method, an appropriate activation method can be selected. Conventional methods can be used.

[0120] Activate the Bifidobacterium longum subsp. infantis Imu-01, for example, using a modified MRS medium (the conventional MRS medium always adds lithium mupirocin and cysteine hydrochloride). The conventional MRS medium includes raw materials such as peptone, yeast, sodium acetate, Tween 80, magnesium sulfate heptahydrate, beef extract, glucose, diammonium citrate, potassium dihydrogen phosphate, manganese sulfate heptahydrate, and water. For the solid MRS medium, an appropriate amount of agar powder can be added to this formula. The ratios of these raw materials can be adjusted according to actual needs. The activation conditions can be conventional. For example, after culturing in an anaerobic incubator at 37 °C for 48 - 72 h, the single colonies of the strain can be directly transferred to a suitable fresh solid medium (such as MRS solid medium). After growing and reproducing in an anaerobic culture at 37 °C for a period of time (48 - 72 h), it is then inoculated into a liquid medium (such as the modified MRS medium). The inoculum size can be selected according to actual needs. For example, inoculate two to three loops into 10 - 50 mL or 15 - 30 mL of the liquid medium, and activate it by culturing at 37 °C in the liquid medium for 18 - 24 h to obtain the activated bacterial liquid.

[0121] The Bifidobacterium longum subsp. infantis Imu-01 described in the present invention can use a variety of human milk oligosaccharides and / or their derivatives as one of the carbon sources or the sole carbon source for growth.

[0122] In this article, human milk oligosaccharides (HMOs) are a class of natural oligosaccharides mainly contained in human milk, which is the third largest solid component in human milk and has unique biological significance and nutritional functions. More than 200 different structures of HMOs have been identified so far, covering three categories: neutral fucosylated, neutral non-fucosylated, and acidic sialylated. In one or more embodiments, the oligosaccharides include 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 3'-sialyllactose (3'-SL), difucosyllactose (DFL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), and / or 6'-sialyllactose (6'-SL). In one or more embodiments, the human milk oligosaccharides are 2'-FL produced by different manufacturers. In one or more embodiments, the human milk oligosaccharides are DFL. In one or more embodiments, the Bifidobacterium longum subsp. infantis Imu-01 can use any one of the following groups of human milk oligosaccharides for growth: (1) 2’-FL; (2) 3’-SL; (3) LNT; (4) DFL; (5) LNnT; (6) 5HMO.

[0123] In this text, 5HMO includes 2'-FL, 3-FL, LNT, 3'-SL, and 6'SL. Any 5HMO human milk oligosaccharide containing 2'-FL, 3-FL, LNT, 3'-SL, and 6'SL in the prior art can be used in the present invention. Exemplarily, 5HMO includes 40 - 70% 2'-FL, 5 - 30% 3-FL, 10 - 40% LNT, 1 - 10% 3'-SL, and 1 - 10% 6'SL. In one embodiment, 5HMO includes 52% 2'-FL, 13% 3-FL, 26% LNT, 4% 3'-SL, and 5% 6'-SL.

[0124] In addition to the above carbon sources, this strain can also grow using conventional carbon sources, such as glucose, sucrose, starch, methanol, ethanol, lactic acid, citric acid, etc. Those skilled in the art should know how to select a carbon source suitable for the growth of the strain.

[0125] The present invention also provides the use of Bifidobacterium longum subsp. infantis Imu-01 with the deposit number CGMCC No: 33792 in regulating the immune level of a subject. The regulation of the subject's immune level includes one or more selected from the following: increasing the organ index, increasing the proliferation level of immune cells, upregulating inflammatory factors, increasing the proportion, quantity, or activity of immune cells. Specifically, the regulation is to increase the organ index of the thymus and / or spleen, promote the proliferation of splenic lymphocytes, enhance the activity of NK cells, upregulate inflammatory factors, increase the proportion of neutrophils, and increase the quantity of immune cells (such as white blood cells, lymphocytes, neutrophils, monocytes). In one or more embodiments, the subject is an immunosuppressed individual.

[0126] The present invention also provides the application of Bifidobacterium longum subsp. infantis Imu-01 with the deposit number CGMCC No: 33792 in the preparation of a microbial preparation that is acid-resistant, bile-salt-resistant, and has adhesion ability.

[0127] The present invention also provides a culture of Bifidobacterium longum subsp. infantis Imu-01 with the deposit number CGMCC No: 33792. This culture contains Bifidobacterium longum subsp. infantis Imu-01 described herein and a culture medium. The culture medium can be a commonly known culture medium for culturing Bifidobacterium in the art, including the culture medium used for preparing the seed solution of Bifidobacterium, and also including the culture medium used for anaerobic fermentation of Bifidobacterium. Any culture medium suitable for Bifidobacterium is included, such as BS medium, MRS medium, BBL medium, and Bifidobacterium agar medium.

[0128] In some embodiments of the present invention, the culture medium is a modified MRS liquid medium (i.e., mupirocin lithium salt and cysteine hydrochloride are added to the conventional MRS medium). The conventional MRS liquid medium contains tryptone, beef extract, yeast extract, glucose, dipotassium hydrogen phosphate, ammonium citrate, sodium acetate, magnesium sulfate, manganese sulfate, Tween 80 and agar. More specifically, the MRS liquid medium contains 10 g / L of peptone, 8 g / L of beef extract, 4 g / L of yeast extract, 20 g / L of glucose, 2 g / L of dipotassium hydrogen phosphate, 2 g / L of ammonium citrate, 5 g / L of sodium acetate, 0.2 g / L of magnesium sulfate, 0.04 g / L of manganese sulfate, 1 g / L of Tween 80 and 15 g / L of agar. When it is necessary to prepare a solid medium, the concentration of agar can be appropriately increased in the above medium, for example, the agar concentration is adjusted to 15 g / L. Generally, the pH value of the culture medium is 6.5 - 7.0, preferably 7.0.

[0129] The present invention also provides a lysate of Bifidobacterium longum subsp. infantis with the preservation number CGMCC 33792. Methods for lysing Bifidobacterium longum subsp. infantis are well known in the art, such as physical lysis methods (ultrasonic lysis, high-pressure homogenization lysis, glass bead grinding method), chemical lysis methods (enzymatic lysis, surfactant lysis), biological lysis methods (phage lysis, autolysis method), combined lysis methods (enzymolysis + ultrasound, chemical + physical). Those skilled in the art can select the lysis method according to the composition and morphological requirements of the lysate.

[0130] The present invention also provides an extract of Bifidobacterium longum subsp. infantis with the preservation number CGMCC 33792 (such as polysaccharides, proteins, nucleic acids, metabolites, etc.). Methods for preparing extracts of Bifidobacterium longum subsp. infantis are well known in the art, including but not limited to: pretreatment of bacterial cells (centrifugation, washing), lysis of bacterial cells to release components (referring to the lysis methods described above, those skilled in the art can select a suitable lysis method according to the target component), separation and purification of the extract (those skilled in the art can select separation techniques according to the properties of the target component, for example: polysaccharides (water extraction and alcohol precipitation method, column chromatography), proteins (salting-out method, chromatography, electrophoresis), nucleic acids (phenol-chloroform method, column extraction method), metabolites (gas chromatography, high-performance liquid chromatography)), concentration and drying (rotary evaporation, ultrafiltration, freeze drying, spray drying). Those skilled in the art know the methods for detecting the purity and activity of the extract.

[0131] The Bifidobacterium longum subsp. infantis Imu-01 described herein can enhance immune function, can grow using a variety of human milk oligosaccharides and / or their derivatives, and has good fermentation ability. In addition, it has good acid resistance, bile salt resistance and adhesion ability. The results of the whole-genome analysis of the strain show that the strain does not contain drug resistance genes and virulence genes.

[0132] Therefore, the present invention also provides a preparation containing Bifidobacterium longum subsp. infantis Imu-01 with the deposit number of CGMCC No: 33792 and / or its culture, lysate or extract. In some embodiments, the preparation is a microbial preparation. In one or more embodiments, the preparation may be in the form of powder, pill, capsule, granule, tablet, liquid preparation or gel.

[0133] The present invention also provides a composition containing Bifidobacterium longum subsp. infantis Imu-01 with the deposit number of CGMCC No: 33792, and the composition is: a composition of Bifidobacterium longum subsp. infantis Imu-01 and prebiotic. The prebiotic may be selected from: fructooligosaccharide, galactooligosaccharide, xylooligosaccharide, isomaltooligosaccharide, inulin, resistant starch, etc. Preferably human milk oligosaccharide. In one or more embodiments, the prebiotic is selected from oligosaccharides, namely selected from 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 3'-sialyllactose (3'-SL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-sialyllactose (6'-SL), and / or difucosyllactose (DFL). In some embodiments, the oligosaccharide is selected from any one of the following groups: (1) 2’-FL; (2) 3’-SL; (3) LNT; (4) DFL; (5) LNnT; (6) 5HMO (2'-FL, 3-FL, LNT, 3'-SL and 6'SL). In one or more embodiments, the composition contains Bifidobacterium longum subsp. infantis Imu-01 and LNnT. In one or more embodiments, the composition can improve immune function.

[0134] The present invention also provides the use of Bifidobacterium longum subsp. infantis Imu-01 with the deposit number of CGMCC No: 33792 in the preparation of a product with immune-enhancing effect. In one or more embodiments, the object of the immune effect is a mammal, preferably a human.

[0135] In the present invention, the product is a food, health product or pharmaceutical composition. In one or more embodiments, the pharmaceutical composition includes but is not limited to drugs for the immune system, such as immune enhancers; further, the forms of the pharmaceutical composition include but are not limited to: powder, tablet, granule, capsule, solution, emulsion, suspension. In one or more embodiments, the food includes but is not limited to: plant-based food, animal-based food, microbial fermented food, processed food, food additives; further, the forms of the food include but are not limited to: food solid beverage, soy product, fruit juice, dairy product, ice cream, candy, biscuit.

[0136] For example, the bacterial suspension suitable for gavage administration can be obtained by culturing, centrifuging, and resuspending with PBS, or can be prepared into a bacterial suspension by adding bacterial powder + PBS. The preparation method of the bacterial powder is known in the art. For example, the fermentation broth of Bifidobacterium longum subsp. infantis with the preservation number of CGMCC No: 33792 is centrifuged, the thalli are collected, a freeze-drying protectant is added to the obtained thalli, and vacuum freeze-drying is performed to obtain a freeze-dried powder. The present invention also provides the bacterial powder prepared by the above method.

[0137] In one or more embodiments, the product is a solid beverage, probiotic oil droplets, chewable tablets, dairy products, formulated milk powder, infant milk powder. Among them, the solid beverage refers to a solid product with no more than 5 grams of moisture per 100 grams of finished product, such as instant coffee, Tang powder, milk tea powder, etc., which has the characteristics of being easy to store and carry; probiotic oil droplets are a product for ingesting probiotics by oral administration, and the oil droplet component can well protect the beneficial bacteria and allow them to survive and play a role in the digestive system; chewable tablets refer to solid candies mainly made of sugar or syrup (powder) and other raw materials and prepared through related processes such as mixing, granulating, and tabletting; dairy products refer to various foods processed mainly from fresh cow (sheep) milk and its products; formulated milk powder is a product that adds various nutritional fortifiers (such as vitamins, minerals, probiotics, DHA, ARA, etc.) on the basis of milk powder, such as pregnant women's milk powder, middle-aged and elderly milk powder, children's growth milk powder; infant milk powder is milk powder specially designed for infants to meet the nutritional needs of infant growth and development, and contains various nutritional components such as protein, fat, carbohydrates, vitamins, and minerals.

[0138] The product can also contain minerals and micronutrients, such as trace elements and vitamins recommended by government agencies such as USRDA. For example, the product can contain one or more of the following micronutrients in a daily dose: calcium, magnesium, phosphorus, iron, zinc, copper, iodine, selenium, vitamin C, vitamin B1, vitamin B6, vitamin B2, niacin, vitamin B12, folic acid, biotin, vitamin D, vitamin E.

[0139] The product can contain at least one other type of other food-grade bacteria. As used herein, food-grade bacteria refer to the bacteria used and are generally considered safe for use in food, such as lactic acid bacteria, Bifidobacterium, and food-grade bacteria are preferably probiotics. The probiotics refer to live microorganisms that are beneficial to the health of the host when ingested in sufficient quantities. The product can further contain at least one prebiotic. The prebiotic refers to a food substance that is expected to promote the growth of probiotics.

[0140] For the purposes of the present invention, a product is considered to contain Bifidobacterium longum subsp. infantis Imu-01 cells that are viable or non-replicating, any cell debris of Bifidobacterium longum subsp. infantis Imu-01, any fraction containing a culture of Bifidobacterium longum subsp. infantis Imu-01, and / or a culture medium used for culturing Bifidobacterium longum subsp. infantis Imu-01 or a part thereof. The fraction refers to different parts obtained by separating, classifying, or grading a culture by a certain method, which may contain at least one of Bifidobacterium longum subsp. infantis Imu-01 cells, cell debris, metabolites, cultures, lysates, and extracts of the culture at different concentrations.

[0141] In the present text, the product may further contain prebiotics, such as human milk oligosaccharides. In one or more embodiments, the oligosaccharides include one or more selected from the following: 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 3'-sialyllactose (3'-SL), difucosyllactose (DFL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-sialyllactose (6'-SL). In some embodiments, the oligosaccharides are selected from any one of the following groups: (1) 2’-FL; (2) 3’-SL; (3) LNT; (4) DFL; (5) LNnT; (6) 5HMO (2'-FL, 3-FL, LNT, 3'-SL, and 6'SL).

[0142] The present invention also provides the use of oligosaccharides (such as LNnT) and Bifidobacterium longum subsp. infantis, a culture, or a microbial preparation according to any one of the embodiments herein in the preparation of a product having a regulated immune level of a target. The regulated immune level of the target includes increasing the proportion of immune cells (such as neutrophils), increasing the number of immune cells (such as neutrophils), or increasing the activity of immune cells (such as NK cells).

[0143] The pharmaceutical composition described herein further comprises pharmaceutically acceptable excipients. The term "pharmaceutically acceptable excipients" refers to carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and the active ingredient, which are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH regulators, surfactants, adjuvants, ionic strength enhancers. For example, pH regulators include, but are not limited to, phosphate buffer; surfactants include, but are not limited to, cationic, anionic or non-ionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

[0144] Preferably, the excipients described herein do not affect the activity of the bacterial cells. For example: diluents / carriers (lactose, microcrystalline cellulose (MCC), starch and derivatives, mannitol, maltodextrin), protectants (lyoprotectants such as trehalose, sucrose, glucose, polyvinylpyrrolidone (PVP), dextran, sodium glutamate, glycine, antioxidants such as vitamin C, glutathione), binders and disintegrants (hydroxypropyl methylcellulose (HPMC), polyvinyl alcohol (PVA), crospovidone (PVPP), sodium carboxymethyl starch (CMS-Na)), coating materials (acrylic resins, hydroxypropyl methylcellulose phthalate, ethylcellulose), lubricants and glidants (magnesium stearate, colloidal silicon dioxide, talc), other functional excipients (pH regulators such as citric acid-sodium citrate buffer pair, osmotic pressure regulators such as sodium chloride, flavoring agents and fragrances such as stevioside, sucralose, fruit essence). An exemplary excipient combination for freeze-dried probiotic powder is trehalose, maltodextrin and mannitol, which form a high viable cell count preparation after freeze-drying. Exemplary excipients for enteric-coated tablets include microcrystalline cellulose, crospovidone, enteric coating materials.

[0145] On the other hand, the present invention also provides a method for screening the high-yield ILA strains, comprising the steps of: (1) collecting samples and subjecting the samples to spread plate isolation; (2) culturing and then isolating and purifying, and observing the colony morphology; (3) preserving the strains; (4) performing qualitative and quantitative screening of ILA production capacity on the strains to be screened by ELISA primary screening and liquid chromatography-tandem mass spectrometry (LC-MS / MS). Among them, the culture medium used for the spread plate isolation in step (1) is the modified MRS agar medium; the culture conditions in step (2) are culturing for 48-72 h under anaerobic culture conditions at 37 °C; the preservation in step (3) is to prepare the purified strains with MRS liquid medium mixed with 10% glycerol and store them in an environment of -80 °C for long-term preservation; the mobile phase used in the liquid chromatography in step (4) includes mobile phase A (0.1% formic acid aqueous solution) and mobile phase B (0.1% formic acid acetonitrile solution). In the screening method, it is also necessary to perform tests after gradient dilution of the standard solution of ILA and tryptophan (Trp), and make a standard curve, and then compare the test results obtained in the above steps with the standard curve to calculate the ILA concentration in the sample. Those skilled in the art should be familiar with the conventional LC-MS / MS test method in step (4).

[0146] The present invention will be further described below in the form of specific embodiments. It should be understood that these embodiments are merely illustrative and not intended to limit the scope of the present invention. The methods and reagents used in the embodiments are conventional methods and reagents in the art unless otherwise specified.

[0147] Embodiment

[0148] Embodiment 1, the ability of Bifidobacterium longum subsp. infantis Imu-01 to produce ILA

[0149] 219 lactic acid bacteria strains preserved in the Mengniu strain library were screened using an ILA ELISA kit (Shanghai Jingkang Biotechnology Co., Ltd., JLC_K5285), and 12 high-yield ILA bifidobacteria strains verified by LC-MS / MS quantification were screened, and Bifidobacterium longum subsp. infantis Imu-01 (isolated from the intestines of healthy infants and young children in Beijing, China) was preferably selected. The Bifidobacterium longum subsp. infantis Imu-01 of the present invention produced 7.39 μg / mL of ILA after overnight culture in the modified MRS liquid medium, which was 1.84 times that of the commercial positive strain Bifidobacterium breve M16V and 2.06 times that of Bifidobacterium longum subsp. longum BB536, and was a high-yield ILA strain (Table 1).

[0150] Table 1 Concentration of ILA in the culture supernatant of the tested strains detected by LC-MS / MS Genus Strain number Sample source pH value OD600 ILA (μg / mL) Bifidobacterium longum subsp. infantis Imu-01 Infant intestinal tract 3.86 1.74 7.39 Bifidobacterium breve M-16V Infant feces 3.80 1.82 4.02 Bifidobacterium longum subsp. infantis MN17683 Infant feces 3.86 1.84 1.45 Bifidobacterium longum subsp. longum BB536 Infant feces 3.86 1.83 3.59 Bifidobacterium bifidum MN15800 Infant feces 3.94 1.74 0.95

[0151] After adding 0.3 mM tryptophan (Trp) to the culture medium, the ability of Bifidobacterium longum subsp. infantis Imu-01 to produce ILA can increase by 30.6% and reach 9.65 μg / mL. This indicates that it can further utilize Trp in the intestine to produce more ILA.

[0152] Table 2 Concentration of ILA in the culture of the tested strain detected by LC-MS / MS (with Trp added) Genus Strain number Sample source pH value OD600 ILA (μg / mL) Increase rate Bifidobacterium longum subsp. infantis Imu-01 Infant intestinal tract 3.81 1.71 9.65 30.6% Bifidobacterium breve M-16V Infant feces 3.90 1.82 5.54 37.8%

[0153] Example 2, Fermentation ability of Bifidobacterium longum subsp. infantis Imu-01

[0154] Using the modified MRS medium containing 0.5% cysteine, under the conditions of 37 °C and strict anaerobic conditions, Bifidobacterium longum subsp. infantis Imu-01 enters the logarithmic growth phase starting from the 2nd hour and reaches the plateau phase at 16 hours. Its fermentation ability within 48 h is very good ( Figure 1 ).

[0155] Example 3, Evaluation of the probiotic characteristics of Bifidobacterium longum subsp. infantis Imu-01

[0156] Bifidobacterium longum subsp. infantis Imu-01 has good acid tolerance. Its survival rate is 29.6% after growing in the medium with pH 3.0 for 12 h, and the survival rate is 64.6% after growing in the medium with pH 4.0 for 12 h ( Figure 2 ). Bifidobacterium longum subsp. infantis Imu-01 has good bile salt tolerance. Its survival rate is about 20% after growing in the medium containing 0.3% bile salt for 12 h, which is comparable to that of the positive strain Bifidobacterium breve M16V ( Figure 3 ). Bifidobacterium longum subsp. infantis Imu-01 has good adhesion ability, and its mucin adhesion ability is significantly better than that of the positive control strain Bifidobacterium breve M-16V ( Figure 4 ).

[0157] In summary, Bifidobacterium longum subsp. infantis Imu-01 is a highly potential probiotic with good acid tolerance, bile salt tolerance and adhesion ability.

[0158] Example 4, 16s sequencing and safety analysis of Bifidobacterium longum subsp. infantis Imu-01

[0159] 16s identification shows that it is Bifidobacterium longum subsp. infantis, and the sequencing result is as shown in SEQ NO ID:1; detected according to the microbial strain hemolysis detection method, the hemolysis reaction of Imu-01 is negative, indicating that Imu-01 is a safe lactic acid bacteria strain ( Figure 5 ).

[0160] Example 5, Immunological Function Study of Bifidobacterium longum subsp. infantis Imu-01 (Animal Experiment)

[0161] Bifidobacterium longum subsp. infantis Imu-01 of the present invention has excellent immunological function. In the immunosuppression experiment (using cyclophosphamide to prepare the immune model), compared with the model group (MOD group), the white blood cell count ( Figure 6 ), lymphocyte count ( Figure 7 ), neutrophil count ( Figure 8 ), and monocyte count ( Figure 9 ) in the blood of mice in the Imu-01 group all increased significantly (p < 0.0001), and there was no difference from those of the normal control group (CON) mice, but they were all significantly higher than those of the positive drug (LH) group (p < 0.05). The results showed that Imu-01 could significantly enhance the immune function of immunosuppressed mice. In addition, the content of butyric acid in the feces of mice in the Imu-01 group increased ( Figure 10 ), indicating that Imu-01 grows and metabolizes in the intestine to produce butyric acid, thus further promoting immune enhancement.

[0162] Example 6, Utilization of Human Milk Oligosaccharides by Bifidobacterium longum subsp. infantis Imu-01

[0163] Bifidobacterium longum subsp. infantis Imu-01 can utilize 2'-fucosyllactose (2'-FL), and the growth-promoting trend of 2'-FL produced by Company A and Company B on Imu-01 is the same, but the fermentation rate is significantly lower than that of glucose ( Figure 11 , where the carbon source in the medium of the Imu-01 group is glucose, and 2'-FL is used to replace glucose in each group containing 2'-FL).

[0164] Bifidobacterium longum subsp. infantis Imu-01 can partially utilize 3'-sialyllactose (3'-SL) for growth ( Figure 12 , where the carbon source in the medium of the Imu-01 group is glucose, and 3'-SL is used to replace glucose in each group containing 3'-SL).

[0165] Bifidobacterium longum subsp. infantis Imu-01 can rapidly utilize lacto-N-tetraose (LNT) for growth, and its fermentation ability is better than that of glucose ( Figure 13 , where the carbon source in the medium of the Imu-01 group is glucose, and LNT is used to replace glucose in each group containing LNT).

[0166] Bifidobacterium longum subsp. infantis Imu-01 can utilize human milk oligosaccharide DFL (DFL) for growth in small amounts ( Figure 14, where the carbon source in the Imu-01 group of culture medium is glucose, and DFL is used to replace glucose in each group containing DFL). DFL is an oligosaccharide in breast milk and is a derivative of 2'-Fucosyllactose (2'-FL), which has the effects of regulating the intestinal flora, promoting the growth of Bifidobacterium, and helps improve intestinal health and immune function.

[0167] Bifidobacterium longum subsp. infantis Imu-01 can rapidly utilize lacto-N-neotetraose (LNnT) for growth, and the growth state is extremely good ( Figure 15 , where the carbon source in the Imu-01 group of culture medium is glucose, and LNnT is used to replace glucose in each group containing LNnT).

[0168] Bifidobacterium longum subsp. infantis Imu-01 can rapidly utilize 5HMOs (2'-FL, 3-FL, LNT, 3'-SL, and 6'-SL) for growth ( Figure 16 , where the carbon source in the Imu-01 group of culture medium is glucose, and 5HMOs is used to replace glucose in each group containing 5HMOs). 5HMO Mix is a mixed oligosaccharide (Chr. Hansen MyOli TM ), containing 5 human milk oligosaccharides: 2'-FL, 3-FL, LNT, 3'-SL, and 6'-SL (52% 2'-FL, 13% 3-FL, 26% LNT, 4% 3'-SL, 5% 6'-SL), and these components act together on the intestinal health, immune system, and neurodevelopment of infants and young children.

[0169] In summary, Bifidobacterium longum subsp. infantis Imu-01 of the present invention can utilize a variety of human milk oligosaccharides. When LNnT and LNT are used as substrates, the fermentation rate is significantly higher than that of glucose. When 5HMOs is used as a substrate, the fermentation rate is also high, followed by DFL and 2'-FL, and it can partially utilize 3'-SL and DFL. It shows that Bifidobacterium longum subsp. infantis Imu-01 can utilize human milk oligosaccharides to promote its growth and proliferation.

[0170] Example 7, Study on the immune function of Bifidobacterium longum subsp. infantis Imu-01 and LNnT (animal experiment)

[0171] In the immunosuppression (preparing an immune model with cyclophosphamide) experiment, compared with the model group, the compound bacterial suspension of Imu-01 and LNnT, with a dose of 1×10 9 cfu / animal / day; LNnT is in PBS solution, with a dose of 500 mg / kg / d), can significantly increase the activity of NK cells in the blood of mice ( Figure 17 , flow cytometer) and the percentage of neutrophils (automatic blood component analyzer) ( Figure 18 ) and the number ( Figure 19). The combination of Imu-01 and LNnT can improve the immune function of immunosuppressed mice.

[0172] Experimental methods

[0173] 1. Strain isolation and culture

[0174] In this study, the collected infant feces were spread and isolated using modified MRS agar medium. The fecal samples were diluted to 10-6 with PBS buffer and then spread. After culturing under anaerobic conditions at 37°C for 48 - 72 h, colonies with different morphologies in each medium were picked for three-zone streak isolation. After culturing under the same conditions for 48 - 72 h, the isolated and purified single colonies were collected in MRS liquid medium for amplification culture, and Gram staining was performed on different strains to record their morphologies and sources. Finally, the purified strains were stored in MRS liquid medium mixed with 10% glycerol at -80°C for long-term preservation.

[0175] The formula is as follows: in 1 L of medium, it contains pancreatic digest of casein: 10 g, yeast extract: 5 g, KH2PO4: 6 g, FeSO4: 0.034 g, MgSO4: 0.075 g, glucose: 20 g, sodium acetic acid: 25 g, sodium citrate: 2 g, MnSO4: 0.12 g, agar (if it is a solid medium): 15 g. The pH value is generally adjusted to 5.5 ± 0.2.

[0176] Modified MRS medium (De Man, Rogosa, and Sharpe Medium) (i.e., adding lithium mupirocin and cysteine hydrochloride to the conventional MRS medium). The conventional MRS formula is as follows: in 1 L of medium, there are peptone: 10 g, beef extract: 8 g, yeast extract: 4 g, glucose: 20 g, dipotassium hydrogen phosphate (K2HPO4): 2 g, ammonium citrate: 2 g, sodium acetate: 5 g, magnesium sulfate (MgSO4): 0.2 g, manganese sulfate (MnSO4): 0.04 g, Tween 80: 1 g, agar (if it is a solid medium): 15 g / L. The pH value is generally adjusted to 6.8 ± 0.2.)

[0177] 2. LC-MS / MS detection

[0178] In this study, liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to quantitatively screen ILA-producing strains. First, standard solutions of ILA and tryptophan (Trp) were prepared. The two standards were accurately weighed using a BT224S electronic balance with a precision of 0.0001 g and dissolved in 1 mL of methanol to obtain a 10 mM stock solution, which was stored at -20 °C for later use. To construct a standard curve, the stock solution was diluted with 20% acetonitrile solution at gradients of 0.05 μM, 0.1 μM, 0.125 μM, 0.25 μM, 0.5 μM, 1 μM, 2.5 μM, and 5 μM to ensure the accuracy and sensitivity of quantitative analysis. For sample preparation, the overnight culture broth was centrifuged at 4000 × g for 10 min (4 °C) to collect the supernatant. 200 μL of the supernatant obtained after centrifugation was added to 800 μL of methanol, vortexed for 10 seconds, and then sonicated for 5 min to extract ILA. Subsequently, it was placed at -20 °C and allowed to stand for 10 min to accelerate precipitation. After 10 min, it was centrifuged again (14000 × g, 10 min, 4 °C), and 500 μL of the supernatant was taken and mixed with 500 μL of ddH2O in a 1.5 mL centrifuge tube for 10 seconds, and then filtered through a 0.22 µm membrane for detection. The sample analysis was performed using an AQUITY UPLC system (equipped with an ACQUITY PREMIER BEH C18 chromatographic column with a specification of 2.1 × 150 mm, 1.7 μm) and a QTRAP 6500 tandem mass spectrometer. Liquid chromatography separation was carried out at a column temperature of 45 °C, with the mobile phase being 0.1% formic acid aqueous solution (phase A) and 0.1% formic acid acetonitrile solution (phase B), a flow rate of 0.3 mL / min, and a gradient elution program of maintaining 85% A from 0 to 0.8 min, decreasing the proportion of phase A from 85% to 5% from 0.8 to 10.5 min, maintaining 5% A from 10.5 to 11.4 min, restoring the proportion of phase A to 85% from 11.4 to 11.5 min, and maintaining 85% A from 11.5 to 12 min. Mass spectrometry detection was performed in the electrospray positive ion mode (ESI+), and quantitative analysis was carried out in the multiple reaction monitoring (MRM) mode. The mass spectrometry parameters included an ion spray voltage of 5000 V, a temperature of 300 °C, a curtain gas (CUR) of 25 psi, ion source gases 1 and 2 (GAS1, GAS2) both being 10 psi, and the collision gas (CAD) set to medium. The MRM transition parameters such as the declustering voltage, collision voltage, and collision cell exit voltage were optimized using a syringe infusion pump to ensure the accuracy and sensitivity of detection. Data acquisition and processing were completed using AB SCIEX Analyst® 1.6 software, and the ILA concentration in the sample was calculated based on the standard curve.

[0179] 3. Detection of Acid Resistance, Bile Salt Resistance, and Adhesive Probiotic Characteristics

[0180] In the acid resistance test, the pH of MRS liquid medium was adjusted to 2, 3, 4, 5 and autoclaved. The activated and cultured bacterial liquid was inoculated into liquid media under different pH conditions at an inoculation amount of 2% (w / v). The medium with a pH of 7 was used as the control group, and it was anaerobically cultured at 37 °C in a growth curve analyzer for 12 hours, and the OD was recorded every 30 minutes. 600nm Absorbance values were used to plot the growth curve, and the survival rates of each strain at different pH values were calculated through the OD 600nm absorbance values. The calculation formula was: survival rate = (OD of the experimental group 600nm ÷ OD of the control group 600nm ) × 100%. The bile salt concentrations used in the bile salt resistance test were 0, 0.15%, 0.3%, 0.6% respectively. Similarly, inoculation was carried out at an inoculation amount of 2%, and it was anaerobically cultured at 37 °C for 12 hours, and the OD 600nm value was monitored and the survival rate was calculated. The calculation formula was: survival rate = (OD of the experimental group 600nm ÷ OD of the control group 600nm ) × 100%.

[0181] The adhesion experiment referred to the method of Hiromi KIMOTO-NIRA et al. (doi:10.1111 / asj.12270) to evaluate the binding ability of each strain to mucin. In this experiment, a 96-well plate was used. Purified porcine gastric mucin (Sigma) was prepared into a 0.5 mg / mL solution, dissolved in 50 mmol / L carbonate / Tween 20 buffer, evenly covered on the surface of the microplate, and incubated at 37 °C to immobilize the mucin. Subsequently, 100 μL of bacterial liquid (0.85% NaCl solution, OD 620nm adjusted to 1.0) was added to each well, incubated at 37 °C for 2 hours to promote adhesion, and washed three times with 200 μL of PBS and 0.05% bicarbonate buffer (pH 9.6) to remove unadhered cells, and fixed overnight at 4 °C. After the fixed wells were washed three times with PBS, they were blocked with PBS containing 1% Tween 20 for 1 hour, and then dried at 55 °C. Subsequently, 100 μL of crystal violet staining solution (1% crystal violet dissolved in 33% acetic acid) was added to each well. After staining for 45 minutes, it was washed twice with PBS, and then 100 μL of 50 mmol / L citrate solution (pH 4.0) was added and incubated at room temperature for 45 minutes to dissolve the staining complex. The absorbance was measured at 595 nm by an enzyme-linked immunosorbent assay to quantitatively evaluate the level of adhered bacteria. The wells without mucin were used as blank controls to correct non-specific adsorption signals.

[0182] 4. Utilization of human milk oligosaccharides

[0183] After the rejuvenated Bifidobacterium longum subsp. infantis Imu-01 was anaerobically cultured at 37 °C for 48 h, it was centrifuged, washed twice with PBS, and then inoculated into MRS liquid medium (with 2% glucose and 2% different human milk oligosaccharides as carbon sources) at an inoculation amount of 1% to evaluate the utilization of different human milk oligosaccharides by Bifidobacterium longum subsp. infantis Imu-01.

[0184] 5. Evaluation of immunosuppressed mice

[0185] 70 male BALB / c mice were maintained at a temperature range of 23 - 25 °C and a humidity of 40 - 60%, with a consistent 12-hour light / dark cycle. After 1 week of adaptation, the mice were randomly assigned to 7 groups, with 10 mice in each group. 60 of the mice were intraperitoneally injected with 80 mg / kg cyclophosphamide (CTX) for three consecutive days to establish an immunosuppressed mouse model. The remaining 10 were the control group. Three days later, probiotics / prebiotics were administered by gavage (the bacterial suspension for gavage was obtained by culturing, centrifuging, and resuspending with PBS, or prepared into a bacterial suspension from bacterial powder + PBS). The grouping was as follows: (1) Control group (CON group, n = 10, 200 μL normal saline), (2) Model group (MOD group, n = 10, CTX + 200 μL normal saline), (3) Positive control group (LH group, n = 10, CTX + levamisole hydrochloride 40 mg / kg / d), (4) CTX + Bifidobacterium longum subsp. infantis Imu-01 (n = 10, 1 × 10 9 CFU / day / mouse), (6) CTX + Bifidobacterium longum subsp. infantis Imu-01 + LNnT (n = 10, 1 × 10 9 CFU CFU / day / mouse, and the dose of LNnT was 500 mg / kg / d). After 4 weeks, the mice were sacrificed, and their spleens, thymuses, blood, colons, and feces were collected.

[0186] The above has detailed the present invention. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although the present invention gives specific implementation examples, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application but are made using conventional techniques known in the art. Some basic features can be applied according to the scope of the following appended claims.

[0187] Partial sequences herein:

[0188] 16S sequencing of Bifidobacterium longum subsp. infantis Imu-01, SEQ ID NO:1

[0189]

Claims

1. Bifidobacterium longum subsp. infantis with the preservation number of CGMCC No. 33792, preserved in the General Microbiology Center of China Microbial Culture Collection Management Committee.

2. A Bifidobacterium longum subsp. infantis ( Bifidobacterium longum subsp. infantis ), whose 16S RNA sequence is shown in SEQ NO ID:

1.

3. The culture of Bifidobacterium longum subsp. infantis according to claim 1 or 2.

4. The culture according to claim 3, characterized in that The culture also contains a culture medium.

5. The culture according to claim 4, wherein The culture medium is a modified MRS liquid medium.

6. A preparation containing Bifidobacterium longum subsp. infantis, its culture, lysate or extract according to claim 1 or 2.

7. The preparation according to claim 6, characterized in that, The preparation is selected from one or more of the following: powder, pill, capsule, granule, tablet, liquid preparation or gel.

8. The use of Bifidobacterium longum subsp. infantis according to claim 1 or 2, the culture according to any one of claims 3-5, and / or the preparation according to claim 6 or 7 in the preparation of a product having the function of regulating the immune level of the subject, increasing the content of butyric acid in the intestine, and improving the utilization of human milk oligosaccharides.

9. The use according to claim 8, characterized in that, The regulation of the immune level of the subject includes one or more of the following: increasing the organ index, increasing the proliferation level of immune cells, up-regulating inflammatory factors, increasing the proportion, number or activity of immune cells, and / or The product is a food, health product or pharmaceutical composition, and / or The product also contains prebiotics.

10. The use according to claim 9, characterized in that, Immune cells include one or more of the following: white blood cells, lymphocytes, neutrophils, monocytes, and / or The prebiotics include human milk oligosaccharides.

11. A product containing Bifidobacterium longum subsp. infantis according to claim 1 or 2, the culture according to any one of claims 3-5, and / or the preparation according to claim 6.

12. The product according to claim 11, wherein, The product is a food, pharmaceutical composition, and / or health product.

13. The product according to claim 12, wherein The food includes: plant-based food, animal-based food, microbial fermentation food, processed food, food additives, and / or The dosage form of the pharmaceutical composition includes powder, tablet, granule, capsule, solution, emulsion, suspension, and / or The dosage form of the health product includes powder, tablet, granule, capsule, solution, emulsion, suspension.

14. The product according to any one of claims 11-13, characterized in that, The product also includes prebiotics.

15. The product according to claim 14, wherein, The prebiotic is human milk oligosaccharide.

16. The product according to claim 15, wherein, The human milk oligosaccharides include one or more of the following: 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 3'-sialyllactose (3'-SL), difucosyllactose (DFL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-sialyllactose (6'-SL).

17. The product according to claim 12, wherein The food includes: dairy products, soy products, probiotic powder, probiotic oil droplets, dietary fiber supplements, nutrition bars, rice flour, fruit puree, fruit and vegetable juice, food solid beverages, fruit juice, ice cream, candies, biscuits, and / or The food also includes raw and auxiliary materials, and the raw and auxiliary materials include additives and / or nutritional fortifiers, and / or The pharmaceutical composition also includes pharmaceutically acceptable excipients.

18. Use of human milk oligosaccharides and one or more selected from the following in the preparation of a product for regulating the immune level of a subject: Bifidobacterium longum subsp. infantis as described in any one of claims 1-2, the culture as described in any one of claims 3-5, and / or the preparation as described in claim 6 or 7.

19. The use according to claim 18, characterized in that, The regulation of the immune level of the subject includes increasing the proportion, number or activity of immune cells in the blood, and / or The human milk oligosaccharides include one or more selected from the following: 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 3'-sialyllactose (3'-SL), difucosyllactose (DFL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-sialyllactose (6'-SL), and / or The product is a food, a pharmaceutical composition, and / or a health product.

20. The use according to claim 19, characterized in that, The immune cells include one or more selected from the following: white blood cells, lymphocytes, neutrophils, monocytes, and / or The human milk oligosaccharides include LNnT.

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