Bifidobacterium longum subsp. infantis imu-01 and application thereof

Abstract

The present application relates to a kind of Bifidobacterium longum infantis subsp.Imu-01 and its application. Specifically, the present application provides Bifidobacterium longum infantis subsp.Imu-01, culture, preparation and preparation of immune function improving product application. The present application provides that Bifidobacterium longum infantis can regulate the immune level of the subject, improve the content of intestinal butyric acid, improve the utilization of human milk oligosaccharide.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microorganisms and food, in particular to a Bifidobacterium longum subsp. infantis and application thereof. BACKGROUND

[0002] According to the definition of the Food and Agriculture Organization of the United Nations and the World Health Organization (FAO / WHO) in 2001, probiotics are a kind of active microorganisms that can produce beneficial effects on the host when sufficient intake. Probiotics regulate the immune system through multiple mechanisms, 1) activating immune cells: stimulating the activity of natural killer cells (NK cells), macrophages and T / B lymphocytes; 2) enhancing barrier function: strengthening the intestinal barrier by increasing mucin secretion and tight junction protein expression of intestinal epithelial cells (Khalighi et al.); 3) regulating inflammatory response: balancing Th1 / Th2 cytokine secretion, reducing pro-inflammatory factor (such as IL-8, TNF-α) level, and relieving excessive immune response; 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 strict adherence to strain specificity, dosage requirements and individual adaptation principles is required.

[0003] Studies have shown that indole-3-lactic acid (ILA) has multiple biological functions and plays an important role in antioxidant, anti-inflammatory, and regulation of intestinal microecology. First, ILA can effectively reduce oxidative stress damage to cells by eliminating reactive oxygen species (ROS) and activating the Nrf2 (Nuclear Factor Erythroid 2-related Factor 2) signaling pathway, increasing the expression of antioxidant enzymes such as glutathione peroxidase and superoxide dismutase, thereby protecting the body's health. Second, ILA has significant anti-inflammatory function, can inhibit the excessive release of pro-inflammatory factors such as TNF-α and IL-1β, and promote the expression of anti-inflammatory factors by activating the AHR (aryl hydrocarbon receptor) signaling pathway, thereby regulating intestinal and systemic inflammatory response. 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 enhance the expression of tight junction proteins such as ZO-1 and Occludin, thereby improving intestinal barrier function, reducing intestinal permeability, and reducing the risk of toxin leakage. Indole-3-lactic acid (ILA) is a derivative of aromatic lactic acid produced by tryptophan metabolism, which is an important indole compound metabolized by intestinal microorganisms. It is derived from the decomposition and metabolic 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 capacity of different microbial strains, screening high-ILA-producing strains is of great value for in-depth study of its functional mechanism and development of functional foods and biological agents based on ILA. Through high-efficiency screening techniques (liquid chromatography-tandem mass spectrometry analysis, cell model evaluation), and animal experiment verification, not only can the metabolic product generation capacity of different strains be quickly evaluated, providing high-quality strain resources and theoretical basis for future clinical trials, but also can provide new strategies and new ideas for the development of functional foods or health products. Therefore, screening and applying high-quality ILA-producing probiotics have important research value and application prospects.

[0005] In addition, due to the influence of geographical environment, genetic genes, and dietary habits, there are significant differences in the composition of intestinal flora among people in different countries around the world. Studies have found that the diversity of intestinal flora in Chinese population is significantly higher than that in western population, and there are significant differences in the composition of dominant intestinal flora between the two. This makes imported probiotic strains not necessarily fully adapt to the intestinal environment of Chinese people, and may result in low colonization efficiency or even no beneficial effect.

[0006] Therefore, it has important practical significance to develop a Chinese pure breast-feeding infant-derived probiotic strain capable of high-yield ILA and effectively improving immunity. SUMMARY

[0007] The application aims to provide a Bifidobacterium longum subsp. infantis strain capable of high-yield ILA and effectively improving immunity of the body, which is applied to related immune products.

[0008] The application provides a Bifidobacterium longum subsp. infantis Imu-01, and a 16S RNA sequence of the Bifidobacterium longum subsp. infantis Imu-01 is shown as SEQ NO ID:1.

[0009] In one or more embodiments, the Bifidobacterium longum subsp. infantis is preserved in the China General Microbiological Culture Collection Center, and is named as Bifidobacterium longum subsp. infantis, and has a preservation number of 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 Bifidobacterium longum subsp. infantis Imu-01 is characterized in that a colony is round, convex, and smooth in edge; the colony is small in size, 1-2 mm in size, and milky white in color, slightly yellowish; and the colony is smooth and moist in surface, and relatively viscous in texture.

[0012] In one or more embodiments, the Bifidobacterium longum subsp. infantis Imu-01 is high in yield 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 grow by using breast milk oligosaccharides and / or derivatives thereof as a carbon source or a sole carbon source.

[0015] In one or more embodiments, the breast milk oligosaccharides are selected from one or more of 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, the B. longum subsp. infantis Imu-01 can grow with 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, the B. longum subsp. infantis Imu-01 has good fermentation capacity, preferably the fermentation capacity is within 48 h.

[0018] In one or more embodiments, the B. longum subsp. infantis Imu-01 has a significantly higher fermentation rate when using LNnT and LNT as substrate than when using glucose as substrate.

[0019] In one or more embodiments, the B. longum subsp. infantis Imu-01 has better acid tolerance, bile salt tolerance and adhesion capacity.

[0020] In one or more embodiments, the B. longum subsp. infantis Imu-01 has better safety.

[0021] In one or more embodiments, the B. longum subsp. infantis Imu-01 produces butyric acid during growth and metabolism, preferably in the intestine.

[0022] The present application also provides a culture medium for culturing the B. longum subsp. infantis described herein, the culture medium comprising a basal medium suitable for B. longum subsp. infantis and a human milk oligosaccharide selected from one or more of 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 culture 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 oligosaccharides in the culture medium is 0.1-10%, preferably 1-3%.

[0026] The present application also provides a method for promoting the proliferation of B. longum subsp. infantis as described herein, comprising adding to the culture medium one or more of the following breast milk oligosaccharides 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).

[0027] The present application also provides a culture of B. longum subsp. infantis Imu-01 having the accession number 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 comprises: MRS medium, mupirocin lithium salt, and cysteine hydrochloride.

[0031] In one or more embodiments, the conventional medium comprises 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 medium comprises 10 g / L of tryptone, 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 application also provides a preparation containing B. longum subsp. infantis Imu-01 having the accession number CGMCC No: 33792, a culture, a lysate or an extract thereof.

[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: a powder, a pill, a capsule, a granule, a tablet, a liquid preparation, or a gel.

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

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

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

[0040] The present application also provides a product comprising B. longum subsp. infantis Imu-01 as described in any of the embodiments herein, a culture as described in any of the embodiments herein, and / or a preparation as described in any of the embodiments herein.

[0041] In one or more embodiments, the B. longum subsp. infantis in the product is a live bacterial suspension, and the dosage is 1 x 10 6 ~1 x 10 12 cfu / d / animal / day (1E6~1E12 cfu / d / animal / day), preferably, the dosage is 1 x 10 8 ~1 x 10 10 cfu / d / animal / day (1E8~1E10 cfu / d / animal / day), more preferably 1 x 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 nutritional enhancers.

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

[0045] In one or more embodiments, the nutritional enhancers include but are not limited to vitamins, minerals, amino acids, fatty acids, dietary fibers.

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

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

[0048] In one or more embodiments, the dosage form of the health product includes, but is not limited to, powders, tablets, granules, capsules, solutions, emulsions, suspensions.

[0049] In one or more embodiments, the dosage form of the pharmaceutical composition includes, but is not limited to, powders, tablets, granules, capsules, solutions, emulsions, suspensions.

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

[0051] In one or more embodiments, the product can further comprise prebiotics, such as human milk oligosaccharides. In one or more embodiments, the human milk oligosaccharides include one or more selected from the group consisting of 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 comprising the B. longum subsp. infantis and LNnT.

[0053] In one or more embodiments, the ratio of B. longum subsp. infantis and LNnT is 1 x 10 6 - 1 x 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 x 10 8 - 1 x 10 10 cfu / d / animal / day: 100-1000 mg / kg / d (1E8~1E10 cfu / d / animal / day: 100-1000 mg / kg / d), more preferably, the ratio is 1 x 10 9 cfu / d / 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 improving immunity.

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

[0056] The present application also provides use of the B. longum subsp. infantis of any of the embodiments herein, the culture of any of the embodiments herein, the preparation of any of the embodiments herein in the preparation of a product for modulating immune level of a subject, for increasing gut butyrate content, for increasing utilization of human milk oligosaccharides, etc.

[0057] In one or more embodiments, the modulating immune level of a subject comprises one or more selected from the group consisting of: increasing organ index, increasing immune cell proliferation level, up-regulating inflammatory factor, increasing immune cell proportion, number or activity.

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

[0059] In one or more embodiments, the immune cell proliferation level is the spleen lymphocyte proliferation level.

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

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

[0062] In one or more embodiments, the immune cell comprises one or more selected from the group consisting of: leukocyte, lymphocyte, neutrophil, monocyte.

[0063] In one or more embodiments, the immune cell is a blood immune cell.

[0064] In one or more embodiments, the increasing immune cell activity comprises increasing NK cell activity.

[0065] In one or more embodiments, the increasing immune cell proportion comprises increasing the proportion of neutrophil.

[0066] In one or more embodiments, the increasing immune cell number comprises increasing the number of one or more immune cells selected from the group consisting of: leukocyte, lymphocyte, neutrophil, monocyte.

[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 product further comprises an ingredient, wherein the ingredient comprises, but is not limited to, an additive and / or a nutritional fortifier.

[0069] In one or more embodiments, the additive comprises, but is not limited to, a flavoring, a stabilizer, a thickener, a preservative, an antioxidant, an emulsifier.

[0070] In one or more embodiments, the nutritional fortifier comprises, but is not limited to, a vitamin, a mineral, an amino acid, a fatty acid, a dietary fiber.

[0071] In one or more embodiments, the type of food product comprises, but is not limited to, a plant-based food product, an animal-based food product, a microbially fermented food product, a processed food product, a food additive.

[0072] In one or more embodiments, the food product comprises, but is not limited to, a dairy product, a soy product, a probiotic powder, a probiotic oil drop, a dietary fiber supplement, a nutrition bar, a rice powder, a fruit puree, a fruit-vegetable juice, a food solid beverage, a fruit juice, an ice cream, a candy, a cookie.

[0073] In one or more embodiments, the dosage form of the health product comprises, but is not limited to, a powder, a tablet, a granule, a capsule, a solution, an emulsion, or a suspension.

[0074] In one or more embodiments, the dosage form of the pharmaceutical composition comprises, but is not limited to, a powder, a tablet, a granule, a capsule, a solution, an emulsion, or a suspension.

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

[0076] In one or more embodiments, the product can further comprise a prebiotic, such as a human milk oligosaccharide. In one or more embodiments, the human milk oligosaccharide comprises one or more selected from the group consisting of 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, a probiotic oil drop, a pressed candy, a dairy product, an infant formula, a baby milk powder.

[0078] The present application also provides the use of a human milk oligosaccharide and one or more selected from the group consisting of the B. longum subsp. infantis of any of the embodiments herein, the culture of any of the embodiments herein, the formulation of any of the embodiments herein in the preparation of a product having a modulated immune level in a subject.

[0079] In one or more embodiments, the human milk oligosaccharide comprises one or more selected from the group consisting of 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 modulating immune level in the subject comprises increasing the proportion, number or activity of immune cells in the blood.

[0081] In one or more embodiments, the increasing immune cell activity comprises increasing NK cell activity.

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

[0083] In one or more embodiments, the increasing immune cell number comprises increasing the number of neutrophils.

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

[0085] In one or more embodiments, the product is a solid beverage, probiotic oil droplet, pressed tablet candy, dairy product, formulated milk powder, infant milk powder.

[0086] The present application also provides a kit comprising (1) a prebiotic and (2) the Bifidobacterium longum subsp. infantis of any of the embodiments herein, the culture of any of the embodiments herein, and / or the formulation of any 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 oligosaccharide comprises one or more selected from the group consisting of 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] Another aspect of the present application provides a method of screening the high-ILA strain, comprising the steps of:

[0089] (1) collecting samples, and performing spread isolation on the samples;

[0090] (2) isolating and purifying after culture, and observing the colony morphology;

[0091] (3) strain preservation;

[0092] (4) ELISA screening and liquid chromatography-tandem mass spectrometry (LC-MS / MS) are used for qualitative and quantitative screening of ILA production capacity of the strains to be screened.

[0093] In one or more embodiments, the coating separation in step (1) uses a modified MRS agar medium; in one or more embodiments, the culture conditions in step (2) are 37°C anaerobic culture for 48-72 h; in one or more embodiments, the preservation in step (3) is to prepare the pure strain after separation using MRS liquid medium mixed with 10% glycerol, and store it in a -80°C environment for long-term preservation; in one or more embodiments, the mobile phase used in 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).

[0094] In one or more embodiments, the screening method also requires testing after gradient dilution of standard solutions of ILA and tryptophan (Trp, Tryptophan), and making a standard curve, and then comparing the test results obtained in the above steps with the standard curve to calculate the ILA concentration in the sample.

[0095] The beneficial effects of the present application are:

[0096] The present application aims to screen a strain with high ILA production and can significantly improve the immune function of pure breastfed infants.

[0097] The Bifidobacterium longum subsp. infantis Imu-01 of the present application has enhanced immune function, and the relevant subject can be a mammal, even a human.

[0098] The Bifidobacterium longum subsp. infantis Imu-01 of the present application is from the intestinal tract of healthy infants and has good safety.

[0099] The Bifidobacterium longum subsp. infantis Imu-01 of the present application can utilize a variety of breast milk oligosaccharides, and the combination of Imu-01 and LNnT has the function of improving immunity.

[0100] The implementation of the present application, the related products of Imu-01 can be live bacteria drugs, drugs, health products, ordinary food, etc.; the form can be powder, liquid or oil drops. BRIEF DESCRIPTION OF DRAWINGS

[0101] Figure 1 The growth curve of Bifidobacterium longum subsp. infantis Imu-01.

[0102] Figure 2 The acid tolerance of Bifidobacterium longum subsp. infantis Imu-01.

[0103] Figure 3 Bifidobacterium longum subsp. infantis Imu-01 bile salt tolerance at 0.3%.

[0104] Figure 4 Bifidobacterium longum subsp. infantis Imu-01 adhesion capacity.

[0105] Figure 5 Bifidobacterium longum subsp. infantis Imu-01 hemolytic plate test results. 1. Negative control bacteria: Listeria innocua CICC 10417; 2. Positive control bacteria: Staphylococcus aureus CICC 10473; 3. Sample: Bifidobacterium longum subsp. infantis Imu-01. Note: Test report number: CICC 25-0284-00499.04-00914

[0106] Figure 6 Bifidobacterium longum subsp. infantis Imu-01 effect on white blood cell levels.

[0107] Figure 7 Bifidobacterium longum subsp. infantis Imu-01 effect on lymphocyte levels.

[0108] Figure 8 Bifidobacterium longum subsp. infantis Imu-01 effect on neutrophil levels.

[0109] Figure 9 Bifidobacterium longum subsp. infantis Imu-01 effect on monocyte levels.

[0110] Figure 10 Bifidobacterium longum subsp. infantis Imu-01 effect on fecal butyrate levels.

[0111] Figure 11 Bifidobacterium longum subsp. infantis Imu-01 utilization of 2’-FL.

[0112] Figure 12 Bifidobacterium longum subsp. infantis Imu-01 utilization of 3’-SL.

[0113] Figure 13 Bifidobacterium longum subsp. infantis Imu-01 utilization of LNT.

[0114] Figure 14 Bifidobacterium longum subsp. infantis Imu-01 utilization of DFL.

[0115] Figure 15Utilization of LNnT by Bifidobacterium longum subsp. infantis Imu-01.

[0116] Figure 16 Utilization of 5HMOs by Bifidobacterium longum subsp. infantis Imu-01.

[0117] Figure 17 Effect of Bifidobacterium longum subsp. infantis Imu-01 + LNnT on NK cell viability.

[0118] Figure 18 Effect of Bifidobacterium longum subsp. infantis Imu-01 + LNnT on neutrophil proportion.

[0119] Figure 19 Effect of Bifidobacterium longum subsp. infantis Imu-01 + LNnT on neutrophil number.

[0120] Figure 20 Colony morphology of Bifidobacterium longum Imu-01. DETAILED DESCRIPTION

[0121] It should be understood that, in the scope of the present application, each of the technical features described above and each of the technical features described in detail below (such as the examples) can be combined with each other to form a preferred technical solution.

[0122] The present application provides a Bifidobacterium longum subsp. infantis, named Imu-01, which was deposited with the China General Microbiological Culture Collection Center (No. 3, Beichen West Road, Haidian District, Beijing, 100101) on March 11, 2025, and was classified and named as Bifidobacterium longum subsp. infantis, with a preservation number of CGMCC No: 33792.

[0123] The colony of Bifidobacterium longum subsp. infantis Imu-01 is round, convex, and smooth-edged; the colony is small, with a size of 1-2 mm, and is milky white with a slight yellowish color; the colony surface is smooth and moist, and the texture is relatively viscous. It is isolated from the intestinal tract of healthy breastfed infants in Beijing, China, and is an infant-derived probiotic. The strain has good acid tolerance, bile salt tolerance, and adhesion capacity, and can produce high levels of indole-3-lactic acid (ILA).

[0124] Strains are generally preserved in the form of microbial agents (such as microbial powder or microbial liquid), and usually need to be activated before use. Depending on the type of strain and the preservation method, an appropriate activation method can be selected. Conventional methods can be used.

[0125] The B. longum subsp. infantis Imu-01 is activated, for example, using a modified MRS medium (regular MRS medium plus mupirocin lithium salt and cysteine hydrochloride). Regular MRS medium includes proteose peptone, yeast, sodium acetate, Tween 80, magnesium sulfate heptahydrate, beef extract, glucose, diammonium citrate, potassium dihydrogen phosphate, manganese sulfate heptahydrate, and water, among other ingredients. Solid MRS medium can include an appropriate amount of agar powder to the formulation. The ingredients can be adjusted according to actual needs. The activation conditions can be conventional, for example, after 48-72 h of incubation in an anaerobic incubator at 37°C, the single colony of the strain can be directly transferred to an appropriate fresh solid medium (e.g., MRS solid medium), and incubated anaerobically at 37°C for a period of time (48-72 h) to grow and multiply, and then inoculated into a liquid medium (e.g., modified MRS medium). The inoculation amount can be selected according to actual needs, for example, two to three circles of inoculation into 10-50 mL or 15-30 mL of liquid medium, and incubated in the liquid medium at 37°C for 18-24 h to obtain the activated bacterial liquid.

[0126] The B. longum subsp. infantis Imu-01 described herein can utilize various human milk oligosaccharides and / or derivatives thereof as one or the only carbon source for growth.

[0127] Herein, human milk oligosaccharides (HMOs) are a class of natural oligosaccharides mainly contained in human milk, which are the third largest solid component in human milk, and have unique biological significance and nutritional functions. More than 200 different structures of HMOs have been identified, covering three major categories of 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 HMOs are 2'-FL produced by different manufacturers. In one or more embodiments, the HMOs are DFL. In one or more embodiments, the B. longum subsp. infantis Imu-01 can utilize any of the following groups of HMOs for growth: (1) 2'-FL; (2) 3'-SL; (3) LNT; (4) DFL; (5) LNnT; (6) 5HMO.

[0128] Herein, the 5HMOs comprise 2'-FL, 3-FL, LNT, 3'-SL and 6'SL. Any of the prior art 5HMOs comprising 2'-FL, 3-FL, LNT, 3'-SL and 6'SL can be used in the present application. Exemplarily, the 5HMOs comprise 40-70% 2'-FL, 5-30% 3-FL, 10-40% LNT, 1-10% 3'-SL and 1-10% 6'SL. In one embodiment, the 5HMOs comprise 52% 2'-FL, 13% 3-FL, 26% LNT, 4% 3'-SL, 5% 6'-SL.

[0129] In addition to the above-mentioned carbon sources, the strain can also utilize conventional carbon sources for growth, such as glucose, sucrose, starch, methanol, ethanol, lactic acid, citric acid, etc., and those skilled in the art should know how to select a suitable carbon source for the growth of the strain.

[0130] The present application also provides the use of B. longum subsp. infantis Imu-01 with the CGMCC No: 33792 for regulating the immune level of a subject. The regulation of the immune level of the subject includes one or more selected from the group consisting of increasing the organ index, increasing the immune cell proliferation level, up-regulating the inflammatory factor, increasing the proportion, number or activity of immune cells. Specifically, the regulation includes increasing the organ index of the thymus and / or spleen, proliferating the splenic lymphocytes, enhancing the activity of NK cells, up-regulating the inflammatory factor, increasing the proportion of neutrophils, and increasing the number of immune cells (e.g., leukocytes, lymphocytes, neutrophils, monocytes). In one or more embodiments, the subject is an immunosuppressed individual.

[0131] The present application also provides the use of B. longum subsp. infantis Imu-01 with the CGMCC No: 33792 for preparing a microbial preparation that is resistant to acid and bile salts and has adhesion ability.

[0132] The present application also provides a culture of B. longum subsp. infantis Imu-01 with the CGMCC No: 33792. The culture contains B. longum subsp. infantis Imu-01 described herein and a culture medium. The culture medium can be a culture medium commonly used in the art for culturing Bifidobacterium, including a culture medium used when preparing a seed solution of Bifidobacterium, and a culture medium used when performing anaerobic fermentation with Bifidobacterium. Any culture medium suitable for Bifidobacterium is included, such as a BS culture medium, an MRS culture medium, a BBL culture medium, and a Bifidobacterium agar culture medium.

[0133] In some embodiments herein, the culture medium is a modified MRS liquid medium (i.e. adding mupirocin lithium salt and cysteine hydrochloride 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 tryptone, 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 a solid culture medium is needed, the concentration of agar in the above-mentioned medium can be appropriately increased, for example, the concentration of agar is adjusted to 15 g / L. Generally, the pH value of the culture medium is 6.5-7.0, preferably 7.0.

[0134] The present application also provides a lysate of B. longum subsp. infantis with the preservation number of CGMCC 33792. Methods for lysing B. longum subsp. infantis are known in the art, such as physical lysing methods (ultrasonic lysing, high-pressure homogenization lysing, glass bead grinding method), chemical lysing methods (enzymatic method, surfactant lysing), biological lysing methods (bacteriophage lysing, autolysis method), combined lysing methods (enzymatic + ultrasonic, chemical + physical). A person skilled in the art can select a lysing method according to the composition and morphological requirements of the lysate.

[0135] The present application also provides an extract (such as polysaccharides, proteins, nucleic acids, metabolites, etc.) of B. longum subsp. infantis with the preservation number of CGMCC 33792. Methods for preparing an extract of B. longum subsp. infantis are known in the art, including but not limited to: pretreatment of bacterial cells (centrifugation, washing), releasing components by lysing bacterial cells (for reference to the lysing methods described above, a person skilled in the art can select a suitable lysing method according to the target component), separation and purification of the extract (a person skilled in the art can select a separation technique according to the properties of the target component, for example: polysaccharides (water extraction and alcohol precipitation method, column chromatography), proteins (salting-out method, chromatography method, 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). A person skilled in the art knows the detection methods for the purity and activity of the extract.

[0136] The B. longum subsp. infantis Imu-01 described herein can enhance immune function, can grow using various human milk oligosaccharides and / or derivatives thereof, and has good fermentation capacity. In addition, it has good acid tolerance, bile salt tolerance and adhesion capacity. Whole genome analysis of the strain shows that the strain does not contain drug resistance genes and virulence genes.

[0137] Thus, provided herein is also a preparation comprising B. longum subsp. infantis Imu-01 having the deposit number CGMCC No: 33792 and / or a culture, lysate or extract thereof. In some embodiments, the preparation is a microbial preparation. In one or more embodiments, the preparation can be a powder, a pill, a capsule, a granule, a tablet, a liquid preparation or a gel.

[0138] The present application also provides a composition comprising B. longum subsp. infantis Imu-01 having the deposit number CGMCC No: 33792, which is a combination of B. longum subsp. infantis Imu-01 and a prebiotic. The prebiotic can be selected from the group consisting of fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, isomaltooligosaccharides, inulin, resistant starch, etc. Preferably, the prebiotic is a human milk oligosaccharide. In one or more embodiments, the prebiotic is selected from oligosaccharides, i.e., selected from 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 3'-sialyllactose (3'-SL), lactose-N-tetraose (LNT), lactose-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 comprises B. longum subsp. infantis Imu-01 and LNnT. In one or more embodiments, the composition is capable of improving immune function.

[0139] The present application also provides a use of B. longum subsp. infantis Imu-01 having the deposit number CGMCC No: 33792 in the manufacture of a product having an improved immune effect. In one or more embodiments, the subject of the immune effect is a mammal, preferably a human.

[0140] Herein, the product is a food, a health product or a pharmaceutical composition. In one or more embodiments, the pharmaceutical composition includes, but is not limited to, an immune system drug, such as an immune enhancer; further, the form of the pharmaceutical composition includes, but is not limited to, a powder, a tablet, a granule, a capsule, a solution, an emulsion, a suspension. In one or more embodiments, the food includes, but is not limited to, a plant food, an animal food, a microbially fermented food, a processed food, a food additive; further, the form of the food includes, but is not limited to, a solid beverage, a bean product, a fruit juice, a dairy product, an ice cream, a candy, a biscuit.

[0141] For example, the bacterial suspension suitable for intragastric administration can be obtained by culturing, centrifuging, resuspending in PBS, or can be prepared by mixing bacterial powder and PBS. The method for preparing bacterial powder is known in the art, for example, centrifuging the fermentation broth of Bifidobacterium longum subsp. infantis with the preservation number of CGMCC No: 33792, collecting the bacterial cells, adding a freeze-drying protective agent to the obtained bacterial cells, and vacuum freeze-drying to obtain a freeze-dried powder. The present application also provides the bacterial powder prepared by the method.

[0142] In one or more embodiments, the product is a solid beverage, probiotic oil droplets, pressed tablet candy, dairy product, formulated milk powder, infant milk powder. Among them, the solid beverage refers to a solid product with water content not higher than 5 grams per 100 grams of finished product, such as instant coffee, fruit juice, milk tea powder, etc., which has the characteristics of easy storage and carrying; probiotic oil droplets are a product for taking probiotics through oral administration, and the oil droplet components can well protect the beneficial bacteria, allowing them to survive in the digestive system and exert their efficacy; pressed tablet candy refers to a solid candy made from sugar or syrup (powder) as the main raw material through mixing, granulating, and pressing molding and other related processes; dairy product refers to various foods made from fresh cow (sheep) milk and its products as the main raw material; formulated milk powder is a product based on milk powder with the addition of various nutritional fortifiers (such as vitamins, minerals, probiotics, DHA, ARA, etc.), such as pregnant woman milk powder, middle-aged and elderly milk powder, and children's growth milk powder; infant milk powder is specially designed for infants to meet their nutritional needs for growth and development, and contains various nutritional ingredients such as protein, fat, carbohydrates, vitamins, and minerals.

[0143] 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 per 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.

[0144] The product can contain at least one other type of other food-grade bacteria. As used herein, food-grade bacteria refers to bacteria used and generally recognized as safe for use in food, such as lactic acid bacteria, Bifidobacterium, and food-grade bacteria are preferably probiotics. The probiotic refers to a live microorganism that is beneficial to the health of the host when taken 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.

[0145] For the purposes of the present invention, a product is considered to comprise B. longum subsp. infantis Imu-01 if it comprises viable or non-replicating B. longum subsp. infantis Imu-01 cells, any cell fragments of B. longum subsp. infantis Imu-01, any fraction comprising a culture of B. longum subsp. infantis Imu-01, and / or a culture medium or a fraction thereof used for culturing B. longum subsp. infantis Imu-01. The fraction refers to different parts of the culture obtained after separation, classification or fractionation of the culture by a method, which can contain at least one of B. longum subsp. infantis Imu-01 cells, cell fragments, metabolites, culture, lysate and extracts of the culture in different concentrations.

[0146] 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).

[0147] The present invention also provides the use of an oligosaccharide (e.g. LNnT) and B. longum subsp. infantis, culture or microbial preparation of any embodiment described herein in the manufacture of a product for modulating immune levels in a subject. The modulating immune levels include increasing the proportion of immune cells (e.g. neutrophils), increasing the number of immune cells (e.g. neutrophils) or increasing the activity of immune cells (e.g. NK cells).

[0148] The pharmaceutical compositions described herein also include pharmaceutically acceptable excipients. The term "pharmaceutically acceptable excipient" refers to a carrier and / or excipient that is compatible with the subject and the active ingredient in a pharmacological and / or physiological sense that is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancers. For example, pH adjusting agents include, but are not limited to, phosphate buffers; 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.

[0149] Preferably, the excipients described herein do not affect the viability of the bacterial cells. For example: diluents / carriers (lactose, microcrystalline cellulose (MCC), starch and derivatives, mannitol, maltodextrin), protective agents (lyoprotectants such as trehalose, sucrose, glucose, polyvinylpyrrolidone (PVP), dextran, sodium glutamate, glycine, antioxidants such as vitamin C, glutathione), binding agents and disintegrating agents (hydroxypropyl methylcellulose (HPMC), polyvinyl alcohol (PVA), cross-linked povidone (PVPP), sodium carboxymethyl starch (CMS-Na)), coating materials (acrylic resin, hydroxypropyl methylcellulose phthalate, ethyl cellulose), lubricants and glidants (magnesium stearate, microfine silica, talc), other functional excipients (pH adjusting agents such as citric acid-sodium citrate buffer pair, osmotic pressure adjusting agents such as sodium chloride, flavoring agents and aromatics such as steviol glycoside, sucralose, fruit essence). An exemplary excipient combination for a probiotic lyophilized powder is trehalose, maltodextrin and mannitol, which, after lyophilization, forms a high viable cell count formulation, and an exemplary excipient for an enteric tablet includes microcrystalline cellulose, cross-linked povidone, enteric coating material.

[0150] Another aspect of the present application also provides a method for screening the high-ILA-producing strain, comprising the steps of: (1) collecting samples, and performing coating separation on the samples; (2) performing culture, separation and purification, and observing the colony morphology; (3) performing strain preservation; (4) performing ILA production capacity qualitative and quantitative screening on the strains to be screened by using ELISA preliminary screening and liquid chromatography-tandem mass spectrometry (LC-MS / MS). In the step (1), the coating separation uses a culture medium of modified MRS agar medium; in the step (2), the culture condition is 37°C anaerobic culture condition for 48-72 h; in the step (3), the preservation is to prepare the strain after the separation and purification by using MRS liquid medium mixed with 10% glycerol, and to store the strain in a -80°C environment for long-term preservation; in the step (4), the mobile phase used in the liquid chromatography includes mobile phase A (0.1% formic acid aqueous solution) and mobile phase B (0.1% formic acid acetonitrile solution). In the screening method, the standard solution of ILA and tryptophan (Trp, Tryptophan) is diluted by gradient, and then tested, and a standard curve is prepared, and then the test results obtained in the above steps are compared with the standard curve, and the ILA concentration in the sample is calculated. The conventional LC-MS / MS test method in the step (4) should be known by those skilled in the art.

[0151] The present application will be further described in the following specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present application. The methods and reagents used in the examples are conventional methods and reagents in the art unless otherwise specified.

[0152] Example

[0153] Example 1, ILA production capacity of B. longum subsp. infantis Imu-01

[0154] The ILA ELISA kit (JLC_K5285, Shanghai Jingantai Biological Engineering Co., Ltd.) was used to screen 219 strains of lactic acid bacteria preserved in the Mengniu strain library, and 12 high-ILA-producing bifidobacteria verified by LC-MS / MS quantitative verification were screened, and B. longum subsp. infantis Imu-01 (isolated from the intestinal tract of healthy infants in Beijing, China) was selected. The amount of ILA produced by the B. longum subsp. infantis Imu-01 of the present application after overnight culture in the modified MRS liquid medium was 7.39 μg / mL, which was 1.84 times that of the commercial positive strain B. breve M16V and 2.06 times that of B. longum subsp. longum BB536, and was a high-ILA-producing strain (Table 1).

[0155] Table 1: LC-MS / MS detection of ILA concentration in culture supernatant of strains

[0156] Species Strain No. Sample Source pH Value OD600 ILA (pg / mL) Bifidobacterium longum subsp. infantis Imu-01 Infantile Intestine 3.86 1.74 7.39 Bifidobacterium breve M-16V Infantile Feces 3.80 1.82 4.02 Bifidobacterium longum subsp. infantis MN17683 Infantile Feces 3.86 1.84 1.45 Bifidobacterium longum subsp. longum BB536 Infantile Feces 3.86 1.83 3.59 Bifidobacterium bifidum MN15800 Infantile Feces 3.94 1.74 0.95

[0157] The ability of B. longum subsp. infantis Imu-01 to produce ILA can be increased by 30.6% to 9.65 μg / mL after adding 0.3 mM of tryptophan (Trp) in the culture medium. This indicates that it can further utilize Trp in the intestinal tract to produce more ILA.

[0158] Table 2 LC-MS / MS detection of ILA concentration in strain culture (add Trp)

[0159] Species Strain No. Sample Source pH Value OD600 ILA (pg / mL) Increase Rate Bifidobacterium longum subsp. infantis Imu-01 Infantile Intestine 3.81 1.71 9.65 30.6% Bifidobacterium breve M-16V Infantile Feces 3.90 1.82 5.54 37.8%

[0160] Example 2, fermentation ability of B. longum subsp. infantis Imu-01

[0161] Using modified MRS medium containing 0.5% cysteine, under strict anaerobic conditions at 37°C, B. longum subsp. infantis Imu-01 enters the logarithmic growth phase from the second hour, reaches the plateau phase at 16 hours, and has very good fermentation ability within 48 hours ( Figure 1 ).

[0162] Example 3, evaluation of the probiotic properties of B. longum subsp. infantis Imu-01

[0163] B. longum subsp. infantis Imu-01 has good acid tolerance, with a survival rate of 29.6% after 12 hours of growth in a medium with pH 3.0, and a survival rate of 64.6% after 12 hours of growth in a medium with pH 4.0 ( Figure 2 ). B. longum subsp. infantis Imu-01 has good bile salt tolerance, with a survival rate of about 20% after 12 hours of growth in a medium containing 0.3% bile salt, which is comparable to the positive strain B. breve M16V ( Figure 3 ). B. longum subsp. infantis Imu-01 has good adhesion ability, and the mucin adhesion ability is significantly better than that of the positive control strain B. breve M-16V ( Figure 4 ).

[0164] In summary, B. longum subsp. infantis Imu-01 is a high-potential probiotic strain with good acid tolerance, bile salt tolerance, and adhesion ability.

[0165] Example 4, 16s sequencing and safety analysis of B. longum subsp. infantis Imu-01

[0166] 16s identification as B. longum subsp. infantis, the sequencing result is shown as SEQ NO ID: 1; according to the microbial strain hemolysis detection method, the hemolysis reaction of Imu-01 is negative, which indicates that Imu-01 is a safe lactic acid bacteria strain ( Figure 5 ).

[0167] Example 5: Immune function study of Bifidobacterium longum infant subspecies Imu-01 (animal experiment)

[0168] The *Bifidobacterium longum* subsp. *infantii* of this invention, Imu-01, exhibits excellent immune function. In an immunosuppression experiment (using cyclophosphamide to create an immune model), compared to the model group (MOD group), the white blood cell count in the Imu-01 group was significantly lower. Figure 6 ), lymphocyte count ( Figure 7 ), neutrophil count ( Figure 8 ) and monocyte count ( Figure 9 The levels of butyric acid and other substances in the feces of mice in the Immu-01 group were significantly increased (p<0.0001), and there was no difference compared with the normal control group (CON) mice, but all were significantly higher than those in the positive drug (LH) group (p<0.05). The results showed that Immu-01 significantly enhanced the immune function of immunosuppressed mice. Furthermore, the butyric acid content in the feces of mice in the Immu-01 group was increased (p<0.0001). Figure 10 This indicates that Imu-01 grows and metabolizes in the intestines to produce butyric acid, thereby further promoting immune enhancement.

[0169] Example 6: Utilization of human milk oligosaccharides by Bifidobacterium longum subsp. infantis Imu-01

[0170] Bifidobacterium longum infantis subspecies Imu-01 can utilize 2'-fucosylated lactose (2'-FL), and the 2'-FL produced by both Company A and Company B showed a consistent trend in promoting the growth of Imu-01, but the fermentation rate was significantly lower than that of glucose (…). Figure 11 In the Imu-01 group, the carbon source in the culture medium was glucose, and in each group containing 2'-FL, 2'-FL replaced glucose.

[0171] Bifidobacterium longum subsp. infantis Imu-01 can partially utilize 3'-sialyl lactose (3'-SL) for growth. Figure 12 In the Imu-01 group, the carbon source in the culture medium was glucose, and in each group containing 3'-SL, 3'-SL was used to replace glucose.

[0172] Bifidobacterium longum subsp. infantis Imu-01 can rapidly utilize lactose-N-tetrasaccharide (LNT) for growth, and its fermentation ability is superior to glucose. Figure 13 In the Imu-01 group, the carbon source in the culture medium was glucose, and in each group containing LNT, LNT replaced glucose.

[0173] Bifidobacterium longum subsp. infantis Imu-01 can utilize small amounts of human milk oligosaccharide DFL (DFL) for growth. Figure 14, the carbon source in Imu-01 group medium is glucose, and in each group containing DFL, DFL replaces glucose). DFL is an oligosaccharide in breast milk, which is a derivative of 2'-Fucosyllactose (2'-FL), has the effect of regulating intestinal flora and promoting the growth of Bifidobacterium, and helps to improve intestinal health and immune function.

[0174] B. longum subsp. infantis Imu-01 can grow rapidly with lacto-N-neotetraose (LNnT) and has an excellent growth state Figure 15 , the carbon source in Imu-01 group medium is glucose, and in each group containing LNnT, LNnT replaces glucose).

[0175] B. longum subsp. infantis Imu-01 can grow rapidly with 5HMOs (2'-FL, 3-FL, LNT, 3'-SL and 6'-SL) Figure 16 , the carbon source in Imu-01 group medium is glucose, and in each group containing 5HMOs, 5HMOs replaces glucose). 5HMO Mix is a mixed oligosaccharide (Kohjin MyOli TM ), containing 5 kinds of breast 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), which jointly act on the intestinal health, immune system and neural development of infants.

[0176] In summary, B. longum subsp. infantis Imu-01 can utilize multiple breast milk oligosaccharides, and the fermentation rate is significantly higher than glucose when LNnT and LNT are substrates, and the fermentation rate is also high when 5HMOs are substrates, followed by DFL and 2'-FL, and can partially utilize 3'-SL and DFL. It is shown that B. longum subsp. infantis Imu-01 can utilize breast milk oligosaccharides to promote its growth and proliferation.

[0177] Example 7, immune function research of B. longum subsp. infantis Imu-01 and LNnT (animal experiment)

[0178] In the immunosuppression experiment (immunization model prepared by cyclophosphamide), compared with the model group, the complex bacterial suspension of Imu-01 and LNnT, the dose is 1×10 9 cfu / each / day; LNnT is PBS solution, the dose is 500 mg / kg / d), can significantly improve the activity of mouse blood NK cells Figure 17 (flow cytometry) and the percentage (automatic blood component analyzer) Figure 18 ) and number Figure 19Imu-01 and LNnT complex can improve the immune function of immunosuppressed mice.

[0179] Experimental methods

[0180] 1. Strain isolation and culture

[0181] In this study, the collected infant feces were plated and isolated using modified MRS agar medium, and the fecal samples were diluted to 10-6 with PBS buffer before plating. After incubation at 37°C under anaerobic conditions for 48-72 h, morphologically different colonies in each culture medium were picked and subjected to three-zone streaking separation. After incubation under the same conditions for 48-72 h, the single colonies after separation and purification were collected and cultured in MRS liquid medium for amplification, and different strains were subjected to gram staining, and their morphology and source were recorded. Finally, the purified strains were preserved in MRS liquid medium mixed with 10% glycerol at -80°C for long-term preservation.

[0182] The formula is: 1L of medium 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 solid medium): 15 g. The pH value is generally adjusted to 5.5 ± 0.2.

[0183] Modified MRS medium (De Man, Rogosa, and Sharpe Medium) (i.e. addition of Mupirocin lithium salt and Cysteine hydrochloride to regular MRS medium). Regular MRS formula contains: Peptone: 10 g, Beef extract: 8 g, Yeast extract: 4 g, Glucose: 20 g, K2HP04: 2 g, Ammonium citrate: 2 g, Sodium acetate: 5 g, MgS04: 0.2 g, MnS04: 0.04 g, Tween 80: 1 g, Agar (if solid medium): 15 g / L. The pH is generally adjusted to 6.8 ± 0.2.

[0184] 2. LC-MS / MS detection

[0185] In this study, liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to screen strains for ILA production and quantification. 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, then dissolved in 1 mL of methanol to obtain a 10 mM stock solution, and stored at -20°C for later use. To construct the standard curve, the stock solution was diluted using a 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 the quantitative analysis. For sample processing, the bacterial solution cultured overnight 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, then ultrasonically treated for 5 min to extract ILA. Subsequently, it was placed at -20 ℃ for 10 min to accelerate precipitation. After 10 min, the supernatant was again centrifuged (14000 × g, 10 min, 4°C), and 500 μL of the supernatant was mixed with 500 μL of ddH2O in a 1.5 mL centrifuge tube for 10 seconds. After filtration through a 0.22 µm membrane, the sample was used for detection. The sample analysis was performed using an AQUITY UPLC system (equipped with an ACQUITY PREMIER BEH C18 column, specifications: 2.1 × 150 mm, 1.7 μm) and a QTRAP 6500 tandem mass spectrometer. Liquid chromatography separation was performed at a column temperature of 45°C, with a mobile phase of 0.1% formic acid in water (phase A) and 0.1% formic acid in acetonitrile (phase B), a flow rate of 0.3 mL / min, and a gradient elution program of 0-0.8 min maintaining 85% A, 0.8-10.5 min A phase ratio decreasing from 85% to 5%, 10.5-11.4 min maintaining 5% A, 11.4-11.5 min A phase ratio returning to 85%, and 11.5-12 min maintaining 85% A. Mass spectrometry detection was performed in positive ion mode (ESI+) using multiple reaction monitoring (MRM) mode for quantitative analysis. Mass spectrometry parameters included ion spray voltage 5000 V, temperature 300°C, gas curtain gas (CUR) 25 psi, ion source gases 1 and 2 (GAS1, GAS2) both 10 psi, and collision gas (CAD) set to medium. The MRM transition parameters, including declustering voltage, collision voltage, and collision cell exit voltage, were optimized by a syringe infusion pump to ensure the accuracy and sensitivity of the 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.

[0186] 3. Detection of acid resistance, bile salt resistance, and adhesive probiotic properties

[0187] In the acid resistance test, the pH of MRS liquid medium was adjusted to 2, 3, 4, 5 and autoclaved. The activated culture was inoculated into the liquid medium with different pH at 2% (w / v) inoculation amount, and the medium with pH 7 was used as the control group, which was placed in a growth curve instrument for anaerobic culture at 37°C for 12 hours, and the OD 600nm value was recorded every 30 minutes. 600nm The absorbance value was used to draw the growth curve, and the survival rate of each strain under different pH was calculated by 12-hour OD 600nm control group OD 600nm ) x 100%. The bile salt concentration used in the bile salt resistance test was 0, 0.15%, 0.3%, and 0.6%, respectively, and the same 2% inoculation amount was inoculated, and the OD 600nm value was monitored and the survival rate was calculated after 12 hours of anaerobic culture at 37°C. The calculation formula is: survival rate = (test group OD 600nm control group OD 600nm ) x 100%.

[0188] The adhesion experiment refers to the method of Hiromi KIMOTO-NIRA et al. (doi: 10.1111 / asj.12270) for evaluating the binding ability of each strain to mucin. The experiment uses a 96-well plate, and purified porcine gastric mucin (Sigma) is prepared into a 0.5 mg / mL solution dissolved in a 50 mmol / L carbonate / Tween 20 buffer, uniformly covers the surface of the microplate, and is incubated at 37°C to fix the mucin. Subsequently, 100 μL of bacterial solution (0.85% NaCl solution, OD 620nm adjusted to 1.0) is 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 washing the fixed wells three times with PBS, they are blocked with 1% Tween 20 in PBS 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) is added to each well, stained for 45 minutes, washed twice with PBS, and then 100 μL of 50 mmol / L citrate solution (pH 4.0) is added, and incubated at room temperature for 45 minutes to dissolve the staining complex. The absorbance is measured at 595 nm by a microplate reader to quantitatively evaluate the level of adhered bacteria, and wells without mucin are used as blank controls to correct non-specific adsorption signals.

[0189] 4. Utilization of human milk oligosaccharides

[0190] After the rejuvenated B. longum subsp. infantis Imu-01 was anaerobically cultured at 37°C for 48 h, it was centrifuged and washed twice with PBS, and then inoculated into MRS liquid medium (2% glucose and 2% different HMOs as carbon sources) at an inoculation amount of 1%. The utilization of different HMOs by B. longum subsp. infantis Imu-01 was evaluated.

[0191] 5. Immunosuppressed mice evaluation

[0192] 70 male BALB / c mice were kept in a temperature range of 23-25 °C, humidity of 40-60%, and consistent 12-hour light / dark cycle. After 1 week of adaptation, the mice were randomly assigned to 7 groups according to body weight, with 10 mice in each group. 60 mice were intraperitoneally injected with 80 mg / kg cyclophosphamide (CTX) for three consecutive days to establish an immunosuppressed mouse model. The remaining 10 mice were the control group. After three days, the mice were gavaged with probiotics / prebiotics (the bacterial suspension for gavage was obtained by culture, centrifugation, and resuspension in PBS, or prepared from bacterial powder + PBS), and the groups were as follows: (1) control group (CON group, n = 10, 200 μL of normal saline), (2) model group (MOD group, n = 10, CTX + 200 μL of normal saline), (3) positive control group (LH group, n = 10, CTX + levamisole hydrochloride 40 mg / kg / d), (4) CTX + B. longum subsp. infantis Imu-01 (n = 10, 1 × 10 9 CFU / d / mouse), (6) CTX + B. longum subsp. infantis Imu-01 + LNnT (n = 10, 1 × 10 9 CFU / d / mouse, and the dose of LNnT was 500 mg / kg / d). After 4 weeks, the mice were sacrificed, and their spleen, thymus, blood, colon, and feces were collected.

[0193] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wider range under equivalent parameters, concentrations and conditions. Although the present application gives a special embodiment, it should be understood that further improvements can be made to the present application. In general, according to the principle of the present application, this application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in this application. Some basic features can be applied within the scope of the following attached claims.

[0194] Some sequences in this paper:

[0195] SEQ ID NO: 1 16s sequencing of Bifidobacterium longum subsp. infantis Imu-01

[0196]

Claims

1. Bifidobacterium longum subsp. infantis having the accession number CGMCC No. 33792 deposited at the China General Microbiological Culture Collection Center. (Bifidobacterium animalis subsp. lactis) 1. Bifidobacterium longum subsp. infantis having the accession number CGMCC No. 33792 deposited at the China General Microbiological Culture Collection Center.​ 2. A culture of Bifidobacterium longum subsp. infantis according to claim 1.

3. The culture of claim 2, wherein, The culture medium in said culture is a modified MRS liquid medium.

4. A preparation comprising Bifidobacterium longum subsp. infantis according to claim 1, or a culture according to claim 2 or 3.

5. The formulation of claim 4, wherein, The preparation is selected from one or more of the following: a powder, a pill, a capsule, a granule, a tablet, a liquid preparation or a gel.

6. Use of Bifidobacterium longum subsp. infantis according to claim 1, of a culture according to claim 2 or 3, and / or of a preparation according to claim 4 or 5, for the manufacture of a health product having the effect of increasing the immune level of a subject.

7. Use according to claim 6, characterized in that, The health product further comprises a prebiotic.

8. Use according to claim 7, characterized in that, The prebiotic is a human milk oligosaccharide.

9. Use of Bifidobacterium longum subsp. infantis according to claim 1, of a culture according to claim 2 or 3, and / or of a preparation according to claim 4 or 5, for the manufacture of a pharmaceutical composition having the effect of increasing the immune level of a subject.

10. Use according to claim 9, characterized in that, The pharmaceutical composition further comprises a prebiotic.

11. Use according to claim 10, characterized in that, The prebiotic is a human milk oligosaccharide.

12. Use of Bifidobacterium longum subsp. infantis according to claim 1, of a culture according to claim 2 or 3, and / or of a preparation according to claim 4 or 5, for the manufacture of a food product.

13. The use according to claim 12, characterized in that, The food product further comprises a prebiotic.

14. The use according to claim 13, characterized in that, The prebiotic is a human milk oligosaccharide.

15. A health product comprising Bifidobacterium longum subsp. infantis according to claim 1, a culture according to claim 2 or 3, and / or a preparation according to claim 4 or 5.

16. The health care product of claim 15, wherein, The dosage form of the health product is selected from a powder, a tablet, a granule, a capsule, a solution, an emulsion, a suspension.

17. The health care product according to claim 15 or 16, wherein The health product further comprises a prebiotic.

18. The health care product of claim 17, wherein, The prebiotic is a human milk oligosaccharide.

19. The health care product of claim 18, wherein, The human milk oligosaccharide is 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), 6'-sialyllactose (6'-SL).

20. A pharmaceutical composition comprising Bifidobacterium longum subsp. infantis according to claim 1, a culture according to any one of claims 2 or 3, and / or a preparation according to claim 4 or 5.

21. The pharmaceutical composition of claim 20, wherein, The dosage form of the pharmaceutical composition is selected from a powder, a tablet, a granule, a capsule, a solution, an emulsion, a suspension.

22. The pharmaceutical composition of claim 20, wherein The pharmaceutical composition further comprises a prebiotic.

23. The pharmaceutical composition of claim 22, wherein, The prebiotic is a human milk oligosaccharide.

24. The pharmaceutical composition of claim 23, wherein, The human milk oligosaccharide is 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), 6'-sialyllactose (6'-SL).

25. The pharmaceutical composition of any one of claims 20-24, wherein, The pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

26. A food or food additive comprising the B. longum subsp. infantis of claim 1, the culture of any one of claims 2 or 3, and / or the preparation of claims 4 or 5.

27. The food product of claim 26, wherein The food is selected from the group consisting of: plant-based food, animal-based food, microbially fermented food.

28. The food product as described in claim 26, characterized in that, The food is selected from the group consisting of processed food.

29. The food product as described in claim 26, characterized in that, The food further comprises a prebiotic.

30. The food product as described in claim 29, characterized in that, The prebiotic is a human milk oligosaccharide.

31. The food product as described in claim 30, characterized in that, The human milk oligosaccharide is selected from one or more of: 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).

32. The food of any one of claims 26-31, wherein, The food is selected from the group consisting of: dairy product, soy product, probiotic powder, oil drop, dietary fiber supplement, nutrition bar, rice flour, fruit puree, fruit and vegetable juice, food solid beverage, candy, cookie, and / or The food further comprises an excipient and / or a food additive.

33. The food product according to any one of claims 26-31, characterized in that, The food further comprises a nutritional fortifier.

34. Use of a human milk oligosaccharide and one or more selected from the group consisting of: the B. longum subsp. infantis of claim 1, the culture of claims 2 or 3, and / or the preparation of claims 4 or 5, in the manufacture of a health product for increasing immune level in a subject.

35. Use of a human milk oligosaccharide and one or more selected from the group consisting of: the B. longum subsp. infantis of claim 1, the culture of claims 2 or 3, and / or the preparation of claims 4 or 5, in the manufacture of a pharmaceutical composition for increasing immune level in a subject.

36. Use of a human milk oligosaccharide and one or more selected from the group consisting of: the B. longum subsp. infantis of claim 1, the culture of claims 2 or 3, and / or the preparation of claims 4 or 5, in the manufacture of a food.

37. Use according to any one of claims 34 to 36, characterised in that The human milk oligosaccharide is selected from one or more of: 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).

38. Use according to any one of claims 34 to 36, characterised in that, The human milk oligosaccharide is LNnT.

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