Bifidobacterium longum BL-113 for improving and promoting growth and development of teenagers, product and application

Abstract

The invention provides bifidobacterium longum BL-113 for improving and promoting the growth and development of teenagers. The bifidobacterium longum provided by the invention can improve or promote the growth of the body of the teenager, improve or promote the absorption and utilization of microelements such as calcium and zinc by the teenager, improve intestinal flora, resist pathogens, help the intelligence development of the teenager and promote the improvement of the immune system of the teenager, and has a huge application prospect in the field of foods or medicines.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a Bifidobacterium longum BL-113 for improving and promoting the growth and development of teenagers, a product and an application thereof. Background Art

[0002] Adolescent children aged 6-18 are in the peak period of height growth. If this opportunity window can be grasped, it is expected to maximize height growth and growth and development. Various studies have shown that promoting the height growth and growth and development of adolescent children can generally be achieved through the following three ways: increasing the levels of hormones and metabolic factors related to growth and development, promoting the absorption of nutritional elements and calcium, and regular exercise.

[0003] The gut microbiome can dynamically regulate host physiology and development, and changes in its abundance and composition can affect host health, growth and development and even lead to the occurrence of diseases. A large number of studies have shown that the gut microbiota can regulate the immune system, host metabolism and endocrine environment, and maintain bone health and growth. Different metabolites produced by the gut microbiota continuously regulate the levels of various metabolic factors, metabolic pathways and the absorption of nutritional elements, thereby affecting bone growth.

[0004] As the main products of the decomposition of indigestible carbohydrates by gut bacteria, short-chain fatty acids (SCFAs), including acetate, propionate, and butyrate. Many recent studies have confirmed that the gut microbiota can play a role in the regulation of bone homeostasis by SCFAs through multiple pathways. First, SCFAs produced by some gut microbiota accelerate the dissolution of minerals and the absorption of calcium ions by lowering the intestinal pH, ultimately achieving the purpose of regulating bone density. Second, SCFAs can induce an increase in insulin-like growth factor-1 (IGF-1), which is a key mediator affecting bone growth and can promote the longitudinal growth of the femur. IGF-1 also has the function of protecting brain neurons and promoting the growth of new motor neurons, and can improve the speed of learning new skills and motor ability. It can also improve the absorption ability of amino acids, glucose, and fatty acids in the human body to promote growth and development. In addition, the activities of histone deacetylases (HDACs) are inhibited by butyrate and propionate, thereby affecting the differentiation ability of Treg cells, ultimately inhibiting osteoclast differentiation, preventing bone resorption, and promoting bone formation. The Wnt signaling pathway plays an important role in bone development and metabolism in animals and humans. Activating this pathway can promote an increase in bone mass. And SCFAs can induce T cells to produce Wnt10b protein, thereby activating the Wnt signaling pathway, reducing osteoblast apoptosis and promoting the production of osteoblasts, and increasing bone mass. On the other hand, gut microbes can polarize colon macrophages into the M1 state, produce endogenous inflammatory cytokines, cause the microbe-induced bystander effect (MIBE), and activate the Wnt / β-catenin signaling pathway with the help of TNF-α. This typical pathway's regulatory effect on osteoblasts will span from human infancy to adulthood.

[0005] The increase in glucagon-like peptide-1 (GLP-1) can inhibit bone resorption and stimulate the proliferation and differentiation of osteoblasts. Secondary bile acids, which can act as agonists of the G protein-coupled bile acid receptor (TGR5), can activate TRG5 to promote the production of GLP-1. And the gut microbiota plays an important role in converting primary bile acids into secondary bile acids. Lipopolysaccharide (LPS) derived from the gut microbiota can upregulate the expression of Sox-9 and inhibit the hypertrophy of growth plate chondrocytes. The gut microbiota can also affect the sex hormone levels in the host body, thereby regulating bone homeostasis and promoting the growth and development of adolescents and children.

[0006] Probiotics are live symbiotic microorganisms that can regulate the intestinal microbiota composition by colonizing the intestine, increase bone mass in children, and promote their growth. On the other hand, probiotics can reduce intestinal permeability, inhibit intestinal inflammation, thereby protecting children's gastrointestinal health, enhancing resistance, and reducing the occurrence of intestinal flora imbalance. Bifidobacterium is a very common probiotic in the human body, which can improve the body's immunity and anti-infection ability, inhibit intestinal putrefactive pathogenic microorganisms, and regulate cell differentiation and apoptosis. With age, the number of Bifidobacterium in the human intestine gradually decreases, and the dominant strains change from Bifidobacterium infantis and Bifidobacterium breve to Bifidobacterium adolescentis and Bifidobacterium longum. Bifidobacterium longum is a probiotic widely used in functional foods. Increasing the intake of Bifidobacterium longum can change the intestinal microbiota composition and maintain the homeostasis of the intestinal barrier.

[0007] Therefore, developing a probiotic that can be used to promote the height growth and development of children and adolescents, maintain intestinal homeostasis, improve exercise ability, and enhance immunity is of great significance for the healthy growth of children and adolescents. Summary of the Invention

[0008] The present invention provides a Bifidobacterium longum and its application in promoting the growth and development, height growth, and enhancing immunity of children and adolescents. This Bifidobacterium longum can maintain intestinal homeostasis, promote the absorption and utilization of nutrient elements, produce a variety of metabolites beneficial to the human body and involved in human metabolism, thereby increasing bone mass and the number of osteoblasts in children and adolescents and promoting their body length growth.

[0009] In the first aspect of the present invention, there is provided a Bifidobacterium longum BL-113 for improving and promoting the growth and development of adolescents, characterized in that the Bifidobacterium longum BL-113 was deposited at the China General Microbiological Culture Collection Center on November 2, 2023, with the deposit address being the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC NO. 28848, and the taxonomic name being Bifidobacterium longum.

[0010] In the second aspect of the present invention, there is provided a preparation method of the Bifidobacterium longum BL-113 according to claim 1, comprising:

[0011] (1) Screening and isolation of the target strain

[0012] The fecal samples from healthy infants who did not take antibiotics were serially diluted in sterile physiological saline. The diluted solutions were selected for spread plating on MRS medium, cultured, and the bacterial morphology was observed and recorded; single colonies with the morphological characteristics of Bifidobacterium colonies were subjected to Gram staining and catalase tests, and microscopic examination was carried out; strains with a positive Gram staining result and a negative catalase test (no bubbles produced) were selected as the preliminary target strains;

[0013] (2) Preliminary identification of strains

[0014] The above-mentioned target strains were added to API 20A medium to prepare a bacterial suspension, and then inoculated onto an API 20A test strip, anaerobically cultured, and additional reagents BCP, XYL, HER, and H2O2 were added for result interpretation. API 20A V3.0 was used for result identification, and the strains identified as Bifidobacterium were further purified;

[0015] (3) Identification of strains

[0016] L-Arabinose, maltose, salicin, sucrose, xylose, and starch were added to the liquid basal medium as carbon sources respectively. The strains preliminarily identified as Bifidobacterium were inoculated into liquid media with different carbon sources for culture, and the acid production of different strains was observed using BTB-MR reagent;

[0017] Strains that could hydrolyze L-arabinose, sucrose, and maltose, partially hydrolyze xylose, and did not hydrolyze starch and salicin were further cultured and then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing identification. The nucleic acid sequence alignment of the sequencing results was carried out in NCBI, and finally the target Bifidobacterium longum BL-113 was obtained.

[0018] In step (1) of the above preparation method:

[0019] The concentration of the diluted solution was: 1×10 -6 ~1×10 -4 mg / ml;

[0020] The culture conditions of MRS medium were to culture at 36±1°C for 48±2 to 72±2 h.

[0021] In step (2) of the above preparation method:

[0022] The conditions for anaerobic culture were to culture at 36±1°C for 48±2 to 72±2 h.

[0023] In step (3) of the above preparation method:

[0024] The formula of the liquid basal medium is 0.5 g of casein peptone, 0.5 g of tryptone, 1.0 g of yeast extract, and 4 ml of salt mixed solution (0.2 g of CaCl2, 0.48 g of magnesium sulfate heptahydrate, 1.0 g of dipotassium hydrogen phosphate, 1.0 g of potassium dihydrogen phosphate, 10.0 g of sodium bicarbonate, and 2.0 g of NaCl are dissolved in water and made up to 1000 ml);

[0025] The culture conditions of the liquid medium are to culture at 36 ± 1 °C for 48 ± 2 to 72 ± 2 h;

[0026] The formula of the BTB-MR reagent is 0.2 g of bromothymol blue (BTB), 0.1 g of methyl red (MR), 300 ml of 95% ethanol, and 200 ml of distilled water;

[0027] The culture conditions for strains that do not hydrolyze starch and salicin are to culture at 36 ± 1 °C for 48 ± 2 to 72 ± 2 h.

[0028] The third aspect of the present invention provides a Bifidobacterium longum BL-113 bacterial powder, which is prepared from the Bifidobacterium longum BL-113 obtained by the above preparation method.

[0029] The fourth aspect of the present invention provides a preparation method of the above Bifidobacterium longum BL-113 bacterial powder, including: inoculating the Bifidobacterium longum BL-113 obtained in step (3) of the above preparation method into MRS liquid medium for culture, collecting the bacterial liquid and centrifuging, rinsing the bacterial sludge obtained by centrifugation, adding a cryoprotectant and then freeze-drying to obtain the Bifidobacterium longum BL-113 bacterial powder. Among them, the culture conditions of the MRS liquid medium are to culture at 36 ± 1 °C for 48 ± 2 to 72 ± 2 h; the cryoprotectant is a composite cryoprotectant composed of 10% - 15% skim milk powder, 20% - 25% trehalose, and 2% - 4% Vc-Na; the mass ratio of the bacterial sludge to the cryoprotectant is 1:(0.6 - 1.5); the freeze-drying conditions are: the first stage: -40 °C, after 4 h of pre-freezing, vacuum is pumped; the second stage: 5 °C, 10 h; the third stage: 15 °C, 10 h, the vacuum degree is 10 Pa, and the thickness is about 0.5 cm.

[0030] The fifth aspect of the present invention provides a Bifidobacterium longum preparation, including the above Bifidobacterium longum BL-113 or the above Bifidobacterium longum BL-113 bacterial powder. The preparation can be a solid bacterial powder or a liquid drink.

[0031] The sixth aspect of the present invention provides the application of the above-mentioned Bifidobacterium longum BL-113, or the above-mentioned Bifidobacterium longum BL-113 bacterial powder, or the above-mentioned Bifidobacterium longum preparation, in a food composition or a pharmaceutical composition for promoting the growth and development of adolescents and children, promoting the body length growth of adolescents and children, anti-obesity in adolescents and children, improving intestinal metabolic disorders in adolescents and children and promoting the normalization of intestinal microbiota composition, enhancing immunity, improving the motor ability and intellectual development of adolescents and children, and facilitating the absorption of trace elements by adolescents and children.

[0032] The Bifidobacterium longum BL-113 provided by the present invention can effectively maintain the intestinal homeostasis of adolescents and children, keep the intestinal microbiota composition at a relatively good level, enhance immunity, and promote the dual growth and development of intelligence and height in children and adolescents. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the detection result of the survival rate of Bifidobacterium longum BL-113 in artificial gastric juice (pH = 3) and artificial intestinal juice (pH = 8) in Example 1.

[0034] Figure 2 It is the change in the intestinal microbiota diversity of Caenorhabditis elegans after being treated with Bifidobacterium longum BL-113 and BL-21 respectively.

[0035] Figure 3 It is the analysis of the abundance at the phylum level of the intestinal microbiota of Caenorhabditis elegans after being treated with Bifidobacterium longum BL-113 and BL-21 respectively.

[0036] Figure 4 It is the change in human body height from 0 to 12 weeks before and after taking Bifidobacterium longum BL-113 or placebo. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] The following will clearly and completely describe the specific embodiments of the present disclosure in conjunction with the accompanying drawings. It should be understood that the embodiments described herein are only a part of the embodiments of the present disclosure, used to illustrate and explain the present disclosure, and do not serve as any limitation to the present disclosure and its application or use.

[0038] Example 1

[0039] Screening, strain identification and preservation of Bifidobacterium longum

[0040] 1. Screening and purification

[0041] (1) Strain screening: Continuously dilute 1 g of fecal samples from healthy infants who have not taken antibiotics in sterile physiological saline until diluted to 10 -6, Select an appropriate concentration of dilution solution for plate coating. The medium used is MRS medium. Incubate at 36±1°C for 48±2 h, observe and record the bacterial morphology. Perform Gram staining and catalase test on single colonies with the colony morphology of Bifidobacterium, and conduct microscopic examination. Select the strains with positive Gram staining results and negative catalase test (no bubbles generated) as the target strains.

[0042] (2) Strain purification: On the MRS medium, streak and purify the above strains multiple times until a single colony with consistent morphology is obtained. Select the single colony whose colony morphology still conforms to the colony morphology characteristics of Bifidobacterium for cultivation, which will be used as the strain for subsequent physiological and biochemical identification.

[0043] Observation of the colony morphology of the above strains found that: the colonies are smooth, soft in texture, milky white, with complete and raised edges.

[0044] 2. Identification

[0045] Extract total DNA from the isolates and sequence the isolated strains through 16S rDNA (Sangon Biotech (Shanghai) Co., Ltd.). Then conduct nucleic acid sequence alignment of the sequencing results in NCBI, and finally obtain the target Bifidobacterium longum BL-113.

[0046] 3. Preservation

[0047] Inoculate the purified Bifidobacterium longum BL-113 into MRS liquid medium and incubate at 36±1°C for 48±2 h. Collect the bacterial liquid and centrifuge it at 8000 g / min for 10 min. Rinse the centrifuged bacterial pellet twice, add a protective agent with a mass ratio of 1:1 to the bacterial pellet, and then perform freeze-drying to obtain the Bifidobacterium longum BL-113 bacterial powder.

[0048] Example 2

[0049] This example describes the performance characteristics of Bifidobacterium longum BL-113.

[0050] 1. Basic situation of Bifidobacterium longum BL-113 cells

[0051] Use API identification to analyze the physiological and biochemical characteristics such as carbohydrate utilization ability and enzyme activity of the above isolated strains. Add a sufficient amount of the above strains to the API 20A medium to prepare a bacterial suspension, then inoculate it onto the API 20A test strip, incubate anaerobically at 36°C for 48 h, add additional reagents BCP, XYL, HER, H2O2 for result interpretation, and use API 20A V3.0 for result identification. The analysis results are shown in Table 1.

[0052] Table 1

[0053]

[0054] "+" indicates the ability to metabolize; "-" indicates the inability to metabolize.

[0055] The results in Table 1 above show that Bifidobacterium longum BL-113 has no spores, the Gram staining result is positive, the catalase test is negative, it can utilize lactose, raffinose, arabinose, sucrose, glucose, maltose, mannitol and salicin, and has a hydrolytic effect on gelatin and esculin.

[0056] 2. Verification of the tolerance and colonization ability of Bifidobacterium longum BL-113 in gastric juice and intestinal juice

[0057] Bifidobacteria are a type of physiologically beneficial bacteria, but their tolerance to acid is generally not strong. In this example, the tolerance of Bifidobacterium longum BL-113 to artificial gastric juice and intestinal juice was tested. At the same time, another Bifidobacterium longum BL-21 with better acid tolerance and the ability to survive through the gastrointestinal tract (purchased from Healthy Probiotics Wecare-probiotics Company) was used for comparison. This strain belongs to Bifidobacterium longum subsp. longum and can be used in the fields of food, dairy products and health products. Similar to the BL-113 strain, it also has the effects of improving the intestinal flora composition (can reduce the Firmicutes / Bacteroidetes ratio), increasing intestinal peristalsis, reducing fat accumulation and anti-obesity.

[0058] (1) Detection of tolerance in gastric juice and intestinal juice environments

[0059] The initial concentrations (i.e., the concentrations at 0 h) and survival rates of the BL-113 strain and the BL-21 strain in artificial gastric juice and artificial intestinal juice are shown in Table 3. The results show that after the BL-113 strain was treated with artificial gastric juice (pH = 3) for 1 h, its survival rate was 85.1%, higher than 55.2% of the control BL-21 strain; after the BL-113 strain was treated with artificial gastric juice (pH = 3) for 3 h, its survival rate was 38.7%, higher than 30.2% of the control BL-21 strain. After the BL-113 strain was treated with intestinal juice (pH = 8) for 1 h, its survival rate was 69.2%, higher than 67.3% of the control BL-21 strain; after the BL-113 strain was treated with intestinal juice (pH = 8) for 3 h, its survival rate was 47.5%, higher than 35.7% of the control BL-21 strain.

[0060] Table 2

[0061]

[0062] (2) Detection of adhesion ability

[0063] Take 200 ul of normal viable HT-29 cell solution to configure the initial number of cells to 1×10 5Prepare HT-29 cell slides with cfu / well for standby. Resuspend Bifidobacterium longum BL-113 and BL21 to be tested in DMEM medium respectively and add them to a 24-well plate containing cell slides. The culture conditions are: incubate at 37 °C and 5% CO2 for 2 h. Wash the monolayer cells 4 times with sterile PBS buffer, fix with methanol for 10 min and perform Gram staining. Observe the adhesion situation through a microscope respectively and record the number of probiotics adhered to 50 cells.

[0064] Table 3

[0065] Test time 0.5h 1h BL-113 62.3 cells / 50 cells 57.4 cells / 50 cells BL-21 59.7 cells / 50 cells 55.2 cells / 50 cells

[0066] The results show that with the increase of time, the number of bacteria adhered to HT-29 cells can still reach 57.4 per 50 cells, indicating that BL-113 has good adhesion ability and better intestinal adhesion compared with the control group.

[0067] The above results indicate that the Bifidobacterium longum BL-113 strain can still survive well after digestion by artificial gastric juice and intestinal juice ( Figure 2 ). Compared with the BL-21 strain, the Bifidobacterium longum BL-113 provided by the present invention has stronger resistance to digestive juice and adhesion ability. Therefore, the BL-113 strain can successfully pass through the digestive tract and survive and colonize in the intestine.

[0068] 3. Toxicology test and safety detection of Bifidobacterium longum BL-113

[0069] (1) Antibiotic resistance test

[0070] To verify the safety of the Bifidobacterium longum BL-113 of the present disclosure, its antibiotic resistance was analyzed. The method for evaluating the antibiotic sensitivity of Bifidobacterium longum BL-113 was selected as the method of SN / T 1944-2007 "Determination of Bacterial Drug Resistance in Animals and Their Products". A total of 10 most common antibiotics were used in this test, including gentamicin (GEN), erythromycin (ERM), streptomycin (STM), chloramphenicol (CP), tetracycline (TET), kanamycin (KAN), vancomycin (VAN), clindamycin (CLM), ampicillin (AMP) and quinupristin / dalfopristin (Q / D). The results show that the Bifidobacterium longum BL-113 strain provided by the present invention is sensitive to the above antibiotics, indicating that the strain provided by the present invention does not contain exogenous antibiotic resistance genes, has good safety, and meets the requirements of the evaluation specification for edible bacterial drug resistance.

[0071] (2) Toxicology experiment

[0072] Animal tests were conducted to verify the safety of the Bifidobacterium longum BL-113 provided by the present invention.

[0073] Experimental group: The original culture solution of Bifidobacterium longum BL-113 was continuously gavaged to healthy male and female mice aged 6 - 8 weeks at a dose of 1.0×10 11 cfu / kg. Control group: Healthy male and female mice aged 6 - 8 weeks were gavaged with 0.85% normal saline at the same dose. Each group was continuously gavaged for 3 days, and the room temperature was maintained at 25 ± 2 °C for 7 days for verification. The observation results are shown in Table 5.

[0074] Table 4

[0075]

[0076] Note: The numbers in Table 4 represent the number of dead animals.

[0077] The results showed that no toxic reactions or deaths were observed in the test mice of both the experimental group and the control group, and no statistically significant differences were found between the two groups (p > 0.05).

[0078] Example 3

[0079] This example illustrates the effects of Bifidobacterium longum BL-113 on maintaining intestinal homeostasis, improving exercise ability, and anti-obesity function.

[0080] Caenorhabditis elegans in vitro model experiment

[0081] There is a high degree of homology between the genes of Caenorhabditis elegans and humans, and there are extensive similarities in metabolism, which is mainly colonized by intestinal bacteria. Therefore, Caenorhabditis elegans is an ideal model for studying the role of human intestinal microorganisms, and using this interspecies model helps to understand the physiological relevance of the human gut microbiota.

[0082] Caenorhabditis elegans fed with 1 ml of Escherichia coli OP50 as bacterial feed was used as a blank control, and Caenorhabditis elegans was fed with 1 ml of Escherichia coli OP50 mixed with 45 mg / L of Bifidobacterium longum BL-113 and Bifidobacterium longum BL-21, respectively. The growth and development of worms fed with different bacterial feeds, as well as the composition and abundance of intestinal microorganisms, were observed.

[0083] (1) Determination of the locomotor performance of Caenorhabditis elegans: 15 worms were randomly selected from different groups and placed in M9 buffer solution. After allowing them to swim freely for 30 s to adapt, their locomotion was observed under a microscope and recorded. The judgment criterion was: A continuous single rapid sinusoidal movement of the worm was recorded as one movement. The results are shown in Table 6.

[0084] (2) Determination of the fat accumulation in Caenorhabditis elegans: Different groups were stained using Oil Red O fat staining method, and the fat accumulation in worms of different groups was observed and recorded. The results are shown in Table 6.

[0085] (3) Determination of the diversity of intestinal flora: Several worms fed with different bacterial diets were selected and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing to obtain the composition and abundance of the intestinal flora of the worms under different conditions.

[0086] (4) Detection of whether the growth and development of Caenorhabditis elegans fed with probiotic-supplemented bacterial diet will be affected by dietary restriction: Using a mature genetic model of dietary restriction, eat-2(ad116), the worm has a mutation in the nicotinic acetylcholine channel, which can reduce food intake. The results show that the growth and development of nematodes fed with probiotic-supplemented bacterial diet will not be affected by dietary restriction. Therefore, no additional dietary control is required for consuming products supplemented with this probiotic.

[0087] Table 5

[0088]

[0089] The results showed that Caenorhabditis elegans fed with Escherichia coli OP50 mixed with Bifidobacterium longum BL-113 had better growth and development than other groups. At the phylum level, the Firmicutes / Bacteroidetes ratio decreased, and the level of Actinobacteria increased in the E. coli OP50+BL-113 group (see Figure 3 ); at the genus level, the levels of Faecalibacterium, Lachnospira, Sutterella, etc. decreased, while the levels of Bifidobacterium, Lactobacillus, Oscillospira, etc. increased. The increase in the levels of these genera also confirmed that Bifidobacterium longum BL-113 can indeed increase the level of SCFA in the intestine, reduce intestinal tissue damage and protect the intestinal barrier.

[0090] Example 4

[0091] This example illustrates the promotion of growth and development, height increase and improvement of immune effect by Bifidobacterium longum BL-113.

[0092] A variety of metabolites produced by intestinal microorganisms have a certain promoting effect on bone growth and growth and development. In particular, SCFA can promote osteoblast production, bone mass increase and bone formation through various pathways, thus promoting the bone development of adolescents and children.

[0093] 1. Verification of the effects of bone growth and height increase in a mouse model

[0094] Preparation: Select 30 two-month-old mice with relatively small body size differences and measure the length of their leg bones. Randomly divide them into a probiotic group, a positive control group, and a blank control group, with 10 mice in each group. Raise them under SPF conditions. During the experiment, the experimental animals drank deionized water and had free access to food. Starting from the second week, the probiotic group was gavaged with 10 mL / kg.bw of mice daily, the positive control group was gavaged with 40 mg / kg.bw of calcium juice (Beijing Landi Pharmaceutical Co., Ltd.) daily, and the blank control group was gavaged with physiological saline daily. Weigh the mice and record their weights every week.

[0095] Determination of SCFA level: After continuous gavage feeding for 4 weeks, anesthetize and sacrifice the mice. Open the chest cavity of the mice, insert a syringe into the right ventricle to draw blood samples and prepare serum using a serum separation tube. Then, use a mouse short-chain fatty acid (SCFA) ELISA detection kit (Shanghai Yanqi Biotechnology Co., Ltd.) to detect the SCFA level in the mouse serum.

[0096] Determination of bone status: After measuring the leg bone length of the mice, take out the bilateral femurs of the experimental mice, place them in an oven at 105 °C and dry them to a constant weight, and measure the density of the two femurs. The bone calcium content of the femur was measured by atomic absorption spectrophotometry, and the results are shown in Table 6.

[0097] Table 6

[0098]

[0099] The results showed that both the probiotic group and the positive control group of mice showed an increase in leg bone length, femoral weight, and bone calcium content. However, the changes in the probiotic group were more significant and the SCFA level in the serum increased significantly. Especially butyrate, its concentration in the serum increased the most, and this metabolite plays a very important role in bone development. The blank control group showed no special performance.

[0100] 2. Clinical verification of the growth-promoting effect

[0101] Recruit 70 subjects aged 6 - 18 years old from adolescents and children, and randomly divide them into a probiotic group and a placebo group for a 12-week controlled trial. This experiment follows the principles of randomization and double-blindness, and none of the selected subjects have taken any form of probiotics or prebiotics within four weeks. The parents or guardians of the subjects have signed the informed consent form. The probiotic group receives 113 sachets of Bifidobacterium longum BL daily, and the placebo group receives placebo (maltodextrin) sachets daily. Refer to the "Technical Specifications for Student Health Examinations" (GB26343 - 2010) to measure the height and weight of the students. And combined with the body mass index (BMI) = weight (kg) / height (m) 2Measurements of height and weight were taken before the start of the test (i.e., week 0) and at week 12 respectively, and the BMI values were calculated. The results are shown in Table 7.

[0102] Table 7

[0103]

[0104] The results showed that after 12 weeks, the body weight and height of the subjects in the probiotic group and the placebo group showed a certain increase. However, the increase in height of the subjects in the probiotic group was more significant, the change in body weight was smaller, and the BMI index remained within the normal range. Therefore, Bifidobacterium longum BL-113 does have the effect of promoting the growth and development of adolescents and children.

[0105] 3. Experimental verification of immune health effects

[0106] An appropriate amount of the original culture solution of Bifidobacterium longum BL-113 was added to the RPIM-1640 medium (containing 10% FBS) and co-cultured with Caco-2 cells. The control group was not cultured with probiotics. The culture conditions were 37 °C and 5% CO2. The expression level of IL-6 in Caco-2 cells after adding probiotics was detected.

[0107] The experimental results found that Bifidobacterium longum BL-113 increased the expression level of IL-6 from 21 ng / ml to 97 ng / ml. Therefore, Bifidobacterium longum BL-113 does have the function of enhancing human immunity.

Claims

1. Bifidobacterium longum BL-113 for improving and promoting the growth and development of adolescents, characterized in that, The Bifidobacterium longum BL-113 was deposited at the China General Microbiological Culture Collection Center on November 2, 2023. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC NO. 28848, and the taxonomic name is Bifidobacterium longum Bifidobacterium longum .

2. A preparation method of Bifidobacterium longum BL-113 according to claim 1, comprising: (1) Screening and isolation of the target strain Continuously dilute fecal samples from healthy infants who have not taken antibiotics in sterile physiological saline, select the dilution for plate coating, use MRS medium as the culture medium, culture, observe and record the bacterial morphology; perform Gram staining and catalase tests on single colonies with the morphological characteristics of Bifidobacterium colonies, and perform microscopy; select the strains with positive Gram staining results and negative catalase test results as the preliminary target strains; (2) Preliminary identification of the strain Add the above-mentioned target strain to API 20 A medium to prepare a bacterial suspension, then inoculate it onto an API 20 A test strip, perform anaerobic culture, add additional reagents BCP, XYL, HER, H2O2 for result interpretation, use API 20 A V3.0 for result identification, and further purify the strains identified as Bifidobacterium; (3) Strain identification Add L-arabinose, maltose, salicin, sucrose, xylose and starch as carbon sources to the liquid basal medium respectively, inoculate the strains preliminarily identified as Bifidobacterium into liquid media with different carbon sources for culture, and use BTB-MR reagent to check the acid production of different strains; Select the strains that can hydrolyze L-arabinose, sucrose, maltose, partially hydrolyze xylose, and do not hydrolyze starch and salicin, culture them further, perform sequencing identification, and perform nucleic acid sequence alignment on the sequencing results in NCBI to finally obtain the target Bifidobacterium longum BL-113.

3. The preparation method according to claim 2, wherein In the step (1): The concentration of the diluent is: 1×10 -6 ~1×10 -4 mg / ml; The culture condition of MRS medium is to culture at 36±1°C for 48±2 - 72±2 h.

4. The preparation method according to claim 2, characterized in that, In the step (2): The condition of anaerobic culture is to culture at 36±1°C for 48±2 - 72±2 h.

5. The preparation method according to claim 2, characterized in that, In the step (3): The formula of the liquid basal medium is 0.5 g of casein peptone, 0.5 g of tryptone, 1.0 g of yeast extract, 4 ml of salt mixture solution (0.2 g of CaCl2, 0.48 g of magnesium sulfate heptahydrate, 1.0 g of dipotassium hydrogen phosphate, 1.0 g of potassium dihydrogen phosphate, 10.0 g of sodium bicarbonate, 2.0 g of NaCl dissolved in water and made up to 1000 ml); The culture condition of the liquid medium is to culture at 36±1°C for 48±2 - 72±2 h; The formula of BTB-MR reagent is 0.2 g of bromothymol blue (BTB), 0.1 g of methyl red (MR), 300 ml of 95% ethanol, 200 ml of distilled water; The culture condition of the strains that do not hydrolyze starch and salicin is to culture at 36±1°C for 48±2 - 72±2 h.

6. A Bifidobacterium longum BL-113 bacterial powder, prepared from Bifidobacterium longum BL-113 obtained by the preparation method according to claims 2 - 3.

7. A method for preparing the Bifidobacterium longum BL-113 bacterial powder according to claim 6, comprising: Inoculate the Bifidobacterium longum BL-113 obtained in step (3) of the preparation method according to claims 2 to 5 into MRS liquid medium for culture, collect the bacterial liquid and perform centrifugation, wash the bacterial sludge obtained by centrifugation, add a cryoprotectant and then perform freeze-drying to obtain the Bifidobacterium longum BL-113 bacterial powder.

8. The preparation method according to claim 7, wherein the culture conditions of the MRS liquid medium are culturing at 36 ± 1 °C for 48 ± 2 to 72 ± 2 h; the cryoprotectant is a composite cryoprotectant composed of 10% - 15% skim milk powder, 20% - 25% trehalose, and 2% - 4% Vc-Na; the mass ratio of the bacterial sludge to the cryoprotectant is 1:(0.6 - 1.5); the freeze-drying conditions are as follows: the first stage: -40 °C, after 4 h of pre-freezing, vacuumize; the second stage: 5 °C, 10 h; the third stage: 15 °C, 10 h, the vacuum degree is 10 Pa, and the thickness is about 0.5 cm.

9. A Bifidobacterium longum preparation, comprising the Bifidobacterium longum BL-113 according to claim 1 or the Bifidobacterium longum BL-113 bacterial powder according to claim 6.

10. Use of the Bifidobacterium longum BL-113 according to claim 1 or the Bifidobacterium longum BL-113 bacterial powder according to claim 6 or the Bifidobacterium longum preparation according to claim 9 in a food composition or a pharmaceutical composition for promoting the growth and development of adolescents and children, promoting the body length increase of adolescents and children, anti-obesity of adolescents and children, improving the intestinal metabolic disorder of adolescents and children and promoting the normalization of the intestinal microbiota composition, enhancing immunity, improving the motor ability and intellectual development of adolescents and children, and promoting the absorption of trace elements by adolescents and children.

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