Lactobacillus mucosae UP-1 for fermentation and its application in improving bone metabolism and promoting growth

Abstract

The present invention relates to Lactobacillus mucosae UP-1 and its application in improving bone metabolism and promoting growth, belonging to the field of microbial technology. The classification and naming of the fermented Lactobacillus mucosae UP-1 is Limosilactobacillus fermentum UP-1, and its preservation unit is the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the preservation number: CGMCC No. 33076; the preservation time: December 16, 2024. The fermented Lactobacillus mucosae UP-1 provided by the present invention can promote the increase of body length and femoral long diameter of the body, improve the trabecular bone density and the level of beneficial metabolite short-chain fatty acids, improve the bone metabolism level, and promote the healthy development of bones by increasing the levels of insulin-like growth factor binding protein-3 (IGFBP3), growth hormone (GH), and insulin-like growth factor (IGF-1) in the serum, so as to achieve the purpose of promoting growth and development and improving nutrient absorption, which helps more scientific dietary intervention and the development of precision probiotics.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to fermented Lactobacillus mucosae UP-1 and its application in improving bone metabolism and promoting growth. Background Art

[0002] Growth and development are governed by different genetic, nutritional, and environmental factors that act systematically on the endocrine system and growth plates. A large number of studies have revealed the important role of the gut microbiota during growth and development. Whether it is physical growth, nervous system maturation, or immune system fortification, the gut microbiota should be regarded as an "organ" that has an important impact on growth and development. The connection between it and growth and development provides a unique approach for improving health and preventing diseases. Compared with mice with normal flora, the growth and development and longitudinal bone growth of germ-free mice are decreased. When germ-free mice are recolonized with gut microbiota, bone formation and long bone length can be improved. Currently, the molecular mechanism by which the gut microbiota promotes bone growth is not clear. In addition to regulating the host growth hormone (GH) / insulin-like growth factor-1 (IGF-1) axis to mediate the host growth and development process, another possible explanation is that gut microbiota helps with the digestion of macronutrients. These studies have emphasized the positive role of gut microbiota in the hormonal and nutritional regulation of bone growth and body growth.

[0003] As important "modulators" of gut microbiota, probiotics are widely used in the fields of health food, dairy processing, animal farming, live bacteria drug development, etc. Their role in promoting body growth is mainly reflected in improving the intestinal environment, enhancing nutrient absorption, regulating the immune system, and inhibiting the reproduction of pathogenic bacteria. Research has found that the cross-regulation of the intestine-bone axis is related to the direct or indirect production of short-chain fatty acids (SCFAs) and other beneficial microbial metabolites by probiotics. These metabolites act on the liver and adipose tissue to induce the production of IGF-1, and IGF-1 in turn acts on osteocytes to affect linear growth, bone mass, and mineralization. At the same time, the metabolites can directly act on bones and muscles to induce the production of local IGF-1. Another study also confirmed this. Supplementation with Lactobacillus rhamnosus GG can indirectly promote the production of SCFAs by gut microbiota, and SCFAs further selectively support the development of peripheral regulatory T cells and then stimulate bone formation, thus improving growth and development performance. More importantly, SCFAs can also play a role in nutritional regulation by stimulating intestinal blood flow and electrolyte uptake. Approximately 10% of the total energy ingested by humans comes from SCFA absorption. In addition, probiotics interact with gut-associated lymphoid tissue (GALT) to stimulate the activity of immune cells (such as macrophages, T cells, and B cells), promote the secretion of anti-inflammatory cytokines (such as IL-10), and at the same time inhibit the overproduction of pro-inflammatory cytokines (such as IL-6, TNF-α). This immunomodulatory effect helps to reduce the inflammatory response and energy consumption, creating good conditions for the normal growth of the body. Obviously, supplementing probiotics is one of the effective strategies to promote host growth. However, currently, there are few studies evaluating the effects of probiotics on the growth and development of normal organisms, and the specific effects of different strains, dose-response relationships, and efficacy in specific populations are still unclear.

[0004] Therefore, developing a probiotic preparation that can promote the growth of normal children has great scientific significance and economic value. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides a fermented Lactobacillus mucosae preparation and its application that can promote digestion, absorption, growth and development, and improve bone metabolism. This strain can effectively promote the physical growth of the body, increase the level of beneficial metabolites in the body, and at the same time improve the bone metabolism level, promote the healthy development of bones, and has a positive effect on the growth, development and nutritional absorption of the body.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] 1. Isolation, identification and safety assessment of probiotics

[0008] The present invention isolates a strain of Lactobacillus mucosae from the breast milk of healthy women (Limosilactobacillus fermentum ). The strain was identified as Lactobacillus mucosae fermentum by phenotypic identification, molecular biology identification, and phylogenetic analysis. Its hemolytic property, antibiotic sensitivity, and bacterial pathogenicity were evaluated, and its safety was confirmed. In addition, its tolerance to gastric juice and intestinal juice was detected, and it was determined that it could reach the intestine alive.

[0009] 2. Growth-promoting effects of probiotics on different female and male mice

[0010] The present invention uses female and male mice as animal models to explore the effects of probiotics on the growth performance and digestion and absorption of the body. The body length changes and body length growth rates of the mice were statistically analyzed, and the changes in serum growth indexes of the mice were detected to evaluate the growth status. The digestion and absorption level was evaluated in combination with the changes in short-chain fatty acids in the mouse intestine.

[0011] The present invention provides Lactobacillus mucosae fermentum UP-1 ( Limosilactobacillus fermentum ), which is deposited with the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, deposit number: CGMCC No. 33076; deposit date: December 16, 2024.

[0012] The present invention also provides a preparation containing Lactobacillus mucosae fermentum UP-1.

[0013] The preparation containing Lactobacillus mucosae fermentum UP-1 is one or more of food raw materials, probiotic powder, food additives, feed additives, drugs, liquid beverages, foods, or health products.

[0014] The preparation containing Lactobacillus mucosae fermentum UP-1 is a solid or liquid preparation; wherein, when the preparation is a liquid preparation, it contains 1.0 × 10 5 CFU / ml~1.0 × 10 12 CFU / ml of Lactobacillus mucosae fermentum UP-1; when the preparation is in solid form, it contains 1.0×10 5 CFU / g~1.0×10 12 CFU / g of Lactobacillus mucosae fermentum UP-1.

[0015] The preparation containing Lactobacillus mucosae fermentum UP-1 further contains excipients, and the excipients include one or more of edible vegetable oil, medium-chain triglyceride, inulin, fructooligosaccharide, resistant dextrin, galactooligosaccharide, skim milk powder, demineralized whey powder, lactoferrin, casein phosphopeptide, vitamins, calcium agents, or hydrolyzed egg yolk powder.

[0016] The present invention also provides a product for promoting digestion and absorption, growth and development, and improving bone metabolism, including the above-mentioned Lactobacillus mucosae fermentum UP-1 or the above-mentioned culture.

[0017] Preferably, the product is a food, a drug or a health product.

[0018] Preferably, the product contains Lactobacillus mucosae UP-1 with viable cell count of not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0019] In an embodiment, promoting the growth of a subject includes one or more of: promoting an increase in the body length of the subject, and promoting an increase in the body length growth rate of the subject.

[0020] In an embodiment, the subject is a mouse. Compared with the prior art, the present invention has the following beneficial effects:

[0021] The Lactobacillus mucosae UP-1 provided by the present invention ( Limosilactobacillus fermentum ) can promote the increase in body length and the long diameter of the femur of the body, improve the trabecular bone density and the level of beneficial metabolite short-chain fatty acids, improve the bone metabolism level, and promote the healthy development of bones by increasing the levels of insulin-like growth factor binding protein-3 (IGFBP3), growth hormone (GH) and insulin-like growth factor (IGF-1) in the serum, so as to achieve the purpose of promoting growth and development and improving nutrient absorption, and contribute to more scientific dietary intervention and the development of precise probiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Shows the colony morphology of Lactobacillus mucosae UP-1.

[0023] Figure 2 Shows the microscopic morphology of Lactobacillus mucosae UP-1 under a scanning electron microscope.

[0024] Figure 3 Shows the phylogenetic evolution tree of Lactobacillus mucosae UP-1.

[0025] Figure 4 Shows the circular map of the chromosomal genome of Lactobacillus mucosae UP-1.

[0026] Figure 5 Shows the gastrointestinal fluid tolerance of Lactobacillus mucosae UP-1.

[0027] Figure 6 Shows the hemolytic assay of Lactobacillus mucosae UP-1.

[0028] Figure 7 Shows the effect of Lactobacillus mucosae UP-1 on the body length of mice. Figure A is for females and Figure B is for males. * indicates P <0.05, ** indicates P <0.01.

[0029] Figure 8 Shows the effect of Lactobacillus mucosae UP-1 on the body length growth rate of mice. Figure A is for females and Figure B is for males. ** indicates P <0.01.

[0030] Figure 9 Shows the effect of Lactobacillus mucosae UP-1 on the long diameter of the femur of mice. Figure A is for females and Figure B is for males. * indicates P <0.05, **** indicates P <0.0001.

[0031] Figure 10 Shows the effect of Lactobacillus mucosae UP-1 on growth factor-binding protein-3 in mice. Figure A is for females and Figure B is for males. *** indicates P <0.001.

[0032] Figure 11 Shows the effect of Lactobacillus mucosae UP-1 on growth hormone in mice. Figure A is for females and Figure B is for males. ** indicates P <0.01, **** indicates P <0.0001.

[0033] Figure 12 Shows the effect of Lactobacillus mucosae UP-1 on growth factors in mice. Figure A is for females and Figure B is for males. ** indicates P <0.01, *** indicates P <0.001.

[0034] Figure 13 Shows the effect of Lactobacillus mucosae UP-1 on the amino-terminal propeptide of type I procollagen in mice. Figure A is for females and Figure B is for males. * indicates P <0.05, *** indicates P <0.001.

[0035] Figure 14 Shows the effect of Lactobacillus mucosae UP-1 on the ratio of the amino-terminal propeptide of type I procollagen to the carboxyl-terminal cross-linked telopeptide of type I collagen in mice. Figure A is for females and Figure B is for males. **** indicates P <0.0001.

[0036] Figure 15 Shows the effect of Lactobacillus mucosae UP-1 on the osteoclast differentiation factor RanKL in the bone marrow of mice. * indicates P <0.05, **** indicates P <0.0001.

[0037] Figure 16Shows the effects of Lactobacillus mucosae UP-1 on bone mass and bone metabolism in female mice. A is the 3D reconstruction diagram of the femur of mice in the Lactobacillus mucosae UP-1 group, B is the 3D reconstruction diagram of the femur of control group mice, C is the bone volume fraction, and * indicates P <0.05.

[0038] Figure 17 Shows the effects of Lactobacillus mucosae UP-1 on bone mass and bone metabolism in male mice. A is the 3D reconstruction diagram of the femur of mice in the Lactobacillus mucosae UP-1 group, B is the 3D reconstruction diagram of the femur of control group mice, C is the bone volume fraction, and * indicates P <0.05.

[0039] Figure 18 Shows the effects of Lactobacillus mucosae UP-1 on pro-inflammatory factors in the mouse colon, and * indicates P <0.05, ** indicates P <0.01, *** indicates P <0.001.

[0040] Figure 19 Shows the effects of Lactobacillus mucosae UP-1 on the levels of short-chain fatty acids in the intestines of female mice, and * indicates P <0.05, ** indicates P <0.01.

[0041] Figure 20 Shows the effects of Lactobacillus mucosae UP-1 on the levels of short-chain fatty acids in the intestines of male mice, and * indicates P <0.05, ** indicates P <0.01.

[0042] Biological deposit

[0043] A strain of Lactobacillus mucosae UP-1 ( Limosilactobacillus fermentum ) is taxonomically named: Lactobacillus mucosae Limosilactobacillus fermentum ; Depositary institution: China General Microbiological Culture Collection Center, Deposit number: CGMCC No. 33076; Deposit date: December 16, 2024; Deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; The test result of this strain is viable. Detailed implementation methods

[0044] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description progresses. However, these examples are merely exemplary and do not impose any limitation on the scope of the present invention. The methods, components, and dosages involved in the following embodiments are all conventional methods known to those skilled in the art, unless otherwise specified. It is worth noting that the raw materials used in the present invention are all ordinary commercially available products. Among them, MRS liquid medium is purchased from Beijing Road & Bridge Technology Co., Ltd.; Lactobacillus mucosae UP-1 is provided by the Key Laboratory of Dairy Biotechnology and Engineering of the Ministry of Education, Inner Mongolia Agricultural University. In the embodiments of the present invention, the mammalian BALB / c mice are conventional model organisms in the prior art and are purchased from SPF (Beijing) Biotechnology Co., Ltd.

[0045] Example 1

[0046] Isolation and identification of Lactobacillus mucosae UP-1 ( Limosilactobacillus fermentum ):

[0047] (1) Isolation and purification

[0048] Lactobacillus mucosae UP-1 was isolated from the breast milk of healthy women in 2018. 0.5 mL of the sample was added to 5 mL of MRS liquid medium and cultured aerobically at 37 °C for 24 h for expansion culture to obtain the expanded sample; the sample solution was gradually diluted to an appropriate gradient (10 6 ~10 7 CFU / mL) using PBS buffer. 100 μL of the gradient dilution was pipetted and spread on MRS solid medium, and cultured aerobically at 37 °C for 48 h to obtain colonies; according to the colony shape, size, edge, and transparency, etc., colonies with typical characteristics of Lactobacillus mucosae on MRS solid medium were selected, and the colonies were picked with an inoculation loop and streaked on MRS solid medium, and cultured aerobically at 37 °C for 24 h, and then the above steps were repeated until pure colonies were obtained.

[0049] (2) Subculture and cryopreservation

[0050] The colonies on MRS solid medium were scraped with an inoculation loop and inoculated into 10 mL of MRS broth medium, cultured at 37 °C for 24 h, and then the sterilized skim milk and the bacterial solution were mixed at a volume ratio of 1:1, mixed well, and stored at -80 °C.

[0051] (3) Identification of the strain

[0052] The fermented Lactobacillus mucosae UP-1 was identified using the FMIC-QO01-001-2015 microbiological detection method for polyphasic bacterial identification. On MRS medium, aerobic culture was carried out at 37 °C for 24 h, and the colony morphology was observed. The colonies of Lactobacillus mucosae UP-1 were white, round, with a moist surface, opaque, and neat edges ( Figure 1 ), and the cells were rod-shaped, 0.4 - 0.6 μm × 0.7 - 1.6 μm, arranged singly or in pairs, and Gram-positive ( Figure 2 ).

[0053] (4) Phylogenetic analysis of the strain

[0054] Fresh fermented Lactobacillus mucosae UP-1 bacterial solution was used as a template in the PCR amplification system for PCR amplification. The genomic DNA of strain UP-1 was extracted using a bacterial genomic DNA extraction kit, and the extracted genomic DNA of strain UP-1 was used as a template for amplification to obtain the 16S rRNA of strain UP-1. The 16S rDNA of strain UP-1 was subjected to homologous sequence alignment using the BLAST software in the NCBI database. The alignment results were used with the MEGA software to construct a phylogenetic tree using the neighbor-joining method, and 1000 similarity replicates were calculated. The isolated and identified strain was named Lactobacillus mucosae UP-1. After 16S rRNA gene sequencing, the sequence of the 16S rRNA gene was obtained as shown in SEQ ID No.1, specifically:

[0055] GTTGGCCCAA TTGATTGATG GTGCTTGCAC CTGATTGATT TTGGTCGCCA

[0056] ACGAGTGGCG GACGGGTGAG TAACACGTAG GTAACCTGCC CAGAAGCGGG

[0057] GGACAACATT TGGAAACAGA TGCTAATACC GCATAACAAC GTTGTTCGCA

[0058] TGAACAACGC TTAAAAGATG GCTTCTCGCT ATCACTTCTG GATGGACCTG

[0059] CGGTGCATTA GCTTGTTGGT GGGGTAACGG CCTACCAAGG CGATGATGCA

[0060] TAGCCGAGTT GAGAGACTGA TCGGCCACAA TGGGACTGAG ACACGGCCCA

[0061] TACTCCTACG GGAGGCAGCA GTAGGGAATC TTCCACAATG GGCGCAAGCC

[0062] TGATGGAGCA ACACCGCGTG AGTGAAGAAG GGTTTCGGCT CGTAAAGCTC

[0063] TGTTGTTAAA GAAGAACACG TATGAGAGTA ACTGTTCATA CGTTGACGGT

[0064] ATTTAACCAG AAAGTCACGG CTAACTACGT GCCAGCAGCC GCGGTAATAC

[0065] GTAGGTGGCA AGCGTTATCC GGATTTATTG GGCGTAAAGA GAGTGCAGGC

[0066] GGTTTTCTAA GTCTGATGTG AAAGCCTTCG GCTTAACCGG AGAAGTGCAT

[0067] CGGAAACTGG ATAACTTGAG TGCAGAAGAG GGTAGTGGAA CTCCATGTGT

[0068] AGCGGTGGAA TGCGTAGATA TATGGAAGAA CACCAGTGGC GAAGGCGGCT

[0069] ACCTGGTCTG CAACTGACGC TGAGACTCGA AAGCATGGGT AGCGAACAGG

[0070] ATTAGATACC CTGGTAGTCC ATGCCGTAAA CGATGAGTGC TAGGTGTTGG

[0071] AGGGTTTCCG CCCTTCAGTG CCGGAGCTAA CGCATTAAGC ACTCCGCCTG

[0072] GGGAGTACGA CCGCAAGGTT GAAACTCAAA GGAATTGACG GGGGCCCGCA

[0073] CAAGCGGTGG AGCATGTGGT TTAATTCGAA GCTACGCGAA GAACCTTACC

[0074] AGGTCTTGAC ATCTTGCGCC AACCCTAGAG ATAGGGCGTT TCCTTCGGGA

[0075] ACGCAATGAC AGGTGGTGCA TGGTCGTCGT CAGCTCGTGT CGTGAGATGT

[0076] TGGGTTAAGT CCCGCAACGA GCGCAACCCT TGTTACTAGT TGCCAGCATT

[0077] AAGTTGGGCA CTCTAGTGAG ACTGCCGGTG ACAAACCGGA GGAAGGTGGG

[0078] GACGACGTCA GATCATCATG CCCCTTATGA CCTGGGCTAC ACACGTGCTA

[0079] CAATGGACGG TACAACGAGT CGCGAACTCG CGAGGGCAAG CAAATCTCTT

[0080] AAAACCGTTC TCAGTTCGGA CTGCAGGCTG CAACTCGCCT GCACGAAGTC

[0081] GGAATCGCTA GTAATCGCGG ATCAGCATGC CGCGGTGAAT ACGTTCCCGG

[0082] GCCTTGTACA CACCGCCCGT CACACCATGA GAGTTTGTAA CACCCAAAGT

[0083] CGGTGGGGTA ACC

[0084] The phylogenetic tree of the UP-1 system is as Figure 3 shown and is identified as Lactobacillus mucosae fermentum ( Limosilactobacillus fermentum), and it belongs to the strains of microorganisms permitted for use in food in the "List of Strains of Microorganisms Permitted for Use in Food". The results of whole-genome sequencing and analysis of the strain showed that the genome of UP-1 contains 1 chromosome (2,253,013 bp) and 1 plasmid (42,454 bp), with a total sequence length of 2,281,393 bp and a GC content of 50.82 ( Figure 4 ).

[0085] Example 2

[0086] Characteristics of Lactobacillus mucosae UP-1

[0087] (1)Detection of tolerance to artificial gastric juice and artificial intestinal juice

[0088] Preparation of artificial gastric juice: 40 ml of sterilized normal PBS solution + 3.0 g / L pepsin (0.3%), then adjust the pH to 2.5 (adjusted with 0.1 mol / L HCl), and filter it through a 0.22-μm filter membrane into a sterilized empty conical flask for use;

[0089] Preparation of artificial intestinal juice: 40 ml of sterilized normal PBS solution + 1.0 g / L trypsin (0.1%) + 1.8% bile salts, then adjust the pH to 8.0 (adjusted with 0.1 mol / L NaOH), and filter it through a 0.22-μm filter membrane into a sterilized empty conical flask for use.

[0090] In this study, the tolerance of UP-1 to digestion in artificial gastric juice at pH 2.5 for 3 hours and in artificial intestinal juice at pH 8.0 for 8 hours was detected. After culturing the UP-1 strain for 24 h, the cells were collected by centrifugation, washed twice with PBS, and 5 mL of PBS was added and shaken evenly to obtain the original bacterial solution. The original bacterial solution was diluted and counted as the value at 0 h; 500 μL of the original bacterial solution was taken into a test tube containing 4.5 mL of gastric juice, cultured in a 37°C water bath for 3 h, taken out and counted; 500 μL of the bacterial solution after 3 h of culture was taken into a test tube containing 4.5 mL of intestinal juice, and counted after being placed in a 37°C water bath for 8 h.

[0091] The results are shown in Figure 5 , and the survival rate of Lactobacillus mucosae UP-1 in artificial gastric juice at pH 2.5 after digestion for 3 hours was 73.26%. Then it was immediately transferred to artificial intestinal juice at pH 8.0 for digestion for 8 hours, and its survival rate was 78.20%. This indicates that Lactobacillus mucosae UP-1 has strong survival ability in artificial gastrointestinal juices.

[0092] Example 3

[0093] Safety evaluation of Lactobacillus mucosae UP-1

[0094] (1)Drug sensitivity test

[0095] According to the antimicrobial susceptibility testing method in Section 2.2.1 of the EFSA 5206-2018 "Guidelines on the Microbiological Characteristics of Feed Additives or Microorganisms Used in the Production of Fermented Products", the resistance of the strain to ampicillin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol was determined. The drug resistance of the strain was expressed as the minimum inhibitory concentration (MIC).

[0096] The MIC test results are shown in Table 1. UP-1 is sensitive to ampicillin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol.

[0097] Table 1 MIC values and drug susceptibility of Lactobacillus mucosae UP-1

[0098] ;

[0099] (2) Antibiotic resistance genes and virulence genes

[0100] The antibiotic resistance genes and virulence genes of Lactobacillus mucosae UP-1 were detected using the FMIC-QO01-024-2020 microbiological test - Detection method for bacterial antibiotic resistance genes and virulence genes. In terms of antibiotic resistance genes, using the CARD database and the RGI software, with the Perfect and Strict algorithms as the screening criteria, no antibiotic resistance-related genes were detected. Using a sequence similarity ≥ 90% and sequence coverage ≥ 60% as the screening criteria, no antibiotic resistance-related genes were detected. In terms of virulence genes, using the experimentally verified setA in the database as the analysis dataset, with a sequence similarity ≥ 80% and sequence coverage ≥ 70% as the screening criteria, no virulence-related genes were detected. Using a sequence similarity ≥ 90% and sequence coverage ≥ 60% as the screening criteria, no virulence-related genes were detected. This indicates that Lactobacillus mucosae UP-1 does not contain exogenous antibiotic resistance genes and virulence genes and is safe for consumption.

[0101] (3) Hemolysis test

[0102] The negative control bacterium used was Listeria innocua ( Listeria innocua ) CICC 10417, and the positive control bacterium used was Staphylococcus aureus ( Staphylococcus aureus ) CICC 10417. Both bacteria were purchased from Beijing Ketuo Hengtong Biotechnology Co., Ltd. According to the SH-QO01-008-2019 microbiological test - Hemolysis test method for microbial strains, the hemolysis phenomenon of UP-1 was detected. As Figure 6 shown, UP-1 was a white, smooth colony and showed no hemolysis on the blood agar plate, indicating that the target strain has no obvious pathogenicity and has a high level of safety.

[0103] (4)Animal pathogenicity detection

[0104] According to the appendix A of the Technical Guidelines for the Safety Inspection and Evaluation of Strains Used in Health Foods (2020 Edition), the animal pathogenicity was detected by the pathogenicity test method for bacteria used in health food raw materials. The results of the intraperitoneal injection test are shown in Table 2 (male) and Table 3 (female). There was no significant difference in the initial and final body weights of male / female mice between the culture group and its corresponding control group ( p >0.05), that is, during the test period, the culture of Lactobacillus mucosae UP-1 had no effect on the body weight of male / female mice, and the culture of Lactobacillus mucosae UP-1 had no adverse effect on the general health of animals. No toxic reaction or death was observed in the test mice (Table 4).

[0105] Table 2 Effects of Lactobacillus mucosae UP-1 culture on the body weight of male mice (intraperitoneal injection)

[0106] ;

[0107] Table 3 Effects of Lactobacillus mucosae UP-1 culture on the body weight of female mice (intraperitoneal injection)

[0108] ;

[0109] Table 4 Acute toxicity of Lactobacillus mucosae UP-1 culture to mice (intraperitoneal injection)

[0110] .

[0111] Example 4

[0112] A kind of Lactobacillus mucosae UP-1 bacterial powder is prepared by a conventional method for preparing bacterial powder, and the steps are as follows:

[0113] (1)Activation of strains

[0114] The strains stored at -80°C were respectively inoculated into the corresponding liquid medium sterilized at 121°C for 15 min, and anaerobically cultured at 37°C for 18-24 h. After such subculture for 1-2 times, the activated strains were obtained.

[0115] (2)Preparation of seed liquid

[0116] The activated strains obtained in step (1) were inoculated into MRS liquid medium and anaerobically cultured at 37°C until the pH value reached 4.5-4.8 and then stopped;

[0117] (3)Inoculation and fermentation

[0118] Inoculate the seed liquid from step (2) into MRS liquid medium at an inoculation ratio of 1‰, and ferment for 18 h under controlled fermentation conditions. The fermentation conditions are controlled as follows: maintain a constant temperature of 30 °C in the early stage of fermentation, and naturally ferment until the pH reaches 5.0; then adjust the fermentation temperature to 37 °C for constant temperature cultivation, and control the pH to remain at 6.0, maintaining aerobic fermentation.

[0119] During the above process, the pH is controlled by adding a neutralizing agent dropwise, and the neutralizing agent is NaOH.

[0120] (4)Freeze-drying

[0121] a. Bacterial cell concentration: Concentrate the bacterial cells in the high-density fermentation broth by centrifugation at 12,000 g.

[0122] b. Add protective agent: Add 5 times the amount of protective agent solution to the concentrated bacterial cell solution. The composition of the protective agent solution is as follows: 14 kg of skim milk powder, 10 kg of lactose, 1.1 kg of vitamin C, 0.8 kg of sodium glutamate, and 1000 L of distilled water.

[0123] c. Drying: Freeze-dry the above-mentioned bacterial cell suspension after adding the protective agent to obtain freeze-dried bacterial powder, and the total viable count in the bacterial powder reaches 1.0×10 11 CFU / g or more.

[0124] The total viable count content of the fermented Lactobacillus mucosae UP-1 bacterial powder is 1×10 11 CFU / g.

[0125] The UP-1 bacterial powder prepared in this example can be used for food, medicine, health products, or animal feed. The food can be ordinary foods or health foods such as fermented milk, cheese, milk-containing beverages, solid beverages, milk powder, etc. Preferably, in the food, the recommended dose of Lactobacillus mucosae UP-1 for human use can be 1.0×10 3 CFU~1.0 ×10 10 CFU / kg body weight / day, more preferably 1.0×10 4 CFU~1.0×10 9 CFU / kg body weight / day.

[0126] Example 5

[0127] Functional characteristics of a fermented Lactobacillus mucosae UP-1 bacterial powder in promoting growth: 1. Experimental method

[0128] (1)Grouping

[0129] Forty-eight 3-week-old BALB / c mice were selected. After all the mice were adaptively fed with basal diet for 1 week, they were randomly divided into a control group and a group fed with fermented Lactobacillus mucosae UP-1 powder, with 24 mice in each group, half male and half female. Among them, the mice in the UP-1 powder treatment group were intragastrically administered with UP-1 bacterial solution with a viable bacteria count of 1×10 9 CFU every day, and the control group was intragastrically administered with an equal amount of normal saline. The intervention lasted for 6 weeks. The experimental animals were raised in an SPF-class barrier environment at a temperature of 20-26 °C and a humidity of 30%-70%, with free access to food and water.

[0130] (2)Phenotypic indicators

[0131] The mental state and activity of the mice were observed at a fixed time every day, and the water intake and food intake of each group of mice were monitored and recorded. The body weight and body length were statistically analyzed once a week.

[0132] (3)Blood collection

[0133] Blood samples of the mice were collected in the 6th week of the experiment: The mice were anesthetized, and blood was collected by the retroorbital venous sinus bleeding method (RBB). Blood was collected using a disposable blood collection tube containing separation gel / coagulant accelerator, and centrifuged at 4 °C and 3000 ×g for 15 min. The supernatant was collected and aliquoted into 200 µL sterile EP tubes and stored at -80 °C.

[0134] (4)Detection of growth indicators

[0135] A kit (ELISA) was used to detect insulin-like growth factor (IGF-1), insulin-like growth factor binding protein-3 (IGFBP3), ghrelin, growth hormone (GH), procollagen type I N-terminal propeptide (PINP), and cross-linked carboxy-terminal telopeptide of type I collagen (CTXI) in the serum to evaluate the effect of UP-1 powder on the growth and development of the host.

[0136] (6)Fecal collection and detection of short-chain fatty acids

[0137] Fecal samples of the mice were collected in the 6th week of the experiment: Each mouse was placed in a sterilized cage box. After the mouse defecated naturally, ≥3 fresh feces were collected with sterile forceps and placed in a 2 mL sterile EP tube, and immediately frozen with liquid nitrogen and stored at -80 °C. After pretreatment of the fecal samples, liquid chromatography-mass spectrometry technology was used to quantitatively detect the extracted short-chain fatty acids.

[0138] 2. Experimental results

[0139] (1)Effect of fermented Lactobacillus mucosae UP-1 on the body length of mice

[0140] After 6 weeks of intervention with UP-1 powder, the body length of the mice was detected, and the results were as follows Figure 7As shown, compared with the control group (NC), the UP-1 bacterial powder significantly increased the body length of female mice ( Figure 7 A) and male mice ( Figure 7 B) (P < 0.05), and the body length growth rate also increased significantly ( Figure 8 , P < 0.05). The long diameter of the femur of mice was measured, and the results were as Figure 9 shown. The femur length of female mice ( Figure 9 A) and male mice ( Figure 9 B) was significantly higher than that of their corresponding control groups. This indicates that the fermented Lactobacillus mucosae UP-1 bacterial powder has the effect of promoting the growth of the femur and body length of mice.

[0141] (2) Effects of fermented Lactobacillus mucosae UP-1 on serum growth indices of mice

[0142] The level of insulin-like growth factor binding protein-3 (IGFBP3) is closely related to growth hormone (GH) and insulin-like growth factor (IGF-1). The three together form the GH-IGF-IGFBP axis, which is the core regulatory system for promoting the growth of children and adolescents. The level of IGFBP-3 is positively correlated with GH and IGF-1, and it is an important biomarker for diagnosing growth and development disorders. Therefore, in this study, the levels of IGF-1, IGFBP3, and GH in the serum of mice were measured. The results were as Figures 10 - 12 shown. In female mice, the levels of IGF-1, IGFBP3, and GH in the serum of the female UP-1 bacterial powder group were significantly higher than those of the female control group ( Figure 10 A, 11A, 12A, P <0.05). In male mice, the levels of IGF-1, IGFBP3, and GH in the serum of the male UP-1 bacterial powder group were also significantly higher than those of the male control group ( Figure 10 B, 11B, 12B, P <0.05). It is speculated that the fermented Lactobacillus mucosae UP-1 bacterial powder can increase the levels of IGF-1, IGFBP3, and GH in the serum of mice by mediating the GH-IGF-IGFBP axis, thereby promoting the growth and development of mice.

[0143] (3) Effects of fermented Lactobacillus mucosae UP-1 on serum bone metabolites of mice

[0144] Propeptide of type I procollagen amino-terminal propeptide (PINP) and cross-linked carboxy-terminal telopeptide of type I collagen (CTXI) are two important biomarkers reflecting bone metabolism, representing bone formation and bone resorption processes respectively. PINP is a by-product of type I collagen synthesis and is released into the blood during the synthesis of type I collagen by osteoblasts. It is a biomarker reflecting bone formation and is commonly used to evaluate bone neogenesis and osteogenic activity. CTXI is a metabolite released into the blood during the degradation of type I collagen and mainly comes from the bone resorption process. It is a biomarker reflecting bone resorption and is commonly used to evaluate osteoclast activity and bone loss. The level of PINP can be used as a positive indicator of bone health during growth and development. A higher PINP means an active bone formation process. The PINP / CTXI ratio can be used to evaluate the state of bone metabolism balance. A higher ratio indicates that bone formation is dominant, which is beneficial for growth and development. In this study, the levels of PINP and the PINP / CTXI ratio in the serum of female mice in the UP-1 probiotic powder group were significantly higher than those in the female control group ( Figure 13 A, 14A, P <0.05). The levels of PINP and the PINP / CTXI ratio in the serum of male mice in the UP-1 probiotic powder group were also significantly higher than those in the male control group ( Figure 13 B, 14B, P <0.05). This indicates that the UP-1 probiotic powder of Lactobacillus mucosae fermentum helps bone formation and promotes the healthy development of bones.

[0145] (4) Effect of Lactobacillus mucosae fermentum UP-1 on the osteoclast differentiation factor RanKL in mice

[0146] RANKL is a key factor in osteoclast formation. It binds to RANK (Receptor Activator of Nuclear Factor κ B) on osteoclast precursor cells and induces their differentiation into mature osteoclasts. Excessive expression of RANKL will lead to increased bone resorption and thus cause osteoporosis. In this study ( Figure 15 ), the level of the osteoclast differentiation factor RanKL in the bone marrow of female mice in the UP-1 probiotic powder group was significantly lower than that in the female control group ( P <0.0001). The level of the osteoclast differentiation factor RanKL in the bone marrow of male mice in the UP-1 probiotic powder group was also significantly lower than that in the male control group ( P <0.05). This indicates that the UP-1 probiotic powder of Lactobacillus mucosae fermentum helps reduce bone resorption, lower the risk of osteoporosis, and thus promote bone health.

[0147] (5) Effect of Lactobacillus mucosae fermentum UP-1 on bone mass and bone metabolism in mice

[0148] To evaluate the effects of Lactobacillus mucosae UP-1 fermentation on bone mass and bone metabolism in mice, this study used micro-CT to detect relevant parameters of the femurs of mice. Bone volume fraction (BV / TV) is a commonly used index for evaluating cortical and cancellous bone mass. For the cancellous bone in the medullary cavity, this ratio can reflect the amount of trabecular bone in different samples. An increase in this value indicates that bone anabolism is greater than catabolism, resulting in increased bone mass, and vice versa, thus indirectly reflecting the status of bone metabolism. The results of this experiment are as follows Figure 16 (female), Figure 17 (male) shown. In the 3D reconstruction model, it was observed that in female mice fermented with Lactobacillus mucosae UP-1 ( Figure 16 A) and male mice ( Figure 17 A), compared with the control group ( Figure 16 B and Figure 17 B), the femurs were filled with a large number of trabecular bones and the gaps were narrow. At the same time, UP-1 also significantly increased the bone volume fraction in female and male mice ( Figure 16 C&17C, P <0.05), indicating that Lactobacillus mucosae UP-1 fermentation can increase the trabecular bone density of the body, enhance bone quality, and promote bone metabolism.

[0149] (6) Effects of Lactobacillus mucosae UP-1 fermentation on pro-inflammatory factors in the colon of mice

[0150] Pro-inflammatory factors are the core regulatory factors of the inflammatory response and play an active role in maintaining the body's immune defense. However, their overexpression may lead to chronic inflammation and related diseases. Therefore, the pro-inflammatory factors tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and interleukin 6 (IL-6) in the colon of mice were detected. The results are as follows Figure 18 shown. The levels of TNF-α, IL-1β, and IL-6 in the colon of female mice in the UP-1 powder group were significantly lower than those in the female control group ( P <0.05), and the levels of TNF-α, IL-1β, and IL-6 in the colon of male mice in the UP-1 powder group were also significantly lower than those in the male control group ( P <0.05). This indicates that the UP-1 powder of Lactobacillus mucosae fermentation helps to reduce the expression of pro-inflammatory factors, inhibit intestinal inflammation, and maintain intestinal health.

[0151] (7) Effects of Lactobacillus mucosae UP-1 fermentation on short-chain fatty acids in the intestines of mice

[0152] Short-chain fatty acids (SCFAs) are the main products of anaerobic fermentation in the colon and are also the main products of protein degradation and amino acid fermentation. They can reflect the feed digestion situation and are also involved in the optimization of various physiological functions of the body and the prevention and treatment of various diseases. Therefore, this experiment detected and analyzed the levels of short-chain fatty acids in the intestines of mice in each group. The results of female mice are as followsFigure 19 As shown, the levels of acetic acid, propionic acid, and butyric acid in the intestines of female mice in the UP-1 bacterial powder group were significantly higher than those in the female control group ( P <0.05). The results of male mice were as Figure 20 shown. The levels of acetic acid and butyric acid in the intestines of male mice in the UP-1 bacterial powder group were significantly higher than those in the male control group ( P <0.05). These results demonstrated the promoting effect of Lactobacillus mucosae UP-1 on intestinal short-chain fatty acids. On the one hand, it reflected that UP-1 promoted the digestion of complex carbohydrates in the intestine, and on the other hand, it indicated the beneficial potential of UP-1 for the physiological functions of the body.

[0153] In summary, the intake of Lactobacillus mucosae UP-1 can improve the levels of insulin-like growth factor-binding protein-3, growth hormone, insulin-like growth factor, and ghrelin in the serum, simultaneously increase the PINP / CTXI ratio and bone volume fraction, increase the trabecular bone density and the levels of intestinal short-chain fatty acids, and reduce the expression of osteoclast differentiation factor RanKL and pro-inflammatory factors, thereby improving the bone metabolism level, promoting the healthy development of bones, and having a positive effect on the growth and development of the body, intestinal immunity, and digestion and absorption. Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than a limitation on the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art shall not depart from the essence and scope of the technical solution of the present invention.

Claims

1. Limosilactobacillus fermentum UP-1, characterized in that, The preservation unit of the fermented Lactobacillus mucosae UP-1 is the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the preservation number: CGMCC No. 33076; the preservation date: December 16, 2024.

2. A preparation, characterized in that, The preparation contains the fermented Lactobacillus mucosae UP-1 described in claim 1.

3. The preparation according to claim 2, characterized in that, The preparation is one or more of probiotic powder, feed additive and medicine.

4. The preparation according to claim 2 or 3, characterized in that, The preparation is a solid or liquid preparation; wherein the preparation contains 1.0×10 5 CFU / mL to 1.0×10 12 CFU / mL or 1.0×10 5 CFU / g to 1.0×10 12 CFU / g of Lactobacillus mucosae UP-1 in fermentation broth.

5. The preparation according to claim 2 or 3, characterized in that, The preparation further contains excipients, and the excipients include one or more of edible vegetable oil, medium-chain triglyceride, inulin, fructooligosaccharide, resistant dextrin, galactooligosaccharide, skim milk powder, demineralized whey powder, lactoferrin, casein phosphopeptide, vitamin, calcium agent or hydrolyzed egg yolk powder.

6. A product, characterized in that, The product contains the fermented Lactobacillus mucosae UP-1 described in claim 1.

7. The product according to claim 6, characterized in that, The product is a medicine, and the product contains Lactobacillus mucosae UP-1 with viable cell count not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

8. Use of the fermented Lactobacillus mucosae UP-1 described in claim 1 in the preparation of a product for improving bone metabolism, promoting body growth and inhibiting intestinal inflammation, and promoting intestinal short-chain fatty acids.

9. The use according to claim 8, wherein promoting body growth includes one or more of promoting body length increase and promoting body length growth rate increase.

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