Application of animal Bifidobacterium lactis subspecies BAPerfectus006 in improving qi deficiency and enhancing immunity

Abstract

The present application relates to the field of microbial technology, and specifically discloses the use of an animal Bifidobacterium lactis subspecies BAPerfectus006 in improving qi deficiency and enhancing immunity. The animal Bifidobacterium lactis subspecies BAPerfectus006 provided in the present application is isolated from the intestine of healthy infants and young children, has no drug resistance, no hemolysis, no pathogenicity, no virulence genes and no transferable drug resistance genes, and is a safe strain. The strain can significantly improve the symptoms of qi deficiency, delay weight loss, and reduce lactic acid accumulation. The improvement effect in the symptoms of qi deficiency, thymus index and spleen index, gripping strength, hanging ability, etc. is better than that of Zhongjing brand Buzhong Yiqi Pills under the same conditions. In addition, the strain also has the effect of enhancing immunity.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and in particular to an application of an animal Bifidobacterium lactis subspecies in improving qi deficiency and enhancing immunity, and specifically to an application of an animal Bifidobacterium lactis subspecies (Bifidobacterium animalissubsp.lactis) BAPerfectus006 in improving qi deficiency and enhancing immunity. Background Art

[0002] Bifidobacterium animalis subsp. lactis is a beneficial microorganism widely used in dairy products and prebiotic products. Phenotypic experiments such as drug sensitivity, hemolysis and cytotoxicity have shown that it has a certain degree of safety. It is recorded in the "Generally Recognized As Safe" (GRAS) list of the U.S. Food and Drug Administration (FDA). The European Food Safety Authority (EFSA) has included this subspecies in the presumption of safety list. It is also recorded in China's "List of Bacteria that can be used in food". It has been widely accepted and used in the food and pharmaceutical industries worldwide.

[0003] Qi deficiency refers to sub-health symptoms such as physical weakness, pale complexion, shortness of breath, weakness in the limbs, dizziness, sweating when moving, and low voice, including the deficiency of Yuan Qi, Zong Qi, and Wei Qi, as well as the decline of Qi's functions of pushing, warming, defending, consolidating, and Qi transformation, often accompanied by the decline or decline of certain functional activities of the body, and the decline of disease resistance. When the human body shows sub-health symptoms such as weight loss, pale complexion, shortness of breath, laziness, fatigue, pale red tongue, white fur, weak pulse, and constipation, people usually choose Western medicine and take Chinese medicine preparations.

[0004] However, it is difficult to fully describe the symptoms of Qi deficiency with quantitative physiological indicators, and it is difficult to find accurate treatment targets. Therefore, the symptoms of Qi deficiency treated by Western medicine often have the problems of treating the symptoms but not the root cause, repeated attacks, and drug side effects. Currently, traditional Chinese medicine is widely used to improve the symptoms of Qi deficiency, but the current Chinese medicines or Chinese medicine preparations for replenishing and nourishing Qi generally have the problems of inconvenience in taking, poor taste, and side effects.

[0005] Probiotics are beneficial microorganisms. A few studies have reported that probiotics can be used to improve qi deficiency, but most of them are studies on the combination of traditional Chinese medicine and probiotics. The application of probiotic single strains in regulating qi deficiency has not been carried out, especially the study on the animal bifidobacterium lactis subspecies in improving qi deficiency. There are many kinds of probiotic products on the domestic market, but most of the probiotic strains used by domestic manufacturers rely on imports. The market lacks a safe and effective food probiotic strain that is particularly suitable for improving the symptoms of qi deficiency. Summary of the invention

[0006] The purpose of the present application is to overcome the shortcomings of the above-mentioned prior art and provide an animal Bifidobacterium lactis subspecies strain suitable for improving qi deficiency constitution and enhancing immunity. The animal Bifidobacterium lactis subspecies strain of the present application is safe and effective, can significantly improve the symptoms of qi deficiency, delay weight loss, increase thymus index and spleen index, improve grip and hanging ability, and reduce serum lactic acid content. The improvement effect is better than that of Zhongjing brand Buzhong Yiqi Pills, and it also has the effect of enhancing immunity, and can be used for food development.

[0007] To achieve the above purpose, the technical solution adopted by this application is:

[0008] The present application provides an application of an animal Bifidobacterium lactis subspecies for improving qi deficiency and enhancing immunity, wherein the animal Bifidobacterium lactis subspecies is animal Bifidobacterium lactis subspecies (Bifidobacterium animalis subsp. lactis) BAPerfectus006, with a preservation number of GDMCC No: 62539, and a preservation classification name of Bifidobacterium animalissubsp. lactis. It was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 13, 2022, and the preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0009] The animal Bifidobacterium lactis subspecies BAPerfectus006 of the present application has no toxic side effects in both in vivo and in vitro experiments, and combined with the results of drug sensitivity test, hemolysis test, mouse pathogenicity test phenotypic experiment, and virulence gene and drug resistance gene analysis, the animal Bifidobacterium lactis subspecies BAPerfectus006 has a negative hemolytic reaction, is non-pathogenic, and has no resistance to common antibiotics. Genetic analysis shows that it has no pathogenic genes and transferable resistance genes, and is a very safe strain.

[0010] Animal Bifidobacterium lactis subspecies BAPerfectus006 does not cause hemolysis of red blood cells, and is safe for oral and intraperitoneal injection in mouse experiments. It complies with the requirements of the "Technical Guidelines for Safety Inspection and Evaluation of Bacteria Used in Health Food Raw Materials (2020 Edition)" issued by the State Administration for Market Regulation. Animal Bifidobacterium lactis subspecies BAPerfectus006 does not contain plasmids, and its functional genes are all on chromosomes, which are not easy to lose and have good functional genetic stability.

[0011] This application also repeatedly confirms and identifies the bacterial species through morphological observation, 16s rDNA identification, physiological and biochemical experiments, and whole genome testing using second-generation plus third-generation gene sequencing detection technology, which is more accurate.

[0012] Compared with other animal Bifidobacterium strains, the animal Bifidobacterium lactis subspecies BAPerfectus006 of the present application has better gastrointestinal fluid tolerance.

[0013] The animal Bifidobacterium lactis subspecies BAPerfectus006 of the present application can improve the symptoms of qi deficiency, and the improvement effects on qi deficiency symptoms, thymus index and spleen index, gripping force and hanging ability are significantly better than Zhongjing brand Buzhong Yiqi Pills, and is a potential probiotic raw material for regulating qi deficiency. In addition, the animal Bifidobacterium lactis subspecies BAPerfectus006 of the present application also has the effect of enhancing immunity.

[0014] As a preferred embodiment of the application described in the present application, the Qi deficiency includes spleen qi deficiency.

[0015] The present application also provides the use of the above-mentioned animal Bifidobacterium lactis subspecies BAPerfectus006 in the preparation of products for improving qi deficiency and enhancing immunity.

[0016] As a preferred embodiment of the application described in the present application, the product includes food or medicine.

[0017] As a preferred embodiment of the application described in this application, the product is used in at least one of the following aspects:

[0018] 1) Significantly improve the symptoms of Qi deficiency; 2) Improve grip and suspension ability; 3) Reduce serum lactic acid content; 4) Increase thymus index and spleen index; 5) Delay weight loss; 6) Improve spleen lymphocyte proliferation ability, half hemolysis value, phagocytic index and macrophage phagocytosis of chicken red blood cells.

[0019] Experimental verification has shown that animal Bifidobacterium lactis subspecies BAPerfectus006 can significantly improve the symptoms of qi deficiency, decreased thymus and spleen indexes, and weight loss in qi deficiency rats modeled after "improper diet + weighted swimming". It can also improve grip and suspension abilities, and remove lactic acid accumulation. This provides a reference for the future development of animal Bifidobacterium lactis subspecies raw materials for improving qi deficiency, especially female qi deficiency, and is also of great significance to the development and utilization of probiotic resources.

[0020] In addition, the use of the animal Bifidobacterium lactis subspecies BAPerfectus006 of the present application helps to enhance immune function. For example, animal Bifidobacterium lactis subspecies BAPerfectus006 can significantly improve the proliferation ability of mouse spleen lymphocytes, half hemolysis value, phagocytic index, macrophage phagocytosis of chicken red blood cells and other properties.

[0021] The present application also provides a food or medicine for improving qi deficiency, wherein the food or medicine includes the above-mentioned animal Bifidobacterium lactis subspecies, wherein the animal Bifidobacterium lactis subspecies is animal Bifidobacterium lactis subspecies BAPerfectus006, with a preservation number of GDMCC No: 62539, and a preservation classification name of Bifidobacterium animalis subsp.lactis. It has been preserved in the Guangdong Provincial Microbiological Culture Collection Center on June 13, 2022, and the preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0022] As a preferred embodiment of the food or medicine described in the present application, the food or medicine further includes auxiliary materials and / or carriers.

[0023] The China Food Industry Association issued the "Probiotic Food" group standard, which stipulates that the total number of live probiotic bacteria in ready-to-eat probiotic foods must be ≥10 7 CFU / g, the animal Bifidobacterium lactis subspecies BAPerfectus006 of the present application can be industrialized, and the number of live bacteria in the original powder is ≥4×10 11 CFU / g, which can meet the requirements of the Standard and has considerable cost.

[0024] Compared with the prior art, this application has the following beneficial effects:

[0025] The present application provides an application of animal bifidobacterium lactis subspecies BAPerfectus006 in improving qi deficiency. The strain has no toxic side effects in both in vitro and in vivo experiments, and has no virulence genes. It is a very safe probiotic that can meet the safety standards of health food raw material strains; the strain is directly collected from healthy human bodies, has stable biological characteristics, has significant gastric acid resistance, intestinal fluid resistance, and oxygen resistance, does not require domestication and rejuvenation processes, and directly enters the preparation process; the present application finds that the strain can significantly improve qi deficiency symptoms, delay weight loss, improve grip and suspension ability, increase thymus index and spleen index, and reduce serum lactic acid content, wherein the effects of improving qi deficiency symptoms, improving grip and suspension ability, increasing thymus index and spleen index, and reducing serum lactic acid are better than Zhongjing brand Buzhong Yiqi Pills under the same conditions, and are a potential raw material for replenishing qi deficiency probiotics. In addition, the strain also has the effect of promoting immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the colony morphology of Bifidobacterium animalis subspecies lactis BAPerfectus006;

[0027] Figure 2 This is a microscopic bacterial morphology of animal Bifidobacterium lactis subspecies BAPerfectus006;

[0028] Figure 3 It is a 16s rDNA sequence alignment diagram of Bifidobacterium animalis lactis subspecies BAPerfectus006;

[0029] Figure 4 This is a comparison diagram of the groEL sequence of Bifidobacterium animalis subspecies lactis BAPerfectus006;

[0030] Figure 5 This is the complete bacterial map of Bifidobacterium animalis subspecies lactis BAPerfectus006;

[0031] Figure 6 This is a hemolysis test diagram of animal Bifidobacterium lactis subspecies BAPerfectus006;

[0032] Figure 7 This is a statistical bar graph of Qi deficiency scores in mice in Example 7;

[0033] Figure 8 This is a statistical bar graph of the mouse thymus index in Example 7;

[0034] Fig. 9 This is a statistical bar graph of the mouse spleen index in Example 7;

[0035] Fig.10 This is a statistical bar graph of serum lactate values ​​of mice at 21 days in Example 7. DETAILED DESCRIPTION

[0036] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0037] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0038] In the following embodiments, BAPerfectus006 and Bifidobacterium animalis subsp. lactis BAPerfectus006 both refer to Bifidobacterium animalis subsp. lactis BAPerfectus006.

[0039] In the following embodiments, the preservation number of Bifidobacterium animalis subsp. lactis F829 is GDMCC No: 64783, the preservation classification name is Bifidobacterium animalis subsp. lactis, and it has been deposited in Guangdong Provincial Microbiological Culture Collection Center on June 21, 2024, and the preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0040] The preservation number of Bifidobacterium animalis subsp. lactis F830 is GDMCC No: 64784, and the preservation classification name is Bifidobacterium animalis subsp. lactis. It was deposited in Guangdong Provincial Microbiological Culture Collection Center on June 21, 2024. The preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0041] Example 1: Isolation and identification of animal Bifidobacterium lactis subspecies BAPerfectus006

[0042] This embodiment provides a method for isolating and identifying animal Bifidobacterium lactis subspecies BAPerfectus006, comprising the following steps:

[0043] 1. Isolation of animal Bifidobacterium lactis subspecies BAPerfectus006:

[0044] Feces of healthy male infants aged 3.5 months were collected as samples. 1 g of sample was added to 10 ml of sterile saline and mixed. The mixture was centrifuged at 800 rpm for 5 min to remove large impurities. 1 ml of the supernatant bacterial suspension was diluted 10 times with sterile saline and the appropriate dilution gradient (10 -5 , 10 -6 , 10 -7 ) was spread on a plate of Bifidobacterium BBL agar medium (Haibo Biotechnology Co., Ltd., Qingdao High-tech Industrial Park, HB0397) and placed in an anaerobic incubator (37°C) for 24 to 48 hours. Use an inoculation loop to select white opaque circular colonies and streak them continuously 3-5 times until they are purified to single colonies. Stain and examine under a microscope according to the instructions of the Gram staining kit. After confirming that they are Gram-positive bacteria, send them for 16s rDNA sequencing and identification. After enrichment culture, the pure colonies were mixed with sterile glycerol (final concentration of 20% glycerol) and stored at -80°C.

[0045] 2. Identification of Bifidobacterium animalis subspecies BAPerfectus006:

[0046] According to the "FMIC-QO01-001-2015 Microbiological Detection of Bacterial Multiphase Identification Detection Method", animal Bifidobacterium lactis subspecies BAPerfectus006 was identified. The results are as follows:

[0047] (1) Morphological observation:

[0048] a. Macroscopic morphology: Animal Bifidobacterium lactis subspecies BAPerfectus006 was cultured in a reinforced Clostridium medium and cultured anaerobically at 37°C for 48 hours. The colonies were white, round, moist, opaque, and had neat edges. Figure 1 .

[0049] b. Microscopic morphology: The bacteria are irregular rod-shaped under the microscope. 0.6-0.9μm×1.1-4.8μm, arranged singly or in pairs, Gram-positive, such as Figure 2 .

[0050] (2) 16S rDNA identification and groEL gene sequence identification:

[0051] The animal Bifidobacterium lactis subspecies BAPerfectus006 described in the present application is isolated from the intestine of an infant, and its DNA is extracted after being cultured in an anaerobic environment at 37°C for 48 hours using a BBL culture medium, and a polymerase chain reaction (PCR) is performed on the genomic DNA using conventional primers for 16S rDNA and the housekeeping gene groEL of the genus Bifidobacterium. The 16S rDNA gene sequence of the strain claimed in the present application is shown in SEQ ID NO: 1.

[0052] The National Center for Biotechnology Information (NCBI) database was used for sequence alignment and phylogenetic analysis. Figure 3 From the result of 16S rDNA, it has 100% similarity with Bifidobacterium animalis subsp. lactis DSM 10140T (CP001606), and can be basically determined to be Bifidobacterium animalis subsp. lactis.

[0053] The groEL sequence of Bifidobacterium animalis subspecies lactis BAPerfectus006 is shown in SEQ ID NO:2.

[0054] In order to further confirm the accuracy of its type, the groEL gene sequence was compared. Figure 4 As shown, the similarity between animal Bifidobacterium lactis subsp. BAPerfectus006 and animal Bifidobacterium lactis subsp. DSM 10140T (CP001606) reached 100.0%, and the similarity between animal Bifidobacterium animalis subsp. ATCC 25527T (CP002567) was 96.9%, so it can be determined that it is indeed animal Bifidobacterium lactis subsp.

[0055] Example 2: Whole genome sequencing of Bifidobacterium animalis subspecies lactis BAPerfectus006

[0056] Combining the second and third generation high-throughput gene sequencing technologies, the full gene sequence of animal Bifidobacterium lactis subspecies BAPerfectus006 was detected, the whole genome map was drawn and the species was identified at the genome level.

[0057] After thawing the animal bifidobacterium lactis subspecies BAPerfectus006 glycerol tube preserved at -80°C, inoculate it into BBL medium, culture it anaerobically at 37°C for 24h, and after passage twice, take the bacterial culture in the logarithmic growth phase and place it in a sterile EP centrifuge tube for centrifugation. After centrifugation at 8000rpm for 5min (4°C), collect the bacterial cells, discard the supernatant, freeze them quickly in liquid nitrogen, wait for the bacterial cells to be frozen through, transfer them into a -80°C refrigerator, and mail them to the testing agency to complete the second and third generation sequencing. The second generation uses the DNBSEQ sequencing platform, and the third generation uses the Nanopore sequencing platform. The library construction and machine sequencing are carried out according to the library construction requirements of the sequencing platform, respectively. After the data is qualified after quality inspection and filtering, the third generation data is calibrated with the second generation data to complete the assembly of the bacterial genome map. The results showed that the genomic DNA of animal Bifidobacterium lactis subspecies BAPerfectus006 was circular, without plasmid, with a genome size of 1.93Mb and a GC content of 60.48%. A total of 9 ribosomal RNAs (rRNAs), 53 transfer RNAs (tRNAs), 1 small RNA (sRNA) and 1628 coding sequences (CDS) were detected. The whole genome map is shown in Figure 5 Based on the comparison of the obtained genome sequence with the recognized NT database, it was determined to be Bifidobacterium animalis subsp. lactis at the whole genome level.

[0058] The animal Bifidobacterium lactis subspecies obtained above was preserved, and the animal Bifidobacterium lactis subspecies was animal Bifidobacterium lactis subspecies BAPerfectus006, with a preservation number of GDMCC No: 62539, and a preservation classification name of Bifidobacterium animalis subsp.lactis. It was preserved in the Guangdong Provincial Microbiological Culture Collection Center on June 13, 2022, and the preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0059] Example 3: Verification of food safety of animal Bifidobacterium lactis subspecies BAPerfectus006

[0060] 1. Drug sensitivity test:

[0061] The experiment was carried out according to the EU standard "EFSA 5206 Guidance on the characterization of miroorganisms used as feed additives or as production or ganisms-2.2". The results showed that animal Bifidobacterium lactis subspecies BAPerfectus006 was sensitive to all antibiotics, as shown in Table 1.

[0062] Table 1 Results of drug sensitivity test of animal Bifidobacterium lactis subspecies BAPerfectus006

[0063] Types of antibiotics MIC breakpoint value (ug / mL) MIC measured value (ug / mL) result Ampicillin (AMP) 2 1.5 sensitive Chloramphenicol (CHL) 4 4 sensitive Clindamycin (CM) 1 0.50 sensitive Erythromycin (ERY) 1 0.5 sensitive Gentamycin (GM) 64 64 sensitive Streptomycin (SM) 128 96 sensitive Tetracycline (TE) 8 8 sensitive Vancomycin (VA) 2 0.50 sensitive

[0064] 2. Hemolytic test:

[0065] After puncture inoculation of blood plate, no transparent circle appeared around the puncture site of animal Bifidobacterium lactis subspecies BAPerfectus006. The hemolytic effect of animal Bifidobacterium lactis subspecies BAPerfectus006 was similar to that of negative control bacteria Listeria innocua CICC10417, and was significantly different from that of positive control bacteria Staphylococcus aureus CICC10473 ( Figure 6 ). The experimental results showed that animal Bifidobacterium lactis subspecies BAPerfectus006 did not cause hemolysis and the result was negative.

[0066] 3. Bacterial pathogenicity experiment:

[0067] Bacterial pathogenicity experiments were conducted according to the method in Appendix A of the "Technical Guidelines for Safety Testing and Evaluation of Bacterial Species Used in Health Food Ingredients (2020 Edition)".

[0068] The results in Table 2 show that the bacterial suspension of animal Bifidobacterium lactis subspecies BAPerfectus006 was injected intraperitoneally once to mice of both sexes, with the amount of bacteria injected per mouse being 1.04×10 7 CFU (bacterial concentration was 5.19×10 7 CFU / mL, injection volume was 0.2mL / animal). During the 21-day observation period, the animals were in normal condition, with no abnormal conditions such as poisoning signs and death.

[0069] Table 2 Results of bacterial pathogenicity experiment in mice (intraperitoneal injection)

[0070]

[0071] The results in Table 3 show that mice of both sexes were given oral administration of Bifidobacterium lactis subspecies BAPerfectus006 suspension for 3 consecutive days, with each oral administration dose of 5.08×10 9 CFU / (kg·BW) and 2.50×10 10 CFU / (kg·BW)(the concentration of bacterial suspension was 2.54×10 8 CFU / mL and 1.25×10 9CFU / mL, the gavage volume was calculated based on the actual body weight of the rats as 20mL / (kg·BW)). During the 21-day observation period, the animals were in normal condition, with no abnormal conditions such as signs of poisoning and death.

[0072] Table 3 Results of bacterial pathogenicity experiment in mice (oral gavage)

[0073]

[0074] According to the bacterial pathogenicity test method used for health food raw materials, the bacterial pathogenicity test result of animal Bifidobacterium lactis subspecies BAPerfectus006 was negative, indicating that animal Bifidobacterium lactis subspecies BAPerfectus006 showed no pathogenicity in animal experiments.

[0075] 4. Genome analysis of Bifidobacterium animalis subspecies BAPerfectus006:

[0076] The genome sequence measured in Example 2 is compared with the latest version of the internationally recognized antibiotic resistance gene database (CARD), and two resistance genes tetracycline antibiotics tet (W / N / W) and rifamycin antibiotics rpoB are annotated. These two potential resistance-related genes are also detected in the animal Bifidobacterium lactis subspecies BB12 genome. To assess the transfer risk of resistance genes, the two resistance genes annotated using NR database and software ABRicate, AMRFinderPlus, Phispy are further analyzed to see if there is a mobile genetic element (MGE), and the results are shown in Table 4, showing that these two genes do not have a transfer risk. Based on animal Bifidobacterium lactis subspecies BB12, it has been included in the "Infant Edible Bacteria List", and its safety has been strongly clinically verified. Rifamycin is not in the scope of antibacterial drugs specified in EFSA5206 and the drug sensitivity test of BAPerfectus006 shows that it is sensitive to tetracycline, so it is believed that the antibiotic resistance of BAPerfectus006 meets the edible safety requirements of health food.

[0077] Table 4 Results of risk assessment of drug resistance gene transfer in animal Bifidobacterium lactis subspecies BAPerfectus006

[0078]

[0079] The genome sequence measured in Example 2 was compared with the latest version of the virulence gene database (VFDB) and sorted by identity. The results of TOP15 are shown in Table 5. The highest homology between animal Bifidobacterium lactis subspecies BAPerfectus006 and the virulence gene DNA sequence is 72.6%, and no suspicious virulence gene (coverage>60% and similarity≥85%) was found. At the same time, animal Bifidobacterium lactis subspecies BAPerfectus006 does not contain plasmid, which embodies the stability and safety of the animal Bifidobacterium lactis subspecies BAPerfectus006 genome.

[0080] Table 5 Sequence alignment results of animal Bifidobacterium lactis subsp. BAPerfectus006 and virulence gene database

[0081] Gene_id Subject_id Identity Align_length Mismatch Gap gene_000668 VFG043550 72.6 537 145 2 gene_001390 VFG006022 69.3 336 94 1 gene_000630 VFG046464 66.7 399 126 3 gene_001388 VFG007661 65.6 291 100 0 gene_000248 VFG009376 63.2 182 64 1 gene_000711 VFG001399 62.7 475 176 1 gene_001400 VFG048789 61.9 415 131 5 gene_001225 VFG026433 61 446 171 2 gene_000356 VFG031736 59.4 229 90 1 gene_001410 VFG016390 59.1 345 123 5 gene_001165 VFG005353 56.1 353 137 8 gene_000678 VFG000079 55.9 814 344 4 gene_001015 VFG005580 55.8 428 183 2 gene_001105 VFG009719 55.6 511 180 10 gene_001090 VFG009931 54.8 748 312 8

[0082] In summary, combined with the experimental results of drug sensitivity test, hemolysis test, mouse bacterial pathogenicity test, and analysis of virulence genes and resistance genes, it can be seen that animal Bifidobacterium lactis subspecies BAPerfectus006 has no resistance to the antibiotics specified by EFSA5206, the hemolysis reaction is negative, the animal experiment is non-pathogenic, and the gene analysis shows no virulence genes and transferable resistance genes. In summary, animal Bifidobacterium lactis subspecies BAPerfectus006 is a safe strain that meets the safety requirements of humans.

[0083] Example 4: Preparation of animal Bifidobacterium lactis subspecies BAPerfectus006 bacterial powder

[0084] This embodiment provides a method for preparing Bifidobacterium animalis subsp. lactis BAPerfectus006 bacterial powder, which is carried out with reference to Example 5 of patent CN117070423B, and comprises the following steps:

[0085] 1. Seed activation: Take one ampoule tube bacterial powder of animal Bifidobacterium lactis subsp. BAPerfectus006, resuscitate it, and then obtain the fermentation seed liquid through primary culture amplification and secondary culture amplification.

[0086] 2. Fermentation culture: inoculate the fermentation seed liquid into a 5-ton fermentation tank filled with 2 tons of fermentation liquid and ferment at 37±2℃.

[0087] 3. Bacteria concentration: Start the centrifuge, and after the centrifuge runs stably, the material enters the centrifuge for centrifugal separation.

[0088] 4. Drying and crushing: freeze-dry the centrifugally concentrated bacterial sludge, and after drying, obtain animal Bifidobacterium lactis subspecies BAPerfectus006 bacterial powder. After production, the moisture content, water activity and viable bacterial count were tested, and the results are shown in Table 6.

[0089] Table 6 Animal Bifidobacterium lactis subspecies BAPerfectus006 bacterial powder production results

[0090] batch <![CDATA[Viable bacteria in bacterial powder (×10 11 CFU / g)]]> Moisture content (%) water activity 20220725001 5.6 7 0.12

[0091] In summary, animal Bifidobacterium lactis subspecies BAPerfectus006 can be industrialized, providing feasibility for realizing the use of the strain.

[0092] Example 5: Test on the tolerance of animal Bifidobacterium lactis subspecies BAPerfectus006 to artificial gastrointestinal fluid

[0093] In this example, two strains of animal Bifidobacterium lactis subspecies [derived from the strain library of Perfect (Guangdong) Daily Necessities Co., Ltd. and sent to the Guangdong Provincial Microbial Culture Collection Center for preservation] were selected for comparison, and the test method was as follows:

[0094] Commercial artificial gastric juice (pH=1.5) and artificial intestinal juice products were purchased, and the pH of the artificial gastric juice was adjusted to 2.5, and then filtered and sterilized with a 0.22 μm filter membrane for use.

[0095] The strain glycerol tube was taken out from the strain bank, thawed at room temperature, and shaken evenly by a vortex oscillator. The strain was streaked on the BBL medium plate with an inoculation loop, and a single colony was obtained by anaerobically culturing at 37°C for 48 hours. The single colony was picked up with an inoculation loop and added to a centrifuge tube containing BBL liquid medium, shaken evenly, and anaerobically cultured at 37°C for about 36 hours. The first generation of bacterial liquid was obtained when the strain grew to the middle and late logarithmic growth period. The third generation of bacterial liquid was expanded according to the same method and then mixed and divided into 4 tubes of equal amount. After centrifugation and discarding the supernatant, the tube was washed twice with physiological saline, and then the tube was resuspended with an equal volume of physiological saline (blank group), artificial gastric juice (pH 3.0), and artificial intestinal juice (pH 6.8) and diluted and coated on 3 plates respectively. After 24 hours, the plate with a colony count in the range of 30-300 was counted, and the survival rate was finally calculated. Survival rate (%) = average number of viable bacteria in the experimental group / average number of viable bacteria in the blank group × 100%. The test results are shown in Table 7.

[0096] Table 7 Artificial gastrointestinal fluid tolerance results

[0097]

[0098]

[0099] After incubating in artificial gastric juice and artificial intestinal juice for 3 hours, the survival rate of animal Bifidobacterium lactis subspecies BAPerfectus006 can still reach 96% to 115%, which is higher than that of the two control strains. This shows that animal Bifidobacterium lactis subspecies BAPerfectus006 has a strong gastrointestinal tolerance.

[0100] Example 6: Experiment on mice using animal Bifidobacterium lactis subspecies BAPerfectus006 to enhance immunity

[0101] 1. Experimental plan:

[0102] With reference to the functional testing methods that help enhance immunity in the "Functional Testing and Evaluation Methods for Health Foods (2022 Edition)" (Draft for Comments), the recommended basic dosage for humans is 2 billion CFU ( / 60kg·BW) per day. 10 times, 30 times, 50 times, and 250 times the recommended human dosage were set as the ultra-low, low, medium, and high dose groups for this experiment, respectively. A total of 240 healthy SPF male mice weighing 18g-22g from Kunming were selected and divided into four batches for the experiment. Each batch was randomly divided into 5 groups, with 12 mice in each batch. The mice were killed for sampling after 31 days of intervention.

[0103] 2. Testing indicators:

[0104] 1) One batch of experiments was conducted to measure the organ / body weight ratio, delayed-type hypersensitivity reaction test, half hemolysis value (HC50) and the number of antibody-producing cells;

[0105] 2) The second batch of experiments conducted carbon clearance experiments;

[0106] 3) Three batches of experiments were conducted on the phagocytosis of chicken red blood cells by mouse peritoneal macrophages;

[0107] 4) Four batches of experiments were conducted on ConA-induced mouse lymphocyte transformation experiments and NK cell activity assays.

[0108] The experimental methods in this embodiment are all referenced from the "Health Food Function Testing and Evaluation Methods (2022 Edition)" (Draft for Comments) which helps to enhance the immune function testing method.

[0109] 3. Data processing:

[0110] SPSS software was used for data processing. Variance analysis was used. The independent sample T test was used for comparison between the sample group and the blank control group. The experimental data were expressed as mean ± standard deviation (Mean ± SD). P < 0.05 was considered to be significantly different from the blank control group. The smaller the P value, the greater the significant difference between the groups. * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, and **** represents P < 0.0001.

[0111] 4. Experimental results:

[0112] 1) Effect of animal Bifidobacterium lactis subspecies BAPerfectus006 on sheep erythrocyte-induced DTH in mice (plantar thickening method).

[0113] Table 8 Effect of animal Bifidobacterium lactis subspecies BAPerfectus006 on delayed-type hypersensitivity

[0114]

[0115] As shown in Table 8, after oral administration of the test substance of Bifidobacterium lactis subspecies BAPerfectus006 to mice for 31 days, compared with the blank control group, all groups could increase the swelling of the mouse paws, among which the high-dose group had a significant difference (P<0.05).

[0116] 2) Effect of animal Bifidobacterium lactis subspecies BAPerfectus006 on the proliferation ability of mouse spleen lymphocytes induced by ConA (MTT method).

[0117] Table 9 Effect of animal Bifidobacterium lactis subspecies BAPerfectus006 on lymphocyte transformation experiment

[0118]

[0119] As shown in Table 9, after oral administration of the test substance Bifidobacterium lactis subspecies BAPerfectus006 to mice for 31 days, compared with the blank control group, all groups can improve the proliferation ability of mouse spleen lymphocytes, among which the medium and high dose groups have significant differences (P<0.05).

[0120] 3) Effect of Bifidobacterium animalis subspecies BAPerfectus006 on serum half-hemolysis value

[0121] Table 10 Effect of animal Bifidobacterium lactis subspecies BAPerfectus006 on half hemolysis value

[0122]

[0123] As shown in Table 10, after oral administration of the test substance of Bifidobacterium lactis subspecies BAPerfectus006 to mice for 31 days, compared with the blank control group, all groups could increase the half hemolysis value of mice, among which the high-dose group had a significant difference (P < 0.05).

[0124] 4) Effect of Bifidobacterium lactis subspecies BAPerfectus006 on carbon clearance ability of mice

[0125] Table 11 Effect of animal Bifidobacterium lactis subspecies BAPerfectus006 on carbon clearance capacity

[0126]

[0127] As shown in Table 11, after oral administration of Bifidobacterium lactis subspecies BAPerfectus006 to animals for 31 days, compared with the blank control group, all groups could increase the phagocytic index of mice, among which the increase in the low-dose and medium-dose groups was significantly different (P<0.05).

[0128] 5) Effect of animal Bifidobacterium lactis subspecies BAPerfectus006 on the phagocytic index of chicken erythrocytes by mouse macrophages

[0129] Table 12 Effect of animal Bifidobacterium lactis subspecies BAPerfectus006 on the phagocytic index of chicken erythrocytes by macrophages

[0130]

[0131]

[0132] As shown in Table 12, after oral administration of the test substance Bifidobacterium lactis subspecies BAPerfectus006 to animals for 31 days, compared with the blank control group, all groups could increase the phagocytic index of chicken red blood cells by mouse macrophages, among which the medium and high dose groups had significant differences (P<0.01, P<0.05).

[0133] In summary, after oral administration of the test substance Bifidobacterium lactis subsp. BAPerfectus006 to animals for 31 days, compared with the blank control group, the low-dose group could improve the carbon clearance phagocytosis ability of mice (P<0.05); the medium-dose group could improve the proliferation ability of mouse spleen lymphocytes (P<0.05) and carbon clearance phagocytosis ability (P<0.05), as well as the phagocytic index of chicken red blood cells by mouse macrophages (P<0.01); the high-dose group could improve the delayed-type hypersensitivity reaction of mice (P<0.05), the proliferation ability of spleen lymphocytes (P<0.05), the half hemolysis value of mice (P<0.05), and the phagocytic index of chicken red blood cells by mouse macrophages (P<0.05).

[0134] The results were judged in accordance with the requirements of the test method for enhancing immunity in the "Health Food Function Evaluation Method (2022 Edition) (Draft for Comments)". After 31 days of sampling, the animal experiment results of Bifidobacterium lactis subspecies BAPerfectus006 that helps to enhance immune function were positive, indicating that it has the function of helping to enhance immunity.

[0135] Example 7: Experiment on improving Qi deficiency in rats using animal Bifidobacterium lactis subspecies BAPerfectus006

[0136] 1. Experimental protocol:

[0137] In this experiment, 24 female SD rats weighing 180 - 220 g and 24 male SD rats weighing 280 - 340 g, which were bred by Zhuhai Baishitong Biotechnology Co., Ltd. [License number: SCXK(Yue)2020 - 0051], were selected.

[0138] Before the start of the experiment, after all rats were adaptively fed for 7 days, they were all adaptively swam for 3 days (the water depth was 60 cm, the temperature was 25 ± 1 °C, and each swim lasted for 5 minutes per day). One day before grouping, lead sheets with a weight of 5% of the body mass were attached to the tails of the rats for exhaustive swimming (when the rat finally sank during swimming and the tip of its nose was immersed in the water and still could not return to the water surface after 10 seconds, it was considered exhaustion). Rats with large differences in the negative gravity exhaustive swimming time (the time from the start of swimming to exhaustion) and rats that could not swim were excluded, and qualified rats with a negative gravity exhaustive swimming time of 10 - 30 minutes were retained. The qualified rats were randomly divided into 4 groups, with 10 rats in each group (5 males and 5 females), namely the normal group, the model group, the positive group (Zhongjing Brand Buzhong Yiqi Pills), and the sample group (Bifidobacterium animalis subsp. lactis BAPerfectus006).

[0139] After the start of the experiment, except for the normal group, the rats in the other groups were modeled using the method of "improper diet + fatigue": at the same time every morning, in a constant temperature water tank (the water depth was 60 cm, the temperature was 25 ± 1 °C), they were subjected to exhaustive swimming with lead sheets with a weight of 5% of the body mass attached to their tails, and after fasting for 1 day, they were given 80 g / kg of SPF - level experimental rat maintenance feed every other day. During the modeling, gavage was carried out simultaneously. Among them, the gavage dose of the positive group was 4.5 g / (kg·d), and the gavage dose of the sample group was 5.14×10 9 CFU viable bacteria / (kg·BW). Both Buzhong Yiqi Pills and Bifidobacterium animalis subsp. lactis BAPerfectus006 were dissolved in normal saline and then gavaged. The normal group and the model group were gavaged with an equal volume of normal saline. The gavage volume for all groups was 1 mL / (100 g·BW), and the drug was administered once a day for 21 consecutive days.

[0140] 2. Detection indicators:

[0141] 1) Body weight: The body weights of the rats were weighed and recorded on the 0th, 7th, 14th, and 21st days of the start of the experiment.

[0142] 2) Qi deficiency score: On the 0th, 7th, 14th, and 21st days of the start of the experiment, the survival, body shape, hair, skin, feces, muscle tension, gait, spirit, respiration, etc. of the animals were recorded, and they were scored from 4 dimensions according to Table 17. The cumulative sum was the total score of the rats' qi deficiency symptoms (0 - 12 points). The symptom quantification score table of the rat model of qi deficiency syndrome is shown in Table 13.

[0143] Table 13 Quantitative score table of symptoms in rat model of Qi deficiency syndrome

[0144]

[0145] 3) Gripping strength: The gripping strength of rats was tested using a gripping strength tester on days 0 and 21 of the experiment.

[0146] 4) Hanging ability: The rats’ two front paws were hung on a horizontally fixed rope (2 mm in diameter and 100 m in length) 1 m above the ground, and the time before landing was recorded. A big fall within 3 s or only one grab was considered a failure. The scoring rules were: 0-4 s was scored as 0 points; 5-9 s was scored as 1 point; 10-14 s was scored as 2 points; 15-19 s was scored as 3 points; 20-24 s was scored as 4 points; 25-29 s was scored as 5 points; and more than 30 s was scored as 6 points.

[0147] 5) Visceral index: Rats were sacrificed on the 21st day after the start of the experiment, and their thymus and spleen were weighed to calculate the thymus index and spleen index. Organ index = organ weight / body weight.

[0148] 6) Serum lactate content: 24 hours after the last exhaustive swimming, blood was collected by tail cutting and serum lactate was measured using a portable lactate detector; blood was collected from the eyeball and stored in a 5 ml centrifuge tube, and then centrifuged at 2,000 r / min for 10 min after resting to preserve the serum.

[0149] 3. Experimental results:

[0150] 1) Weight:

[0151] As shown in Tables 14 and 15, during the 0-21 days experiment, the body weight of male and female rats in the normal group showed an increasing trend, while the body weight of rats in the model group, positive group and sample group showed a decreasing trend, but the degree of decrease in the positive group and sample group was lower than that in the model group. Among them, the effect of improving the weight loss of female rats was relatively good, indicating that animal Bifidobacterium lactis subspecies BAPerfectus006 had the effect of delaying the weight loss of female rats with Qi deficiency during the 0-21 days of the experiment.

[0152] Table 14 Effect of animal Bifidobacterium lactis subspecies BAPerfectus006 on body weight of male rats

[0153]

[0154] Table 15 Effect of animal Bifidobacterium lactis subspecies BAPerfectus006 on body weight of female rats

[0155]

[0156] 2) Qi deficiency score:

[0157] like Figure 7As shown, from day 0 to day 21 of the experiment, the scores of the normal group were all 0, and no symptoms of Qi deficiency were shown. The Qi deficiency scores of the model group gradually increased, and the symptoms of Qi deficiency increased significantly (P < 0.01), indicating that the model was successfully established.

[0158] From the Qi deficiency scores of male rats, compared with the model group (0, 2.20±0.45, 4.80±1.10, 7.60±1.14), there were no significant differences in the positive group (0, 2.00±0.00, 2.80±0.84, 3.20±1.79) and the sample group (0, 1.80±0.45, 1.80±0.45, 2.00±0.00) on days 0 and 7, and the Qi deficiency scores on days 14 and 21 were significantly reduced (P<0.01), indicating that the Qi deficiency symptoms were significantly improved, among which the sample group The effect was better than that of the positive group; from the perspective of the Qi deficiency score of the female rat group, compared with the model group (0, 2.60±1.14, 5.75±1.50, 8.80±0.45), there was no significant difference between the positive group (0, 2.60±0.55, 2.80±0.84, 4.00±0.00) and the sample group (0, 3.00±00, 3.20±0.45, 3.00±0.00) on days 0 and 7, but the Qi deficiency score was significantly reduced on days 14 and 21 (P<0.01), and the symptoms of Qi deficiency were improved.

[0159] The above results show that during the 0-21 days of the experiment, when the intervention was continued for 14 to 21 days, the symptoms of qi deficiency in the positive group and the sample group were significantly improved, and compared with the positive group, the improvement effect of the sample group was better, indicating that animal Bifidobacterium lactis subspecies BAPerfectus006 has a significant effect on improving qi deficiency, and the effect is better than Zhongjing brand Buzhong Yiqi Pills.

[0160] 3) Grip:

[0161] Table 16 Effect of animal Bifidobacterium lactis subspecies BAPerfectus006 on the grip strength of rats

[0162]

[0163] As shown in Table 16, from the 0th to 21st day of the experiment, from the change value of the gripping force of the male rat group, the gripping force of the normal group increased by an average of (3.74±2.31)N, while the gripping force of the model group decreased by (0.32±2.77)N, which was significantly reduced (P<0.05). Compared with the model group, the gripping force of the positive group and the sample group showed an upward trend after 21 days, among which the improvement of the sample group was significant (P<0.05), and the effect was better than that of the positive group; from the change value of the gripping force of the female rat group, the gripping force of the normal group increased by (1.08±2.33)N, and the gripping force of the model group decreased by (2.30±2.00)N compared with the normal group. Compared with the model group, the gripping force of the positive group and the sample group showed an upward trend after 21 days, among which the positive group had a better effect. Combining the total average change values ​​of females and males, compared with the normal group, the gripping force of the model group decreased by (1.31±2.51)N (P<0.01). Compared with the model group, the grip strength of the positive group and the sample group was significantly improved after 21 days (P<0.05, P<0.01), and the intervention effect of the sample group was better.

[0164] The above results show that during the 0-21 days of the experiment, the gripping strength of the rats in the Qi deficiency model group was significantly reduced compared with the normal group. After continuous consumption for 21 days, Zhongjing Buzhong Yiqi Pills and animal Bifidobacterium lactis subsp. BAPerfectus006 significantly improved the low gripping strength of male Qi deficiency rats, and the intervention effect of animal Bifidobacterium lactis subsp. BAPerfectus006 was better.

[0165] 4) Suspension capacity:

[0166] Table 17 Effect of animal Bifidobacterium lactis subspecies BAPerfectus006 on the scores of the rat hanging test

[0167]

[0168] As shown in Table 17, on the 21st day of the experiment, the hanging test scores of the male rat group showed a downward trend compared with the normal group. Compared with the model group, the positive group and the sample group showed an upward trend, among which the sample group had a better effect, which was significant (P<0.01); from the hanging test scores of the female rat group, the model group showed a downward trend, and compared with the model group, the hanging test scores of the positive group and the sample group increased, among which the sample group had a significant effect (P<0.01). Combining the total average scores of male and female rats, the trend is the same as above.

[0169] The above results show that after 21 days of intervention, Zhongjing Buzhong Yiqi Pills and animal Bifidobacterium lactis subsp. BAPerfectus006 can effectively improve the hanging ability of rats with qi deficiency, and animal Bifidobacterium lactis subsp. BAPerfectus006 has a better improvement effect.

[0170] 5) Visceral Index:

[0171] like Figure 8 As shown in the figure, on the 21st day of the experiment, compared with the normal group (0.097±0.010), the thymus index of the model group decreased to (0.057±0.007) (P<0.01), and the positive group and sample group also showed a downward trend, which were (0.050±0.018) and (0.038±0.011), respectively. Compared with the normal group (0.117±0.012), the thymus index of the model group decreased to Compared with the model group, the positive group and the sample group showed an upward trend, which were (0.052±0.009 and (0.063±0.015), respectively. The sample group had a better effect on improving the thymus index, which was significant (P<0.01), indicating that animal Bifidobacterium lactis subspecies BAPerfectus006 can significantly improve the thymus index of female rats with qi deficiency, and the effect is better than that of Zhongjing brand Buzhong Yiqi Pills.

[0172] like Fig. 9 As shown in the figure, on the 21st day of the experiment, compared with the normal group (0.188±0.030), the model group showed a downward trend of (0.169±0.012), with no significant difference; the positive group and the sample group also showed a downward trend, (0.177±0.020) and (0.166±0.012), respectively, but there was no significant difference. From the spleen index of the female rat group, compared with the normal group (0.230±0.032), the thymus index of the model group decreased to (0.120±0.027) (P<0.01). Compared with the model group, both the positive group and the sample group showed an upward trend, which were (0.052±0.009) and (0.063±0.015), respectively. The sample group had a better effect in improving the spleen index, which was significant (P<0.01), indicating that animal Bifidobacterium lactis subspecies BAPerfectus006 can significantly improve the spleen index of female rats with qi deficiency, and the effect is better than Zhongjing brand Buzhong Yiqi Pills.

[0173] 6) Serum lactate content:

[0174] like Fig.10As shown in the figure, on the 21st day of the experiment, the serum lactate value of the male rat group was (0.96±0.09)mmol / L in the normal group. Compared with the normal group, the serum lactate of the model group was (1.54±0.29)mmol / L, which showed an upward trend. Compared with the model group, the serum lactate of the positive group and the sample group showed a downward trend, which were (1.48±0.33)mmol / L and (1.22±0.18)mmol / L, respectively. From the perspective of serum lactate of the female rat group, compared with the normal group (1.02±0.15)mmol / L, the model group was significantly increased to (2.26±0.68)mmol / L (P<0.01); compared with the model group, the positive group was significantly decreased to (1.02±0.40)mmol / L (P<0.01), and the sample group was significantly decreased to (1.20±0.48)mmol / L (P<0.01). It shows that after 21 days of intervention, animal Bifidobacterium lactis subspecies BAPerfectus006 can effectively reduce the serum lactic acid content of female rats with qi deficiency.

[0175] Figure 7-10 The statistical differences in the results were compared with those in the model group, and the statistical symbols mean:

[0176] *P<0.05, **P<0.01, ***P<0.001, ***P<0.0001.

[0177] From the above results, it can be seen that animal Bifidobacterium lactis subspecies BAPerfectus006 can significantly improve the symptoms of qi deficiency in qi deficiency rats, delay weight loss, improve grip and hanging ability, increase thymus index and spleen index, and reduce serum lactic acid content. Among them, the effects of improving qi deficiency symptoms, improving grip and hanging ability, increasing the thymus index and spleen index of female qi deficiency rats, and reducing serum lactic acid in female qi deficiency rats are better than Zhongjing brand Buzhong Yiqi Pills under the same conditions.

[0178] Combined with drug sensitivity test, hemolytic test, mouse pathogenicity test phenotype experimental test results and virulence gene and drug resistance gene analysis, the animal bifidobacterium lactis subspecies BAPerfectus006 hemolytic reaction of the present application is negative, non-pathogenic, non-resistant to common antibiotics, and gene analysis shows no pathogenicity gene and transferable drug resistance gene, which is a very safe strain. And animal bifidobacterium lactis subspecies BAPerfectus006 does not contain plasmid, and functional genes are all on chromosomes, which are not easy to lose, and functional genetic stability is good. The animal bifidobacterium lactis subspecies BAPerfectus006 in vivo and in vitro experiments of the present application all show no toxic side effects, and no virulence gene, which is a very safe probiotic, can meet the safety standards of health food raw material strains. The present application first discovered the application of animal Bifidobacterium lactis subspecies in improving the symptoms of qi deficiency, and the effects of improving the symptoms of qi deficiency, improving grip and hanging ability, increasing thymus index and spleen index, and reducing serum lactic acid are better than those of Zhongjing brand Buzhong Yiqi Pills under the same conditions. It is a better probiotic raw material for improving qi deficiency, and has a better effect of enhancing immunity. The animal Bifidobacterium lactis subspecies BAPerfectus006 of the present application is directly collected from healthy human bodies, has stable biological characteristics, and has significant resistance to gastrointestinal fluid, and does not require domestication and rejuvenation processes, and can be directly used in the preparation process.

[0179] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. Use of a strain of Bifidobacterium lactis subsp. animalis in the preparation of a product for improving qi deficiency and enhancing immunity, characterized in that: The animal Bifidobacterium lactis subspecies is animal Bifidobacterium lactis subspecies (Bifidobacterium animalissubsp.lactis) BAPerfectus006, with a preservation number of GDMCC No: 62539. It was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 13, 2022, and the preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The use according to claim 1, characterized in that The Qi deficiency includes spleen qi deficiency.

3. The use according to claim 1, characterized in that The product includes a food or a medicine.

4. The use according to claim 1, characterized in that The product is used in at least one of the following aspects: 1) Significantly improve the symptoms of Qi deficiency; 2) Improve grip and suspension ability; 3) Reduce serum lactic acid content; 4) Increase thymus index and spleen index; 5) Delay weight loss; 6) Improve spleen lymphocyte proliferation ability, half hemolysis value, phagocytic index and macrophage phagocytosis of chicken red blood cells.

5. A food or medicine for improving qi deficiency, characterized in that: Including the animal Bifidobacterium lactis subspecies as described in claim 1, the animal Bifidobacterium lactis subspecies is animal Bifidobacterium lactis subspecies (Bifidobacterium animalissubsp.lactis) BAPerfectus006, the preservation number is GDMCCNo: 62539, and it has been preserved in the Guangdong Provincial Microbiological Culture Collection Center on June 13, 2022, and the preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

6. The food or medicine according to claim 5, characterized in that The food or medicine further comprises auxiliary materials and / or carriers.

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