CN110959792B

CN110959792B - Solid beverage containing bifidobacterium lactis and application thereof

Info

Publication number: CN110959792B
Application number: CN201811161153.9A
Authority: CN
Inventors: 洪维鍊; 刘伟贤; 赵雯; 孙婷; 何剑; 陈醇
Original assignee: Inner Mongolia Yili Industrial Group Co Ltd
Current assignee: Inner Mongolia Yili Industrial Group Co Ltd
Priority date: 2018-09-30
Filing date: 2018-09-30
Publication date: 2022-03-29
Anticipated expiration: 2038-09-30
Also published as: CN110959792A

Abstract

The invention discloses a solid beverage containing bifidobacterium lactis and application thereof. Specifically, the invention provides a solid beverage composition which contains Bifidobacterium lactis (Bifidobacterium lactis) or active substances thereof with the preservation number of CGMCC No. 15650. The solid beverage has the effects of preventing osteoporosis and the like.

Description

Solid beverage containing bifidobacterium lactis and application thereof

Technical Field

The invention relates to a solid beverage containing probiotics and application thereof, in particular to a solid beverage containing bifidobacterium lactis and application thereof in the aspects of preventing osteoporosis and the like.

Background

Osteoporosis is a systemic metabolic bone disease characterized by a decrease in bone mass and destruction of the bone microstructure. It is manifested by increased bone fragility and susceptibility to fracture. Currently about 2 million people worldwide suffer from osteoporosis. The incidence of hip fractures due to osteoporosis is increasing worldwide, and the elderly population is mostly affected by the risk of fractures. The total number of disabilities per year is 580 million counted worldwide. 51% of this number are due to fractures, occurring primarily in europe and america. Thus, osteoporotic fractures are considered to be a significant cause of mortality and morbidity in developed countries. The most common type of osteoporosis is associated with postmenopausal women aged 50 and older. Of the elderly over

age

50, 1/3 women and 1/5 men are threatened by osteoporosis. The survey results of the Ministry of health of China from 2002 to 2005 show that the prevalence rate of osteoporosis is 8.8%, and the prevalence rate of chronic diseases of famous Chinese residents is the third place. Therapeutic agents for osteoporosis are generally drugs such as injections of hormones, calcium supplements, and bone resorption inhibitors. After long-term administration of drugs or hormones, the drugs or hormones can cause obvious side effects on the organism.

Bifidobacterium lactis (Bifidobacterium lactis) has been reported to have various health effects such as immunomodulation and intestinal health, and has been reported to be used for solid beverages.

However, there are few technical reports on the use of bifidobacterium lactis for the treatment and/or prevention of osteoporosis.

Disclosure of Invention

The invention provides Bifidobacterium lactis (Bifidobacterium lactis) with an effect of treating and/or preventing osteoporosis, and accordingly provides a solid beverage containing the Bifidobacterium lactis or active substances thereof.

In one aspect, the present invention provides a Bifidobacterium lactis (Bifidobacterium lactis) which can be used for treating or preventing osteoporosis and has the effects of increasing calcium ions and/or phosphorus ions in blood. The lack of acid resistance and gastrointestinal fluid resistance is a common property of bifidobacteria, which results in bifidobacteria being difficult to reach and colonize the gut via gastric juices. The Bifidobacterium lactis (Bifidobacterium lactis) provided by the invention has gastric acid resistance and intestinal juice resistance, the survival rate of viable bacteria is more than 62% when the Bifidobacterium lactis is treated in gastric acid solution with pH of 2.5 for 30min, and the survival rate of viable bacteria is more than 61% when the Bifidobacterium lactis is treated for 2 hours; the survival rate of viable bacteria is more than 70 percent after the intestinal juice with pH6.8 is treated for 2 hours.

In the present invention, Bifidobacterium lactis (Bifidobacterium lactis) is provided and named BL-99. The strain has been preserved in China general microbiological culture Collection center (CGMCC) (address: No. 3 Xilu-Beijing province No.1, Beijing Korean district, Ministry of China microbiology institute) 26.04.2018, and is named after classification: bifidobacterium lactis (Bifidobacterium lactis); the preservation number is CGMCC No. 15650.

The study of the invention finds that the bifidobacterium lactis BL-99 (namely the bifidobacterium lactis with the preservation number of CGMCC No. 15650) can be used for treating or preventing osteoporosis, and has the function of improving the concentration of calcium ions and/or phosphorus ions in blood, and the study specifically comprises the following steps: significantly reduce the loss of bone mass caused by estrogen deficiency; the blood calcium and phosphorus concentrations are improved; the amount of osteoclast in vivo and the absorption effect of osteoclast on bone are inhibited by adjusting the proportion of OPG/RANKL; the expression of the protein of the bone anabolism related factor gene, such as alkaline phosphatase and osteocalcin, is promoted, so that the level of the protein is improved, and the formation of new bone is promoted.

The research of the invention also finds that the bifidobacterium lactis BL-99 can be used for enhancing the immune response of an organism, and specifically comprises the following components: the carbon clearance index of the mouse can be improved; improving half hemolysis value of the mouse; increasing the number of mouse antibody-producing cells; activating NK cell activity; delayed immunoreaction is positive; the phagocytic rate and phagocytic index of macrophages are increased.

The study of the invention also finds that the bifidobacterium lactis BL-99 disclosed by the invention has the capacity of promoting the growth of intestinal bifidobacteria and lactic acid bacteria, can inhibit the growth of vibrio desulfovibrio and/or enterobacter in the intestinal tract, and particularly can inhibit the growth of helicobacter pylori and/or escherichia-shigella. In addition, mouse experiments show that the strain has no oral acute toxicity and no antibiotic tolerance, and can be safely used for food processing.

Further, the present invention provides a solid beverage composition comprising Bifidobacterium lactis (Bifidobacterium lactis) or an active substance thereof having a storage number of CGMCC No. 15650.

According to a specific embodiment of the present invention, in the solid beverage composition of the present invention, the active substance is a fermentation culture product of bifidobacterium lactis with a preservation number of CGMCC No.15650, including but not limited to: a fermentation culture containing bacterial cells, a fermentation supernatant from which the bacterial cells have been removed, or an active substance extracted from the fermentation culture. Bifidobacterium lactis BL-99 of the present invention can be cultured by anaerobic fermentation in a culture medium for Bifidobacterium lactis (e.g., TPY medium, BBL medium, etc.) which is commonly used in the art. The optimal fermentation temperature is 35-38 ℃, and the optimal fermentation time is 7-24 h. The invention also provides a preparation method of the fermentation culture product of the bifidobacterium lactis BL-99, which comprises the step of carrying out anaerobic culture on the strain in a liquid fermentation culture medium to obtain a fermentation liquid containing the strain. The fermentation liquor can be directly used as a liquid bacterial preparation or further concentrated, and the fermentation liquor can be dried to prepare bacterial powder, or the bacterial powder is prepared by separating thalli from the fermentation liquor. The liquid bacterial preparation of the present invention may be a liquid bacterial preparation prepared by suspending the bacterial cells in a culture medium, a buffer solution, deionized water or other solvent. The BL-99 liquid bacterium preparation or the solid bacterium preparation (bacterium powder) can be stored in a viable bacterium form and has better stability in a storage period. The BL-99 liquid bacteria preparation or solid bacteria preparation (bacteria powder) of the present invention can also be preserved in the form of inactivated dead bacteria. The bacterial preparation can be used for preparing the solid beverage.

According to a specific embodiment of the invention, in the solid beverage composition of the invention, the content of the bifidobacterium lactis with the preservation number of CGMCC No.15650 in the beverage composition is 1.0 x10³CFU～1.0×10¹⁰CFU/kg body weight/day, or 0.001. mu.g to 100mg/kg body weight/day, preferably 0.01. mu.g to 10mg/kg body weight/day in terms of the weight of the cells.

According to a specific embodiment of the invention, in the solid beverage composition, the content of the bifidobacterium lactis with the preservation number of CGMCC No.15650 in the solid beverage is 0.1-50% by taking the total weight of the solid beverage as 100%.

According to a specific embodiment of the invention, the solid beverage composition of the invention can comprise some conventional components of the existing probiotic solid beverage besides the bifidobacterium lactis with the preservation number of CGMCC No. 15650. In some embodiments of the present invention, the raw material composition of the solid beverage of the present invention further comprises one or more of resistant dextrin, fructo-oligosaccharide, lactitol, vitamins, and calcium carbonate.

According to some more specific embodiments of the present invention, the solid beverage composition of the present invention comprises the following raw materials, based on the total weight of the solid beverage as 100%: 0.1-50% of bifidobacterium lactis or active substances thereof with the preservation number of CGMCC No. 15650; 31.26-58.05% of resistant dextrin; 4.9-9.1% of fructo-oligosaccharide; 4.9-9.1% of lactitol; 0.7-1.3% of vitamin C; 0.7-1.3% of calcium carbonate; and (4) the balance of fruit powder.

In another aspect, the invention also provides the application of the solid beverage composition in preparing a solid beverage with the effect of preventing osteoporosis.

In another aspect, the invention also provides the application of the solid beverage composition in preparing the solid beverage with the effect of regulating the balance of gastrointestinal flora.

On the other hand, the invention also provides application of the solid beverage composition in preparing a solid beverage with the effect of improving immunity.

The preparation method of the solid beverage can be carried out by referring to the existing preparation technology of the probiotic solid beverage. In general, the solid beverage of the present invention can be obtained by uniformly mixing the components of the composition.

According to a specific embodiment of the invention, the bifidobacterium lactis with the preservation number of CGMCC No.15650 can exist in the solid beverage in a live form or in an inactivated form. According to a specific embodiment of the invention, the bifidobacterium lactis with the preservation number of CGMCC No.15650 can be in an inactivated form in the solid beverage with the effect of regulating the gastrointestinal flora.

The solid beverage provided by the invention has the corresponding effects of preventing and treating osteoporosis, regulating the balance of gastrointestinal flora, improving immunity and the like due to the inclusion of the bifidobacterium lactis BL-99.

Drawings

Figure 1A shows the change in body weight of animals before and after intervention with bifidobacterium lactis BL-99. Figure 1B shows the change in uterine weight of animals before and after intervention with bifidobacterium lactis BL-99.

Fig. 2A shows the outcome of post-HE staining for bifidobacterium lactis BL-99. Fig. 2B shows the change in percentage of trabecular area before and after intervention with bifidobacterium lactis BL-99.

Fig. 3A shows results of tibial TRAP staining following bifidobacterium lactis BL-99 intervention. FIG. 3B shows the change in the percentage of osteoclasts on the bone surface (OcS/BS) before and after intervention by Bifidobacterium lactis BL-99.

FIGS. 4A to 4D show the changes in serum calcium, serum phosphorus, serum osteocalcin, type I collagen C-terminal peptide after the Bifidobacterium lactis BL-99 is dried, respectively.

Fig. 5A-5E show the effect of bifidobacterium lactis BL-99 intervention on the regulation of genes associated with bone metabolism.

FIG. 6 is a schematic view of a fermentation process according to an embodiment of the present invention.

Microbial preservation of the patent procedure:

bifidobacterium lactis BL-99 of the present invention:

the preservation date is as follows: 26/04/2018;

the preservation unit: china general microbiological culture Collection center (CGMCC);

the address of the depository: xilu No.1 Hospital No. 3, the institute of microbiology, China academy of sciences, Beijing, Chaoyang

The preservation number is: CGMCC No. 15650;

and (3) classification and naming: bifidobacterium lactis (Bifidobacterium lactis).

Detailed Description

For a more clear understanding of the technical features, objects and advantages of the present invention, reference is now made to the following detailed description taken in conjunction with the accompanying specific embodiments, and the technical solutions of the present invention are described, it being understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.

Unless specifically defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art. Unless otherwise indicated, all numbers expressing quantities of ingredients, cell culture, processing conditions, and so forth used in the present disclosure are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters are approximations and may vary depending upon the desired properties sought to be obtained by the present invention. The term "at least" preceding a series of elements is to be understood as referring to each element in the series, unless otherwise indicated.

In various examples of the invention, experimental data are expressed as Mean ± s.e.m. data were counted using PRISM version 5.0(GraphPad, San Diego, CA, USA). Differences between groups were counted using a one-way ANOVA followed by Tukery's multiple compliance test. There were significant statistical differences at P < 0.05.

Example 1: bifidobacterium lactis BL-99 and performance measurement thereof

The bifidobacterium lactis BL-99 of the invention is obtained from Shanghai Bingzhui GmbH and is separated from the intestinal tract of the infant. The strain has been preserved in China general microbiological culture Collection center (CGMCC) (address: No. 3 Xilu-Beijing province No.1, Beijing Korean district, Ministry of China microbiology institute) 26.04.2018, and is named after classification: bifidobacterium lactis (Bifidobacterium lactis); the preservation number is CGMCC No. 15650.

1. Taxonomical characterization of Bifidobacterium lactis BL-99

The results of the physical and chemical tests are as follows:

16S rRNA Gene sequence sequencing results (SEQ ID No. 1):

GCTCCCCCACAAGGGTCGGGCCACCGGCTTCGGGTGCTACCCACTTTCATGACTTGACGGGCGGTGTGTACAAGGCCCGGGAACGCATTCACCGCGGCGTTGCTGATCCGCGATTACTAGCGACTCCGCCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGACCGGTTTTCAGCGATCCGCCCCACGTCACCGTGTCGCACCGCGTTGTACCGGCCATTGTAGCATGCGTGAAGCCCTGGACGTAAGGGGCATGATGATCTGACGTCATCCCCACCTTCCTCCGAGTTGACCCCGGCGGTCCCACATGAGTTCCCGGCATCACCCGCTGGCAACATGCGGCGAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTGAACCGGCCCCGAAGGGAAACCGTGTCTCCACGGCGATCCGGCACATGTCAAGCCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCGCATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTTCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGATGCTTAACGCGTTGGCTCCGACACGGGACCCGTGGAAAGGGCCCCACATCCAGCATCCACCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTGACGGCCCAGAGACCTGCCTTCGCCATTGGTGTTCTTCCCGATATCTACACATTCCACCGTTACACCGGGAATTCCAGTCTCCCCTACCGCACTCCAGCCCGCCCGTACCCGGCGCAGATCCACCGTTAGGCGATGGACTTTCACACCGGACGCGACGAACCGCCTACGAGCCCTTTACGCCCAATAAATCCGGATAACGCTCGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTATTCGAACAATCCACTCAACACGGCCGAAACCGTGCCTTGCCCTTGAACAAAAGCGGTTTACAACCCGAAGGCCTCCATCCCGCACGCGGCGTCGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTATCTCAGTCCCAATGTGGCCGGTCACCCTCTCAGGCCGGCTACCCGTCAACGCCTTGGTGGGCCATCACCCCGCCAACAAGCTGATAGGACGCGACCCCATCCCATGCCGCAAAAGCATTTCCCACCCCACCATGCGATGGAGCGGAGCATCCGGTATTACCACCCGTTTCCAGGAGCTATTCCGGTGCACAGGGCAGGTTGGTCACGCATTACTCACCCGTTCGCCACTCTCACCCCGACAGCAAGCTGCCAGGGATCCCGTTCGACT

2. tolerance of bifidobacterium lactis BL-99 to artificial gastric juice and intestinal juice

Bifidobacteria are genera that are generally not acid-fast. In this example, the tolerance of bifidobacterium lactis BL-99 of the present invention to artificial gastric juice and intestinal juice was tested, and bifidobacterium lactis which had been recognized in the art as having excellent acid resistance and survived through the gastrointestinal tract

For comparison.

The test method comprises the following steps: culturing Bifidobacterium lactis BL-99 strain in MRS liquid culture medium at 37 deg.C for 16 hr, centrifuging at 4 deg.C and 2500rpm for 10min, and collecting thallus.

Respectively culturing the strains to be tested in artificial gastric juice and artificial small intestine juice, processing at 37 ℃ for 0, 30min and 2h, and then performing viable count analysis to evaluate the acid resistance and intestinal juice resistance of the strains according to the survival rate. Survival rate (viable cell count after treatment/viable cell count at time 0) × 100%.

The survival rate detection result of the bacterial strain in artificial gastric acid (pH2.5) is shown in Table 1, the survival rate of the viable bacteria is 7.04% when BB-12 is treated in the artificial gastric acid (pH2.5) for 30min, and the survival rate of the viable bacteria is only 1.64% after 2 hours of treatment; the survival rate of the live bacteria of the bifidobacterium lactis BL-99 is 62.60 percent when the bifidobacterium lactis BL-99 is treated in artificial gastric acid (pH2.5) for 30min, and the survival rate of the live bacteria is 61.83 percent when the bifidobacterium lactis BL-99 is treated for 2 hours. The bifidobacterium lactis BL-99 disclosed by the invention has excellent gastric acid resistance and can smoothly pass through the stomach to reach the intestinal tract to play a probiotic role.

TABLE 1 survival rate of the strains in artificial gastric acid (pH2.5)

The survival rate of the strain in the artificial small intestine solution (pH6.8) is tested and shown in Table 2. The data show that the viable bacteria survival rate of BB-12 in artificial small intestine solution (pH6.8) for 2 hours is only 28.95%; the viable bacteria survival rate of the bifidobacterium lactis BL-99 is 70.23 percent when the bifidobacterium lactis BL-99 is treated in artificial gastric acid (pH2.5) for 2 hours. The bifidobacterium lactis BL-99 disclosed by the invention has excellent intestinal juice resistance and can survive and colonize in intestinal tracts.

TABLE 2 survival rate of the strains in artificial intestinal juice (pH6.8)

3. Toxicity experiment and safety detection of bifidobacterium lactis BL-99

Inoculating the bifidobacterium lactis BL-99 of the invention into a BBL liquid culture medium, carrying out anaerobic culture for 48 +/-2 hours at 36 +/-1 ℃, and counting the viable count of the bifidobacterium lactis BL-99 in the culture solution to be 3.7 multiplied by 10⁸cfu/mL, stock solutions and 5-fold concentrates of the cultures were continuously gavaged to 20.0mL/kg BW for 3 days and observed for 7 days. The experiment was performed with a control group of 5-fold concentrated solution and a stock solution of the medium. The test result shows that: the BBL culture stock solution and 5-fold concentrated solution of Bifidobacterium lactis BL-99 had no statistical effect on the weight gain of mice (p > 0.05) compared with the respective control group, and no toxic reaction or death of the tested mice was observed.

The antibiotic sensitivity of the bifidobacterium lactis BL-99 is evaluated by adopting an SN/T1944-2007 method of determination of bacterial resistance in animals and products thereof. The evaluation results show that the bifidobacterium lactis BL-99 is sensitive to Ampicillin Ampicillin, penicillin G Penicillin G, Erythromycin Erythromycin, Chloramphenicol Chloramphenicol, Clindamycin Clindamycin, Vancomycin Vancomycin, Tetracycline and the like. Meets the requirements of European Food Safety Authority (European Food Safety Authority) on the evaluation specification of the resistance of the edible bacteria. The bifidobacterium lactis BL-99 does not contain exogenous antibiotic resistance genes and is safe to eat.

4. Detection of efficacy of preventing and treating osteoporosis

4.1 Deovariectomized rat animal models and probiotic intervention

Culturing Bifidobacterium lactis BL-99 strain in MRS liquid culture medium at 37 deg.C for 16 hr, centrifuging at 4 deg.C and 2500rpm for 10min, collecting thallus, washing with Phosphate Buffer Solution (PBS), freeze drying, and storing at-18 deg.C or below. Is used for the experimental study of the efficacy of preventing and treating osteoporosis.

85 adult female SD rats with the age of 17 weeks with the body weight of 200-300 g. The rats were randomly divided into 3 groups of 10 rats each. 20 rats were subjected to ovariectomy, and the remaining 10 rats were subjected to sham surgery. Rats were exposed to light/dark for 12h daily at room temperature around 25 ℃ with free access to water. After 12 weeks of surgical intervention, animals in the model investigation group are sacrificed, samples of uterus, thighbone, shinbone and the like are collected, and osteoporosis related indexes such as uterus coefficient, bone microstructure morphology, bone structure model parameters and the like are measured.

After the animal after operation has a rest for two weeks, the animal begins to be administered by gavage, and the treatment course is once a day for 12 weeks continuously. Feeding BL-99 probiotics to ovariectomized rats, and feeding distilled water to a sham operation group and an ovariectomized blank control group; the animals were dosed as follows:

the specific grouping of animals is as follows:

group 1-sham blank control (sham), 10, blank solvent;

group 2-ovariectomized blank control group (OVX), 10, blank solvent;

group 3-Deovariectomized + Probiotics BL-99 group, 10, Probiotics dose 10⁹CFU/mL。

The body weight changes of the animals before and after the intervention are shown in fig. 1A. After successful model establishment, the weight of the sham-operated group was significantly lower than that of each of the other ovariectomized groups, consistent with significant weight gain in postmenopausal women. Each intervention group had some increase in body weight during the 12 week intervention period. Compared with the ovariectomized blank group and the ovariectomized probiotic BL-99 intervention group, the weight of the ovariectomized blank group has no significant difference before and after the intervention.

After 12 weeks of dry prognosis, the animals were sacrificed, the weight of the uterus was collected, weighed and recorded, and the uterine coefficient (i.e., the ratio of the weight of the uterus to the weight of the body) was calculated. The experimental results (fig. 1B) show that there is a very significant difference in uterine coefficients between the ovariectomized OVX group and the sham operated group (P <0.001vs. sham operated), indicating significant atrophy of the uterus following ovariectomy with a decrease in vivo estrogen. And the ovariectomized blank group and the ovariectomized + probiotic BL-99 intervention group have no influence on the weight of the uterus, which indicates that the ovariectomized blank group and the ovariectomized + probiotic BL-99 intervention group have no estrogen-like side effect.

4.2 bone histomorphometry

The proximal tibia 1/3 is cut along the sagittal plane and the material is selected at 1X 0.5cm³Soaking in 10% EDTA/PBS (PH7.4) decalcifying liquid for about 1 week, changing liquid once every 3 days, dehydrating conventionally, embedding paraffin, staining by HE along sagittal plane (thickness 4 μm), determining total tissue area, trabecular area and total girth of trabecular, and calculating trabecular area percentage, trabecular number, trabecular thickness and trabecular separation degree by calculation formula. The bone tissue slices are also used for observing the appearance, arrangement, morphological structure integrity and the like of the trabeculae.

After probiotic BL-99 intervenes for 12 weeks, HE staining results (figure 2A) show that the trabecular bone of the sham operation group is closely arranged and has a complete structure; and compared with the false operation group, the area percentage of the trabecular bone is obviously reduced (P <0.001vs. the false operation). Compared with the OVX blank group, the probiotic BL-99 group can increase the trabecular bone area percentage by about 12.5% (OVX: 16.8 +/-2.5%, OVX + BL-99:18.9 +/-1.8) (P <0.05vs. OVX) (figure 2B), and the probiotic BL-99 can inhibit bone loss caused by estrogen deficiency and has a certain protection effect on bones.

Osteoclasts are the major cells responsible for bone resorption in the body and play an important role in bone development, growth, repair, and remodeling. Osteoclasts are derived from the monocyte-macrophage system and are specialized terminally differentiated cells that can be formed into large multinucleated cells from their mononuclear precursor cells by cell fusion. Osteoclasts correspond functionally to osteoblasts. The two are synergistic and play an important role in the development and formation of bones. Highly expressed Tartrate Resistant Acid Phosphatase (TRAP) is one of the major markers of osteoclasts. Results of tibial TRAP staining are shown in fig. 3A, with positive staining as a result of staining wine red to the osteoclast cytoplasm. The number of osteoclasts stained by TRAP on the surface of tibia in the rats in the OVX blank group was significantly higher than that in the sham-operated rats. Compared with OVX blank rats, the number of TRAP-stained osteoclasts in the probiotic BL-99 group is significantly reduced by about 17.9% (OVX: 22.3 + -1.1%, OVX + BL-99:18.3 + -0.6) (P <0.05vs. In vivo, estrogen can inhibit the activity of osteoclast, and induce the apoptosis of osteoclast so as to play the role of bone resorption resistance. In OVX animals, the inhibitory effect on osteoclasts was lost due to significantly lower estrogen levels, the number of osteoclasts and the ability to resorb bone were significantly increased (fig. 3B), and the final result was a significant decrease in cancellous bone mass. The results from fig. 2A, 2B and 3A, 3B suggest that BL-99 intervention inhibits OVX-induced loss of bone mass, possibly by reducing the number of osteoclasts.

4.3 measurement of Biochemical indicators

The biochemical indicators measured include blood calcium, blood phosphorus, serum Osteocalcin (OCN), type I collagen C-terminal peptide (C-terminal peptides of type I collagen, CTX-I). The method adopts atomic absorption spectrophotometry to measure the blood calcium and the blood phosphorus, and the serum sample is directly measured. The kit for detection comprises: calcum

Test(REF 0155-225)，Phosphorus

Test (REF 0830-. However, compared with the OVX blank group, the probiotic BL-99 dry group can obviously improve high serum calcium ions (OVX: 2.28 +/-0.02 mg/dl, OVX + BL-99:2.49 +/-0.03 mg/dl) by about 10 percent and phosphorus ions (OVX: 1.02 +/-0.08 mg/dl, OVX + BL-99:1.41 +/-0.11 mg/dl) by about 40 percent.

Serum osteocalcin is an active polypeptide secreted by osteoblasts and plays an important role in regulating bone metabolism, with levels reflecting osteoblast activity. The C-terminal peptide of type I collagen is a small fragment of type I collagen after degradation, and the content and change of the C-terminal peptide can evaluate the bone resorption state. Serum osteocalcin and type I collagen C-terminal peptide were measured using an ELASA kit, and the measurement was performed according to the instructions in the kit. The kit used for detection is as follows: the Rat Osteocalcin ELISA Kit, Rat C-telopeptide of Collagen alpha-1(I) chain ELISA Kit, was manufactured as SAB (SAbiosciences, USA).

As shown in FIGS. 4A-4D, compared with the OVX blank group, the probiotic BL-99 dried group significantly increased serum osteocalcin levels by about 44.6% (OVX: 68.6 + -16.4 pg/dl, OVX + BL-99:92.6 + -24.3 pg/dl) and decreased serum type I-collagen C-terminal peptide by about 14.6%, but had no statistical significance.

4.4 detection of bone specimen PCR

This example continues to study and examine gene expression in bone specimens associated with osteoclastogenesis and osteoblastogenesis. Probiotic BL-99 intervenes in the same manner as in example 2. After total RNA was extracted from bone tissue using Trizol, the RNA was reverse transcribed into cDNA using a reverse transcription kit, followed by PCR amplification using different gene primers (see table below).

Compared with an OVX blank control group, the probiotic BL-99 intervention group can obviously improve the gene expression of Osteoprotegerin (OPG) without obviously influencing the gene expression of RANKL, so that the ratio of OPG/RANKL is increased. RANKL binds to RANK receptors on the surface of osteoclasts, promotes differentiation and activation of osteoclasts, and inhibits apoptosis thereof; osteoprotegerin OPG prevents binding of RANKL to RANK, thereby preventing osteoclast activation, inhibiting osteoclast function, reducing bone resorption, and acting as a negative regulator. The ratio of OPG/RANKL suggests that osteoclast levels are regulated in vivo. The research finds that the expression ratio of OPG/RANKL gene is increased after BL-99 intervention, the ratio is increased by about 75% from 1 to 1.75 of an ovariectomized blank group, and the result indicates that BL-99 has an obvious effect of inhibiting osteoclast formation.

In addition, as shown in fig. 5A to 5E: compared with an OVX blank group, the BL-99 intervention group can improve the gene expression level of osteocalcin by about 1.1 times (OVX: 0.908 +/-0.107, OVX + BL-99: 2.075 +/-0.643) and alkaline phosphatase by about 37.8% (OVX: 0.990 +/-0.217, OVX + BL-99: 1.364 +/-0.513), and the expression water of the two genes is closely related to the bone formation capacity, so that BL-99 intervention can promote the formation of new bone and antagonize the loss of bone mass caused by OVX by promoting the expression of related genes of the bone formation.

The results of the above studies confirm that: the bifidobacterium lactis BL-99 can obviously inhibit the loss of bone mass caused by ovariectomy or low estrogen, and improve the blood calcium and the blood phosphorus.

5. Immunomodulatory activity assay

Culturing Bifidobacterium lactis BL-99 strain in MRS liquid culture medium at 37 deg.C for 16 hr, centrifuging at 4 deg.C and 2500rpm for 10min, collecting thallus, washing with Phosphate Buffer Solution (PBS), freeze drying, and storing at-18 deg.C or below. The experimental studies used in this example.

700 healthy male BALB/C mice, 6-8 weeks old, 16-18g, were provided by the laboratory animal technology, Inc., Viton, Beijing. The animal is bred in animal laboratories of the occupational health and poisoning control institute of the Chinese disease prevention and control center: maintaining the room temperature (25 + -2 deg.C), relative humidity (55 + -2)%, and lighting for 12h/12h, and freely eating and drinking.

Animals were randomly divided into 5 groups for each experimental study of this example, 140 mice per group, 1 normal control group per group, and 1 dose group per probiotic sample.

The test sample is administered to the mouse in an intragastric manner for 28 days, the intragastric volume is 0.2mL/10g once a day, and the control group is administered with distilled water by intragastric administration.

According to the probiotic sample information, the dosage of each test sample to be given to the mice is calculated by referring to the daily human body demand and the conversion coefficient of the dosage of 70Kg adults and 20g mice, the dosage of BB-12 group is 2.36mg/Kg, and the dosage of BL-99 group is 6.34 mg/Kg.

5.1 monocyte-macrophage function

5.1.1 carbon Clearance test

Animals were continuously given the sample for 28 days, weighed, injected with India ink in the tail vein, and 20. mu.L of blood was collected at 2min and 10min after the injection, added to 2M of L0.1% sodium carbonate solution, and measured for OD at 600 nm. Mice were sacrificed, livers and spleens were removed, blood stains on the surfaces of the visceral organs were blotted with filter paper and weighed.

The phagocytosis index is used for expressing the carbon clearance capacity of the mouse, and the calculation is carried out according to a formula

The results of the phagocytic index of carbon clearance experiments are shown in table 3. The results show that the phagocytosis index of the mouse carbon clearance test of the BL-99 group is lower than that of the control group, and the mouse carbon clearance test of the BB-12 has no significant difference compared with the control group (p is more than 0.05).

TABLE 3 phagocytic index results in carbon clearance experiments

Group by group	Animal number (only)	Phagocytic index	P-value compared with control group
				Control
	8	7.64±0.62	--
				BB-12 group	8	7.56±0.61	0.902
BL-99 group	9	6.43±0.55	0.039*

5.1.2 measurement of organ/body weight ratio

Weighing the initial weight and the final weight of the mouse 28 days after sample administration respectively, dislocating and killing the mouse, taking the spleen and the thymus, removing fascia, sucking blood stains on the surfaces of organs by using filter paper, weighing, and calculating the ratio of the spleen to the body weight and the ratio of the thymus to the body weight.

The results are shown in Table 4. After the test sample is given, compared with the control group, the spleen/body weight ratio and the thymus/body weight ratio of the BL-99 and BB-12 groups have no significant difference, which indicates that the samples BL-99 and BB-12 have no influence on the spleen/thymus of the mice.

TABLE 4 change in organ/body weight ratio of mice

5.2 humoral immune assay

5.2.1 serum hemolysin half maximal hemolysis value (HC)₅₀) Measurement of (2)

Animals were immunized by intraperitoneal injection of 0.2mL SRBC per mouse 28 days after serial dosing. After 4 days, the eyeball is removed, blood is taken out of the 1.5mL centrifugal tube, the centrifugal tube is placed for about 1h at 4 ℃ to ensure that serum is fully separated out, and the centrifugal tube is centrifuged at 2000r/min for 10min to collect the serum. Serum was diluted 100-fold with SA buffer. Adding diluted serum into 96-well plate, adding 100 μ L of the diluted serum into each well, sequentially adding 10% (v/v) SRBC50 μ L and complement 100 μ L (diluted with SA solution at a ratio of 1: 8), placing in 37 deg.C constant temperature water bath, keeping the temperature for 30min, and centrifuging at 1500r/min for 10 min. Then 50 mu L of supernatant is taken from each sample well and blank control well, added into another 96-well culture plate, added with 150 mu L of Wen Chi reagent, provided with half of hemolysis wells simultaneously, added with 12.5 mu L of 10% (v/v) SRBC, added with the Wechi Chi reagent to 200 mu L, fully and uniformly mixed by using a shaking period, placed for 10min, and measured with a full-automatic enzyme standard instrument at 540 to obtain the optical density value of each well.

The amount of hemolysin is expressed as the half hemolysis value (HC50) and is calculated according to the following formula:

the results are shown in Table 5. As can be seen from Table 5, the hemolytic value of HC50 was increased in half (p < 0.05) for both BL-99 and BB-12 groups compared to the control group, and BL-99 group was higher than BB-12 group.

TABLE 5 values of the median hemolysis HC₅₀Results

Group of	Animal number (only)	HC₅₀	p value
				Control	14	51.07±2.17	--
BB-12 group	14	66.31±3.66	0.000^**
				BL-99 group	14	67.43±4.04	0.000^**

5.2.2 detection of antibody-producing cells

Mixing the mouse spleen cell suspension immunized by Sheep Red Blood Cells (SRBC) with a certain amount of SRBC, and dissolving the SRBC around the spleen cells secreting the antibody in the presence of complement to form macroscopic plaques, wherein the number of the hemolytic plaques can reflect the number of antibody-producing cells.

Animals were immunized by intraperitoneal injection of 0.2mL SRBC per mouse 28 days after serial dosing. Mice immunized with SRBC for 4 days were sacrificed, and the spleen was removed and prepared into 5X10⁶Individual cells/mL of cell suspension. Heating agarose for dissolving, mixing with equal amount of double Hank's solution, subpackaging into small tubes with 0.5mL per tube, adding 20% (V/V, prepared with normal saline) into the tubes, pressing to obtain 50 μ L SRBC and 200 μ L spleen cell suspension, rapidly mixing, pouring onto six-well plate with agarose thin layer, coagulating with agar, placing into carbon dioxide incubator for continuous incubation for 1h, adding complement (1:10) diluted with SA buffer solution, continuously incubating for 2h, and counting number of hemolytic plaques.

The results of the number of antibody-producing cells are shown in Table 6, in which the sample group and the control group showed significant difference (p < 0.05) between BL-99 and BB-12 groups compared to the control group, and BL-99 group compared to the control group showed significant difference (p > 0.05).

TABLE 6 results of the number of antibody-producing cells

Group of	Animal number (only)	Number of hemolytic plaques/10⁶Spleen cell	p value
				Control	11	18.64±1.91	--
BB-12 group	10	23.30±3.13	0.003**
				BL-99 group	9	28.67±2.87	0.000**

5.3NK cell Activity assay

5.3.1 ConA-induced mouse lymphocyte transformation experiments

After 28 days of continuous animal sampling, mice were sacrificed, after sterilization in a beaker of 75% alcohol, spleens were aseptically taken, placed in a small plate containing 3cm × 3cm four layers of gauze (autoclaved), an appropriate amount of sterile Hank's solution was added, splenomeles were crushed with gauze, the spleens were gently crushed with an elbow walk on tiptoe to prepare a single cell suspension, washed 2 times with Hank's solution, centrifuged at 1000rpm for 10min each time, then the cells were suspended in 2mL of complete culture solution, the number of viable cells was counted, and the cell concentration was adjusted to 5 × 10⁶one/mL. The cell suspension was added to the 24-well culture medium in two wells, 1mL per well, and 75. mu.L of LCoA solution (equivalent to 7.5. mu.g/mL) was added to one well, and the other well was used as a control and incubated at 37 ℃ in 5% CO2 for 72 hours. 4 hours before the end of the culture, 0.7mL of the supernatant RPMI1640 medium containing no calf serum was gently aspirated from each well, and 50. mu.L of MTT (5mg/mL) was added to each well, and the culture was continued for 4 hours. The culture is finishedThen, 1mL of acidic isopropanol is added into each hole, and the mixture is uniformly blown to ensure that the purple crystals are completely dissolved. Then, the cells were plated in 96-well plates, each well was plated in 2 wells as a parallel sample, and the optical density was measured at a wavelength of 570nm using an enzyme-linked immunosorbent assay. The proliferative capacity of lymphocytes is expressed as the optical density value of ConA plus wells minus the optical density value of ConA minus wells.

The results are shown in Table 7. As can be seen from Table 7, the BB-12 group was significantly higher than the control group (p < 0.05), while BL-99 was not significantly changed from the control group (p > 0.05).

TABLE 7 results of mouse splenic lymphocyte transformation experiments

5.3.2NK cell Activity assay

The animals were continuously administered for 28 days, and the target cell YAC-1 was subcultured 24h before the start of the experiment, washed 2 times with Hank's solution before use, and the cell concentration was adjusted to 1X 10 with 10% calf serum-containing RPMI1640 complete medium⁵one/mL (target cells). Sudden death of cervical dislocation of mouse, aseptically taking spleen, preparing into suspension of skin cells, washing with Hank's solution for 2 times, centrifuging at 1000rpm for 10min, resuspending with 2mL RPMI1640 containing 10% calf serum, staining with trypan blue viable cells for counting (viable cell number should be above 95%), adjusting cell concentration to 1 × 10⁷number/mL (effector cells) to give an effective target ratio of 100: 1. 100 mu L of each of the target cells and the effector cells are taken and added into a U-shaped 96-well culture plate, 100 mu L of each of the target cells and the culture solution is added into a natural release hole of the target cells, 100 mu L of each of the target cells and 1% NP40 is added into a maximum release hole of the target cells, and three parallel holes are formed in the above. 37 ℃ and 5% CO₂Culturing for 4h in an incubator, centrifuging the 96-well culture plate at 1500rpm for 5min, sucking 100 microliter of supernatant per well, placing the supernatant in a flat-bottomed 96-well culture plate, simultaneously adding 100 microliter of LDH matrix solution, reacting for 3min, adding 30 microliter of 1mol/L HCL solution per well to terminate the reaction, measuring an OD value at 490nm of a microplate reader, and calculating the NK activity according to the following formula:

the results are shown in Table 8. As can be seen from Table 8, the NK cell activity of BL-99 was higher than that of the control group and BB-12, and the difference was significant.

TABLE 8 NK cell Activity results

Group of	Animal number (only)	Cell Activity (%)	P value
				Control	14	30.70±3.31	--
BB-12 group	14	33.38±4.17	0.078
				BL-99 group	14	47.92±5.63	0.000**

5.4 cellular immune response

5.4.1 delayed type hypersensitivity

After the animals were continuously administered for 28 days, 0.2mL of SRBC (v/v, prepared with physiological saline) was intraperitoneally injected with 2% volume of SRBC per rat, and after 4 days of sensitization, the thickness of the left hind toe was measured, and the same site was measured 3 times to obtain an average value. Then 20% SRBC 20. mu.L was injected subcutaneously at the measurement site, left rear toe thickness was measured at 24h after injection, and the same site was averaged 3 times to express the degree of DTH as the difference in toe thickness before and after challenge (toe swelling degree).

The results are shown in Table 9. As can be seen from Table 9, the toe thickness of each group of mice was at the same level before SRBC challenge, and the toe swelling of the mice occurred 24 hours after SRBC total, and the swelling degree was expressed by the toe thickness difference before and after the challenge. The BB-12 and BL-99 groups were found to be significantly higher than the control group (p < 0.05) by statistical analysis.

TABLE 9 late allergic toe swelling results in mice

5.4.2 mouse peritoneal macrophage phagocytosis assay

After animals are continuously fed with samples for 28 days, injecting 0.2mL of 2% SRBC into the abdominal cavity of each mouse 4 days before the completion of the gavage to activate the macrophages of the mice, killing the mice by cervical dislocation on the same day of the experiment, injecting 3mL of Hank's solution added with calf serum into the abdominal cavity, gently massaging the abdominal cavity for 20 times to fully wash out the macrophages of the abdominal cavity, then cutting a small opening on the abdominal wall, sucking 0.5mL of mixed solution of the abdominal cavity washing solution, and adding the mixed solution into an agar ring of a glass slide. Placing in an incubator and incubating for 15-20min at 37 ℃. After the incubation is finished, the non-adherent cells are quickly washed out by normal saline, fixed in methanol solution for 1min, and stained by Giemsa for 15 min. Washed clean with distilled water, air dried, and counted by a 40 × microscope for phagocytosis rate and phagocytosis index. The phagocytosis rate is the percentage of phagocytes which phagocytize chicken erythrocytes in every 100 phagocytes; the phagocytosis index is the average number of chicken erythrocytes phagocytosed per phagocyte.

The results were calculated as follows:

the results are shown in Table 10. The result of phagocytosis rate and phagocytosis index of the phagocytes shows that the result of the phagocytosis experiment of the BB-12 group is negative if the phagocytosis rate and phagocytosis index of the BB-12 group are not different from those of the control group; the phagocytosis rate and the phagocytosis index of the BL-99 group are higher than those of the control group, and the phagocytosis experiment result of the macrophages of the BL-99 group is positive.

TABLE 10 macrophage phagocytosis Rate and phagocytosis index results

Group of	Animal number (only)	Phagocytosis ratio (%)	p value	Phagocytic index	p value
						Control	14	22.55±1.58	--	0.42±0.04	--
BB-12 group	14	22.28±2.75	0.799	0.47±0.04	0.055
						BL-99 group	14	25.48±2.86	0.005**	0.53±0.04	0.000**

The results prove that the isolated strain Bifidobacterium lactis BL-99 can improve the immune organ index, the non-specific immune function and the specific immune function characteristic of an organism, thereby achieving the purpose of improving the activity of the organism in immunity.

6. Intestinal flora regulating effect

6.1 see "health food inspection and evaluation technical criteria-decision criteria for regulating intestinal flora function", the effect of bifidobacterium lactis of the invention on intestinal regulation is determined.

Culturing Bifidobacterium lactis BL-99 strain in MRS liquid culture medium at 37 deg.C for 16 hr, centrifuging at 4 deg.C and 2500rpm for 10min, collecting thallus, washing with Phosphate Buffer Solution (PBS), freeze drying, and storing at below-18 deg.C. The experimental studies used in this example.

36 healthy SPF-grade BABL/c mice were taken, weighing 18-22g (supplied by Beijing Huafukang Biotech, Inc.). After 3 days of acclimatization, they were randomly divided into 3 groups of 12 individuals, i.e., placebo group, sample group. And (3) separately perfusing sterile water (the perfused volume is 0.2mL/10g) dissolved with Bifidobacterium lactis BL-99 powder into the stomach of each group of animals, and perfusing sterile water with the same volume into the stomach of a blank control group. The preparation is administered 1 time per day by continuous feeding or intragastric administration for 14 days. And (3) gavage metering: 1.3X 10⁷CFU/ml(2 multiplied by 10 according to the human body demand⁹CFU/d, human and mouse scaling factor 0.0026). Taking sterilized centrifuge tubes, numbering, adaptively feeding, collecting 2-3 mice feces of about 100mg per mouse under sterile condition, and transferring to a sterile operating room under low temperature condition for detecting flora. At the end of the experiment, the mouse feces were collected again. The mice are numbered in groups by picric acid, weighed respectively on the 8 th day and the 14 th day of the administration of the test substance, the gavage amount of the mice is calculated, and the mice are weighed 1 time at the end of the experiment. And (3) counting colonies: preparing selective culture medium according to the strain to be identified, sterilizing the strain to be detected and corresponding culture medium as shown in Table 11, shaking up, cooling to 45-50 ℃, and pouring into a flat plate for later use.

TABLE 11 strains to be tested and corresponding selection media

Bacterial strain to be tested	Selective medium
		Enterobacter	Eosin Methylene Blue (EMB) agar
Enterococcus	Sodium azide-crystal violet-esculin agar
		Bifidobacterium	BBL agar
Lactobacillus strain	LBS agar
		Clostridium perfringens	Tryptone-sulfite-cycloserine (TSC) agar

The collected mouse feces are placed in a sterilization tube filled with 0.5mL of physiological saline to prepare bacterial suspension, and the suspension is shaken for 1min before use. Sucking 0.1mL of the bacterial suspension by using a 0.1mL micropipette, slowly injecting the bacterial suspension into 0.9mL of sterilized physiological saline, and shaking or repeatedly blowing to uniformly mix the bacterial suspension to prepare the bacterial suspension with the ratio of 1: 10. Another 0.1mL micropipette tip was taken and diluted by 10-fold gradient according to this method to 10-7 g/mL. Two consecutive suitable dilutions were selected according to the viable count of the species to be identified, 10. mu.L of bacterial suspension was pipetted using a 10. mu.L micropipette for each dilution, surface-coated on selective agar plates, and cultured under the culture conditions shown in Table 12. The colony counting method is carried out by referring to GB 4789.2-2010 food safety national standard food microbiology test colony total number determination.

TABLE 12 culture medium for testing intestinal flora and identification method

Data statistics were performed using SPSS 17.0. Comparing the change conditions of the bifidobacterium, the lactobacillus, the enterococcus and the enterobacter before and after the experiment and between groups, wherein the comparative change of the test group before and after the experiment has significance, and the animal test result of the test sample can be judged to be positive according to any one of the following conditions. First, the number of bifidobacteria or lactic acid bacteria in the feces is significantly increased, the number of clostridia is reduced or not significantly changed, and the number of enterococci and enterobacteria is not significantly changed. Secondly, the bifidobacteria or the lactic acid bacteria in the excrement are obviously increased, the clostridia are reduced or have no obvious change, and the enterococci and the enterobacteria are obviously increased, but the increase amplitude is lower than that of the bifidobacteria or the lactic acid bacteria.

The results of the weight change of the animals during the experiment are shown in Table 13. In the experimental period, the animal characteristics are normal, no adverse reaction occurs after the test substance is given, and the body weights of two groups of animals do not have obvious difference in the experimental period.

TABLE 13 weight changes of animals

Numbering	Grouping	Number of animals	Initial body weight (g)	Middle-term body weight (g)	Terminal body weight (g)
						1	Control group	14	21.89±1.25	22.14±0.87	21.24±0.87
2	BL-99	14	23.36±0.77	22.41±0.93	22.59±1.53
						3	BB-12	14	22.31±1.07	22.00±1.55

Tables 14 to 18 record the changes of different bacteria in the intestinal tracts of animals before and after the test, respectively. From tables 6 to 10, it can be seen that bifidobacterium lactis BL-99 can significantly promote the growth of bifidobacterium and lactic acid bacteria, has no significant influence on enterobacteria, enterococcus and clostridium perfringens, can be judged according to the health food inspection and evaluation technical specification-judgment standard for regulating intestinal flora function, and bifidobacterium lactis BL-99 in the research has the effect of regulating intestinal flora. TABLE 14 Change in Bifidobacterium longum in animal intestinal before and after test (LgCFU/g)

TABLE 15 Change of Lactobacillus in intestinal tract of animals before and after test (LgCFU/g)

TABLE 16 Change of Enterobacter enterobacter in animal intestinal before and after test (LgCFU/g)

TABLE 17 Change in enterococcus in animal gut before and after test (LgCFU/g)

TABLE 18 Change of Clostridium perfringens of animal gut before and after the test (LgCFU/g)

6.2 comparison of intestinal flora regulating effects of different doses of active Bifidobacterium lactis and inactivated strains

In this example, intestinal flora regulating effects of different doses of active bifidobacterium lactis BL-99 and inactivated strains were respectively detected.

Viable bacteria sample: weighing 1g viable bacteria sample according to sample specification, and suspending to 40ml with PBS solution, wherein viable bacteria concentration is 2.5x10⁹CFU/ml。

High dose group: calculated according to the gavage amount of 0.2ml/10g of the mice, the gavage amount of 20g of the mice is 0.4ml, and the gavage dose of 10 high-dose group mice is⁹CFU/20g。

The medium dose group: respectively adding 5ml of the high-dose suspension into PBS to reach 50ml of constant volume, calculating according to the gavage amount of 0.2ml/10g of mice, the gavage amount of 20g of mice is 0.4ml, and the gavage amount of 10 medium-dose group mice is 10⁸CFU/20g。

Low dose group: adding PBS into 5ml of the medium-dose group suspension respectively to reach a constant volume of 50ml, calculating according to the gavage amount of 0.2ml/10g of the mice, wherein the gavage amount of 20g of the mice is 0.4ml, and the gavage amount of the low-dose group mice is 10⁷CFU/20g。

Dead bacteria samples: weighing 1g viable bacteria sample according to sample specification, and suspending to 40ml with PBS solution, i.e. viable bacteria concentration is 2.5x10⁹CFU/ml. The concentration is 2.5x10⁹And (3) killing the CFU/ml live bacteria sample bacteria liquid for 20min at 100 ℃. The sample preparation method of the high, medium and low dose groups is the same as that of the viable bacteria group.

The BABL/c mice of 6 weeks old are raised in a clean animal room with temperature of 22 ℃, humidity of 10-60%, 12-hour illumination of alternating light and shade, and fed with standard feed and freely drinking water. Adaptive feeding was performed for 5 days, and 182 mice were randomly divided into 13 groups of 14 mice each, and the grouping is shown in Table 19.

TABLE 19 Experimental groups for regulating intestinal flora

Before beginning the gavage, the feces of each mouse were collected under sterile conditions, labeled, stored at-20 ℃ and tested for intestinal flora. The test was performed by gavage of 0.2ml/10g of each test substance, the control group was performed by PBS for 1-14 days, and the test groups were performed by gavage of test substances at the corresponding doses according to Table 19. Mice were weighed once a week and gavage was adjusted according to body weight. Collecting feces of each mouse under sterile condition after 14 days, marking, storing at-20 deg.C, and detecting intestinal flora.

Before and after the experiment, the body weight of each group of mice has no obvious difference. At phylum level, the relative abundance of Firmicutes (Firmicutes) in the intestinal flora of mice increased and the relative abundance of Bacteroidetes (Bacteroidetes) and Proteobacteria (Proteobacteria) decreased after supplementation with different doses of probiotics. Researches show that the ratio of firmicutes to bacteroidetes has strong correlation with human intestinal diseases, and obese patients tend to reduce the ratio of the firmicutes to the bacteroides. Patients with enteritis and intestinal stress syndrome tend to have higher Proteobacteria (Proteobacteria) abundance.

The effect of BL-99 on gut flora at the genus level is shown in table 20.

TABLE 20 Effect of BL-99 on intestinal flora

At the genus level, BL-99 in the probiotic group mouse intestinal flora significantly increased the relative abundance of Lactobacillus (Lactobacillus) in the mouse intestinal tract compared to the control group, and BL-99 low dose group increased Lactobacillus (Lactobacillus) most significantly. The BL-99 dead bacteria high-dose group has obvious inhibition effect on desulfurization vibrio (Desulfovibrio) and enterobacter (enterobacter).

The inhibitory effect of BL-99 on the pathogenic bacteria helicobacter pylori, Escherichia coli-Shigella is shown in Table 21.

TABLE 21 inhibitory Effect of BL-99 on pathogenic bacteria

The pathogenic bacteria are analyzed, and the result also shows that the BL-99 low-dose group has a remarkable inhibiting effect on Helicobacter pylori (Helicobacter), all groups have an inhibiting effect on Escherichia-Shigella (Escherichia-Shigella), and the killing effect is better.

The experiment shows that the balance of intestinal flora can be adjusted by supplementing BL-99 in the low-dose group, the growth of beneficial bacteria is promoted, harmful bacteria and even pathogenic bacteria are inhibited, and the dead bacteria are also effective, so that the BL-99 or BL-99 dead bacteria in the low-dose group can play a potential health role.

Example 2: solid beverage containing bifidobacterium lactis BL-99

Referring to the fermentation process flow shown in fig. 6, bifidobacterium lactis BL-99 (i.e. bifidobacterium lactis with the preservation number of CGMCC No. 15650) provided by the invention is subjected to anaerobic culture in a TPY liquid culture medium. TPY liquid Medium (g/L): 10.0 parts of hydrolyzed casein, 5.0 parts of soytone, 2.0 parts of yeast powder, 5.0 parts of glucose, 0.5 part of L-cysteine, 2.0 parts of dipotassium phosphate, 0.5 part of magnesium chloride, 0.25 part of zinc sulfate, 0.15 part of calcium chloride, 0.0001 part of ferric chloride, and 801.0 parts of Tween, wherein the pH value is 6.5 +/-0.1. The fermentation broth after primary and secondary amplification culture is centrifuged at 2500rpm for 10min at 4 ℃ to collect the thallus. Freeze drying the collected thallus to obtain BL-99 active bacteria powder, and storing at-18 deg.c below.

The solid beverage of this example, solid beverage product specifications, were formulated according to the following ingredients in table 22: 2000 mg/bar.

TABLE 22 solid beverage ingredient table

Batching table	The gram quantity (mg) is required for each bag	Percent (%)
			BL-99 bacterial powder	126.9190	6.346
Resistant dextrins	893.0810	44.654
			Fructo-oligosaccharide	140.0000	7.000
Lactitol	140.0000	7.000
			Vitamin C	20.0000	1.000
Pineapple fruit powder	660.0000	33.000
			Calcium silicate	20.0000	1.000
Total of	2000	100

The solid beverage of the present example was prepared by mixing the above ingredients uniformly. Evaluation experiments show that the flavor of the solid beverage has no obvious difference from the flavor of the solid beverage without BL-99 bacteria.

Sequence listing

<110> Inmunogu Yili industry group GmbH

<120> solid beverage containing bifidobacterium lactis and application thereof

<130> GAI18CN5036

<160> 11

<170> SIPOSequenceListing 1.0

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<213> Bifidobacterium lactis (Bifidobacterium lactis)

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tgaaccggcc ccgaagggaa accgtgtctc cacggcgatc cggcacatgt caagcccagg 480

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ggctccgaca cgggacccgt ggaaagggcc ccacatccag catccaccgt ttacggcgtg 660

gactaccagg gtatctaatc ctgttcgctc cccacgcttt cgctcctcag cgtcagtgac 720

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caccgggaat tccagtctcc cctaccgcac tccagcccgc ccgtacccgg cgcagatcca 840

ccgttaggcg atggactttc acaccggacg cgacgaaccg cctacgagcc ctttacgccc 900

aataaatccg gataacgctc gcaccctacg tattaccgcg gctgctggca cgtagttagc 960

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<213> Artificial Sequence (Artificial Sequence)

<220>

Claims

1. A solid beverage composition comprises Bifidobacterium lactis (active substances) with a preservation number of CGMCC No.15650 or active substances thereof, wherein the content of the Bifidobacterium lactis with the preservation number of CGMCC No.15650 in the solid beverage is 0.1-50% by taking the total weight of the solid beverage as 100%.

2. A solid beverage composition comprises Bifidobacterium lactis (CGMCC No. 15650) or its active substances, and the raw materials of the solid beverage further comprise one or more of resistant dextrin, fructo-oligosaccharide, lactitol, vitamins and calcium carbonate.

3. A solid beverage composition according to claim 1 or 2, wherein the active substance is a fermentation culture product of bifidobacterium lactis with a accession number of CGMCC number 15650.

4. A solid beverage composition according to claim 1 or 2, wherein the Bifidobacterium lactis having a storage number of CGMCC number 15650 is contained in the beverage composition in an amount of 1.0X 10³CFU~1.0×10¹⁰CFU/kg body weight/day, or 0.001-100 mg/kg body weight/day in terms of the weight of the thallus.

5. The solid beverage composition according to claim 4, wherein the Bifidobacterium lactis having a storage number of CGMCC number 15650 is contained in the beverage composition in an amount of 0.01 to 10mg/kg of body weight per day in terms of the weight of the cells.

6. The solid beverage composition according to claim 2, wherein the content of the bifidobacterium lactis with the preservation number of CGMCC number 15650 in the solid beverage is 0.1-50% by weight based on 100% of the total weight of the solid beverage.

7. The solid beverage composition according to claim 2, which comprises the following raw materials by weight percentage based on the total weight of the solid beverage as 100 percent:

0.1-50% of bifidobacterium lactis or active substances thereof with the preservation number of CGMCC number 15650;

31.26-58.05% of resistant dextrin;

4.9-9.1% of fructo-oligosaccharide;

4.9-9.1% of lactitol;

0.7-1.3% of vitamin C;

0.7-1.3% of calcium carbonate;

and (4) the balance of fruit powder.

8. Use of the solid beverage composition according to any one of claims 1 to 7 for preparing a solid beverage having osteoporosis prevention effect.

9. Use of the solid beverage composition according to any one of claims 1 to 7 for preparing a solid beverage having the efficacy of regulating the balance of gastrointestinal flora.

10. Use of the solid beverage composition according to any one of claims 1 to 7 in the preparation of a solid beverage with immunity enhancing effect.

11. Use according to claim 8 or 9 or 10, wherein bifidobacterium lactis having a accession number CGMCC number 15650 is present in the solid beverage in viable or inactivated form.