WO2020063553A1 - 一种乳双歧杆菌bl-99及其应用 - Google Patents

一种乳双歧杆菌bl-99及其应用 Download PDF

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WO2020063553A1
WO2020063553A1 PCT/CN2019/107392 CN2019107392W WO2020063553A1 WO 2020063553 A1 WO2020063553 A1 WO 2020063553A1 CN 2019107392 W CN2019107392 W CN 2019107392W WO 2020063553 A1 WO2020063553 A1 WO 2020063553A1
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bifidobacterium lactis
composition
preparation
increasing
group
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PCT/CN2019/107392
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English (en)
French (fr)
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刘伟贤
赵雯
洪维鍊
孙婷
司徒文佑
冯昊天
韦斯科姆·菲利普·A
李昊虬
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内蒙古伊利实业集团股份有限公司
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Priority claimed from CN201811161053.6A external-priority patent/CN110964655B/zh
Priority claimed from CN201811161108.3A external-priority patent/CN110964657B/zh
Priority claimed from CN201811161107.9A external-priority patent/CN110964656B/zh
Application filed by 内蒙古伊利实业集团股份有限公司 filed Critical 内蒙古伊利实业集团股份有限公司
Priority to EP19867197.6A priority Critical patent/EP3812452A4/en
Publication of WO2020063553A1 publication Critical patent/WO2020063553A1/zh
Priority to US17/217,305 priority patent/US11298382B2/en
Priority to US17/685,974 priority patent/US11911423B2/en
Priority to US17/685,908 priority patent/US11911422B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the field of microorganism technology, in particular to a strain of Bifidobacterium lactis, which has the properties of resistance to gastric acid and intestinal fluid, can significantly promote the growth of bifidobacteria and lactic acid bacteria, has immunomodulatory activity, and has the ability to prevent bone Porphyria, improve blood calcium and / or phosphorus.
  • probiotics such as Bifidobacterium lactis have multiple functions, such as regulating intestinal disorders, enhancing intestinal immune function, and inhibiting allergic reactions.
  • the World Health Organization's definition of probiotic products is that food contains a sufficient number of live microorganisms, and through various processes of food processing, and after entering the human intestine, it can still maintain an appropriate number of live bacteria and bacterial activity. Therefore, it is generally considered that it is necessary for the strain to be able to maintain a relatively stable number of viable bacteria after the production of bacterial powder, the production and processing of products, and the stress of gastric acid and intestinal fluid through the human gastrointestinal tract.
  • An object of the present invention is to provide a new Bifidobacterium lactis and its use.
  • the present invention provides a Bifidobacterium lactis, which has gastric acid resistance, and has a viable survival rate of more than 62% when treated in a gastric acid solution of pH 2.5 for 30 minutes, and a viable survival rate of 2 hours after treatment 61% or more.
  • the Bifidobacterium lactis provided by the invention also has intestinal fluid resistance, and the survival rate of viable bacteria is more than 70% after being treated in the small intestinal fluid at pH 6.8 for 2 hours.
  • the provided Bifidobacterium lactis is named BL-99.
  • the strain was deposited on April 26, 2018 at the China General Microbial Strain Collection Management Center CGMCC (Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), classified and named: Bifidorum Bifidobacterium lactis; deposit number CGMCC No. 15650.
  • the Bifidobacterium lactis BL-99 (ie, Bifidobacterium lactis deposited under CGMCC No. 15650) of the present invention has the ability to significantly promote the growth of intestinal bifidobacteria and lactic acid bacteria, and can inhibit The growth of Desulfovibrio and / or Enterobacter in the intestine, especially curatively inhibits the growth of H. pylori and / or Escherichia-Shigella, has a significant effect on regulating the gastrointestinal flora.
  • the Bifidobacterium lactis BL-99 (ie, Bifidobacterium lactis deposited under CGMCC No. 15650) of the present invention can be used to enhance the body's immune response, and can be used to improve the triggering caused by irregular diet and increased work pressure. Problems such as low immunity. Specifically include: (1) can increase the carbon clearance index of mice; (2) increase the half hemolysis value of mice; (3) increase the number of mouse antibody-producing cells; (4) activate NK cell activity; (5) delayed immunity Positive response; (6) The phagocytic rate and phagocytic index of macrophages increased.
  • the research of the present invention has found that the Bifidobacterium lactis BL-99 of the present invention can be used for treating or preventing osteoporosis (there is little in the prior art about the use of Bifidobacterium lactis for treating and / or preventing osteoporosis Technical report), and has the effects of increasing blood calcium and / or phosphorus ion concentration, including: (1) significantly reducing bone loss caused by estrogen deficiency; (2) increasing blood calcium and blood phosphorus concentrations; ( 3) By adjusting the ratio of OPG / RANKL, the number of osteoclasts in the body can be inhibited, and its resorption effect on bone can be inhibited; (4) Proteins such as alkaline phosphatase and bone calcium are promoted by promoting the expression of bone anabolic factors The expression of hormones increases the level of its protein to promote the formation of new bone.
  • the Bifidobacterium lactis BL-99 of the present invention can be cultured by anaerobic fermentation in a Bifidobacterium lactis medium (such as TPY medium, BBL medium, etc.) commonly used in the field.
  • a Bifidobacterium lactis medium such as TPY medium, BBL medium, etc.
  • the best fermentation temperature is 35 ⁇ 38 °C, and the best fermentation time is 7 ⁇ 24h.
  • the invention also provides a method for preparing a Bifidobacterium lactis BL-99 bacterial preparation, which comprises anaerobic culturing the strain in a liquid fermentation medium to obtain a fermentation liquid containing bacterial cells.
  • the fermentation broth can be directly or further concentrated to be used as a liquid bacterial preparation, or the fermentation broth can be dried to prepare bacterial powder, or the bacterial body can be separated from the fermentation broth to prepare bacterial powder.
  • the liquid bacterial preparation of the present invention may be a liquid preparation in which the bacterial weight is suspended in a solvent such as a culture solution, a buffer solution, or deionized water.
  • the BL-99 liquid bacteria preparation or solid bacteria preparation (bacteria powder) of the present invention can be stored in the form of live bacteria, and has better stability during the storage period.
  • the BL-99 liquid bacteria preparation or solid bacteria preparation (bacteria powder) of the present invention can also be stored in the form of inactivated dead bacteria.
  • the bacterial preparation can be used in the production of food, feed or medicine. Experiments in mice have shown that the strain has no oral acute toxicity, no antibiotic resistance, and is safe for food processing.
  • the present invention provides a Bifidobacterium lactis, and its deposit number is CGMCC No. 15650.
  • the present invention also provides a Bifidobacterium lactis preparation, which contains the Bifidobacterium lactis according to the present invention and is a solid or liquid bacterial preparation.
  • the Bifidobacterium lactis preparation of the present invention may be in a viable form or an inactivated form.
  • the present invention also provides an application of the Bifidobacterium lactis or the Bifidobacterium lactis preparation in preparing a composition having an effect of regulating intestinal flora, the composition including a food composition , Feed composition or pharmaceutical composition.
  • the present invention also provides the use of the Bifidobacterium lactis or the Bifidobacterium lactis preparation in the preparation of a composition having the effect of increasing the number of Bifidobacterium and / or lactic acid bacteria in the intestine.
  • the composition includes a food composition, a feed composition, or a pharmaceutical composition.
  • the present invention also provides a combination of the Bifidobacterium lactis or the Bifidobacterium lactis preparation having the effect of inhibiting Desulfovibrio and / or Enterorhabdus in the intestinal tract.
  • a food composition the composition includes a food composition, a feed composition, or a pharmaceutical composition.
  • the present invention also provides the said Bifidobacterium lactis or the said Bifidobacterium lactis preparation in preparation for inhibiting Helicobacter and / or Escherichia-Shigella (Escherichia -Shigella) efficacy composition, including a food composition, a feed composition or a pharmaceutical composition.
  • Escherichia-Shigella Escherichia-Shigella
  • the present invention also provides an application of the Bifidobacterium lactis or the Bifidobacterium lactis preparation in the preparation of a composition for improving the immune activity of the body, the composition including a food composition , Feed composition or pharmaceutical composition.
  • the present invention also provides the use of the Bifidobacterium lactis or the Bifidobacterium lactis preparation in the preparation of a composition for improving the carbon clearance index of the body, the composition including a food composition , Feed composition or pharmaceutical composition.
  • the present invention also provides an application of the Bifidobacterium lactis or the Bifidobacterium lactis preparation in the preparation of a composition for improving the half hemolysis value of the body, the composition including a food composition , Feed composition or pharmaceutical composition.
  • the present invention also provides an application of the Bifidobacterium lactis or the Bifidobacterium lactis preparation in preparing a composition for increasing the number of antibody-producing cells in the body, the composition including a food combination Food, feed composition or pharmaceutical composition.
  • the present invention also provides an application of the Bifidobacterium lactis or the Bifidobacterium lactis preparation in preparing a composition for activating NK cell activity of the body, the composition including a food composition , Feed composition or pharmaceutical composition.
  • the present invention also provides an application of the Bifidobacterium lactis or the Bifidobacterium lactis preparation in preparing a composition for increasing phagocytic rate and phagocytic index of macrophages in the body, the combination
  • the composition includes a food composition, a feed composition, or a pharmaceutical composition.
  • the present invention also provides an application of the Bifidobacterium lactis or the Bifidobacterium lactis preparation in preparing a composition for preventing and treating osteoporosis, the composition including a food composition, Feed composition or pharmaceutical composition.
  • the present invention also provides use of the Bifidobacterium lactis or the Bifidobacterium lactis preparation in the preparation of a composition for reducing bone mass caused by estrogen deficiency, the composition comprising Food composition, feed composition or pharmaceutical composition.
  • the present invention also provides the use of the Bifidobacterium lactis or the Bifidobacterium lactis preparation in the preparation of a composition for improving blood calcium and / or blood phosphorus, the composition comprising Food composition, feed composition or pharmaceutical composition.
  • the present invention also provides an application of the Bifidobacterium lactis or the Bifidobacterium lactis preparation in preparing a composition for inhibiting the number of osteoclasts in the body, the composition comprising a food combination Food, feed composition or pharmaceutical composition.
  • the present invention also provides the use of the Bifidobacterium lactis or the Bifidobacterium lactis preparation in the preparation of a composition for promoting the expression protein of a gene related to bone anabolic metabolism, the combination
  • the composition includes a food composition, a feed composition, or a pharmaceutical composition.
  • the expressed protein of the bone anabolic metabolism-related factor gene includes alkaline phosphatase and / or osteocalcin and the like.
  • the composition of the present invention can be used for animals or humans.
  • the composition may also include conventional feed ingredients in the art.
  • an appropriate amount of an excipient may be included, and the excipient may be an excipient, a diluent, a filler, an absorption enhancer, and the like.
  • the Bifidobacterium lactis of the present invention can be produced according to the food containing Bifidobacterium lactis in the prior art, and the composition can adopt different forms according to the needs of the recipient. Examples include powders, lozenges, granulations, microcapsules, liquid preparations, and the like.
  • the present invention also provides a food, which comprises the Bifidobacterium lactis or the Bifidobacterium lactis preparation; preferably, the food is a fermented dairy product (for example, fermented milk, flavored fermented milk, fermented milk beverage, etc.), cheese, milk-containing beverage, solid beverage or milk powder.
  • a fermented dairy product for example, fermented milk, flavored fermented milk, fermented milk beverage, etc.
  • cheese cheese
  • milk-containing beverage solid beverage or milk powder.
  • the present invention also provides a medicament, which comprises the Bifidobacterium lactis or the Bifidobacterium lactis preparation.
  • a medicament which comprises the Bifidobacterium lactis or the Bifidobacterium lactis preparation.
  • the specific formulation and production method of the medicine can be performed with reference to the medicine containing Bifidobacterium lactis in the prior art.
  • the present invention also provides a feed, which comprises the Bifidobacterium lactis or the Bifidobacterium lactis preparation.
  • a feed which comprises the Bifidobacterium lactis or the Bifidobacterium lactis preparation.
  • the specific formula and production method of the feed can be performed with reference to the feed containing probiotics in the prior art, and the Bifidobacterium lactis or bacteria preparation of the present invention can be added as a probiotic supplement.
  • the suggested dosage of Bifidobacterium lactis BL-99 for animal or human use may be 1.0 ⁇ 10 3 CFU to 1.0 ⁇ 10 10 CFU / kg body weight / day.
  • the recommended dosage for Bifidobacterium lactis BL-99 for animal or human use may be 0.001 ⁇ g to 100 mg / kg body weight / day, more preferably 0.01, based on the weight of the bacteria. ⁇ g ⁇ 10mg / kg body weight / day.
  • the food, medicine or feed of the present invention contains the Bifidobacterium lactis BL-99 and has corresponding effects of regulating the gastrointestinal flora, improving immunity and / or preventing osteoporosis.
  • the present invention also provides a method for regulating the gastrointestinal flora, which method comprises administering to a subject an effective amount of the Bifidobacterium lactis BL-99 or the Bifidobacterium lactis according to the present invention.
  • the regulation of the gastrointestinal flora includes: increasing the number of bifidobacteria and / or lactic acid bacteria in the intestine, inhibiting Desulfovibrio and / or Enterorhabdus in the intestine, and / or inhibiting the pylorus helix Helicobacter and / or Escherichia-Shigella.
  • the present invention also provides a method for improving the body's immune activity, which method comprises administering to a subject an effective amount of the Bifidobacterium lactis BL-99 or the Bifidobacterium lactis preparation.
  • the improvement of the body's immune activity includes: increasing the body's carbon clearance index, increasing the body's half hemolysis value, increasing the body's antibody-producing cells, activating the body's NK cell activity, and / or increasing the body's macrophage phagocytic rate and phagocytic index.
  • the present invention also provides a method for preventing and / or treating osteoporosis, the method comprising administering to a subject an effective amount of the Bifidobacterium lactis BL-99 according to the present invention or the lactobacillus Bifidobacterium preparation.
  • the prevention and / or treatment of osteoporosis includes: reducing the loss of bone mass caused by estrogen deficiency, increasing blood calcium and / or blood phosphorus, inhibiting the number of osteoclasts in the body, and / or promoting bone anabolic metabolism Factor gene expression protein.
  • the expressed protein of the bone anabolic metabolism factor gene includes alkaline phosphatase and / or osteocalcin.
  • the present invention provides a Bifidobacterium lactis BL-99, which is deposited with CGMCC No. 15650.
  • the Bifidobacterium lactis has gastric acid resistance and intestinal fluid resistance, and regulates the stomach.
  • the intestinal flora in particular, it can inhibit Desulfovibrio and / or Enterobacter in the intestine, curatively inhibit Helicobacter pylori and / or Escherichia-Shigella, and can be used for preparing Food, medicine and feed of the bacterial flora; and the bacterium has the ability to enhance humoral immunity and activate NK cells, which can be used to improve the problem of low immune capacity caused by irregular diet and increased work pressure; in addition, the bacterium The fungus has significant effects in preventing and treating osteoporosis. The fungus can be used for food, feed or medical purposes, and has a wide application prospect.
  • FIG. 1A shows the weight change of animals before and after intervention with Bifidobacterium lactis BL-99 in Example 4.
  • FIG. 1A shows the weight change of animals before and after intervention with Bifidobacterium lactis BL-99 in Example 4.
  • FIG. 1B shows changes in uterine weight of animals before and after intervention with Bifidobacterium lactis BL-99 in Example 4.
  • FIG. 2A shows the results of HE staining in Example 4.
  • FIG. 2B shows the change in the percentage of trabecular bone area before and after intervention with Bifidobacterium lactis BL-99 in Example 4.
  • FIG. 3A shows the results of tibia TRAP staining in Example 4.
  • FIG. 3B shows the change of the percentage of osteoclasts on the bone surface (OcS / BS) before and after intervention with Bifidobacterium lactis BL-99 in Example 4.
  • FIG. 3B shows the change of the percentage of osteoclasts on the bone surface (OcS / BS) before and after intervention with Bifidobacterium lactis BL-99 in Example 4.
  • Figures 4A-4D show changes in blood calcium, blood phosphorus, serum osteocalcin, and C-terminal peptide of type I collagen after Bifidobacterium lactis BL-99 intervention.
  • 5A-5E show the effects of B. lactis BL-99 intervention on genes related to bone metabolism regulation.
  • FIG. 6 is a schematic diagram of a fermentation process in a specific embodiment of the present invention.
  • Bifidobacterium lactis BL-99 of the present invention Bifidobacterium lactis BL-99 of the present invention:
  • CGMCC General Microbial Center of China Microbial Collection Management Committee
  • the experimental data is expressed as Mean ⁇ S.E.M.
  • the data is calculated using PRISM version 5.0 (GraphPad, San Diego, CA, USA). Differences between groups were calculated using one-way ANOVA followed Tukery ’s multiple comparison test. There were significant statistical differences at P ⁇ 0.05.
  • Example 1 Bifidobacterium lactis BL-99 and its performance determination
  • the Bifidobacterium lactis BL-99 of the present invention is from Shanghai Jiaotong University Onli Co., Ltd. and is isolated from the intestine of infants.
  • the strain was deposited on April 26, 2018 at the China General Microbial Strain Collection Management Center CGMCC (Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), classified and named: Bifidorum Bifidobacterium lactis; deposit number CGMCC No. 15650.
  • Bifidobacterium is a genus that is generally not acid resistant.
  • the tolerance of artificial gastric juice and intestinal fluid of Bifidobacterium lactis BL-99 of the present invention was tested, and at the same time, Bifidobacterium lactis, which is recognized in the art as having excellent acid resistance and can survive through the gastrointestinal tract As a comparison.
  • Test method Bifidobacterium lactis BL-99 strain was cultured in MRS liquid culture medium at 37 ° C for 16 hours, and then centrifuged at 4 ° C and 2500 rpm for 10 minutes to collect bacterial cells.
  • the test strains were cultured in artificial gastric juice and artificial small intestinal juice, and treated at 37 ° C for 0, 30 min, and 2 h, and the live bacteria count analysis was performed to evaluate the acid and intestinal fluid resistance of the strains by survival rate.
  • Survival rate (the number of viable bacteria after treatment / the number of viable bacteria at time 0) ⁇ 100%.
  • the test results of the survival rate of the strains in artificial gastric acid (pH 2.5) are shown in Table 1.
  • the survival rate of live bacteria was 7.04%, and the survival rate of live bacteria was 2 hours after treatment. Only 1.64%; and the Bifidobacterium lactis BL-99 of the present invention was 62.60% in viable bacteria when treated in artificial gastric acid (pH 2.5) for 30 minutes, and 61.83% in 2 hours. It is shown that the Bifidobacterium lactis BL-99 of the present invention has excellent gastric acid resistance and can smoothly pass through the stomach to the intestine to exert a probiotic effect.
  • the Bifidobacterium lactis BL-99 of the present invention was inoculated in a BBL liquid medium, and anaerobic culture was performed at 36 ⁇ 1 ° C for 48 ⁇ 2 hours, and the number of viable Bifidobacterium lactis BL-99 in the culture solution was 3.7 ⁇ 10 8 cfu / mL, the stock solution of the culture and 5-fold concentrated solution were orally administered to the test mice at 20.0 mL / kg BW for 3 consecutive days and observed for 7 days. The experiment set the medium stock solution and the 5-fold concentrated solution control group.
  • test results showed that the BBL culture stock solution and 5-fold concentrated solution of Bifidobacterium lactis BL-99 had no statistically significant effect on the weight gain of mice compared with the respective control group (p> 0.05), and no observation was made at the same time. The test mice had a toxic reaction or died.
  • B. lactis BL-99 was sensitive to ampicillin Ampicillin, penicillin G, penicillin G, erythromycin Erythromycin, chloramphenicol Chloramphenicol, clindamycin Clindamycin, vancomycin Vancomycin, and tetracycline Tetracycline. Meet the requirements of the European Food Safety Commission (European Food Safety Authority) for the evaluation of bacterial resistance to food bacteria.
  • Bifidobacterium lactis BL-99 does not contain exogenous antibiotic resistance genes and is safe to eat.
  • Example 2 Analysis of intestinal flora regulation effect of active Bifidobacterium lactis
  • This example intends to confirm the effect of the Bifidobacterium lactis of the present invention on intestinal regulation.
  • the principle and procedure refer to "Technical Specification for Inspection and Evaluation of Health Foods-Criteria for Regulating the Function of the Intestinal Microflora”.
  • Bifidobacterium lactis BL-99 strain was cultured in MRS liquid culture medium at 37 ° C for 16 hours, and then centrifuged at 4 ° C and 2500 rpm for 10 min. The cells were collected, washed with phosphate buffered saline (PBS), and freeze-dried. Store below -18 ° C. Used for various experimental studies of this embodiment.
  • PBS phosphate buffered saline
  • mice Thirty-six healthy SPF-grade BABL / c mice were taken and weighed 18-22 g (provided by Beijing Huafukang Biotechnology Co., Ltd.). After 3 days of adaptive breeding, they were randomly divided into 3 groups, 12 in each group, namely the blank control group and the sample group. To each group of animals, sterile water (gavage volume 0.2mL / 10g) in which Bifidobacterium lactis BL-99 bacterial powder was dissolved was administered orally, and the same volume of sterile water was administered to the blank control group. Once a day, feed or gavage continuously for 14 days.
  • Gavage measurement 1.3 ⁇ 10 7 CFU / ml (according to the human requirement is 2 ⁇ 10 9 CFU / d, the conversion factor of human and mouse is 0.0026). Take sterilized centrifuge tubes, number them, collect mouse feces under aseptic conditions after adaptive feeding, and 2-3 capsules each, about 100mg, transfer to aseptic operation room at low temperature for bacterial detection. At the end of the experiment, mouse feces were collected again. After the mice were numbered with picric acid, they were weighed on the 8th and 14th days after the administration of the test substance, and the amount of intragastric administration was calculated. The mice were weighed once at the end of the experiment. Colony count: Prepare a selective medium according to the strain to be identified, the strain to be tested and the corresponding medium are as shown in Table 3, sterilize, shake well, cool to 45 ° -50 ° C and pour the plate for later use.
  • EMB Enterobacter Eosin blue
  • BBL Enterococcus Sodium azide-crystal violet-escin
  • Bifidobacterium BBL agar Lactobacillus LBS agar
  • TSC Tryptone-sulfite-cycloserine
  • mice The collected feces of the mice were placed in a sterilized tube filled with 0.5 mL of physiological saline to prepare a bacterial suspension, and shaken for 1 min before use.
  • Another 0.1mL micropipette pipette tip was taken, and according to this method, a 10-fold gradient dilution was performed until 10-7 g / ml. According to the number of live bacteria of the strain to be identified, two consecutive appropriate dilutions are selected.
  • Each dilution uses a 10 ⁇ L micropipette to pipette 10 ⁇ L of the bacteria suspension, and the surface is coated on a selective agar plate. The culture conditions are shown.
  • the colony counting method is referred to the "GB 4789.2-2010 National Food Safety Standard Food Microbiological Determination of Total Colony Determination”.
  • Tables 6 to 10 record the changes in different intestinal bacteria of the animals before and after the test. It can be seen from Tables 6 to 10 that Bifidobacterium lactis BL-99 can significantly promote the growth of Bifidobacterium and Lactobacillus, and has no significant effect on Enterobacter, Enterococcus, Clostridium perfringens. According to health food inspection and Evaluation technical specifications-the criterion for regulating the function of the intestinal flora can determine that the Bifidobacterium lactis BL-99 in this study has the effect of regulating the intestinal flora.
  • Live bacteria samples According to the sample specifications, weigh 1g of live bacteria samples and suspend to 40ml with PBS solution, that is, the live bacteria concentration is 2.5x10 9 CFU / ml.
  • High dose group calculated in accordance with 0.2ml / 10g mouse gavaged volume, mice fed an amount of 20g 0.4ml gavage dose of high dose group mice 10 9 CFU / 20g.
  • Medium-dose group 5ml high-dose suspension was added to PBS to make up to 50ml. Calculated according to 0.2ml / 10g of mice, 20g mice were given 0.4ml, and medium-dose mice were given 10 8 CFU / 20g.
  • Low-dose group 5ml medium-dose group suspensions were added to PBS to make up to 50ml, calculated according to 0.2ml / 10g of mice, 20g mice were given 0.4ml, and low-dose mice were given 10 7 CFU / 20g.
  • Dead bacteria samples According to the sample specifications, weigh 1g of live bacteria samples and suspend to 40ml with PBS solution, that is, the live bacteria concentration is 2.5x10 9 CFU / ml.
  • the bacterial solution of the live bacteria sample at a concentration of 2.5 ⁇ 10 9 CFU / ml was heat-killed at 100 ° C. for 20 min.
  • the sample allocation method of the high, middle and low dose groups is the same as the above live bacteria group.
  • 6-week-old BABL / c mice were housed in a clean-grade animal room at a temperature of 22 ° C and a humidity of 10-60%, alternating light and dark lighting for 12 hours, standard feed feeding and free drinking water.
  • 182 mice were randomly divided into 13 groups, 14 in each group. The grouping conditions are shown in Table 11.
  • each test substance was administered at 0.2ml / 10g
  • the control group was administered with PBS for 1-14 days
  • the experimental group was administered with the corresponding dosage of test substance according to Table 2.
  • Mice were weighed once a week and the amount of gavage was adjusted according to body weight. After 14 days, the feces of each mouse were collected under aseptic conditions, labeled, and stored at -20 ° C to detect the intestinal flora.
  • BL-99 in the intestinal flora of mice in the probiotic group can significantly increase the relative abundance of Lactobacillus in the intestine of mice, and the low-dose group of BL-99 has a relatively The increase in Lactobacillus was the most significant.
  • the high-dose BL-99 dead bacteria group had significant inhibitory effects on Desulfovibrio and Enterorhabdus.
  • Bifidobacterium lactis BL-99 strain was cultured in MRS liquid culture medium at 37 ° C for 16 hours, and then centrifuged at 4 ° C and 2500 rpm for 10 minutes to collect bacterial cells. After washing with phosphate buffered saline (PBS), freeze-dry, -18 ° C Save the following. Used for various experimental studies of this embodiment.
  • PBS phosphate buffered saline
  • the animals were randomly divided into 5 large groups, which were used for each experimental study in this example. Each large group had 140 mice, and a normal control group was set up in each large group, and one set of each probiotic sample was set up. Dose group.
  • test samples were administered to mice by gavage for 28 days, once a day.
  • the volume of gavage was 0.2mL / 10g, and the control group was given gavage with distilled water.
  • the dose of each test sample to be administered to the mice Based on the probiotic sample information, referring to the daily human demand and the conversion factor for the dosage of 70Kg adults and 20g mice, calculate the dose of each test sample to be administered to the mice.
  • the dose in the BB-12 group is 2.36mg / kg
  • the dose of BL-99 group was 6.34mg / kg.
  • mice were sampled continuously for 28 days, weighed, and Indian ink was injected into the tail vein. At 2 min and 10 min after the ink was injected, 20 ⁇ L of blood was taken, added to a 2M l0.1% sodium carbonate solution, and the OD value was measured at 600 nm. The mice were sacrificed, the liver and spleen were taken, the blood on the surface of the organs was blotted with filter paper, and weighed.
  • mice The ability of mice to clear carbon is expressed by the phagocytic index, which is calculated according to the formula
  • the phagocytosis index results of the carbon clearance experiment are shown in Table 14. The results showed that the BL-99 group was lower than the control group, and the phagocytosis index of the BB-12 mouse carbon clearance test was not significantly different from that of the control group (p> 0.05).
  • mice The initial weight and the weight of the mice were weighed 28 days after the samples were given as the initial weight and the final weight.
  • the mice were sacrificed by dislocation, the spleen and thymus were removed, the fascia was removed, and the blood on the surface of the organ was dried with filter paper. Weight ratio and thymus, weight ratio.
  • each mouse was injected intraperitoneally with 0.2 mL of SRBC for immunization. After 4 days, the eyeballs were removed and blood was collected in a 1.5 mL centrifuge tube, which was left at 4 ° C for about 1 hour to allow the serum to be analyzed, and centrifuged at 2000 r / min for 10 minutes to collect the serum. The serum was diluted 100-fold with SA buffer.
  • the amount of hemolysin is expressed as the half hemolysis value (HC50) and calculated according to the following formula:
  • SRBC sheep red blood cells
  • each mouse was injected intraperitoneally with 0.2 mL of SRBC for immunization.
  • Mice immunized with SRBC for 4 days were sacrificed, and the spleen was removed to prepare a cell suspension of 5 ⁇ 10 6 cells / mL.
  • the agarose is dissolved by heating, it is mixed with an equal amount of double Hank's solution, divided into small test tubes, 0.5mL per tube, and then 20% (V / V, configured with physiological saline) is added to the tube to accumulate 50 ⁇ L of SRBC, and the spleen cells are suspended.
  • the number of antibody-producing cells is shown in Table 17. Compared with the control group, the BL-99 and BB-12 groups were significantly higher than the control group (p ⁇ 0.05), and the BL-99 group showed a higher Significant difference (p> 0.05).
  • the animals were sacrificed for 28 consecutive days, and the mice were sacrificed. After being sterilized in a 75% alcohol beaker, the spleen was aseptically removed and placed in a small dish filled with 4 cm gauze (autoclaved) of 3 cm ⁇ 3 cm. Hank's solution, spleen beads with gauze, gently spleen with elbow to make a single cell suspension, wash twice with Hank's solution, centrifuge at 1000rpm for 10min each time, and then suspend the cells in 2mL of complete In the culture medium, the number of viable cells was counted, and the cell concentration was adjusted to 5 ⁇ 10 6 cells / mL. The cell suspension was divided into two wells and added to a 24-well culture office. Each well was 1mL.
  • each well is divided into 2 wells as a parallel sample, and the optical density value is measured with an enzyme-linked immunoassay detector at a wavelength of 570nm.
  • the ability of lymphocytes to increase value is expressed by the optical density value of the ConA well minus the optical density value of the non-ConA well.
  • the target cell YAC-1 was subcultured 24 hours before the experiment.
  • the cells were washed twice with Hank's solution before use, and the cell concentration was adjusted to 1 ⁇ 10 5 with RPMI1640 complete culture solution containing 10% calf serum.
  • Cells / mL target cells. Sudden death of mouse cervical dislocation, spleen was taken aseptically, made into a skin cell suspension, washed twice with Hank's solution, centrifuged at 1000 rpm for 10 min, and resuspended in 2 mL of RPMI 1640 complete medium containing 10% calf serum.
  • Trypan blue live cell staining count (the number of live cells should be above 95%), adjust the cell concentration to 1 ⁇ 10 7 cells / mL (effect cells), and make the effect target ratio to 100: 1.
  • the target cells will release 100 ⁇ L each of the target cells and the culture medium, and the target cells will release 100 ⁇ L each of the target cells and 1% NP40.
  • Three parallel holes Incubate for 4 h at 37 ° C in a 5% CO 2 incubator. Centrifuge the 96-well culture plate at 1500 rpm for 5 minutes.
  • each rat was injected intraperitoneally with 2% hematocrit SRBC (v / v, prepared with normal saline) SRBC 0.2mL. After sensitization for 4 days, the thickness of the left hind toe was measured, and the same site was measured 3 times. average value. Then, 20% SRBC was injected subcutaneously at the measurement site, 20 ⁇ L, and the thickness of the left hind toe was measured at 24 hours after the injection. The same site was measured 3 times to take the average value. The difference in toe thickness before and after the attack (toe swelling) was used to express DTH. Degree.
  • mice in each group were at the same level. After 24 hours of SRBC, the toes of the mice swelled. The degree of swelling was expressed by the difference between the toe thickness before and after the challenge.
  • the BB-12 and BL-99 groups were significantly higher than the control group (p ⁇ 0.05).
  • mice After 28 consecutive days of animal feeding, each mouse was injected intraperitoneally with 0.2 mL 2% SRBC to activate mouse macrophages 4 days before the end of gavage.
  • the mice were sacrificed by cervical dislocation on the day of the experiment, and Hank's with calf serum was injected intraperitoneally. 3mL / solution, gently rub the abdomen 20 times to fully wash out the peritoneal macrophages, then cut a small opening in the abdominal wall, draw 0.5mL of the peritoneal wash solution, and add it to the agar circle of the slide. Place in an incubator at 37 ° C for 15-20min.
  • the phagocytic rate is the percentage of phagocytic cells that phagocytose chicken red blood cells per 100 phagocytic cells; the phagocytic index is the average number of phagocytic chicken red blood cells per phagocytic cell. The result is calculated as:
  • Bifidobacterium lactis BL-99 strain was cultured in MRS liquid culture medium at 37 ° C for 16 hours, and then centrifuged at 4 ° C and 2500 rpm for 10 minutes to collect bacterial cells. After washing with phosphate buffered saline (PBS), freeze-dry, -18 ° C Save the following. It is used for the experimental study of Examples 2 to 5 of the present invention.
  • PBS phosphate buffered saline
  • Rats There were 85 female 17-week-old adult SD rats weighing 200-300g. Rats were randomly divided into 3 groups of 10 rats each. Twenty rats underwent ovariectomy and the remaining 10 rats underwent sham surgery. Rats were exposed to light / dark for 12h every day, room temperature was about 25 °C, and they were free to drink water. Twelve weeks after the intervention, the animals in the model study group were sacrificed, and samples such as uterus, femur, and tibia were collected, and osteoporosis-related indexes such as uterine coefficient, bone microstructure and bone structure model parameters were measured.
  • test substance was administered by gavage.
  • the course of treatment was once a day for 12 weeks.
  • Ovariectomized rats were fed with BL-99 probiotics, and the sham-operated group and the ovary-removed blank control group were fed with distilled water; the specific administration of the animals was as follows. The specific grouping of animals is shown in Table 22 below.
  • the probiotic BL-99 group can increase the bone trabecular area percentage by about 12.5% (OVX: 16.8 ⁇ 2.5%, OVX + BL-99: 18.9 ⁇ 1.8) (P ⁇ 0.05vs.OVX) ( Figure 2B), suggesting that the probiotic BL-99 can inhibit bone loss caused by estrogen deficiency and has a certain protective effect on bone.
  • Osteoclasts are the main cells responsible for bone resorption in the body and play an important role in bone development, growth, repair, and reconstruction. Osteoclasts are derived from the monocyte-macrophage system, which is a special type of terminally differentiated cells. It can form huge multinucleated cells from their monocyte precursor cells through cell fusion. Osteoclasts correspond functionally to osteoblasts. The two work together to play an important role in the development and formation of bones. Highly expressed tartrate resistant acid phosphatase (TRAP) is one of the main signs of osteoclasts. The tibia TRAP staining results are shown in FIG. 3A. The positive staining results were that the cytoplasm of osteoclasts was stained with wine red.
  • TRAP tartrate resistant acid phosphatase
  • the number of TRAP stained osteoclasts in the OVX blank group rats was significantly higher than that in the sham group. Compared with the OVX blank group rats, the number of TRAP-stained osteoclasts in the probiotic BL-99 group decreased significantly by about 17.9% (OVX: 22.3 ⁇ 1.1%, OVX + BL-99: 18.3 ⁇ 0.6) (P ⁇ 0.05) vs. OVX).
  • In vivo estrogen can inhibit the activity of osteoclasts, and at the same time induce osteoclasts to play an anti-bone resorption role.
  • the biochemical parameters measured included blood calcium, blood phosphorus, serum osteocalcin (OCN), and C-terminal telopeptides of type I collagen (CTX-I).
  • Atomic absorption spectrophotometry was used to determine blood calcium and blood phosphorus, and serum samples were directly determined.
  • Test kits used include: Calcium Test (REF 0155-225), Phosphorus Test (REF 0830-125), the manufacturers are Stanbio Laboratory (NorthMainBoerne, FX, USA) as shown in Figure 4A-4D. Compared with the sham operation group, the blood calcium and phosphorus levels of the OVX blank group were reduced. The reduction in calcium was not statistically significant.
  • the probiotic BL-99 intervention group can significantly increase high serum calcium (OVX: 2.28 ⁇ 0.02mg / dl, OVX + BL-99: 2.49 ⁇ 0.03mg / dl), an increase of about 10 %, And phosphorus ions (OVX: 1.02 ⁇ 0.08 mg / dl, OVX + BL-99: 1.41 ⁇ 0.11 mg / dl), which increased by about 40%.
  • Serum osteocalcin is an active polypeptide secreted by osteoblasts, which plays an important role in regulating bone metabolism, and its level reflects osteoblast activity.
  • Type I collagen C-terminal peptide is a small fragment of type I collagen after degradation, and its content and changes can evaluate the state of bone resorption.
  • Serum osteocalcin and type I collagen C-terminal peptide were measured using the ELASA kit, and the measurement method was performed according to the instructions in the kit.
  • the test kits used are as follows: Rat Osteocalcin ELISA Kit, Rat C-telopeptide of Collagen alpha-1 (I) chain ELISA Kit, the manufacturers are SAB (SABiosciences, USA).
  • the probiotic BL-99 intervention group can significantly increase the serum osteocalcin level by about 44.6% (OVX: 68.6 ⁇ 16.4pg / dl, OVX + BL-99: 92.6 ⁇ 24.3 pg / dl), reducing serum C-terminal peptide of type I collagen by about 14.6%, but there was no statistical significance.
  • the reverse transcription kit After extracting total RNA from bone tissue using Trizol, the reverse transcription kit reverse-transcribes the RNA into cDNA, and then uses different gene primers (such as Table 23) for PCR amplification.
  • the probiotic BL-99 intervention group can significantly increase the gene expression of osteoprotectin (OPG), but has no significant effect on the gene expression of RANKL, thus increasing the OPG / RANKL ratio.
  • RANKL binds to the RANK receptor on the surface of osteoclasts, promotes the differentiation and activation of osteoclasts and inhibits their apoptosis; OPG prevents the binding of RANKL and RANK, thereby preventing the activation of osteoclasts and inhibiting osteoclasts Cell functions reduce bone resorption and play a negative regulatory role.
  • the OPG / RANKL ratio suggests that the level of osteoclasts is regulated in vivo.
  • the BL-99 intervention group can increase osteocalcin by about 1.1 times (OVX: 0.908 ⁇ 0.107, OVX + BL-99: 2.075 ⁇ 0.643) and alkaline Phosphatase gene expression level of about 37.8% (OVX: 0.990 ⁇ 0.217, OVX + BL-99: 1.364 ⁇ 0.513), the expression of these two genes is closely related to the ability of bone formation, so BL-99 intervention can be passed Promote the expression of osteogenic related genes to promote the formation of new bone and antagonize the loss of bone mass caused by OVX.
  • Example 5 Preparation of BL-99 bacterial powder and its use in food production
  • the Bifidobacterium lactis BL-99 (that is, the Bifidobacterium lactis with a deposit number of CGMCC No. 15650) provided by the present invention is anaerobic cultured in a TPY liquid medium.
  • TPY liquid culture medium g / L: hydrolyzed casein 10.0, soybean mash 5.0, yeast powder 2.0, glucose 5.0, L-cysteine 0.5, dipotassium phosphate 2.0, magnesium chloride 0.5, zinc sulfate 0.25, calcium chloride 0.15, ferric chloride 0.0001, Tween 80 1.0, pH 6.5 ⁇ 0.1.
  • the BL-99 bacterial powder prepared in this embodiment can be used for food, feed, or medical purposes.
  • the food may be, for example, ordinary food such as fermented milk, cheese, milk-containing beverage, solid beverage, milk powder, or health food.
  • the recommended dosage of Bifidobacterium lactis BL-99 for human use may be 1.0 ⁇ 10 3 CFU to 1.0 ⁇ 10 10 CFU / kg body weight / day, and more preferably 1.0 ⁇ 10 4 CFU ⁇ 1.0 ⁇ 10 9 CFU / kg body weight / day.
  • the recommended dosage for Bifidobacterium lactis BL-99 for human use may be 0.001 ⁇ g to 100 mg / kg body weight / day, more preferably 0.01 ⁇ g to 10 mg / kg body weight / day, and still more preferably 0.01 ⁇ g to 1 mg / kg. Weight / day.

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Abstract

提供了一种可提高免疫力的乳双歧杆菌(Bifidobacteriumlactis)BL-99及其应用。所述乳双歧杆菌BL-99，于2018年4月26日保藏于中国普通微生物菌种保藏管理中心CGMCC，保藏编号为CGMCC No.15650，其具有耐胃酸和耐肠液性能，能显著促进双歧杆菌和乳酸菌的生长，且能显著提高抗体生成细胞和半数溶血值HC 50，激活NK细胞活性，可以用于制备提高免疫力的食品等，并具有预防骨质疏松、提高血液的钙离子和/或磷离子等功效。

Description

一种乳双歧杆菌BL-99及其应用 技术领域
本发明涉及微生物技术领域，尤其是涉及一株乳双歧杆菌(Bifidobacterium lactis)，其具有耐胃酸和耐肠液性能，能显著促进双歧杆菌和乳酸菌的生长，具有免疫调节活性，并具有预防骨质疏松、提高血液的钙离子和/或磷离子等功效。
背景技术
研究指出益生菌如乳双歧杆菌(Bifidobacterium lactis)具有多种功能，如调节肠道失调、增强肠道免疫功能，抑制过敏反应等。世界卫生组织对益生菌产品的定义为食品中含有充足数量的活的微生物，经过食品加工的各个过程，以及进入人体肠道以后，仍能保持适当的活菌数量和菌活性。因此通常认为菌株在菌粉的制作、产品的生产加工以及经人体胃肠道的胃酸、肠液胁迫后，菌株应能够保持较为稳定的活菌数量是有必要的。
然而，不抗酸、不耐受胃肠液是双歧杆菌属的普遍性能，这将导致双歧杆菌很难通过胃液到达肠道并在肠道中定殖。因此，筛选具有耐胃酸和耐肠液性能的双歧杆菌益生菌是领域中的一个重要研究方向。
发明内容
本发明的一个目的在于提供了一种新的乳双歧杆菌及其用途。
一方面，本发明提供了一种乳双歧杆菌(Bifidobacterium lactis)，其具有耐胃酸性能，在pH2.5的胃酸液中处理30min时活菌存活率62％以上，处理2小时活菌存活率61％以上。本发明提供的乳双歧杆菌(Bifidobacterium lactis)还具有耐肠液性能，在pH6.8的小肠液中处理2小时活菌存活率70％以上。本发明中将所提供的乳双歧杆菌(Bifidobacterium lactis)命名为BL-99。该菌株已于2018年04月26日保藏于中国普通微生物菌种保藏管理中心CGMCC(地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所)，分类命名：乳双歧杆菌(Bifidobacterium lactis)；保藏编号为CGMCC No.15650。
本发明的研究发现，本发明的乳双歧杆菌BL-99(即保藏编号为CGMCC No.15650的乳双歧杆菌)单菌即具有显著促进肠道双歧杆菌和乳酸菌增长的能力，能够抑制肠道中脱硫弧菌和/或肠杆菌属的生长，特别是可治病性抑制幽门螺旋杆菌和/或埃希氏菌-志贺菌属的生长，具有显著的调节胃肠道菌群功效。
并且，本发明的乳双歧杆菌BL-99(即保藏编号为CGMCC No.15650的乳双歧杆菌) 可以用于增强机体的免疫反映，能用于改善由于饮食不规律和工作压力增加而引发的免疫能力低下等问题。具体包括：(1)能够提高小鼠碳廓清指数；(2)提高小鼠半数溶血值；(3)提高小鼠抗体生成细胞数；(4)激活NK细胞活性；(5)迟发性免疫反应阳性；(6)巨噬细胞吞噬率与吞噬指数增加。
此外，本发明的研究发现，本发明的乳双歧杆菌BL-99可以用于治疗或预防骨质疏松(现有技术中鲜有关于乳双歧杆菌用于治疗和/或预防骨质疏松的技术报道)，并具有提高血液的钙离子和/或磷离子浓度等功效，具体包括：(1)显著降低雌激素缺乏引起的骨量的丢失；(2)提升血钙和血磷浓度；(3)通过调节OPG/RANKL的比例，来抑制体内破骨细胞的数量，抑制其对骨的吸收作用；(4)通过促进骨合成代谢相关因子基因的表达蛋白，如碱性磷酸酶和骨钙素的表达，提高其蛋白的水平从而来促进新骨的形成。
本发明的乳双歧杆菌BL-99，其可在领域中常用的乳双歧杆菌培养基(例如TPY培养基、BBL培养基等)中厌氧发酵培养。最佳发酵温度35～38℃，最佳发酵时间7～24h。本发明同时提供了一种乳双歧杆菌BL-99菌制剂的制作方法，其是将所述的菌株在液态发酵培养基中厌氧培养，获得包含菌体的发酵液。发酵液可直接或进一步浓缩后作为液态菌制剂，也可将发酵液干燥后制备菌粉，或是从发酵液中分离出菌体制备菌粉。本发明的液态菌制剂也可以是将菌体重悬于培养液、缓冲液或去离子水等溶剂后的液态制剂。本发明的BL-99液态菌制剂或固态菌制剂(菌粉)可以以活菌形式保存，在保藏期具有较好的稳定性。本发明的BL-99液态菌制剂或固态菌制剂(菌粉)也可以以灭活的死菌形式保存。所述的菌制剂可以用于食品、饲料或药品的生产。小鼠实验表明该菌株无口服急性毒性，无抗生素耐受，安全可以用于食品加工。
从而，一方面，本发明提供了一种乳双歧杆菌(Bifidobacterium lactis)，其保藏编号为CGMCC No.15650。
另一方面，本发明还提供了一种乳双歧杆菌菌制剂，该菌制剂中含有本发明所述的乳双歧杆菌，为固态或液态菌制剂。本发明的乳双歧杆菌菌制剂，可以为活菌形式或灭活形式。
另一方面，本发明还提供了所述的乳双歧杆菌或所述的乳双歧杆菌菌制剂在制备具有调节肠道菌群功效的组合物中的应用，所述组合物包括食品组合物、饲料组合物或药品组合物。
另一方面，本发明还提供了所述的乳双歧杆菌或所述的乳双歧杆菌菌制剂在制备具 有增加肠道中双歧杆菌和/或乳酸菌数量的功效的组合物中的应用，所述组合物包括食品组合物、饲料组合物或药品组合物。
另一方面，本发明还提供了所述的乳双歧杆菌或所述的乳双歧杆菌菌制剂在制备具有抑制肠道中脱硫弧菌(Desulfovibrio)和/或肠杆菌属(Enterorhabdus)功效的组合物中的应用，所述组合物包括食品组合物、饲料组合物或药品组合物。
另一方面，本发明还提供了所述的乳双歧杆菌或所述的乳双歧杆菌菌制剂在制备具有抑制幽门螺旋杆菌(Helicobacter)和/或埃希氏菌-志贺菌属(Escherichia-Shigella)功效的组合物中的应用，所述组合物包括食品组合物、饲料组合物或药品组合物。
另一方面，本发明还提供了所述的乳双歧杆菌或所述的乳双歧杆菌菌制剂在制备用于提高机体免疫力活性的组合物中的应用，所述组合物包括食品组合物、饲料组合物或药品组合物。
另一方面，本发明还提供了所述的乳双歧杆菌或所述的乳双歧杆菌菌制剂在制备用于提高机体碳廓清指数的组合物中的应用，所述组合物包括食品组合物、饲料组合物或药品组合物。
另一方面，本发明还提供了所述的乳双歧杆菌或所述的乳双歧杆菌菌制剂在制备用于提高机体半数溶血值的组合物中的应用，所述组合物包括食品组合物、饲料组合物或药品组合物。
另一方面，本发明还提供了所述的乳双歧杆菌或所述的乳双歧杆菌菌制剂在制备用于提高机体抗体生成细胞数的组合物中的应用，所述组合物包括食品组合物、饲料组合物或药品组合物。
另一方面，本发明还提供了所述的乳双歧杆菌或所述的乳双歧杆菌菌制剂在制备用于激活机体NK细胞活性的组合物中的应用，所述组合物包括食品组合物、饲料组合物或药品组合物。
另一方面，本发明还提供了所述的乳双歧杆菌或所述的乳双歧杆菌菌制剂在制备用于增加机体巨噬细胞吞噬率与吞噬指数的组合物中的应用，所述组合物包括食品组合物、饲料组合物或药品组合物。
另一方面，本发明还提供了所述的乳双歧杆菌或所述的乳双歧杆菌菌制剂在制备用于防治骨质疏松的组合物中的应用，所述组合物包括食品组合物、饲料组合物或药品组合物。
另一方面，本发明还提供了所述的乳双歧杆菌或所述的乳双歧杆菌菌制剂在制备用于降低雌激素缺乏引起的骨量的组合物中的应用，所述组合物包括食品组合物、饲料组 合物或药品组合物。
另一方面，本发明还提供了所述的乳双歧杆菌或所述的乳双歧杆菌菌制剂在制备用于提升血钙和/或血磷的组合物中的应用，所述组合物包括食品组合物、饲料组合物或药品组合物。
另一方面，本发明还提供了所述的乳双歧杆菌或所述的乳双歧杆菌菌制剂在制备用于抑制体内破骨细胞数量的组合物中的应用，所述组合物包括食品组合物、饲料组合物或药品组合物。
另一方面，本发明还提供了所述的乳双歧杆菌或所述的乳双歧杆菌菌制剂在制备用于促进骨合成代谢相关因子基因的表达蛋白的组合物中的应用，所述组合物包括食品组合物、饲料组合物或药品组合物。具体地，所述骨合成代谢相关因子基因的表达蛋白包括碱性磷酸酶和/或骨钙素等。
本发明的组合物可使用于动物或是人类。所述组合物还可包括所属领域中的常规用料组分。例如，对于药物组合物，可包括适量的辅料，所述辅料可以为赋型剂、稀释剂、填充剂、吸收促进剂等。对于食品组合物，本发明的乳双歧杆菌可以按照现有技术中含乳双歧杆菌的食品进行生产，所述组合物可根据受施予者的需要，而采用不同形态。例如粉剂、锭剂、造粒、微胶囊、液体制剂等。
在本发明的一些具体实施方案中，本发明还提供了一种食品，其中包含所述的乳双歧杆菌或所述的乳双歧杆菌菌制剂；优选地，所述食品为发酵乳制品(例如发酵乳、风味发酵乳、发酵乳饮料等)、乳酪、含乳饮料、固体饮料或乳粉。食品的具体配方和生产方法可参照现有技术中含乳双歧杆菌的食品进行。
在本发明的另一些具体实施方案中，本发明还提供了一种药物，其中包含所述的乳双歧杆菌或所述的乳双歧杆菌菌制剂。药物的具体配方和生产方法可参照现有技术中含乳双歧杆菌的药物进行。
在本发明的另一些具体实施方案中，本发明还提供了一种饲料，其中包含所述的乳双歧杆菌或所述的乳双歧杆菌菌制剂。饲料的具体配方和生产方法可参照现有技术中含益生菌的饲料进行，本发明的乳双歧杆菌或菌制剂作为益生菌补充剂添加即可。
优选地，在所述食品、药物或饲料中，乳双歧杆菌BL-99供动物或人类使用的建议剂量可为1.0×10 3CFU～1.0×10 10CFU/kg体重/天。或者，在所述食品、药物或饲料中，乳双歧杆菌BL-99以菌体的重量计，供动物或人类使用的建议剂量可为0.001μg～100mg/kg体重/天，更优选为0.01μg～10mg/kg体重/天。本发明的食品、药物或饲料，因包含所述的乳双歧杆菌BL-99而具有相应的调节胃肠道菌群、提高免疫力和/或防治骨质疏松的 功效。
另一方面，本发明还提供了一种调节胃肠道菌群的方法，该方法包括给予受试者有效量的本发明所述的乳双歧杆菌BL-99或所述的乳双歧杆菌菌制剂。具体地，所述调节胃肠道菌群包括：增加肠道中双歧杆菌和/或乳酸菌数量，抑制肠道中脱硫弧菌(Desulfovibrio)和/或肠杆菌属(Enterorhabdus)，和/或抑制幽门螺旋杆菌(Helicobacter)和/或埃希氏菌-志贺菌属(Escherichia-Shigella)。
另一方面，本发明还提供了一种提高机体免疫力活性的方法，该方法包括给予受试者有效量的本发明所述的乳双歧杆菌BL-99或所述的乳双歧杆菌菌制剂。具体地，所述提高机体免疫力活性包括：提高机体碳廓清指数，提高机体半数溶血值，提高机体抗体生成细胞数，激活机体NK细胞活性，和/或增加机体巨噬细胞吞噬率与吞噬指数。
另一方面，本发明还提供了一种预防和/或治疗骨质疏松的方法，该方法包括给予受试者有效量的本发明所述的乳双歧杆菌BL-99或所述的乳双歧杆菌菌制剂。具体地，所述预防和/或治疗骨质疏松包括：降低雌激素缺乏引起的骨量的丢失，提升血钙和/或血磷，抑制体内破骨细胞数量，和/或促进骨合成代谢相关因子基因的表达蛋白。优选地，所述骨合成代谢相关因子基因的表达蛋白包括碱性磷酸酶和/或骨钙素。
综上所述，本发明提供了一种乳双歧杆菌BL-99，其保藏编号为CGMCC No.15650的乳双歧杆菌，该乳双歧杆菌具有耐胃酸和耐肠液性能，并具有调节胃肠道菌群功效，特别是能够抑制肠道中脱硫弧菌和/或肠杆菌属，治病性抑制幽门螺旋杆菌和/或埃希氏菌-志贺菌属，可以用于制备具有调节肠道菌群的食品、药物及饲料等；并且，该菌具有增强体液免疫、激活NK细胞的能力，能用于改善由于饮食不规律和工作压力增加而引发的免疫能力低下等问题；此外，该菌具有显著的防治骨质疏松的功效，该菌可用于食品、饲料或医药用途，具有广泛的应用前景。
附图说明
图1A显示实施例4中乳双歧杆菌BL-99干预前后动物的体重变化。
图1B显示实施例4中乳双歧杆菌BL-99干预前后动物子宫重量变化。
图2A显示实施例4中HE染色结果。
图2B显示实施例4中乳双歧杆菌BL-99干预前后骨小梁面积百分率的变化。
图3A显示实施例4中胫骨TRAP染色结果。
图3B显示实施例4中乳双歧杆菌BL-99干预前后破骨细胞占骨表面百分比(OcS/BS)的变化。
图4A-图4D分别显示乳双歧杆菌BL-99干预后血钙、血磷、血清骨钙素、I-型胶原C端肽变化。
图5A-图5E显示乳双歧杆菌BL-99干预对调节骨代谢相关基因的影响。
图6为本发明一具体实施例中的发酵工艺流程示意图。
用于专利程序的微生物保存：
本发明的乳双歧杆菌BL-99：
保藏日期：2018年04月26日；
保藏单位：中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)；
保藏单位地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所
保藏编号：CGMCC No.15650；
分类命名：乳双歧杆菌(Bifidobacterium lactis)。
具体实施方式
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现结合具体实施例及对本发明的技术方案进行以下详细说明，应理解这些实例仅用于说明本发明而不用于限制本发明的范围。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
除非另外专门定义，本文使用的所有技术和科学术语都与相关领域普通技术人员的通常理解具有相同的含义。除非另有说明，本发明中使用的所有表示成分、细胞培养、处理条件等的量的数字应当理解为在所有条件下受到术语“约”的修饰。因此，除非另有相反的说明，数值参数为近似值，并且可以根据通过本发明试图获得的期望特性而变化。除非另有说明，一系列元素之前的术语“至少”应当理解为指该系列中的每个元素。
本发明的各实施例中，实验数据表示为Mean±S.E.M.数据采用PRISM version 5.0(GraphPad,San Diego,CA,USA)进行统计。组间差异采用one-way ANOVA跟随Tukery’s multiple comparison test的方法进行统计。P<0.05时具有显著的统计学差异。
实施例1：乳双歧杆菌BL-99及其性能测定
本发明的乳双歧杆菌BL-99，来自上海交大昂立股份有限公司，是自婴儿肠道中分离得到的。该菌株已于2018年04月26日保藏于中国普通微生物菌种保藏管理中心CGMCC(地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所)，分类命名：乳双歧杆菌(Bifidobacterium lactis)；保藏编号为CGMCC No.15650。
1.乳双歧杆菌BL-99的分类学特征
理化试验结果:
Figure PCTCN2019107392-appb-000001
16S rRNA基因序列测序结果(SEQ ID No.1)：
Figure PCTCN2019107392-appb-000002
2.乳双歧杆菌BL-99的人工胃液、肠液耐受性
双歧杆菌为通常不抗酸的菌属。本实施例中，测试了本发明的乳双歧杆菌BL-99的人工胃液、肠液耐受性，同时以目前领域中公认耐酸性能极好、可以通过胃肠道存活的乳双歧杆菌
Figure PCTCN2019107392-appb-000003
作为对比。
测试方法：将乳双歧杆菌BL-99菌株于MRS液体培养基中37℃培养16小时后，于4℃、2500rpm下离心10min，收集菌体。
将待测菌株分别在人工胃液、人工小肠液中培养，37℃处理0、30min、2h后进行活菌计数分析，以存活率评价菌株的耐酸及耐肠液性能。存活率＝(处理后的活菌数/0时刻活菌数)×100％。
菌株在人工胃酸(pH2.5)中的存活率检测结果如表1所示，BB-12在人工胃酸(pH2.5)中处理30min时活菌存活率7.04％，处理2小时活菌存活率仅1.64％；而本发明的乳双歧杆菌BL-99在人工胃酸(pH2.5)中处理30min时活菌存活率62.60％，处理2小时活菌存活率61.83％。表明本发明的乳双歧杆菌BL-99具有优异的耐胃酸能力，能较为顺利地通过胃到达肠道发挥益生作用。
表1、菌株在人工胃酸(pH2.5)中的存活率
Figure PCTCN2019107392-appb-000004
菌株在人工小肠液(pH6.8)中的存活率检测结果参见表2。数据显示，BB-12在人工小肠液(pH6.8)中处理2小时活菌存活率仅28.95％；而本发明的乳双歧杆菌BL-99在人工胃酸(pH2.5)中处理2小时活菌存活率70.23％。表明本发明的乳双歧杆菌BL-99具有优异的耐肠液能力，可以在肠道内存活并定殖。
表2、菌株在人工小肠液(pH6.8)中的存活率
Figure PCTCN2019107392-appb-000005
3.乳双歧杆菌BL-99的的毒力实验及安全性检测
将本发明的乳双歧杆菌BL-99接种于BBL液体培养基中，36±1℃厌氧培养48±2小时，计数培养液中乳双歧杆菌BL-99活菌数为3.7×10 8cfu/mL，将培养物的原液和5倍浓缩液，经口以20.0mL/kg BW给受试小鼠连续灌胃3天，观察7天。试验设培养基原液和5倍浓缩液对照组。试验结果表明：乳双歧杆菌BL-99的BBL培养物原液和5倍浓缩液 组与各自对照组相比，对小鼠体重增长的影响无统计学意义(p＞0.05)，同时未观察到受试小鼠有毒性反应或死亡。
采用SN/T 1944-2007《动物及其制品中细菌耐药性的测定》方法，评估乳双歧杆菌BL-99的抗生素敏感性能。评价结果显示，乳双歧杆菌BL-99对氨苄西林Ampicillin、青霉素G PenicillinG、红霉素Erythromycin、氯霉素Chloramphenicol、克林霉素Clindamycin、万古霉素Vancomycin和四环素Tetracycline等敏感。符合欧洲食品安全委员会(European Food Safety Authority)对食用细菌耐药性评价规范中的要求。乳双歧杆菌BL-99不含外源抗生素耐药基因，食用安全。
实施例2：活性乳双歧杆菌肠道菌群调节效果分析
本实施例意图证实本发明的乳双歧杆菌在肠道调节方面的效果，其原理和步骤参见“保健食品检验与评价技术规范-调节肠道菌群功能判定标准”。
将乳双歧杆菌BL-99菌株于MRS液体培养基中37℃培养16小时后，于4℃、2500rpm下离心10min，收集菌体，磷酸盐缓冲液(PBS)洗涤后冷冻干燥，所得菌粉-18℃以下保存。用于本实施例的各项实验研究。
取36只健康SPF级BABL/c小鼠，体重18-22g(由北京华阜康生物科技有限责任公司提供)。适应性饲养3天后，将其随机分为3组，每组12只，即空白对照组，样品组。向每组动物分别灌胃溶解了乳双歧杆菌BL-99菌粉的无菌水(灌胃体积0.2mL/10g)，空白对照组灌胃相同体积的无菌水。每天1次，连续饲喂或灌胃14天。灌胃计量：1.3×10 7CFU/ml(按人体需要量为2×10 9CFU/d，人体和小鼠换算系数0.0026进行换算)。取灭菌离心管，编号，适应性喂养后于无菌条件下采集小鼠粪便，每只2-3粒，约100mg，低温条件转移至无菌操作间进行菌群的检测。实验结束时，再次采集小鼠粪便。使用苦味酸对小鼠进行分组编号后，分别于给予受试物第8天、第14天称重，计算小鼠灌胃量，实验结束时称重1次。菌落计数：根据待鉴定菌种配制选择性培养基，待测菌种及相应培养基如表3，灭菌，摇匀，冷至45℃-50℃倾注平板，备用。
表3、待测菌种及对应选择性培养基
待测菌种 选择性培养基
肠杆菌 伊红美蓝(EMB)琼脂
肠球菌 叠氮钠-结晶紫-七叶苷琼脂
双歧杆菌 BBL琼脂
乳酸杆菌 LBS琼脂
产气荚膜梭菌 胰胨-亚硫酸盐-环丝氨酸(TSC)琼脂
将采集的小鼠粪便，置于装有0.5mL生理盐水的灭菌管中，配制成菌悬液，使用前 震荡1min。使用0.1mL微量移液器吸取0.1mL菌悬液，缓慢注于0.9mL灭菌生理盐水中，震荡或反复吹打使其混匀，制成1:10的菌悬液。另取0.1mL微量移液器吸头，依此方法，进行10倍梯度稀释，直至10-7g/ml。根据待鉴定菌种的活菌数，选择两个连续的适宜稀释度，每个稀释度使用10μL微量移液器吸取10μL菌悬液，在选择性琼脂平板上进行表面涂布，按表4所示培养条件进行培养。菌落计数方法参考《GB 4789.2-2010食品安全国家标准食品微生物学检验菌落总数测定》进行。
表4、肠道菌群检验用培养基及鉴定方法
项目 培养基 培养条件
肠杆菌 伊红美蓝琼脂 36℃±1℃培养24h
肠球菌 叠氮钠-结晶紫-七叶苷琼脂 36℃±1℃培养48h
双歧杆菌 BBL琼脂 36℃±1℃培养48h，厌氧培养
乳酸菌 LBs琼脂 36℃±1℃培养48h
产气荚膜梭菌 TSC琼脂 36℃±1℃培养24h，厌氧培养
采用SPSS17.0进行数据统计。比较实验前后自身及组间双歧杆菌、乳酸菌、肠球菌、肠杆菌的变化情况，试验组实验前后自身比较变化有显著性，符合以下任意一项，可以判定该受试样品动物实验结果阳性。第一、粪便中双歧杆菌或者乳酸菌明显增加，梭菌减少或无明显变化，肠球菌、肠杆菌无明显变化。第二、粪便中双歧杆菌或者乳酸菌明显增加，梭菌减少或无明显变化，肠球菌、肠杆菌明显增加，但增加的幅度低于双歧杆菌或者乳酸菌增加的幅度。
实验期间动物的体重变化结果如表5所示。在实验期内，动物表征正常，给予受试物后未出现任何不良反应，实验周期内，两组动物体重并未出现显著差异。
表5、动物的体重变化
编号 分组 动物数 初始体重(g) 中期体重(g) 终末体重(g)
1 对照组 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
表6～表10分别记录了受试前后动物肠道不同菌的变化。从表6～表10可以看出，乳双歧杆菌BL-99能显著促进双歧杆菌和乳酸菌的生长，对于肠杆菌、肠球菌、产气荚膜梭菌无显著影响，根据保健食品检验与评价技术规范-调节肠道菌群功能判定标准可以判定，本研究中乳双歧杆菌BL-99具有调节肠道菌群的功效。
表6、受试前后动物肠道双歧杆菌的变化(LgCFU/g)
Figure PCTCN2019107392-appb-000006
表7、受试前后动物肠道乳杆菌的变化(LgCFU/g)
Figure PCTCN2019107392-appb-000007
表8、受试前后动物肠道肠杆菌的变化(LgCFU/g)
Figure PCTCN2019107392-appb-000008
表9、受试前后动物肠道肠球菌的变化(LgCFU/g)
Figure PCTCN2019107392-appb-000009
表10、受试前后动物肠道荚膜梭菌的变化(LgCFU/g)
Figure PCTCN2019107392-appb-000010
不同剂量活性乳双歧杆菌及灭活菌种的肠道菌群调节效果比较
分别检测不同剂量活性乳双歧杆菌BL-99及灭活菌种的肠道菌群调节效果。
活菌样品：依据样品规格，称取1g活菌样品，使用PBS溶液悬浮至40ml，即活菌浓度均为2.5x10 9CFU/ml。
高剂量组：按照小鼠0.2ml/10g灌胃量计算，20g小鼠灌胃量为0.4ml，高剂量组小鼠灌胃剂量为10 9CFU/20g。
中剂量组：分别取5ml高剂量悬浮液加入PBS定容至50ml，按照小鼠0.2ml/10g灌胃量计算，20g小鼠灌胃量为0.4ml，中剂量组小鼠灌胃剂量为10 8CFU/20g。
低剂量组：分别取5ml中剂量组悬浮液加入PBS定容至50ml，按照小鼠0.2ml/10g灌胃量计算，20g小鼠灌胃量为0.4ml，低剂量组小鼠灌胃剂量为10 7CFU/20g。
死菌样品：依据样品规格，称取1g活菌样品，使用PBS溶液悬浮至40ml，即活菌浓度为2.5x10 9CFU/ml。将浓度为2.5x10 9CFU/ml的活菌样品菌液100℃热杀死20min。 其高、中、低剂量组样品配置方法与上述活菌组相同。
6周龄BABL/c小鼠饲养于清洁级动物房，温度22℃，湿度10-60％，12小时明暗交替照明，标准饲料喂养，自由饮水。适应性喂养5天，182只小鼠随机分为13组，每组14只，分组情况见表11。
表11、调节肠道菌群实验分组
Figure PCTCN2019107392-appb-000011
开始灌胃前，无菌条件下采集每只小鼠粪便，标记，-20℃保存，检测肠道菌群。实验按照0.2ml/10g灌胃量给予各受试物，对照组1-14天给予PBS，实验组分别依据表2灌胃给予相应剂量的受试物。小鼠每周称重一次，根据体重调整灌胃量。14天后无菌条件下采集每只小鼠粪便，标记，-20℃保存，检测肠道菌群。
实验前后，各组小鼠体重无显著差异。在门的水平，补充不同剂量的益生菌后，小鼠肠道菌群中厚壁菌门(Firmicutes)相对丰度增加而拟杆菌门(Bacteroidetes)、变形菌门(Proteobacteria)相对丰度降低。研究表明，厚壁菌门与拟杆菌门的比值与人体肠道疾病具有较强的相关性，肥胖病人倾向于二者比值降低。而肠炎、肠应激综合症患者则倾向于具有较高的变形菌门(Proteobacteria)丰度。
BL-99在属的水平对肠道菌群的影响参见表12。
表12、BL-99对肠道菌群的影响
Figure PCTCN2019107392-appb-000012
在属的水平，控制组相比，益生菌组小鼠肠道菌群中BL-99可显著增加小鼠肠道中的乳酸杆菌属(Lactobacillus)相对丰度，且BL-99低剂量组对乳酸杆菌(Lactobacillus)的增加程度最为显著。而BL-99死菌高剂量组对脱硫弧菌(Desulfovibrio)、肠杆菌属(Enterorhabdus)具有显著抑制作用。
BL-99对致病菌幽门螺杆菌、埃希氏菌-志贺氏菌属的抑制效果参见表13。
表13、BL-99对致病菌的抑制效果
Figure PCTCN2019107392-appb-000013
对致病菌进行分析，结果同样表明，BL-99低剂量组对幽门螺旋杆菌(Helicobacter)具有显著抑制作用，所有组别均对埃希氏菌-志贺菌属(Escherichia-Shigella)具有抑菌作用，且死菌效果更优。
上述实验表明，补充低剂量组的BL-99即可以调节肠道菌群平衡，促进有益菌生长，抑制有害菌甚至是致病菌，且死菌同样有效，表明补充低剂量组的BL-99或BL-99死菌既可以起到潜在的健康的功效。
实施例3：免疫调节活性分析
将乳双歧杆菌BL-99菌株于MRS液体培养基中37℃培养16小时后，于4℃、2500rpm下离心10min，收集菌体，磷酸盐缓冲液(PBS)洗涤后冷冻干燥，-18℃以下保存。用于本实施例的各项实验研究。
健康雄性BALB/C小鼠700只，6-8周龄，16-18g，由北京维通利华实验动物技术有限公司提供。饲养于中国疾病预防控制中心职业卫生与中毒控制所动物实验室：维持室温(25±2℃)，相对湿度(55±2)％，12h/12h光照，自由进食和饮水。
动物随机分为5个大组，分别用于本实施例的各项试验研究，每个大组140只小鼠，每个大组中设立1个正常对照组，每种益生菌样品设立1个剂量组。
以灌胃的方式给予小鼠受试样品28天，每天灌胃一次，灌胃体积为0.2mL/10g，对照组灌胃给予蒸馏水。
依据益生菌样品信息，参考每日人体需求量及70Kg成人与20g小鼠使用剂量的换算系数，计算出需给予小鼠的各受试样品的剂量，BB-12组的剂量为2.36mg/kg，BL-99组的剂量为6.34mg/kg。
1.单核-巨噬细胞功能
1.1碳廓清实验
动物连续给样28天，称体重，尾静脉注射印度墨汁，注入墨汁后第2min及第10min，取血20μL，加入2M的l0.1％碳酸钠溶液中，600nm处测定OD值。将小鼠处死，取肝脏和脾脏，用滤纸吸干脏器表面血污，称重。
以吞噬指数表示小鼠碳廓清的能力，按照公式进行计算
Figure PCTCN2019107392-appb-000014
Figure PCTCN2019107392-appb-000015
碳廓清实验的吞噬指数结果见表14。结果显示，BL-99组低于对照组，BB-12的小鼠碳廓清实验的吞噬指数与对照组相比无显著差异(p＞0.05)。
表14、碳廓清实验中吞噬指数结果
组次 动物数(只) 吞噬指数 与对照组相比较的p值
对照 8 7.64±0.62 --
BB-12组 8 7.56±0.61 0.902
BL-99组 9 6.43±0.55 0.039*
1.2脏器、体重比值测定
将小鼠初始体重和给样28天后称重分别作为初始体重和终体重，小鼠脱臼处死，取脾脏和胸腺，去尽筋膜，用滤纸吸干脏器表面血污，称重，计算脾脏、体重比值和胸腺、体重比值。
结果见表15。给予受试样品后，与对照组相比，BL-99与BB-12组脾脏/体重比值、胸腺/体重比值与对照组均无显著性差异，说明样品BL-99与BB-12对小鼠脾脏/胸腺未造成影响。
表15、小鼠脏器/体重比值变化
组别 动物数(只) 脾脏/体重比值(mg/g) p值 胸腺/体重比值(mg/g) p值
对照 14 13.46±0.56 -- 10.25±0.61 --
BB-12组 14 13.01±0.52 0.100 10.22±0.49 0.875
BL-99组 14 13.60±0.65 0.614 10.40±0.36 0.418
2.体液免疫实验
2.1血清溶血素半数溶血值(HC 50)的测定
动物连续给样28天后，经腹腔对每只小鼠注射SRBC0.2mL进行免疫。4天后，摘除眼球取血于1.5mL离心管内，4℃放置约1h，使血清充分析出，2000r/min离心10min 收集血清。取血清用SA缓冲液稀释100倍。将稀释后的血清加入96孔板，每孔100μL，再依次加入10％(v/v)SRBC50μL，补体100μL(用SA溶液按1:8稀释)，置37℃恒温水浴中保温30min，1500r/min离心10min。然后样品孔和空白对照孔各取上清液50μL，加入另一个96孔培养板内，加文奇氏试剂150μL，同时设半数溶血孔，加入10％(v/v)SRBC12.5μL，再加文奇氏试剂至200μL，用震荡期充分混匀，放置10min后，与540处用全自动酶标仪测定各孔光密度值。
溶血素的量以半数溶血值(HC50)表示，按以下公式计算：
Figure PCTCN2019107392-appb-000016
结果见表16。从表16可以看出，与对照组相比，BL-99与BB-12组半数溶血值HC50均有升高(p＜0.05)，且BL-99组高于BB-12组。
表16、半数溶血值HC 50结果
组别 动物数(只) HC 50 p值
对照 14 51.07±2.17 --
BB-12组 14 66.31±3.66 0.000 **
BL-99组 14 67.43±4.04 0.000 **
2.2抗体生成细胞检测实验
经过绵羊红细胞(SRBC)免疫的小鼠脾细胞悬液与一定量的SRBC混合，在补体参与下，使分泌抗体的脾细胞周围的SRBC溶解，形成肉眼可见的空斑，溶血空斑数可反映抗体生成细胞数。
动物连续给样28天后，经腹腔对每只小鼠注射SRBC0.2mL进行免疫。将SRBC免疫4天后的小鼠处死，取脾，制成5×10 6个细胞/mL的细胞悬液。将琼脂糖加热溶解后，与等量双倍的Hank’s液混合，分装小试管，每管0.5mL，再向管内加20％(V/V，用生理盐水配置)压积SRBC50μL，脾细胞悬液200μL，迅速混匀，倾倒至已刷琼脂糖薄层的六孔板上，带琼脂凝固后，放入二氧化碳培养箱中继续孵育1h，然后加入SA缓冲液稀释的补体(1:10)，继续孵育2h，计数溶血空斑数。
抗体生成细胞数结果如表17所示，样品组与对照组相比，BL-99与BB-12组显著高于对照组(p＜0.05)，且BL-99组与对照组相比呈现出显著性差异(p＞0.05)。
表17、抗体生成细胞数结果
组别 动物数(只) 溶血空斑数/10 6脾细胞 p值
对照 11 18.64±1.91 --
BB-12组 10 23.30±3.13 0.003**
BL-99组 9 28.67±2.87 0.000**
3.NK细胞活性试验
3.1ConA诱导的小鼠淋巴细胞转化实验
动物连续给样28天后，将小鼠处死，在75％酒精的烧杯中消毒后，无菌取脾，置于装有3cm×3cm四层纱布(高压灭菌)的小平皿中，加入适量无菌Hank’s液，用纱布将脾宝珠，用弯头蹑轻轻地将脾磨碎，制成单细胞悬液，用Hank’s液洗2次，每次1000rpm离心10min，然后将细胞悬浮于2mL的完全培养液中，计数活细胞数，调整细胞浓度为5×10 6个/mL。再将细胞悬液分两孔加入24孔培养办中，每孔1mL，在其中一孔加入75μLConA液(相当于7.5μg/mL)，另一孔作为对照，置于5％CO2，37℃培养72h。培养结束前4h，每孔轻轻吸去上清液0.7mL不含小牛血清的RPMI 1640培养液，同时加入MTT(5mg/mL)50μL/孔，继续培养4h。培养结束后，每孔加入1mL酸性异丙醇，吹打均匀，使紫色结晶完全溶解。然后分装到96孔培养板中，每个孔分装2孔作为平行样，用酶联免疫检测仪，以570nm波长测定光密度值。淋巴细胞的增值能力用加ConA孔的光密度值减去不加ConA孔的光密度值表示。
结果见表18。由表18可以得出，与对照组相比，BB-12组显著高于对照组(p＜0.05)，而BL-99与对照组相比无显著性变化(p＞0.05)。
表18、小鼠脾淋巴细胞转化实验结果
Figure PCTCN2019107392-appb-000017
3.2NK细胞活性测定
动物连续给样28天，开始实验前24h将靶细胞YAC-1进行传代培养，用前以Hank’s液洗2次，用含10％小牛血清的RPMI1640完全培养液调整细胞浓度为1×10 5个/mL(靶细胞)。小鼠颈椎脱臼猝死，无菌取脾，制成皮细胞悬液，用Hank’s液洗2次，1000rpm离心10min，再用2mL含10％含小牛血清的RPMI 1640完全培养基重悬，用台盼蓝活细胞染色计数(活细胞数应在95％以上)，调整细胞浓度为1×10 7个/mL(效应细胞)，使效靶比为100:1。取靶细胞和效应细胞各100μL，加入U型96孔培养板中，靶细胞自然释放孔加靶细胞和培养液各100μL，靶细胞最大释放孔加靶细胞和1％NP40各100μL，上述均设三个平行孔。37℃，5％CO 2培养箱中培养4h，将96孔培养板以1500rpm离心5min，每孔吸取上清100μL置平底96孔培养板中，同时加入LDH基质液100μL，反应3min，然后每孔加入1mol/L的HCL溶液30μL终止反应，在酶标仪490nm处测OD值，NK活性按下式计算：
Figure PCTCN2019107392-appb-000018
结果见表19。从表19可以看出，BL-99的NK细胞活性高于对照组与BB-12，且差异具有显著性。
表19、NK细胞活性结果
组别 动物数(只) 细胞活性(％) P值
对照 14 30.70±3.31 --
BB-12组 14 33.38±4.17 0.078
BL-99组 14 47.92±5.63 0.000**
4.细胞免疫反应
4.1迟发型变态反应
动物连续给样28天后，每鼠腹腔注射2％压积SRBC(v/v，用生理盐水配制)SRBC0.2mL，致敏4天后，测量左后足趾部厚度，同一部位测量3次，取平均值。然后在测量部位皮下注射20％SRBC 20μL，注射后于24h测量左后足趾部厚度，同一部位测量3次取平均值，以攻击前后足趾厚度的差值(足趾肿胀度)来表示DTH的程度。
结果见表20。从表20可以看出，SRBC攻击前，各组小鼠的足趾厚度均处于同一水平，SRBC共计24h后，小鼠足趾部出现肿胀，肿胀度使用攻击前后足趾部厚度差值表示。通过统计分析发现，与对照组相比，BB-12与BL-99组显著高于对照组(p＜0.05)。
表20、小鼠迟发型变态反应足趾肿胀结果
Figure PCTCN2019107392-appb-000019
4.2小鼠腹腔巨噬细胞吞噬实验
动物连续给样28天后，灌胃结束前4天给每只小鼠腹腔注射0.2mL 2％SRBC激活小鼠巨噬细胞，实验当天用颈椎脱臼法处死小鼠，腹腔注射加小牛血清的Hank’s液3mL/只，轻轻按揉腹部20次，以充分洗出腹腔巨噬细胞，然后将腹壁剪开一个小口，吸取腹腔洗液0.5mL混合液，加入玻片的琼脂圈内。放置孵箱内37℃孵育15-20min。孵育结束后迅速用生理盐水将未贴壁细胞冲净，于甲醇液中固定1min，Giemsa染色15min。用蒸馏水冲洗干净，晾干，用40×显微镜计数吞噬率和吞噬指数。吞噬率为每100个吞噬细胞中，吞噬鸡红细胞的吞噬细胞所占的百分率；吞噬指数为平均每个吞噬细胞吞噬鸡红细胞的个数。结果按下式计算：
Figure PCTCN2019107392-appb-000020
Figure PCTCN2019107392-appb-000021
结果见表21。从吞噬细胞吞噬率和吞噬指数结果可以得出，BB-12组吞噬率和吞噬指数与对照组相比无差异，则BB-12组巨噬细胞吞噬实验结果阴性；BL-99组吞噬率和吞噬指数均高于对照组，则BL-99组巨噬细胞吞噬实验结果阳性。
表21、巨噬细胞吞噬率及吞噬指数结果
组别 动物数(只) 吞噬率(％) p值 吞噬指数 p值
对照 14 22.55±1.58 -- 0.42±0.04 --
BB-12组 14 22.28±2.75 0.799 0.47±0.04 0.055
BL-99组 14 25.48±2.86 0.005** 0.53±0.04 0.000**
以上结果证实，本发明的分离菌株乳双歧杆菌BL-99能够提高机体的免疫脏器指数、非特异性免疫功能和特异性免疫功能特性，从而达到提高机体免疫里的活性。
实施例4：预治骨质疏松功效分析
1.去卵巢大鼠动物模型和益生菌干预
将乳双歧杆菌BL-99菌株于MRS液体培养基中37℃培养16小时后，于4℃、2500rpm下离心10min，收集菌体，磷酸盐缓冲液(PBS)洗涤后冷冻干燥，-18℃以下保存。用于本发明实施例2-实施例5的实验研究。
17周龄雌性成年SD大鼠85只，体重200-300g。大鼠随机分成3组，每组10只。20只大鼠进行去卵巢手术，剩余10只大鼠进行假手术。大鼠每天12h光照/黑暗，室温在25℃左右，自由饮水。术干预12周后，处死模型考察组动物，收集子宫、股骨、胫骨等样品，并测定子宫系数、骨微观结构形态、骨结构模型参数等骨质疏松相关指标。
术后动物休息两周后开始灌胃给予受试物，疗程为每天给药一次，连续12周。去卵巢大鼠喂食BL-99益生菌，假手术组和去卵巢空白对照组喂食蒸馏水；动物具体给药情况如下。动物具体分组情况如下表22。
表22
编号 组别 动物数(只) 益生菌剂量
1 假手术空白对照组(假手术) 10 空白溶剂
2 去卵巢空白对照组(OVX) 10 空白溶剂
3 去卵巢+益生菌BL-99 10 10 9CFU/mL
动物在干预前后的体重变化见图1A。模型建立成功后，假手术假手术组的体重明显低于其他各去卵巢组，这与绝经后女性的体重明显增加相一致。各干预组在12周干预期 间体重都有了一定程度的增加。去卵巢空白组与去卵巢+益生菌BL-99干预组比较体重在干预前后无显著差异。
12周干预后，处死动物，收集、称量并记录子宫的重量，计算子宫系数(即子宫重量与体重的比值)。实验结果(图1B)显示，去卵巢OVX组与假手术假手术组的子宫系数有极其显著的差异(P<0.001vs.假手术)，说明去卵巢后，随着体内雌激素的减少，子宫显著的萎缩。而去卵巢空白组和去卵巢+益生菌BL-99干预组对子宫的重量均无影响，说明其无雌激素样副作用。
2.骨组织形态计量学测定
取左侧胫骨近端1/3处，沿矢状面纵向切开，取材约1×0.5×0.5cm 3，在脱钙液10％EDTA/PBS(pH7.4)中浸泡至完全脱钙，约1周左右，每3天换液一次，然后常规脱水，石蜡包埋，沿矢状面(厚度4μm)，HE染色，用病理图像分析仪可测定总组织面积、骨小梁面积、骨小梁总周长，再用计算公式换算出骨小梁面积百分率、骨小梁数目、骨小梁厚度和骨小梁分离度。骨组织切片还观察骨小梁外观、排列及形态结构完整性等情况。
益生菌BL-99干预12周以后，HE染色结果(图2A)，假手术组骨小梁排列紧密,结构完整；而OVX空白组骨小梁排列松散稀疏，结构不完整，与假手术组比，骨小梁面积百分比显著减低(P<0.001vs.假手术)。而与OVX空白组相比较，益生菌BL-99组能增加骨小梁面积百分比约12.5％(OVX：16.8±2.5％，OVX+BL-99:18.9±1.8)(P<0.05vs.OVX)(图2B)，提示益生菌BL-99能抑制由雌激素缺乏导致的骨丢失，对骨有一定的保护作用。
破骨细胞是体内负责骨吸收的主要细胞，在骨发育、生长、修复、重建中具有重要的作用。破骨细胞来源于单核－巨噬细胞系统，是一种特殊的终末分化细胞，它可由其单核前体细胞通过细胞融合形成巨大的多核细胞。破骨细胞与成骨细胞在功能上相对应。二者协同，在骨骼的发育和形成过程中发挥重要作用。高表达的抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase，TRAP)是破骨细胞主要标志之一。胫骨TRAP染色结果如图3A所示，染色阳性结果为定位于破骨细胞胞浆被染成酒红色。OVX空白组的大鼠胫骨表面TRAP染色的破骨细胞数目显著高于假手术组大鼠。与OVX空白组大鼠相比较，益生菌BL-99组TRAP染色破骨细胞的数量出现显著降低约17.9％(OVX：22.3±1.1％，OVX+BL-99:18.3±0.6)(P<0.05vs.OVX)。在体内雌激素能抑制破骨细胞的活性，同时诱导破骨细胞的凋亡进而发挥抗骨吸收作。在OVX的动物中，由于雌激素的水平明显低下，导致其对破骨细胞的抑制作用缺失，破骨细胞的数量和骨吸收的能力明显增加(图3B)，最终的后果就是松质骨骨量的明显减少。而从图2A、图2B和图3A、图3B的结果提示BL-99干预能抑制OVX引起的骨量的丢失，可能是通过减少破骨细胞的数量。
3.生化指标的测定
测定的生化指标包括血钙、血磷、血清骨钙素(Osteocalcin,OCN)、I-型胶原C端肽(C-terminal telopeptides of type I collagen,CTX-I)。采用原子吸收分光光度法测定血钙、血磷，血清样品直接测定。检测所用试剂盒包括：Calcium
Figure PCTCN2019107392-appb-000022
Test(REF 0155-225)，Phosphorus
Figure PCTCN2019107392-appb-000023
Test(REF 0830-125)，生产商均为Stanbio Laboratory(NorthMainBoerne,FX,USA)如图4A-图4D所示，与假手术组对比，OVX空白组的血钙和血磷水平降低，虽然血钙的降低没有统计学意义。但与OVX空白组相比较，益生菌BL-99干预组能显著提升高血清钙离子(OVX：2.28±0.02mg/dl，OVX+BL-99:2.49±0.03mg/dl)，升高约10％，和磷离子(OVX：1.02±0.08mg/dl，OVX+BL-99:1.41±0.11mg/dl)，升高约40％。
血清骨钙素是由成骨细胞分泌的一种活性多肽，在调节骨代谢中起重要的作用，其水平反映成骨细胞活性。I型胶原C端肽是I型胶原降解后的小片段，其含量和变化可评价骨吸收状态。血清骨钙素和I型胶原C末端肽采用ELASA试剂盒测定，测定方法按照试剂盒中说明书进行。检测所用试剂盒如下：Rat Osteocalcin ELISA Kit，Rat C-telopeptide of Collagen alpha-1(I)chain ELISA Kit，生产商均为SAB(SABiosciences,USA)。
如图4A-图4D所示与OVX空白组相比较，益生菌BL-99干预组能显著提升血清骨钙素的水平约44.6％(OVX：68.6±16.4pg/dl，OVX+BL-99:92.6±24.3pg/dl)，降低血清I-型胶原C端肽约14.6％，但没有统计学的意义。
4.骨标本PCR的检测
本实施例继续研究和检测骨标本中与破骨细胞形成和成骨细胞形成相关的基因表达。益生菌BL-99干预方式同“4.1去卵巢大鼠动物模型和益生菌干预”。
骨组织利用Trizol提取总RNA后，反转录试剂盒将RNA反转录为cDNA，之后用不同基因引物(如表23)进行PCR扩增。
表23
Figure PCTCN2019107392-appb-000024
与OVX空白对照组相比，益生菌BL-99干预组能显著提升骨保护素(OPG)的基因表达，而对RANKL的基因表达没有显著的影响，从而挺高了OPG/RANKL的比值。RANKL与破骨细胞表面上的RANK受体结合，促进破骨细胞的分化和激活，并抑制其凋亡；骨保护素OPG阻止RANKL与RANK的结合，从而阻止破骨细胞的激活，抑制破骨细胞的功能，减少骨吸收，起负调节作用。OPG/RANKL的比值提示体内调控破骨细胞的水平。本研究发现BL-99干预后OPG/RANKL基因表达比值升高，从去卵巢空白组的1到1.75，提升比值约75％，此结果提示BL-99有明显的抑制破骨细胞形成的作用。另外，如图5A-图5E所示：与OVX空白组比较，BL-99干预组可以提高骨钙素约1.1倍(OVX：0.908±0.107，OVX+BL-99：2.075±0.643)和碱性磷酸酶约37.8％(OVX：0.990±0.217，OVX+BL-99：1.364±0.513)的基因表达水平，这两个基因的表达水品与骨形成的能力密切相关，因此BL-99干预可通过促进成骨的相关基因的表达，来促进新骨的形成而拮抗OVX引起的骨量的丢失。
以上研究结果证实：本发明的乳双歧杆菌BL-99能显著抑制去卵巢或雌激素低下导致的骨量的丢失，提升血钙和血磷。
实施例5：BL-99菌粉的制备及其用于食品的生产
参照图6所示的发酵工艺流程，将本发明提供的乳双歧杆菌BL-99(即保藏编号为CGMCC No.15650的乳双歧杆菌)在TPY液体培养基中进行厌氧培养。TPY液体培养基(g/L)：水解酪蛋白10.0，大豆胨5.0，酵母粉2.0，葡萄糖5.0，L-半胱氨酸0.5，磷酸氢二钾2.0，氯化镁0.5，硫酸锌0.25，氯化钙0.15，氯化铁0.0001，吐温80 1.0，pH值6.5±0.1。经一级、二级并扩大培养后的发酵液，于4℃、2500rpm下离心10min，收集菌体，冷冻干燥，得到BL-99菌粉，-18℃以下保存。
本实施例所制得的BL-99菌粉，可以用于食品、饲料或医药用途。所述食品例如可以是发酵乳、乳酪、含乳饮料、固体饮料、乳粉等普通食品或保健食品。优选地，在所述食品中，乳双歧杆菌BL-99供人类使用的建议的剂量可为1.0×10 3CFU～1.0×10 10CFU/kg体重/天，更优选为1.0×10 4CFU～1.0×10 9CFU/kg体重/天。或者，乳双歧杆菌BL-99供人类使用的建议的剂量可为0.001μg～100mg/kg体重/天，更优选为0.01μg～10mg/kg体重/天，进一步优选为0.01μg～1mg/kg体重/天。

Claims (15)

  1. 一种乳双歧杆菌(Bifidobacterium lactis)，其具有耐胃酸性能，在pH2.5的胃酸液中处理30min时活菌存活率62％以上，处理2小时活菌存活率61％以上。
  2. 根据权利要求1所述的乳双歧杆菌，其具有耐肠液性能，在pH6.8的小肠液中处理2小时活菌存活率70％以上。
  3. 一种乳双歧杆菌(Bifidobacterium lactis)，其保藏编号为CGMCC No.15650。
  4. 一种乳双歧杆菌菌制剂，该菌制剂中含有权利要求1～3任一项所述的乳双歧杆菌，其为活菌形式或灭活形式的固态菌粉，或者为活菌形式或灭活形式的液态态菌制剂。
  5. 权利要求1～3任一项所述的乳双歧杆菌或权利要求4所述的乳双歧杆菌菌制剂在制备具有调节胃肠道菌群功效的组合物中的应用，所述组合物包括食品组合物、饲料组合物或药品组合物。
  6. 根据权利要求5所述的应用，其中，所述调节胃肠道菌群包括：增加肠道中双歧杆菌和/或乳酸菌数量，抑制肠道中脱硫弧菌(Desulfovibrio)和/或肠杆菌属(Enterorhabdus)，和/或抑制幽门螺旋杆菌(Helicobacter)和/或埃希氏菌-志贺菌属(Escherichia-Shigella)。
  7. 权利要求1～3任一项所述的乳双歧杆菌或权利要求4所述的乳双歧杆菌菌制剂在制备用于提高机体免疫力活性的组合物中的应用，所述组合物包括食品组合物、饲料组合物或药品组合物。
  8. 根据权利要求7所述的应用，其中，所述提高机体免疫力活性包括：提高机体碳廓清指数，提高机体半数溶血值，提高机体抗体生成细胞数，激活机体NK细胞活性，和/或增加机体巨噬细胞吞噬率与吞噬指数。
  9. 权利要求1～3任一项所述的乳双歧杆菌或权利要求4所述的乳双歧杆菌菌制剂在制备用于预防和/或治疗骨质疏松的组合物中的应用，所述组合物包括食品组合物、饲料组合物或药品组合物。
  10. 根据权利要求9所述的应用，其中，所述预防和/或治疗骨质疏松包括：降低雌激素缺乏引起的骨量的丢失，提升血钙和/或血磷，抑制体内破骨细胞数量，和/或促进骨合成代谢相关因子基因的表达蛋白；
    优选地，所述骨合成代谢相关因子基因的表达蛋白包括碱性磷酸酶和/或骨钙素。
  11. 一种食品，其中包含权利要求1～3任一项所述的乳双歧杆菌或权利要求4所述的乳双歧杆菌菌制剂；优选地，所述食品为发酵乳制品、乳酪、含乳饮料、固体饮料或 乳粉。
  12. 一种药品，其中包含权利要求1～3任一项所述的乳双歧杆菌或权利要求4所述的乳双歧杆菌菌制剂。
  13. 一种调节胃肠道菌群的方法，该方法包括给予受试者有效量的权利要求1～3任一项所述的乳双歧杆菌或权利要求4所述的乳双歧杆菌菌制剂；
    具体地，所述调节胃肠道菌群包括：增加肠道中双歧杆菌和/或乳酸菌数量，抑制肠道中脱硫弧菌(Desulfovibrio)和/或肠杆菌属(Enterorhabdus)，和/或抑制幽门螺旋杆菌(Helicobacter)和/或埃希氏菌-志贺菌属(Escherichia-Shigella)。
  14. 一种提高机体免疫力活性的方法，该方法包括给予受试者有效量的权利要求1～3任一项所述的乳双歧杆菌或权利要求4所述的乳双歧杆菌菌制剂；
    具体地，所述提高机体免疫力活性包括：提高机体碳廓清指数，提高机体半数溶血值，提高机体抗体生成细胞数，激活机体NK细胞活性，和/或增加机体巨噬细胞吞噬率与吞噬指数。
  15. 一种预防和/或治疗骨质疏松的方法，该方法包括给予受试者有效量的权利要求1～3任一项所述的乳双歧杆菌或权利要求4所述的乳双歧杆菌菌制剂；
    具体地，所述预防和/或治疗骨质疏松包括：降低雌激素缺乏引起的骨量的丢失，提升血钙和/或血磷，抑制体内破骨细胞数量，和/或促进骨合成代谢相关因子基因的表达蛋白；
    优选地，所述骨合成代谢相关因子基因的表达蛋白包括碱性磷酸酶和/或骨钙素。
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US11298382B2 (en) 2018-09-30 2022-04-12 Inner Mongolia Yili Industrial Group Co., Ltd. Bifidobacterium lactis BL-99 and application thereof
WO2023098764A1 (zh) * 2021-11-30 2023-06-08 内蒙古伊利实业集团股份有限公司 乳双歧杆菌bl-99在预防和/或改善胃炎中的应用

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