WO2023165607A1 - 一株促进生长与生殖发育的高富集有机锌动物双歧杆菌 - Google Patents

一株促进生长与生殖发育的高富集有机锌动物双歧杆菌 Download PDF

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WO2023165607A1
WO2023165607A1 PCT/CN2023/079598 CN2023079598W WO2023165607A1 WO 2023165607 A1 WO2023165607 A1 WO 2023165607A1 CN 2023079598 W CN2023079598 W CN 2023079598W WO 2023165607 A1 WO2023165607 A1 WO 2023165607A1
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zinc
ccfm1230
bifidobacterium
bifidobacterium animalis
rich
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French (fr)
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毛丙永
崔树茂
韩欣然
唐鑫
张秋香
赵建新
陈卫
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江南大学
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • 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
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/02—Nutrients, e.g. vitamins, minerals
    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/51—Bifidobacterium
    • A23V2400/515—Animalis
    • 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 a high-enrichment organozinc animal bifidobacterium that promotes growth and reproductive development, and belongs to the technical field of microorganisms.
  • Zinc is an essential trace element to maintain the normal growth and development of the human body.
  • the human body contains 2-3g of zinc, nearly 90% of which is found in the muscles and bones. Healthy adults take in about 10-15mg of zinc from the diet every day, and the general absorption rate is 20-30%.
  • Zinc deficiency is considered to be a prevalent nutritional deficiency worldwide, affecting approximately 31% of the global population and is prevalent in both developed and developing countries, especially in developing countries. Zinc deficiency can lead to growth retardation and hypogonadism. Mild to moderate zinc deficiency is common in the world. At present, in some impoverished areas of my country, due to the low intake of animal food, the access to zinc-rich food is limited, and plant food is the main food after birth. Phytate in the diet will inhibit the absorption of zinc, resulting in long-term zinc deficiency during the growth and development period, affecting growth and development.
  • Zinc supplementation is currently achieved mainly through additional intake of products containing organic or inorganic zinc.
  • inorganic zinc ZnO, ZnSO 4 , ZnCl 2 , etc.
  • simple organic zinc zinc gluconate, zinc acetate, zinc propionate, etc.
  • organic zinc amino acid chelated zinc, protein complex zinc, etc.
  • inorganic salts to the medium enables microorganisms to enrich mineral ions, enrich trace elements on the cell surface or transport them to the intracellular storage. They can be combined with amino acids, It exists in the form of complexes of proteins, lipids and polysaccharides, thereby realizing the transformation of inorganic trace elements into organic trace elements, and providing a good dietary source for the human body to supplement organic trace elements.
  • most of the strains used to enrich zinc by microorganisms are yeasts, and bifidobacteria are rarely used.
  • zinc-rich bifidobacteria and bifidobacteria, as a common class of microorganisms that are beneficial to human health, also have the function of enriching metal ions.
  • bifidobacteria have more probiotic properties, and zinc enrichment with bifidobacteria may be of higher value than yeast enrichment.
  • Zinc-rich probiotics can regulate gut microbes while supplementing zinc. As a new dietary zinc source containing active probiotics, zinc-enriched probiotics have more advantages than probiotics or other zinc supplements and are worth exploring.
  • a Bifidobacterium animalis CCFM1230 with high zinc-rich content and high organic biological content was finally screened out.
  • the zinc content of its dry bacterial powder can reach 3.8mg/g and organic zinc The conversion rate can reach 95.3%.
  • the zinc content of other strains was not more than 2.5mg/g.
  • Zhang Qingsong and others screened 6 strains of Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium animalis, and Bifidobacterium breve, and the enrichment of zinc ranged from 0.064mg/g to 0.50mg/g .
  • Leonardi et al. determined the enrichment of zinc by 5 strains of bifidobacteria (Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium pseudosmall chain), and found that the amount of zinc enriched by bifidobacteria was between 0.975mg/g and 2.08 Between mg/g.
  • the bifidobacterium zinc content described by Hu Wenfeng et al. is 0.35 mg/g.
  • the zinc content of Bacillus subtilis described by Wang Baowei et al. is 2.51 mg/g.
  • Bifidobacteria in the above-mentioned literatures have low zinc enrichment content, which cannot achieve the ideal effect of high zinc enrichment and high organic zinc conversion rate of the patent strain.
  • the present invention provides a Bifidobacterium animalis CCFM1230 highly enriched in organic zinc, named after taxonomy: Bifidobacterium animalis, which was preserved in the Guangdong Microbial Culture Collection Center on February 11, 2022.
  • the deposit number is GDMCC No: 62248, and the deposit address is 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou City.
  • the animal Bifidobacterium CCFM1230 has the following characteristics:
  • the strain was small, white, and opaque colonies after being cultured on the MRS medium for 48 hours;
  • Inorganic zinc can be efficiently enriched and converted into organic zinc, which can be better absorbed and utilized by the body;
  • the zinc content per gram of bacterial powder can reach more than 3.8 mg, the conversion rate of organic zinc can reach 95.3%, and the number of viable bacteria can reach more than 4.73 ⁇ 10 9 CFU/g.
  • the present invention also provides a probiotic preparation containing the Bifidobacterium animalis CCFM1230, or the cells obtained after the Bifidobacterium animalis CCFM1230 is cultured with zinc enrichment, or a cell lysate containing organic zinc.
  • the organic zinc content in the probiotic preparation per g or per mL is ⁇ 3605.45 ⁇ g.
  • each g or each mL of the probiotic preparation contains ⁇ 1 ⁇ 10 10 CFU/g or ⁇ 1 ⁇ 10 10 CFU/mL Bifidobacterium animalis CCFM1230 or the zinc-rich culture obtained cell.
  • the cells include but not limited to living cells or dead cells; the dead cells include but not limited to cells that are naturally inactivated or cells that have been inactivated.
  • the zinc-enriched culture is to cultivate the Bifidobacterium animalis CCFM1230 in a zinc-enriched medium to a bacterial cell number ⁇ 1 ⁇ 10 8 CFU/mL.
  • the zinc-rich culture is to cultivate the Bifidobacterium animalis CCFM1230 in a zinc-rich medium. Raising for a period of time; the zinc ion concentration in the zinc-rich medium is 200-500 mg/L.
  • the Bifidobacterium animalis CCFM1230 is further dried after zinc-enriched culture; the drying methods include but not limited to: vacuum freeze drying, spray drying, vacuum drying, and fluidized bed drying.
  • the present invention also provides a preparation method of zinc-rich Bifidobacterium animalis CCFM1230, said method comprising the following steps:
  • the bacterial liquid was centrifuged at 8000 g for 20 min at 4° C., and the wet bacterial cells were rinsed twice with pure water to obtain zinc-rich Bifidobacterium longum sludge.
  • the concentration of zinc ions in the zinc-rich liquid medium in the step (2) is 200-500 mg/L.
  • the zinc-rich liquid medium contains: glucose 20-30g/L, nitrogen source 15-25g/L (the mass ratio of yeast extract powder and peptone is 1:2), anhydrous sodium acetate 2g /L, diammonium hydrogen citrate 2g/L, K 2 HPO 4 3H 2 O 2.6g/L, MgSO 4 7H 2 O 0.1g/L, MnSO 4 H 2 O 0.05g/L, Tween- 801g/L, cysteine 0.5g/L, zinc sulfate (added according to the conversion of zinc ion concentration of 200-500mg/L).
  • the zinc-rich bifidobacterium animal sludge is further dried to obtain a bifidobacterium animal powder highly enriched in organic zinc.
  • the zinc-rich bifidobacterium animal sludge is further subjected to any drying treatment; the drying includes but is not limited to spray drying, vacuum drying, fluidized bed drying or vacuum freeze drying.
  • the zinc-rich Bifidobacterium animalis sludge is inactivated and then subjected to any drying process to obtain the Bifidobacterium animalis CCFM1230 bacterial powder with high organic zinc enrichment without cell activity; the drying Use egg whites or dextrins as fillers, or use no fillers at all.
  • the invention also provides the zinc-enriched bacteria powder prepared by the method.
  • the present invention also provides the application of the animal bifidobacterium CCFM1230 or the probiotic preparation in the preparation of food, medicine or health products.
  • the present invention also provides the application of the animal bifidobacterium CCFM1230 or the probiotic preparation in promoting the growth and reproductive development of young mammals.
  • the present invention provides a strain of bifidobacterium animalis capable of high enrichment of organic zinc, which can enrich and absorb inorganic zinc and The bacteria are transformed into biological state zinc. After the strain is enriched with zinc, the zinc content per gram of bacterial powder can reach more than 3.8mg, and the content of organic zinc can reach 95.3%. The number of viable bacteria in bacterial powder can reach 4.73 ⁇ 10 9 CFU/ more than g. Regardless of whether it is active or not, the organic zinc enriched and produced by the strain can be better absorbed and utilized by the body, and can effectively promote the growth and reproductive development of mammals. biological material deposit
  • Bifidobacterium animalis (Bifidobacterium animalis) CCFM1230, named Bifidobacterium animalis, has been preserved in Guangdong Microbial Culture Collection Center on February 11, 2022.
  • the preservation number is GDMCC No: 62248, and the preservation address is 100 Xianlie Middle Road, Guangzhou City 5th Floor, Building 59, No. 1 Courtyard.
  • Figure 1 shows the effect of zinc supplementation on ALP activity in rat serum; Note: different letters represent significant differences between groups (*p ⁇ 0.05, **p ⁇ 0.01);
  • Figure 2 shows the effect of zinc supplementation on the concentration of insulin-like growth factor and testosterone in rat serum; Note: Different letters represent significant differences between groups (*p ⁇ 0.05, **p ⁇ 0.01).
  • Zinc sulfate (product number: 10024018, CAS: 7446-20-0) involved in the following examples was purchased from Sinopharm Chemical Reagent Co., Ltd.; nitric acid (product number: yb2-308, CAS: 7697-37-2) was purchased from Sinopharm Chemical Reagent Co., Ltd.
  • MRS liquid medium peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, glucose 20g/L, anhydrous sodium acetate 2g/L, diammonium hydrogen citrate 2g/L, K 2 HPO 4 3H 2 O 2.6g/L, MgSO 4 7H 2 O 0.1g/L, MnSO 4 H 2 O 0.05g/L, Tween-801g/L, cysteine 0.5g/L, distilled water 1000mL.
  • MRS solid medium peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, glucose 20g/L, anhydrous sodium acetate 2g/L, diammonium hydrogen citrate 2g/L, K 2 HPO 4 3H 2 O 2.6g/L, MgSO 4 7H 2 O 0.1g/L, MnSO 4 H 2 O 0.05g/L, Tween-801g/L, cysteine 0.5g/L, agar 20g/L L, distilled water 1000mL.
  • Zinc-rich liquid medium glucose 20-30g/L, nitrogen source 15-25g/L (the mass ratio of yeast extract powder and peptone is 1:2), anhydrous sodium acetate 2g/L, diammonium hydrogen citrate 2g/L L, K 2 HPO 4 3H 2 O 2.6g/L, MgSO 4 7H 2 O 0.1g/L, MnSO 4 H 2 O 0.05g/L, Tween-801g/L, cysteine 0.5g /L, distilled water 1000mL, zinc sulfate (added according to the conversion of zinc ion concentration of 200-500mg/L).
  • Example 1 Screening, strain identification and preservation of animal bifidobacteria
  • the isolated strains with strong zinc-enrichment ability were subjected to PCR amplification of 16S rDNA, and the PCR products were sent to Yingwei Jieji (Shanghai) Trading Co., Ltd. for sequencing, and the results obtained by sequencing were compared in NCBI for nucleic acid sequence. Finally, a strain of Bifidobacterium animalis was obtained, which was named as Bifidobacterium animalis CCFM1230.
  • Embodiment 2 the preparation method of zinc-rich animal Bifidobacterium
  • step (2) The seed bacterial solution obtained in step (1) is inserted into a fermentation bottle containing a zinc-rich liquid medium at an inoculum size of 2% (v/v) and cultivated for 12-18 hours.
  • step (3) Centrifuge the bacterial liquid after the fermentation of step (2) at 8000g for 20min at 4°C, take the wet bacterial cells and rinse them twice with pure water, use skim milk with a mass fraction of 13% as a freeze-drying protective agent, and wash The final wet bacteria and freeze-drying protective agent were mixed at a mass ratio of 1:1, and then freeze-dried to obtain Bifidobacterium animalis powder with high zinc enrichment.
  • the number of viable bacteria was 4.73 ⁇ 10 9 CFU/g bacteria powder.
  • the organic zinc content in the powder can reach more than 3605.45 ⁇ g.
  • zinc-rich Bifidobacterium animalis can also be inactivated and dried to prepare bacteria powder.
  • the drying method can be spray drying, vacuum drying, fluidized bed drying or vacuum freeze drying.
  • the inactivated high zinc-enriched Bifidobacterium animalis powder can be prepared as follows: after the fermentation of the aforementioned step (2), centrifuge the bacterial liquid at 8000g for 20min at 4°C, take the wet bacterial cells and wash them with pure water Rinse twice, and put the washed wet bacteria into Line spray drying or vacuum drying or fluidized bed or vacuum freeze-drying, use albumen or dextrin as filler (the mass ratio of bacterium slime and filler solution is 1:1, and filler solution is whey protein or collagen of mass fraction 13%) Protein or soybean protein or dextrin solution) to obtain inactive high-enrichment zinc bifidobacterium animal powder, the content of organic zinc in the bacterial powder can reach more than 3605.45 ⁇ g.
  • Example 3 Zinc content of zinc-rich animal bifidobacteria and detection of organic zinc
  • Zinc standard stock solution 1000mg/L: Accurately weigh 1.2447g (accurate to 0.0001g) of zinc oxide, add a small amount of nitric acid solution with a volume fraction of 50%, heat to dissolve, transfer to a 1000mL volumetric flask after cooling, add water to the mark, mix uniform.
  • 2Zinc standard intermediate solution (10mg/L) Accurately draw 0.5mL of zinc standard stock solution (1000mg/L) into a 50mL volumetric flask, add nitric acid solution with a volume fraction of 5% to the mark, and mix well.
  • 3Zinc standard series solutions Accurately draw 0mL, 0.5mL, 1mL, 2mL, 4mL, and 5mL of zinc standard intermediate solutions into 50mL volumetric flasks, add nitric acid solution with a volume fraction of 5% to the mark, and mix well.
  • the mass concentrations of the zinc standard series solutions are 0mg/L, 0.1mg/L, 0.2mg/L, 0.4mg/L, 0.8mg/L, 1mg/L respectively.
  • the zinc content in the sample was measured by flame atomic absorption spectrometry, and the detection result was 3.8mg/g.
  • the supernatant obtained by centrifugation was collected for measuring the content of water-soluble zinc (ie, inorganic zinc) on the cell surface of zinc-rich Bifidobacterium animalis.
  • Add 45mL of 10mmol/L EDTA solution to the precipitation then adjust the pH value of the EDTA solution to 6.5 with dilute acid or dilute alkali, and then use a 50mL volumetric flask to adjust the pH value of the EDTA solution to 6.5.
  • transfer the zinc-enriched Bifidobacterium animalis solution to a beaker, stir slowly and uniformly with a glass rod for 5-10 minutes, and centrifuge at 8000 r/min at room temperature for 15 minutes after stirring.
  • the supernatant obtained by centrifugation was collected for Measuring the zinc content of the cell wall polysaccharides and protein complexes of zinc-rich Bifidobacterium animalis.
  • the second centrifugal sedimentation was used to measure the zinc content of organic macromolecules or small molecules bound in the cells of Zn-rich Bifidobacterium animalis.
  • Organic degree (cell wall polysaccharide and protein complexed zinc content + intracellular organic macromolecules or small molecular bound zinc content) / total zinc content
  • the zinc content in each component is measured by flame atomic absorption spectrometry, and the detection results are as follows:
  • the total zinc content of the zinc-rich Bifidobacterium animalis CCFM1230 bacterial powder reaches 3.8 mg/g, indicating that the Bifidobacterium animalis has a strong ability to enrich zinc.
  • the content of inorganic zinc is 4.7%, which shows that the assimilation effect of the animal bifidobacterium on inorganic zinc is better.
  • 8.0% of zinc is bound to macromolecules such as polysaccharides and proteins in the cell wall in the form of organisms; 87.2% of zinc is bound to organic macromolecules or small molecules in Bifidobacterium animalis cells.
  • the results showed that the total zinc content in the bacterial powder prepared from the inactivated bacteria slime was 3.8 mg/g, and the inorganic state zinc content was 4.7 mg/g. %, the organic state zinc content is 95.3%.
  • Bifidobacterium bifidum O4 Bifidobacterium adolescentis W5, Bifidobacterium adolescentis HuNan-2016 MRS 11-2, Bifidobacterium animalis HuNan-2016 22-3, Bifidobacterium breve HuNan-2016 49-7, Bifidobacterium breve HuNan-2016 49-7, Bifidobacterium GuXi-2016 6-7, Lactobacillus reuteri 138-1, and Lactobacillus bulgaricus MJ-1 were published in the paper "Zinc-enriching characteristics of lactic acid bacteria and the alleviation effect of zinc-enriched lactic acid bacteria on colitis in mice"; Bifidobacterium breve WC 0421, Bifidobacterium breve WC 0480, Bifidobacterium breve WC 04
  • the bacterial strains shown in Table 3 were cultivated according to the same method as in Example 2, and the zinc content after 18 hours of cultivation was detected. Among them, Bifidobacterium breve F-JS-ZJ-1-M5, Lactobacillus rhamnosus DG11-1, Lactobacillus plantarum NFM11, Lactobacillus casei RS-2-1, Lactobacillus fermentum NT65-2 were obtained by self-screening Zinc-rich strains.
  • Example 4 Effects of zinc-rich Bifidobacterium longum on the growth and reproductive development of male rat pups
  • Modeling Select 40 young male SD rats at the age of 3 weeks, and randomly divide them into 4 groups: normal group, zinc deficiency group, inorganic zinc group, and zinc-enriched bifidus group, with 5 rats in each group.
  • Zinc deficiency group, inorganic zinc group, and zinc-rich animal bifidus group were fed with zinc deficiency feed TP0690-01G (1ppm) (ordered from Nantong Trofe Feed Technology Co., Ltd.) for one week for zinc deficiency modeling, and the normal group was fed as control feed.
  • Intervention From the second week, 1.0 mL of gavage was administered.
  • the zinc oxide solution of 0.7mg Zn/daily dose was administered into the stomach while feeding the zinc-deficient feed;
  • Zinc-rich animal bifidus group is fed with zinc-deficient feed while bacterial suspension of 0.7mg Zn/ dose per day (will be based on zinc
  • the bacterium powder prepared by the embodiment 2 of content meter is dissolved in normal saline) and carries out gavage;
  • the zinc-deficient control group was fed with zinc-deficient feed and given an equal volume of normal saline;
  • the normal control group was fed the control feed and gavaged the same volume of normal saline for two weeks.

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Abstract

提供一株促进生长与生殖发育的高富集有机锌动物双歧杆菌，属于微生物技术领域。筛选得到的动物双歧杆菌(Bifidobacterium animalis)CCFM1230能够将无机锌高效富集，转化为有机锌，被机体更好的吸收利用，且有效促进生长与生殖发育。经动物实验证明，富锌动物双歧杆菌能够促进雄性大鼠幼鼠的生长与生殖发育，发挥较无机锌更高的生物活性，达到补充锌的生理要求。使用动物双歧杆菌CCFM1230可对无机锌进行高效富集，用于制备促进生长与生殖发育的益生菌制剂，并在食品或医药领域具有巨大的应用前景。

Description

一株促进生长与生殖发育的高富集有机锌动物双歧杆菌 技术领域
本发明涉及一株促进生长与生殖发育的高富集有机锌动物双歧杆菌，属于微生物技术领域。
背景技术
锌是维持人体正常生长发育所必需的微量元素。人体含有2-3g锌，其中近90％存在于肌肉和骨骼中。健康成人每天从饮食中摄取约10-15mg锌，一般吸收率为20-30％。锌缺乏症被认为是世界范围内普遍存在的营养缺乏症，影响了全球约31％的人口，在发达国家和发展中国家都流行，尤其是在发展中国家。缺锌会导致生长迟缓和性腺机能减退。轻度至中度缺锌在世界范围内普遍存在，目前在我国一些贫困地区，由于动物性食物的摄入量低，对富锌的食物获取有限，出生后以植物性食物为主，植物性饮食中的植酸盐等会抑制锌的吸收，造成生长发育期长期锌缺乏，影响生长发育。
由于锌无法在人体内储存，为了维持机体所有功能的正常进行，需要每天补充锌，这就极易造成人体缺锌的状况发生。目前补锌主要是通过额外摄入含有机或无机锌的产品来实现的。市场上的锌补充剂分为三种：无机锌(ZnO、ZnSO4、ZnCl2等)、简单有机锌(葡萄糖酸锌、醋酸锌、丙酸锌等)和有机锌(氨基酸螯合锌、蛋白质络合锌等)。不同补锌剂的吸收效率不同。有研究表明，有机锌比无机锌更容易被人体吸收。无机锌吸收率低，副作用明显。单纯有机锌比无机锌吸收率高，但仍存在一定的刺激胃肠道等副作用。有机锌以人工合成为主，相对安全，但合成较为复杂。
微生物富集锌是近几十年来研究的热点，向培养基中添加无机盐使得微生物能够富集矿物离子，将微量元素富集在细胞表面或将它们转运到细胞内储存，它们以与氨基酸、蛋白质、脂类和多糖的复合物的形式存在，从而实现无机态微量元素向有机态微量元素的转化，为人体补充有机态微量元素提供了一个良好的膳食来源。目前利用微生物富集锌元素所用的菌株大多是酵母菌，较少用到双歧杆菌。相比之下，关于富锌双歧杆菌的报道很少，而双歧杆菌作为一类常见的并且对人体健康有益的微生物，同样具有富集金属离子的功能。而且双歧杆菌具有更多的益生特性，用双歧杆菌富集锌可能比用酵母菌富集锌具有更高的价值。富锌益生菌在补充锌的同时还可以调节肠道微生物。作为一种新型的含有活性益生菌的膳食锌源，富锌益生菌比益生菌或其他锌补充剂具有更多的优势，值得探索。与单独补充无机锌相比，有必要对富锌益生菌高效富集无机锌，转化为有机锌进行研究，开发出一种价格低廉、制备 方法简单、安全可靠且补锌更高效的锌补充剂。
通过在培养过程中添加无机锌(非吸附)，最终筛选出一株富锌量高且有机生物态含量高的动物双歧杆菌CCFM1230，其干菌粉锌含量能够达到3.8mg/g且有机锌转化率可达到95.3％。其余菌株的锌含量均不超过2.5mg/g。张青松等人对6株两歧双歧杆菌、青春双歧杆菌、动物双歧杆菌、短双歧杆菌进行了筛选，对锌的富集量在0.064mg/g～0.50mg/g之间不等。Leonardi等人测定了5株双歧杆菌(短双歧杆菌、婴儿双歧杆菌、假小链双歧杆菌)对锌的富集，发现双歧杆菌富集锌的量在0.975mg/g～2.08mg/g之间。在公开号为CN101971921B的专利申请文本中，胡文锋等人所述的双歧杆菌锌含量为0.35mg/g。在公开号为CN108220208B的专利申请文本中，王宝维等人所述的枯草芽孢杆菌锌含量为2.51mg/g。以上所述文献中双歧杆菌对锌的富集含量均较低，无法达到本专利菌株高富锌量、高有机锌转化率的理想效果。
发明内容
本发明提供了一株高富集有机锌的动物双歧杆菌CCFM1230，分类学命名为：动物双歧杆菌(Bifidobacterium animalis)，已于2022年2月11日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No：62248，保藏地址为广州市先烈中路100号大院59号楼5楼。
所述动物双歧杆菌CCFM1230具有如下特征：
(1)该菌株在MRS培养基上培养48h后呈小的、白色、不透明菌落；
(2)可将无机锌高效富集，转化为有机锌，能够被机体更好的吸收利用；
(3)菌株经过富锌发酵后，每克菌粉锌含量能够达到3.8mg以上，有机锌转化率可达到95.3％，活菌数能够达到4.73×109CFU/g以上。
本发明还提供了含有所述动物双歧杆菌CCFM1230，或含有动物双歧杆菌CCFM1230经富锌培养后所获得的细胞，或含有机锌的细胞裂解物的益生菌制剂。
在一种实施方式中，每g或每mL所述益生菌制剂中的有机锌含量≥3605.45μg。
在一种实施方式中，每g或每mL益生菌制剂中含有≥1×1010CFU/g或≥1×1010CFU/mL动物双歧杆菌CCFM1230或所述经富锌培养后所获得的细胞。
在一种实施方式中，所述细胞包括但不限于活细胞或死细胞；所述死细胞包括但不限于自然失去活性的细胞或经灭活处理后的细胞。
在一种实施方式中，所述富锌培养是将所述动物双歧杆菌CCFM1230在富锌培养基中培养至菌体数量≥1×108CFU/mL。
在一种实施方式中，所述富锌培养是将所述动物双歧杆菌CCFM1230在富锌培养基中培 养一段时间；所述富锌培养基中的锌离子浓度为200～500mg/L。
在一种实施方式中，所述动物双歧杆菌CCFM1230经富锌培养后还经过干燥处理；干燥处理的方式包括但不限于：真空冷冻干燥、喷雾干燥、真空干燥、流化床干燥。
本发明还提供富锌动物双歧杆菌CCFM1230的制备方法，所述方法包括如下步骤：
(1)将所述的动物双歧杆菌在改良MRS固体培养基上划线，平板于37℃倒置培养36～48h，挑取单菌落接入到改良MRS液体培养基中于37℃培养24h。再以2％(v/v)的接种量接入到改良MRS液体培养基中37℃培养12～18h作为后续培养的种子菌液；
(2)将动物双歧杆菌的种子菌液以2％(v/v)接种至富锌液体培养基中培养12～18h；
(3)发酵完成后将菌液在4℃条件下8000g离心20min，取湿菌体用纯水漂洗2次，得到富锌长双歧杆菌菌泥。
在一种实施方式中，所述步骤(2)中所述的富锌液体培养基中锌离子浓度为200～500mg/L。
在一种实施方式中，所述富锌液体培养基含有：葡萄糖20-30g/L，氮源15-25g/L(酵母浸粉、蛋白胨的质量比为1:2)、无水乙酸钠2g/L、柠檬酸氢二铵2g/L、K2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4·H2O 0.05g/L、吐温-801g/L、半胱氨酸0.5g/L、硫酸锌(按锌离子浓度为200～500mg/L换算添加)。
在一种实施方式中，所述富锌动物双歧杆菌菌泥还经过干燥处理，得到高富集有机锌的动物双歧杆菌菌粉。
在一种实施方式中，所述富锌动物双歧杆菌菌泥还经过任意干燥处理；所述干燥包括但不限于喷雾干燥、真空干燥、流化床干燥或真空冷冻干燥。
在一种实施方式中，所述富锌动物双歧杆菌菌泥经过灭活后再经任意干燥方式处理，得到无细胞活性的高富集有机锌的动物双歧杆菌CCFM1230菌粉；所述干燥使用蛋白或糊精作填充剂，或不使用任何填充剂。
本发明还提供应用所述方法制备的富锌菌粉。
本发明还提供所述动物双歧杆菌CCFM1230或所述益生菌制剂在制备食品、药品或保健品中的应用。
本发明还提供所述动物双歧杆菌CCFM1230或所述益生菌制剂在促进幼年哺乳动物生长和生殖发育方面的应用。
有益效果：
本发明提供了一株可高富集有机锌的动物双歧杆菌，该菌株能够将无机锌富集吸收并在 菌体内转化为生物态锌，该菌株经过富锌培养后，每克菌粉锌含量能够达到3.8mg以上，有机锌含量可达到95.3％；菌粉中活菌数能够达到4.73×109CFU/g以上。无论有无活性，经该菌株富集生产的有机锌能够被机体更好的吸收利用，可有效促进哺乳动物的生长与生殖发育。生物材料保藏
动物双歧杆菌(Bifidobacterium animalis)CCFM1230，分类命名为Bifidobacterium animalis，已于2022年2月11日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No：62248，保藏地址为广州市先烈中路100号大院59号楼5楼。
附图说明
图1为补锌对大鼠血清中ALP活性的影响；注：不同字母代表组别间具有显著性差异(*p<0.05，**p<0.01)；
图2为补锌对大鼠血清中胰岛素样生长因子和睾酮浓度的影响；注：不同字母代表组别间具有显著性差异(*p<0.05，**p<0.01)。
具体实施方式
下面结合具体实施例对本发明进行进一步的阐述。
下述实施例中涉及的硫酸锌(产品货号：10024018、CAS：7446-20-0)购自国药集团化学试剂有限公司；硝酸(产品货号：yb2-308、CAS：7697-37-2)购于国药集团化学试剂有限公司。
下述实施例中涉及的培养基如下：
改良MRS液体培养基：蛋白胨10g/L、牛肉膏10g/L、酵母提取物5g/L、葡萄糖20g/L、无水乙酸钠2g/L、柠檬酸氢二铵2g/L、K2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4·H2O 0.05g/L、吐温-801g/L、半胱氨酸0.5g/L、蒸馏水1000mL。
改良MRS固体培养基：蛋白胨10g/L、牛肉膏10g/L、酵母提取物5g/L、葡萄糖20g/L、无水乙酸钠2g/L、柠檬酸氢二铵2g/L、K2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4·H2O 0.05g/L、吐温-801g/L、半胱氨酸0.5g/L、琼脂20g/L、蒸馏水1000mL。
富锌液体培养基：葡萄糖20-30g/L，氮源15-25g/L(酵母浸粉、蛋白胨的质量比为1:2)、无水乙酸钠2g/L、柠檬酸氢二铵2g/L、K2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4·H2O 0.05g/L、吐温-801g/L、半胱氨酸0.5g/L、蒸馏水1000mL、硫酸锌(按锌离子浓度为200～500mg/L换算添加)。
实施例1：动物双歧杆菌的筛选、菌种鉴定及保存
1、筛选
以来源于上海市的婴儿粪便为样本，用无菌生理盐水进行10倍梯度稀释至10-6，然后分别取100μL稀释倍数为10-4、10-5、10-6的稀释液于改良MRS固体培养基上进行平板涂布，37℃培养48h，观察并记录菌落形态；挑取改良MRS固体培养基上不同形态的菌落进行划线分离，经37℃培养48h后，再次挑取改良MRS固体培养基上不同形态的单菌落进行划线分离，直至得到形态一致的纯的单菌落；挑取改良MRS固体培养基上的纯菌落接种于含有硫酸锌的富锌液体培养基中，37℃培养18h；将菌液转移至无菌离心管中，8000g离心10min后弃去上层培养基，得到的菌泥漂洗2遍后冷冻干燥，得到富锌菌粉。使用原子吸收分光光度计检测菌粉中的锌含量，选出富锌能力较强的菌株。
2、鉴定
将分离得到的富集锌能力较强的菌株进行PCR扩增16S rDNA，PCR产物送至英潍捷基(上海)贸易有限公司进行测序，将测序得到的结果在NCBI中进行核酸序列比对，最终得到1株动物双歧杆菌，命名为动物双歧杆菌(Bifidobacterium animalis)CCFM1230。
3、保存
将动物双歧杆菌(Bifidobacterium animalis)CCFM1230接种于改良MRS液体培养基中，37℃培养18h；取1mL菌液于无菌离心管中，8000g离心10min后弃去上层培养基，菌泥重悬于30％甘油溶液中置于-80℃中保藏。
实施例2：富锌动物双歧杆菌的制备方法
(1)将实施例1筛选获得的动物双歧杆菌CCFM1230在改良MRS固体培养基上划线，平板于37℃倒置培养48h；挑取单菌落接入MRS液体培养基中于37℃培养24h；以2％(v/v)的接种量接入到改良MRS液体培养基中37℃培养12-18h作为后续培养的种子菌液。
(2)将步骤(1)获得的种子菌液以2％(v/v)的接种量接入到含富锌液体培养基的发酵瓶中培养12-18h。
(3)将步骤(2)发酵完成后的菌液在4℃条件下8000g离心20min，取湿菌体用纯水漂洗2次，以质量分数13％的脱脂乳作为冻干保护剂，将洗涤后的湿菌体与冻干保护剂以质量比1:1混匀，冷冻干燥，得到高富集锌的动物双歧杆菌菌粉，活菌数为4.73×109CFU/g菌粉，菌粉中的有机锌含量可达3605.45μg以上。
可选地，还可将富锌动物双歧杆菌灭活、经干燥处理制备菌粉，干燥方式可选用喷雾干燥或真空干燥或流化床干燥或真空冷冻干燥。
可选地，灭活高富集锌动物双歧杆菌菌粉可按如下方法制备：将前述步骤(2)发酵完成后将菌液在4℃条件下8000g离心20min，取湿菌体用纯水漂洗2次，将洗涤后的湿菌体进 行喷雾干燥或真空干燥或流化床或真空冷冻干燥，使用蛋白或糊精作填充剂(菌泥与填充剂溶液质量比为1:1，填充剂溶液为质量分数13％的乳清蛋白或胶原蛋白或大豆蛋白或糊精溶液)，得到无活性的高富集锌动物双歧杆菌菌粉，菌粉中的有机锌含量可达3605.45μg以上。
实施例3：富锌动物双歧杆菌锌含量及有机锌检测
1、富锌动物双歧杆菌锌含量检测
(1)微波消解
称取实施例2制备的动物双歧杆菌(Bifidobacterium animalis)CCFM1230菌粉样品0.1g～0.15g于微波消解罐中，加入5mL硝酸后，进行微波消解。冷却后取出消解罐，在电热板上于140℃～160℃赶酸至1mL左右。消解罐放冷后，将消化液转移至25mL容量瓶中，用少量水洗涤消解罐2次～3次，合并洗涤液于容量瓶中，用水定容至刻度，混匀备用。同时做试剂空白试验。
(2)标准溶液配制
①锌标准储备液(1000mg/L)：准确称取1.2447g(精确至0.0001g)氧化锌，加少量体积分数50％的硝酸溶液，加热溶解，冷却后移入1000mL容量瓶，加水至刻度，混匀。
②锌标准中间液(10mg/L)准确吸取锌标准储备液(1000mg/L)0.5mL于50mL容量瓶中，加体积分数为5％的硝酸溶液至刻度，混匀。
③锌标准系列溶液：分别准确吸取锌标准中间液0mL、0.5mL、1mL、2mL、4mL、和5mL于50mL容量瓶中，加体积分数为5％的硝酸溶液至刻度，混匀。此锌标准系列溶液的质量浓度分别为0mg/L、0.1mg/L、0.2mg/L、0.4mg/L、0.8mg/L、1mg/L。
参照中华人民共和国国家标准GB 5009.14-2017中的第一种方法火焰原子吸收光谱法测量样品中的锌含量，其检测结果为3.8mg/g。
2、富锌动物双歧杆菌有机锌分析
准确称取0.5g的富锌动物双歧杆菌CCFM1230菌粉于烧杯中，加入45mL的蒸馏水，然后用稀酸或稀碱调节蒸馏水pH值至6.5，调好后用50mL容量瓶定容，定容蒸馏水的pH值也调至6.5，定容毕，将富锌动物双歧杆菌溶液转移至烧杯中，用玻璃棒缓慢匀速搅拌5～10min，搅拌毕，用8000r/min于室温离心15min。收集离心获得的上清液用于测量富锌动物双歧杆菌细胞表面水溶性锌(即无机锌)含量。沉淀中加入45mL的10mmol/L EDTA溶液，然后用稀酸或稀碱调节EDTA溶液pH值至6.5，调好后用50mL容量瓶定容，定容的EDTA溶液pH值也调至6.5，定容毕，将富锌动物双歧杆菌溶液转移至烧杯中，用玻璃棒缓慢匀速搅拌5～10min，搅拌毕，于8000r/min室温离心15min。收集离心获得的上清液用于 测量富锌动物双歧杆菌细胞壁多糖及蛋白质络合的锌含量。第二次离心沉淀用于测量富锌动物双歧杆菌细胞内有机大分子或小分子结合锌含量。
有机化程度＝(细胞壁多糖及蛋白质络合的锌含量+细胞内有机大分子或小分子结合锌含量)/总锌含量
参照中华人民共和国国家标准GB 5009.14-2017中的第一种方法火焰原子吸收光谱法测量各组分中的锌含量，其检测结果如下：
表1富锌动物双歧杆菌CCFM1230菌粉锌含量分析
由此可见，富锌动物双歧杆菌CCFM1230菌粉总锌含量达3.8mg/g，表明该动物双歧杆菌对锌的富集能力较强。无机态锌含量为4.7％，说明该动物双歧杆菌对无机锌的同化效果较好。有8.0％的锌以有机体的形式结合在细胞壁的多糖和蛋白质等大分子上；87.2％的锌与动物双歧杆菌细胞内有机大分子或小分子结合。采用相同方法检测无活性的高富集锌动物双歧杆菌菌粉中有机锌含量，结果显示灭活后的菌泥制备的菌粉中总锌含量为3.8mg/g，无机态锌含量为4.7％，有机态锌含量为95.3％。
对比例1：不同富锌双歧杆菌的富锌量和有机化程度
检索并收集不同来源的益生菌富锌培养后的锌含量。其中，两歧双歧杆菌O4、青春双歧杆菌W5、青春双歧杆菌HuNan-2016 MRS 11-2、动物双歧杆菌HuNan-2016 22-3、短双歧杆菌HuNan-2016 49-7、短双歧杆菌GuXi-2016 6-7、罗伊氏乳杆菌138-1、保加利亚乳杆菌MJ-1公开于论文《乳酸菌对锌的富集特性及富锌乳酸菌对小鼠结肠炎的缓解作用》；短双歧杆菌WC 0421、短双歧杆菌WC 0480、短双歧杆菌WC 0481、婴儿双歧杆菌WC 0460、假小链双歧杆菌WC 0455公开于论文《Zinc Uptake by Lactic Acid Bacteria》中；枯草芽孢杆菌NZ56公开于公开号为CN 108220208 B的专利中。
表2不同富锌双歧杆菌的锌含量对比

按照实施例2相同的方法培养表3所示的菌株，并检测培养18h后的锌含量。其中，短双歧杆菌F-JS-ZJ-1-M5、鼠李糖乳杆菌DG11-1、植物乳杆菌NFM11、干酪乳杆菌RS-2-1、发酵乳杆菌NT65-2为自行筛选获得的富锌菌株。
表3不同富锌双歧杆菌有机化程度对比
以上为已有文献或专利中所述的菌株，其对锌的富集含量和有机锌转化率均相对较低，无法达到本发明菌株的高富锌量、高有机锌含量的理想效果。
实施例4：富锌长双歧杆菌对雄性大鼠幼鼠生长及生殖发育的影响
1、造模：选取3周龄雄性SD大鼠幼鼠40只，随机分为正常组、缺锌组、无机锌组、富锌动物双歧组4组，每组5只。缺锌组、无机锌组、富锌动物双歧组用缺锌饲料TP0690-01G(1ppm)(订购于南通特洛菲饲料科技有限公司)缺锌喂养一周进行缺锌造模，正常组喂食对照饲料。
2、干预：从第2周开始按照1.0mL的灌胃量进行灌胃。
无机锌组在饲喂缺锌饲料的同时按照每日0.7mg Zn/只剂量的氧化锌溶液进行灌胃；
富锌动物双歧组在饲喂缺锌饲料的同时按照每日0.7mg Zn/只剂量的菌悬液(将按照锌 含量计的实施例2制备的菌粉溶于生理盐水中)进行灌胃；
缺锌对照组饲喂缺锌饲料并灌胃等体积的生理盐水；
正常对照组饲喂对照饲料并灌胃等体积的生理盐水，灌胃两周。
3、实验结果：饲养期间定期观察大鼠反应，活动情况，精神状况，毛发变化，拍照记录大鼠形态变化，最后一次灌胃后24h收集粪便，然后夜间禁食。次日对大鼠实施安乐死，采集血液、肝脏、睾丸、肾、胰。肝脏、睾丸、肾、胰组织在液氮中速冻并保存在-80℃。将血样吸入促凝管，离心后收集血清。样品在-20℃冷冻保存直至分析。我们的结果表明缺锌对幼鼠的生长和生殖发育有负面影响。不同的锌补充剂由于吸收利用的差异，对身体各项指标的恢复能力也不同。富锌动物双歧杆菌CCFM1230的生物利用度要高于无机锌，能够被机体更好的吸收利用。
表4大鼠身长、身宽
注：不同字母代表组别间具有显著性差异(p<0.05)
表5大鼠睾丸重量
注：不同字母代表组别间具有显著性差异(p<0.05)
表6大鼠组织中的锌含量
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (10)

  1. 一株高富集有机锌的动物双歧杆菌(Bifidobacterium animalis)CCFM1230，已于2022年2月11日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No：62248。
  2. 一种益生菌制剂，其特征在于，含有(a)、(b)或(c)：
    (a)权利要求1所述的动物双歧杆菌CCFM1230；
    (b)权利要求1所述动物双歧杆菌CCFM1230经富锌培养后的细胞；
    (c)权利要求1所述动物双歧杆菌CCFM1230经富锌培养得到的含有机锌的细胞裂解物。
  3. 根据权利要求2所述的益生菌制剂，其特征在于，所述细胞包括但不限于活细胞或死细胞；所述死细胞包括但不限于自然失去活性的细胞或经灭活处理后的细胞。
  4. 根据权利要求2或3所述的益生菌制剂，其特征在于，每g或每mL益生菌制剂中含有≥1×1010CFU/g或≥1×1010CFU/mL动物双歧杆菌CCFM1230或所述经富锌培养后所获得的细胞。
  5. 根据权利要求2所述的益生菌制剂，其特征在于，所述富锌培养是将所述动物双歧杆菌CCFM1230在富锌培养基中培养至菌体数量≥1×108CFU/mL。
  6. 一种高富集有机锌的动物双歧杆菌CCFM1230的制备方法，其特征在于，将所述动物双歧杆菌CCFM1230在富锌培养基中培养至菌体数量≥1×108CFU/mL；所述富锌培养基中锌离子浓度为200～500mg/L。
  7. 根据权利要求6所述的方法，其特征在于，所述富锌培养基含有：葡萄糖20-30g/L，氮源15-25g/L、无水乙酸钠2g/L、柠檬酸氢二铵2g/L、K2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4·H2O 0.05g/L、吐温-80 1g/L、半胱氨酸0.5g/L、硫酸锌；所述氮源包括质量比为1:2的酵母浸粉和蛋白胨。
  8. 根据权利要求6或7所述的方法，其特征在于，所述富锌动物双歧杆菌菌泥还经过干燥处理。
  9. 权利要求1所述的动物双歧杆菌CCFM1230或权利要求2～5任一所述的益生菌制剂在制备食品、药品或保健品中的应用。
  10. 权利要求1所述的动物双歧杆菌CCFM1230或权利要求2～5任一所述的益生菌制剂在促进幼年哺乳动物生长和生殖发育方面的应用。
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