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WO2022057741A1 - 动物双歧杆菌及其选育方法和应用 - Google Patents

动物双歧杆菌及其选育方法和应用 Download PDF

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WO2022057741A1
WO2022057741A1 PCT/CN2021/117750 CN2021117750W WO2022057741A1 WO 2022057741 A1 WO2022057741 A1 WO 2022057741A1 CN 2021117750 W CN2021117750 W CN 2021117750W WO 2022057741 A1 WO2022057741 A1 WO 2022057741A1
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bifidobacterium
strain
group
bifidobacterium animalis
bifidobacteria
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PCT/CN2021/117750
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French (fr)
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徐轲
栾庆刚
彭跃
屈倩
王培亮
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山东得益乳业股份有限公司
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    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
    • 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
    • C12N1/02—Separating microorganisms from their culture media
    • 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
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/51—Bifidobacterium
    • A23V2400/515—Animalis
    • 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

Definitions

  • the invention relates to an animal bifidobacteria, a breeding method and application thereof, and belongs to the technical field of Bifidobacterium lactis.
  • Probiotics are a class of active microorganisms that can improve the host's micro-ecological balance and exert beneficial effects on the human body. Probiotics colonize the human body, such as the intestinal tract, and play an important physiological role in the digestion and absorption of nutrients, the growth and development of the body, immunity and antagonism. In a normal adult, the gut contains more than 1,000 species of microorganisms, which can be divided into three categories: origin flora, opportunistic pathogens and transit bacteria.
  • the flora of origin is the dominant intestinal bacteria with immunomodulatory effects; opportunistic pathogens are harmless when the intestinal flora is balanced, but can be harmful to the human body under certain conditions; harmful bacteria, the number of such bacteria is small under normal circumstances, and the bacteria It can cause disease when the group is out of balance. People with constipation and diarrhea are often accompanied by an imbalance of intestinal flora.
  • Active probiotics with intestinal optimization function or yogurt products loaded with such active probiotics can also improve the human intestinal environment, relieve or eliminate constipation and diarrhea. It is a better choice for regulating and optimizing human intestinal health.
  • low-temperature yogurt mainly functions from the microorganisms added and proliferated in the production of yogurt, especially functional probiotics; It is easier to keep active in medium and low temperature yogurt is a good carrier for the survival of probiotics.
  • the core of functional probiotics lies in the selection of strains, the verification and strengthening of their functions, and only strains that have been scientifically verified can play a safe and effective role in functional health.
  • the purpose of the present invention is to provide a kind of animal bifidobacteria, which can not only improve the intestinal health of the population, but also has the potential to enhance immunity; meanwhile, the present invention provides a simple and easy breeding method and application, and the efficiency High and low cost.
  • the animal Bifidobacterium of the present invention is Bifidobacterium animalis IU-100 in the genus Bifidobacterium, and the animal Bifidobacterium has been preserved in China Microbial Species on September 5, 2016 The General Microbiology Center of the Preservation and Management Committee, Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, classified as Bifidobacterium animalis, and deposited as CGMCC No.12942.
  • Bifidobacterium animal strains are the Bifidobacterium strains obtained by selecting and purifying the fecal samples of the elderly with a high number of intestinal bifidobacteria through years of research on the intestinal flora of long-lived elderly people in Bama, Guangxi, China.
  • the breeding method of described animal bifidobacteria comprises the following steps:
  • Bifidobacterium animalis IU-100 is obtained by carrying out strain identification of the above-selected Bifidobacterium pure culture.
  • the animal bifidobacteria is used for preparing yogurt products.
  • the present invention has the following beneficial effects:
  • the animal Bifidobacterium strain selected by the present invention has an excellent effect of improving the intestinal health of the population, and is specifically manifested in that by improving the intestinal microenvironment of the human body, the function of moistening the intestines and defecation of the human body is realized;
  • the animal Bifidobacterium strain selected by the present invention also has the potential to enhance immunity.
  • Group M As shown in Table 1, all the people in group M are from Jiazhuan Township, where the long-lived elderly are concentrated in the suburbs of Bama.
  • Group S is the direct or collateral descendants of Group M, who also live in Jiazhuan Township, and Group C is from Bama County Welfare Institute. And Baxu Urban Village.
  • group M is the longevity old people living in Jiazhuan Township, the suburb of Bama, group S is the generation of longevity old people living in the suburb of Bama, and group C is the old people living in the county town of Bama.
  • the 8 centenarians in group M came from villages such as Ping'an Village and Poyue Village, Jiazhuan Township. Among them, there were 6 longevity elders in Ping'an Village. The survey results showed that 7 centenarians could still perform housework and even farming activities, confirming that the aging symptoms of the long-lived elderly were delayed.
  • each elderly person was given a numbered, weighed and sterilized 50mL sampling tube and an ice box.
  • an additional sampling tube containing 20mL of sterilized anaerobic diluent is issued for the isolation and screening of Bifidobacterium strains from long-lived elderly.
  • Each elderly person collects a fresh fecal sample and packs it in a collection tube (the elderly in group M divide the sample into two sampling tubes), put it in the ice box immediately, and notify the sample collector, and the sample will be transported back to the local experiment within 4 hours room.
  • the fecal samples were soaked in anaerobic diluent for bacterial isolation and culture immediately; another sample tube was stored in liquid nitrogen, then transferred to a dry ice incubator, airlifted back to Beijing, stored at -80°C, and processed within 7 days complete.
  • CGMCC General Microbiology Center
  • Safety evaluation is a prerequisite for the application of probiotics, and Bifidobacterium animalis IU-100 is a potential probiotic that has been extensively studied.
  • the present invention conducts in vitro and in vivo safety evaluation of the strain, and provides a basic guarantee for the application of the strain.
  • Antibiotic susceptibility and biogenic amine production of IU-100 were evaluated in vitro; oral toxicity and translocation risk of IU-100 were evaluated in vivo.
  • strain IU-100 had no antibiotic-susceptibility peculiar to the strain, which ruled out the risk of lateral transfer of antibiotic resistance; no biogenic amine was found to be produced by the strain after culturing it in decarboxylase medium; 9. After 10 10 cfu/mouse was fed for 28 days, the mice did not have adverse reactions, and the strain was not found in blood, kidney and liver. The study found no unsafe factors for strain IU-100.
  • Bifidobacterium animalis IU-100 isolated from the long-lived old man in Bama; Staphylococcus aureus ATCC25923 was purchased from China General Microorganism Collection.
  • mice SPF grade Balb/C mice, 6 to 8 weeks old, female. Raised in SPF animal room: temperature 22 ⁇ 2°C, humidity 55% ⁇ 2%, 12h light/dark cycle, free water and food intake. Mice were randomly divided into 10 groups (10 mice in each group), and the groups were as follows:
  • each mouse was intragastrically administered 2 ⁇ 10 8 , 2 ⁇ 10 9 and 2 ⁇ 10 10 cfu/mouse for 28 days (0.2 mL per mouse per day);
  • Control group gavage with normal saline
  • intraperitoneal injection group (3 groups): inject bacterial suspension with a concentration of 2 ⁇ 10 9 cfu/mouse, 0.2 mL per mouse;
  • Intraperitoneal injection control group (3 groups): inject normal saline, 0.2 mL per mouse.
  • mice The three groups of intraperitoneal injection group and control group were sacrificed at 2, 5, and 10 days after injection, respectively, to detect the translocation of strains. During the experiment, the general physical signs, behavior, poisoning performance and death of the mice were observed every day, and the weight and food intake of the mice were recorded every week.
  • mice Bacterial translocation in blood, liver, spleen and kidney of mice was analyzed. 50uL blood was spread on MRS plate and BHI plate, MRS and BHI were cultured anaerobic and aerobic respectively at 37°C for 48h, and the number of colonies on the plate was counted. Tissue samples were suspended in sterile physiological saline at 1 g/mL, and after homogenization, 100 uL were spread on MRS plates and BHI plates respectively, and cultured in the same way as blood samples. Colony growth was recorded as positive.
  • Blood routine indexes reflect the side effects of experimental strains in blood, and blood biochemical indexes reflect nutritional deficiencies and metabolic imbalances. Changes in these indicators usually precede other clinical symptoms.
  • Strain IU-100 has no antibiotic resistance other than species-specific resistance, and there is no risk of antibiotic transfer; the amine production experiment did not find the production of biogenic amines. Oral administration of 10 8 , 10 9 , and 10 10 cfu/day in mice did not develop translocation in blood, liver, spleen and kidney for 28 days; it showed high safety of translocation. The safety evaluation of Bifidobacterium animalis IU-100 found no unsafe factors of the strain.
  • the tolerance of IU-100 was evaluated by comparing the laboratory strain IU-100 with the commercial strain BB12.
  • the OD 600nm value of IU-100 increased by about 2.0 and 0.5 when the bile salt concentration was 0% and 0.1%, respectively; when the bile salt concentration was 0.2% and 0.3%, there was basically no increase.
  • IU-100 is similar to the commercial strain BB12 in acid resistance, bile salt resistance and artificial gastrointestinal fluid resistance, and has the potential to develop into a commercial strain.
  • the experimental animals of the present invention are: Balb/c female mice, 6-8 weeks old, weighing 18-22 g, SPF grade, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • Detection methods include:
  • mice in each group were fasted for 16 hours.
  • the model control group, the positive control group and the three dose groups were given compound diphenoxylate (10 mg/kgBW) by gavage, and the blank control group was given distilled water.
  • the mice in the blank control group and the model control group were gavaged with ink. They were kept in single cages and fed with normal drinking water. From the time of filling the ink, the time of the first black stool, the number and weight of the black stool and the water content of the feces of each animal were recorded within 5 hours.
  • the upper end of the intestinal tube from the pylorus and the lower end to the ileocecal area was cut, placed on a tray, and the small intestine was gently pulled into a straight line. , from the pylorus to the front of the ink is the "ink advance length".
  • Ink propelling rate % ink propelling length (cm)/total length of small intestine (cm) ⁇ 100%
  • IU-100 bacterial strains were orally gavaged in experimental mice, and after 7 days, the influence of defecation time, particle, weight and fecal water content on constipation model mice was measured, and the results are shown in the following table:
  • the experimental animals were given IU100 viable bacteria for 15 days, and the small intestine propulsion test was performed.
  • the emptying period lasted a total of 10 days. During the period, volunteers could not drink any fermented products (including yogurt, cheese, active lactic acid bacteria drinks, etc.), but could drink milk. Day 10 ⁇ 1 was the time of the first stool sample collection.
  • the drinking period lasts for a total of 20 days. During the period, the volunteers were not allowed to drink any fermented products (including yogurt, cheese, active lactic acid bacteria drinks, etc.) except for drinking a bottle of IU-100 yogurt within one hour after lunch every day. In addition, it is necessary to fill in the meal statistics form on time every day. Day 10 ⁇ 1 was the time of the second stool sample collection, and day 20 ⁇ 1 was the time of the third stool sample collection, and the body weight was recorded.
  • any fermented products including yogurt, cheese, active lactic acid bacteria drinks, etc.
  • the preparation method of the fermented milk rich in Bifidobacterium lactis IU-100 in the present invention comprises the following steps:
  • the raw milk is heated up to 60°C, and uniformly mixed white granulated sugar, agar, whey protein concentrate, and starch material are added, the homogenization and sterilization are completed, and the temperature is lowered to 43°C;
  • step (3) the fermented milk obtained in step (2) is cooled to 4° C. after demulsification, and then transferred to an environment with a temperature of 4° C. for refrigerating and ripening for 8 hours to obtain Bifidobacterium lactis IU- 100 of fermented milk.
  • Ordinary yogurt solidified yogurt, 200g/bottle, fermented strains: Streptococcus thermophilus and Lactobacillus bulgaricus.
  • Bifidobacterium-containing yogurt group coagulated yogurt, 200g/bottle, fermented strains: Streptococcus thermophilus, Lactobacillus bulgaricus, Bifidobacterium animalis (maintain more than 1 ⁇ 10 8 CFU/ml during the experiment).
  • the number of bowel movements per day decreased by 0.41 times/day in the group taking regular yogurt, and the number of bowel movements improved in 9.5% of the people.
  • the frequency of defecation increased by 0.4 times/day in the group of people who took yogurt containing bifidobacteria, and 33.3% of the population improved the frequency of defecation.
  • the difference was significant, indicating that taking yogurt containing bifidobacteria, the frequency of defecation was significantly improved. .
  • the Bristol stool score is an important indicator for evaluating the health status of stool, of which 4 is the most ideal score, and the closer to 4, the higher the health.
  • the table above shows the changes in Bristol score before and after 20 days of consumption of yogurt. There is no significant difference between groups A and B. Among them, the improvement rate of fecal morphogenesis in group A was 21.4%, while that in group B was 46.5. There was a significant difference between the two. The results show that yogurt containing bifidobacteria can significantly improve the fecal morphology of the population compared with ordinary yogurt.
  • Table 10 counts the proportion of people with ideal feces (4 servings) before and after taking yogurt for 20 days.
  • the proportion of people with ideal feces (4 servings) in group A only increased by 2.4%
  • the proportion of people in group B with ideal feces (4 servings) increased by only 2.4%.
  • the yogurt containing bifidobacteria can significantly improve the fecal morphology of the crowd.
  • Table 11 counts the changes in the defecation somatosensory state of the crowd before and after taking yogurt for 20 days. It is subdivided into six aspects, including defecation pain, defecation incompleteness, defecation effort, defecation time, unsuccessful defecation times, and defecation discomfort. It shows that before and after taking yogurt, there is no significant change in defecation body sensation, and there is no significant change in yogurt in groups A and B, which may be related to the fact that the experimental population itself has no defecation disorder.
  • the experiment selected 110 healthy adults for the population experiment, and at the end of the experiment there were 90 valid populations.
  • the results showed that compared with ordinary yogurt, taking yogurt containing Bifidobacterium IU-100 did not significantly affect the state of defecation, but the frequency of defecation and the score of stool morphology were significantly improved.
  • Raw milk Shandong Deyi Dairy Co., Ltd. Gaoqing Collins raw milk, white sugar, agar, starch, whey protein concentrate: protein content 34.5%, pectin, strains (commercial freeze-dried strains, Streptococcus thermophilus , Lactobacillus delbrueckii subsp. bulgaricus), IU-100 Bifidobacterium lactis (Shandong Deyi Dairy Co., Ltd. own strain).
  • DHP-9272 type electric heating constant temperature incubator constant temperature water bath, JHG-Q54-P70 high pressure homogenizer, ZHJH-1109B sterile operating table, LDZX-40CI type vertical automatic electric heating pressure steam sterilizer, Haier refrigerator, IKA RW20digital Digital mixer.
  • Acidity Determined according to GB5413.34-2010 Determination of acidity in milk and other dairy products.
  • Fat determined according to the Soxhlet extraction method in GB5009.6-2016.
  • Microorganisms test according to GB4789.18-2010.
  • IU100 probiotic yogurt stops acidity 75°T
  • Escherichia coli 5, 2, 0, 0;
  • the number of viable bacteria in 1 day was 9.2 ⁇ 10 7 CFU/g;
  • the number of viable bacteria in 7 days was 8.8 ⁇ 10 7 CFU/g;
  • the number of viable cells in 21 days was 5.4 ⁇ 10 7 CFU/g.

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Abstract

一种动物双歧杆菌及其选育方法和应用，所述动物双歧杆菌为双歧杆菌属中的动物双歧杆菌IU-100，保藏编号为CGMCC No.12942，是通过持续多年对中国广西巴马地区长寿老人肠道菌群的研究，选择肠道双歧杆菌数量高的老人粪便样品，利用选择培养法分离纯化得到的双歧杆菌菌株，不仅能改善人群肠道健康状况，还具有潜在的提升免疫力作用；同时提供了一种简单易行的选育方法和应用，效率高，成本低。

Description

动物双歧杆菌及其选育方法和应用 技术领域
本发明涉及一种动物双歧杆菌及其选育方法和应用，属于乳双歧杆菌技术领域。
背景技术
益生菌是一类能够改善宿主微生态平衡、发挥对人体有益作用的活性微生物，广泛存在于人和动物的肠道、母乳、传统发酵食品等处。益生菌定殖于人体，如肠道内，在营养物质的消化与吸收、机体的生长与发育、免疫与拮抗等方面均发挥着重要的生理作用。一个正常成人体内，肠道含微生物种属超过1000种，具体可分为三大类：原籍菌群、条件致病菌和过路菌。原籍菌群为具有免疫调节等作用的肠道优势菌；条件致病菌在肠道菌群平衡时无害，特定条件下会对人体有害；有害菌，正常情况下该类菌数量少，菌群失衡时可致病。便秘、腹泻等人群常伴随着肠道菌群的失衡。
随着社会发展，人民大众对自身的健康日益重视，通过运动和健康饮食的身体维护已成为目前主要的保健方式。但繁忙的工作、不规律的生活作息、孕期的身体变化等因素导致目前人群肠道健康的异常，如便秘和腹泻，成为各年龄段人群均具有且较为主要的亚健康表现。通过药物可以治疗肠道异常，但是会影响肠道的微生态平衡，并且对大多数人来说服药并非一种健康并且持续的方式。
具有肠道优化功能的活性益生菌或者载有此类活性益生菌的酸奶制品也具有改善人体肠道环境、缓解或消除便秘和腹泻的作用，不仅不会破坏人体肠道微环境，还具有恢复和改善的作用，是一种调节优化人体肠道健康的更佳选择。
此外，低温酸奶作为一种集美味与健康一体的食物，其主要功能性来自于酸奶生产中添加和增殖的微生物，特别是具有功能性的益生菌；益生菌在低温酸奶这种营养丰富的食品中更用容易保持活性，低温酸奶是益生菌存活的良好载体。
功能益生菌的核心在于菌种的选择、功能的验证和强化，只有通过科学验证的菌种才能安全有效的起到相应的功能健康作用。
发明内容
本发明的目的在于提供一种动物双歧杆菌，其不仅能改善人群肠道健康状况，还具有潜在的提升免疫力作用；同时本发明提供了一种简单易行的选育方法和应用，效率高，成本低。
本发明所述的动物双歧杆菌，该菌株为双歧杆菌(Bifidobacterium)属中的动物双歧杆菌 IU-100，该动物双歧杆菌已于2016年9月5日，保藏至中国微生物菌种保藏管理委员会普通微生物中心，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，分类命名为动物双歧杆菌(Bifidobacterium animalis)，保藏编号为CGMCC No.12942。
动物双歧杆菌菌株是通过持续多年对中国广西巴马地区长寿老人肠道菌群的研究，选择肠道双歧杆菌数量高的老人粪便样品，利用选择培养法分离纯化得到的双歧杆菌菌株。
用所述的动物双歧杆菌改善人群肠道健康状况。
所述的动物双歧杆菌的选育方法，包括以下步骤：
(1)将浸泡于厌氧稀释液的长寿老人粪便样品漩涡振荡制备粪便悬液，以厌氧稀释液连续10倍梯度稀释，使用NPNL-X-Gal选择性培养基倾注平板，在厌氧袋中配合厌氧产气包37℃培养40-60h，分离潜在双歧杆菌；
(2)从平板上挑取蓝色菌落，经过革兰氏染色镜检后，选取革兰氏阳性，菌体呈不规则杆菌的菌落，继续分离纯化2-3次，直到分离出潜在双歧杆菌纯培养物；
(3)将上述分离出的若干双歧杆菌纯培养物进行细胞粘附实验，优选出粘附能力优良的菌株；
(4)将上述优选出的双歧杆菌纯培养物进行菌种鉴定，获得动物双歧杆菌IU-100。
所述的动物双歧杆菌，应用于制备酸奶产品。
与现有技术相比，本发明具有以下有益效果：
(1)本发明选育出的动物双歧杆菌菌株，具有优异的改善人群肠道健康的功效，具体表现为通过改善人体肠道微环境，实现人体润肠通便的功能；
(2)本发明选育出的动物双歧杆菌菌株，还具有潜在的提升免疫力作用。
具体实施方式
下面结合实施例对本发明作进一步的说明，但并不限制本发明的实施。
实施例1
样品采集：
于2007年4月赴广西壮族自治区巴马县，对长寿老人生活状况进行调查并采集了老人粪便样品。
如表1所示，M组所有人群来自巴马郊区长寿老人集中的甲篆乡，S组为M组直系或旁系子一代人群，同样生活于甲篆乡，C组来自巴马县城福利院及巴徐城中村。
其中M组为居住于巴马郊区甲篆乡的长寿老人，S组为同居住于巴马郊区长寿老人子一代人群，C组为居住于巴马县城的老人。
表1 采样人群分组情况
Figure PCTCN2021117750-appb-000001
M组中8名百岁老人来自甲篆乡平安村、坡月村等村落，其中平安村长寿老人最为集中，为6名。调查结果显示，7名百岁老人仍然可以进行家务甚至农耕活动，证实长寿老人机体衰老表征被延缓。
采样前每名老人发放已经编号、称重、灭菌的50mL采样管，同时发放冰盒一只。对于Group M老人额外发放一只内含有20mL灭菌厌氧稀释液采样管，用于长寿老人源双歧杆菌菌株的分离筛选。
每名老人自行采集一份新鲜粪便样本装于采集管(M组老人将样品分装于两只采样管)，立即装入冰盒，并通知样品收集人员，样品在4小时内运回当地实验室。
浸泡于厌氧稀释液粪便样品立即进行菌株分离培养；另一支样品管置于液氮保存，之后转入干冰保温箱，空运回北京，置于-80℃保存，并于7天之内处理完毕。
实施例2
动物双歧杆菌的分子生物学菌种鉴定：
1、利用细菌基因组提取试剂盒提取潜在双歧杆菌菌株基因组DNA。
2、使用27F、1541R引物PCR扩增细菌16S rDNA全序列，产物经DNA纯化试剂盒回收纯化后送至Invitrogen(北京)公司进行测序。
3、将测序结果输入NCBI数据库，经过BLASTn同源性比对，鉴定菌株种属。
测序结果为：
Figure PCTCN2021117750-appb-000002
Figure PCTCN2021117750-appb-000003
4、比对：
将上述16s rDNA序列在NCBI上进行比对，发现该序列与动物双歧杆菌亚种菌株2601(Bifidobacterium animalis subsp.lactis strain 2601)的16s rDNA序列同源性为99％，E value值为0。因此，可确定该分离菌株即为动物双歧杆菌。
5、保藏：
我们随后将该菌株送到中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)进行保藏，保藏编号为CGMCC No.12942。
实施例3
菌种安全性评价：
安全性评价是益生菌应用的前提，Bifidobacterium animalis IU-100是一株广泛研究的潜在益生菌。本发明对该菌株进行体外及体内的安全性评价，为菌株应用提供基础保障。
通过体外实验评价了IU-100的抗生素抗敏感性和生物胺的生成；体内实验评价了IU-100的经口毒性及易位风险。
结果表明，菌株IU-100不存在菌株特有的抗生素敏感型，排除了抗生素抗性横向转移的风险；经过在脱羧酶培养基中培养未发现菌株产生生物胺；灌胃试验发现，10 8、10 9、10 10cfu/小鼠28天饲喂后，小鼠未发生不良反应，在血液、肾脏、肝脏未发现该菌株。研究未发现菌株IU-100的不安全因素。
下面针对动物实验及细菌位移实验进行详述。
1.实验材料
菌株：Bifidobacterium animalis IU-100，分离自巴马长寿老人；Staphylococcus aureus ATCC25923购自中国普通微生物保藏中心。
2.实验方法
(1)动物实验设计：
SPF级Balb/C小鼠，6到8周龄，雌性。饲养于SPF级动物房：温度22±2℃，湿度55％±2％，12h灯照/黑暗循环，自由饮水和摄食。小鼠随机分为10组(每组10只)，分组如下：
灌胃组(3组)，每只小鼠分别以每天2×10 8,2×10 9和2×10 10cfu/鼠灌胃28d(每只小鼠每天0.2mL)；
对照组，用生理盐水灌胃；腹腔注射组(3组)：注射浓度为2×10 9cfu/鼠的菌悬液，每只小鼠0.2mL；
腹腔注射对照组(3组)：注射生理盐水，每只小鼠0.2mL。
三组腹腔注射组及对照组分别于注射后2、5、10天处死，检测菌株易位情况。实验期间每天观察小鼠的一般表观体征、行为、中毒表现和死亡情况，每周记录小鼠的体重、摄食量。
(2)细菌移位能力评价：
分析小鼠血、肝脏、脾脏及肾脏的细菌易位情况。50uL血涂布于MRS平板及BHI平板，MRS及BHI于37℃下分别厌氧及有氧培养48h，计数平板上的菌落数。组织样品，以1g/mL悬于无菌生理盐水，匀浆后取100uL分别涂布于MRS平板及BHI平板，并于血样同样方法培养。有菌落生长即记为阳性。
(3)统计分析试验结果数据用均值±标准差(±SD)表示。不同处理方法之间均值比较采用单因素方差分析进行差异显著性检验，不同处理之间移位率比较采用卡方检验。所有的统计 分析都采用统计软件SPSS17.0版，显著性水平为P<0.05。
3.结果分析
(1)口服IU-100
血常规指标反映实验菌株在血液中的副作用，血生化指标反映营养不足及代谢失衡的状况。这些指标的变化通常早于其他的临床症状。
本实验中实验组和对照组血常规和血生化指标未发生任何显著变化，说明实验菌株对实验动物无不利影响。
表2 经口灌胃组血常规指标(n＝10)
Figure PCTCN2021117750-appb-000004
Figure PCTCN2021117750-appb-000005
表3 经口灌胃组小鼠被试菌转移到肝、脾、肾和血液的移位率
Figure PCTCN2021117750-appb-000006
4.结论
菌株IU-100不存在种属特有耐性以外的抗生素耐受性，不存在抗生素转移的风险；产胺实验未发现生物胺的产生。口服10 8、10 9、10 10cfu/天的小鼠28天未发生血液、肝脏、脾脏和肾脏的易位；表现为较高的易位安全性。动物双歧杆菌IU-100安全性评价未发现菌株的不安全因素。
实施例4
菌种耐受能力评价：
将实验室的菌株IU-100与商业菌株BB12作比较，对IU-100的耐受性进行评价。
采用对IU-100和BB12进行耐酸性实验、耐胆盐性实验以及模拟人工胃肠液实验，用平板计数和生长曲线的形式来评价IU-100的耐受性。
根据实验结果可得出以下结论：
在0-3h内，IU-100在pH6.5、3.0和2.5的条件下存活数均保持原有数量级；
在pH2.0的条件下存活数下降约2.5个数量级。
在不同条件下处理14h后，IU-100在胆盐浓度为0％和0.1％时，OD 600nm值分别增长约2.0和0.5；胆盐浓度为0.2％、0.3％时，基本无增长。
IU-100在人工胃液和人工肠液中分别培养2h和6h后，存活数均上升约1个数量级。
IU-100在耐酸性、耐胆盐性及耐人工胃肠液性能方面与商业菌株BB12相似，有发展成为商业菌株的潜能。
实施例5
动物实验(润肠通便小鼠实验)：
动物实验力在证明动物双歧杆菌IU100菌株对便秘小鼠具有润肠通便作用。
本发明的实验动物为：Balb/c雌性小鼠，6-8周龄，体重18-22g，SPF级，购自北京维通利华实验动物技术有限公司。
检测方法包括：
(1)排便时间、粪便粒数和粪便重量的测定：给受试样品7日后，各组小鼠禁食不禁水16小时。模型对照组，阳性对照组和三个剂量组小鼠灌胃给予复方地芬诺酯(10mg/kgBW)，空白对照组给蒸馏水。给复方地芬诺酯0.5小时后，空白对照组和模型对照组小鼠用墨汁灌胃，处理组给予含受试样品的墨汁(含5％的活性炭粉、10％阿拉伯树胶)，动物均单笼饲养，正常饮水进食。从灌墨汁开始，记录每只动物首粒排黑便时间、5小时内排黑便粒数及重量及粪便含水率。
(2)小肠推进试验：给受试样品15天后，各组小鼠禁食不禁水16小时。模型对照组和处理组灌胃给予复方地芬诺酯(5mg/kgBW)，空白对照组给蒸馏水。给复方地芬诺酯后0.5小时后，处理组分别给予含受试样品的墨汁(含5％的活性炭粉、10％阿拉伯树胶)，对照组给墨汁灌胃。25分钟后立即脱颈椎处死动物，打开腹腔分离肠系膜，剪取上端自幽门、下端至回盲部的肠管，置于托盘上，轻轻将小肠拉成直线，测量肠管长度为“小肠总长度”，从幽门至墨汁前沿为“墨汁推进长度”。
按下式计算墨汁推进率：
墨汁推进率％＝墨汁推进长度(cm)/小肠总长度(cm)×100％
结果与分析：
1.排便时间、粪便粒数和粪便重量
实验小鼠经口灌胃IU-100菌株，7日后测定对便秘模型小鼠排便时间、颗粒、重量及粪 便含水量的影响，结果见下表：
表4 IU-100对便秘老鼠首次排便时间、颗粒、重量及粪便含水量的影响
Figure PCTCN2021117750-appb-000007
▲与空白组对比， ▲▲p<0.01；*与模型组对比，*p<0.05；**p<0.01； ■与阳性对照比较 ■p<0.05
与空白对照组比较，模型组动物首次排黑便时间、排便颗粒和排便重量显著性差异，说明便秘小鼠模型成立。不同剂量IU100菌液均缩短了便秘小鼠的首次排便时间，增加小鼠排便颗粒和粪便重量，尤其是中剂量组样品，首次排黑便时间与模型组有显著性差异，作用效果优于阳性对照菌株。
2.小肠推进试验研究结果
试验动物给IU100活菌15日，做小肠推进试验，各组小鼠肠道的墨汁推进率见下表。
表5 IU100对小鼠小肠运动的影响
Figure PCTCN2021117750-appb-000008
Figure PCTCN2021117750-appb-000009
▲与空白组对比， ▲▲p<0.01；*与模型组对比，*p<0.05；**p<0.01。
结果表明，模型组墨汁推进率显著低于空白组，小鼠便秘模型成立。IU100给药15日，各剂量组小鼠墨汁推进率均显著高于模型组，其中低、高剂量组达到极显著水平，与空白对照接近。IU100组小鼠与阳性对照组比较无差异。研究结果表明IU100菌株能显著增进便秘小鼠小肠蠕动，促进肠内容物的推进，对便秘小鼠具有润肠通便功能。
最终结果表明：IU100给药7日，可明显改善便秘小鼠首次排黑便时间、增加排黑便粒数和重量，给药15日，可以显著提高便秘小鼠小肠墨汁推进率。表明动物双歧杆菌IU100菌株对便秘小鼠具有润肠通便作用。
实施例6
含动物双歧杆菌酸奶对健康成人肠道改善作用：
1.实验流程
本实验共分为以下三个阶段：
(1)志愿者初筛：
筛选出符合实验要求的志愿者。
(2)排空期：
排空期共持续10天。期间，志愿者不可饮用任何发酵制品(包括酸奶、奶酪、活性乳酸菌饮料等)，但可饮用牛奶。第10±1天为第一次粪便样品采集时间。
(3)酸奶饮用期：
饮用期共持续20天。期间，志愿者除每天中午饭后一小时内饮用一瓶IU-100酸奶外，不可饮用任何发酵制品(包括酸奶、奶酪、活性乳酸菌饮料等)。此外，需要每天按时填写膳食统计表。第10±1天为第二次粪便样品采集时间，第20±1天为第三次粪便样品采集时间，记录体重。
本发明中富含乳双歧杆菌IU-100的发酵乳的制备方法包括以下步骤：
(1)生牛乳升温至60℃，加入混合均匀的白砂糖、琼脂、浓缩乳清蛋白、淀粉化料，均质杀菌完毕，降温至43℃；
(2)向步骤(1)处理过的原料乳接种嗜热链球菌，德氏乳杆菌保加利亚亚种，IU-100乳双歧杆菌，搅拌混合均匀，在温度43℃下，静置发酵至滴定酸度大于70°T，得发酵乳；
(3)将步骤(2)制得的发酵乳破乳后冷却至4℃，灌装后转入温度为4℃的环境中进行冷藏后熟8h，即可得富含乳双歧杆菌IU-100的发酵乳。
2.实验样本及人群分组
经过筛选，入组人群共计110人，期间由于流感、服药等因素退出20人，人群分组情况如下表所示：
表6
组别 男 女 共计人数 平均年龄 实验结束有效人数
普通酸奶对照组-A组 12 40 52 23.2±1.4 42
含双歧杆菌酸奶组-B组 23 35 58 25.3±5.0 48
实验样本：
(1)普通酸奶：凝固型酸奶，200g/瓶，发酵菌种：嗜热链球菌和保加利亚乳杆菌。
(2)含双歧杆菌酸奶组：凝固型酸奶，200g/瓶，发酵菌种：嗜热链球菌、保加利亚乳杆菌、动物双歧杆菌(实验期内保持1×10 8CFU/ml以上)。
3.排便状况评分标准
表7
Figure PCTCN2021117750-appb-000010
4.实验结果
表8 排便次数统计
Figure PCTCN2021117750-appb-000011
服用普通酸奶的人群，每日排便次数下降0.41次/天，9.5％的人群排便次数改善。而服用 含双歧杆菌酸奶的人群，排便次数增加了0.4次/天，33.3％的人群排便次数改善，与对照A组相比，差异显著，说明服用含双歧杆菌的酸奶，排便次数显著改善。
表9 布里斯托粪便评分(4分为最理想评分，越接近4分越理想)
Figure PCTCN2021117750-appb-000012
布里斯托粪便评分是评价粪便健康状况的重要指标，其中4分为最理想评分，越接近4分说明健康程度越高。上表统计了人群服用酸奶20天前后，布里斯托评分的变化，前后对比来看，A、B两组没有显著差异。其中A组中粪便形态发生改善的比例21.4％，而B组为46.5，二者相比差异显著。结果表明，含双歧杆菌的酸奶相比普通酸奶，能显著改善人群的粪便形态。
表10 理想粪便(4分)形状人员比例
分组 0天 20天 改变
A组 35.7％(15/42) 38.1％(16/42) 2.4％(1/42)
B组 37.5％(18/48) 52.0％(26/48) 16.6％(8/48)
统计分析 P>0.05 P>0.05 P<0.05
表10统计了人群服用酸奶20天前后理想粪便(4份)人员的比例，A组中理想粪便(4份)人员的比例仅仅提高了2.4％，B组中理想粪便(4份)人员的比例提高了16.7％，与对照组差异显著，结果表明，含双歧杆菌的酸奶相比普通酸奶，能显著改善人群的粪便形态。
表11 排便体感相关症状
Figure PCTCN2021117750-appb-000013
Figure PCTCN2021117750-appb-000014
表11统计了人群服用酸奶20天前后，人群排便体感状态的前后变化，共细分为排便痛感、排便不尽感、排便用力程度、排便时间、不成功次数及排便不适等6个方面，结果表明服用酸奶前后，排便体感没有显著变化，A、B两组酸奶也无显著变化，这可能由于实验人群本身没有排便障碍有关。
综上所述，实验选取了健康成人110人进行人群实验，实验结束时有效人群90人。经20天的双盲口服实验，结果表明与普通酸奶相比，服用含双歧杆菌IU-100的酸奶，排便体感状态并无显著影响，而排便次数及粪便形态评分显著改善。
实施例7
含双歧杆菌IU-100的益生菌酸奶制作：
1、材料与仪器
生牛乳：山东得益乳业股份有限公司高青牧场生牛乳，白砂糖，琼脂，淀粉，浓缩乳清蛋白：蛋白含量34.5％，果胶，菌种(市售冻干菌种，嗜热链球菌、德氏乳杆菌保加利亚亚种)，IU-100乳双歧杆菌(山东得益乳业股份有限公司自有菌种)。
DHP-9272型电热恒温培养箱、恒温水浴锅、JHG-Q54-P70高压均质机、ZHJH-1109B无菌操作台、LDZX-40CI型立式自动电热压力蒸汽灭菌锅、海尔冰箱、IKA RW20digital数显型搅拌器。
2、样品的制备
(1)0.1mol/L NaOH标准溶液的制备：按照GB/T 601-2016化学试剂标准滴定溶液的制备进行制备。
3、工艺流程
产品制作的工艺流程如下所示：
Figure PCTCN2021117750-appb-000015
4、理化及微生物指标的测定
酸度：按照GB5413.34-2010中乳及其他乳制品中酸度的测定进行测定。
脂肪：按照GB5009.6-2016中索氏抽提法进行测定。
蛋白质：按照GB2009.5-2016中凯氏定氮法进行测定。
微生物：按照GB4789.18-2010进行检验。
5、检测结果
IU100益生菌酸奶终止酸度75°T；
脂肪含量3.7g/100g；
蛋白质含量3.0g/100g；
霉菌：未检出；
酵母菌：未检出；
致病菌：5，0，0，-；
大肠杆菌：5，2，0，0；
各项指标均符合GB19302-2010的要求。
货架期内IU100益生菌酸奶中乳双歧杆菌活菌数：
1天活菌数为9.2×10 7CFU/g；
7天活菌数为8.8×10 7CFU/g；
21天活菌数为5.4×10 7CFU/g。

Claims (4)

  1. 一种动物双歧杆菌，其特征在于：该菌株为双歧杆菌(Bifidobacterium)属中的动物双歧杆菌IU-100，该菌株的保藏编号为CGMCC No.12942。
  2. 根据权利要求1所述的动物双歧杆菌，其特征在于：用所述的动物双歧杆菌改善人群肠道健康状况。
  3. 一种权利要求1所述的动物双歧杆菌的选育方法，其特征在于：包括以下步骤：
    (1)将浸泡于厌氧稀释液的长寿老人粪便样品漩涡振荡制备粪便悬液，以厌氧稀释液连续10倍梯度稀释，使用NPNL-X-Gal选择性培养基倾注平板，在厌氧袋中配合厌氧产气包37℃培养40-60h，分离潜在双歧杆菌；
    (2)从平板上挑取蓝色菌落，经过革兰氏染色镜检后，选取革兰氏阳性，菌体呈不规则杆菌的菌落，继续分离纯化2-3次，直到分离出潜在双歧杆菌纯培养物；
    (3)将上述分离出的若干双歧杆菌纯培养物进行细胞粘附实验，优选出粘附能力优良的菌株；
    (4)将上述优选出的双歧杆菌纯培养物进行菌种鉴定，获得动物双歧杆菌IU-100。
  4. 一种权利要求1所述的动物双歧杆菌的应用，其特征在于：应用于制备酸奶产品。
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