WO2022110281A1 - 两歧双歧杆菌i771、其分离纯化方法及应用 - Google Patents

两歧双歧杆菌i771、其分离纯化方法及应用 Download PDF

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WO2022110281A1
WO2022110281A1 PCT/CN2020/134211 CN2020134211W WO2022110281A1 WO 2022110281 A1 WO2022110281 A1 WO 2022110281A1 CN 2020134211 W CN2020134211 W CN 2020134211W WO 2022110281 A1 WO2022110281 A1 WO 2022110281A1
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bifidobacterium bifidum
parts
weight
culture
medium
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French (fr)
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张栋
魏立华
袁庆彬
王世杰
冯丽莉
薛玉玲
荀一萍
封肖颖
刘尧尧
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君乐宝乳业集团有限公司
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    • 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 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.
    • 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
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • 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
    • 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
    • A23V2400/517—Bifidum
    • 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 belongs to the field of bioengineering, and in particular relates to a strain, a screening method and application thereof, in particular to a Bifidobacterium bifidum i771, a separation and purification method and application thereof.
  • Depression is the fourth largest disease in the world. The popularization, prevention and treatment of depression needs urgent attention. The prevention and treatment of depression has been listed as the priority of national mental health work.
  • Pathogen infection can activate the immune system and release toxins and cytokines, thereby affecting host behavior, metabolism, and neurotransmitter function. The released cytokines can induce neuroendocrine and central nervous system chemical changes, thereby causing depression.
  • lactic acid bacteria may achieve ameliorating effects on depression through multiple pathways and multiple targets. Lactic acid bacteria can improve depression by inhibiting and replacing the adhesion of pathogenic bacteria to epithelial cells through colonization resistance, production of bacteriocins, and enhancement of intestinal barrier to compete with pathogenic bacteria for epithelial cell receptors.
  • Cortisol is a natural hormone secreted by the human adrenal glands. This hormone-secreting gland is located above each kidney. Studies have linked high and low levels of cortisol with depression, and people with clinical depression tend to produce too much cortisol. Blood cortisol levels fluctuate naturally in healthy people, but many depressed patients do not have this natural variation. Cortisol levels are normally highest in the morning and lowest in the evening. And many people with depression have cortisol levels that don't decrease throughout the day. Too much of this hormone can make people feel sad, lonely, and depressed. Patients may also feel low energy or listless.
  • Bifidobacterium is the main flora in the intestinal tract of humans and other animals, accounting for more than 90% of the total bacteria in breast-fed infants. It has the characteristics of high GC content, high nutritional value, strict anaerobicity, and bifurcation of cell ends. , is often added to food.
  • the relationship between bifidobacteria and cortisol is rarely studied; Chinese invention patent CN110331119A, "Bifidobacterium bifidum CCFM1063 and its application" discloses the effect of Bifidobacterium bifidum CCFM1063 on the abundance of other bacterial genera in the intestinal tract.
  • the association study between bifidobacteria and cortisol has not been carried out.
  • the purpose of the present invention is to provide a kind of Bifidobacterium bifidum i771, which is isolated and screened from the feces of breastfed infants or young children, and the bacteria have been deposited in the China Microorganism Culture Collection on May 20, 2020 The General Microbiology Center of the Management Committee, whose address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCCNO.19855, and the Latin name is Bifidobacterium bifidum;
  • Another object of the present invention is to provide the above-mentioned Bifidobacterium bifidum i771 separation and purification method
  • Another object of the present invention is to provide the application of the above-mentioned Bifidobacterium bifidum i771.
  • the bacteria can be used as an active ingredient in beverages, foods, medicines or probiotic products for treating and/or relieving depression, and can also be used to prepare fermented dairy products. Increased yogurt preference.
  • a Bifidobacterium bifidum i771 is preserved in the General Microbiology Center of the China Microorganism Culture Collection Management Committee, and the preservation number is CGMCC NO.19855.
  • the 16SrDNA sequence of the Bifidobacterium bifidum i771 is as follows:
  • the tuf gene sequence of the Bifidobacterium bifidum i771 is:
  • the Bifidobacterium bifidum i771 is isolated and screened from the feces of breast-fed infants or young children.
  • the present invention also provides a method for separating and purifying the Bifidobacterium bifidum i771, which is performed in the following order of steps:
  • the modified MRS solid medium Take the modified MRS solid medium, melt it, and pour it into the first to fifth petri dishes respectively. After cooling and complete solidification, get the medium D1 ⁇ D5, and draw 0.1mL of each of the bacterial suspensions C1 ⁇ C5 and add them one by one. Correspondingly coated on the culture medium D1-D5, then inverted the culture dish, placed in the environment of 35-40 °C for anaerobic culture for 62-82 hours, and observed the colony growth, wherein, the modified MRS solid medium was: The solid medium obtained by adding 15% by mass of agar to every 1000 mL of the improved MRS liquid medium;
  • step S4 As the limitation of the above-mentioned separation and purification method, the following steps are also included after the step S4:
  • the modified MRS liquid medium is composed of 10 parts by weight of casein peptone, 10 parts by weight of beef extract, 5 parts by weight of yeast extract, 20 parts by weight of glucose, 5 parts by weight of acetic acid Sodium, 2 parts by weight of diamine citrate, 1 part by weight of Tween-80, 2 parts by weight of K 2 HPO 4 , 0.2 part by weight of MgSO 4 ⁇ 7H 2 O, 0.05 part by weight of MnSO 4 ⁇ 7H 2 O, 0.5 part by weight of half Cystine and 1000 parts by volume of distilled water are composed; wherein, the corresponding proportional relationship between parts by weight and parts by volume is g:mL.
  • the bacteriological characteristics of the Bifidobacterium bifidum i771 of the present invention are as follows:
  • experimental project result experimental project result a positive Cell morphology polymorphic rod oxidase - contact enzyme -
  • the present invention also provides the application of the above-mentioned Bifidobacterium bifidum i771 as an active ingredient in beverages, foods, medicines or probiotic products for treating and/or relieving depression.
  • the bacteria are used for the preparation of fermented dairy products.
  • the probiotic product is an inoculum prepared from the Bifidobacterium bifidum i771, or the probiotic product is prepared from the Bifidobacterium bifidum i771, Lactobacillus paracasei N1115, rhamnosus A bacterial preparation prepared by mixing Lactobacillus saccharomyces X253 and animal bifidobacteria subsp. i797.
  • Bifidobacterium bifidum i771 provided by the present invention can effectively alleviate the damage of cortisol to human neuroblastoma cells (SH-SY5Y), and then can effectively regulate the level of cortisol in the body to treat and/or alleviate depression disease;
  • Bifidobacterium bifidum i771 provided by the present invention can increase the aroma of fermented milk and improve the preference of yogurt, and the bacterial strain can also regulate the balance of intestinal flora and maintain intestinal stability.
  • strain of the present invention is suitable for the application of active ingredients in beverages, foods, medicines or probiotic products for treating and/or relieving depression.
  • Embodiment 1 Bifidobacterium bifidum i771 and its separation and purification method
  • Bifidobacterium bifidum i771 is isolated and screened from the intestinal flora or feces of breast-fed infants or young children. This strain can regulate the balance of intestinal flora and promote physical health; this strain is preserved in the China Microbial Culture Collection and Management The General Microbiology Center of the Committee, the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is May 20, 2020, the deposit number is CGMCC NO.19855, and the Latin name is Bifidobacterium bifidum.
  • the separation and purification method is carried out according to the following sequence of steps:
  • sample A Take the solution of sample A, add it to the modified MRS liquid medium, and culture it anaerobically for 62-82 hours at 35-40°C to obtain culture solution B.
  • the components of the improved MRS liquid medium include: casein peptone, beef extract, yeast extract, glucose, sodium acetate, diamine citrate, Tween-80, K 2 HPO 4 , MgSO 4 ⁇ 7H 2 O, MnSO 4 7H 2 O, cysteine, distilled water; wherein, casein peptone, beef extract, yeast extract, glucose, sodium acetate, diamine citrate, Tween-80, K 2 HPO 4 , MgSO 4 7H 2 O,
  • the dosage ratio relationship of MnSO 4 ⁇ 7H 2 O, cysteine and distilled water is 10g: 10g: 5g: 20g: 5g: 2g: 1g: 2g: 0.2g: 0.05g: 0.5g: 1000mL.
  • the modified MRS liquid medium is an MRS liquid medium supplemented with 0.5 ⁇ by mass of cysteine; the volume ratio of sample A to the modified MRS liquid medium is 1:10-100.
  • the dilution gradient is 10-1 , 10-2 , 10-3 , 10-4 , 10-5 times, corresponding to the obtained bacteria Suspension C1 ⁇ C5;
  • the modified MRS solid medium is a solid medium obtained by adding 15% by mass of agar to every 1000 mL of the modified MRS liquid medium;
  • the single colony E is streaked onto the new improved MRS solid medium, and cultured in an anaerobic environment at 35 to 40°C for 62 to 82 hours, and cultured three times in a row to obtain a pure culture F, which is the bifurcation.
  • the preservation method of the Bifidobacterium bifidum i771 strain is as follows: the pure culture F is mixed with sterile glycerol with a mass fraction of 50% according to a ratio of 1:1, placed in a strain preservation tube, and after mixing, Store at -80 ⁇ 70°C, and inoculate it to the slant of a new modified MRS solid medium test tube for temporary storage.
  • Example 1 The Bifidobacterium bifidum strain isolated and purified in Example 1 was cultured anaerobically at 37° C. for 48 hours, and the strain characteristics were observed and tested respectively.
  • the basic features of Bifidobacterium bifidum i771 provided by Example 1 are shown in Table 1:
  • experimental project result experimental project result a positive Cell morphology polymorphic rod oxidase - contact enzyme -
  • strains were identified by molecular biology, through DNA extraction, PCR amplification, 16Sr DNA sequencing, and finally determined to be Bifidobacterium bifidum i771 through sequence comparison. in,
  • the SH-SY5Y cells in good condition were seeded in a 96-well plate containing 200 ⁇ L of RPMI 1640 complete medium (10% fbs) per well at an inoculation amount of 5 ⁇ 10 4 per well, and cultured until the cells were confluent at the bottom of 60-80 cells. %.
  • the blank control group, the cortisol intervention group and the experimental group were set respectively, with 3 repetitions in each group. In the cortisol intervention group and the experimental group, 300 ⁇ mol/L cortisol was used to stimulate SH-SY5Y cells, and the cells were treated at 37°C for 24 h to complete the preparation of depression model cells.
  • the depression model cells were intervened with Bifidobacterium bifidum i771 at three doses of 10 7 , 10 6 and 10 5 CFU respectively, and the intervention was performed at 37°C for 4 hours. Finally, cell viability was detected by CCK8 assay.
  • CCK8 cell viability detection method inoculate three cell suspensions (100 ⁇ L/well) with a concentration of 2 ⁇ 10 4 cells/mL in a 96-well plate; place the culture plate in an incubator for pre-culture (37°C, 5% CO 2 ); after inoculating cells, add 10 ⁇ L CCK8 solution to each well at 0h, 24h, 48h, 72h, and 96h respectively; incubate the culture plate in an incubator for 2 hours, and measure the absorbance at 450nm with a microplate reader.
  • Table 3 The experimental results are shown in Table 3:
  • Bifidobacterium bifidum i771 has the effect of alleviating the cell activity of the depression model, and high, medium and low doses have the ability to alleviate cortisol-induced cell damage.
  • Bifidobacterium bifidum i771 has a dose-dependent effect on alleviating cell damage, and has a more obvious effect in the high-dose group.
  • the sensory evaluation method is the overall taste preference using a 9-point intensity scale (9 ⁇ 1 means like ⁇ dislike, ⁇ 5 means not liked), and other sensory characteristics using a 10-point intensity scale (10 ⁇ 0 means extremely strong ⁇ imperceptible). Adopt this method to carry out overall preference, aroma, viscosity, powdery feeling, cohesiveness evaluation to above-mentioned fermented milk sample, and the evaluation results are shown in Table 4:
  • Bifidobacterium bifidum i771 can be used as an active ingredient in beverages, foods, medicines or probiotic products for treating and/or relieving depression.
  • it can be used to prepare cereals and their derivatives, fermented meat products, probiotic products, formula milk powder with intestinal regulation and relieve depression; it can also be used to prepare beverages and fermented yogurt with intestinal regulation function; It is used to prepare medicines with intestinal regulating function, and can be made into capsules, powders, pills, oral liquids or sprays.
  • Bifidobacterium bifidum i771 When Bifidobacterium bifidum i771 is used as a probiotic product, it can be a probiotic product containing only a single species of Bifidobacterium bifidum i771, or it can be a probiotic product composed of Bifidobacterium bifidum i771, Lactobacillus paracasei N1115, rhamnose milk
  • the compound probiotic product is prepared by mixing Bacillus X253 and animal bifidobacteria subsp. i797, and the dosage form of the compound probiotic product can be compound probiotic microcapsule powder.

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Abstract

本发明属于生物工程领域，公开了一种两歧双歧杆菌i771、其分离纯化方法及应用，该菌株是从婴儿粪便中筛选出来的，保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.19855；该两歧双歧杆菌i771是通过对母乳喂养婴儿或幼儿粪便中的目标菌株进行富集、筛选、不断纯化的方法获得的。本发明获得的菌株能够调节肠道菌群平衡、治疗或缓解抑郁症；同时在酸奶发酵方面还能够增加发酵乳香气，提高酸奶喜好度；该菌可用于制备发酵乳制品以及作为治疗和/或缓解抑郁症的药物中活性成分的应用。

Description

两歧双歧杆菌i771、其分离纯化方法及应用 技术领域
本发明属于生物工程领域，尤其涉及一种菌株、其筛选方法及应用，具体地说，是一种两歧双歧杆菌i771、其分离纯化方法及应用。
背景技术
抑郁症是世界第四大疾病，对抑郁症的科普、防范、治疗工作亟待重视，抑郁症防治已被列入全国精神卫生工作重点。病原菌感染可激活免疫系统，释放毒素和细胞因子，从而影响宿主行为、代谢及神经递质功能，释放的细胞因子可诱导神经内分泌与中枢神经化学改变，从而引发抑郁。现有技术中表明乳酸菌可能通过多途径、多靶点实现对抑郁症的改善作用。乳酸菌可通过定植抗力、产生细菌素及增强肠道屏障与病原菌竞争上皮细胞受体，抑制和取代病原菌黏附到上皮细胞，从而改善抑郁。
皮质醇是一种由人体肾上腺分泌产生的天然激素。该荷尔蒙分泌腺体位于每片肾的上方。研究已证实皮质醇水平高低与抑郁存在关联，并且临床抑郁症患者往往产生太多皮质醇。健康者的血液皮质醇水平会自然波动，但许多抑郁病人没有这种自然变化。皮质醇水平正常情况下在早上最高，晚上最低。而许多抑郁症患者的皮质醇水平一整天都不会减少。这种激素过多会使人感觉悲伤，孤独和抑郁。患者还会觉得精力不足或无精打采。
双歧杆菌是人和其他动物肠道内的主要菌群，在母乳喂养的婴儿体内占总菌数的90％以上，其具有高GC含量、营养价值高、严格厌氧、细胞末端分叉等特点，常被添加于食品中。而双歧杆菌与皮质醇之间的关系却少有研究；中国发明专利CN110331119A、“两歧双歧杆菌CCFM1063及其应用”公开了两歧双歧杆菌 CCFM1063对肠道内其他菌属的丰度的影响，进而起到减少焦虑抑郁和肠炎等疾病发生的功能，但并未进行双歧杆菌与皮质醇之间的关联研究。
发明内容
本发明的目的，是要提供一种两歧双歧杆菌i771，它是从母乳喂养的婴儿或幼儿粪便中分离筛选出来的，该菌已于2020年5月20日保藏于中国微生物菌种保藏管理委员会普通微生物中心，地址是北京市朝阳区北辰西路1号院3号，保藏编号为CGMCCNO.19855，拉丁文名称是Bifidobacteriumbifidum；
本发明的另外一个目的，是提供上述的两歧双歧杆菌i771分离纯化方法；
本发明还有一个目的，是提供上述两歧双歧杆菌i771的应用。该菌可作为治疗和/或缓解抑郁症的饮品、食品、药品或益生菌制品中活性成分的应用，还可用于制备发酵乳制品，与常规酸奶相比制备的发酵酸奶乳香气分数更高，提高了酸奶喜好度。
为实现上述目的，本发明所采用的技术方案是：
一种两歧双歧杆菌i771，保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCCNO.19855。
作为本发明的限定，所述两歧双歧杆菌i771的16SrDNA序列如下：
Figure PCTCN2020134211-appb-000001
Figure PCTCN2020134211-appb-000002
作为本发明的另一种限定，所述两歧双歧杆菌i771的tuf基因序列为：
Figure PCTCN2020134211-appb-000003
Figure PCTCN2020134211-appb-000004
作为本发明的第三种限定，所述两歧双歧杆菌i771是从母乳喂养的婴儿或幼儿粪便中分离筛选出来的。
本发明也提供了所述两歧双歧杆菌i771的分离纯化方法，按照以下步骤顺序进行：
S1.取母乳喂养的婴儿或幼儿粪便，加入到浓度为0.9％的无菌生理盐水中混匀，得到样品A溶液；
S2.将所述样品A溶液加入到改良MRS液体培养基中，于35～40℃条件下厌氧培养62～82h，得到培养液B，其中，所述样品A溶液与所述改良MRS液体培养基的体积比为1:10～100；
S3.将所述培养液B用浓度为0.9％的无菌生理盐水以10倍梯度倍增稀释，稀释梯度依次为10 -1、10 -2、10 -3、10 -4、10 -5倍，对应得到菌悬液C1～C5；
取改良MRS固体培养基，融化后，分别倒入第一至第五培养皿中，待冷却、完全凝固后，得培养基D1～D5，分别吸取菌悬液C1～C5各0.1mL并一一对应涂布到所述培养基D1～D5上，然后倒置所述培养皿，置于35～40℃环境下厌氧培养62～82h，观察菌落生长情况，其中，所述改良MRS固体培养基为每1000mL所述改良MRS液体培养基中添加15％质量份数的琼脂所得固体培养基；
待所述培养基D1～D5上出现典型菌落后，挑选出典型的单菌落E；
S4.将所述单菌落E划线接种到新的所述改良MRS固体培养基上，35～40℃厌氧环境下培养62～82h，连续培养三次，得纯培养物F，即为所述两歧双歧杆菌i771的菌株。
作为上述分离纯化方法的限定，所述步骤S4后还包括以下步骤：
S5.将所述纯培养物F(即两歧双歧杆菌i771的菌株))与无菌的质量份数为50％的甘油按照1:1的比例混合，在-80～-70℃环境下保存，同时接种至新的所述改良MRS固体培养基试管斜面用于临时保存。
作为上述分离纯化方法的另一种限定，所述步骤S2中，改良MRS液体培养基由10重量份酪蛋白胨、10重量份牛肉膏、5重量份酵母膏、20重量份葡萄糖、5重量份乙酸钠、2重量份柠檬酸二胺、1重量份吐温-80、2重量份K 2HPO 4、0.2重量份MgSO 4·7H 2O、0.05重量份MnSO 4·7H 2O、0.5重量份半胱氨酸和1000体积份蒸馏水组成；其中，重量份与体积份的对应比例关系为g:mL。
本发明的所述两歧双歧杆菌i771细菌学特性如下：
(1)菌株特性实验
将上述所分离纯化出的菌株于37℃厌氧培养48h，分别进行菌株特性的观察和实验，实验结果证明两歧双歧杆菌i771的基本特征如下：
实验项目 结果 实验项目 结果
革兰氏染色 阳性 细胞形态 多形态杆状
氧化酶 - 接触酶 -
注：“-”表示无相关基本特征。
(2)糖发酵特性实验
将上述分离纯化菌株挑取单菌落进行平板划线，37℃厌氧培养24h，传代一次，取菌悬液接种至糖发酵管中，37℃厌氧培养48h，观察颜色变化。实验结果证明两歧双歧杆菌i771的糖发酵特性如下：
实验项目 结果 实验项目 结果 实验项目 结果
甘油 - 甘露醇 - 松三糖 -
赤藓醇 - 山梨醇 - 棉子糖 -
D-阿拉伯糖 - α-甲基-甘露糖甙 - 淀粉 -
L-阿拉伯糖 - α-甲基-葡萄糖甙 - 糖原 -
D-核糖 - N-乙酰-葡糖胺 - 木糖醇 -
D-木糖 - 苦杏仁甙 - 龙胆二糖 +
L-木糖 - 熊果甙 - D-松二糖 -
阿东醇 - 七叶灵 - D-来苏糖 -
β-甲基-D-木糖甙 - 水杨苷 - D-塔格糖 -
D-半乳糖 + 纤维二糖 - D-岩藻糖 -
D-葡萄糖 + 麦芽糖 - L-岩藻糖 -
D-果糖 + 乳糖 + D-阿拉伯糖醇 -
D-甘露糖 - 蜜二糖 - L-阿拉伯糖醇 -
L-山梨糖 - 蔗糖 - 葡萄糖酸盐 -
L-鼠李糖 - 海藻糖 - 2-酮基-葡萄糖酸盐 -
卫茅醇 - 菊糖 - 肌醇 -
注：“+”表示发酵利用；“-”表示不发酵利用。
(3)分子生物学鉴定
将上述菌株进行分子生物学鉴定，通过DNA提取、PCR扩增，16SrDNA测序。其中：
①两歧双歧杆菌i771的16Sr DNA测序结果如下：
Figure PCTCN2020134211-appb-000005
Figure PCTCN2020134211-appb-000006
②两歧双歧杆菌i771的tuf基因序列为：
Figure PCTCN2020134211-appb-000007
Figure PCTCN2020134211-appb-000008
上述基因序列比对最终确定为两歧双歧杆菌i771。
本发明还提供了上述两歧双歧杆菌i771的应用，该菌作为治疗和/或缓解抑郁症的饮品、食品、药品或益生菌制品中活性成分的应用。
作为上述应用的限定，该菌用于制备发酵乳制品。
作为上述应用的另一种限定，所述益生菌制品为由所述两歧双歧杆菌i771制成的菌剂，或为由所述两歧双歧杆菌i771、副干酪乳杆菌N1115、鼠李糖乳杆菌X253和动物双歧乳亚种i797混合制备而成的菌剂。
由于采用了上述的技术方案，本发明与现有技术相比所取得的有益效果是：
(1)本发明提供的两歧双歧杆菌i771可以有效缓解皮质醇对于人神经母细胞瘤细胞(SH-SY5Y)的损伤，进而可以有效调控皮质醇在体内的水平而治疗和/或缓解抑郁症；
(2)本发明提供的两歧双歧杆菌i771可以增加发酵乳香气，提高酸奶喜好度，该菌株还能够调节肠道菌群平衡，维持肠道稳定。
(3)本发明该菌株适用于治疗和/或缓解抑郁症的饮品、食品、药品或益生菌制品中活性成分的应用。
本发明下面将结合具体实施例作进一步详细说明。
具体实施方式
以下实施例只用于说明本发明，而并不限定本发明。
实施例1两歧双歧杆菌i771及其分离纯化方法
一、菌种信息
两歧双歧杆菌i771是从母乳喂养的婴儿或幼儿肠道菌群或粪便中分离筛选出来的，该菌株能够调节肠道菌群平衡，促进身体健康；该菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地址是北京市朝阳区北辰西路1号院3号，保藏日期是2020年5月20日，保藏编号为CGMCC NO.19855，拉丁文名称是Bifidobacterium bifidum。
二、两歧双歧杆菌i771及其分离纯化方法
该分离纯化方法按照以下步骤顺序进行：
步骤1.采集样品
取母乳喂养的婴儿或幼儿粪便，然后加入到浓度为0.9％的无菌生理盐水中并充分混匀，得到样品A溶液。
步骤2.样品富集
取样品A溶液，加入到改良MRS液体培养基中，于35～40℃条件下厌氧培养62～82h，得到培养液B。
所述改良MRS液体培养基的组成成分包括：酪蛋白胨、牛肉膏、酵母膏、葡萄糖、乙酸钠、柠檬酸二胺、吐温-80、K 2HPO 4、MgSO 4·7H 2O、MnSO 4·7H 2O、半胱氨酸、蒸馏水；其中，酪蛋白胨、牛肉膏、酵母膏、葡萄糖、乙酸钠、柠檬酸二胺、吐温-80、K 2HPO 4、MgSO 4·7H 2O、MnSO 4·7H 2O、半胱氨酸、蒸馏水的用量比例关系为10g：10g：5g：20g：5g：2g：1g：2g：0.2g：0.05g：0.5g：1000mL。
所述改良MRS液体培养基是添加有0.5‰质量份数半胱氨酸的MRS液体培养基；样品A与所述改良MRS液体培养基的体积比为1:10～100。
步骤3.菌株分离筛选
取培养液B，用浓度为0.9％的无菌生理盐水以10倍梯度倍增稀释，稀释梯度依次为10 -1、10 -2、10 -3、10 -4、10 -5倍，对应得到菌悬液C1～C5；
取改良MRS固体培养基，融化后，分别倒入第一至第五培养皿中，待冷却、 完全凝固后，得培养基D1～D5，分别吸取菌悬液C1～C5各0.1mL并一一对应涂布到培养基D1～D5上，然后倒置培养皿，置于35～40℃环境下厌氧培养62～82h，观察菌落生长情况；待平板出现典型菌落后，挑选出典型的单菌落E；
所述改良MRS固体培养基为每1000mL改良MRS液体培养基中添加15％质量份数的琼脂所得固体培养基；
步骤4.菌株纯化
将所述单菌落E划线接种到新的所述改良MRS固体培养基上，35～40℃厌氧环境下培养62～82h，连续培养三次，得纯培养物F，即为所述两歧双歧杆菌i771的菌株；
步骤5.菌株保存
所述两歧双歧杆菌i771菌株的保存方法如下：将纯培养物F与无菌的质量份数为50％的甘油按照1:1的比例混合，置于菌种保存管内，混匀后在-80～70℃环境下保存，同时接种至新的改良MRS固体培养基试管斜面用于临时保存。
实施例2两歧双歧杆菌i771的细菌学特征
一.菌株特性实验
将实施例1所分离纯化出的两歧双歧杆菌菌株37℃厌氧培养48h，分别进行菌株特性的观察和实验。实施例1所提供的两歧双歧杆菌i771的基本特征如表1：
表1.两歧双歧杆菌i771基本特征
实验项目 结果 实验项目 结果
革兰氏染色 阳性 细胞形态 多形态杆状
氧化酶 - 接触酶 -
注：“-”表示无相关基本特征。
二.糖发酵特性实验
将上述分离纯化菌株挑取单菌落进行平板划线，37℃厌氧培养36～48h，传代一次，挑取单菌落接种至MRS液体培养基后厌氧培养36～48h，取菌悬液4mL， 12000rpm离心2min，去上清，用灭菌生理盐水洗涤两次。按API50试剂盒说明操作(将洗涤后的菌体沉淀接种至API50试剂盒提供的培养基内，充分吹吸混匀后，分装至糖各个糖发酵孔内，37℃眼样培养培养48h，观察颜色变化。)，具体结果见表2：
表2.两歧双歧杆菌i771的糖发酵特性
实验项目 结果 实验项目 结果 实验项目 结果
甘油 - 甘露醇 - 松三糖 -
赤藓醇 - 山梨醇 - 棉子糖 -
D-阿拉伯糖 - α-甲基-甘露糖甙 - 淀粉 -
L-阿拉伯糖 - α-甲基-葡萄糖甙 - 糖原 -
D-核糖 - N-乙酰-葡糖胺 - 木糖醇 -
D-木糖 - 苦杏仁甙 - 龙胆二糖 +
L-木糖 - 熊果甙 - D-松二糖 -
阿东醇 - 七叶灵 - D-来苏糖 -
β-甲基-D-木糖甙 - 水杨苷 - D-塔格糖 -
D-半乳糖 + 纤维二糖 - D-岩藻糖 -
D-葡萄糖 + 麦芽糖 - L-岩藻糖 -
D-果糖 + 乳糖 + D-阿拉伯糖醇 -
D-甘露糖 - 蜜二糖 - L-阿拉伯糖醇 -
L-山梨糖 - 蔗糖 - 葡萄糖酸盐 -
L-鼠李糖 - 海藻糖 - 2-酮基-葡萄糖酸盐 -
卫茅醇 - 菊糖 - 肌醇 -
注：“+”表示发酵利用；“-”表示不发酵利用。
三.分子生物学鉴定
将上述菌株进行分子生物学鉴定，通过DNA提取、PCR扩增、16Sr DNA测序，经序列比对最终确定为两歧双歧杆菌i771。其中，
①两歧双歧杆菌i771的16Sr DNA测序结果如下：
Figure PCTCN2020134211-appb-000009
Figure PCTCN2020134211-appb-000010
②两歧双歧杆菌i771的tuf基因序列为：
Figure PCTCN2020134211-appb-000011
实施例3两歧双歧杆菌i771对抑郁症细胞模型干预实验
以每孔5×10 4的接种量，将状态良好的SH-SY5Y细胞接种于每孔含200μL RPMI 1640完全培养基(10％fbs)的96孔板中，培养至细胞铺满底部60～80％。分别设置空白对照组、皮质醇干预组及实验组，每组3个重复。皮质醇干预组和实验组采用300μmol/L的皮质醇刺激SH-SY5Y细胞，37℃处理24h，完成抑郁模型细胞制备。分别用10 7、10 6、10 5CFU三个计量的两岐双歧杆菌i771干预抑郁模型细胞，37℃干预4h。最后，采用CCK8法检测细胞活性。
CCK8法细胞活性检测方法：在96孔板中接种浓度为2×10 4个/mL的三种细胞悬液(100μL/孔)；将培养板放在培养箱中预培养(37℃，5％CO 2)；接种细 胞后分别于0h、24h、48h、72h、96h向每孔加入10μLCCK8溶液；将培养板在培养箱内孵育2小时，用酶标仪测定在450nm处的吸光度。实验结果如表3：
表3.抑郁细胞模型干预试验中细胞活性对比
组别 空白组 皮质醇处理组 10 5活菌数处理组 10 6活菌数处理组 10 7活菌数处理组
细胞活性 0.245 0.184 0.221 0.227 0.242
由上表所示的实验结果可知，抑郁细胞模型干预试验初步证明：两歧双歧杆菌i771具有缓解抑郁模型细胞活性的功效，高、中、低剂量都具有缓解皮质醇诱导的细胞损伤的能力，而两歧双歧杆菌i771对缓解细胞损伤具有剂量依赖性，在高剂量组下有更明显的效果。
实施例4发酵乳感官测试
称取鲜牛乳930g，加入白砂糖70g，在水浴中加热搅拌使其充分融化，65℃(20MPa)均质，将均质好的配料95℃(10min)灭菌，降温至40℃左右，将两歧双歧杆菌i771菌种子液按10 6CFU/mL的接种量与市售家庭酸奶发酵剂(接种量0.07g/kg)共同接入，37℃发酵至pH4.5终止发酵，放入4℃冷藏24h后熟处理，同时设置对照组，以市售家庭酸奶发酵剂作为对照(接种量0.07g/kg)召集专家级感官评价人员10人对其进行综合评价。
感官评价方法为整体口感喜好度用9点强度标度法(9→1表示喜欢→不喜欢，＜5表示不被喜欢)，其它感官特性用10点强度标度法(10→0表示极强→察觉不到)。采用此方法对上述发酵乳样品进行整体喜好度、香气、黏度、粉感、粘聚性评价，评价结果见表4:
表4.发酵乳感官评价结果
  整体喜好度 香气 黏度 粉感 粘聚性评价
对照组 6.02 5.50 5.35 2.00 4.05
实验组 6.60 6.20 5.33 1.89 4.02
由表4所示的评价结果可知：两歧双歧杆菌I771的添加明显提高了发酵乳的喜好度评分和香气评分，同时在黏度、粉感、粘聚性评价中与对照组没有出现明显差异。
实施例5两歧双歧杆菌i771的应用
两歧双歧杆菌i771可以作为治疗和/或缓解抑郁症的饮品、食品、药品或益生菌制品中活性成分的应用。例如可以用于制备具有肠道调节、缓解抑郁情绪的谷类食物及其衍生物、发酵肉制品、益生菌制品、配方奶粉；还可以用于制备具有肠道调节功能的饮料、发酵酸奶；也可以用于制备具有肠道调节功能的药品，制成胶囊、粉末、药丸、口服液或者喷雾均可。
两歧双歧杆菌i771作为益生菌制品时可以是只包含两歧双歧杆菌i771单一菌种的益生菌制品，也可以是由两歧双歧杆菌i771、副干酪乳杆菌N1115、鼠李糖乳杆菌X253和动物双歧乳亚种i797混合制备而成的复合益生菌制品，该复合益生菌制品的剂型可以是复合益生菌微胶囊粉。

Claims (10)

  1. 一种两歧双歧杆菌i771，其特征在于：该菌保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.19855。
  2. 根据权利要求1所述的两歧双歧杆菌i771，其特征在于：该菌的16Sr DNA序列如下：
    GGGGTTAGGCCACCGGCTTCGGGTGCTGCCCACTTTCATGACTTGACGGGCGGTGTGTACAAGGCCCGGGAACGCATTCACCGCGGCGTTGCTGATCCGCGATTACTAGCGACTCCGCCTTCACGGAGCCGGGTTGCAGGCTCCGATCCGAACTGAGACCGGTTTTCAGGGATCCGCTCCCAATTGTCGGCCCAATGGTCTGCATTCGCCGCTGTAGCCGGCCATTGTAGACATGCGGTGAAGCCCTGGACGTAAGGGGCATGATGATCTGACGTCATCCCCACCTTCCTCCGAGTTAACCCCGGCGGTCCCCCGTGAGGTTCCCACCATAACGTGCTGGCAACACGGGGCGAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTGAACCCGCCCCCGAAGGGAAACGCCATCTCTGGCGTCGTCGGGAACATGTCAAGCCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCGCATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTTCTTTGAGTTTTAGCCTTGCGGCCCGTACTCCCCAGGCGGGACGCTTAACGCGTTAGCTCCGACACGGAACACGTGGAACGTGCCCCACATCCAGCGTCCACCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTGACGGCCCAGAGACCTGCCTTCGCCATCGGTGTTCTTCCCGATATCTACACATTCCACCGTTACACCGGGAATTCCAGTCTCCCCTACCGCACTCCAGCCCGCCCGTACCCGGCGCAGATCCACCGTTAAGCGATGGACTTTCACACCGGACGCGACGAGCCGCCTACGAGCCCTTTACGCCCAATAAATCCGGATAACGCTTGCGCCCTACGTATTACTCGCGGCTGCTGGCACGTAGTTAGCCGGCGCTTATTCGAAAGGTACACTCACCCGAAGGCTTGCTCCCAAACAAAAGAGGTTTACAACCCGAAGGCCTCCATCCCTCACGCGGCGTCGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTATCTCAGTCCCAATGTGGCCGGTCGCCCTCTCAGGCCGGCTACCCGTCGAAGCCTTGGTGAGCCGTTACCTCACCAACAAGCTGATAGGACGCGACCCCATCCCACGCCGATAGAATCTTTCCCACAATCACATGCGATCATGTGGAACATCCGGCATTACCACCCGTTTCCAGGAGCTATTCCGGAGCATGGGGCAGGTC GGTCACGCATTACTCACCCGTTCGCCACTCTCACCACCAAGCAAGCTTGATGGA。
  3. 根据权利要求1所述的两歧双歧杆菌i771，其特征在于：该菌的tuf基因序列为：
    GTGGTCGCCGTCGATGTAGTAGCTCAGTTCGGCGATGCCGTCGTATCCGGCCCGCTGATGGAATTTCGAAAGCGAGCCAAACCCCACGACGGTGGCTTCGGGACCTTCGATGACGACGACCGGATACTTGGCGCGTGGCCGATGGGAGTCGACCCACGCACGACGGCTTTCCAGAGACACCGGTTCAAGGTCGGCGGACGCTCCCCCACGGATCACCGCCTCGTTGTATATGTCAGCGATGGCCGGAACGTCGGCGTCAACGGCCGCGCGGATGGTATAGGACATGTCTTCAAGCATAACAACCGCCCTGCCGGCGGGATAGGGCGGGTGAGACTGGTGTCATCGGTGCATGATGCGCGATTCGCCTCGATCCGCCGACAGAAACGTGGACAACGGCCGCACAATCACGAAATGCGGGAGTTGTCCAGAACGAACACGATACAACTGCCGCAAAACAGCCCTTTGCAGCAGTTATCCAGC。
  4. 根据权利要求1～3中任一项所述的两歧双歧杆菌i771，其特征在于：该菌是从母乳喂养的婴儿或幼儿粪便中分离筛选出来的。
  5. 根据权利要求1～4中任一项所述的两歧双歧杆菌i771的分离纯化方法，其特征在于：该分离纯化方法按照以下步骤顺序进行：
    S1.取母乳喂养的婴儿或幼儿粪便，加入到浓度为0.9％的无菌生理盐水中混匀，得到样品A溶液；
    S2.将所述样品A溶液加入到改良MRS液体培养基中，于35～40℃条件下厌氧培养62～82h，得到培养液B，其中，所述样品A溶液与所述改良MRS液体培养基的体积比为1:10～100；
    S3.将所述培养液B用浓度为0.9％的无菌生理盐水以10倍梯度倍增稀释，稀释梯度依次为10 -1、10 -2、10 -3、10 -4、10 -5倍，对应得到菌悬液C1～C5；
    取改良MRS固体培养基，融化后，分别倒入第一至第五培养皿中，待冷却、完全凝固后，得培养基D1～D5，分别吸取菌悬液C1～C5各0.1mL并一一对应涂布到所述培养基D1～D5上，然后倒置所述培养皿，置于35～40℃环境下厌氧 培养62～82h，观察菌落生长情况，其中，所述改良MRS固体培养基为每1000mL所述改良MRS液体培养基中添加15％质量份数的琼脂所得固体培养基；
    待所述培养基D1～D5上出现典型菌落后，挑选出典型的单菌落E；
    S4.将所述单菌落E划线接种到新的所述改良MRS固体培养基上，35～40℃厌氧环境下培养62～82h，连续培养三次，得纯培养物F，即为所述两歧双歧杆菌i771的菌株。
  6. 根据权利要求5所述的两歧双歧杆菌i771的分离纯化方法，其特征在于：所述步骤S4后还包括以下步骤：
    S5.将所述纯培养物F，与无菌的质量份数为50％的甘油，按照1:1的比例混合，在-80～-70℃环境下保存，同时接种至新的所述改良MRS固体培养基试管斜面用于临时保存。
  7. 根据权利要求5或6所述的两歧双歧杆菌i771的分离纯化方法，其特征在于：所述步骤S2中，改良MRS液体培养基由10重量份酪蛋白胨、10重量份牛肉膏、5重量份酵母膏、20重量份葡萄糖、5重量份乙酸钠、2重量份柠檬酸二胺、1重量份吐温-80、2重量份K 2HPO 4、0.2重量份MgSO 4·7H 2O、0.05重量份MnSO 4·7H 2O、0.5重量份半胱氨酸和1000体积份蒸馏水组成；其中，重量份与体积份的对应比例关系为g:mL。
  8. 根据权利要求1～4中任一项所述的两歧双歧杆菌i771的应用，其特征在于：该菌作为治疗和/或缓解抑郁症的饮品、食品、药品或益生菌制品中活性成分的应用。
  9. 根据权利要求8所述的两歧双歧杆菌i771的应用，其特征在于：该菌用于制备发酵乳制品。
  10. 根据权利要求8所述的两歧双歧杆菌i771的应用，其特征在于：所述益生菌制品为由所述两歧双歧杆菌i771制备的菌剂，或为由所述两歧双歧杆菌i771、副干酪乳杆菌N1115、鼠李糖乳杆菌X253和动物双歧乳亚种i797混合制备而成的菌剂。
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