WO2020037532A1 - 短双歧杆菌ccfm1025、其发酵食品及其应用 - Google Patents

短双歧杆菌ccfm1025、其发酵食品及其应用 Download PDF

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WO2020037532A1
WO2020037532A1 PCT/CN2018/101685 CN2018101685W WO2020037532A1 WO 2020037532 A1 WO2020037532 A1 WO 2020037532A1 CN 2018101685 W CN2018101685 W CN 2018101685W WO 2020037532 A1 WO2020037532 A1 WO 2020037532A1
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ccfm1025
mice
bifidobacterium breve
increase
intestinal
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陈卫
王刚
田培郡
赵建新
张灏
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Jiangnan University
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    • 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
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • 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
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • 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
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • 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; 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
    • 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
    • A23V2400/519—Breve
    • 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 belongs to the technical field of microorganisms, and particularly relates to Bifidobacterium breve CCFM1025, a fermented food thereof, and an application thereof.
  • Depression is also called depressive disorder, including major depression, bipolar disorder, seasonal emotional disorders, postpartum depression, and so on.
  • depressive disorder including major depression, bipolar disorder, seasonal emotional disorders, postpartum depression, and so on.
  • the pathogenesis of depression has not been clarified.
  • the inconsistent response of different depression patients to treatment makes it a growing global burden of disease.
  • the monoamine neurotransmitter disorder is the main research direction and has been confirmed by a large number of cases. It is mainly manifested in neurotransmitter levels such as serotonin (5-HT), dopamine, and norepinephrine in the cerebral synaptic space. reduce.
  • neurotransmitter levels such as serotonin (5-HT), dopamine, and norepinephrine in the cerebral synaptic space. reduce.
  • 5-HT serotonin
  • dopamine dopamine
  • norepinephrine in the cerebral synaptic space. reduce.
  • GC glucocorticoids
  • Hippocampal injury further aggravates neuroendocrine abnormalities and exacerbates depression symptoms.
  • antidepressants currently used in the clinic are mainly focused on increasing monoamine neurotransmitter levels (mainly 5-HT) in the synaptic cleft of the brain.
  • monoamine neurotransmitter levels mainly 5-HT
  • selective serotonin reuptake inhibitors (SSRI) and serotonin and norepinephrine reuptake inhibitors (SNRI) are the current first-line antidepressants, accounting for more than 70% of clinical medications.
  • the current clinical medication has the following two problems: First, the onset of the drug has a delay of 2-4 weeks.
  • Brain-gut axis is a new concept proposed in recent years. As a two-way communication system between intestinal bacteria and the brain, it mainly regulates brain functions and behaviors through neural, endocrine and immune pathways.
  • the intestine contains a large number of intestinal microorganisms (about 10 14 to 10 15 ), which is the largest micro-ecosystem of the human body.
  • the normal communication between the intestinal flora and its metabolites and the host is necessary to maintain the health of the host.
  • Disorders of the intestinal microecology are related to many diseases, including diabetes, obesity, inflammatory bowel disease, neurodegenerative diseases and tumors.
  • the regulation of intestinal flora through probiotics has also become a new way to treat neurological diseases.
  • the present invention overcomes the shortcomings in the prior art and provides Bifidobacterium breve CCFM1025 (Bifidobacterium breve), which was deposited at the Guangdong Provincial Center for Microbial Strains on June 11, 2018, and deposited at It is the Guangdong Provincial Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No. 60386.
  • the present invention overcomes the shortcomings in the prior art and provides a fermented food.
  • the present invention provides the following technical solutions, wherein the fermented food is produced by fermentation using Bifidobacterium breve CCFM1025, and the fermented food includes solid food, liquid food, and semi-solid food.
  • the fermented food includes dairy products, soybean products, fruit and vegetable products
  • the dairy products include milk, sour cream, cheese
  • the fruit and vegetable products include cucumber, carrot, Beet, celery, cabbage products.
  • the present invention overcomes the shortcomings in the prior art, and provides the application of Bifidobacterium breve CCFM1025 in the preparation of probiotic bacteria colonized in vivo.
  • the present invention overcomes the shortcomings in the prior art, and provides the application of Bifidobacterium breve CCFM1025 in the preparation of antidepressant, anti-inflammatory bowel disease, anti-obesity drugs and health products.
  • the bifidobacterium breve CCFM1025 can improve the depression-like behavior of depressed mice and the brain of the depressed mice Serotonin (5-HT), serotonin (5-HTP), and brain-derived neurotrophic factor (BDNF), reduce corticosterone levels in the serum of depressed mice;
  • the Bifidobacterium breve CCFM1025 can increase the level of serotonin in the serum of depressed mice and improve the function of gastrointestinal motility;
  • the Bifidobacterium breve CCFM1025 can improve the intestinal flora disorder of depressed mice and reduce the intestinal Veillonellaceae ), Increasing the abundance of Bifidobacterium and Allobaculum, increasing the alpha diversity of the intestinal flora, reducing the incidence of inflammatory bowel disease and obesity;
  • the present invention overcomes the deficiencies in the prior art, and provides the application of the fermented food according to claim 2 or 3 in the preparation of functional foods with anti-depression, anti-inflammatory bowel disease, and anti-obesity.
  • the Bifidobacterium breve CCFM1025 can improve the depression-like behavior of depressed mice, and increase -Levels of serotonin, serotonin, and brain-derived neurotrophic factors, reducing corticosterone levels in the serum of depressed mice; the Bifidobacterium breve CCFM1025 can increase the levels of serotonin in the serum of depressed mice, and improve Gastrointestinal peristalsis function; the Bifidobacterium breve CCFM1025 can improve the intestinal flora of depressed mice, reduce the abundance of Veillonellaceae, and increase Bifidobacterium and Mycoplasma The abundance of the family (Allobaculum) improves the intestinal flora ⁇ -diversity and reduces the occurrence of inflammatory bowel disease and obesity; the Bifidobacterium breve can increase try
  • the present invention provides the following technical solutions:
  • CCFM1025 can significantly alleviate the depression-like behavior of mice, and the evaluation indicators include forced swimming test, sugar water preference test and platform jumping test; taking CCFM1025 can significantly improve the hippocampus of depressed mice Levels of 5-hydroxytryptophan and serotonin in the brain, while significantly increasing the level of BDNF in the prefrontal cortex of the brain; taking CCFM1025 can significantly reduce the levels of corticosterone in the serum of depressed mice and relieve the hyperfunction of HPA caused by depression; Taking CCFM1025 can increase serotonin levels in serum tissues of depressed mice and improve their intestinal motility.
  • CCFM1025 can improve the intestinal flora disorder caused by depression, increase the ⁇ -diversity of the intestinal flora, reduce the abundance of the intestinal Veillonellaceae, increase the Bifidobacterium and mycoplasma The abundance of the family (Allobaculum) and normalizes the intestinal flora, reducing the incidence of inflammatory bowel disease and obesity.
  • CCFM1025 can increase the mRNA level of tryptophan hydroxylase 1 in simulated intestinal chromaffin cells (RIN14B cells), and increase the secretion of 5-hydroxytryptophan in the cells, which can specifically stimulate the intestine Channel chromaffin cells produce serotonin to provide precursors for serotonin synthesis in the brain.
  • the Bifidobacterium breve CCFM1025 can be used to prepare foods, health products and medicines with antidepressant functions, and has a very wide application prospect.
  • Fig. 1 is a schematic diagram of the behavioral changes of mice after six weeks of intervention in depressed mice.
  • FIG 2 Six-week intervention of depressed mice with serotonin (5-HT, figure a) and 5-hydroxytryptophan (5-HTP, figure b) in the hippocampus, and brain-derived nerves in the prefrontal cortex Schematic diagram of changes in the levels of nutritional factors (BDNF, Figure c); * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001 (vs model group).
  • Figure 3 is a schematic diagram of corticosterone levels in the serum of mice after six weeks of intervention with depressed mice; ** P ⁇ 0.01, *** P ⁇ 0.001 (vs model group).
  • Figure 4 is a schematic diagram of 5-HT levels in the serum of mice after six weeks of intervention with the strain; ** P ⁇ 0.01 (vs model group).
  • Fig. 5 is a schematic diagram of ⁇ -diversity changes of intestinal flora of mice after six weeks of intervention of the depressed mice by the strain; * P ⁇ 0.05 (vs model group).
  • FIG. 6 is a schematic diagram of ⁇ -diversity changes of intestinal flora of mice after six weeks of intervention of depressed mice by the strain.
  • FIG. 7 is a schematic diagram showing changes in Veillonellaceae, Bifidobacterium, and Allobaculum in the intestinal tract of mice after six weeks of intervention with depressed mice; of which, * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001.
  • FIG. 8 is a schematic diagram of changes in the intracellular tryptophan hydroxylase 1 mRNA levels and the 5-hydroxytryptophan content in the cell supernatant after RIN14B cells were stimulated by the strain; of which * P ⁇ 0.05, ** P ⁇ 0.01 .
  • an embodiment or “an embodiment” referred to herein refers to a particular feature, structure, or characteristic that can be included in at least one implementation of the present invention.
  • the appearances of "in one embodiment” in various places in this specification do not all refer to the same embodiment, nor are they separate or selectively mutually exclusive embodiments.
  • the Bifidobacterium Brevii CCFM1025 (Bifidobacterium breve) of the present invention was deposited at the Guangdong Provincial Center for Microbial Strains on June 11, 2018. Therefore, the accession number is GDMCC No. 60386.
  • the Bifidobacterium breve CCFM1025 has the following biological characteristics:
  • Bacterial characteristics Gram-staining-positive, non-forming bacilli, non-moving bacteria.
  • the minimum growth temperature of the strain is 15 ° C
  • the maximum growth temperature is 45 ° C
  • the best growth is at a temperature of 35-37 ° C
  • the optimal growth pH is 6.5
  • the culture enters a stable period after 18h;
  • the extraction method of Bifidobacterium breve CCFM1025 is:
  • step (a) (1) culturing the lactic acid bacteria obtained in step (a) in a liquid mMRS culture medium for 24 hours, and then taking 1 mL of the culture and centrifuging at 8000 rpm for 2 minutes;
  • centrifuge at 12000rpm for 5-10min take the supernatant 200 ⁇ L; add 400 ⁇ L ice ethanol or ice isopropanol to 200uL The supernatant was left at 20 ° C for 1 hour, centrifuged at 12000rpm for 5-10min, and the supernatant was discarded; 500 ⁇ L of 70% (volume percentage) ice ethanol was added to resuspend the pellet, and the supernatant was centrifuged at 12000rpm for 1-3min, and the supernatant was discarded; Or let it air dry; 50 ⁇ L ddH 2 O redissolve the precipitate for PCR;
  • the obtained PCR product is sent to a professional sequencing company, and the obtained sequencing result is searched and similarly compared with GeneBank using BLAST, and identified as Bifidobacterium breve.
  • the extracted whole genome was sent to a professional sequencing company, and the whole genome of the bacteria was sequenced by a second-generation sequencer.
  • the obtained sequence results were searched and similarly compared in GeneBank using BLAST.
  • the sequencing results were identified as belonging to Bifidobacterium breve A newly discovered strain. Strains were stored at -80 ° C until use.
  • Example 1 Bifidobacterium breve CCFM1025 can significantly improve behavioral indicators of depressed mice
  • mice Thirty-two 6-week-old male C57BL / 6J mice were selected. After one week of adaptation, they were randomly divided into four groups according to body weight: control group, model group, fluoxetine intervention group, CCFM1025 intervention group, each group containing 8 mice. Animal grouping and processing methods are shown in Table 1.
  • Chronic unpredictable stress depression mouse model 1-2 stimuli are randomly used daily, and the timing of each stimulus is randomly determined to avoid circadian rhythm. Each method does not exceed three for five weeks. Stimulating factors include: (1) fasting for 24h; (2) water-free + empty bottle stimulation for 24h; (3) tail clamping for 3min; (4) wet litter for 24h; (5) braking for 1 to 2h; (6) 45 ° Tilt cage box for 24h; (7) continuous light for 24h; (8) no litter for 24h; (9) forced swimming for 15 minutes; (10) orphanage for 24h.
  • Lactobacillus gavage Take the second-generation activated bifidobacterium CCFM1025 and incubate at 37 ° C for 24h, centrifuge at 3 ° C and 8000r / min for 3min to collect the bacteria, discard the supernatant and resuspend the bacteria with 5% sterilized skim emulsion
  • concentration of lactic acid bacteria was 5 ⁇ 10 9 CFU / mL.
  • the gavage volume was 0.2 mL / piece / day.
  • the forced swimming test is a behavioral despair experiment, and it is a classic experimental model for evaluating the antidepressant effect of drugs.
  • the experimental bucket was filled with about 20 cm of clear water, and the water temperature was about 24 ⁇ 1 °.
  • Each mouse was subjected to a 15-minute swimming training test 24 hours before the official experiment. For formal experiments, each mouse was tested for 6 minutes. Use the camera to record the entire experiment. Record the immobility (floating) time, that is, the limbs are not moving or only the hind limbs are slightly moving.
  • the experimental results are shown in Figure 1a.
  • the mice in the depression group significantly reduced their swimming time in the water, showing behavioral despair. And taking CCFM1025 can significantly improve this phenomenon, indicating that depression symptoms in mice are reduced.
  • the sugar water preference test is a model for detecting a lack of pleasure in depression.
  • two identical drinking bottles were placed in the cage to allow the mice to adaptively drink water for at least 3 days. After acclimation, one of the bottles was replaced with an aqueous solution containing 1% sucrose. The water and sucrose solution intake was measured by weighing the bottle. Change the position of two bottles daily to reduce drinking preferences due to different amounts of water.
  • the first phase is the electric shock training phase.
  • the mice were placed on the platform of the jumping platform reaction box, and the electric grid at the bottom of the reaction box was energized (38V). When the mice jumped off the platform, they received an electric shock.
  • the training time is 3 minutes. If individual mice do not jump off the platform within 3 minutes, they will be artificially kicked off to ensure that all animals have been injured by electric shock, causing nociceptive memory. After training, return to the original cage.
  • the second phase (24h after electric shock training) is the memory reproduction test phase. Put the mouse on the platform of the reaction box. The test time is 3 minutes. This test is not powered. Record the time from when the mouse jumps from the platform to the first jump (time spent on the platform), that is, the platform incubation period.
  • the mouse has not jumped off the platform at 180s. Try to avoid human interference with animal behavior throughout the experiment.
  • the experimental results show (Figure 1c) that the depression latency of depressed mice is significantly increased, which represents an increase in depression and anxiety, and taking CCFM1025 can alleviate their anxiety.
  • Example 2 CCFM1025 can significantly increase neurotransmitter levels in the brain of depressed mice
  • mice in Example 2 were euthanized at the sixth weekend, the brain tissues of the mice were taken, and the hippocampus and prefrontal cortex were separated on ice. Take fresh seahorse and prefrontal cortex tissues of a certain quality (weight not less than 50mg), add 9 times the volume of sterile PBS buffer (equivalent to 1g tissue plus 9ml homogenate), and homogenize with a tissue homogenizer. After the tissue fluid was centrifuged at 3000g and 20min, the supernatant was collected, and the 5-HT and BDNF contents were detected by an ELISA kit.
  • sterile PBS buffer equivalent to 1g tissue plus 9ml homogenate
  • the supernatant of the hippocampal tissue fluid was added with an equal volume of 5% perchloric acid to precipitate the protein, centrifuged at 10,000 g for 10 min, and the supernatant was filtered through a 0.22 ⁇ m aqueous filter, and then subjected to high performance liquid chromatography-fluorescence detection (HPLC-FLD) for 5- Determination of HTP content.
  • HPLC-FLD high performance liquid chromatography-fluorescence detection
  • the experimental results are shown in Figure 2. The results show that taking CCFM1025 can significantly reverse the reduction of 5-HT, 5-HTP and BDNF levels in the hippocampus caused by stress. Among them, CCFM1025 improved hippocampal 5-HTP and 5-HT to the same extent as fluoxetine, and significantly improved the BDNF in prefrontal cortex better than fluoxetine.
  • Example 3 Bifidobacterium breve CCFM1025 can reduce corticosterone levels in serum of mice
  • mice in Example 2 were sacrificed euthanized on the sixth weekend, and the blood of the mice was collected and centrifuged at 1000 g for 15 min to obtain serum.
  • ELISA kit was used to detect the content of corticosterone in serum.
  • the experimental results show ( Figure 3) that, due to persistent chronic stress, the hypothalamus-pituitary-adrenal axis (HPA) hyperfunction occurs in depressed mice, and the serum corticosterone level is significantly increased. Taking CCFM1025 can significantly reduce the serum of depressed mice Corticosterone levels, relieve HPA hyperthyroidism, and show good antidepressant efficacy.
  • HPA hypothalamus-pituitary-adrenal axis
  • Example 4 Bifidobacterium breve CCFM1025 can increase serum 5-HT levels in depressed mice
  • the serum in Example 4 was taken, and the 5-HT content in the serum was detected using an ELISA kit.
  • 5-HT in peripheral tissues is important for maintaining normal intestinal motility.
  • 5-HT in peripheral tissues can stimulate 5-HT2 receptors in gastrointestinal smooth muscle or 5-HT4 receptors in ganglion cells in the intestinal wall, causing gastrointestinal smooth muscle contraction, increasing gastrointestinal tension, and intestinal motility ;
  • the reduction of 5-HT in peripheral tissues is directly related to diseases such as constipation and gastrointestinal discomfort.
  • the experimental results show ( Figure 4) that 5-HT in peripheral tissues of depressed mice is significantly reduced, and intestinal motility is impaired.
  • taking fluoxetine has an antidepressant effect, it does not improve the intestinal function of depressed mice.
  • taking CCFM1025 can not only relieve the symptoms of depression, but also significantly increase the level of 5-HT in serum, restore it to normal levels, and restore the normal intestinal motility of depressed mice.
  • Example 5 Regulation of Bifidobacterium Brevibacterium CCFM1025 on the intestinal flora of depressed mice and its multifunctional regulation
  • the fresh feces from the sixth weekend of the mice in Example 2 were taken, and the total DNA in the feces samples of the mice was extracted using the MP feces kit.
  • the specific operation steps are as follows mainly with reference to the kit instructions.
  • the mouse feces genome was used as a template, and the upstream primer 520F (5′-AYTGGGYDTAAAGNG-3 ′) and the downstream primer 802R (5′-TACNVGGGTATCTAATCC-3 ′) were used as primers to amplify the V3-V4 region fragment of 16S rDNA.
  • the length of the target fragment was It is about 247bp.
  • a 2.0% agarose gel was prepared and electrophoresed at 120V for 40 minutes. After running the gel, the target bands were quickly cut under UV light.
  • the QIAquick Gel Extraction Kit gel recovery kit instructions to perform the target strip gel recovery.
  • the DNA concentration of the samples was measured according to the Qubit DNA 3.0 kit, and then the library was constructed according to the TurSeq DNA LT Sample Preparation Kit and its instructions. Finally, the MiSeq Regent Kit and its instructions were used to determine the concentration on the Illumina Miseq sequencer.
  • Veillonellaceae includes 6 genera and 25 species, which are Gram-negative, anaerobic Or microaerobic and cocci. Some of these bacteria are conditional pathogens, which are common in microbial infections in humans and animals, and are also found in severe osteoarthritis and endocarditis. Taking CCFM1025 can also increase the abundance of Bifidobacterium and Allobaculum. Among them, Bifidobacterium has a variety of important physiological functions such as biological barriers, nutritional effects, anti-tumor, immune enhancement, gastrointestinal function improvement, anti-aging, etc., and is a kind of physiological beneficial bacteria.
  • Mycoplasmaceae can produce short-chain fatty acids, especially butyric acid, which can not only provide oxidative energy to intestinal epithelial cells, but also maintain important water and electrolyte balance, regulate intestinal flora balance, and regulate intestinal barrier function.
  • short-chain fatty acids can exert anti-inflammatory effects through the two signaling pathways of the G protein-coupled receptors (GPCRs) activation pathway and the histone deacetylases (HDACs) inhibition pathway, and have an effect on inflammation Inflammatory bowel disease (IBD) and obesity have significant improvement effects.
  • GPCRs G protein-coupled receptors
  • HDACs histone deacetylases
  • RIN14B cells were seeded at a density of 4 ⁇ 10 5 / mL in a 24-well plate and incubated for 72 h. Discard the medium, wash cells with HBSS (1 mL) containing 0.1% bovine serum albumin (BSA) and 2 ⁇ M fluoxetine, and add 1 mL of HBSS suspension containing CCFM1025 (control group is HBSS without bacteria), 37 Incubate at °C for 20min, collect the supernatant, centrifuge at 6000g for 5min to remove the precipitate, and freeze the supernatant at -80 °C for measurement. HPLC-FLD was used to detect 5-HTP in the cell supernatant (Reference Example 3).
  • TPH1 mRNA measurement The adherent cells in the above steps were washed three times with HBSS, 1 mL of Trizol was added, and incubated on ice for 5-10 min. The cells were detached by pipetting, and the lysate was transferred to an enzyme-free EP tube. Total cellular RNA was extracted by conventional methods, and cDNA was synthesized according to the instructions of the reverse transcription kit (Prime Script RT reagent Kit gDNAEraser, Takara). The synthesized cDNA samples were tested for concentration and purity (A260 / A280) by ultra-micro spectrophotometer (NanoDrop 2000C), and stored at -80 ° C until use.
  • CCFM1025 stimulated RIN14B cells, the mRNA level of TPH1 in the cells was significantly increased. Accordingly, the amount of 5-HTP secreted by the cells was also significantly increased.
  • 5-HTP secreted by intestinal chromaffin cells can enter the blood circulation and pass through the blood-brain barrier to provide precursors for 5-HT synthesis in the brain. Therefore, CCFM1025 can specifically stimulate 5-HTP secretion by intestinal chromaffin cells to promote 5-HT synthesis in the brain and achieve antidepressant function.
  • Example: 7 Using the Bifidobacterium breve CCFM1025 of the present invention to produce a fermented food containing the bacterium
  • Fresh vegetables are washed and squeezed, followed by instant high-temperature sterilization. After being heat-sterilized at 140 ° C for 2 seconds, the temperature is immediately reduced to 37 ° C, and then the Bifidobacterium brevis CCFM1025 fungicide fermentation agent prepared by the present invention is used. , So that its concentration reaches 10 6 CFU / mL or more, and it is stored under refrigeration at a temperature of 4 ° C., so that a fruit and vegetable beverage containing the live bifidobacterium CCFM1025 bacteria of the present invention is obtained.
  • fermented foods can be prepared by using Bifidobacterium breve CCFM1025 for fermentation production, and the fermented foods include solid food, liquid food, and semi-solid food.
  • the fermented food includes dairy products, soy products, fruit and vegetable products, the dairy products include milk, sour cream, cheese; and the fruit and vegetable products include cucumber, carrot, beet, celery, and cabbage products.
  • the fermented food can improve the depression-like behavior of depressed mice, increase the levels of serotonin, 5-hydroxytryptophan and brain-derived neurotrophic factor in the brain of depressed mice, and reduce the level of corticosterone in the serum of depressed mice
  • the fermented food can improve the serotonin level in the serum of depressed mice and improve the gastrointestinal motility;
  • the fermented food can improve the intestinal flora disturbance of the depressed mice and reduce the intestinal Weirongcocaceae ( Veillonellaceae), increase the abundance of Bifidobacterium and Allobaculum, increase the alpha diversity of the intestinal flora, reduce the incidence of inflammatory bowel disease and obesity; can improve the simulated intestinal chromaffin
  • the level of tryptophan hydroxylase 1 mRNA in cells (RIN14B cells), and increase the secretion of 5-hydroxytryptophan in the cells can specifically stimulate the intestinal chromaffin cells to produce 5-hydroxytryptophan to thereby Provide precursors for the

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Abstract

短双歧杆菌CCFM1025、其发酵食品及其在制备抗抑郁、抗炎症性肠病、抗肥胖的药物、保健品和功能性食品中的应用。

Description

短双歧杆菌CCFM1025、其发酵食品及其应用 技术领域
本发明属于微生物技术领域，具体涉及短双歧杆菌CCFM1025、其发酵食品及其应用。
背景技术
抑郁症又称抑郁障碍，包括重度抑郁症、双相情感障碍、季节性情绪失调、产后抑郁等。作为一种异质性疾病，抑郁症的发病机制至今尚未明确阐明。不同抑郁症患者对治疗的反应具有不一致性，使其成为越来越严重的全球性疾病负担。目前世界上有超过3.5亿的抑郁症患者，预计到2030年，抑郁症将成为全球第一大疾病。
目前关于抑郁症的机制有两大解释，包括脑中单胺类神经递质失调和下丘脑-垂体-肾上腺轴(HPA)功能异常。其中单胺类神经递质失调是主要研究的方向，也被大量的病例证实，主要表现为大脑神经突触间隙的5-羟色胺(5-HT)、多巴胺、去甲肾上腺素等神经递质水平降低。此外，作为一种持续的慢性应激状态，抑郁症能够导致HPA轴持续激活，分泌过量糖皮质激素(GC)，导致海马体长期受到过量的GC攻击，损害海马神经元的结构和功能。海马损伤又进一步加重神经内分泌异常，加重抑郁症状。
基于已有的发病机制，目前用于临床的抗抑郁药物，主要集中在提高大脑突触间隙的单胺类神经递质水平(主要是5-HT)。其中选择性5-羟色胺重摄取抑制剂(SSRI)和5-羟色胺和去甲肾上腺素重摄取抑制剂(SNRI)是目前的一线抗抑郁药物，占临床用药的70％以上。但目前的临床用药，存在以下两个问题：第一，药物起效有2-4周的延迟，原因是服用SSRI增加突触间隙的5-HT浓度后，5-HT会反作用于突触前5-HT1A受体，产生负反馈抑制，导致突触前5-HT的合成和分泌减少。持续服药2-4周，使5-HT1A受体脱敏后，SSRI才能真正起效。而这2-4周，在临床上会导致患者服药依从性降低，大大增加自杀风险。第二，大部分抗抑郁药物不具备预防作用，且长期服药会产生非特异性腹痛、便秘、腹泻、消化不良、胃胀气等副作用。因此，探索新的抗抑郁方法显得尤为重要，也显示出十分广阔的市场潜力。
“脑肠轴”是近年来提出的一个新概念，作为肠内细菌与大脑间的双向通信系统，主要通过神经途径、内分泌途径和免疫途径来调节大脑的功能以及行 为。肠道内包含大量肠道微生物(约10 14-10 15个)，是人体最大的微生态系统。肠道菌群及其代谢产物与宿主之间的正常交流，对于维持宿主的健康是必要的。肠道微生态的紊乱与许多疾病相关，包括糖尿病、肥胖症、炎性肠病、神经退行性疾病和肿瘤等。通过益生菌调节肠道菌群也成为治疗神经类疾病的一种新途径。
因此，筛选出一种能够调节肠道菌群，并能有效缓解抑郁的益生菌显得十分必要。对于深入挖掘益生菌的功能，开发具有更高保健价值的益生菌具有十分重要的意义。同时为利用膳食策略缓解抑郁症开辟出新的途径和解决方案。
发明内容
本部分的目的在于概述本发明的实施例的一些方面以及简要介绍一些较佳实施例。在本部分以及本申请的说明书摘要和发明名称中可能会做些简化或省略以避免使本部分、说明书摘要和发明名称的目的模糊，而这种简化或省略不能用于限制本发明的范围。
鉴于上述的技术缺陷，提出了本发明。
因此，作为本发明其中一个方面，本发明克服现有技术中存在的不足，提供短双歧杆菌CCFM1025(Bifidobacterium breve)，于2018年6月11日保藏于广东省微生物菌种保藏中心，保藏地址为广州市先烈中路100号大院59号楼5楼广东省微生物研究所，保藏编号为GDMCC No.60386。
作为本发明另一个方面，本发明克服现有技术中存在的不足，提供一种发酵食品。
为解决上述技术问题，本发明提供如下技术方案，其中：所述发酵食品为使用短双歧杆菌CCFM1025发酵生产制得，所述发酵食品包括固态食品、液态食品、半固态食品。
作为本发明所述发酵食品的一种优选方法，其中：所述发酵食品包括乳制品、豆制品、果蔬制品，所述乳制品包括牛奶、酸奶油、干酪；所述果蔬制品包括黄瓜、胡萝卜、甜菜、芹菜、圆白菜制品。
作为本发明另一个方面，本发明克服现有技术中存在的不足，提供短双歧杆菌CCFM1025在制备体内定植益生菌中的应用。
作为本发明另一个方面，本发明克服现有技术中存在的不足，提供短双歧杆菌CCFM1025在制备抗抑郁、抗炎症性肠病、抗肥胖药物和保健品中的应 用。
作为本发明所述短双歧杆菌CCFM1025在制备体内定植益生菌中的应用的一种优选方案，其中：所述短双歧杆菌CCFM1025能够改善抑郁小鼠的抑郁样行为、提高抑郁小鼠脑中的5-羟色胺(5-HT)、5-羟色氨酸(5-HTP)和脑源神经营养因子(BDNF)的水平、降低抑郁小鼠血清中的皮质酮水平；所述短双歧杆菌CCFM1025能够提高抑郁小鼠血清中的5-羟色胺水平，改善胃肠道蠕动功能；所述短双歧杆菌CCFM1025能够改善改善抑郁小鼠的肠道菌群紊乱，降低肠道韦荣球菌科(Veillonellaceae)的丰度，提高双歧杆菌属(Bifidobacterium)和支原体科(Allobaculum)的丰度，提高肠道菌群α-多样性，减少炎症性肠病和肥胖的发生；所述短双歧杆菌能够提高模拟肠嗜铬细胞(RIN14B细胞)中色氨酸羟化酶1的mRNA水平，并提高该细胞5-羟色氨酸的分泌量，能够特异性地通过刺激肠道嗜铬细胞产生5-羟色氨酸从而为脑中5-羟色胺的合成提供前体物质。
作为本发明另一个方面，本发明克服现有技术中存在的不足，提供权利要求2或3所述的发酵食品在制备抗抑郁、抗炎症性肠病、抗肥胖功能性食品中的应用。
作为本发明所述发酵食品在制备抗抑郁功能性食品中的应用的一种优选方案，其中：所述短双歧杆菌CCFM1025能够改善抑郁小鼠的抑郁样行为、提高抑郁小鼠脑中的5-羟色胺、5-羟色氨酸和脑源神经营养因子的水平、降低抑郁小鼠血清中的皮质酮水平；所述短双歧杆菌CCFM1025能够提高抑郁小鼠血清中的5-羟色胺水平，改善胃肠道蠕动功能；所述短双歧杆菌CCFM1025能够改善改善抑郁小鼠的肠道菌群紊乱，降低肠道韦荣球菌科(Veillonellaceae)的丰度，提高双歧杆菌属(Bifidobacterium)和支原体科(Allobaculum)的丰度，提高肠道菌群α-多样性，减少炎症性肠病和肥胖的发生；所述短双歧杆菌能够提高模拟肠嗜铬细胞(RIN14B细胞)中色氨酸羟化酶1的mRNA水平，并提高该细胞5-羟色氨酸的分泌量，能够特异性地通过刺激肠道嗜铬细胞产生5-羟色氨酸从而为脑中5-羟色胺的合成提供前体物质。
为解决上述技术问题，本发明提供了如下技术方案：
本发明的有益效果：在抑郁模型小鼠实验中，服用CCFM1025能够显著缓解小鼠的抑郁样行为，评估指标包括强迫游泳实验、糖水偏好实验和跳台实 验；服用CCFM1025能够显著提高抑郁小鼠海马体中的5-羟色氨酸和5-羟色胺的水平，同时显著提高大脑前额叶皮质中BDNF的水平；服用CCFM1025能够显著降低抑郁小鼠血清中的皮质酮水平，缓解抑郁引起的HPA功能亢进；服用CCFM1025能够提高抑郁小鼠血清组织中的5-羟色胺水平，改善其肠道蠕动功能。服用CCFM1025能够改善抑郁引起的肠道菌群紊乱，增加肠道菌群的α-多样性，还能够降低肠道韦荣球菌科(Veillonellaceae)的丰度、提高双歧杆菌属(Bifidobacterium)和支原体科(Allobaculum)的丰度，并使肠道菌群趋于正常化，减少炎症性肠病和肥胖的发生。体外实验结果表明，CCFM1025能够提高模拟肠嗜铬细胞(RIN14B细胞)中色氨酸羟化酶1的mRNA水平，并提高该细胞5-羟色氨酸的分泌量，能够特异性地通过刺激肠道嗜铬细胞产生5-羟色氨酸从而为脑中5-羟色胺的合成提供前体物质。
本发明所述的短双歧杆菌CCFM1025可用于制备具有抗抑郁功能的食品、保健品和药品，具有非常广泛的应用前景。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其它的附图。其中：
图1为本菌株干预抑郁小鼠六周后，小鼠的行为学变化示意图。(a)强迫游泳实验；(b)糖水偏好实验；(c)跳台实验；其中*P<0.05，**P<0.01，***P<0.001(vs模型组)。
图2为本菌株干预抑郁小鼠六周后，海马组织中5-羟色胺(5-HT，图a)和5-羟色氨酸(5-HTP，图b)、前额叶皮质中脑源神经营养因子(BDNF，图c)的水平变化示意图；其中*P<0.05，**P<0.01，***P<0.001(vs模型组)。
图3为本菌株干预抑郁小鼠六周后，小鼠血清中皮质酮水平示意图；其中**P<0.01，***P<0.001(vs模型组)。
图4为本菌株干预抑郁小鼠六周后，小鼠血清中5-HT水平示意图；其中**P<0.01(vs模型组)。
图5为本菌株干预抑郁小鼠六周后，小鼠肠道菌群α-多样性变化示意图；其中*P<0.05(vs模型组)。
图6为本菌株干预抑郁小鼠六周后，小鼠肠道菌群β-多样性变化示意图。
图7为本菌株干预抑郁小鼠六周后，小鼠肠道中韦荣球菌科(Veillonellaceae)、双歧杆菌属(Bifidobacterium)和支原体科(Allobaculum)的变化示意图；其中*P<0.05，**P<0.01，***P<0.001。
图8为本菌株刺激RIN14B细胞后，细胞内色氨酸羟化酶1的mRNA水平和细胞上清中5-羟色氨酸含量的变化示意图；其中其中*P<0.05，**P<0.01。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合具体实施例对本发明的具体实施方式做详细的说明。
在下面的描述中阐述了很多具体细节以便于充分理解本发明，但是本发明还可以采用其他不同于在此描述的其它方式来实施，本领域技术人员可以在不违背本发明内涵的情况下做类似推广，因此本发明不受下面公开的具体实施例的限制。
其次，此处所称的“一个实施例”或“实施例”是指可包含于本发明至少一个实现方式中的特定特征、结构或特性。在本说明书中不同地方出现的“在一个实施例中”并非均指同一个实施例，也不是单独的或选择性的与其他实施例互相排斥的实施例。
本发明的短双歧杆菌CCFM1025(Bifidobacterium breve)，于2018年6月11日保藏于广东省微生物菌种保藏中心，保藏地址为广州市先烈中路100号大院59号楼5楼广东省微生物研究所，保藏编号为GDMCC No.60386。
所述短双歧杆菌CCFM1025具有以下生物学特性：
(1)菌体特征：呈革兰氏染色阳性，不形成抱子，不运动的细菌。
(2)菌落特征：菌落小，乳白色，圆形，边缘整齐，微凸起，不透明，表面湿润光滑；
(3)生长特性：该菌株的最低生长温度为15℃，最高生长温度为45℃，在温度35-37℃下生长最佳，最适生长pH为6.5，培养18h后进入稳定期；
(4)在抑郁小鼠模型中能够显著改善小鼠的行为学表现；
(5)在抑郁小鼠模型中能够提高小鼠脑中的5-HT、5-HTP和BDNF的水平；
(6)在抑郁小鼠模型中能降低小鼠血清皮质酮的水平；
(7)在抑郁小鼠模型中能提高抑郁小鼠血清组织中的5-HT水平，改善其肠道蠕动功能；
(8)能降低抑郁小鼠肠道中韦荣球菌科(Veillonellaceae)的丰度、提高双歧杆菌属(Bifidobacterium)和支原体科(Allobaculum)的丰度，增加肠道菌群的α-多样性，改善抑郁引起的肠道菌群紊乱，减少炎症性肠病和肥胖的发生。
所述短双歧杆菌CCFM1025的提取方法为：
(一)双歧杆菌的分离筛选：
(l)取1g健康成年人的新鲜粪便。梯度稀释后涂布于mMRS固体培养基，置于厌氧环境下37℃培养72小时；
(2)观察记录菌落形态，挑取菌落划线纯化；
(3)在mMRS液体培养基中，37℃培养48小时，所得菌落进行革兰氏染色，记录菌落形态。
(4)弃除菌落中的革兰氏阴性菌菌株和革兰氏阳性球菌，挑选得到革兰氏阳性杆菌。
(5)过氧化氢酶分析后，弃除过氧化氢酶阳性菌株，保留过氧化氢酶阴性菌株。
(二)双歧杆菌的初步鉴定：果糖-6-磷酸盐磷酸酮酶测定法
(1)将步骤(一)所筛选得到的乳酸菌在液体mMRS培养液中培养24h，然后取lmL培养物8000rpm离心2min；
(2)用含0.05％(质量百分数)半胱氨酸盐酸盐的pH6.5的0.05M KH2PO4溶液洗涤两次；
(3)重悬于200μL添加了0.25％(质量百分数)Triton X-100的上述磷酸盐缓冲液；
(4)添加50μL浓度为6mg/mL氟化钠和10mg/mL碘乙酸钠的混合液以及50μL浓度为80mg/mL的果糖-6-磷酸，37℃孵育1h；
(5)添加300μL浓度为0.139g/mL、pH 6.5的盐酸羟胺，并于室温放置10min；
(6)分别添加200μL15％(质量百分数)的三氯乙酸和4M HCI；
(7)添加200μL含有5％(质量百分数)三氯化铁的0.1M HCI，体系迅 速变为红色，即为F6PPK阳性，初步断定其为双歧杆菌。
(三)双歧杆菌的分子生物学鉴定：
(l)单菌基因组抽提：将步骤(二)所筛选得到的双歧杆菌培养过夜，取培养过夜的菌悬液lmL于1.5mL离心管，10000rpm离心2min，弃上清得菌体；用lmL无菌水吹洗菌体后，10000rpm离心2min，弃上清得菌体；加入200μL SDS裂解液，80℃水浴30min；加入酚-氯仿溶液200μL于菌体裂解液中，其中酚-氯仿溶液的组成成分及体积比为Tris饱和酚:氯仿:异戊醇＝25:24:1，颠倒混匀后，12000rpm离心5-10min，取上清200μL；加入400μL冰乙醇或冰异丙醇于200uL上清中，﹣20℃静置1h，12000rpm离心5-10min，弃上清；加入500μL70％(体积百分数)冰乙醇重悬沉淀，12000rpm离心1-3min，弃上清；60℃烘箱烘干，或者自然晾干；50μL ddH 2O重溶沉淀以备PCR；
(2)16S rDNA PCR：
A.细菌16S rDNA 50μLPCR反应体系：
10×Taq buffer，5μL；dNTP，5μL；27F，0.5μL；1492R，0.5μL；Taq酶，0.5μL；模板，0.5μL；ddH 2O，38μL。
B.PCR条件：
95℃5min；95℃10s；55℃30s；72℃30s；step2-4 30×；72℃5min；12℃2min；
C.制备1％琼脂糖凝胶，之后将PCR产物与10000×loading buffer混合，上样量2μL，120V跑30min，然后进行凝胶成像；
D.将得到PCR产物送专业测序公司，将得到的测序结果与使用BLAST在GeneBank中进行搜索和相似性比对，鉴定为短双歧杆菌。
(3)全基因组测序
将提取的全基因组送专业测序公司，利用二代测序仪对菌的全基因组进行测序，将得到的序列结果使用BLAST在GeneBank中进行搜索和相似性比对，测序结果鉴定为属于短双歧杆菌的一种新发现的菌株。菌株-80℃保藏备用。
实施例1：短双歧杆菌CCFM1025能够显著改善抑郁小鼠的行为学指标
取6周龄雄性C57BL/6J小鼠32只，适应环境一周后，根据体重随机分为四组：对照组、模型组、氟西汀干预组、CCFM1025干预组，每组含8只小鼠。动物分组及处理方法见表1。
表1 动物实验分组及处理方法
Figure PCTCN2018101685-appb-000001
慢性不可预知应激抑郁小鼠模型：每天随机采用1-2种刺激，每天刺激的时间随机决定，避免昼夜节律。每种方法不超过三次，为期五周。刺激因素包括：(1)禁食24h；(2)禁水+空瓶刺激24h；(3)夹尾3min；(4)潮湿垫料24h；(5)制动1～2h；(6)45°倾斜笼盒24h；(7)持续光照24h；(8)无垫料24h；(9)强迫游泳15分钟；(10)孤养24h。
乳酸菌灌胃剂：取活化2代的双歧杆菌CCFM1025并于37℃下培养至24h，于4℃、8000r/min离心3min收集菌体，弃去上清并用5％灭菌脱脂乳液重悬菌体，使乳酸菌浓度达到5×10 9CFU/mL。灌胃体积为0.2mL/只/天。
第五周开始，停止每日的慢性不可预知应激以及药物和益生菌的干预，同时对所有小鼠进行行为学测试。包括强迫游泳实验、悬尾实验、糖水偏好实验、旷场实验和跳台实验。具体实施方法和结果如下：
(1)强迫游泳实验：
强迫游泳实验是一种行为绝望实验法，是用于评价药物抗抑郁作用的经典实验模型。实验桶内装入大约20cm高的清水，水温在24±1°左右，在正式实验前24小时，每只小鼠均进行15分钟的游泳训练测试。正式实验时，每只小鼠进行6分钟测试。使用摄像头对实验全程录像。记录不动(漂浮)时间，即四肢不动或仅后肢轻微运动。实验结果如图1a所示，抑郁组小鼠在水中的游泳时间显著降低，表现出行为绝望的特点。而服用CCFM1025能够显著改善这一现象，表明小鼠抑郁症状减轻。
(2)糖水偏好实验：
糖水偏好测试是检测抑郁症快感缺失的模型。在开始测试之前，笼内放置 两个相同的饮水瓶，使小鼠适应性饮水至少3天。适应结束后，将其中一瓶水替换为含有1％蔗糖的水溶液。通过称量瓶子重量来检测水和蔗糖溶液的摄入量。每天更换两个瓶子的位置，以减少因水量不同引起的饮水偏好。蔗糖偏好计算公式为：蔗糖偏好＝V(蔗糖溶液)/[V(蔗糖溶液)+V(水)]×100％，总共测试3天，取平均值。实验结果如图1b所示，抑郁小鼠的糖水偏好程度显著下降，服用CCFM1025后，小鼠恢复至正常的糖水偏好程度，表明CCFM1025能缓解抑郁导致的快感缺失。
(3)跳台实验：
第一阶段为电击训练阶段。将小鼠放入跳台反应箱的平台上，反应箱底部电栅通电(38V)，当小鼠从平台上跳下时，即遭受一次电击刺激。训练时间为3min，若个别小鼠3分钟内没有跳下平台，则人为将其赶下，确保所有动物均遭受到电击伤害，造成伤害感受性记忆。训练完毕后放回原笼饲养。第二阶段(电击训练后24h)为记忆再现测试阶段。将小鼠放入反应箱平台上，测试时间为3分钟，本次测试不通电，记录小鼠从站上平台到第1次跳下的时间(在平台上停留的时间)即跳台潜伏期，如果小鼠一直未跳下平台则以180s计算。整个实验过程中尽量避免对动物行为的人为干扰。实验结果表明(图1c)，抑郁小鼠的跳台潜伏期显著升高，代表抑郁和焦虑程度增加，而服用CCFM1025能够缓解其焦虑程度。
实施例2：CCFM1025能显著提高抑郁小鼠脑内神经递质水平
实施例2中的小鼠于第六周末安乐处死，取小鼠脑组织，并于冰上分离海马和前额叶皮质。分别取一定质量的新鲜海马和前额叶皮质组织(重量不低于50mg)，加入9倍体积的无菌PBS缓冲液(相当于1g组织加9ml的匀浆液)，用组织匀浆器进行匀浆，组织液经过3000g、20min离心后取上清，用ELISA试剂盒检测5-HT和BDNF含量。海马组织液上清加入等体积的5％高氯酸沉淀蛋白，10000g离心10min，吸取上清液经0.22μm水系滤膜过滤后，采用高效液相色谱-荧光检测法(HPLC-FLD)进行5-HTP的含量测定。色谱柱采用岛津Intertsil ODS-3(5μm，4.6mm×250mm)，流动相A为0.1mol/L的NaAc(其中含有0.1mmol/L的EDTA-2Na)，pH5.1。流动相B为甲醇，流动相A：B＝85:15，流速1.0mL/min，荧光检测激发波长为290nm，发射波长为330nm，进样量为10μL。实验结果如图2所示，结果表明，服用CCFM1025能够显著 逆转应激造成的海马中5-HT、5-HTP和前额叶皮质中BDNF水平的降低。其中，CCFM1025对海马5-HTP和5-HT的改善程度和氟西汀相当，对前额叶皮质中BDNF的改善效果显著优于氟西汀。
实施例3：短双歧杆菌CCFM1025能够降低小鼠血清中皮质酮水平
实施例2中的小鼠于第六周末安乐处死，收集小鼠血液，1000g离心15min获得血清。用ELISA试剂盒检测血清中皮质酮的含量。实验结果表明(图3)，抑郁小鼠由于持续的慢性应激，出现下丘脑-垂体-肾上腺轴(HPA)功能亢进，血清中皮质酮水平显著升高，服用CCFM1025能显著降低抑郁小鼠血清中的皮质酮水平，缓解HPA亢进，显示出良好的抗抑郁功效。
实施例4：短双歧杆菌CCFM1025能够提高抑郁小鼠血清5-HT水平
取实施例4中的血清，用ELISA试剂盒检测血清中的5-HT含量。外周组织中的5-HT对维持正常的肠道蠕动功能具有重要意义。外周组织的5-HT能够激动胃肠道平滑肌5-HT2受体，或作用于肠壁内神经节细胞5-HT4受体，引起胃肠道平滑肌收缩，使胃肠道张力增加，肠蠕动加快；外周组织5-HT的减少与便秘、胃肠道不适等疾病有直接的关联。实验结果表明(图4)，抑郁小鼠外周组织5-HT显著降低，肠道蠕动功能受损。服用氟西汀虽然有抗抑郁效果，但无法改善抑郁小鼠的肠道功能。而服用CCFM1025不仅能够缓解抑郁症状，还能显著提高血清中5-HT的含量，使之恢复至正常水平，恢复抑郁小鼠的正常肠道蠕动功能。
实施例5：短双歧杆菌CCFM1025对抑郁小鼠肠道菌群的调节作用及其多功能调节作用
取实施例2中小鼠第六周末的新鲜粪便，采用MP的粪便试剂盒提取小鼠粪便样品中总DNA。具体操作步骤如下主要参照试剂盒说明书进行。以小鼠粪便基因组为模板，以上游引物520F(5′-AYTGGGYDTAAAGNG-3′)、下游引物802R(5′-TACNVGGGTATCTAATCC-3′)为引物扩增16S rDNA的V3-V4区片段，目的片段长度为247bp左右。PCR反应结束，将观察到目的条带的所有PCR样品再次进行电泳，配制2.0％琼脂糖凝胶，于120V条件下电泳40min，跑胶结束后，在紫外灯下快速进行目的条带的切割。按照QIAquick Gel Extraction Kit胶回收试剂盒说明书进行目的条带胶回收。根据Qubit DNA3.0试剂盒检测样品DNA浓度，随后根据TurSeq DNA LT Sample Preparation Kit及其说明构建文库，最后根据MiSeq Regent Kit及其说明经过Illumina Miseq测序仪上机测定。测序完成后，剔除掉序列长度＜200bp、引物序列、不能拼接的单序列，按照重叠碱基>10bp且 无错配的标准拼接序列。将相似度大于97％序列定义为一个分类单元(Operational Taxonomic Unit，OTU)，通过Ribosomal Database Project(RDP)
Figure PCTCN2018101685-appb-000002
Bayesclassifier来确定物种。计算样品的α-多样性、β-多样性，用来评估样品的菌群多样性。其中α-多样性用chao1和PD whole tree指数来表征，结果显示(图5)，抑郁小鼠的肠道菌群，α-多样性降低，表明抑郁伴随着一定程度的肠道菌群紊乱。服用CCFM1025能显著上调肠道菌群的α-多样性，改善肠道菌群的物种丰度程度。β-多样性用主坐标分析(PCoA)进行评估(图6)，结果表明抑郁小鼠肠道菌群与正常小鼠存在显著差异，小鼠服用CCFM1025后其肠道菌群与抑郁组小鼠的菌群实现一定程度的分离，并有向正常小鼠菌群转化的趋势。
此外，韦荣球菌科(Veillonellaceae)丰度在抑郁小鼠中显著提高，而服用CCFM1025能够显著逆转这一现象；韦荣球菌科包括6个属和25个种，为革兰氏阴性、厌氧或微需氧球菌和球杆菌。其中部分菌为条件致病菌，常见于人和动物的微生物感染疾病中，在重度骨关节炎和心内膜炎中也有发现。服用CCFM1025还能增加双歧杆菌属(Bifidobacterium)和支原体科(Allobaculum)的丰度。其中，双歧杆菌属具有生物屏障、营养作用、抗肿瘤、增强免疫、改善胃肠道功能、抗衰老等多种重要的生理功能，是一种生理性有益菌。支原体科能够产生短链脂肪酸，尤其是丁酸，不仅能够为肠上皮细胞氧化供能，而且还有维持水电解质平衡、调节肠道菌群平衡、调节肠道屏障功能等重要作用。此外，短链脂肪酸能通过G蛋白偶联受体(G protein-coupled receptors，GPCRs)激活途径和组蛋白脱乙酰基酶(histone deacetylases,HDACs)抑制途径两条信号通路发挥抗炎作用，对炎症性肠病(Inflammatory bowel disease，IBD)和肥胖均具有显著的改善作用。以上结果表明CCFM1025在抗抑郁功能的基础上，还同时具备调节肠道菌群、调节免疫及肠道屏障、减少炎症性肠病和肥胖发生的多重功能。
实施例6：双歧杆菌CCFM1025对RIN14B细胞5-HTP合成的影响
细胞上清中5-HTP的测定：RIN14B细胞以4×10 5/mL的密度种在24孔板，孵育72h。弃去培养基，用含有0.1％牛血清白蛋白(BSA)和2μM氟西汀的HBSS(1mL)清洗细胞，加入1mL含有CCFM1025的HBSS悬液(对照组为不含菌体的HBSS)，37℃孵育20min，收集上清，6000g离心5min去除沉淀，取上清液冷冻于-80℃待测。HPLC-FLD检测细胞上清中的5-HTP(参考实施例3)。
TPH1的mRNA测定：上述步骤中的贴壁细胞用HBSS洗涤三次，加入1mL Trizol，置于冰上孵育5-10min，吹打使细胞脱落，将裂解液转移至无酶EP管。采用常规方法提取细胞总RNA，根据反转录试剂盒的说明进行cDNA的合成(Prime Script RT reagent Kit gDNAEraser，Takara)。合成的cDNA样品经过超微量分光光度计(NanoDrop 2000C)检测其浓度和纯度(A260/A280)，-80℃保存待用。用荧光染料SYBR Green super mix(Qiagen,Germany)混合样本，PCR体系为5μL mix、1μL cDNA、1μL正向和反向引物，用dd水补至总体积为10μL。在实时荧光定量基因扩增仪器CFX96 TMReal-Time System(Bio-Rad，USA)上进行检测，每个样本设立3个平行孔，并以管家基因β-Actin为内参，所得结果用2 -ΔΔCq的方法进行分析；所用的引物序列见表2。
表2 qPCR引物序列
Figure PCTCN2018101685-appb-000003
结果表明，CCFM1025刺激RIN14B细胞后，细胞内TPH1的mRNA水平显著提高。相应地，细胞分泌5-HTP的量也显著提高。肠嗜铬细胞分泌的5-HTP能够进入血液循环，并通过血脑屏障，为脑中5-HT的合成提供前体物质。因此CCFM1025能够特异性地通过刺激肠嗜铬细胞分泌5-HTP从而促进大脑中5-HT的合成，实现抗抑郁功能。
实施例:7：利用本发明短双歧杆菌CCFM1025制造含该菌的发酵食品
选用新鲜蔬菜洗净后榨汁，接着进行高温瞬间灭菌，在温度140℃下高温热杀菌2秒后，立即降温至37℃，再接入本发明制备的短双歧杆菌CCFM1025菌剂发酵剂，使其浓度达到10 6CFU/mL以上，在温度4℃下冷藏保存，于是得到含有本发明短双歧杆菌CCFM1025活菌的果蔬饮料。
利用本发明能够使用短双歧杆菌CCFM1025发酵生产制备其他发酵食品，所述发酵食品包括固态食品、液态食品、半固态食品。所述发酵食品包括乳制品、豆制品、果蔬制品，所述乳制品包括牛奶、酸奶油、干酪；所述果蔬制品包括黄瓜、胡萝卜、甜菜、芹菜、圆白菜制品。
所述发酵食品能够改善抑郁小鼠的抑郁样行为、提高抑郁小鼠脑中的5-羟色胺、5-羟色氨酸和脑源神经营养因子的水平、降低抑郁小鼠血清中的皮质 酮水平；所述发酵食品能够提高抑郁小鼠血清中的5-羟色胺水平，改善胃肠道蠕动功能；所述发酵食品能够改善改善抑郁小鼠的肠道菌群紊乱，降低肠道韦荣球菌科(Veillonellaceae)的丰度，提高双歧杆菌属(Bifidobacterium)和支原体科(Allobaculum)的丰度，提高肠道菌群α-多样性，减少炎症性肠病和肥胖的发生；能够提高模拟肠嗜铬细胞(RIN14B细胞)中色氨酸羟化酶1的mRNA水平，并提高该细胞5-羟色氨酸的分泌量，能够特异性地通过刺激肠道嗜铬细胞产生5-羟色氨酸从而为脑中5-羟色胺的合成提供前体物质。
应说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，其均应涵盖在本发明的权利要求范围当中。

Claims (8)

  1. 短双歧杆菌CCFM1025(Bifidobacterium breve)，于2018年6月11日保藏于广东省微生物菌种保藏中心，保藏地址为广州市先烈中路100号大院59号楼5楼广东省微生物研究所，保藏编号为GDMCC No.60386。
  2. 一种发酵食品，其特征在于：所述发酵食品为使用短双歧杆菌CCFM1025发酵生产制得，所述发酵食品包括固态食品、液态食品、半固态食品。
  3. 如权利要求2所述的发酵食品，其特征在于：所述发酵食品包括乳制品、豆制品、果蔬制品，所述乳制品包括牛奶、酸奶油、干酪；所述果蔬制品包括黄瓜、胡萝卜、甜菜、芹菜、圆白菜制品。
  4. 短双歧杆菌CCFM1025在制备体内定植益生菌中的应用。
  5. 短双歧杆菌CCFM1025在制备抗抑郁、抗炎症性肠病、抗肥胖药物和保健品中的应用。
  6. 如权利要求5所述的应用，其特征在于：所述短双歧杆菌CCFM1025能够改善抑郁小鼠的抑郁样行为、提高抑郁小鼠脑中的5-羟色胺(5-HT)、5-羟色氨酸(5-HTP)和脑源神经营养因子(BDNF)的水平、降低抑郁小鼠血清中的皮质酮水平；所述短双歧杆菌CCFM1025能够提高抑郁小鼠血清中的5-羟色胺水平，改善胃肠道蠕动功能；所述短双歧杆菌CCFM1025能够改善改善抑郁小鼠的肠道菌群紊乱，降低肠道韦荣球菌科(Veillonellaceae)的丰度，提高双歧杆菌属(Bifidobacterium)和支原体科(Allobaculum)的丰度，提高肠道菌群α-多样性，减少炎症性肠病和肥胖的发生；所述短双歧杆菌能够提高模拟肠嗜铬细胞(即RIN14B细胞)中色氨酸羟化酶1(TPH1)的mRNA水平，并提高该细胞5-羟色氨酸的分泌量，能够特异性地通过刺激肠道嗜铬细胞产生5-羟色氨酸从而为脑中5-羟色胺的合成提供前体物质。
  7. 权利要求2或3所述的发酵食品在制备抗抑郁、抗炎症性肠病、抗肥胖的功能性食品中的应用。
  8. 如权利要求7所述的应用，其特征在于：所述短双歧杆菌CCFM1025能够改善抑郁小鼠的抑郁样行为、提高抑郁小鼠脑中的5-羟色胺、5-羟色氨酸和脑源神经营养因子的水平、降低抑郁小鼠血清中的皮质酮水平；所述短双歧杆菌CCFM1025能够提高抑郁小鼠血清中的5-羟色胺水平，改善胃肠道蠕动功能；所述短双歧杆菌CCFM1025能够改善改善抑郁小鼠的肠道菌群紊乱，降低肠道韦荣球菌科(Veillonellaceae)的丰度，提高双歧杆菌属(Bifidobacterium)和支 原体科(Allobaculum)的丰度，提高肠道菌群α-多样性，减少炎症性肠病和肥胖的发生；所述短双歧杆菌能够提高模拟肠嗜铬细胞(RIN14B细胞)中色氨酸羟化酶1的mRNA水平，并提高该细胞5-羟色氨酸的分泌量，能够特异性地通过刺激肠道嗜铬细胞产生5-羟色氨酸从而为脑中5-羟色胺的合成提供前体物质。
PCT/CN2018/101685 2018-08-22 2018-08-22 短双歧杆菌ccfm1025、其发酵食品及其应用 Ceased WO2020037532A1 (zh)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118792182A (zh) * 2023-04-12 2024-10-18 合肥瀚微生物科技有限公司 一株短发酵剂乳杆菌及其应用
CN120098834A (zh) * 2025-02-24 2025-06-06 上海市儿童医院 一株缓解炎症性肠病的母乳源短双歧杆菌及其用途

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114533765B (zh) * 2022-03-17 2023-08-25 江南大学 短双歧杆菌ccfm1025在调节幼年神经发育中的应用
KR102693040B1 (ko) * 2022-12-15 2024-08-09 한림대학교 산학협력단 비피도박테리움 브레베 ckdb002 균주를 포함하는 염증성 질환의 예방 및 치료용 조성물
CN117305171B (zh) * 2023-09-28 2026-02-24 江南大学 一株短双歧杆菌及其应用
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105985918A (zh) * 2014-10-31 2016-10-05 安徽锦乔生物科技有限公司 抗肥胖的乳酸菌菌株及其在食品组成物以及医药组成物中的应用
CN107109348A (zh) * 2014-08-29 2017-08-29 科.汉森有限公司 益生菌青春双歧杆菌菌株

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107109348A (zh) * 2014-08-29 2017-08-29 科.汉森有限公司 益生菌青春双歧杆菌菌株
CN105985918A (zh) * 2014-10-31 2016-10-05 安徽锦乔生物科技有限公司 抗肥胖的乳酸菌菌株及其在食品组成物以及医药组成物中的应用

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118792182A (zh) * 2023-04-12 2024-10-18 合肥瀚微生物科技有限公司 一株短发酵剂乳杆菌及其应用
CN120098834A (zh) * 2025-02-24 2025-06-06 上海市儿童医院 一株缓解炎症性肠病的母乳源短双歧杆菌及其用途

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