WO2020238870A1 - 缓解抑郁的乳酸片球菌ccfm6432、其发酵食品及其应用 - Google Patents

缓解抑郁的乳酸片球菌ccfm6432、其发酵食品及其应用 Download PDF

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WO2020238870A1
WO2020238870A1 PCT/CN2020/092193 CN2020092193W WO2020238870A1 WO 2020238870 A1 WO2020238870 A1 WO 2020238870A1 CN 2020092193 W CN2020092193 W CN 2020092193W WO 2020238870 A1 WO2020238870 A1 WO 2020238870A1
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ccfm6432
fermented food
pediococcus
food
lactis
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PCT/CN2020/092193
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French (fr)
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王刚
陈卫
田培郡
赵建新
张灏
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江南大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
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    • 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
    • A23C11/00—Milk substitutes, e.g. coffee whitener compositions
    • A23C11/02—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins
    • A23C11/10—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins
    • A23C11/103—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins containing only proteins from pulses, oilseeds or nuts, e.g. nut milk
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    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • A23C9/1236—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt using Leuconostoc, Pediococcus or Streptococcus sp. other than Streptococcus Thermophilus; Artificial sour buttermilk in general
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    • 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
    • A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
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    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/38—Other non-alcoholic beverages
    • A23L2/382—Other non-alcoholic beverages fermented
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • 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
    • 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
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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    • 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
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    • 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
    • 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
    • A23V2400/41—Pediococcus
    • A23V2400/413—Acidilactici
    • A—HUMAN NECESSITIES
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the present invention relates to the field of microbial technology, in particular to Pediococcus lactis CCFM6432 which relieves depression, its fermented food and its application.
  • Depression is characterized by significant and lasting depression. Currently, more than 350 million people worldwide suffer from depression, and the incidence has continued to rise in recent years. At present, antidepressants that are widely used in clinical treatment, such as Selective Serotonin Reuptake Inhibitor (SSRI), generally have shortcomings such as slow onset, inconsistent treatment effects, and large side effects, making the treatment of depression serious The limitations.
  • SSRI Selective Serotonin Reuptake Inhibitor
  • the human intestine contains more than 1,000 kinds of microorganisms, with a total of about 10 14 to 10 15 and its mass can reach 1 to 1.5 kilograms.
  • the total number of coding genes of these gut microbes exceeds 3.3 million, which is about 100 times the total number of human coding genes. Therefore, the gut microbes are considered to be the second genome of the human body.
  • the gut microbial genome and human genome interact with environmental factors to affect a variety of important physiological functions of the host.
  • 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.
  • probiotics As a dietary supplement, probiotics have been widely accepted by consumers. At present, most studies on the function of probiotics are focused on improving gastrointestinal function, regulating nutrition metabolism and immunity. A large number of scientific research and clinical experiments have proved that probiotics can significantly improve constipation, enteritis, lactose intolerance, anti-infection, inflammation, allergies, and glucose and lipid metabolism disorders. With the gradual maturity of the "brain-gut axis" theory, the use of probiotics to regulate the intestinal flora and improve neurological function has become a new method for the treatment of depression.
  • the present invention overcomes the deficiencies in the prior art and provides a Pediococcus acidilactici CCFM6432, which was deposited in the Guangdong Provincial Microbial Culture Collection on April 25, 2019.
  • the address is Guangdong Institute of Microbiology, 5th Floor, Building 59, Dayuan No. 100, Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No. 60638.
  • the Pediococcus acidilactici CCFM6432 has the following characteristics:
  • the second objective of the present invention is to provide a probiotic preparation containing the Pediococcus acidilactici CCFM6432.
  • the concentration of Pediococcus lactis ⁇ 1 ⁇ 10 5 CFU/mL.
  • the concentration of Pediococcus lactis ⁇ 1 ⁇ 10 5 CFU/g.
  • the third object of the present invention is to provide a fermented food, which is produced by fermentation using Pediococcus lactis CCFM6432.
  • the fermented food includes 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
  • the fruit and vegetable products include cucumbers, carrots, beets, Celery and cabbage products.
  • the fermented food is prepared after being fermented with Pediococcus lactis CCFM6432 at the beginning of fermentation.
  • the fourth objective of the present invention is to provide the application of Pediococcus lactis CCFM6432 in preparing probiotics for in vivo colonization.
  • the application is as a host to prepare probiotics to be colonized in vivo.
  • the application is to culture the Pediococcus lactis CCFM6432 in an MRS liquid medium at 37°C for 48 hours to obtain a cell culture solution of Pediococcus lactis CCFM6432.
  • the cell culture fluid is used to prepare a probiotic product.
  • the fifth object of the present invention is to provide the application of Pediococcus lactis CCFM6432 in the preparation of functional foods, medicines and health products for anti-depression, anti-inflammatory bowel disease, anti-obesity and anti-type II diabetes.
  • the drug further contains a pharmaceutically acceptable carrier.
  • the fifth object of the present invention is to provide the application of the fermented food in the preparation of anti-depressant, anti-inflammatory, anti-inflammatory bowel disease, anti-obesity and anti-type I diabetes functional food.
  • the Pediococcus lactis CCFM6432 can alleviate depression-like behavior in depressed mice and increase the neurotransmitter serotonin in the brain Content, promote the biosynthesis of 5-hydroxytryptophan, a neurotransmitter precursor in peripheral tissues; the Pediococcus lactis CCFM6432 can inhibit the excessive secretion of corticotropin releasing factor in the hypothalamus, reduce the level of corticosterone in the serum, and relieve "Hypothalamus-pituitary-adrenal axis" hyperfunction; the Pediococcus lactis CCFM6432 can regulate the host's immunity, increase the proportion of splenic regulatory T cells, and reduce serum pro-inflammatory factors IL-1 ⁇ , IL-6 and TNF- ⁇ The concentration of Pediococcus lactis CCFM6432 can also improve the diversity of intestinal f
  • the beneficial effects of the present invention in the experiment of depression model mice, taking Pediococcus lactis CCFM6432 of the present invention can reduce the depression-like behavior of mice caused by stress and increase the neurotransmitter (5-HT) content of the brain tissue of depressed mice , Increase the biosynthesis of the neurotransmitter precursor (5-HTP) in peripheral tissues, inhibit the excessive secretion of the hypothalamic corticotropin releasing factor (CRF), and reduce the level of corticosterone in the serum; the fermented food can also regulate the host Improve the immunity of spleen, increase the proportion of regulatory T cells in the spleen, and reduce the concentration of pro-inflammatory factors IL-1 ⁇ , IL-6 and TNF- ⁇ in the serum; in addition, the fermented food can improve the diversity of intestinal flora and restore stress The resulting intestinal flora disorder, increase the abundance of beneficial bacteria (Bacillus, Faecococcus), increase the content of intestinal butyric acid, reduce the risk of inflammatory bowel disease
  • the Pediococcus lactis CCFM6432 of the present invention can be used to prepare foods, health products and medicines with the functions of anti-depression, anti-inflammatory, anti-inflammatory bowel disease, anti-obesity and anti-type I diabetes, and has very broad application prospects .
  • Pediococcus acidilactici CCFM6432 (Pediococcus acidilactici), classified and named Pediococcus acidilactici, was deposited in the Guangdong Provincial Microbial Culture Collection on April 25, 2019, and the deposit number is GDMCC No. 60638.
  • Figure 1 shows the core gene analysis of CCFM6432 and Pediococcus lactis model strain DSM20284;
  • Figure 2 shows the performance of mice in the elevated plus maze.
  • A the number of times to enter the open arm;
  • B the residence time in the open arm;
  • C the movement track of the mouse in the plus maze; where *P ⁇ 0.05, **P ⁇ 0.01.
  • Figure 3 shows the performance of mice in the tail suspension experiment and the sugar water preference experiment.
  • A The time the mice were still in the tail suspension experiment;
  • B The consumption ratio of the mice to sugar water; where *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
  • Figure 4 shows the effect of CCFM6432 on the neurotransmitter 5-HT content in mouse brain tissue.
  • A The concentration of 5-HT in the prefrontal cortex;
  • B The concentration of 5-HT in the brainstem; where *P ⁇ 0.05, ****P ⁇ 0.0001.
  • Figure 5 shows the effect of CCFM6432 on the synthesis and secretion of 5-HT precursor in the mouse intestine.
  • A The expression level of tryptophan hydroxylase 1 (Tph1) mRNA in enterochromaffin cells;
  • B the content of 5-HTP secreted by enterochromaffin cells; where *P ⁇ 0.05, ****P ⁇ 0.0001.
  • Figure 6 shows the alleviating effect of CCFM6432 on hyperHPA in depressed mice.
  • A The level of corticotropin releasing factor in the hypothalamus;
  • B the level of corticosterone in the serum.
  • Figure 7 shows the effect of CCFM6432 on the proportion of regulatory T cells in the spleen of depressed mice. Where *P ⁇ 0.05, **P ⁇ 0.01.
  • Figure 8 shows the effect of CCFM6432 on the level of inflammatory factors in the serum of depressed mice.
  • A The content of IL-1 ⁇ in serum
  • B The content of IL-6 in serum
  • C The content of TNF- ⁇ in serum.
  • Figure 9 shows the effect of CCFM6432 on the intestinal flora of mice.
  • A Shannon index
  • B Simpson index
  • C PCoA scatter plot
  • D Actinomycota abundance
  • E Proteobacteria abundance
  • F Actinomycota and Proteobacteria Door ratio.
  • Figure 10 shows the effect of CCFM6432 on intestinal probiotics and their metabolites.
  • A Bacillus abundance
  • B Coprococcus abundance
  • C butyric acid content in feces.
  • the “one embodiment” or “embodiment” referred to herein refers to a specific feature, structure, or characteristic that can be included in at least one implementation of the present invention.
  • the appearances of "in one embodiment” in different places in this specification do not all refer to the same embodiment, nor are they separate or selectively mutually exclusive embodiments with other embodiments.
  • centrifuge at 12000rpm for 5-10min After mixing upside down, centrifuge at 12000rpm for 5-10min, and take 200 ⁇ L of supernatant; add 400 ⁇ L of ice ethanol or ice isopropanol to In 200uL supernatant, let stand at -20°C for 1h, centrifuge at 12000rpm for 5-10min, discard the supernatant; add 500 ⁇ L of 70% (volume percentage) ice ethanol to resuspend the pellet, centrifuge at 12000rpm for 1-3min, discard the supernatant; oven dry at 60°C , Or air dry naturally; 50 ⁇ L ddH 2 O re-dissolves the precipitate for PCR;
  • the sequencing result is identified as belonging to Pediococcus lactis A newly discovered strain.
  • the strain is stored at -80°C for later use.
  • the results of the comparative genome analysis with the model strain Pediococcus acidilactici DSM 20284 are shown in Table 1.
  • the core gene analysis results are shown in Figure 1. The number of core genes shared by the two strains is 1816, the number of unique genes for CCFM6432 is 54 and the number of unique genes for DSM20284 is 49.
  • Bacterial characteristics it is Gram stain positive, does not form spores, and does not move bacteria.
  • Colony characteristics The colony is milky white, round, with neat edges, slightly convex, opaque, and moist and smooth;
  • mice Thirty-two 6-week-old male C57BL/6J mice were selected. After one week of adaptation to the environment, they were randomly divided into four groups according to their body weight: normal control group, depression model group, drug intervention group, CCFM6432 intervention group, each containing 8 mice. Animal grouping and treatment methods are shown in Table 2.
  • Chronic unpredictable stress depression mouse model 1-2 kinds of stimuli are randomly used every day, and the time of stimulus every day is randomly determined to avoid circadian rhythms. Each method does not exceed three times and lasts for five weeks. Stimulating factors include: (1) fasting for 24 hours; (2) water-free + empty bottle stimulation for 24 hours; (3) tail clamping for 3 minutes; (4) wet padding for 24 hours; (5) braking for 1 to 2 hours; ° Tilt the cage for 24h; (7) Continuous light for 24h; (8) No litter for 24h; (9) Forced swimming for 15 minutes; (10) Orphan for 24h.
  • Lactic acid bacteria gavage take the activated second-generation Pediococcus lactis CCFM6432 and culture it at 37°C for 24h, centrifuge at 4°C and 8000r/min for 3min to collect the bacteria, discard the supernatant and resuspend the bacteria in 5% sterilized degreasing emulsion
  • concentration of lactic acid bacteria reaches 5 ⁇ 10 9 CFU/mL.
  • the gavage volume is 0.2 mL/head/day.
  • mice were placed into the maze from the central grid to the closed arm, and their activities within 10 minutes were recorded.
  • Observation indicators include: open arm entry times (two front melons must enter the arm), open arm stay time, closed arm entry times, and closed arm stay time. Calculate the proportion of open arm residence time, the proportion of open arm entry times, and the total number of entries in the elevated plus maze.
  • the mouse was taken out, the arms were cleaned, and alcohol was sprayed to remove the odor, and the next experiment was carried out. The results are shown in Figure 2.
  • the number of times and residence time of depressed mice entering the open arms were significantly reduced. Taking CCFM6432 can significantly improve this symptom. Fluoxetine has no effect on the depression-like behavior of mice in the plus maze.
  • the tail suspension experiment is similar to the forced swimming experiment, and it is also a behavioral despair model.
  • the rear 1/3 of the tail of the mouse was fixed with tape and hung on the bracket, the head was 30cm away from the table, and the camera was taken.
  • the background of the camera was in obvious contrast with the color of the mouse's coat.
  • the timing was stopped after 6 minutes, and the immobility time of the mice in the last four minutes (3-6 minutes) was counted using the small animal behavior analysis software. Among them, the immobile state means that the animal gives up actively struggling and is completely immobile.
  • the results of the experiment are shown in Figure 3A.
  • the depressed mice have a significant increase in the immobility time in the tail suspension experiment. Taking CCFM6432 can reduce their immobility time and relieve their depression, and the effect of CCFM6432 is better than the antidepressant fluoxetine Ting.
  • the sugar water preference test is a model for detecting anhedonia in depression. Before starting the test, two identical drinking bottles were placed in the cage to allow the mice to drink adaptively for at least 3 days. After the adaptation, one of the bottles of water was replaced with an aqueous solution containing 1% sucrose. Check the intake of water and sucrose solution by weighing the bottle. Change the positions of the two bottles every day to reduce drinking preferences due to different amounts of water.
  • the experimental results are shown in Figure 3B. Depressed mice have a significant decrease in sugar preference. After taking CCFM6432, the mice return to their normal sugar preference, indicating that CCFM6432 can relieve the anhedonia caused by depression.
  • Example 3 CCFM6432 increases neurotransmitter (5-HT) 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 brainstem and prefrontal cortex were separated on ice.
  • Take a certain quality of fresh brain stem and prefrontal cortex tissue (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
  • the supernatant was taken from the pulp and tissue fluid, and an equal volume of 5% perchloric acid was added to precipitate the protein.
  • Centrifuge at 10000g for 10min. After filtering the supernatant through a 0.22 ⁇ m water filter membrane, the 5-HT was detected by ELISA Content.
  • the results of the experiment are shown in Figure 4.
  • the results show that taking CCFM6432 can significantly reverse the decrease of 5-HT levels in the brain stem and prefrontal cortex caused by chronic stress.
  • CCFM6432 is better than fluoxetine in improving 5-HT in the brain stem and prefrontal cortex.
  • Research reports have shown that, as a selective 5-HT reuptake inhibitor, fluoxetine has inconsistencies in its ability to function in different individuals, and there is a 2-4 week delay in onset.
  • the results of this experiment show that after taking Pediococcus lactis CCFM6432 for five weeks, the content of 5-HT in the brain can be significantly increased, there is no delayed onset of action, and it shows a good potential for antidepressant treatment.
  • Example 4 Pediococcus lactis CCFM6432 stimulates enterochromaffin cells to secrete 5-HTP
  • TPH1 tryptophan hydroxylase 1
  • the adherent cells in the above steps are washed three times with HBSS, 1mL Trizol is added, and the cells are incubated on ice for 5-10min. The cells are detached by pipetting and the lysate is transferred to no Enzyme EP tube.
  • the total cell RNA was extracted by conventional methods, and cDNA synthesis was performed according to the instructions of the reverse transcription kit (Prime Script RT reagent Kit gDNA Eraser, Takara).
  • the synthesized cDNA sample was tested for its concentration and purity (A260/A280) by an ultra-micro spectrophotometer (NanoDrop 2000C), and stored at -80°C until use.
  • the samples were mixed with the fluorescent dye SYBR Green super mix (Qiagen, Germany), and the PCR system was 5 ⁇ L mix, 1 ⁇ L cDNA, 1 ⁇ L forward and reverse primers, and dd water was used to make up the total volume to 10 ⁇ L.
  • the detection was carried out on the real-time fluorescent quantitative gene amplification instrument CFX96 TM Real-Time System (Bio-Rad, USA). Three parallel wells were set up for each sample, and the housekeeping gene ⁇ -Actin was used as the internal reference. The result was obtained with 2- ⁇ Cq Method for analysis; the primer sequences used are shown in Table 3.
  • Example 5 Pediococcus lactis CCFM6432 can relieve HPA hyperfunction in depressed mice
  • mice in Example 2 were euthanized at the end of the sixth week, and their blood was collected and centrifuged at 1000 g for 15 minutes to obtain serum. Take mouse brain tissue and separate hypothalamus on ice to prepare tissue homogenate (refer to Example 2). ELISA kit was used to detect the content of corticosterone in serum and the content of corticotropin releasing factor (CRF) in hypothalamus.
  • the experimental results show ( Figure 6) that due to continuous chronic stress, depressed mice have hyperfunction of the hypothalamus-pituitary-adrenal axis (HPA), the CRF released by the hypothalamus increases significantly, and the serum corticosterone concentration rises significantly. Taking CCFM6432 can significantly inhibit the release of CRF and reduce the level of serum corticosterone, thereby alleviating HPA hyperactivity and showing good antidepressant effect.
  • HPA hypothalamus-pituitary-adrenal axis
  • mice in Example 2 were euthanized at the end of the sixth week.
  • Fresh mouse spleens (-50 mg) were taken, 5 mL of pre-cooled PBS buffer was added, cut, ground, and filtered with 200 mesh nylon to obtain a tissue cell suspension.
  • Tregs regulatory T cells
  • eBioscience Mouse Regulatory T Cell Staining Kit Invitrogen Corporation, Carlsbad, CA, USA.
  • Tregs CD4+CD25+Foxp3+
  • the levels of IL-1 ⁇ , IL-6 and TNF- ⁇ in serum were detected by ELISA kit.
  • Figure 7 Chronic stress can significantly reduce the proportion of Tregs in the spleen of mice, which is the impaired immunity of mice; at the same time, the proinflammatory factors IL-1 ⁇ , IL-6 and TNF in the serum of mice The - ⁇ content increased significantly ( Figure 8), indicating that depressed mice were accompanied by systemic inflammation.
  • Both fluoxetine and CCFM6432 can significantly improve the immune status of depressed mice and reduce the degree of inflammation. In particular, compared with fluoxetine, taking CCFM6432 can significantly reduce the content of TNF- ⁇ in serum and has a better anti-inflammatory effect.
  • Example 7 Regulation of Pediococcus lactis CCFM6432 on the intestinal flora and metabolites of depression mice
  • 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.
  • CCFM6432 can significantly reverse this phenomenon and restore the structure of intestinal flora to normal ( Figure 9D, E, F) ; ⁇ -diversity was evaluated by principal co-ordinates analysis (PCoA) ( Figure 9C). It can also be seen from the results that both fluoxetine and CCFM6432 intervention can make the structure of the mouse intestinal flora tend to normalization. In addition, taking CCFM6432 can significantly increase the abundance of Bacillus ( Figure 10A). Bacillus is an important type of lactic acid-producing bacteria in the host, which plays a very important role in maintaining normal intestinal motility and relieving constipation.
  • Bacillus such as Bacillus coagulans and Bacillus subtilis
  • Bacillus coagulans and Bacillus subtilis can secrete bacteriocins, inhibit the growth of intestinal pathogenic bacteria, and prevent the occurrence of Inflammatory bowel disease (IBD).
  • IBD Inflammatory bowel disease
  • CCFM6432 can also significantly increase the abundance of Coprococcus in the intestinal tract of depressed mice ( Figure 10B).
  • Coprococcus is an important butyric acid-producing bacteria in the intestine.
  • Butyric acid can not only provide energy for intestinal epithelial cell oxidation , Improve the intestinal barrier function, maintain the immune homeostasis of the intestinal mucosa, but also through the G protein-coupled receptors (G protein-coupled receptors, GPCRs) activation pathway and histone deacetylases (histone deacetylases, HDACs) inhibition Through two signal pathways, it induces the expression of downstream glucose and lipid metabolism regulators, such as glucagon-like polypeptide-1 (GLP-1) and tyrosine (PYY), which can significantly improve type II diabetes and obesity .
  • GLP-1 glucagon-like polypeptide-1
  • PYY tyrosine
  • CCFM6432 Take 500uL of the supernatant to the gas phase vial, and detect the content of short-chain fatty acids (acetic acid, butyric acid, isobutyric acid, butyric acid, etc.) by GC-MS using selective ion sweeping. The results are shown in Figure 10C. Taking Pediococcus lactis CCFM6432 can improve the decrease in butyric acid levels caused by depression, which further proves that CCFM6432 can not only relieve depression, but also can relieve and prevent type II diabetes and obesity by adjusting the intestinal butyric acid content And other diseases.
  • short-chain fatty acids acetic acid, butyric acid, isobutyric acid, butyric acid, etc.
  • mMRS medium MRS medium + 0.05% cysteine hydrochloride
  • the survival rate is the ratio of the logarithm of the number of viable bacteria in the culture solution at the time of sampling to the logarithm of the number of viable bacteria at the 0th hour, expressed in %.
  • Take 1 mL of the culture solution of Pediococcus lactis CCFM6432 and add 9 mL of artificial simulated intestinal fluid (containing 0.3% bovine bile salt, 1% trypsin, pH 8.0 mMRS medium), and anaerobic culture at 37°C, respectively at 0h and 0.5 Samples were taken at h, 1h and 2h, and the plate colonies were counted by pouring and culturing with mMRS agar medium, and the number of viable bacteria was determined and the survival rate was calculated.
  • the survival rate is the ratio of the logarithm of the number of viable bacteria in the culture solution at the time of sampling to the logarithm of the number of viable bacteria at the 0th hour, expressed in %.
  • the experimental results are shown in Table 4 and Table 5. The results show that Pediococcus lactis CCFM6432 has a good tolerance to artificial gastrointestinal fluid.
  • Example 9 Use of Pediococcus lactis CCFM6432 of the present invention to manufacture fermented food
  • the invention can use Pediococcus longiformis CCFM6432 to ferment and produce other fermented foods, and the fermented foods include solid foods, liquid foods, and semi-solid foods.
  • 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, and cabbage products.
  • fermented foods were verified according to the methods in Examples 2-7. The results showed that the fermented foods obtained can alleviate depression-like behaviors in mice caused by stress and increase neurotransmitter (5-HT) in the brain tissue of depressed mice.
  • 5-HT neurotransmitter precursors
  • the fermented food can also Regulate the host’s immunity, increase the proportion of regulatory T cells in the spleen, and reduce the concentration of pro-inflammatory factors IL-1 ⁇ , IL-6 and TNF- ⁇ in the serum; in addition, the fermented food can improve the diversity of intestinal flora and restore
  • the intestinal flora disorder caused by stress increases the abundance of beneficial bacteria (Bacillus, Faecococcus), increases the content of butyric acid in the intestine, and reduces the risk of inflammatory bowel disease, obesity and type II diabetes.

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Abstract

提供了缓解抑郁的乳酸片球菌CCFM6432、其发酵食品及其应用。所述的乳酸片球菌CCFM6432能够用于制备具有抗抑郁、抗炎症、抗炎症性肠病、抗肥胖和抗I型糖尿病等的功能的食品、保健品和药品，还提供发酵食品，所述发酵食品使用乳酸片球菌CCFM6432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。

Description

缓解抑郁的乳酸片球菌CCFM6432、其发酵食品及其应用 技术领域
本发明涉及属于微生物技术领域，具体涉及缓解抑郁的乳酸片球菌CCFM6432、其发酵食品及其应用。
背景技术
抑郁症以显著而持久的情绪低落为主要临床特征。目前全球有超过3.5亿人患有抑郁症，且近年来发病率持续上升。目前被广泛应用于临床治疗的抗抑郁药物，如选择性五羟色胺重摄取抑制剂(Selective Serotonin Reuptake Inhibitor，SSRI)普遍存在起效缓慢、治疗效果不一致、副作用大等缺点，使得抑郁症的治疗存在严重的局限性。
人体肠道内含有超过1000多种微生物，总数约为10 14～10 15个，其质量可达1～1.5千克。这些肠道微生物编码基因的总数超过330万，约为人类自身编码基因总数的100倍，因此肠道微生物又被认为是人体的第二基因组。肠道微生物基因组与人体基因组一起，通过与环境因素的相互作用，影响宿主的多种重要的生理功能。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。
许多临床研究显示，抑郁症患者的肠道菌群与健康对照组相比存在显著差异，包括多样性降低、有益菌类群丰度减少等。此外，抑郁症常伴随有便秘、腹泻等胃肠道的功能异常，这可能于肠道菌群的紊乱、抗抑郁药物的副作用相关。神经功能异常的动物和健康对照动物的肠道菌群之间也存在显著差异，并被多种抑郁模型所证实，包括嗅球切除模型、母婴分离模型、社会应激模型、慢性不可预知性应激模型等。这些研究均说明，肠道菌群在抑郁症的发生发展中发挥着至关重要的作用。
益生菌作为膳食补充剂，已被消费者广泛接受。目前，大部分关于益生菌功能的研究都集中在改善胃肠道功能、调节营养代谢和免疫等方面。大量的科学研究和临床实验已经证明，益生菌对便秘、肠炎、乳糖不耐、抗感染、炎症、过敏、糖脂代谢紊乱均具有显著改善作用。随着“脑肠轴”理论的逐渐成熟，通过益生菌来调节肠道菌群进而改善神经功能，成为治疗抑郁症的一种新的手段。某些双歧杆菌和乳杆菌对神经功能的调节作用也已被动物研究和临床研究所证实，例如Lactobacillus helveticus R0052、Bifidobacterium longum R0175等。
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目的，本发明克服现有技术中存在的不足，提供一种乳酸片球菌(Pediococcus acidilactici)CCFM6432，于2019年4月25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.60638。
在一种实施方式中，所述乳酸片球菌(Pediococcus acidilactici)CCFM6432具有如下特性：
(1)减轻抑郁小鼠的抑郁样行为；
(2)提高抑郁小鼠脑中神经递质5-羟色胺含量；
(3)促进抑郁小鼠外周组织神经递质前体5-羟色氨酸的生物合成；
(4)抑制抑郁小鼠下丘脑促肾上腺皮质激素释放因子的过度分泌，降低抑郁小鼠血清中皮质酮的水平，从而缓解“下丘脑-垂体-肾上腺轴”功能亢进；
(5)调节宿主的免疫力，提高脾脏调节性T细胞比例，降低血清中促炎因子IL-1β、IL-6和TNF-α的浓度；
(6)改善宿主肠道菌群多样性，恢复应激造成的肠道菌群紊乱，提高芽孢杆菌属、粪球菌属的丰度、提高肠道丁酸的含量、减少炎症性肠病、肥胖和II型糖尿病发生的风险。
本发明的第二个目的是提供一种益生菌制剂，所述益生菌制剂含有所述乳酸片球菌(Pediococcus acidilactici)CCFM6432。
在一种实施方式中，所述乳酸片球菌的浓度≥1×10 5CFU/mL。
在一种实施方式中，所述乳酸片球菌的浓度≥1×10 5CFU/g。
本发明的第三个目的是提供一种发酵食品，所述发酵食品为使用乳酸片球菌CCFM6432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实施方法，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。
在一种实施方式中，所述发酵食品是在发酵初始接入所述乳酸片球菌CCFM6432进行发酵后制得。
本发明的第四个目的是提供乳酸片球菌CCFM6432在制备体内定植益生菌中的应用。
在一种实施方式中，所述应用是作为宿主，制备体内定植的益生菌。
在一种实施方式中，所述应用是将所述乳酸片球菌CCFM6432在MRS液体培养基中，37℃培养48小时，获得乳酸片球菌CCFM6432细胞培养液。
在一种实施方式中，用所述细胞培养液制备益生菌产品。
本发明的第五个目的是提供乳酸片球菌CCFM6432在制备抗抑郁、抗炎症性肠病、抗肥胖和抗II型糖尿病的功能性食品、药物和保健品中的应用。
在一种实施方式中，所述药物还含有药学上可接受的载体。
本发明的第五个目的是提供所述的发酵食品在制备抗抑郁、抗炎症、抗炎症性肠病、抗肥胖和抗I型糖尿病等的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：所述乳酸片球菌CCFM6432能够减轻抑郁小鼠的抑郁样行为、提高脑中神经递质5-羟色胺含量、促进外周组织神经递质前体5-羟色氨酸的生物合成；所述乳酸片球菌CCFM6432能够抑制下丘脑促肾上腺皮质激素释放因子的过度分泌，降低血清中皮质酮的水平，从而缓解“下丘脑-垂体-肾上腺轴”功能亢进；所述乳酸片球菌CCFM6432能够调节宿主的免疫力，提高脾脏调节性T细胞比例，降低血清中促炎因子IL-1β、IL-6和TNF-α的浓度；所述乳酸片球菌CCFM6432还能够善肠道菌群多样性，恢复应激造成的肠道菌群紊乱，提高芽孢杆菌属、粪球菌属的丰度、提高肠道丁酸的含量、减少炎症性肠病、肥胖和II型糖尿病发生的风险。
本发明的有益效果：在抑郁模型小鼠实验中，服用本发明的乳酸片球菌CCFM6432能够减轻应激造成的小鼠的抑郁样行为、提高抑郁小鼠脑组织神经递质(5-HT)含量、增加外周组织神经递质前体(5-HTP)的生物合成、抑制下丘脑促肾上腺皮质激素释放因子(CRF)的过度分泌、降低血清中的皮质酮水平；所述发酵食品还能调节宿主的免疫力、提高脾脏调节性T细胞比例、降低血清中促炎因子IL-1β、IL-6和TNF-α的浓度；此外，所述发酵食品能够改善肠道菌群多样性、恢复应激造成的肠道菌群紊乱、提高有益菌的丰度(芽孢杆菌属、粪球菌属)、提高肠道丁酸的含量、减少炎症性肠病、肥胖和II型糖尿病发生的风险。
本发明所述的乳酸片球菌CCFM6432能够用于制备具有抗抑郁、抗炎症、抗炎症性肠病、抗肥胖和抗I型糖尿病等的功能的食品、保健品和药品，具有非常广泛的应用前景。
生物材料保藏
乳酸片球菌CCFM6432(Pediococcus acidilactici)，分类命名为Pediococcus acidilactici，于2019年4月25日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No.60638。
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CCFM6432与乳酸片球菌模式菌株DSM20284的核心基因分析；
图2为小鼠在高架十字迷宫中的表现。(A)进入开放臂的次数；(B)在开放臂中的停留时间；(C)小鼠在十字迷宫中的行动轨迹；其中*P<0.05，**P<0.01。
图3为小鼠在悬尾实验和糖水偏好实验中的表现。(A)小鼠在悬尾实验中静止不动的时间；(B)小鼠对糖水的消耗比例；其中*P<0.05，**P<0.01，***P<0.001。
图4为CCFM6432对小鼠脑组织中神经递质5-HT含量的影响。(A)前额叶皮质中5-HT的浓度；(B)脑干中5-HT的浓度；其中*P<0.05，****P<0.0001。
图5为CCFM6432对小鼠肠道5-HT前体合成和分泌的影响。(A)肠嗜铬细胞中色氨酸羟化酶1(Tph1)mRNA的表达量；(B)肠嗜铬细胞分泌的5-HTP的含量；其中*P<0.05，****P<0.0001。
图6为CCFM6432对抑郁小鼠HPA亢进的缓解作用。(A)下丘脑中促肾上腺皮质激素释放因子的水平；(B)血清中皮质酮的水平。其中*P<0.05，**P<0.01，***P<0.001。
图7为CCFM6432对抑郁小鼠脾脏调节性T细胞比例的影响。其中*P<0.05，**P<0.01。
图8为CCFM6432对抑郁小鼠血清中炎症因子水平的影响。(A)血清中IL-1β的含量；(B)血清中IL-6的含量；(C)血清中TNF-α的含量。
图9为CCFM6432对小鼠肠道菌群的影响。(A)香浓指数；(B)辛普森指数；(C)PCoA散点图；(D)放线菌门丰度；(E)变形菌门丰度；(F)放线菌门和变形菌门比例。其中**P<0.01，***P<0.001。
图10为CCFM6432对肠道益生菌属及代谢产物的影响。(A)芽孢杆菌属(Bacillus)丰度；(B)粪球菌属(Coprococcus)丰度；(C)粪便中丁酸含量。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。
实施例1乳酸片球菌的筛选
(一)乳酸片球菌CCFM6432的分离筛选：
(l)取1g健康人的新鲜粪便。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MRS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)乳酸片球菌的分子生物学鉴定：
(l)单菌基因组抽提：将步骤(二)所筛选得到的乳酸片球菌培养过夜，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l mL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，鉴定为乳酸片球菌。
(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乳酸片球菌的一种新发现的菌株。菌株-80℃保藏备用。与模式菌株Pediococcus acidilactici DSM 20284的比较基因组分析结果见表1；核心基因分析结果见图1，两株共有核心基因数为1816，CCFM6432特有基因数为54，DSM20284特有基因数为49。
表1与模式菌株Pediococcus acidilactici DSM 20284的比较基因组分析结果
Figure PCTCN2020092193-appb-000001
(三)对乳酸片球菌CCFM6432的生物学特性进行研究，结果显示，所述乳酸片球菌CCFM6432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：菌落呈乳白色，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。
实施例2：乳酸片球菌CCFM6432减少小鼠的抑郁样行为
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：正常对照组、抑郁模型组、药物干预组、CCFM6432干预组，每组含8只小鼠。动物分组及处理方法见表2。
表2动物实验分组及处理方法
Figure PCTCN2020092193-appb-000002
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代的乳酸片球菌CCFM6432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。包括高架十字迷宫实验、悬尾实验和糖水偏好实验。具体实施方法和结果如下：
(1)高架十字迷宫实验
实验开始时将小鼠从中央格面向闭合臂放入迷宫，记录10分钟内的活动情况。观察指标包括：开放臂进入次数(必须有两只前瓜进入臂内)，开放臂停留时间，闭合臂进入次数，闭合臂停留时间。计算开放臂停留时间比例，开放臂进入次数比例，高架十字迷宫中总进入次数。实验完成后将小鼠取出，将两臂清理干净，喷洒酒精除去气味，进行下一只实验。结果如图2所示，抑郁小鼠进入开放臂的次数和停留时间均显著降低，服用CCFM6432能够显著改善这一症状，氟西汀对小鼠在十字迷宫中的抑郁样行为无改善作用。
(2)悬尾实验
悬尾实验与强迫游泳实验类似，也是一种行为绝望模型。将小鼠尾部后1/3处用胶带固定，悬挂于支架上，头部距离台面30cm，进行摄像，摄像背景与小鼠毛色呈明显反差。计时6min后停止，利用小动物行为学分析软件对小鼠后四分钟(3-6min)的不动时间进行统计。其中，不动状态是指动物放弃主动挣扎，处于完全不动的状态。实验结果如图3A所示，抑郁小鼠在悬尾实验中静止不动的时间显著增加，而服用CCFM6432能够降低其不动时间，缓解其抑郁状态，且CCFM6432的效果优于抗抑郁药物氟西汀。
(3)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3B所示，抑郁小鼠的糖水偏好程度显著下降，服用CCFM6432后，小鼠恢复至正常的糖水偏好程度，表明CCFM6432能缓 解抑郁导致的快感缺失。
实施例3：CCFM6432提高抑郁小鼠脑中神经递质(5-HT)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，并于冰上分离脑干和前额叶皮质。分别取一定质量的新鲜脑干和前额叶皮质组织(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，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ELISA试剂盒检测5-HT的含量。实验结果如图4所示，结果表明，服用CCFM6432能够显著逆转慢性应激造成的脑干和前额叶皮质中5-HT水平的降低。其中，CCFM6432对脑干和前额叶皮质中5-HT的改善程度优于氟西汀。研究报道表明，作为选择性5-HT重摄取抑制剂，氟西汀在不同个体内发挥作用的能力具有不一致性，且存在2-4周的起效延迟期。本实验结果表明，服用乳酸片球菌CCFM6432五周后，即可显著提高脑内5-HT的含量，不存在起效延迟的现象，显示出良好的抗抑郁治疗潜力。
实施例4：乳酸片球菌CCFM6432刺激肠嗜铬细胞分泌5-HTP
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CCFM6432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)。
色氨酸羟化酶1(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 gDNA Eraser，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 TM Real-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3。
表3 qPCR引物序列
Figure PCTCN2020092193-appb-000003
Figure PCTCN2020092193-appb-000004
结果表明(图5)，CCFM6432刺激RIN14B细胞后，细胞内Tph1的mRNA水平显著提高，分别提高2.5和近40倍。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CCFM6432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5：乳酸片球菌CCFM6432能够缓解抑郁小鼠HPA功能亢进
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。取小鼠脑组织，并于冰上分离下丘脑，制备组织匀浆液(参考实施例2)。用ELISA试剂盒检测血清中皮质酮的含量和下丘脑中促肾上腺皮质激素释放因子(corticotropin releasing factor，CRF)的含量。实验结果表明(图6)，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，下丘脑释放的CRF显著增多，并导致血清中皮质酮浓度显著上升，服用CCFM6432能显著抑制CRF的释放，降低血清皮质酮水平，从而缓解HPA亢进，显示出良好的抗抑郁功效。
实施例6：乳酸片球菌对抑郁小鼠的免疫调节作用
实施例2中的小鼠于第六周末安乐处死，取新鲜小鼠脾脏(～50mg)，加入5mL预冷的PBS缓冲液，剪碎、研磨、用200目尼龙晒过滤得到组织细胞悬液。加入2mL红细胞裂解液裂解4min，室温下离心去除裂解液(300×g，5min)，所得细胞沉淀用预冷的PBS洗两遍，用一定量的PBS重悬，得到淋巴细胞悬浮液。调节性T细胞(Regulatory cells，Tregs)的抗体标记按照eBioscience Mouse Regulatory T Cell Staining Kit(Invitrogen Corporation,Carlsbad,CA,USA)的说明书进行操作。采用流式细胞仪对Tregs(CD4+CD25+Foxp3+)进行检测。血清中IL-1β、IL-6和TNF-α的含量采用ELISA试剂盒进行检测。实验结果如图7所示，慢性应激可导致小鼠脾脏的Tregs比例显著减少，是小鼠免疫能力受损；与此同时，小鼠血清中促炎因子IL-1β、IL-6和TNF-α的含量显著上升(图8)，说明抑郁小鼠伴随着系统性炎症。氟西汀和CCFM6432均能显著改善抑郁小鼠的免疫状态，降低炎症程度。特别地，相比与氟西汀，服用CCFM6432能够显著降低血清中TNF-α的含量，具有更好的抗炎效果。
实施例7：乳酸片球菌CCFM6432对抑郁小鼠肠道菌群及代谢物的调节
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 PCTCN2020092193-appb-000005
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-多样性用香浓指数(shanno index)和辛普森指数(simpson index)来表征，结果显示(图9A、B)，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CCFM6432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和均匀程度。在门水平上，抑郁小鼠肠道菌群结构与正常小鼠存在显著差异，尤其是变形菌门和放线菌门的丰度。抑郁小鼠放线菌门丰度增加，放线菌门和变形菌门比例显著升高，而服用CCFM6432能够显著逆转这一现象，使肠道菌群结构恢复正常(图9D、E、F)；β-多样性用主坐标分析(principal co-ordinates analysis，PCoA)进行评估(图9C)，从结果也可以看出，氟西汀和CCFM6432干预均能使小鼠肠道菌群结构趋于正常化。此外，服用CCFM6432能够显著提高芽孢杆菌属(Bacillus)的丰度(图10A)。芽孢杆菌属是宿主体内一类重要的乳酸产生菌，对维持肠道正常蠕动、缓解便秘具有十分重要的作用。此外，芽孢杆菌属中部分种(如凝结芽孢杆菌、枯草芽孢杆菌)能够分泌细菌素，抑制肠道致病菌的生长，预防对炎症性肠病(Inflammatory bowel disease，IBD)的发生。服用CCFM6432还能够显著提高抑郁小鼠肠道粪球菌属(Coprococcus)的丰度(图10B)，粪球菌属是肠道中重要的丁酸产生菌，丁酸能够不仅能够为肠上皮细胞氧化供能，改善肠道屏障功能，维持肠粘膜免疫稳态，而且还能通过G蛋白偶联受体(G protein-coupled receptors，GPCRs)激活途径和组蛋白去乙酰基化酶(histone deacetylases,HDACs)抑制途径两条信号通路，诱导下游糖脂代谢调节因子的表达，如胰高血糖素样多肽-1(GLP-1)、酪酪肽(PYY)，对II型糖尿病、肥胖均具有显著的改善作用。
为了验证肠道菌群变化对肠道丁酸含量的影响，取对应的小鼠粪便样本，加入500uL饱和NaCl，震荡均匀；加入40uL 10％的硫酸，震荡均匀；加入1mL乙醚，震荡均匀；18000g，4℃ 离心15min，取上清至2mL EP管，加入0.25g无水硫酸钠。18000g，4℃离心15min。取500uL上清液至气相小瓶，通过GC-MS以选择离子扫面的方式检测短链脂肪酸的含量(乙酸、丁酸、异丁酸、丁酸等)。结果如图10C所示，服用乳酸片球菌CCFM6432能够改善抑郁导致的丁酸水平降低，进一步证明了CCFM6432不仅能够缓解抑郁，还可以通过调节肠道丁酸含量，发挥缓解和预防II型糖尿病、肥胖等疾病的作用。
实施例8：
将冷冻保存的乳酸片球菌CCFM6432接种于mMRS培养基(MRS培养基+0.05％半胱氨酸盐酸盐)中，在温度37℃厌氧培养48h，再经mMRS培养液传代培养2～3次后，取1mL乳酸片球菌CCFM6432的培养液，与9.0mL pH 2.5人工模拟胃液(含1％胃蛋白酶、pH＝2.5的mMRS培养基)混合，并在37℃下厌氧培养，分别在0h、0.5h、1h和2h时取样，用mMRS琼脂培养基浇注培养进行平板菌落计数，测定活菌数并计算其存活率。
存活率是在该培养液中在取样时的活菌数对数值与在第0h时活菌数对数值之比，以％表示。取1mL乳酸片球菌CCFM6432的培养液加入9mL人工模拟肠液(含0.3％牛胆盐、1％胰蛋白酶、pH＝8.0的mMRS培养基)中，在37℃下厌氧培养，分别在0h、0.5h、1h和2h时取样，用mMRS琼脂培养基浇注培养进行平板菌落计数，测定活菌数并计算其存活率。存活率是在该培养液中在取样时的活菌数对数值与在第0h时活菌数对数值之比，以％表示。实验结果如表4和表5所示。结果表明，乳酸片球菌CCFM6432对人工胃肠液具有较好的耐受性。
表4乳酸片球菌CCFM6432在人工模拟胃液中的耐受性
Figure PCTCN2020092193-appb-000006
表5乳酸片球菌CCFM6432在人工模拟肠液中的耐受性
Figure PCTCN2020092193-appb-000007
实施例9利用本发明乳酸片球菌CCFM6432制造发酵食品
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乳酸片球菌CCFM6432菌剂发酵剂，使其浓度达到10 8CFU/mL以上，在温度4℃下冷藏保存，得到含有本发明乳酸片球菌CCFM6432活菌的果蔬饮 料。
利用本发明能够使用长乳酸片球菌CCFM6432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。
按照实施例2～7的方法对发酵食品的效果进行验证，结果显示，制备获得的发酵食品能够减轻应激造成的小鼠的抑郁样行为、提高抑郁小鼠脑组织神经递质(5-HT)含量，增加外周组织神经递质前体(5-HTP)的生物合成，抑制下丘脑促肾上腺皮质激素释放因子(CRF)的过度分泌，降低血清中的皮质酮水平；所述发酵食品还能调节宿主的免疫力，提高脾脏调节性T细胞比例，降低血清中促炎因子IL-1β、IL-6和TNF-α的浓度；此外，所述发酵食品能够改善肠道菌群多样性，恢复应激造成的肠道菌群紊乱，提高有益菌的丰度(芽孢杆菌属、粪球菌属)，提高肠道丁酸的含量，减少炎症性肠病、肥胖和II型糖尿病等发生的风险。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (15)

  1. 乳酸片球菌(Pediococcus acidilactici)，其特征在于：所述乳酸片球菌于2019年4月25日保藏于广东省微生物菌种保藏中心，其保藏编号为GDMCC No.60638。
  2. 含有权利要求1所述乳酸片球菌的益生菌制剂。
  3. 根据权利要求2所述的益生菌制剂，其特征在于，乳酸片球菌的浓度≥1×10 5CFU/mL或1×10 5CFU/g。
  4. 一种发酵食品，其特征在于，所述发酵食品使用乳酸片球菌CCFM6432发酵生产制得，所述发酵食品为固态食品、液态食品或半固态食品。
  5. 根据权利要求4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。
  6. 根据权利要求4所述的发酵食品，其特征在于，所述果蔬制品包括黄瓜、胡萝卜、甜菜、芹菜、圆白菜制品。
  7. 根据权利要求4～6任一所述的发酵食品，其特征在于，所述发酵食品是在发酵初始接入所述乳酸片球菌CCFM6432进行发酵后制得。
  8. 权利要求1所述的乳酸片球菌CCFM6432在制备体内定植益生菌的应用。
  9. 权利要求1所述的乳酸片球菌CCFM6432在制备抗抑郁、抗炎症、抗炎症性肠病、抗肥胖和抗II型糖尿病的产品中的应用。
  10. 根据权利要求9所述的应用，其特征在于，用于制备具有(a)～(e)至少一种功能的产品：
    (a)减轻抑郁患者的抑郁样行为、提高脑中神经递质5-羟色胺含量、促进外周组织神经递质前体5-羟色氨酸的生物合成；
    (b)抑制下丘脑促肾上腺皮质激素释放因子的过度分泌、降低血清中皮质酮的水平、缓解“下丘脑-垂体-肾上腺轴”功能亢进；
    (c)调节宿主的免疫力；
    (d)提高脾脏调节性T细胞比例；
    (e)降低血清中促炎因子IL-1β、IL-6和/或TNF-α的浓度。
  11. 根据权利要求9或10所述的应用，其特征在于，还用于改善肠道菌群多样性、恢复应激造成的肠道菌群紊乱、提高芽孢杆菌属、粪球菌属的丰度、提高肠道丁酸的含量、减少炎症性肠病、肥胖和II型糖尿病发生的风险。
  12. 根据权利要求9～11任一所述的应用，其特征在于，所述产品为药物。
  13. 根据权利要求12所述的药物，其特征在于，还含有药学上可接受的载体或辅料。
  14. 根据权利要求9～11任一所述的应用，其特征在于，所述产品为功能性食品。
  15. 权利要求2或3所述的益生菌制剂，或权利要求4～7任一所述的发酵食品在制备抗抑郁、抗炎症、抗炎症性肠病、抗肥胖和抗II型糖尿病的功能性食品中的应用。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117305187A (zh) * 2023-11-24 2023-12-29 四川厌氧生物科技有限责任公司 一种改善肠道健康状况的乳酸片球菌及其应用

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110079485B (zh) * 2019-05-31 2020-09-25 江南大学 缓解抑郁的乳酸片球菌ccfm6432、其发酵食品及其应用
CN111109359B (zh) * 2019-12-29 2022-09-02 江南大学 多功能乳酸片球菌ccfm1105、其发酵食品及应用
CN111743159B (zh) * 2020-07-17 2022-09-27 江南大学 复合微生物制剂及缓解抑郁和便秘的应用
CN114717148B (zh) * 2022-03-17 2023-08-25 江南大学 缓解抑郁情绪的副干酪乳杆菌ccfm1229及其应用
CN115404187B (zh) * 2022-09-15 2023-07-14 广州能靓生物技术有限公司 一种体重管理及减压的益生菌组合物
CN117568242B (zh) * 2024-01-15 2024-04-16 山东中科嘉亿生物工程有限公司 改善女性乳腺炎的乳酸片球菌jypa-30及其菌剂、应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5786297A (en) * 1980-11-19 1982-05-29 Nippon Zeon Co Ltd Preparation of s-adenosyl-l-methionine
CN103764154A (zh) * 2011-06-10 2014-04-30 普洛特拉有限公司 包含片球菌属的药物组合物和减轻胃肠综合征的症状的方法
CN109182225A (zh) * 2018-10-30 2019-01-11 吉林省农业科学院 一株乳酸片球菌及其在抗动脉粥样硬化中的应用
CN109803666A (zh) * 2015-11-30 2019-05-24 医迈霖科技公司 包括益生菌的组合物及其使用方法
CN110079485A (zh) * 2019-05-31 2019-08-02 江南大学 缓解抑郁的乳酸片球菌ccfm6432、其发酵食品及其应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3373943A1 (en) * 2015-11-10 2018-09-19 Elizabeth Mckenna Control of cellular redox levels
US20170368135A1 (en) * 2016-06-27 2017-12-28 Vitaliva, Llc Herbal drug composition containing probiotic and optionally prebiotic and/or active pharmaceutical ingredient
CN106957810B (zh) * 2017-05-22 2020-05-05 哈尔滨中科生物工程有限公司 一种乳酸片球菌及其用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5786297A (en) * 1980-11-19 1982-05-29 Nippon Zeon Co Ltd Preparation of s-adenosyl-l-methionine
CN103764154A (zh) * 2011-06-10 2014-04-30 普洛特拉有限公司 包含片球菌属的药物组合物和减轻胃肠综合征的症状的方法
CN109803666A (zh) * 2015-11-30 2019-05-24 医迈霖科技公司 包括益生菌的组合物及其使用方法
CN109182225A (zh) * 2018-10-30 2019-01-11 吉林省农业科学院 一株乳酸片球菌及其在抗动脉粥样硬化中的应用
CN110079485A (zh) * 2019-05-31 2019-08-02 江南大学 缓解抑郁的乳酸片球菌ccfm6432、其发酵食品及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
P. FORSYTHE, W. A. KUNZE: "Voices from within: gut microbes and the CNS", CELL. MOL. LIFE SCI., vol. 70,, no. 1,, 27 May 2012 (2012-05-27), XP035157538, ISSN: 1420-9071, DOI: 20200810112042A *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117305187A (zh) * 2023-11-24 2023-12-29 四川厌氧生物科技有限责任公司 一种改善肠道健康状况的乳酸片球菌及其应用
CN117305187B (zh) * 2023-11-24 2024-02-06 四川厌氧生物科技有限责任公司 一种改善肠道健康状况的乳酸片球菌及其应用

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