WO2020259564A1 - 缓解pfoa毒害作用的多功能乳杆菌及其应用 - Google Patents

缓解pfoa毒害作用的多功能乳杆菌及其应用 Download PDF

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WO2020259564A1
WO2020259564A1 PCT/CN2020/098036 CN2020098036W WO2020259564A1 WO 2020259564 A1 WO2020259564 A1 WO 2020259564A1 CN 2020098036 W CN2020098036 W CN 2020098036W WO 2020259564 A1 WO2020259564 A1 WO 2020259564A1
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pfoa
lactobacillus
mice
ccfm1051
group
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PCT/CN2020/098036
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French (fr)
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王刚
陈卫
梁席
孔庆敏
翟齐啸
陆文伟
崔树茂
杨波
毛丙永
赵建新
张灏
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江南大学
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Priority claimed from CN201910575996.1A external-priority patent/CN110684682B/zh
Priority claimed from CN201910574811.5A external-priority patent/CN110229769B/zh
Priority claimed from CN201910575089.7A external-priority patent/CN110226630B/zh
Application filed by 江南大学 filed Critical 江南大学
Publication of WO2020259564A1 publication Critical patent/WO2020259564A1/zh
Priority to US17/562,081 priority Critical patent/US20220152130A1/en

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    • 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
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
    • A23L19/20—Products from fruits or vegetables; Preparation or treatment thereof by pickling, e.g. sauerkraut or pickles
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • C12R2001/225—Lactobacillus
    • 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
    • C12R2001/225—Lactobacillus
    • C12R2001/245—Lactobacillus casei

Definitions

  • the present invention relates to a multifunctional Lactobacillus that alleviates the toxic effect of PFOA and its application, and specifically relates to the multifunctional Lactobacillus fermentum CCFM1051, Lactobacillus casei CCFM1052, Lactobacillus brucelli CCFM1053 and any of the above-mentioned strains or combinations thereof, which alleviate the toxic effect of PFOA Application belongs to the field of microbial technology.
  • Perfluorinated compounds have been widely used in various industries due to their hydrophobic and oleophobic properties, as well as good thermal, chemical and biological stability. Used in ready-to-wear (such as waterproof, anti-fouling outdoor clothing) and home textiles (such as carpets, furniture fabrics, etc.), take-out food containers, personal care products (such as dental floss) and fire fighting foam, etc. Among them, PFOA (PFOA), as one of the final conversion products of many fluorine compounds, can be enriched along the food chain. PFOA has been detected in various environmental media around the world, such as water, soil, atmosphere, dust, etc., as well as in animals and humans, and the half-life in humans is 2-3 years. Therefore, more and more researchers have received Attention.
  • PFOA PFOA
  • PFOA blood PFOA content of exposed population and possible health effects
  • exposure to PFOA may be associated with the increase in blood total cholesterol concentration, the increase in liver enzyme ALT concentration and the decrease in birth weight.
  • PFOA exposure is associated with reduced vaccination response.
  • PFOA may affect the body's liver function, lipid metabolism and immune function.
  • the effects in humans have been clearly discovered in mammals.
  • PFOA has multiple toxic effects such as liver toxicity, immune toxicity, reproductive toxicity, developmental toxicity, neurotoxicity and so on.
  • PFOA can cause hepatomegaly, and at the same time can induce oxidative stress in the liver tissue of mice, and increase the abnormal increase of free radicals, which may be the main cause of liver damage.
  • PFOA exposure causes varying degrees of damage to multiple immune organs of the immune system of aquatic animals and rodents, resulting in the atrophy and aging of the immune organs, the spleen and thymus, and obviously interferes with the expression of interleukin in the zebrafish spleen, and the apoptosis and decline of lymphocytes.
  • the high-dose PFOA exposure increased the peripheral blood inflammatory factor IL-4, and the IFN- ⁇ was significantly lower than that of the asthma model group, that is, induced Th2-type immune response and aggravated lung inflammation.
  • the first objective of the present invention is to provide any of the following lactobacilli:
  • Lactobacillus fermentum CCFM1051 classified and named Lactobacillus fermentum, has been deposited at the Guangdong Provincial Microbial Culture Collection on April 29, 2019, and the deposit number is GDMCC No: 60649;
  • Lactobacillus casei CCFM1052 classified as Lactobacillus casei, has been deposited in the Guangdong Provincial Microbial Culture Collection on April 29, 2019, and the deposit number is GDMCC No: 60650;
  • Lactobacillus buchneri CCFM1053 classified as Lactobacillus buchneri, has been deposited in the Guangdong Provincial Microbial Culture Collection on April 29, 2019, and the deposit number is GDMCC No: 60651.
  • the second object of the present invention is to provide a composition containing the Lactobacillus.
  • the composition is food, medicine or health care product.
  • the food is a fermented food, which is produced by fermentation using the Lactobacillus fermentum CCFM1051, Lactobacillus casei CCFM1052, Lactobacillus brucei CCFM1053, or a mixture of multiple lactobacilli.
  • the fermented food includes solid food, liquid food or semi-solid food.
  • the fermented food includes dairy products, soybean products, fruit and vegetable products.
  • the food is a functional food.
  • the dairy product includes milk, sour cream, and cheese; the fruit and vegetable product includes cucumber, carrot, beet, celery, and cabbage products.
  • the composition is a starter; the starter is a liquid starter or a solid starter.
  • the number of lactobacilli in the starter is ⁇ 1 ⁇ 10 8 CFU/mL.
  • the number of lactobacilli in the starter is ⁇ 1 ⁇ 10 8 CFU/g.
  • the drug contains a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier includes but is not limited to excipients and diluents;
  • the excipients include but are not limited to lactose, dextrose, sucrose, sorbitol, mannitol, Starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methylcellulose;
  • the diluent includes but not Limited to: saline or syrup.
  • the drug has at least one of the following uses:
  • the third object of the present invention is to provide the Lactobacillus fermentum CCFM1051, Lactobacillus casei CCFM1052, Lactobacillus brucei CCFM1053, alone or in combination, used to adsorb PFOA, improve spleen atrophy caused by PFOA exposure, and reduce serum levels after PFOA exposure.
  • ALT alanine aminotransferase
  • ⁇ -GT ⁇ -glutamyl transpeptidase
  • the present invention screened and obtained 3 strains of Lactobacillus with PFOA adsorption capacity.
  • the strains have high adsorption of PFOA and have the potential to alleviate PFOA-related metabolic diseases. They can be used to prepare medicines or health products for the treatment or alleviation of liver diseases and metabolic diseases.
  • the three Lactobacillus strains of the present invention have the following biological characteristics:
  • Lactobacillus fermentum CCFM1051 1. Lactobacillus fermentum CCFM1051:
  • Bacterial characteristics Gram-positive, spherical cells, 0.8-1.0 ⁇ m in diameter, no flagella, no spores;
  • Colony characteristics the colony is milky white, the edges are neat, spherical, convex, opaque, and the surface is moist and smooth;
  • Lactobacillus fermentum CCFM1051 can significantly improve spleen atrophy in mice exposed to PFOA;
  • Lactobacillus fermentum CCFM1051 can significantly reduce the level of IL-4 in the serum of mice exposed to PFOA;
  • Lactobacillus fermentum CCFM1051 can significantly reduce the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and ⁇ -glutamyl transpeptidase ( ⁇ -GT) in the serum of mice exposed to PFOA;
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • ⁇ -GT ⁇ -glutamyl transpeptidase
  • Lactobacillus fermentum CCFM1051 can significantly reduce the abundance of S24-7 and Lactobacillus in the intestine of PFOA-exposed mice, increase the abundance of Bacteroides and Eubacteriaceae, and improve the intestinal tract caused by PFOA exposure Disorders, reduce the occurrence of liver disease, hypertension and obesity.
  • Lactobacillus fermentum CCFM1051 can significantly increase the fecal water content of constipated mice and the discharge time of the first black stool, and significantly improve the constipation of mice.
  • Lactobacillus fermentum CCFM1051 can significantly improve the proliferation of INS-1 cells and the expression of MafA gene under high glucose, and alleviate PFOA-related diabetes.
  • Lactobacillus casei CCFM1052 Lactobacillus casei CCFM1052
  • Bacterial characteristics Gram-positive, spherical cells, 0.8-1.0 ⁇ m in diameter, no flagella, no spores;
  • Colony characteristics the colony is milky white, the edges are neat, spherical, convex, opaque, and the surface is moist and smooth;
  • Lactobacillus casei CCFM1052 can significantly improve spleen atrophy in mice exposed to PFOA;
  • Lactobacillus casei CCFM1052 can significantly reduce the levels of alanine aminotransferase (ALT) and ⁇ -glutamyl transpeptidase ( ⁇ -GT) in the serum of mice exposed to PFOA;
  • ALT alanine aminotransferase
  • ⁇ -GT ⁇ -glutamyl transpeptidase
  • Lactobacillus casei CCFM1052 can significantly reduce the activity of superoxide dismutase SOD and the content of malondialdehyde MDA in liver homogenate of PFOA-exposed mice;
  • Lactobacillus casei CCFM1052 can significantly reduce the abundance of Allobaculum in the intestine of PFOA-exposed mice, increase the abundance of Clostridiaceae, Adlercreutzia, Bacteroides and Holdmania, improve the intestinal disorders caused by PFOA exposure, and reduce liver disease And metabolic diseases;
  • Lactobacillus casei CCFM1052 can significantly increase the content of acetic acid and butyric acid in the intestines of PFOA-exposed mice, and improve the metabolism disorder of intestinal flora caused by PFOA exposure;
  • Lactobacillus casei CCFM1052 can significantly increase the fecal water content of constipated mice and the time for the first black stool to pass;
  • Lactobacillus casei CCFM1052 can significantly improve the proliferation of INS-1 cells and the expression of MafA gene under high glucose, and can alleviate PFOA-related diabetes.
  • Lactobacillus brucei CCFM1053 has the following biological characteristics:
  • Bacterial characteristics Gram-positive, spherical cells, 0.8-1.0 ⁇ m in diameter, no flagella, no spores;
  • Colony characteristics the colony is milky white, the edges are neat, spherical, convex, opaque, and the surface is moist and smooth;
  • Lactobacillus brucei CCFM1053 can significantly improve the spleen atrophy of mice exposed to PFOA;
  • Lactobacillus brucei CCFM1053 can significantly reduce the levels of ALT, AST and ⁇ -GT in the serum of mice exposed to PFOA;
  • Lactobacillus brucei CCFM1053 can significantly reduce the levels of MDA and GSH in the liver of mice exposed to PFOA;
  • Lactobacillus brucei CCFM1053 can significantly reduce the content of TNF- ⁇ in the serum of mice exposed to PFOA;
  • Lactobacillus brucei CCFM1053 can significantly reduce the abundance of Allobaculum in the intestine of PFOA-exposed mice, increase the abundance of Bacteroides and Eubacteriaceae, and improve the intestinal tract caused by PFOA exposure Disorder, reduce the occurrence of liver disease;
  • Lactobacillus brucei CCFM1053 can significantly increase the fecal water content and discharge time of the first black stool in constipated mice, and relieve the constipation of mice;
  • Lactobacillus brucei CCFM1053 can significantly improve the proliferation of INS-1 cells and the expression of MafA gene under high glucose, and alleviate PFOA-related diabetes.
  • Lactobacillus fermentum CCFM1051 classified and named Lactobacillus fermentum, has been deposited in the Guangdong Provincial Microbial Culture Collection on April 29, 2019, and the deposit number is GDMCC No: 60649.
  • Lactobacillus casei CCFM1052 classified and named as Lactobacillus casei, has been deposited in the Guangdong Provincial Microbial Culture Collection on April 29, 2019, and the deposit number is GDMCC No: 60650.
  • Lactobacillus buchneri CCFM1053 classified and named Lactobacillus buchneri, has been deposited in the Guangdong Provincial Microbial Culture Collection on April 29, 2019, and the deposit number is GDMCC No: 60651.
  • Figure 1 shows that different lactic acid bacteria were resuspended in PFOA at a concentration of 10mg/L in vitro, after being shaken at 37°C and 150rpm for 6 hours, and passed through a 0.22 ⁇ m water-based filter membrane into the ultra-high performance liquid chromatography-mass spectrometer.
  • Figure 2 shows the ability of different lactic acid bacteria to scavenge diphenyltrinitrophenylhydrazine free radicals (DPPH), hydroxyl free radicals and reducing abilities in vitro.
  • DPPH diphenyltrinitrophenylhydrazine free radicals
  • Figure 3 is a graph showing changes in the ratio of spleen of mice exposed to PFOA after 10 days of intervention with different substances. Where *P ⁇ 0.05 (vs model group).
  • Figure 4 is a schematic diagram of IL-4 in the serum of mice exposed to PFOA after 10 days of intervention with different substances. Among them, *P ⁇ 0.05, **P ⁇ 0.01 (vs model group).
  • Figure 5 is a schematic diagram showing the levels of ALT, AST and ⁇ -GT in the serum of mice exposed to PFOA after 10 days of intervention with different substances. Among them, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001 (vs model group).
  • Figure 6 is a schematic diagram of changes in the ⁇ diversity of the intestinal flora of mice exposed to PFOA after 10 days of intervention with different substances; where *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001 (vs model group).
  • Figure 7 is a schematic diagram showing the changes in the abundance of S24-7 family, Lactobacillus, Bacteroides and Eubacteriaceae in the intestines of mice exposed to PFOA after 10 days of intervention with different substances; where *P ⁇ 0.05, **P ⁇ 0.01 (vs model group).
  • Figure 8 shows the improvement of fecal water content in mice with constipation after intervention of different substances; *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001 (vs model group).
  • Figure 9 shows the reduction of the discharge time of the first black stool in constipated mice after the intervention of different substances; *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001 (vs model group).
  • Figure 10 shows the effects of different strains on the proliferation of INS-1 cells under high glucose.
  • Figure 11 shows the effects of different strains on the MafA gene expression of INS-1 cells under high glucose.
  • Figure 12 is a graph showing changes in the spleen coefficient of mice exposed to PFOA after 10 days of intervention with different substances. Among them, *P ⁇ 0.05, **P ⁇ 0.01 (vs model group).
  • Figure 13 is a schematic diagram showing the levels of alanine aminotransferase (ALT) and ⁇ -glutamyl transpeptidase ( ⁇ -GT) in the serum of mice exposed to PFOA after 10 days of intervention with different substances. Among them, *P ⁇ 0.05, **P ⁇ 0.01, ****P ⁇ 0.0001 (vs model group).
  • ALT alanine aminotransferase
  • ⁇ -GT ⁇ -glutamyl transpeptidase
  • Figure 14 shows the changes in superoxide dismutase SOD activity and MDA content in liver homogenate of mice exposed to PFOA after 10 days of intervention with different substances; where *P ⁇ 0.05 (vs model group).
  • Figure 15 is a schematic diagram of the ⁇ diversity analysis of mouse gut strains after 10 days of intervention with different substances after mice are exposed to PFOA, and the changes in the abundance of Clostridiaceae, Adlercreutzia, Allobaculum, Bacteroides and Holdmania in the flora; where *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001 (vs model group).
  • Figure 16 shows the changes in the content of acetic acid and butyric acid in the intestine of mice exposed to PFOA after 10 days of intervention with different substances; where *P ⁇ 0.05 (vs model group).
  • Figure 17 shows the improvement of fecal water content of constipated mice after the intervention of the strain of the present invention.
  • Figure 18 shows the reduction of the time to first black stool in constipated mice after the intervention of the strain of the present invention.
  • Figure 19 shows the effect of the strain of the present invention on the proliferation of INS-1 cells under the action of high glucose.
  • Figure 20 shows the effect of the strain of the present invention on the expression of MafA gene in INS-1 cells under high glucose.
  • Figure 21 shows that different strains were resuspended in PFOA at a concentration of 10 mg/L in vitro, after being shaken at 37°C and 150 rpm for 6 hours, and passed through a 0.22 ⁇ m water-based filter membrane into the ultra-high performance liquid chromatography-mass spectrometer.
  • Figure 22 is a graph showing the change of spleen ratio of mice exposed to PFOA after 10 days of intervention with different substances; where *P ⁇ 0.05 (vs model group).
  • Figure 23 is a schematic diagram of the levels of ALT, AST and ⁇ -GT in the serum of mice exposed to PFOA after 10 days of intervention with different substances; where *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, *** *P ⁇ 0.0001 (vs model group).
  • Figure 24 is a schematic diagram showing the changes in GSH activity and MDA content in the liver of mice exposed to PFOA after 10 days of intervention with different substances.
  • Figure 25 is a schematic diagram of the changes in the ⁇ diversity of the intestinal flora of mice exposed to PFOA after 10 days of intervention with different substances; where *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001 (vs model group).
  • Figure 26 is a schematic diagram showing the changes in the abundance of Eubacteriaceae, Bacteroides and Allobaculum in the intestines of mice exposed to PFOA after 10 days of intervention with different substances; where *P ⁇ 0.05, **P ⁇ 0.01 (vs model group).
  • Figure 27 shows the improvement of fecal water content in constipated mice after intervention of different substances; where *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001 (vs model group).
  • Figure 28 shows the reduction of the discharge time of the first black stool in constipated mice after intervention of different substances; where *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001 (vs model group).
  • Figure 29 shows the effects of different substances on the proliferation of INS-1 cells under high glucose.
  • Figure 30 shows the effects of different substances on the MafA gene expression in INS-1 cells under high glucose.
  • step (1) Culture the lactic acid bacteria selected in step (1) in liquid sorbitol MRS culture medium for 24 hours, and then take 1 mL of the culture and centrifuge at 8000 rpm for 2 minutes;
  • step (2) Cultivate the lactic acid bacteria selected in step (2) overnight, take 1 mL of the bacterial suspension cultured overnight in a 1.5 mL centrifuge tube, centrifuge at 10000 rpm for 2 minutes, discard the supernatant to obtain the bacteria;
  • the Lactobacillus fermentum CCFM1051 and the control strain were purified and cultured by activation, inoculated into MRS liquid medium at 1% (v/v) inoculum, and cultured at 37°C for 18 hours. Then the cells were collected by centrifugation at 8000r/min for 5min, the precipitate was cleaned up with saline and then centrifuged at 8000r/min for 5min to remove the precipitation to obtain live cells, namely wet cells. Suspend the wet bacteria weight in a 10mg/L PFOA solution, and make the final bacterial cell concentration reach 1g dry bacteria/L (suspend the wet bacteria weight in ultrapure water without PFOA as a blank control).
  • PFOA adsorption capacity After the adsorption experiment, the sample solution was centrifuged at 8000r/min for 5 minutes and filtered with a 0.22 ⁇ m water membrane. The PFOA concentration was determined by UPLC-MS with Waters SYNAPT MS system, using Acquity UPLC BEH c18 column (2.1 ⁇ 100mm, 1.7 ⁇ m, Waters Co.), column temperature 35°C, injection volume 1 ⁇ L. Using 100% (v/v) acetonitrile solution (solution A) and 0.1% (v/v) formic acid aqueous solution (solution B) as eluents, gradient cleaning was performed, and the flow rate was 0.3 mL/min.
  • Mass spectrometry conditions ionization source is ESI source; MRM detection; MS+ detection; Capillary (capillary); 3.0kV; Conc (cone): 40.00V; Source Temperature: 120°C; Desolvation (desolvation) temperature : 400°C; Conc Gas Flow: 50L/h; Desolvation Gas Flow: 700L/h.
  • the gas flow rate is 0.1ml/min; the proton ratio scan range: 100-2000; the scan time is 1s, and the interval is 0.061s.
  • the results were analyzed by MassLynxV4.1 (Waters Company); the amount of PFOA adsorbed by lactic acid bacteria was calculated based on the difference in the concentration of PFOA before and after adsorption. The measurement results are shown in Figure 1.
  • the adsorption capacity of CCFM1051 to 10mg/L PFOA is 57.5% ⁇ 1.5%, and the adsorption capacity of other Lactobacillus fermentum to PFOA is less
  • Example 3 In vitro, it has good scavenging ability of diphenyltrinitrophenylhydrazine free radical (DPPH), scavenging ability of hydroxyl free radical and reducing ability
  • DPPH diphenyltrinitrophenylhydrazine free radical
  • DPPH radical scavenging rate (%) [1-A 517 (sample)/A 517 (control)] ⁇ 100%.
  • Mixture 1 1 mL of H 2 O 2 to "Mixture 1", and in a water bath at 37°C for 1.5 hours, measure the absorbance at 536 nm, which is expressed as A 536 (sample).
  • a 536 bladed
  • Hydroxyl radical scavenging rate (%) [A 536 (sample)-A 536 (blank)]/[A 536 (control)-A 536 (blank)] ⁇ 100%
  • the DPPH scavenging ability of CCFM1051 is 57 ⁇ 4.2%, the scavenging ability of DPPH of other lactic acid bacteria is less than 50%, and the scavenging ability of hydroxyl radicals is 32 ⁇ 4.5%, while the scavenging capacity of hydroxyl radicals of other lactic acid bacteria is less than 30%.
  • the reducing ability is 14 ⁇ 1.8%, and the reducing ability of other lactic acid bacteria is less than 13%.
  • Lactobacillus fermentum CCFM1051 significantly improved spleen atrophy in mice exposed to PFOA
  • mice Fifty 6-week-old male C57BL/6J mice were adapted to the environment for one week and were randomly divided into five groups according to their body weight: control group, model group, quercetin intervention group, Lactobacillus fermentum CCFM1051 intervention group, LGG intervention group, each group Containing 10 mice, the animal grouping and processing methods are shown in Table 2.
  • mice were weighed on the 13th day and then euthanized.
  • the spleens were taken out and weighed to calculate the organ coefficient.
  • the organ coefficient of the mouse spleen was calculated according to the following formula:
  • Spleen organ coefficient wet spleen weight/weight of the mouse before euthanasia.
  • Lactobacillus fermentum CCFM1051 significantly reduces the IL-4 content in the serum of mice exposed to PFOA
  • mice in Example 4 were euthanized on the 13th day.
  • the serum was collected and centrifuged at 3000g for 15 minutes to obtain the serum, and the content of IL-4 in the serum was detected with an ELISA kit.
  • the results show that taking Lactobacillus fermentum CCFM1051 can significantly improve the immune damage caused by PFOA in mice ( Figure 4), restore the serum IL-4 content to normal levels, and the effect is better than quercetin.
  • Lactobacillus fermentum CCFM1051 significantly reduced the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and ⁇ -glutamyl transpeptidase ( ⁇ -GT) in the serum of mice exposed to PFOA
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • ⁇ -GT ⁇ -glutamyl transpeptidase
  • Lactobacillus fermentum CCFM1051 significantly reduced the abundance of S24-7 and Lactobacillus in the intestines of PFOA-exposed mice, increased the abundance of Bacteroides, Eubaceriaceae, and Eubacteriaceae, improved intestinal disorders caused by PFOA exposure, and reduced liver disease, hypertension and obesity occur
  • the fresh feces of the mice on the 12th day in Example 4 were taken, and the total DNA in the feces samples of the mice was extracted with the stool kit of MP.
  • the specific operation steps are as follows, mainly referring to the kit instructions.
  • the upstream primer 520F (5'-AYTGGGYDTAAAGNG-3', SEQ ID NO. 1) and the downstream primer 802R (5'-TACNVGGGTATCTAATCC-3', SEQ ID NO. 2) were used as primers for amplification.
  • the V3-V4 region fragment of 16S rDNA, the length of the target fragment is about 247bp.
  • S24-7 family and Lactobacillus was significantly increased in PFOA-infected mice, and taking Lactobacillus fermentum CCFM1051 can significantly reverse this situation;
  • S24-7 is highly localized in the gastrointestinal tract of warm-blooded animals , Gram-negative non-exercise anaerobic microorganisms, capable of fermenting a variety of carbohydrates, are related to the occurrence and development of non-alcoholic fatty liver and hypertension.
  • Lactobacillus is part of the normal gastrointestinal tract and genitourinary tract, and is a common probiotic.
  • the abundance of PFOA model group and infection model increased.
  • Lactobacillus may have feedback after PFOA exposure The phenomenon of regulation. Taking Lactobacillus fermentum CCFM1051 can also significantly increase the abundance of Bacteroides and Eubacteriaceae in PFOA-infected mice (Figure 7).
  • Bacteroides also known as Bacteroides, is a genus of the Bacteroides family. It is a gram-negative, non-spore-free, obligate anaerobic small bacterium. Bacteroides normally inhabit the intestines, oral cavity, upper respiratory tract and reproductive tract of humans and animals. Bacteroides are a large number of normal flora in humans and animals, accounting for more than 1/4 of adult intestinal flora.
  • Eubacteriaceae is related to hepatic encephalopathy, that is, the recovery of the dysfunctional gut-liver-brain axis in cirrhosis, and its abundance is significantly reduced after the bile duct bypass operation in severely obese patients.
  • Lactobacillus fermentum CCFM1051 not only relieves the toxicity of PFOA, but also has the ability to regulate intestinal flora, regulate immunity and intestinal barrier, and reduce the occurrence of liver disease, hypertension and obesity.
  • mice 40 SPF male BALB/c mice (20-25g) were randomly divided into 5 groups: blank control group, constipation model control group, Lactobacillus fermentum CCFM1051 intervention group, Lactobacillus plantarum control group and phenolphthalein treatment group.
  • the group contains 10 mice.
  • the lyophilized Lactobacillus fermentum CCFM1051 bacterial powder was resuspended in skimmed milk powder to prepare a bacterial suspension with a concentration of 4.0 ⁇ 10 9 CFU/mL. 14 days before the experiment, mice in the intervention group were fed with 0.25 mL of Lactobacillus fermentum CCFM1051 skimmed milk suspension (4.0 ⁇ 10 9 CFU/mL) every day.
  • the Lactobacillus plantarum group was fed with the same amount of L.plantarum ST-III, and the rest The 3 groups were fed the same amount of non-bacterial skim milk.
  • the negative control group was given intragastrically with 0.25 mL of normal saline, and the remaining four groups were given intragastrically with 0.25 mL of 1 mg/mL loperamide solution to ensure that the intragastric dose of loperamide in mice was 10 mg/kg BW .
  • mice in the Lactobacillus fermentum CCFM1051 intervention group were fed 0.25 mL of Lactobacillus fermentum CCFM1051 (4.0 ⁇ 10 9 CFU/mL), and the phenolphthalein treatment control group Gavage 0.25 mL of 7 mg/mL phenolphthalein solution to ensure that the gavage amount of phenolphthalein in mice is 70 mg/kg BW.
  • the Lactobacillus plantarum group was given 0.25 mL of L.plantarum ST-III (4.0 ⁇ 10 9 CFU/mL).
  • mice feces were collected every day to calculate the water content of mouse feces.
  • the water content of feces was calculated as follows:
  • Fecal moisture content (%) (wet weight of feces-dry weight of feces)/wet weight of feces ⁇ 100.
  • mice On the morning of the 17th day, except for the blank control group, all the other groups were given loperamide by intragastric administration. After 1 hour, all mice were intragastrically administered with 0.25 mL of activated charcoal gum arabic solution, and then each mouse was placed separately In a clean stainless steel cage covered with absorbent paper, record the time (min) from the start of gavage of activated carbon to the discharge of the first black stool, as the time for the first black stool to be used to evaluate the effect of Lactobacillus fermentum CCFM1051 on constipation in mice During the alleviating effect, the mice eat and drink freely. The results are shown in Figure 8 and Figure 9.
  • Lactobacillus fermentum CCFM1051 can relieve constipation, increase the water content of feces, and shorten the discharge time of the first black stool, and its effect is better than that of Lactobacillus plantarum ST-III.
  • Lactobacillus fermentum CCFM1051 can promote the proliferation and Maf A mRNA expression of INS-1 cells induced by high glucose
  • the experiment was divided into 5 groups: normal group (normal culture medium containing 11.1mmol/L glucose), high sugar group (high sugar culture medium containing 22.2mmol/L glucose), rosiglitazone group (high glucose culture medium +80 ⁇ mol/ L rosiglitazone), CCFM1051 group (high-sugar culture medium + 1 ⁇ 10 9 CFU/mL CCFM1051 bacterial liquid) LGG group (high-sugar culture medium + 1 ⁇ 10 9 CFU/mL LGG bacterial liquid).
  • Inoculate INS-1 cells (number: BH-AC0530) in RPMI-1640 (containing 11.1mmol/L glucose, 10% FBS, 50 ⁇ mol/L 2-mercaptoethanol, 1mmol/L pyruvate, 10mmol/L HEPES) culture medium Incubate in a 37°C, 5% CO 2 incubator.
  • CCK-8 method to detect cell proliferation Digest and centrifuge the cells in good condition and inoculate them on a 96-well plate with approximately 5 ⁇ 10 3 cells in each well. The peripheral wells of the plate are not inoculated with cells. To prevent edge effects, add PBS to it at the same time Solution. After the cells adhere to the wall, RPMI-1640 medium containing 0.5% fetal bovine serum is added to each well for 24h synchronous treatment. At the end of synchronization, add the corresponding culture medium to each well for 48 hours according to the group, set three multiple wells for each group, and set the zero adjustment hole at the same time.
  • the old medium is aspirated, washed twice with PBS, 180 ⁇ L serum-free medium and 20 ⁇ L CCK-8 solution are added, and incubated for 3-4 h. After the incubation, the absorbance value of each well was measured at 450 nm using a microplate reader.
  • Centrifuge at 4°C at 12000rpm for 15min draw about 0.4ml of the supernatant, transfer to another enzyme-free EP tube, add 0.5mL of isopropanol, mix by inversion, and let it stand for 10min at room temperature.
  • Centrifuge at 4°C at 12000 rpm for 10 min carefully discard the supernatant, add 1.0 mL of 75% ethanol and mix by inversion.
  • Add 20 ⁇ L of DEPC treated water to dissolve and store at 80°C until use. Determine the concentration and quality of RNA, and perform reverse transcription according to the reverse transcription kit instructions.
  • the cDNA obtained by reverse transcription was detected by q RT-PCR, and the MafA specific primers: F: 5'-atcactctgcccaccatcac-3' (SEQ ID NO. 3), R: 5'-atgacctcctccttgctgaa-3' (SEQ ID NO. 4).
  • the PCR system is: F (10 ⁇ M), 0.50 ⁇ L; R (10 ⁇ M), 0.50 ⁇ L; cDNA template, 1.00 ⁇ L; dd H 2 O, 3.00 ⁇ L; mix, 5.00 ⁇ L.
  • PCR program 95°C, 2min;
  • the relative gene expression analysis is performed by the 2- ⁇ CT method.
  • the CFX Manager software was used to analyze the expression level of the target gene in each group of rat INS-1 cells, and then the expression level of the normal group was set as 1, and the other groups were compared with each other to calculate the gene expression level of each group.
  • the CCK-8 method test results are shown in Figure 10. Compared with the normal group, the cell growth of the high glucose group was significantly reduced (P ⁇ 0.05), and the cell proliferation of the rosiglitazone control group was significantly increased (P ⁇ 0.05). 0.05), the cell proliferation status of CCFM1051 group was significantly increased compared with high glucose group (P ⁇ 0.05).
  • Maf A mRNA The expression of Maf A mRNA is shown in Figure 11.
  • the expression of MafA mRNA in the cells of the high glucose group was significantly lower than that of the normal group (P ⁇ 0.05), while the expression of Maf A mRNA in the rosiglitazone positive control group and the CCFM1051 group was higher.
  • the high glucose group increased significantly (P ⁇ 0.05).
  • the concentration is increased to 10 6 CFU/mL or more, and it is stored under refrigeration at a temperature of 4° C., thereby obtaining a fruit and vegetable beverage containing the live Bifidobacterium breve CCFM1051 of the present invention.
  • the fruits and vegetables include cucumber, carrot, beet, celery or cabbage products.
  • Lactobacillus fermentum CCFM1051 is inoculated into raw materials of dairy products or soybean products to prepare fermented dairy products or fermented soybean products; the dairy products include milk, sour cream, and cheese.
  • the fermented food prepared by CCFM1051 can significantly improve the spleen atrophy of mice caused by PFOA exposure; significantly reduce the serum interleukin 4 (IL-4) content of PFOA-exposed mice; significantly increase High levels of ALT, AST and ⁇ -GT in the serum of PFOA-exposed mice; improve the intestinal flora disorder of PFOA-exposed mice, reduce the abundance of S24-7 and Lactobacillus in the intestine, increase the abundance of Bacteroides and Eubacteriaceae, and Normalize the intestinal flora and reduce the incidence of liver disease, hypertension and obesity.
  • IL-4 serum interleukin 4
  • Taking the fermented food containing Lactobacillus fermentum CCFM1051 can significantly increase the water content of the feces and the discharge time of the first black stool in constipated mice, and significantly improve the constipation of mice.
  • Cell experiments show that fermented food containing Lactobacillus fermentum CCFM1051 can significantly improve the proliferation of INS-1 cells and the expression of MafA gene under high glucose, and alleviate PFOA-related diabetes.
  • In vitro experiments show that Lactobacillus fermentum CCFM1051 can adsorb PFOA well, can effectively scavenge diphenyltrinitrophenylhydrazine radical (DPPH), scavenge hydroxyl radicals, and exhibit good reducing ability.
  • DPPH diphenyltrinitrophenylhydrazine radical
  • the present invention screens out probiotics that have high adsorption capacity for PFOA and are not colonized in the human body and have high antioxidant capacity, which can not only inhibit the oxidative stress caused by PFOA, but also can fundamentally eliminate PFOA in the human body.
  • Lactobacillus fermentum CCFM1051 can be used to prepare foods, health products and medicines that can relieve the toxicity of PFOA, and has a very broad application prospect.
  • step (1) Culture the lactic acid bacteria selected in step (1) in liquid sorbitol MRS culture medium for 24 hours, and then take 1 mL of the culture and centrifuge at 8000 rpm for 2 minutes;
  • step (2) Cultivate the lactic acid bacteria selected in step (2) overnight, take 1 mL of the bacterial suspension cultured overnight in a 1.5 mL centrifuge tube, centrifuge at 10000 rpm for 2 minutes, discard the supernatant to obtain the bacteria;
  • 10 ⁇ Taq buffer 5 ⁇ L; dNTP, 5 ⁇ L; 27F, 0.5 ⁇ L; 1492R, 0.5 ⁇ L; Taq enzyme, 0.5 ⁇ L; template, 0.5 ⁇ L; ddH2O, 38 ⁇ L.
  • Example 12 Ability of different lactic acid bacteria to adsorb PFOA in vitro
  • Lactobacillus casei CCFM1052 and other lactic acid bacteria as a control were purified and cultured, inoculated into MRS liquid medium at 1% (v/v) inoculum, and cultured at 37°C for 18h. Then the cells were collected by centrifugation at 8000r/min for 5min, the precipitate was cleaned up with saline and then centrifuged at 8000r/min for 5min to remove the precipitation to obtain live cells, namely wet cells. Suspend the wet bacteria weight in a 10mg/LPFOA solution, and make the final bacterial cell concentration reach 1g dry bacteria/L (suspend the wet bacteria weight in ultrapure water without PFOA as a blank control).
  • mice Fifty 6-week-old male C57BL/6J mice were adapted to the environment for a week and were randomly divided into five groups according to their body weight: control group, model group, quercetin intervention group, Lactobacillus casei 15-7 intervention group, LGG intervention group, Each group contains 10 mice, and the animal grouping and processing methods are the same as in Example 4.
  • Lactobacillus casei CCFM1052 significantly reduced the levels of alanine aminotransferase (ALT) and ⁇ -glutamyl transpeptidase ( ⁇ -GT) in the serum of mice exposed to PFOA;
  • mice in Example 12 were weighed on the 13th day and then euthanized. After blood was taken from the orbit, the serum was obtained by centrifugation at 3000 rpm/min for 15 minutes, and the alanine aminotransferase (ALT) and ⁇ -glutamine in the serum were detected by an automatic biochemical analyzer The content of acyl transpeptidase ( ⁇ -GT). ALT mainly exists in the soluble part of the hepatocyte protoplasm. Increased ALT activity indicates that hepatocytes are destroyed and cell membrane permeability is enhanced.
  • ⁇ -GT acyl transpeptidase
  • Lactobacillus casei CCFM1052 can significantly reduce the activity of superoxide dismutase SOD and the content of malondialdehyde MDA in liver homogenate of PFOA-exposed mice
  • liver tissue On ice, add pre-cooled physiological saline in a ratio of 1:9, homogenize the tissue to prepare a 10% liver homogenate, centrifuge at 4000 ⁇ g for 15 minutes to obtain the supernatant. Determine the protein concentration of liver homogenate according to the instructions. And calculate the SOD content according to the following formula:
  • SOD inhibition rate (%) [(A control-A control blank)-(A determination-A determination blank)]/(A control-A control blank)
  • SOD activity (U/mL) SOD inhibition rate ⁇ 50% ⁇ reaction system dilution factor ⁇ sample dilution factor before testing
  • Lactobacillus casei CCFM1052 significantly reduced the abundance of Allobaculum in the intestine of PFOA-exposed mice, increased the abundance of Clostridiaceae, Adlercreutzia, and Bacteroides, improved intestinal disorders caused by PFOA exposure, and reduced liver diseases and metabolic diseases happened
  • the fresh stool of the mouse on the 12th day in Example 12 was taken, and the total DNA in the mouse stool sample was extracted using the stool kit of MP.
  • the specific operation steps are as follows, mainly referring to the kit instructions.
  • the upstream primer 520F (5′-AYTGGGYDTAAAGNG-3′, SEQ ID NO. 1) and the downstream primer 802R (5′-TACNVGGGTATCTAATCC-3′, SEQ ID NO. 2) were used as primers for amplification
  • the V3-V4 region fragment of 16S rDNA, the length of the target fragment is about 247bp.
  • the sequence with a similarity greater than 97% is defined as a taxonomic unit (Operational Taxonomic Unit, OTU), through the Ribosomal Database Project (RDP) Bayesclassifier to determine the species.
  • OTU Operaational Taxonomic Unit
  • RDP Ribosomal Database Project
  • Figure 15 shows that the abundance of Allobaculum increased in the intestines of mice in the PFOA model group, and the administration of Lactobacillus casei CCFM1052 can significantly reverse this situation.
  • the abundance of Allobaculum also increased in the Gulf War disease, and it was The activation of TLR4 induced by leaky gut and systemic endotoxemia further induced neuroinflammation and gastrointestinal disorders of Gulf War disease.
  • the increased abundance of Allobaculum may be used as one of the risk indicators of female hepatocellular carcinoma, and it is one of the key variables before the occurrence of host cancer caused by carcinogen exposure.
  • Clostridium is the largest type of Firmicutes, representing the most diverse bacteria in the human microbiota.
  • Clostridium is a type of anaerobic, gram-positive, spore-forming coryneform bacteria. Play a role in regulating the immune balance of the gastrointestinal tract. In non-alcoholic fatty liver disease, a decrease in the abundance of the Clostridiaceae family was found. Comparing the intestinal flora of patients with chronic functional constipation and the healthy control group, it was found that the abundance of the Clostridiaceae family in the healthy control group was significantly increased. Therefore, taking cheese milk stick CCFM1052 reduced the occurrence and development of non-alcoholic fatty liver disease and constipation.
  • Bacteroides also known as Bacteroides, is a genus of the Bacteroides family.
  • Bacteroides normally inhabit the intestines, oral cavity, upper respiratory tract and reproductive tract of humans and animals. Bacteroides are a large number of normal flora in humans and animals, accounting for more than 1/4 of adult intestinal flora. It is a source of nutrition for intestinal bacteria; it can regulate the expression of a variety of host genes, including those involved in nutrient absorption, mucosal barrier strengthening, and angiogenesis; activate T cell-dependent immune responses; affect Paneth cell protein expression; restriction Colonization of pathogens in the gastrointestinal tract. In clinical studies of metabolic diseases, there is an inverse relationship with obesity and related metabolic disease parameters.
  • Adlercreutzia was originally found in human feces and can produce short-chain fatty acids. It is a class of anti-inflammatory microorganisms. It is reduced in the intestinal microbiota of patients with primary sclerosing cholangitis and multiple sclerosis.
  • Traditional Chinese medicine is used In the process of type 2 diabetes rats, its hyperglycemia, lipid metabolism dysfunction and inflammation have been significantly improved, and the abundance of Adlercreutzia has increased significantly, and it shows that Adlercreutzia and type 2 diabetes are related indicators closely related.
  • Lactobacillus casei CCFM1052 can increase the content of acetic acid and butyric acid in the intestine of PFOA-exposed mice
  • Example 12 Take the fresh feces of the mouse on the 12th day in Example 12, weigh 100mg of feces into 2mL EP tube; add 500uL saturated NaCl, shake evenly (after soaking for 30min, tissue grinder 70Hz/30s, shake and break 3 times); add 40uL 10% sulfuric acid , Vortex and vortex for 30s; add 1mL ether, vortex and vortex, then centrifuge at 18000 ⁇ g for 15min, centrifuge at 4°C; after centrifugation, take the supernatant, transfer to a new 2mL EP tube, add 0.25g anhydrous sodium sulfate; centrifuge at 18000 ⁇ g Centrifuge for 15 min at 4°C; take 500 uL of supernatant to a gas phase vial and put it on the machine.
  • SCFA provides energy for intestinal mucosal cells, can maintain the integrity of the intestinal barrier, regulate inflammation, and inhibit the growth of pathogenic bacteria.
  • Acetic acid can maintain the integrity of the epithelium when Caco-2 cells invaded by dorsal hemorrhagic E. coli, and can also increase the secretion of host antibacterial peptides to exert antibacterial effects.
  • Butyric acid is the main energy source of intestinal epithelial cells. It can not only provide energy for intestinal epithelial cells oxidation, maintain water and electrolyte balance, regulate the balance of intestinal flora, and regulate the intestinal barrier function.
  • GPCRs G protein-coupled receptors
  • HDACs histone deacetylases
  • Figure 16 shows that the determination of short-chain fatty acids in feces shows that Lactobacillus casei CCFM1052 can significantly increase the acetic acid content in the intestines of PFOA-exposed mice (P ⁇ 0.05), restore the acetic acid content to the normal content, and have a positive effect on the butyrate content A better improvement trend will help maintain the microbial balance in the intestinal tract.
  • mice 40 SPF male BALB/c mice (20-25g) were randomly divided into 5 groups: blank control group, constipation model control group, Lactobacillus casei CCFM1052 intervention group, Lactobacillus plantarum control group and phenolphthalein treatment control group. Each group contains 10 mice.
  • the freeze-dried Lactobacillus casei CCFM1052 bacterial powder was resuspended in skimmed milk powder to prepare a bacterial suspension with a concentration of 4.0 ⁇ 10 9 CFU/mL.
  • mice in the intervention group were fed with 0.25 mL of Lactobacillus casei CCFM1052 skimmed milk suspension with a concentration of 4.0 ⁇ 10 9 CFU/mL, and the same amount of L.plantarum ST-III in the Lactobacillus plantarum group , The other 3 groups were fed the same amount of non-bacterial skim milk.
  • the negative control group was given intragastrically with 0.25 mL of normal saline, and the remaining four groups were given intragastrically with 0.25 mL of 1 mg/mL loperamide solution to ensure that the intragastric dose of loperamide in mice was 10 mg/kg BW .
  • the negative control group and the constipation model control group were gavaged with skimmed milk.
  • the mice in the Lactobacillus casei CCFM1052 intervention group were fed with 4.0 ⁇ 10 9 CFU/mL Lactobacillus casei CCFM1052 0.25 mL, phenolphthalein treatment control
  • the group was given 0.25mL, 7mg/mL phenolphthalein solution to ensure that the mice were given 70mg/kg BW.
  • the Lactobacillus plantarum group was given 0.25 mL of L.plantarum ST-III at 4.0 ⁇ 10 9 CFU/mL.
  • Fecal moisture content (%) (wet weight of feces-dry weight of feces)/wet weight of feces ⁇ 100. On the morning of the 17th day, except for the blank control group, all the other groups were given loperamide by intragastric administration.
  • mice After 1 hour, all mice were intragastrically administered with 0.25 mL of activated charcoal gum arabic solution, and then each mouse was placed separately In a clean stainless steel cage covered with absorbent paper, record the time (min) from the start of gavage of activated carbon to the discharge of the first black stool, which is used to evaluate the effect of Lactobacillus casei CCFM1052 on mouse constipation During the alleviating effect, the mice eat and drink freely.
  • Figure 17 shows the measurement results of fecal water content in mice, showing that the fecal water content in the constipation model group was significantly reduced, while the phenolphthalein treatment group was able to significantly increase the water content in the feces of constipated mice (P ⁇ 0.001), Lactobacillus casei
  • the effect of CCFM1052 on improving the water content in the stool of constipated mice (P ⁇ 0.01) is better than that of the control strain Lactobacillus plantarum ST-III (P ⁇ 0.05).
  • the results of the first black stool in mice showed (Figure 18) that the time of the first black stool in the constipation model group was significantly delayed.
  • the phenolphthalein treatment group significantly shortened the defecation time of the constipated mice (P ⁇ 0.001).
  • Lactobacillus casei CCFM1052 had Like phenolphthalein, it has an excellent effect of shortening the discharge time of the first black stool (P ⁇ 0.001). Lactobacillus plantarum ST-III also significantly shortens the discharge time of the first black stool (P ⁇ 0.05), but the effect is obviously not as good as that of Lactobacillus casei CCFM1052 .
  • Lactobacillus casei CCFM1052 promotes the proliferation and Maf A mRNA expression of INS-1 cells induced by high glucose
  • the experiment was divided into 5 groups: normal group (normal culture medium containing 11.1mmol/L glucose), high sugar group (high sugar culture medium containing 22.2mmol/L glucose), rosiglitazone group (high glucose culture medium +80 ⁇ mol/ L
  • CCFM1052 group high-sugar culture medium + 1*10 9 CFU/mL CCFM1052 bacterial liquid
  • LGG group high-sugar culture medium + 1*10 9 CFU/mL LGG bacterial liquid
  • INS-1 cells (number: BH-AC0530) in RPMI-1640 medium (containing 11.1mmol/L glucose, 10% FBS, 50 ⁇ mol/L 2-mercaptoethanol, 1mmol/L pyruvate, 10mmol/L HEPES) And put it in a 37°C, 5% CO 2 incubator.
  • RPMI-1640 medium containing 11.1mmol/L glucose, 10% FBS, 50 ⁇ mol/L 2-mercaptoethanol, 1mmol/L pyruvate, 10mmol/L HEPES
  • CCK-8 method to detect cell proliferation Digest and centrifuge the cells in good condition and inoculate them on a 96-well plate with approximately 5 ⁇ 10 3 cells in each well. The peripheral wells of the plate are not inoculated with cells. To prevent edge effects, add PBS to it at the same time Solution. After the cells adhere to the wall, RPMI-1640 medium containing 0.5% fetal bovine serum is added to each well for 24h synchronous treatment. At the end of synchronization, add the corresponding culture medium to each well for 48 hours according to the group, set three multiple wells for each group, and set the zero adjustment hole at the same time.
  • the old medium is aspirated, washed twice with PBS, 180 ⁇ L serum-free medium and 20 ⁇ L CCK-8 solution are added, and incubated for 3-4 h. After the incubation, the absorbance value of each well was measured at 450 nm using a microplate reader.
  • Centrifuge at 4°C at 12000rpm for 15min draw about 0.4ml of the supernatant, transfer to another enzyme-free EP tube, add 0.5mL of isopropanol, mix by inversion, and let it stand for 10min at room temperature.
  • Centrifuge at 4°C at 12000 rpm for 10 min carefully discard the supernatant, add 1.0 mL of 75% ethanol and mix by inversion.
  • Add 20 ⁇ L of DEPC treated water to dissolve and store at 80°C until use. Determine the concentration and quality of RNA, and perform reverse transcription according to the reverse transcription kit instructions.
  • the cDNA obtained by reverse transcription was detected by q RT-PCR, and the MafA specific primers: F: 5'-atcactctgcccaccatcac-3' (SEQ ID NO. 3), R: 5'-atgacctcctccttgctgaa-3' (SEQ ID NO. 4).
  • the PCR system is: F (10 ⁇ M), 0.50 ⁇ L; R (10 ⁇ M), 0.50 ⁇ L; cDNA template, 1.00 ⁇ L; dd H 2 O, 3.00 ⁇ L; mix, 5.00 ⁇ L.
  • PCR program 95°C, 2min;
  • the relative gene expression analysis is performed by the 2- ⁇ CT method.
  • the CFX Manager software was used to analyze the expression level of the target gene in each group of rat INS-1 cells, and then the expression level of the normal group was set as 1, and the other groups were compared with each other to calculate the gene expression level of each group.
  • Maf A mRNA The expression of Maf A mRNA is shown in Figure 20.
  • the expression of MafA mRNA in the cells of the high glucose group was significantly lower than that in the normal group (P ⁇ 0.05), while the expression of MafA mRNA in the rosiglitazone positive control group and CCFM1052 group was higher.
  • the high glucose group increased significantly (P ⁇ 0.05).
  • Example 19 Utilizing Lactobacillus casei CCFM1052 of the present invention to manufacture fermented food containing the bacteria
  • the fruits and vegetables include cucumber, carrot, beet, celery or cabbage products.
  • Lactobacillus casei CCFM1052 is inoculated into the raw materials of dairy products or soybean products to prepare fermented dairy products or fermented soybean products; the fermented food includes fermented dairy products or fermented soybean products; the dairy products include milk, sour cream, and cheese.
  • Lactobacillus casei CCFM1052 can significantly alleviate liver toxicity and intestinal flora imbalance caused by perfluorooctanoic acid (PFOA) exposure, and can significantly relieve constipation Lactobacillus casei CCFM1052 and its uses; containing cheese milk
  • PFOA perfluorooctanoic acid
  • the fermented food of Bacillus CCFM1052 can highly adsorb PFOA in vitro and is not colonized in the intestine. It can significantly alleviate the liver oxidative stress damage and serum biochemical indicators caused by PFOA, significantly improve the spleen atrophy caused by PFOA exposure, and significantly improve the PFOA exposure.
  • intestinal microorganisms such as the Clostridiaceae, Adlercreutzia, Allobaculum and Bacteroides in the intestinal tract.
  • the fermented food containing Lactobacillus casei CCFM1052 can significantly increase the proliferation of INS-1 cells and the expression of MafA gene under the action of high sugar, and has the potential to alleviate PFOA-related diabetes.
  • the Lactobacillus casei CCFM1052 of the present invention is used for preparing drug combinations and fermented foods for alleviating PFOA toxicity and constipation, reducing the occurrence of liver diseases, metabolic diseases and potential carcinogenicity, and has broad application prospects.
  • step (1) Culture the lactic acid bacteria selected in step (1) in liquid sorbitol MRS culture medium for 24 hours, and then take 1 mL of the culture and centrifuge at 8000 rpm for 2 minutes;
  • step (2) Cultivate the lactic acid bacteria selected in step (2) overnight, take 1 mL of the bacterial suspension cultured overnight in a 1.5 mL centrifuge tube, centrifuge at 10000 rpm for 2 minutes, discard the supernatant to obtain the bacteria;
  • 10 ⁇ Taq buffer 5 ⁇ L; dNTP, 5 ⁇ L; 27F, 0.5 ⁇ L; 1492R, 0.5 ⁇ L; Taq enzyme, 0.5 ⁇ L; template, 0.5 ⁇ L; ddH2O, 38 ⁇ L.
  • Example 21 Lactobacillus brucei CCFM1053 has good PFOA adsorption capacity
  • Lactobacillus brucei CCFM1053 and other lactic acid bacteria as a control were purified and activated. They were inoculated into MRS liquid medium at 1% (v/v) inoculum and cultured at 37°C for 18 hours. Then the cells were collected by centrifugation at 8000r/min for 5min, the precipitate was cleaned up with saline and then centrifuged at 8000r/min for 5min to remove the precipitation to obtain live cells, namely wet cells. Suspend the wet bacteria weight in a 10mg/LPFOA solution, and make the final bacterial cell concentration reach 1g dry bacteria/L (suspend the wet bacteria weight in ultrapure water without PFOA as a blank control).
  • the measurement results are shown in Figure 21.
  • the adsorption rate of CCFM1053 to 10mg/L PFOA is 67.5% ⁇ 1.2%, and the PFOA adsorption rate of the remaining lactic acid bacteria is less than 40%.
  • Example 22 Lactobacillus brucei CCFM1053 significantly improved spleen atrophy in mice exposed to PFOA
  • mice Fifty 6-week-old male C57BL/6J mice were adapted to the environment for one week and randomly divided into five groups according to their body weight: control group, model group, quercetin intervention group, Lactobacillus brucei CCFM1053 intervention group, and LGG intervention group. Each group contains 10 mice, and the animal grouping and processing methods are the same as in Example 4.
  • Lactobacillus brucei CCFM1053 significantly reduces the levels of ALT, AST and ⁇ -GT in the serum of mice exposed to PFOA
  • ALT mainly exists in the soluble part of the hepatocyte protoplasm. Increased ALT activity indicates that hepatocytes are destroyed and cell membrane permeability is enhanced. AST mainly exists in the mitochondria of liver cells, and the increase of AST activity indicates mitochondrial damage.
  • Figure 23 shows that taking Lactobacillus brucei CCFM1053 can significantly reduce the content of ALT, AST and ⁇ -GT in the serum of mice exposed to PFOA. It shows that taking Lactobacillus brucei CCFM1053 can significantly alleviate the damage to the structure and function of mouse liver cell membrane caused by PFOA.
  • Lactobacillus brucei CCFM1053 can significantly reduce the levels of MDA and GSH in the liver of mice exposed to PFOA
  • the mouse liver in Example 22 was taken to make a 10% homogenate, and the level of MDA and GSH in the liver was measured using a kit purchased from Nanjing Jiancheng Institute.
  • GSH is an important antioxidant enzyme in the body, which has an important role in scavenging ROS.
  • MDA is the final product of ROS in the lipid peroxidation process, which can directly reflect the level of lipid peroxidation.
  • Figure 24 shows that taking Lactobacillus brucei CCFM1053 can significantly reduce the content of MDA and GSH in the liver of PFOA-exposed mice. It shows that Lactobacillus brucei CCFM1053 can effectively improve the liver oxidative stress damage caused by PFOA.
  • Lactobacillus brucei CCFM1053 significantly reduced the abundance of Allobaculum in the intestine of PFOA-exposed mice, increased the abundance of Bacteroides and Eubacteriaceae, improved intestinal disorders caused by PFOA exposure, and reduced liver disease occur
  • the fresh stool of the mouse on the 12th day in Example 22 was taken, and the total DNA in the mouse stool sample was extracted with the stool kit of MP.
  • the specific operation steps are as follows, mainly referring to the kit instructions.
  • the upstream primer 520F (5'-AYTGGGYDTAAAGNG-3', SEQ ID NO. 1) and the downstream primer 802R (5'-TACNVGGGTATCTAATCC-3', SEQ ID NO. 2) were used as primers for amplification.
  • the V3-V4 region fragment of 16S rDNA, the length of the target fragment is about 247bp.
  • Bacteroides also known as Bacteroides, is a genus of the Bacteroides family. It is a gram-negative, non-spore-free, obligate anaerobic small bacterium. Bacteroides normally inhabit the intestines, oral cavity, upper respiratory tract and reproductive tract of humans and animals. Bacteroides are a large number of normal flora in humans and animals, accounting for more than 1/4 of adult intestinal flora.
  • Eubacteriaceae is related to hepatic encephalopathy, that is, the recovery of the dysfunctional gut-liver-brain axis in cirrhosis, and its abundance is significantly reduced after the bile duct bypass operation in severely obese patients. Taking Lactobacillus brucei CCFM1053 can significantly reduce the abundance of Allobaculum in the intestines of PFOA-exposed mice.
  • the increased abundance of Allobaculum may be used as one of the risk indicators for female hepatocellular carcinoma, and it is a pre-carcinogen exposure to host cancer.
  • One of the key variables The above results indicate that Lactobacillus brucei CCFM1053 not only relieves the toxicity of PFOA, but also has the ability to regulate the intestinal flora, regulate immunity and intestinal barrier, and reduce the occurrence of liver disease.
  • mice Take 40 SPF male BALB/c mice (20-25g) and randomly divide them into 5 groups: blank control group, constipation model control group, Lactobacillus brucei CCFM1053 intervention group, Lactobacillus plantarum control group and phenolphthalein treatment control group , Each group contains 10 mice.
  • mice in the intervention group were fed with 0.25 mL of Lactobacillus brucei CCFM1053 skimmed milk suspension at a concentration of 4.0 ⁇ 10 9 CFU/mL, and the same amount of L.plantarum ST- III.
  • the remaining 3 groups were fed the same amount of non-bacterial skim milk.
  • the negative control group was given intragastrically with 0.25 mL of normal saline, and the remaining four groups were given intragastrically with 0.25 mL of 1 mg/mL loperamide solution to ensure that the intragastric dose of loperamide in mice was 10 mg/kg BW .
  • the negative control group and the constipation model control group were gavaged with skimmed milk, and the mice in the intervention group of Lactobacillus brucei CCFM1053 were fed with a concentration of 4.0 ⁇ 10 9 CFU/mL Lactobacillus brucei CCFM1053 0.25 mL, phenolphthalein
  • the treatment control group was given 0.25mL, 7mg/mL phenolphthalein solution to ensure that the mice were given 70mg/kg BW.
  • the Lactobacillus plantarum group was given 0.25 mL of L.plantarum ST-III at 4.0 ⁇ 10 9 CFU/mL.
  • mice feces were collected every day to calculate the water content of mouse feces.
  • the water content of feces was calculated as follows:
  • Fecal moisture content (%) (wet weight of feces-dry weight of feces)/wet weight of feces ⁇ 100.
  • Lactobacillus brucei CCFM1053 can relieve constipation, increase the water content of feces, and make the water content of feces slightly higher than the control group.
  • Lactobacillus brucei CCFM1053 can promote the proliferation and Maf A mRNA expression of INS-1 cells induced by high glucose
  • the experiment was divided into 5 groups: normal group (normal culture medium containing 11.1mmol/L glucose), high sugar group (high sugar culture medium containing 22.2mmol/L glucose), rosiglitazone group (high glucose culture medium +80 ⁇ mol/ L rosiglitazone), CCFM1053 group (high sugar culture medium + 1*10 9 CFU/mL CCFM1053 bacterial liquid) LGG group (high sugar culture medium + 1*10 9 CFU/mL LGG bacterial liquid).
  • normal group normal culture medium containing 11.1mmol/L glucose
  • high sugar group high sugar culture medium containing 22.2mmol/L glucose
  • rosiglitazone group high glucose culture medium +80 ⁇ mol/ L rosiglitazone
  • CCFM1053 group high sugar culture medium + 1*10 9 CFU/mL CCFM1053 bacterial liquid
  • LGG group high sugar culture medium + 1*10 9 CFU/mL LGG bacterial liquid.
  • INS-1 cells (number: BH-AC0530) in RPMI-1640 medium (containing 11.1mmol/L glucose, 10% FBS, 50 ⁇ mol/L 2-mercaptoethanol, 1mmol/L pyruvate, 10mmol/L HEPES) And put it in a 37°C, 5% CO 2 incubator.
  • RPMI-1640 medium containing 11.1mmol/L glucose, 10% FBS, 50 ⁇ mol/L 2-mercaptoethanol, 1mmol/L pyruvate, 10mmol/L HEPES
  • CCK-8 method to detect cell proliferation Digest and centrifuge the cells in good condition and inoculate them on a 96-well plate with approximately 5 ⁇ 10 3 cells in each well. The peripheral wells of the plate are not inoculated with cells. To prevent edge effects, add PBS to it at the same time Solution. After the cells adhere to the wall, RPMI-1640 medium containing 0.5% fetal bovine serum is added to each well for 24h synchronous treatment. At the end of synchronization, add the corresponding culture medium to each well for 48 hours according to the group, set three multiple wells for each group, and set the zero adjustment hole at the same time.
  • the old medium is aspirated, washed twice with PBS, 180 ⁇ L serum-free medium and 20 ⁇ L CCK-8 solution are added, and incubated for 3-4 h. After the incubation, the absorbance value of each well was measured at 450 nm using a microplate reader.
  • Centrifuge at 4°C at 12000rpm for 15min draw about 0.4ml of the supernatant, transfer to another enzyme-free EP tube, add 0.5mL of isopropanol, mix by inversion, and let it stand for 10min at room temperature.
  • Centrifuge at 4°C at 12000 rpm for 10 min carefully discard the supernatant, add 1.0 mL of 75% ethanol and mix by inversion.
  • Add 20 ⁇ L of DEPC treated water to dissolve and store at 80°C until use. Determine the concentration and quality of RNA, and perform reverse transcription according to the reverse transcription kit instructions.
  • the cDNA obtained by reverse transcription was detected by q RT-PCR, and the MafA specific primers: F: 5'-atcactctgcccaccatcac-3' (SEQ ID NO. 3), R: 5'-atgacctcctccttgctgaa-3' (SEQ ID NO. 4).
  • the PCR system is: F (10 ⁇ M), 0.50 ⁇ L; R (10 ⁇ M), 0.50 ⁇ L; cDNA template, 1.00 ⁇ L; dd H 2 O, 3.00 ⁇ L; mix, 5.00 ⁇ L.
  • PCR program 95°C, 2min;
  • the relative gene expression analysis is performed by the 2- ⁇ CT method.
  • the CFX Manager software was used to analyze the expression level of the target gene in each group of rat INS-1 cells, and then the expression level of the normal group was set as 1, and the other groups were compared with each other to calculate the gene expression level of each group.
  • the CCK-8 test results are shown in Figure 29. Compared with the normal group, the cell growth of the high glucose group was significantly reduced (P ⁇ 0.05), and the cell proliferation of the rosiglitazone control group was significantly increased (P ⁇ 0.05). 0.05), the cell proliferation status of CCFM1053 group was significantly increased compared with high glucose group (P ⁇ 0.05).
  • the expression of Maf A mRNA is shown in Figure 30.
  • the expression of MafA mRNA in the cells of the high glucose group was significantly lower than that in the normal group (P ⁇ 0.05), while the expression of MafA mRNA in the rosiglitazone positive control group and CCFM1053 group was higher.
  • the high glucose group increased significantly (P ⁇ 0.05).
  • Example 28 Using Lactobacillus brucei CCFM1053 to manufacture fermented food
  • the fruits and vegetables include cucumber, carrot, beet, celery or cabbage products.
  • Lactobacillus brucei CCFM1053 is inoculated into raw materials of dairy products or soybean products to prepare fermented dairy products or fermented soybean products; the dairy products include milk, sour cream, and cheese.
  • the fermented food prepared by taking Lactobacillus brucei CCFM1053 can significantly improve the spleen atrophy of mice caused by PFOA exposure; significantly increase the TNF- ⁇ content in the serum of mice exposed to PFOA; The Lactobacillus brucei CCFM1053 significantly increased the content of ALT, AST and ⁇ -GT in the serum of PFOA-exposed mice; the Lactobacillus brucei CCFM1053 was able to reduce the content of MDA in the liver of PFOA-exposed mice to normal levels, and Reduced GSH activity.
  • the Lactobacillus brucei CCFM1053 can significantly increase the fecal water content and discharge time of the first black stool in constipated mice, and relieve the constipation of mice. Lactobacillus brucei CCFM1053 can also increase the proliferation of INS-1 cells and the expression of MafA gene under high glucose, and alleviate PFOA-related diabetes.
  • Lactobacillus fermentum CCFM1051 was purified and activated and cultured, inoculated into MRS liquid medium at 1% (v/v) inoculum, and cultured at 37°C for 18h. Then centrifuge at 8000r/min for 5min to collect the bacteria.
  • the cell of Lactobacillus fermentum CCFM1051 is mixed with a pharmaceutically acceptable carrier to prepare a medicine.
  • the pharmaceutically acceptable carrier includes but is not limited to excipients, which include but are not limited to lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, Tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methylcellulose.
  • excipients include but are not limited to lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, Tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methylcellulose.
  • the pharmaceutically acceptable carrier includes but is not limited to prebiotics; the prebiotics are selected from one or more of fructooligosaccharides, galactooligosaccharides and lactitol.
  • Lactobacillus casei CCFM1052 was purified and activated and cultured, inoculated into MRS liquid medium at 1% (v/v) inoculum, and cultured at 37°C for 18h. Then centrifuge at 8000r/min for 5min to collect the bacteria.
  • the cell of Lactobacillus casei CCFM1052 is mixed with a pharmaceutically acceptable carrier to prepare a medicine.
  • the pharmaceutically acceptable carrier includes but is not limited to excipients, which include but are not limited to lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, Tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methylcellulose.
  • excipients include but are not limited to lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, Tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methylcellulose.
  • the pharmaceutically acceptable carrier includes but is not limited to prebiotics; the prebiotics are selected from one or more of fructooligosaccharides, galactooligosaccharides and lactitol.
  • Lactobacillus brucei CCFM1053 was purified and activated and cultured, inoculated into MRS liquid medium at 1% (v/v) inoculum, and cultured at 37°C for 18 hours. Then centrifuge at 8000r/min for 5min to collect the bacteria.
  • the cell of Lactobacillus brucei CCFM1053 is mixed with a pharmaceutically acceptable carrier to prepare a medicine.
  • the pharmaceutically acceptable carrier includes but is not limited to excipients, which include but are not limited to lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, Tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methylcellulose.
  • excipients include but are not limited to lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, Tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methylcellulose.
  • the pharmaceutically acceptable carrier includes but is not limited to prebiotics; the prebiotics are selected from one or more of fructooligosaccharides, galactooligosaccharides and lactitol.
  • Lactobacillus fermentum CCFM1051, Lactobacillus casei CCFM1052, and Lactobacillus brucei CCFM1053 were respectively prepared according to the following methods. After a single colony was activated in the liquid medium, it was inoculated into the MRS liquid medium at 1% (v/v) inoculum and cultured at 37°C for 18 hours. Then centrifuge at 8000r/min for 5min to collect the bacteria.
  • Lactobacillus fermentum CCFM1051, Lactobacillus casei CCFM1052, and Lactobacillus brucei CCFM1053 are combined with other pharmacologically or food-permitted probiotics to prepare medicines.
  • the drug may also contain a pharmaceutically acceptable carrier, including but not limited to excipients and diluents.
  • excipients include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose , Polyvinylpyrrolidone, cellulose, water, syrup and methylcellulose.
  • the diluent includes, but is not limited to: physiological saline or syrup.
  • Lactobacillus fermentum CCFM1051, Lactobacillus casei CCFM1052, and Lactobacillus brucei CCFM1053 were respectively prepared according to the following methods. After a single colony was activated in the liquid medium, it was inoculated into the MRS liquid medium at 1% (v/v) inoculum and cultured at 37° C. for 18 hours to prepare the starter.
  • the cytoprotective agent includes, but is not limited to: glycerin, DMSO, ethylene glycol, propylene glycol, acetamide, methanol, polyvinylpyrrolidone (PVP), sucrose, polyethylene glycol, dextran, albumin, hydroxyethyl starch One or more of them.

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Abstract

一种缓解PFOA毒害作用的多功能乳杆菌及其应用，属于微生物技术领域。发酵乳杆菌CCFM1051、干酪乳杆菌CCFM1052、布氏乳杆菌CCFM1053对PFOA具有高吸附作用，能够显著缓解PFOA造成的肝脏氧化应激损伤和血清生化指标，改善PFOA暴露造成的脾脏萎缩，改善因PFOA暴露导致的肠道内微生物的失调，改善因PFOA暴露造成的肠道菌群代谢紊乱，显著提高了肠道中乙酸和丙酸的含量，提高便秘小鼠的粪便含水量和首粒黑便时间，提高高糖作用下INS-1细胞的增殖和MafA基因的表达，具有缓解PFOA相关糖尿病的潜力，减少肝病、代谢类疾病和潜在的致癌性的发生。

Description

缓解PFOA毒害作用的多功能乳杆菌及其应用 技术领域
本发明涉及缓解PFOA毒害作用的多功能乳杆菌及其应用，具体涉及缓解PFOA毒害作用的多功能发酵乳杆菌CCFM1051、干酪乳杆菌CCFM1052、布氏乳杆菌CCFM1053及上述任一菌株或其组合物的应用，属于微生物技术领域。
背景技术
全氟化合物由于其疏水疏油特性，并且具有良好的热稳定性，化学稳定性和生物稳定性，在各行各业受到了广泛的应用。用于成衣(如防水，防污户外服饰)及家居纺织品(如地毯，家具布料等)，外卖食品容器，个人护理产品(如牙线)以及灭火泡沫等等。而其中PFOA(PFOA)作为是多种氟类化合物的最终转化产物之一，可以随着食物链富集。在全球各种环境介质，例如水，土壤，大气层，灰尘等，以及动物人体内中均检测到PFOA的存在，并且在人体内的半衰期为2-3年，因此受到了研究学者越来越多的重视。在2013年就作为持久，累积和毒性化学物质被《化学品注册、评估、授权和限制法规》(REACH法规)纳入《高度关注物质候选名单》，在2017年正式列入REACH法规，在欧盟各国实施限制。然而，在部分国家PFOA仍在大量使用，且环境中残留的PFOA还会在未来很长一段时间对整个生态系统造成持久影响。
暴露人群血液中PFOA含量与可能的健康影响之间的关联研究发现，暴露于PFOA可能与血液中总胆固醇浓度的升高，肝酶ALT浓度上升与出生体重降低有较明显的关系。而且已经发现PFOA暴露与疫苗接种反应减少存在关联。这些迹象表明，PFOA可能影响人体的肝脏功能，脂代谢与免疫功能。而在人体中出现的影响在哺乳动物体内已经被明确发现，PFOA具有肝脏毒性，免疫毒性，生殖毒性，发育毒性，神经毒性等多种毒性作用。PFOA可以导致肝肿大，同时可诱发小鼠肝组织氧化应激，使自由基异常增多，可能是造成肝脏损伤的主要原因。PFOA暴露对水生动物和啮齿动物免疫系统的多个免疫器官均产生不同程度的损伤，造成免疫器官脾脏和胸腺的萎缩老化，明显干扰斑马鱼脾脏白介素的表达，淋巴细胞的凋亡与衰退。在对哮喘小鼠的暴露实验中，高剂量PFOA暴露相对哮喘模型组外周血炎症因子IL-4升高，IFN-γ明显较低，即诱导Th2型免疫反应加剧肺部炎症。
目前缓解PFOA毒性的方法多是从具有高抗氧化活性的天然化学物质着手，研究中有缓解作用的如枸杞多糖、桑色素、三羟异黄酮、番茄红素等。但这些天然物质都存在价格昂贵且难以获得，此外由于人体的承受能力，大量的摄入对人体是否存在的潜在危害尚不得知。 因此，寻找一种能够有效缓解PFOA毒性，并且对人体不存在其他可能的有害作用的有效方法显然十分有必要。
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。
本发明的第一个目的是提供如下任一乳杆菌：
(a)发酵乳杆菌CCFM1051，分类命名为Lactobacillus fermentum，已于2019年4月29日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No：60649；
(b)干酪乳杆菌CCFM1052，分类命名为Lactobacillus casei，已于2019年4月29日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No：60650；
(c)布氏乳杆菌CCFM1053，分类命名为Lactobacillus buchneri，已于2019年4月29日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No：60651。
本发明的第二个目的是提供含有所述乳杆菌的组合物。
在一种实施方式中，所述组合物为食品、药物或保健品。
在一种实施方式中，所述食品为发酵食品，是使用所述发酵乳杆菌CCFM1051、干酪乳杆菌CCFM1052、布氏乳杆菌CCFM1053或其中多种乳杆菌的混合物发酵生产制得。
在一种实施方式中，所述发酵食品包括固态食品、液态食品或半固态食品。
在一种实施方式中，所述发酵食品包括乳制品、豆制品、果蔬制品。
在一种实施方式中，所述食品为功能性食品。
在一种实施方式中，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，所述组合物为发酵剂；所述发酵剂为液态发酵剂或固态发酵剂。
在一种实施方式中，所述发酵剂中的乳杆菌数量≥1×10 8CFU/mL。
在一种实施方式中，所述发酵剂中的乳杆菌数量≥1×10 8CFU/g。
在一种实施方式中，所述药物含有药学上可接受的载体。
在一种实施方式中，所述药学上可接受的载体包括但不限于赋形剂、稀释剂；所述赋形剂包括但不限于乳糖，右旋糖，蔗糖，山梨糖醇，甘露醇，淀粉，阿拉伯树胶，磷酸钙，藻酸盐，黄蓍胶，明胶，硅酸钙，微晶纤维素，聚乙烯吡咯烷酮，纤维素，水，糖浆和甲基纤维素；所述稀释剂包括但不限于：生理盐水或糖浆。
在一种实施方式中，所述药物具有如下至少一种用途：
(a)缓解PFOA的毒性作用；
(b)预防、治疗或缓解便秘；
(c)抗肝病或减少肝病的发生；
(d)抗高血压或降低血压；
(e)抗肥胖或改善肥胖引起的代谢类疾病；
(f)提高高糖作用下INS-1细胞的增殖和MafA基因的表达，或缓解PFOA相关糖尿病。
本发明的第三个目的是提供所述发酵乳杆菌CCFM1051、干酪乳杆菌CCFM1052、布氏乳杆菌CCFM1053在单独或组合用于吸附PFOA、改善因PFOA暴露造成的脾脏萎缩、降低PFOA暴露后血清中谷丙转氨酶(ALT)和γ-谷氨酰转肽酶(γ-GT)的含量、改善PFOA暴露后的肠道菌群紊乱、降低肠道中Allobaculum属的丰度、升高Clostridiaceae科、Adlercreutzia属、Bacteroides属和Holdmania属微生物的丰度、减少肝病和代谢类疾病的发生、改善因PFOA暴露造成的肠道菌群代谢紊乱、升高肠道中乙酸和丁酸的含量、提高便秘患者的粪便含水量和首粒黑便时间，或预防/治疗便秘方面的应用。
有益效果：本发明筛选获得了3株具有PFOA吸附能力的乳杆菌，菌株对PFOA具有高吸附，具有缓解PFOA相关代谢疾病的潜力，可用于制备治疗或缓解肝病、代谢类疾病的药物或保健品。本发明的3株乳杆菌分别具有如下的生物学特性：
1、发酵乳杆菌CCFM1051：
(1)菌体特征：革兰氏阳性，细胞球状，直径0.8～1.0μm，无鞭毛，无芽孢；
(2)菌落特征：菌落乳白色，边缘整齐，球状，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)体外具有良好的PFOA吸附能力；
(5)体外具有良好的清除二苯基三硝基苯肼自由基(DPPH)能力，清除羟自由基能力和还原能力；
(6)发酵乳杆菌CCFM1051能显著改善PFOA暴露小鼠脾脏萎缩；
(7)发酵乳杆菌CCFM1051能够显著降低PFOA暴露小鼠血清中IL-4的含量；
(8)发酵乳杆菌CCFM1051能显著降低PFOA暴露小鼠血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)和γ-谷氨酰转肽酶(γ-GT)的水平；
(9)发酵乳杆菌CCFM1051能显著降低PFOA暴露小鼠肠道中S24-7科和乳酸菌属 (Lactobacillus)的丰度，提高拟杆菌属(Bacteroides)和Eubacteriaceae科丰度，改善PFOA暴露造成的肠道紊乱，减少肝病、高血压和肥胖的发生。
(10)发酵乳杆菌CCFM1051能够显著提高便秘小鼠粪便含水量和首粒黑便排出时间，明显改善小鼠的便秘情况。
(11)发酵乳杆菌CCFM1051能够显著改善高糖作用下INS-1细胞的增殖和MafA基因的表达，缓解PFOA相关糖尿病。
2、干酪乳杆菌CCFM1052：
(1)菌体特征：革兰氏阳性，细胞球状，直径0.8～1.0μm，无鞭毛，无芽孢；
(2)菌落特征：菌落乳白色，边缘整齐，球状，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)体外具有良好的PFOA吸附能力；
(5)干酪乳杆菌CCFM1052能显著改善PFOA暴露小鼠脾脏萎缩；
(6)干酪乳杆菌CCFM1052能显著降低PFOA暴露小鼠血清中谷丙转氨酶(ALT)和γ-谷氨酰转肽酶(γ-GT)的含量；
(7)干酪乳杆菌CCFM1052能显著降低PFOA暴露小鼠肝匀浆中超氧化物歧化酶SOD的活性和丙二醛MDA的含量；
(8)干酪乳杆菌CCFM1052能显著降低PFOA暴露小鼠肠道中Allobaculum属的丰度，升高Clostridiaceae科、Adlercreutzia属、Bacteroides属和Holdmania属的丰度，改善PFOA暴露造成的肠道紊乱，减少肝病和代谢类疾病的发生；
(9)干酪乳杆菌CCFM1052能显著升高PFOA暴露小鼠肠道中乙酸和丁酸的含量，改善PFOA暴露造成的肠道菌群代谢紊乱；
(10)干酪乳杆菌CCFM1052能显著提高便秘小鼠粪便含水量以及首粒排黑便的时间；
(11)干酪乳杆菌CCFM1052能够显著改善高糖作用下INS-1细胞的增殖和MafA基因的表达，能够缓解PFOA相关糖尿病。
3、布氏乳杆菌CCFM1053具有以下生物学特性：
(1)菌体特征：革兰氏阳性，细胞球状，直径0.8～1.0μm，无鞭毛，无芽孢；
(2)菌落特征：菌落乳白色，边缘整齐，球状，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)体外具有良好的PFOA吸附能力；
(5)布氏乳杆菌CCFM1053能显著改善PFOA暴露小鼠脾脏萎缩；
(6)布氏乳杆菌CCFM1053能显著降低PFOA暴露小鼠血清中ALT、AST、γ-GT的水平；
(7)布氏乳杆菌CCFM1053能显著降低PFOA暴露小鼠肝脏中MDA和GSH的水平；
(8)布氏乳杆菌CCFM1053能够显著降低PFOA暴露小鼠血清中TNF-α的含量；
(9)布氏乳杆菌CCFM1053能显著降低PFOA暴露小鼠肠道中的丰度Allobaculum属的丰度，提高拟杆菌属(Bacteroides)和优杆菌科(Eubacteriaceae)丰度，改善PFOA暴露造成的肠道紊乱，减少肝病的发生；
(10)布氏乳杆菌CCFM1053能显著提高便秘小鼠的粪便含水量和首粒黑便排出时间，缓解小鼠的便秘情况；
(11)布氏乳杆菌CCFM1053能够显著改善高糖作用下INS-1细胞的增殖和MafA基因的表达，缓解PFOA相关糖尿病。
生物材料保藏
发酵乳杆菌CCFM1051，分类命名为Lactobacillus fermentum，已于2019年4月29日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No：60649。
干酪乳杆菌CCFM1052，分类命名为Lactobacillus casei，已于2019年4月29日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No：60650。
布氏乳杆菌CCFM1053，分类命名为Lactobacillus buchneri，已于2019年4月29日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No：60651。
附图说明
图1为不同乳酸菌在体外重悬于浓度为10mg/L PFOA中经过37℃，150rpm摇床6h后，过0.22μm水系滤膜进入超高效液相色谱质谱联用仪中，吸附前后PFOA浓度变化示意图。
图2为不同乳酸菌在体外清除二苯基三硝基苯肼自由基(DPPH)能力，清除羟自由基能力、还原能力。
图3为不同物质干预10天后小鼠暴露于PFOA，小鼠脾脏比的变化图。其中*P<0.05(vs模型组)。
图4为不同物质干预10天后小鼠暴露于PFOA，小鼠血清中白介素4示意图。其中*P<0.05，**P<0.01(vs模型组)。
图5为不同物质干预10天后小鼠暴露于PFOA，小鼠血清中ALT、AST、γ-GT水平示 意图。其中*P<0.05，**P<0.01，***P<0.001，****P<0.0001(vs模型组)。
图6为不同物质干预10天后小鼠暴露于PFOA，小鼠肠道菌群α多样性变化示意图；其中*P<0.05，**P<0.01，***P<0.001(vs模型组)。
图7为不同物质干预10天后小鼠暴露于PFOA，小鼠肠道中S24-7科、乳酸菌属(Lactobacillus)、拟杆菌属(Bacteroides)和Eubacteriaceae科丰度的变化示意图；其中*P<0.05，**P<0.01(vs模型组)。
图8为不同物质干预后便秘小鼠粪便含水量的改善情况；其中*P<0.05，**P<0.01，***P<0.001(vs模型组)。
图9为不同物质干预后便秘小鼠首粒黑便排出时间的降低情况；其中*P<0.05，**P<0.01，***P<0.001(vs模型组)。
图10是不同菌株对高糖作用下INS-1细胞增值情况的影响。
图11是不同菌株对高糖作用下INS-1细胞MafA基因表达的影响。
图12为不同物质干预10天后小鼠暴露于PFOA，小鼠脾脏系数的变化图。其中*P<0.05，**P<0.01(vs模型组)。
图13为不同物质干预10天后小鼠暴露于PFOA，小鼠血清中谷丙转氨酶(ALT)和γ-谷氨酰转肽酶(γ-GT)水平示意图。其中*P<0.05，**P<0.01，****P<0.0001(vs模型组)。
图14为不同物质干预10天后小鼠暴露于PFOA，小鼠肝匀浆中超氧化物歧化酶SOD活性和MDA含量的变化；其中*P<0.05(vs模型组)。
图15为不同物质干预10天后小鼠暴露于PFOA后，小鼠肠道菌株α多样性分析，及菌群中Clostridiaceae科、Adlercreutzia属、Allobaculum属、Bacteroides属和Holdmania属丰度的变化示意图；其中*P<0.05，**P<0.01，***P<0.001(vs模型组)。
图16为不同物质干预10天后小鼠暴露于PFOA，小鼠肠道中乙酸和丁酸含量的变化；其中*P<0.05(vs模型组)。
图17为本发明菌株干预后，便秘小鼠粪便含水量的改善情况；其中*P<0.05，**P<0.01，***P<0.001(vs模型组)。
图18为本发明菌株干预后，便秘小鼠首粒排黑便时间的降低情况；其中*P<0.05，***P<0.001(vs模型组)。
图19是本发明菌株对高糖作用下INS-1细胞增值情况的影响。
图20是本发明菌株对高糖作用下INS-1细胞MafA基因表达的影响。
图21为不同菌株在体外重悬于浓度为10mg/L PFOA中经过37℃，150rpm摇床6h后， 过0.22μm水系滤膜进入超高效液相色谱质谱联用仪中，吸附前后PFOA浓度变化示意图。
图22为不同物质干预10天后小鼠暴露于PFOA的脾脏比变化图；其中*P<0.05(vs模型组)。
图23为不同物质干预10天后小鼠暴露于PFOA，小鼠血清中ALT、AST、γ-GT水平示意图；其中*P<0.05，**P<0.01，***P<0.001，****P<0.0001(vs模型组)。
图24为不同物质干预10天后暴露于PFOA，小鼠肝脏中GSH活性和MDA含量的变化示意图。
图25为不同物质干预10天后小鼠暴露于PFOA，小鼠肠道菌群α多样性变化示意图；其中*P<0.05，**P<0.01，***P<0.001(vs模型组)。
图26为不同物质干预10天后小鼠暴露于PFOA，小鼠肠道中Eubacteriaceae科、拟杆菌属(Bacteroides)和Allobaculum属丰度的变化示意图；其中*P<0.05，**P<0.01(vs模型组)。
图27为不同物质干预后便秘小鼠粪便含水量的改善情况；其中*P<0.05，**P<0.01，***P<0.001(vs模型组)。
图28为不同物质干预后便秘小鼠首粒黑便排出时间的降低情况；其中*P<0.05，**P<0.01，***P<0.001(vs模型组)。
图29是不同物质对高糖作用下INS-1细胞增值情况的影响。
图30是不同物质对高糖作用下INS-1细胞MafA基因表达的影响。
具体实施方式
实施例1发酵乳杆菌CCFM1051的筛选与鉴定
(一)乳酸菌的分离筛选：
(l)取1g健康成年人的新鲜粪便。将样品在含有山梨醇MRS培养基中35℃富集12h；
(2)将富集样品进行梯度稀释后涂布于添加了0.02％嗅甲酚紫的MRS固体平板上，培养24-48h；
(3)选取变色圈明显，并且符合乳酸菌基本形态的单菌落进行平板划线纯化，筛选分离出乳酸菌；
(4)将上述单菌落培养于液体MRS培养液中培养24h后进行革兰氏染色，选取革兰氏阳性菌进行后续试验。
(二)乳杆菌的初步鉴定：溶钙圈测定法
(l)将步骤(一)所筛选得到的乳酸菌在液体山梨醇MRS培养液中培养24h，然后取l mL培养物8000rpm离心2min；
(2)用0.05M KH 2PO 4溶液洗涤两次；
(3)将所得菌泥重悬，划线在山梨醇MRS-0.75％CaCO 3的固体培养基上，培养24h；
(4)选取溶钙圈明显，且呈凸面圆形、细密色白、无菌丝体的菌落，革兰氏染色后显微镜观察菌体为杆状即初步判定为乳杆菌。
(三)发酵乳杆菌的分子生物学鉴定：
(l)单菌基因组抽提：
A.将步骤(二)所筛选得到的乳酸菌培养过夜，取培养过夜的菌悬液l mL于1.5mL离心管，10000rpm离心2min，弃上清得菌体；
B.用l mL无菌水吹洗菌体后，10000rpm离心2min，弃上清得菌体；
C.加入200μLSDS裂解液，80℃水浴30min；
D.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；
E.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；
F.加入500μL70％(体积百分数)冰乙醇重悬沉淀，12000rpm离心1-3min，弃上清；
G.60℃烘箱烘干，或者自然晾干；
H.50μLddH 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；
(3)制备1％琼脂糖凝胶，之后将PCR产物与10×loading buffer混合，上样量5μL，120V跑30min，然后进行凝胶成像；
(4)将16S rDNA的PCR产物进行测序分析，将得到的序列结果使用BLAST在GeneBank中进行搜索和相似性比对，选取测序结果鉴定为属于发酵乳杆菌的一种新发现的菌株，-80℃保藏备用。
实施例2不同乳酸菌体外吸附PFOA的能力
对发酵乳杆菌CCFM1051及对照菌株进行纯化和活化培养，按1％(v/v)接种量接种于 MRS液体培养基中，37℃培养18h。然后在8000r/min离心5min收集菌体，取沉淀用生理盐水清理后继续在8000r/min离心5min，去沉淀得到活菌体细胞，即湿菌体。将湿菌体重悬于10mg/L PFOA溶液中，并使最终菌体浓度达到1g干菌体/L(将湿菌体重悬于不含PFOA的超纯水中作为空白对照)。使用0.1M的NaOH或HCl溶液将含菌液的PFOA溶液的pH迅速调整至3.0，添加少量的NaOH或HCl(少于0.5ml)其离子强度对PFOA吸附的影响可以忽略。随后将装有100ml样液的250ml锥形瓶置于37℃、150rpm摇床培养，6h后取样测定，2次平行试验取平均值。
PFOA吸附量的测定：吸附实验后，样液在8000r/min离心5min，并用0.22μm的水膜过滤，PFOA浓度用具有Waters SYNAPT MS系统的UPLC-MS测定，采用Acquity UPLC BEH c18柱(2.1×100mm，1.7μm，Waters Co.)，柱温35℃，进样量1μL。用100％(v/v)的乙腈溶液(溶液A)和0.1％(v/v)甲酸水溶液(溶液B)作为洗脱液，进行梯度清洗，流速是0.3mL/min。
表1梯度洗脱条件
t/min 0-0.5 0.5-5.0 5.0-7.0 7.0-7.5
溶剂A比例 70％ 70-100％ 100％ 100-70％
质谱条件：电离源为ESI源；MRM检测；MS+检测；Capillary(毛细管)；3.0kV；Conc(椎体)：40.00V；Source Temperature(放射源温度)：120℃；Desolvation(去溶剂化)温度：400℃；Conc Gas Flow：50L/h；Desolvation Gas Flow：700L/h.气体流速为0.1ml/min；质子比扫描范围：100-2000；扫面时间1s，间隔0.061s。结果用MassLynxV4.1(Waters公司)分析；根据吸附前后PFOA的浓度差异计算乳酸菌对PFOA的吸附量。测定结果列于图1，CCFM1051对10mg/L的PFOA的吸附量为57.5％±1.5％，其余发酵乳杆菌对PFOA的吸附量不及40％。
实施例3体外具有良好的清除二苯基三硝基苯肼自由基(DPPH)能力，清除羟自由基能力和还原能力
将1mL乳酸菌完整细胞悬液与1mL新鲜配制的DPPH无水乙醇溶液(0.2mmol/L)充分混匀后，37℃条件下避光反应30min。将DPPH与PBS(pH7.2)混合作为对照样品，同样条件培养。7000×g离心10min后，在517nm测定吸光度，并按照以下公式计算乳酸菌清除DPPH自由基的能力：
DPPH自由基清除率(％)＝[1-A 517(样品)/A 517(对照)]×100％。
将1mL1,10-邻二氮菲1mLPBS(pH7.2)，1mL乳酸菌完整细胞悬液或以及1mLFeSO 4 混合均匀(称为“混合物1”)。向“混合物1”中加入1mLH 2O 2，37℃条件下水浴1.5h，在536nm测定吸光度，表示为A 536(样品)。将“混合物1”中的完整细胞悬液换成同体积的蒸馏水，以同样条件培养并检测，表示为A 536(空白)。将向“混合物1”加入的H 2O 2换成同体积的蒸馏水，以同样条件培养并检测，表示为A 536(对照)。按照以下公式计算乳酸菌清除羟自由基的能力：
羟自由基清除率(％)＝[A 536(样品)-A 536(空白)]/[A 536(对照)-A 536(空白)]×100％
将0.5mL乳酸菌完整细胞悬液与同体积的铁氰化钾(1％)及PBS缓冲液(pH6.6)混合，振荡使体系均匀。将蒸馏水与铁氰化钾及PBS混合作为空白对照。混合体系在50℃条件下培养20min，快速冷却，并加入0.5mL10％的三氯乙酸。在2000×g离心5min后，取1mL上清与1mL0.1％的氯化铁混合并反应10min。随后在700nm波长下测定吸光度，并使用半胱氨酸(Cysteine)作为表征还原力的标准。结果见图2，CCFM1051的DPPH清除能力为57±4.2％，其他乳酸菌的DPPH的清除能力不及50％，羟自由基清除能力为32±4.5％，而其他乳酸菌的羟自由基清除能力不及30％，还原能力为14±1.8％，其他乳酸菌的还原能力不及13％。
实施例4发酵乳杆菌CCFM1051显著改善PFOA暴露小鼠脾脏萎缩
6周龄雄性C57BL/6J小鼠50只，适应环境一周后，根据体重随机分为五组：对照组、模型组、槲皮素干预组、发酵乳杆菌CCFM1051干预组、LGG干预组，每组含10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 PCTCN2020098036-appb-000001
将小鼠于第13天称体重，随后安乐处死，取出脾脏称湿重以计算脏器系数，按以下公式计算小鼠脾脏的脏器系数：
脾脏脏器系数＝脾脏湿重/安乐死前小鼠体重。
结果如图3所示，服用发酵乳杆菌CCFM1051能够显著逆转由于PFOA染毒造成的小鼠脾脏萎缩，效果优于柚皮素。
实施例5发酵乳杆菌CCFM1051显著降低PFOA暴露小鼠血清中IL-4的含量
实施例4中的小鼠于第13天安乐处死。收集血清，3000g离心15min获得血清，用ELISA试剂盒检测血清中IL-4的含量。结果表明服用发酵乳杆菌CCFM1051能显著改善PFOA染毒造成的小鼠免疫损伤(图4)，使血清IL-4含量恢复至正常水平，效果优于槲皮素。
实施例6发酵乳杆菌CCFM1051显著降低PFOA暴露小鼠血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)和γ-谷氨酰转肽酶(γ-GT)的水平
取实施例5中的血清，采用全自动生化分析仪检测血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)和γ-谷氨酰转肽酶(γ-GT)的含量。ALT主要存在于肝细胞原浆的可溶部分，ALT活性升高提示肝细胞遭到破坏，细胞膜通透性增强。AST主要存在于肝细胞线粒体中，AST活性提高提示线粒体损伤。结果表明(图5)，服用发酵乳杆菌CCFM1051能够显著降低PFOA暴露小鼠血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)和γ-谷氨酰转肽酶(γ-GT)的含量。表明服用发酵乳杆菌CCFM1051能够显著缓解PFOA造成的小鼠肝细胞膜结构和功能的损伤，使ALT由PFOA模型组的68.8±15.2U/L降低至44.5±12.4U/L，使AST由PFOA模型组的240.7±48.2U/L降低至208.6±13.4，使γ-GT由PFOA模型组的310.5±80.3U/L降低至215±15.4U/L。
实施例7发酵乳杆菌CCFM1051显著降低PFOA暴露小鼠肠道中S24-7和Lactobacillus的丰度，提高Bacteroides、Eubacteriaceae和Eubacteriaceae丰度，改善PFOA暴露造成的肠道紊乱，减少肝病、高血压和肥胖的发生
取实施例4中小鼠第12天的新鲜粪便，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′，SEQ ID NO.1)、下游引物802R(5′-TACNVGGGTATCTAATCC-3′，SEQ ID NO.2)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 PCTCN2020098036-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observed species指数来表征，结果(图6)显示PFOA模型组小鼠的肠道菌群α多样性升高，说明PFOA暴露会伴随着一定程度的肠道紊乱。服用发酵乳杆菌CCFM1051能明显降低肠道菌群的α多样性，改善肠道紊乱情况。
此外，S24-7科和乳酸杆菌(Lactobacillus)丰度在PFOA染毒小鼠中显著提高，而服用发酵乳杆菌CCFM1051能够显著的逆转这一情况；S24-7高度定位于恒温动物的胃肠道，革兰氏阴性非运动型厌氧型微生物，能够发酵多种碳水化合物，和非酒精性脂肪肝和高血压的发生发展有关。乳酸杆菌(Lactobacillus)是正常胃肠道和泌尿生殖器的一部分，是常见的益生菌，在乳酸菌预防实验中PFOA模型组和染毒模型出现丰度上升的情况，乳酸菌可能在PFOA暴露后出现了反馈调节的现象。服用发酵乳杆菌CCFM1051还能显著提高PFOA染毒小鼠中拟杆菌属(Bacteroides)和Eubacteriaceae科的丰度(图7)。拟杆菌属(Bacteroides)又称类杆菌属，是拟杆菌科的一属，为革兰氏染色阴性、无芽孢、专性厌氧的小杆菌。拟杆菌属正常寄居于人和动物的肠道、口腔、上呼吸道和生殖道。拟杆菌是人和动物体内大量存在的正常菌群，约占成年个体肠道菌群的1/4以上。是肠道细菌的营养来源；能够调节多种宿主基因的表达，包括那些涉及营养吸收，黏膜屏障强化和血管因子生成的基因；激活T细胞依赖性免疫应答；影响潘氏细胞蛋白的表达；限制病原体在胃肠道的定植。而Eubacteriaceae与肝性脑病，即肝硬化中功能失调的肠-肝-脑轴的恢复有关，在严重肥胖患者的胆肠道旁路术后其丰度显著减少。以上结果表明发酵乳杆菌CCFM1051在缓解PFOA毒性的基础上，还同时具备调节肠道菌群、调节免疫及肠道屏障、减少肝病、高血压和肥胖的发生。
实施例8发酵乳杆菌CCFM1051对小鼠便秘的缓解作用
将SPF级雄性BALB/c小鼠40只(20-25g)，随机分为5组：空白对照组、便秘模型对照组、发酵乳杆菌CCFM1051干预组、植物乳杆菌对照组和酚酞治疗组，每组含小鼠10只。
将发酵乳杆菌CCFM1051冻干菌粉重悬于脱脂乳粉中，制成浓度为4.0×10 9CFU/mL的菌悬液。实验前14天每天给干预组小鼠灌喂0.25mL的发酵乳杆菌CCFM1051脱脂乳悬液(4.0×10 9CFU/mL)，植物乳杆菌组灌胃等量的L.plantarum ST-III，其余3组灌喂等量的不含菌的脱脂乳。试验第15-17天，阴性对照组灌胃0.25mL生理盐水，其余四组灌胃0.25mL， 1mg/mL的洛哌丁胺溶液，确保小鼠洛哌丁胺的灌胃量为10mg/kgBW。
灌胃结束1h后，阴性对照组和便秘模型对照组灌胃脱脂乳，发酵乳杆菌CCFM1051干预组小鼠灌喂0.25mL的发酵乳杆菌CCFM1051(4.0×10 9CFU/mL)，酚酞治疗对照组灌胃0.25mL的7mg/mL的酚酞溶液，确保小鼠酚酞灌胃量为70mg/kgBW。植物乳杆菌组灌胃0.25mL的L.plantarum ST-III(4.0×10 9CFU/mL)。
期间的每天收集小鼠粪便，用于小鼠粪便含水量的计算，按下式计算粪便含水量：
粪便含水量(％)＝(粪便湿重-粪便干重)/粪便湿重×100。
第17天上午除空白对照组灌胃生理盐水，其余组均灌胃洛哌丁胺，灌胃1h后，对所有小鼠灌胃活性炭阿拉伯树胶水溶液0.25mL，然后将每只小鼠单独放置在一个干净的铺有吸水纸的不锈钢笼中，记录从灌胃活性炭开始到第一粒黑便排出的时间(min)，作为首粒排黑便时间，用于评价发酵乳杆菌CCFM1051对小鼠便秘的缓解作用，期间小鼠自由进食和饮水。结果见图8和图9。如图8所示，发酵乳杆菌CCFM1051能够缓解便秘，提高粪便的含水量，并缩短首粒黑便的排出时间，其效果优于植物乳杆菌ST-III。
实施例9发酵乳杆菌CCFM1051可促进高糖诱导的INS-1细胞的增殖及Maf A mRNA表达
实验分成5组：正常组(含11.1mmol/L葡萄糖的普通培养液)，高糖组(含22.2mmol/L葡萄糖的高糖培养液)，罗格列酮组(高糖培养液+80μmol/L的罗格列酮)，CCFM1051组(高糖培养液+含1×10 9CFU/mL CCFM1051菌液)LGG组(高糖培养液+含1×10 9CFU/mL LGG菌液)。
将INS-1细胞(编号：BH-AC0530)接种于RPMI-1640(含11.1mmol/L葡萄糖，10％FBS，50μmol/L 2-巯基乙醇，1mmol/L丙酮酸，10mmol/L HEPES)培养液中，并在37℃，5％CO 2的培养箱中培养。
CCK-8法检测细胞增殖：将状态良好的细胞消化离心并接种于96孔板上，各孔约5×10 3个细胞，板的周边孔不接种细胞，为防止边缘效应同时向其中加入PBS溶液。待细胞贴壁，各孔中加入含0.5％胎牛血清的RPMI-1640培养基，同步化处理24h。同步化结束，依据分组向各孔加入相应培养基培养48h，每组设三个复孔，同时设置调零孔。药物干预结束，吸去旧培养基，PBS清洗2次，加入180μL无血清培养基和20μL CCK-8溶液，孵育3-4h。孵育结束，使用酶标仪于450nm下测定各孔吸光度值。
Maf A mRNA表达的测定：Trizol法提取RNA，吸弃6孔板中原培养液，同时用预冷的PBS清洗2次，各孔中分别加入1.0mL Trizol裂解细胞并将含细胞的裂解液转至无酶EP管，移液枪吹打至无明显沉淀静置5min。向各EP管加入0.2mL三氯甲烷，剧烈震荡15s，室温放置2-3min。4℃，12000rpm离心15min，吸取上清0.4m L左右，转入到另一无 酶EP管中，加入0.5mL的异丙醇，颠倒混匀，室温静置10min。4℃，12000rpm离心10min，小心弃去上清，加入1.0mL 75％乙醇并颠倒混匀。4℃，12000rpm离心5min，弃上清，室温干燥2-5分钟。加入20μL DEPC处理水溶解，保存于80℃待用。测定RNA的浓度和质量，并按照反转录试剂盒说明书进行反转录。反转录得到的cDNA进行q RT-PCR检测，其中MafA特异性引物：F:5'-atcactctgcccaccatcac-3'(SEQ ID NO.3)，R:5'-atgacctcctccttgctgaa-3'(SEQ ID NO.4)。PCR体系为：F(10μM),0.50μL；R(10μM),0.50μL；cDNA模板，1.00μL；dd H 2O，3.00μL；mix，5.00μL。PCR程序：95℃，2min；
(95℃，30sec；60℃，30sec；72℃，20sec)×35；72℃，5min；目的基因经过Real-time PCR检测后，采用2 -△△CT法进行相对基因表达分析。先用CFX Manager软件分析各组大鼠INS-1细胞目标基因的表达量，再以正常组表达量为1，其他各组与之相比较，计算各组基因表达水平。
CCK-8法检测结果如图10所示，与正常组相比，高糖作用组细胞生长明显降低(P<0.05)，罗格列酮对照组细胞增殖较高糖组有明显增加(P<0.05)，CCFM1051组与高糖组相比细胞增殖状况也明显增加(P<0.05)。
Maf A mRNA表达情况如图11显示，高糖作用组细胞的MafA mRNA的表达量明显低于正常组(P<0.05)，而罗格列酮阳性对照组和CCFM1051组的Maf A mRNA表达量较高糖作用组明显上升(P<0.05)。
实施例10利用发酵乳杆菌CCFM1051制备发酵食品
(1)制备果蔬饮料
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发酵乳杆菌CCFM1051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CCFM1051活菌的果蔬饮料。所述果蔬包括黄瓜、胡萝卜、甜菜、芹菜或圆白菜制品。
(2)制备发酵乳制品
将发酵乳杆菌CCFM1051接种至乳制品或豆制品的原料中，制备发酵乳制品或发酵豆制品；所述乳制品包括牛奶、酸奶油、干酪。
(3)将发酵乳杆菌CCFM1051接种至固态、半固态或液态原料中发酵，制备固态发酵食品、液态发酵食品、半固态发酵食品。
与发酵乳杆菌CCFM1051菌体的应用效果类似，服用CCFM1051制备的发酵食品能够显著改善因PFOA暴露造成的小鼠脾脏萎缩；显著降低PFOA暴露小鼠血清中白介素4(IL-4) 含量；显著升高PFOA暴露小鼠血清中ALT、AST、γ-GT的含量；改善PFOA暴露小鼠的肠道菌群紊乱，降低肠道中S24-7和Lactobacillus的丰度，升高Bacteroides和Eubacteriaceae丰度，并使肠道菌群趋于正常化，减少肝病、高血压和肥胖的发生。服用含发酵乳杆菌CCFM1051的发酵食品能够显著提高便秘小鼠粪便含水量和首粒黑便排出时间，明显改善小鼠的便秘情况。细胞实验表明，含发酵乳杆菌CCFM1051的发酵食品能够显著提高高糖作用下INS-1细胞的增殖情况和MafA基因的表达，缓解PFOA相关糖尿病。体外实验表明，发酵乳杆菌CCFM1051能够良好的吸附PFOA,能够有效地清除二苯基三硝基苯肼自由基(DPPH)、清除羟自由基、表现出良好的还原能力。
本发明筛选出对PFOA具有高吸附能力且不在人体中定植并且具有高抗氧化能力的益生菌，不仅能够抑制PFOA造成的氧化应激，而且能够从根本上清除人体内的PFOA。发酵乳杆菌CCFM1051可用于制备具有缓解PFOA毒性的食品、保健品和药品，具有非常广泛的应用前景。
实施例11干酪乳杆菌CCFM1052的筛选和鉴定
(一)乳酸菌的分离筛选：
(l)取1g健康成年人的新鲜粪便。将样品在含有山梨醇MRS培养基中35℃富集12h；
(2)将富集样品进行梯度稀释后涂布于添加了0.02％嗅甲酚紫的MRS固体平板上，培养24-48h；
(3)选取变色圈明显，并且符合乳酸菌基本形态的单菌落进行平板划线纯化，筛选分离出乳酸菌；
(4)将上述单菌落培养于液体MRS培养液中培养24h后进行革兰氏染色，选取革兰氏阳性菌进行后续试验。
(二)乳杆菌的初步鉴定：溶钙圈测定法
(l)将步骤(一)所筛选得到的乳酸菌在液体山梨醇MRS培养液中培养24h，然后取l mL培养物8000rpm离心2min；
(2)用0.05M KH2PO4溶液洗涤两次；
(3)将所得菌泥重悬，划线在山梨醇MRS-0.75％CaCO3的固体培养基上，培养24h；
(4)选取溶钙圈明显，且呈凸面圆形、细密色白、无菌丝体的菌落，革兰氏染色后显微镜观察菌体为杆状即初步判定为乳杆菌。
(三)干酪乳杆菌的分子生物学鉴定：
(l)单菌基因组抽提：
A.将步骤(二)所筛选得到的乳酸菌培养过夜，取培养过夜的菌悬液l mL于1.5mL离心管，10000rpm离心2min，弃上清得菌体；
B.用l mL无菌水吹洗菌体后，10000rpm离心2min，弃上清得菌体；
C.加入200μLSDS裂解液，80℃水浴30min；
D.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；
E.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；
F.加入500μL70％(体积百分数)冰乙醇重悬沉淀，12000rpm离心1-3min，弃上清；
G.60℃烘箱烘干，或者自然晾干；
H.50μLddH2O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；ddH2O，38μL。
B.PCR条件：
95℃5min；95℃10s；55℃30s；72℃30s；step2-4 30×；72℃5min；12℃2min；
(3)制备1％琼脂糖凝胶，之后将PCR产物与10×loading buffer混合，上样量5μL，120V跑30min，然后进行凝胶成像；
(4)将16S rDNA的PCR产物进行测序分析，将得到的序列结果使用BLAST在GeneBank中进行搜索和相似性比对，选取测序结果鉴定为属于干酪乳杆菌的一种新发现的菌株，-80℃保藏备用。
实施例12不同乳酸菌体外吸附PFOA的能力
对干酪乳杆菌CCFM1052和作为对照的其它乳酸菌进行纯化和活化培养，按1％(v/v)接种量接种于MRS液体培养基中，37℃培养18h。然后在8000r/min离心5min收集菌体，取沉淀用生理盐水清理后继续在8000r/min离心5min，去沉淀得到活菌体细胞，即湿菌体。将湿菌体重悬于10mg/LPFOA溶液中，并使最终菌体浓度达到1g干菌体/L(将湿菌体重悬于不含PFOA的超纯水中作为空白对照)。使用0.1M的NaOH或HCl溶液将含菌液的PFOA溶液的pH迅速调整至3.0，添加少量的NaOH或HCl(少于0.5ml)其离子强度对PFOA吸附 的影响可以忽略。随后将装有100ml样液的250ml锥形瓶置于37℃、150rpm摇床培养，6h后取样测定，2次平行试验取平均值。测定方法同实施例2所示，结果如表3所示，CCFM1052对10mg/L的PFOA的吸附量为60％±3.2％，其余发酵乳杆菌对PFOA的吸附量不及45％。
表3不同菌株对PFOA的吸附率
Figure PCTCN2020098036-appb-000003
6周龄雄性C57BL/6J小鼠50只，适应环境一周后，根据体重随机分为五组：对照组、模型组、槲皮素干预组、干酪乳杆菌15-7干预组、LGG干预组，每组含10只小鼠，动物分组及处理方法同实施例4。
结果如图12所示，结果表明服用干酪乳杆菌CCFM1052能够显著逆转由于PFOA染毒造成的小鼠脾脏萎缩。
实施例13干酪乳杆菌CCFM1052显著降低PFOA暴露小鼠血清中谷丙转氨酶(ALT)和γ-谷氨酰转肽酶(γ-GT)的水平；
将实施例12中的小鼠于第13天称体重，随后安乐处死，眼眶取血后3000rpm/min离心15min得到血清，采用全自动生化分析仪检测血清中谷丙转氨酶(ALT)和γ-谷氨酰转肽酶(γ-GT)的含量。ALT主要存在于肝细胞原浆的可溶部分，ALT活性升高提示肝细胞遭到破坏，细胞膜通透性增强。结果表明(图13)，服用干酪乳杆菌CCFM1052能够显著降低PFOA暴露小鼠血清中谷丙转氨酶(ALT)和γ-谷氨酰转肽酶(γ-GT)的含量。表明服用干酪乳杆菌8-9能够显著缓解PFOA造成的小鼠肝细胞膜结构和功能的损伤。使ALT由PFOA模型组的68.8±15.2U/L降低至55.2±11.8U/L，使γ-GT由PFOA模型组的310.5±80.3U/L降低至180.3±18.9U/L。
实施例14干酪乳杆菌CCFM1052能显著降低PFOA暴露小鼠肝匀浆中超氧化物歧化酶SOD的活性和丙二醛MDA的含量
将实施例12中的小鼠于第13天称体重，随后安乐处死，取肝脏。在冰上称取一定重量的肝脏组织，按1:9的比例加入预冷的生理盐水，组织匀浆后制得10％肝脏匀浆液，4000×g离心15分钟取上清液。按说明书要求测定肝脏匀浆液的蛋白浓度含量。并根据如下公式计算 MDA含量：MDA含量(nmol/mgprot)＝[(测定OD值-对照OD值)/(标准OD值-空白OD值)]×标准品浓度(nmol/mL)÷待测样本蛋白浓度(mgprot/mL)
在冰上称取一定重量的肝脏组织，按1:9的比例加入预冷的生理盐水，组织匀浆后制得10％肝脏匀浆液，4000×g离心15分钟取上清液。按说明书要求测定肝脏匀浆液的蛋白浓度含量。并根据如下公式计算SOD含量：
SOD抑制率(％)＝[(A对照-A对照空白)-(A测定-A测定空白)]/(A对照-A对照空白)
SOD活力(U/mL)＝SOD抑制率÷50％×反应体系稀释倍数×样本测试前稀释倍数
结果表明(图14)，服用干酪乳杆菌CCFM1052能够显著降低PFOA暴露小鼠肝匀浆中超氧化物歧化酶SOD的活性和丙二醛MDA的含量。正常情况下，细胞中活性氧(Reactive oxygen species，ROS)生成和消除都受到机体有效的控制，处于动态平衡状态，如果细胞发生氧化应激失衡，ROS累积能够损伤DNA、蛋白质及脂质等，SOD是体内重要的抗氧化酶。丙二醛MDA是脂质过氧化物降解的主要产物，其含量高低间接反映了机体细胞受自由基攻击的严重程度。服用干酪乳杆菌CCFM1052能够显著降低PFOA暴露小鼠受到的自由基攻击，显著缓解肝组织的氧化应激水平，降低PFOA暴露小鼠的肝脏损伤。
实施例15干酪乳杆菌CCFM1052显著降低PFOA暴露小鼠肠道中Allobaculum属的丰度，升高Clostridiaceae科、Adlercreutzia属和Bacteroides属的丰度，改善PFOA暴露造成的肠道紊乱，减少肝病和代谢类疾病的发生
取实施例12中小鼠第12天的新鲜粪便，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′,SEQ ID NO.1)、下游引物802R(5′-TACNVGGGTATCTAATCC-3′,SEQ ID NO.2)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 PCTCN2020098036-appb-000004
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指数来表征，图15结果显示模型组小鼠的肠道菌群α多样性升高，说明PFOA暴露会伴随着一定程度的肠道紊乱。服用干酪乳杆菌CCFM1052能明显降低肠道菌群的α多样性，改善肠道紊乱情况。确定主要肠道微生物组成在各种疾病做的关键作用对于预防或逆转生态失调从而预防或控制疾病具有重要意义。
图15显示，Allobaculum属的丰度在PFOA模型组小鼠肠道升高，而服用干酪乳杆菌CCFM1052能够显著的逆转这一情况，Allobaculum属的丰度在海湾战争疾病中同样出现上升，并与肠道漏肠和全身内毒素血症诱导的TLR4活化进一步诱导了海湾战争疾病的神经炎症和胃肠道紊乱。以往研究Allobaculum属丰度增加有可能作为雌性肝细胞癌风险指标之一，并且是致癌物暴露引起的宿主癌症发生前的关键变量之一。梭菌属是厚壁菌门中最大的一类，代表了人体微生物群中最多样化的细菌，梭菌是一类厌氧、革兰氏阳性、孢子形成的棒状细菌。在监管胃肠道免疫平衡中发挥作用。在非酒精性脂肪性肝病中发现了Clostridiaceae科丰度的下降，在慢性功能性便秘患者与健康对照组肠道菌群对比发现，Clostridiaceae科在健康对照组肠道中丰度明显增多。因此服用干酪乳杆局CCFM1052降低了非酒精性脂肪性肝病和便秘的发生发展。拟杆菌属(Bacteroides)又称类杆菌属，是拟杆菌科的一属，为革兰氏染色阴性、无芽孢、专性厌氧的小杆菌。拟杆菌属正常寄居于人和动物的肠道、口腔、上呼吸道和生殖道。拟杆菌是人和动物体内大量存在的正常菌群，约占成年个体肠道菌群的1/4以上。是肠道细菌的营养来源；能够调节多种宿主基因的表达，包括那些涉及营养吸收，黏膜屏障强化和血管因子生成的基因；激活T细胞依赖性免疫应答；影响潘氏细胞蛋白的表达；限制病原体在胃肠道的定植。在代谢疾病临床研究中与肥胖和相关的代谢疾病参数呈反向关系，高糖饮食的动物实验中，高糖饮食组小鼠中拟杆菌属的丰度明显低于正常组。Adlercreutzia属最初在人类粪便中发现，能够产生短链脂肪酸，是一类抗炎菌的微生物，在原发性硬化性胆管炎患者和多发性硬化症的肠道微生物群中减少，在用传统中药对二型糖尿病大鼠的过程中，其高血糖、脂质代谢功能障碍和炎症得到了明显的改善，而Adlercreutzia属的丰度出现显著性升高，并显示Adlercreutzia属与二型糖尿病的相关指标密切相关。
实施例16干酪乳杆菌CCFM1052能升高PFOA暴露小鼠肠道中中乙酸和丁酸的含量
取实施例12中小鼠第12天的新鲜粪便，称取粪便100mg至2mLEP管；加入500uL饱和NaCl，震荡均匀(浸泡30min后，组织研磨仪70Hz/30s,震荡破碎3次)；加入40uL10％硫酸，漩涡震荡均匀30s；加入1mL乙醚，旋涡震荡均匀，随后18000×g离心15min，4℃离心；离心结束取上清，转移至新的2mLEP管，加入0.25g无水硫酸钠；18000×g离心15min， 4℃离心；取500uL上清至气相小瓶，上机。
SCFA为肠粘膜细胞提供能量，可以维持肠屏障的完整性，调节炎症反应，抑制病原菌增值。乙酸可以使Caco-2细胞在背出血性大肠杆菌侵袭时维持上皮得完整性，此外还可以增加宿主抗菌肽的分泌从而发挥抗菌作用。而丁酸是肠上皮细胞的主要能量来源，不仅能够为肠上皮细胞氧化供能，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。而且在毫摩尔浓度下就能发挥抑制肿瘤细胞增值、分化和诱导凋亡的作用，能够激活钠离子/葡萄糖联合载体基因(SLC5A8)的表达，使其发挥诱导细胞凋亡的作用。此外，口服丁酸盐有利于糖尿病的进程。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)具有显著的改善作用。图16为粪便中短链脂肪酸的测定显示，干酪乳杆菌CCFM1052能显著升高PFOA暴露小鼠肠道中乙酸的含量(P<0.05)，使乙酸含量恢复至正常含量，并对丁酸的含量具有较好的提高趋势，有助于维护肠道中的微生物平衡。
实施例17干酪乳杆菌CCFM1052对小鼠便秘的缓解作用
取SPF级雄性BALB/c小鼠40只(20-25g)，随机分为5组：空白对照组、便秘模型对照组、干酪乳杆菌CCFM1052干预组、植物乳杆菌对照组和酚酞治疗对照组，每组含小鼠10只。
将干酪乳杆菌CCFM1052冻干菌粉重悬于脱脂乳粉中，制成浓度为4.0×10 9CFU/mL的菌悬液。实验前14天每天给干预组小鼠灌喂制备的浓度为4.0×10 9CFU/mL的干酪乳杆菌CCFM1052脱脂乳悬液0.25mL，植物乳杆菌组灌胃等量的L.plantarum ST-III，其余3组灌喂等量的不含菌的脱脂乳。试验第15-17天，阴性对照组灌胃0.25mL生理盐水，其余四组灌胃0.25mL，1mg/mL的洛哌丁胺溶液，确保小鼠洛哌丁胺的灌胃量为10mg/kgBW。
灌胃结束1h后，阴性对照组和便秘模型对照组灌胃脱脂乳，干酪乳杆菌CCFM1052干预组小鼠灌喂制备的浓度4.0×10 9CFU/mL的干酪乳杆菌CCFM1052 0.25mL，酚酞治疗对照组灌胃0.25mL，7mg/mL的酚酞溶液，确保小鼠酚酞灌胃量为70mg/kgBW。植物乳杆菌组灌胃0.25mL，4.0×10 9CFU/mL的L.plantarum ST-III。
实验期间的每天收集小鼠粪便，用于小鼠粪便含水量的计算，按下式计算粪便含水量。粪便含水量(％)＝(粪便湿重-粪便干重)/粪便湿重×100。第17天上午除空白对照组灌胃生理盐水，其余组均灌胃洛哌丁胺，灌胃1h后，对所有小鼠灌胃活性炭阿拉伯树胶水溶液0.25mL，然后将每只小鼠单独放置在一个干净的铺有吸水纸的不锈钢笼中，记录从灌胃活性炭开始到第一粒黑便排出的时间(min)，作为首粒排黑便时间，用于评价干酪乳杆菌 CCFM1052对小鼠便秘的缓解作用，期间小鼠自由进食和饮水。
图17为小鼠粪便含水量的测定结果，显示便秘模型组中的粪便含水量显著降低，而酚酞治疗组能够极显著的提高便秘小鼠粪便中的含水量(P<0.001)，干酪乳杆菌CCFM1052对便秘小鼠粪便中含水量的提高效果(P<0.01)优于对照菌株植物乳杆菌ST-III(P<0.05)。小鼠首粒排黑便结果表明(图18)，便秘模型组小鼠首粒黑便时间显著推迟，酚酞治疗组显著缩短了便秘小鼠的排便时间(P<0.001)，干酪乳杆菌CCFM1052具有和酚酞一样优秀的缩短首粒黑便排出时间的效果(P<0.001)，植物乳杆菌ST-III同样显著缩短了首粒黑便排出时间(P<0.05)，但效果明显不如干酪乳杆菌CCFM1052。
实施例18干酪乳杆菌CCFM1052促进高糖诱导的INS-1细胞的增殖及Maf A mRNA表达
实验分成5组：正常组(含11.1mmol/L葡萄糖的普通培养液)，高糖组(含22.2mmol/L葡萄糖的高糖培养液)，罗格列酮组(高糖培养液+80μmol/L
的罗格列酮)，CCFM1052组(高糖培养液+含1*10 9CFU/mL CCFM1052菌液)LGG组(高糖培养液+含1*10 9CFU/mL LGG菌液)。
将INS-1细胞(编号：BH-AC0530)培养于RPMI-1640培养液(含11.1mmol/L葡萄糖，10％FBS，50μmol/L 2-巯基乙醇，1mmol/L丙酮酸，10mmol/L HEPES)中，并放入37℃，5％CO 2的培养箱中。
CCK-8法检测细胞增殖：将状态良好的细胞消化离心并接种于96孔板上，各孔约5×10 3个细胞，板的周边孔不接种细胞，为防止边缘效应同时向其中加入PBS溶液。待细胞贴壁，各孔中加入含0.5％胎牛血清的RPMI-1640培养基，同步化处理24h。同步化结束，依据分组向各孔加入相应培养基培养48h，每组设三个复孔，同时设置调零孔。药物干预结束，吸去旧培养基，PBS清洗2次，加入180μL无血清培养基和20μL CCK-8溶液，孵育3-4h。孵育结束，使用酶标仪于450nm下测定各孔吸光度值。
Maf A mRNA表达的测定：Trizol法提取RNA，吸弃6孔板中原培养液，同时用预冷的PBS清洗2次，各孔中分别加入1.0mL Trizol裂解细胞并将含细胞的裂解液转至无酶EP管，移液枪吹打至无明显沉淀静置5min。向各EP管加入0.2mL三氯甲烷，剧烈震荡15s，室温放置2-3min。4℃，12000rpm离心15min，吸取上清0.4m L左右，转入到另一无酶EP管中，加入0.5mL的异丙醇，颠倒混匀，室温静置10min。4℃，12000rpm离心10min，小心弃去上清，加入1.0mL 75％乙醇并颠倒混匀。4℃，12000rpm离心5min，弃上清，室温干燥2-5分钟。加入20μL DEPC处理水溶解，保存于80℃待用。测定RNA的浓度和质量，并按照反转录试剂盒说明书进行反转录。反转录得到的cDNA进行q RT-PCR检测，其中MafA特异性引物：F:5'-atcactctgcccaccatcac-3'(SEQ ID NO.3)， R:5'-atgacctcctccttgctgaa-3'(SEQ ID NO.4)。PCR体系为：F(10μM),0.50μL；R(10μM),0.50μL；c DNA模板，1.00μL；dd H 2O，3.00μL；mix，5.00μL。PCR程序：95℃，2min；
(95℃，30sec；60℃，30sec；72℃，20sec)*35；72℃，5min；目的基因经过Real-time PCR检测后，采用2 -△△CT法进行相对基因表达分析。先用CFX Manager软件分析各组大鼠INS-1细胞目标基因的表达量，再以正常组表达量为1，其他各组与之相比较，计算各组基因表达水平。
CCK-8法检测结果如图19所示，与正常组相比，高糖作用组细胞生长明显降低(P<0.05)，罗格列酮对照组细胞增殖较高糖组有明显增加(P<0.05)，CCFM1052组与高糖组相比细胞增殖状况也明显增加(P<0.05)。
Maf A mRNA表达情况如图20显示，高糖作用组细胞的MafA mRNA的表达量明显低于正常组(P<0.05)，而罗格列酮阳性对照组和CCFM1052组的Maf A mRNA表达量较高糖作用组明显上升(P<0.05)。
实施例19利用本发明干酪乳杆菌CCFM1052制造含该菌的发酵食品
(1)制备果蔬饮料
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℃，再接种本发明制备的干酪乳杆菌CCFM1052，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干酪乳杆菌CCFM1052活菌的果蔬饮料。所述果蔬包括黄瓜、胡萝卜、甜菜、芹菜或圆白菜制品。
(2)制备发酵乳制品
将干酪乳杆菌CCFM1052接种至乳制品或豆制品的原料中，制备发酵乳制品或发酵豆制品；所述发酵食品包括发酵乳制品或发酵豆制品；所述乳制品包括牛奶、酸奶油、干酪。
(3)将干酪乳杆菌CCFM1052接种至固态、半固态或液态原料中发酵，制备固态发酵食品、液态发酵食品、半固态发酵食品。
与干酪乳杆菌CCFM1052的应用效果类似，干酪乳杆菌CCFM1052能够显著缓解全氟辛酸(PFOA)暴露造成的肝脏毒性和肠道菌群失调，并能够显著缓解便秘的干酪乳杆菌CCFM1052及其用途；含干酪乳杆菌CCFM1052的发酵食品能够在体外对PFOA具有高吸附作用，不在肠道内定植，显著缓解PFOA造成的肝脏氧化应激损伤和血清生化指标，显著改善PFOA暴露造成的脾脏萎缩，显著改善因PFOA暴露导致的肠道内Clostridiaceae科、Adlercreutzia属、Allobaculum属和Bacteroides属等肠道微生物的失调。显著改善因PFOA暴露造成的肠道菌群代谢紊乱，显著提高肠道中乙酸和丙酸的含量。并能显著提高便秘小鼠的 粪便含水量和首粒黑便时间。此外，含干酪乳杆菌CCFM1052的发酵食品可显著提高高糖作用下INS-1细胞的增殖和MafA基因的表达，具有缓解PFOA相关糖尿病的潜力。
本发明的干酪乳杆菌CCFM1052用于制备缓解PFOA毒性及便秘的药物组合及发酵食品，减少肝病、代谢类疾病和潜在的致癌性的发生，具有广泛的应用前景。
实施例20布氏乳杆菌CCFM1053的筛选和鉴定
(一)乳酸菌的分离筛选：
(l)取100μL泡菜水。将样品在含有山梨醇MRS培养基中35℃富集12h；
(2)将富集样品进行梯度稀释后涂布于添加了0.02％嗅甲酚紫的MRS固体平板上，培养24-48h；
(3)选取变色圈明显，并且符合乳酸菌基本形态的单菌落进行平板划线纯化，筛选分离出乳酸菌；
(4)将上述单菌落培养于液体MRS培养液中培养24h后进行革兰氏染色，选取革兰氏阳性菌进行后续试验。
(二)乳杆菌的初步鉴定：溶钙圈测定法
(l)将步骤(一)所筛选得到的乳酸菌在液体山梨醇MRS培养液中培养24h，然后取l mL培养物8000rpm离心2min；
(2)用0.05M KH2PO4溶液洗涤两次；
(3)将所得菌泥重悬，划线在山梨醇MRS-0.75％CaCO3的固体培养基上，培养24h；
(4)选取溶钙圈明显，且呈凸面圆形、细密色白、无菌丝体的菌落，革兰氏染色后显微镜观察菌体为杆状即初步判定为乳杆菌。
(三)布氏乳杆菌的分子生物学鉴定：
(l)单菌基因组抽提：
A.将步骤(二)所筛选得到的乳酸菌培养过夜，取培养过夜的菌悬液l mL于1.5mL离心管，10000rpm离心2min，弃上清得菌体；
B.用l mL无菌水吹洗菌体后，10000rpm离心2min，弃上清得菌体；
C.加入200μLSDS裂解液，80℃水浴30min；
D.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；
E.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；
F.加入500μL70％(体积百分数)冰乙醇重悬沉淀，12000rpm离心1-3min，弃上清；
G.60℃烘箱烘干，或者自然晾干；
H.50μLddH2O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；ddH2O，38μL。
B.PCR条件：
95℃5min；95℃10s；55℃30s；72℃30s；step2-4 30×；72℃5min；12℃2min；
(3)制备1％琼脂糖凝胶，之后将PCR产物与10×loading buffer混合，上样量5μL，120V跑30min，然后进行凝胶成像；
(4)将16S rDNA的PCR产物进行测序分析，将得到的序列结果使用BLAST在GeneBank中进行搜索和相似性比对，选取测序结果鉴定为属于布氏乳杆菌的一种新发现的菌株，-80℃保藏备用。
实施例21布氏乳杆菌CCFM1053具有良好的PFOA吸附能力
对布氏乳杆菌CCFM1053和作为对照的其它乳酸菌进行纯化和活化培养，按1％(v/v)接种量接种于MRS液体培养基中，37℃培养18h。然后在8000r/min离心5min收集菌体，取沉淀用生理盐水清理后继续在8000r/min离心5min，去沉淀得到活菌体细胞，即湿菌体。将湿菌体重悬于10mg/LPFOA溶液中，并使最终菌体浓度达到1g干菌体/L(将湿菌体重悬于不含PFOA的超纯水中作为空白对照)。使用0.1M的NaOH或HCl溶液将含菌液的PFOA溶液的pH迅速调整至3.0，添加少量的NaOH或HCl(少于0.5ml)其离子强度对PFOA吸附的影响可以忽略。随后将装有100ml样液的250ml锥形瓶置于37℃、150rpm摇床培养，6h后取样测定，2次平行试验取平均值。
按照实施例2公开的方法进行测定，根据吸附前后PFOA的浓度差异计算乳酸菌对PFOA的吸附量。测定结果列于图21，CCFM1053对10mg/L的PFOA的吸附率为67.5％±1.2％，其余乳酸菌的PFOA吸附率不及40％。
实施例22布氏乳杆菌CCFM1053显著改善PFOA暴露小鼠脾脏萎缩
将6周龄雄性C57BL/6J小鼠50只，适应环境一周后，根据体重随机分为五组：对照组、模型组、槲皮素干预组、布氏乳杆菌CCFM1053干预组、LGG干预组，每组含10只小鼠，动物分组及处理方法同实施例4。
结果如图22所示，结果表明服用布氏乳杆菌CCFM1053能够显著逆转由于PFOA染毒造成的小鼠脾脏萎缩。
实施例23布氏乳杆菌CCFM1053显著降低PFOA暴露小鼠血清中ALT、AST和γ-GT的水平
取实施例22中的血清，采用全自动生化分析仪检测血清中ALT、AST和γ-GT的含量。ALT主要存在于肝细胞原浆的可溶部分，ALT活性升高提示肝细胞遭到破坏，细胞膜通透性增强。AST主要存在于肝细胞线粒体中，AST活性提高提示线粒体损伤。结果表明(图23)，服用布氏乳杆菌CCFM1053能够显著降低PFOA暴露小鼠血清中ALT、AST和γ-GT的含量。表明服用布氏乳杆菌CCFM1053能够显著缓解PFOA造成的小鼠肝细胞膜结构和功能的损伤。使ALT由PFOA模型组的68.8±15.2U/L降低至38.3±8.9U/L，使AST由PFOA模型组的240.7±48.2U/L降低至170.5±9.8U/L，使γ-GT由PFOA模型组的310.5±80.3U/L降低至160.3±20.4U/L。
实施例24布氏乳杆菌CCFM1053能显著降低PFOA暴露小鼠肝脏中MDA和GSH的水平
取实施例22中的小鼠肝脏制成10％匀浆，采用购买自南京建成研究所的试剂盒检测肝脏中MDA和GSH的水平。GSH是体内重要的抗氧化酶，对ROS具有重要的清除作用。MDA是脂质过氧化过程中ROS的终产物，可直接反应脂质过氧化的水平。结果表明(图24)，服用布氏乳杆菌CCFM1053能够显著降低PFOA暴露小鼠肝脏中MDA和GSH的含量。说明布氏乳杆菌CCFM1053对PFOA造成的肝脏氧化应激损伤能起到有效的改善效果。
实施例25布氏乳杆菌CCFM1053显著降低PFOA暴露小鼠肠道中的丰度Allobaculum属的丰度，提高拟杆菌属(Bacteroides)和Eubacteriaceae科丰度，改善PFOA暴露造成的肠道紊乱，减少肝病的发生
取实施例22中小鼠第12天的新鲜粪便，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′，SEQ ID NO.1)、下游引物802R(5′-TACNVGGGTATCTAATCC-3′,SEQ ID NO.2)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 PCTCN2020098036-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chao1和shannon指数来表征，图25结果显示模型组小鼠的肠道菌群α多样性升高，说明PFOA暴露会伴随着一定程度的肠道紊乱。服用布氏乳杆菌CCFM1053能明显降低肠道菌群的α多样性，改善肠道紊乱情况。
此外，图26结果显示，服用布氏乳杆菌CCFM1053还能显著提高PFOA染毒小鼠中拟杆菌属(Bacteroides)和Eubacteriaceae科的丰度。拟杆菌属(Bacteroides)又称类杆菌属，是拟杆菌科的一属，为革兰氏染色阴性、无芽孢、专性厌氧的小杆菌。拟杆菌属正常寄居于人和动物的肠道、口腔、上呼吸道和生殖道。拟杆菌是人和动物体内大量存在的正常菌群，约占成年个体肠道菌群的1/4以上。是肠道细菌的营养来源；能够调节多种宿主基因的表达，包括那些涉及营养吸收，黏膜屏障强化和血管因子生成的基因；激活T细胞依赖性免疫应答；影响潘氏细胞蛋白的表达；限制病原体在胃肠道的定植。而Eubacteriaceae与肝性脑病，即肝硬化中功能失调的肠-肝-脑轴的恢复有关，在严重肥胖患者的胆肠道旁路术后其丰度显著减少。服用布氏乳杆菌CCFM1053能显著降低PFOA暴露小鼠肠道中Allobaculum属的丰度，Allobaculum属丰度增加有可能作为雌性肝细胞癌风险指标之一，并且是致癌物暴露引起的宿主癌症发生前的关键变量之一。以上结果表明布氏乳杆菌CCFM1053在缓解PFOA毒性的基础上，还同时具备调节肠道菌群、调节免疫及肠道屏障、减少肝病的发生。
实施例26布氏乳杆菌CCFM1053对小鼠便秘的缓解作用
取SPF级雄性BALB/c小鼠40只(20-25g)，随机分为5组：空白对照组、便秘模型对照组、布氏乳杆菌CCFM1053干预组、植物乳杆菌对照组和酚酞治疗对照组，每组含小鼠10只。
将布氏乳杆菌CCFM1053冻干菌粉重悬于脱脂乳粉中，制成浓度为4.0×10 9CFU/mL的菌悬液。实验前14天每天给干预组小鼠灌喂制备的浓度为4.0×10 9CFU/mL的布氏乳杆菌CCFM1053脱脂乳悬液0.25mL，植物乳杆菌组灌胃等量的L.plantarum ST-III，其余3组灌喂等量的不含菌的脱脂乳。试验第15-17天，阴性对照组灌胃0.25mL生理盐水，其余四组灌胃0.25mL，1mg/mL的洛哌丁胺溶液，确保小鼠洛哌丁胺的灌胃量为10mg/kgBW。
灌胃结束1h后，阴性对照组和便秘模型对照组灌胃脱脂乳，布氏乳杆菌CCFM1053干预组小鼠灌喂制备的浓度4.0×10 9CFU/mL的布氏乳杆菌CCFM1053 0.25mL，酚酞治疗对照组灌胃0.25mL，7mg/mL的酚酞溶液，确保小鼠酚酞灌胃量为70mg/kgBW。植物乳杆菌组 灌胃0.25mL，4.0×10 9CFU/mL的L.plantarum ST-III。
实验期间的每天收集小鼠粪便，用于小鼠粪便含水量的计算，按下式计算粪便含水量：
粪便含水量(％)＝(粪便湿重-粪便干重)/粪便湿重×100。
结果见图27，布氏乳杆菌CCFM1053能够缓解便秘，提高粪便的含水量，并使粪便含水量略高于对照组。
第17天上午除空白对照组灌胃生理盐水，其余组均灌胃洛哌丁胺，灌胃1h后，对所有小鼠灌胃活性炭阿拉伯树胶水溶液0.25mL，然后将每只小鼠单独放置在一个干净的铺有吸水纸的不锈钢笼中，记录从灌胃活性炭开始到第一粒黑便排出的时间(min)，作为首粒排黑便时间，用于评价布氏乳杆菌CCFM1053对小鼠便秘的缓解作用，期间小鼠自由进食和饮水。结果如图28，布氏乳杆菌CCFM1053能够缩短首粒黑便的排出时间，其效果优于植物乳杆菌ST-III。
实施例27布氏乳杆菌CCFM1053可促进高糖诱导的INS-1细胞的增殖及Maf A mRNA表达
实验分成5组：正常组(含11.1mmol/L葡萄糖的普通培养液)，高糖组(含22.2mmol/L葡萄糖的高糖培养液)，罗格列酮组(高糖培养液+80μmol/L的罗格列酮)，CCFM1053组(高糖培养液+含1*10 9CFU/mL CCFM1053菌液)LGG组(高糖培养液+含1*10 9CFU/mL LGG菌液)。
将INS-1细胞(编号：BH-AC0530)培养于RPMI-1640培养液(含11.1mmol/L葡萄糖，10％FBS，50μmol/L 2-巯基乙醇，1mmol/L丙酮酸，10mmol/L HEPES)中，并放入37℃，5％CO 2的培养箱中。
CCK-8法检测细胞增殖：将状态良好的细胞消化离心并接种于96孔板上，各孔约5×10 3个细胞，板的周边孔不接种细胞，为防止边缘效应同时向其中加入PBS溶液。待细胞贴壁，各孔中加入含0.5％胎牛血清的RPMI-1640培养基，同步化处理24h。同步化结束，依据分组向各孔加入相应培养基培养48h，每组设三个复孔，同时设置调零孔。药物干预结束，吸去旧培养基，PBS清洗2次，加入180μL无血清培养基和20μL CCK-8溶液，孵育3-4h。孵育结束，使用酶标仪于450nm下测定各孔吸光度值。
Maf A mRNA表达的测定：Trizol法提取RNA，吸弃6孔板中原培养液，同时用预冷的PBS清洗2次，各孔中分别加入1.0mL Trizol裂解细胞并将含细胞的裂解液转至无酶EP管，移液枪吹打至无明显沉淀静置5min。向各EP管加入0.2mL三氯甲烷，剧烈震荡15s，室温放置2-3min。4℃，12000rpm离心15min，吸取上清0.4m L左右，转入到另一无酶EP管中，加入0.5mL的异丙醇，颠倒混匀，室温静置10min。4℃，12000rpm离心10min， 小心弃去上清，加入1.0mL 75％乙醇并颠倒混匀。4℃，12000rpm离心5min，弃上清，室温干燥2-5分钟。加入20μL DEPC处理水溶解，保存于80℃待用。测定RNA的浓度和质量，并按照反转录试剂盒说明书进行反转录。反转录得到的cDNA进行q RT-PCR检测，其中MafA特异性引物：F:5'-atcactctgcccaccatcac-3'(SEQ ID NO.3)，R:5'-atgacctcctccttgctgaa-3'(SEQ ID NO.4)。PCR体系为：F(10μM),0.50μL；R(10μM),0.50μL；c DNA模板，1.00μL；dd H 2O，3.00μL；mix，5.00μL。PCR程序：95℃，2min；
(95℃，30sec；60℃，30sec；72℃，20sec)*35；72℃，5min；目的基因经过Real-time PCR检测后，采用2 -△△CT法进行相对基因表达分析。先用CFX Manager软件分析各组大鼠INS-1细胞目标基因的表达量，再以正常组表达量为1，其他各组与之相比较，计算各组基因表达水平。
CCK-8法检测结果如图29所示，与正常组相比，高糖作用组细胞生长明显降低(P<0.05)，罗格列酮对照组细胞增殖较高糖组有明显增加(P<0.05)，CCFM1053组与高糖组相比细胞增殖状况也明显增加(P<0.05)。
Maf A mRNA表达情况如图30显示，高糖作用组细胞的MafA mRNA的表达量明显低于正常组(P<0.05)，而罗格列酮阳性对照组和CCFM1053组的Maf A mRNA表达量较高糖作用组明显上升(P<0.05)。
实施例28利用布氏乳杆菌CCFM1053制造发酵食品
(1)制备果蔬饮料
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布氏乳杆菌CCFM1053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布氏乳杆菌CCFM1053活菌的果蔬饮料。所述果蔬包括黄瓜、胡萝卜、甜菜、芹菜或圆白菜制品。
(2)制备发酵乳制品
将布氏乳杆菌CCFM1053接种至乳制品或豆制品的原料中，制备发酵乳制品或发酵豆制品；所述乳制品包括牛奶、酸奶油、干酪。
(3)将布氏乳杆菌CCFM1053接种至固态、半固态或液态原料中发酵，制备固态发酵食品、液态发酵食品、半固态发酵食品。
与布氏乳杆菌CCFM1053菌体的应用效果类似，服用布氏乳杆菌CCFM1053制备的发酵食品能够显著改善因PFOA暴露造成的小鼠脾脏萎缩；显著升高PFOA暴露小鼠血清中TNF-α含量；所述布氏乳杆菌CCFM1053显著升高PFOA暴露小鼠血清中ALT、AST、γ-GT的含 量；所述布氏乳杆菌CCFM1053能够将PFOA暴露小鼠肝脏中MDA的含量降低到正常水平，并降低了GSH活性。明显改善肠道菌群紊乱，降低肠道中Allobaculum属的丰度，升高拟杆菌属(Bacteroides)和Eubacteriaceae科的丰度，减少肝病的发生。所述布氏乳杆菌CCFM1053能显著提高便秘小鼠的粪便含水量和首粒黑便排出时间，缓解小鼠的便秘情况。布氏乳杆菌CCFM1053还能够提高高糖作用下INS-1细胞的增殖和MafA基因的表达、缓解PFOA相关糖尿病。
实施例29含发酵乳杆菌CCFM1051的药物的制备
将发酵乳杆菌CCFM1051进行纯化和活化培养，按1％(v/v)接种量接种于MRS液体培养基中，37℃培养18h。然后在8000r/min离心5min收集菌体。
将发酵乳杆菌CCFM1051菌体细胞与药学上可接受的载体混合，制备药物。
所述药学上可接受的载体包括但不限于赋形剂，赋形剂包括但不限于乳糖，右旋糖，蔗糖，山梨糖醇，甘露醇，淀粉，阿拉伯树胶，磷酸钙，藻酸盐，黄蓍胶，明胶，硅酸钙，微晶纤维素，聚乙烯吡咯烷酮，纤维素，水，糖浆和甲基纤维素。
所述药学上可接受的载体包括但不限于益生元；所述益生元选自低聚果糖、低聚半乳糖和乳糖醇中的一种或几种。
实施例30含干酪乳杆菌CCFM1052的药物的制备
将干酪乳杆菌CCFM1052进行纯化和活化培养，按1％(v/v)接种量接种于MRS液体培养基中，37℃培养18h。然后在8000r/min离心5min收集菌体。
将干酪乳杆菌CCFM1052菌体细胞与药学上可接受的载体混合，制备药物。
所述药学上可接受的载体包括但不限于赋形剂，赋形剂包括但不限于乳糖，右旋糖，蔗糖，山梨糖醇，甘露醇，淀粉，阿拉伯树胶，磷酸钙，藻酸盐，黄蓍胶，明胶，硅酸钙，微晶纤维素，聚乙烯吡咯烷酮，纤维素，水，糖浆和甲基纤维素。
所述药学上可接受的载体包括但不限于益生元；所述益生元选自低聚果糖、低聚半乳糖和乳糖醇中的一种或几种。
实施例31含布氏乳杆菌CCFM1053的药物的制备
将布氏乳杆菌CCFM1053进行纯化和活化培养，按1％(v/v)接种量接种于MRS液体培养基中，37℃培养18h。然后在8000r/min离心5min收集菌体。
将布氏乳杆菌CCFM1053菌体细胞与药学上可接受的载体混合，制备药物。
所述药学上可接受的载体包括但不限于赋形剂，赋形剂包括但不限于乳糖，右旋糖，蔗糖，山梨糖醇，甘露醇，淀粉，阿拉伯树胶，磷酸钙，藻酸盐，黄蓍胶，明胶，硅酸钙，微 晶纤维素，聚乙烯吡咯烷酮，纤维素，水，糖浆和甲基纤维素。
所述药学上可接受的载体包括但不限于益生元；所述益生元选自低聚果糖、低聚半乳糖和乳糖醇中的一种或几种。
实施例32含益生菌的药物的制备
将发酵乳杆菌CCFM1051、干酪乳杆菌CCFM1052、布氏乳杆菌CCFM1053分别按照如下方法制备菌体细胞。将单菌落在液体培养基中活化后，按1％(v/v)接种量接种于MRS液体培养基中，37℃培养18h。然后在8000r/min离心5min收集菌体。
将发酵乳杆菌CCFM1051、干酪乳杆菌CCFM1052、布氏乳杆菌CCFM1053中的一种或多种乳杆菌与其它药学或食品上允许的益生菌组合，制备药物。
所述药物还可含有药学上可接受的载体，包括但不限于赋形剂、稀释剂。
所述赋形剂包括但不限于乳糖，右旋糖，蔗糖，山梨糖醇，甘露醇，淀粉，阿拉伯树胶，磷酸钙，藻酸盐，黄蓍胶，明胶，硅酸钙，微晶纤维素，聚乙烯吡咯烷酮，纤维素，水，糖浆和甲基纤维素。
所述稀释剂包括但不限于：生理盐水或糖浆。
实施例33含益生菌的发酵剂的制备
将发酵乳杆菌CCFM1051、干酪乳杆菌CCFM1052、布氏乳杆菌CCFM1053分别按照如下方法制备菌体细胞。将单菌落在液体培养基中活化后，按1％(v/v)接种量接种于MRS液体培养基中，37℃培养18h，制得发酵剂。
或将上述细胞培养液离心，收集乳杆菌细胞，加入细胞保护剂，使益生菌的浓度≥1×10 8CFU/mL。
可选地，对加入细胞保护剂的细胞悬液进行真空冷冻干燥，制备获得粉末状发酵剂，使益生菌的浓度≥1×10 8CFU/g。
所述细胞保护剂包括但不限于：甘油、DMSO、乙二醇、丙二醇、乙酰胺、甲醇、聚乙烯吡咯烷酮(PVP)、蔗糖、聚乙二醇、葡聚糖、白蛋白、羟乙基淀粉中的一种或多种的混合。
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 (18)

  1. 乳杆菌，其特征在于，为(a)或(b)或(c)：
    (a)发酵乳杆菌CCFM1051，分类命名为Lactobacillus fermentum，已于2019年4月29日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No：60649；
    (b)干酪乳杆菌CCFM1052，分类命名为Lactobacillus casei，已于2019年4月29日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No：60650；
    (c)布氏乳杆菌CCFM1053，分类命名为Lactobacillus buchneri，已于2019年4月29日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No：60651。
  2. 含有权利要求1所述乳杆菌的组合物。
  3. 一种发酵食品，其特征在于，所述发酵食品为使用发酵乳杆菌CCFM1051发酵生产制得。
  4. 根据权利要求3所述的发酵食品，其特征在于，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2或3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。
  6. 根据权利要求5所述的发酵食品，其特征在于，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。
  7. 权利要求1所述的发酵乳杆菌CCFM1051、干酪乳杆菌CCFM1052或布氏乳杆菌CCFM1053在制备体内非定植益生菌中的应用。
  8. 含有权利要求1所述乳杆菌的发酵剂，其特征在于，所述发酵剂中的乳杆菌数量≥1×10 8CFU/mL或≥1×10 8CFU/g。
  9. 一种药物，其特征在于，含有权利要求1所述的发酵乳杆菌CCFM1051、干酪乳杆菌CCFM1052或布氏乳杆菌CCFM1053中的至少一种乳杆菌。
  10. 根据权利要求9所述的药物，其特征在于，还含有药学上可接受的载体。
  11. 根据权利要求10所述的药物，其特征在于，所述药学上可接受的载体包括但不限于赋形剂、稀释剂；
    所述赋形剂包括但不限于乳糖，右旋糖，蔗糖，山梨糖醇，甘露醇，淀粉，阿拉伯树胶，磷酸钙，藻酸盐，黄蓍胶，明胶，硅酸钙，微晶纤维素，聚乙烯吡咯烷酮，纤维素，水，糖浆和甲基纤维素；
    所述稀释剂包括但不限于：生理盐水或糖浆。
  12. 根据权利要求9～11任一所述的药物，其特征在于，所述药物具有如下至少一种用途：
    (a)缓解PFOA的毒性作用；
    (b)预防、治疗或缓解便秘；
    (c)抗肝病或减少肝病的发生；
    (d)抗高血压或降低血压；
    (e)抗肥胖或改善肥胖引起的代谢类疾病；
    (f)提高高糖作用下INS-1细胞的增殖和MafA基因的表达，或缓解PFOA相关糖尿病。
  13. 权利要求1所述的乳杆菌在制备缓解PFOA的毒性作用、预防和治疗便秘、抗肝病、抗高血压、抗肥胖的药物和保健品中的应用。
  14. 根据权利要求13所述的应用，其特征在于，所述药物和保健品能够吸附PFOA、清除二苯基三硝基苯肼自由基、清除羟自由基、抗氧化、改善因PFOA暴露造成的脾脏萎缩、降低PFOA暴露后血清中IL-4含量、升高PFOA暴露后血清中谷丙转氨酶、谷草转氨酶、γ-谷氨酰转肽酶的含量、改善PFOA暴露后的肠道菌群紊乱、降低肠道中S24-7科、乳酸菌属的丰度、升高拟杆菌属和Eubacteriaceae科的丰度，减少肝病、高血压和肥胖的发生、提高便秘患者粪便含水量和首粒黑便排出时间、改善便秘情况。
  15. 根据权利要求13所述的应用，其特征在于，所述药物和保健品具有提高高糖作用下INS-1细胞的增殖和MafA基因的表达、缓解PFOA相关糖尿病的功能。
  16. 权利要求2～6任一所述的发酵食品在制备缓解PFOA毒性、抗肝病、抗高血压、抗肥胖的功能性食品中的应用。
  17. 根据权利要求16所述的应用，其特征在于，所述功能性食品用于吸附PFOA、清除二苯基三硝基苯肼自由基、清除羟自由基、抗氧化、改善因PFOA暴露造成的脾脏萎缩、降低PFOA暴露后血清中IL-4含量、升高PFOA暴露后血清中谷丙转氨酶、谷草转氨酶、γ-谷氨酰转肽酶的含量、改善PFOA暴露后的肠道菌群紊乱、降低肠道中S24-7科、乳酸菌属的丰度、升高拟杆菌属和Eubacteriaceae科的丰度，减少肝病、高血压和肥胖的发生、提高便秘患者粪便含水量和首粒黑便排出时间、改善便秘情况。
  18. 根据权利要求16所述的应用，其特征在于，所述功能性食品能够提高高糖作用下INS-1细胞的增殖和MafA基因的表达、缓解PFOA相关糖尿病。
PCT/CN2020/098036 2019-06-28 2020-06-24 缓解pfoa毒害作用的多功能乳杆菌及其应用 WO2020259564A1 (zh)

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