CN110684682A - Multifunctional lactobacillus casei CCFM1052 capable of relieving PFOA toxic effect, fermented food and application thereof - Google Patents

Multifunctional lactobacillus casei CCFM1052 capable of relieving PFOA toxic effect, fermented food and application thereof Download PDF

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CN110684682A
CN110684682A CN201910575996.1A CN201910575996A CN110684682A CN 110684682 A CN110684682 A CN 110684682A CN 201910575996 A CN201910575996 A CN 201910575996A CN 110684682 A CN110684682 A CN 110684682A
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pfoa
ccfm1052
lactobacillus casei
exposure
intestinal
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CN110684682B (en
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陈卫
王刚
梁席
赵建新
张灏
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Jiangnan University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C13/00—Cream; Cream preparations; Making thereof
    • A23C13/12—Cream preparations
    • A23C13/16—Cream preparations containing, or treated with, microorganisms, enzymes, or antibiotics; Sour cream
    • A23C13/165—Making sour cream by chemical or physical means only
    • 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
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/10—Laxatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/143—Fermentum
    • 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

Abstract

The strain has high adsorption effect on PFOA, is not fixedly planted in intestinal tracts, obviously relieves liver oxidative stress injury and serum biochemical indexes caused by PFOA, obviously improves spleen atrophy caused by PFOA exposure, improves the imbalance of microorganisms in the intestinal tracts caused by the PFOA exposure, improves the metabolic disturbance of intestinal flora caused by the PFOA exposure, obviously improves the content of acetic acid and propionic acid in the intestinal tracts, improves the water content of excrement and the first-grain stool time of constipation mice, improves the proliferation of INS-1 cells and the expression of MafA genes under the action of high sugar, has the potential of relieving PFOA-related diabetes, reduces liver diseases, metabolic diseases and potential carcinogenicity, and has wide application prospect.

Description

Multifunctional lactobacillus casei CCFM1052 capable of relieving PFOA toxic effect, fermented food and application thereof
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to multifunctional lactobacillus casei CCFM1052 for relieving PFOA toxic action, fermented food thereof and application thereof.
Background
The perfluoro compound has good thermal stability, chemical stability and biological stability due to the hydrophobic and oleophobic characteristics, and is widely applied to various industries. For use in apparel (e.g., water-resistant, stain-resistant outdoor apparel) and household textiles (e.g., carpets, upholstery, etc.), take-away food containers, personal care products (e.g., dental floss), and fire-fighting foams, among others. And PFOA as one of the final transformation products of many fluorine compounds can be enriched along the food chain. The presence of PFOA is detected in various environmental media worldwide, such as water, soil, the atmosphere, dust, etc., and in animals, and the half-life in humans is 2-3 years, and thus has been receiving increasing attention from researchers. The persistent, cumulative and toxic chemical substances were included in the list of candidates for substances of high interest by the regulations on registration, evaluation, authorization and restriction of chemicals (REACH regulations) in 2013, the REACH regulations were officially listed in 2017, and restrictions were enforced in countries of the european union. However, PFOA is still in heavy use in some countries and residual PFOA in the environment may also have a lasting effect on the entire ecosystem for a long time in the future.
Research on the correlation between the PFOA content in blood of the exposed people and possible health influence shows that the PFOA exposure may have a more obvious relationship with the increase of total cholesterol concentration in blood and the increase of ALT concentration of liver enzyme and the reduction of birth weight. Also PFOA exposure has been found to be associated with a reduced vaccination response. These signs suggest that PFOA may affect liver function, lipid metabolism and immune function in humans. The effects occurring in the human body have been found clearly in mammals, and PFOA has various toxic effects such as hepatotoxicity, immunotoxicity, reproductive toxicity, developmental toxicity, neurotoxicity, and the like. PFOA can cause hepatomegaly and induce mouse liver tissue oxidative stress to increase free radicals, possibly leading to liver injury. PFOA exposure causes damage to a plurality of immune organs of the immune system of aquatic animals and rodents to different degrees, so that atrophy and aging of spleen and thymus of the immune organs are caused, and the expression of splenic interleukins of zebra fish and apoptosis and decline of lymphocytes are obviously interfered. In an exposure experiment of asthmatic mice, the high-dose PFOA exposure is higher than that of peripheral blood inflammatory factor IL-4 in an asthmatic model group, and IFN-gamma is obviously lower, namely Th2 type immune response is induced to aggravate pulmonary inflammation.
At present, methods for relieving PFOA toxicity are mostly based on natural chemical substances with high antioxidant activity, such as lycium barbarum polysaccharide, morin, trihydroxyisoflavone, lycopene and the like which have relieving effects in research. However, these natural substances are expensive and difficult to obtain, and in addition, the potential hazard to the human body from the large intake is unknown due to the human body's tolerance. Therefore, it is clearly necessary to find an effective way to effectively alleviate PFOA toxicity without the potential for other harmful effects on the human body.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made in view of the above-mentioned technical drawbacks.
Therefore, as one aspect of the invention, the invention overcomes the defects in the prior art, and provides lactobacillus casei CCFM1052 which is preserved in Guangdong province microorganism culture collection center and is positioned at No. 59, No. 5, of the Michelia furiosa No. 100, Guangzhou city, with the preservation number of GDMCC No: 60650.
in order to solve the technical problems, the invention provides the following technical scheme: lactobacillus casei CCFM1052 with a collection number GDMCC No: 60650.
as another aspect of the present invention, the present invention overcomes the disadvantages of the prior art and provides a fermented food: the fermented food is prepared by fermenting with Lactobacillus casei CCFM1052, and comprises solid food, liquid food, and semi-solid food.
As a preferable embodiment of the fermented food of the present invention: the fermented food comprises dairy products, bean products and fruit and vegetable products, wherein the dairy products comprise milk, sour cream and cheese; the fruit and vegetable products comprise cucumber, carrot, beet, celery and cabbage products.
As another aspect of the invention, the invention overcomes the defects in the prior art and provides the application of the lactobacillus casei CCFM1052 in preparing non-colonized probiotics in vivo.
As another aspect of the invention, the invention overcomes the defects in the prior art and provides the application of lactobacillus casei CCFM1052 in preparing medicines and health products for relieving the toxic effect of PFOA, preventing and treating constipation, resisting liver diseases and antimetabolite diseases.
As a preferred embodiment of the application of the present invention: the lactobacillus casei CCFM1052 can adsorb PFOA, improve spleen atrophy caused by PFOA exposure, reduce the content of glutamic pyruvic transaminase (ALT) and gamma-glutamyl transpeptidase (gamma-GT) in serum after PFOA exposure, improve intestinal flora disturbance after PFOA exposure, reduce the abundance of Allobaculum in intestines, increase the abundance of Clostridiaceae, Adlercutzia, Bacteroides and Holdmania, reduce the occurrence of liver diseases and metabolic diseases, improve the intestinal flora metabolic disturbance caused by PFOA exposure, increase the content of acetic acid and butyric acid in the intestines, improve the water content of excrement and the first-grain defecation time of constipation patients, and prevent and treat constipation.
The lactobacillus casei CCFM1052 can also improve the proliferation of INS-1 cells and the expression of MafA genes under the action of high sugar, and relieve PFOA related diabetes.
As another aspect of the invention, the invention overcomes the defects in the prior art and provides the application of the fermented food in preparing functional foods for relieving PFOA toxicity, preventing and treating constipation, and resisting liver diseases and antimetabolite diseases. The lactobacillus casei CCFM1052 can adsorb PFOA, improve spleen atrophy caused by PFOA exposure, reduce the content of glutamic pyruvic transaminase (ALT) and gamma-glutamyl transpeptidase (gamma-GT) in serum after PFOA exposure, improve intestinal flora disturbance after PFOA exposure, reduce the abundance of Allobaculum in intestines, increase the abundance of Clostridiaceae, Adlercutzia, Bacteroides and Holdmania, reduce the occurrence of liver diseases and metabolic diseases, improve the intestinal flora metabolic disturbance caused by PFOA exposure, increase the content of acetic acid and butyric acid in the intestines, improve the water content of excrement and the first-grain defecation time of constipation patients, and prevent and treat constipation. The lactobacillus casei CCFM1052 can also improve the proliferation of INS-1 cells and the expression of MafA genes under the action of high sugar, and relieve PFOA related diabetes.
The invention has the beneficial effects that: in PFOA model mouse experiments, spleen atrophy of mice caused by PFOA exposure can be obviously improved by taking lactobacillus casei CCFM 1052; the content of alanine Aminotransferase (ALT) and gamma-glutamyl transpeptidase (gamma-GT) in the serum of the mice exposed by PFOA is obviously increased by taking lactobacillus casei CCFM 1052; the administration of lactobacillus casei CCFM1052 can improve the intestinal flora disturbance of PFOA exposed mice, reduce the abundance of Allobaculum in the intestines, and increase the abundance of Clostridiaceae, Adlercutzia, Bacteroides and Holdmania; the lactobacillus casei CCFM1052 is taken to improve the metabolic disturbance of intestinal flora caused by PFOA exposure, increase the content of acetic acid and butyric acid in the intestinal tract, lead the intestinal flora and the metabolism to tend to be normalized and reduce the occurrence of liver diseases and metabolic diseases. The water content of the excrement and the first black excrement time of a constipation mouse are obviously improved by taking the lactobacillus casei CCFM1052, and the constipation is effectively prevented and treated. Cell experiments show that intervention of lactobacillus casei CCFM1052 can obviously improve the proliferation condition of INS-1 cells and the expression of MafA genes under the action of high sugar, and has the potential of relieving PFOA-related diabetes. In vitro experiments show that the lactobacillus casei CCFM1052 can well adsorb PFOA.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
FIG. 1 is a schematic diagram of the concentration change of PFOA before and after adsorption of the bacterial strain in vitro after being resuspended in 10mg/LPFOA, passed through a 0.22 mu m water system filter membrane, and then passed through a shaking table at 37 ℃ and 150rpm for 6 h.
FIG. 2 is a graph showing the change in the spleen coefficient of mice exposed to PFOA 10 days after the intervention with the strain of the present invention. Wherein P <0.05, P <0.01(vs model group).
FIG. 3 is a graph showing the levels of alanine Aminotransferase (ALT) and gamma-glutamyl transpeptidase (gamma-GT) in the serum of mice exposed to PFOA 10 days after the intervention of the strain of the present invention. Wherein P <0.05, P <0.01, P <0.0001(vs model group).
FIG. 4 shows the change of SOD activity and MDA content of superoxide dismutase in liver homogenate of mice exposed to PFOA after 10 days of intervention of the strain of the invention; where P <0.05(vs model group).
FIG. 5 is a graph showing the exposure of mice to PFOA after 10 days of intervention with the strain of the present invention, the analysis of alpha diversity of the strains in the mouse intestinal tract, and the changes in abundance of the genera Clostridium, Adlercreutzia, Allobaculum, Bacteroides and Holdmania in the flora; wherein P <0.05, P <0.01, P <0.001(vs model group).
FIG. 6 shows the change of acetic acid and butyric acid contents in the intestinal tracts of mice exposed to PFOA after 10 days of intervention by the strain of the present invention; where P <0.05(vs model group).
FIG. 7 shows the improvement of the water content of feces of constipation-treated mice after the strain of the present invention is dried. Wherein P <0.05, P <0.01, P <0.001(vs model group).
FIG. 8 shows the decrease of the first-grain defecation time of constipation mice after the strain of the present invention is dried. Wherein P <0.05, P <0.001(vs model group).
FIG. 9 shows the effect of the strains of the invention on INS-1 cell proliferation under high sugar;
FIG. 10 shows the effect of the strains of the invention on INS-1 cell MafA gene expression under high sugar.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
The lactobacillus casei CCFM1052 is preserved in Guangdong province microorganism strain preservation center, and the preservation number is GDMCC No: 60650. the preservation date is 2019, 4 months and 29 days, and the taxonomic name is Lactobacillus casei.
The lactobacillus casei has the following biological characteristics:
(1) the characteristics of the thallus are as follows: gram-positive, spherical cell, diameter of 0.8-1.0 μm, no flagellum, no spore;
(2) colony characteristics: the colony is milky white, the edge is neat, spherical, convex and non-transparent, and the surface is moist and smooth;
(3) growth characteristics: the minimum growth temperature of the strain is 15 ℃, the maximum growth temperature is 45 ℃, the strain grows optimally at the temperature of 35-37 ℃, the optimum growth pH is 6.5, and the strain enters a stable period after being cultured for 18 hours;
(4) has good PFOA adsorption capacity in vitro;
(5) lactobacillus casei CCFM1052 can obviously improve spleen atrophy of mice exposed to PFOA;
(6) the lactobacillus casei CCFM1052 can obviously reduce the contents of alanine Aminotransferase (ALT) and gamma-glutamyl transpeptidase (gamma-GT) in the serum of a mouse exposed by PFOA;
(7) the lactobacillus casei CCFM1052 can obviously reduce the activity of superoxide dismutase SOD and the content of malondialdehyde MDA in the liver homogenate of the mice exposed by PFOA;
(8) lactobacillus casei CCFM1052 can obviously reduce the abundance of Allobaculum in the intestinal tract of mice exposed by PFOA, increase the abundance of Clostridiaceae, Adlercreutzia, Bacteroides and Holdmania, improve the intestinal tract disorder caused by PFOA exposure and reduce the occurrence of liver diseases and metabolic diseases.
(9) Lactobacillus casei CCFM1052 can obviously increase the content of acetic acid and butyric acid in the intestinal tract of a mouse exposed by PFOA and improve the metabolic disturbance of intestinal flora caused by PFOA exposure.
(10) Lactobacillus casei CCFM1052 can obviously improve the water content of the feces of constipated mice and the time for the first grain to defecate.
(11) Lactobacillus casei CCFM1052 can obviously improve the proliferation of INS-1 cells and the expression of MafA genes under the action of high sugar, and can relieve PFOA-related diabetes.
The extraction method of the lactobacillus casei CCFM1052 comprises the following steps:
separating and screening lactic acid bacteria:
(l) 1g of fresh faeces of healthy adults were taken. Enriching a sample in a culture medium containing sorbitol MRS for 12h at 35 ℃;
(2) performing gradient dilution on the enriched sample, coating the enriched sample on an MRS solid plate added with 0.02% of olcresol purple, and culturing for 24-48 h;
(3) selecting single bacterial colony with obvious color changing ring and according with the basic shape of the lactobacillus to perform plate streaking purification, and screening and separating the lactobacillus;
(4) and culturing the single colony in a liquid MRS culture solution for 24h, performing gram staining, and selecting gram-positive bacteria for subsequent tests.
(II) preliminary identification of lactobacillus: caldolytic ring assay
(l) Culturing the lactobacillus obtained by screening in the step (I) in a liquid sorbitol MRS culture solution for 24h, and then centrifuging the lmL culture at 8000rpm for 2 min;
(2) washing twice with 0.05M KH2PO4 solution;
(3) resuspending the obtained bacterial sludge, streaking on a solid culture medium of sorbitol MRS-0.75% CaCO3, and culturing for 24 h;
(4) selecting bacterial colonies which are obvious in calcium-dissolving ring, round in convex surface, fine, dense, white and sterile mycelia, and preliminarily determining lactobacillus by observing the bacteria in a rod shape through a microscope after gram staining.
(III) molecular biological identification of lactobacillus casei:
(l) Extracting a single-bacterium genome:
A. culturing the lactic acid bacteria obtained by screening in the step (II) overnight, taking the overnight-cultured bacterial suspension lmL in a 1.5mL centrifuge tube, centrifuging at 10000rpm for 2min, and removing the supernatant to obtain thalli;
B. purging the thallus with lmL sterile water, centrifuging at 10000rpm for 2min, and removing the supernatant to obtain thallus;
C. adding 200 μ LSDS lysate, and water-bathing at 80 deg.C for 30 min;
D. adding 200 μ L of phenol-chloroform solution into the thallus lysate, wherein the phenol-chloroform solution comprises Tris saturated phenol, chloroform and isoamylol at a volume ratio of 25:24:1, mixing, centrifuging at 12000rpm for 5-10min, and collecting 200 μ L of supernatant;
E. adding 400 μ L of glacial ethanol or glacial isopropanol into 200uL of supernatant, standing at-20 deg.C for 1h, centrifuging at 12000rpm for 5-10min, and discarding the supernatant;
F. adding 500 μ L70% (volume percentage) of glacial ethanol, resuspending the precipitate, centrifuging at 12000rpm for 1-3min, and discarding the supernatant;
drying in an oven at G.60 ℃ or naturally airing;
h.50. mu.L ddH2O pellet was re-solubilized for PCR;
(2)16S rDNA PCR
A. bacterial 16S rDNA 50 μ LPCR reaction:
10 × Taq buffer, 5 μ L; dNTP, 5. mu.L; 27F, 0.5 μ L; 1492R, 0.5 μ L; taq enzyme, 0.5. mu.L; template, 0.5 μ L; ddH2O, 38 μ L.
PCR conditions:
95℃5min；95℃10s；55℃30s；72℃30s；step2-430×；72℃5min； 12℃2min；
(3) preparing 1% agarose gel, mixing the PCR product with 10 × loading buffer, loading the sample by 5 μ L, running at 120V for 30min, and performing gel imaging;
(4) and (3) sequencing the PCR product of the 16S rDNA, searching and comparing the obtained sequence result in GeneBank by using BLAST, selecting a newly discovered strain identified as belonging to lactobacillus casei as a sequencing result, and preserving at-80 ℃ for later use.
Example 1: has good PFOA adsorption capacity in vitro;
the lactobacillus casei CCFM1052 is purified and activated by the thallus adsorption, inoculated into an MRS liquid culture medium according to the inoculation amount of 1% (v/v), and cultured for 18h at 37 ℃. Then centrifuging at 8000r/min for 5min to collect thallus, collecting precipitate, cleaning with physiological saline, centrifuging at 8000r/min for 5min, and removing precipitate to obtain viable thallus cell, i.e. wet thallus. The wet cells were resuspended in 10mg/LPFOA solution to a final cell concentration of 1g dry cells/L (the wet cells were resuspended in PFOA-free ultrapure water as a blank control). The pH of the PFOA solution containing the inoculum solution was rapidly adjusted to 3.0 using 0.1M NaOH or HCl solution, and the effect of the ionic strength on PFOA adsorption was negligible by adding a small amount of NaOH or HCl (less than 0.5 ml). Subsequently, a 250ml conical flask containing 100ml of the sample solution was subjected to shake cultivation at 37 ℃ and 150rpm, and after 6 hours, sampling was performed, and 2 parallel experiments were averaged.
Measurement of PFOA adsorption amount: after the adsorption experiment, the sample was centrifuged at 8000r/min for 5min and filtered with a 0.22 μm water membrane, PFOA concentration was measured with UPLC-MS with Waters SYNAPT MS system using an acquisition UPLC BEH c18 column (2.1X 100mm, 1.7 μm, Waters Co.), column temperature 35 ℃ and sample size 1 μ L. Gradient washing was carried out using 100% (v/v) acetonitrile solution (solution A) and 0.1% (v/v) aqueous formic acid solution (solution B) as eluents at a flow rate of 0.3 mL/min.
TABLE 1 gradient elution conditions
Figure RE-GDA0002228731820000091
Mass spectrum conditions: the ionization source is an ESI source; MRM detection; MS + detection; capillary (Capillary); 3.0 kV; conc (vertebral body): 40.00V; source Temperature: 120 ℃; desolvation (Desolvation) temperature: 400 ℃; conc Gas Flow: 50L/h; desolvation Gas Flow: 700L/h, gas flow rate of 0.1 ml/min; proton ratio scan range: 100-; scan time 1s, interval 0.061 s. The results were analyzed with MassLynxV4.1(Waters Corp.); and calculating the PFOA adsorption amount of the lactic acid bacteria according to the concentration difference of the PFOA before and after adsorption. The results of the measurement are shown in FIG. 1.
Example 2: lactobacillus casei CCFM1052 can obviously improve spleen atrophy of mice exposed to PFOA;
50 male C57BL/6J mice at 6 weeks of age were randomized into five groups according to body weight after acclimation for one week: control group, model group, quercetin intervention group, Lactobacillus casei 15-7 intervention group, and LGG intervention group, each group contains 10 mice, and animal grouping and processing method are shown in Table 2
TABLE 2 animal experiment grouping and processing method
Figure RE-GDA0002228731820000092
Figure RE-GDA0002228731820000101
The mice in example 2 were weighed on day 13 and then euthanized, spleens were removed and wet weighed to calculate organ coefficients, and organ coefficients of mouse spleens were calculated according to the following formula:
spleen organ coefficient ═ spleen wet weight/euthanasia mouse body weight
The experimental results are shown in figure 2, and the results show that the spleen atrophy of mice caused by PFOA contamination can be remarkably reversed by taking lactobacillus casei 8-9.
Example 3: lactobacillus casei CCFM1052 can significantly reduce the levels of alanine Aminotransferase (ALT) and gamma-glutamyl transpeptidase (gamma-GT) in the serum of PFOA-exposed mice;
the mice in example 3 were weighed on day 13, then euthanized, and serum was obtained by centrifugation at 3000rpm/min for 15min after orbital bleeding, and the serum was assayed for alanine Aminotransferase (ALT) and gamma-glutamyl transpeptidase (gamma-GT) levels using a fully automated biochemical analyzer. ALT is mainly present in the soluble part of the hepatic cell protoplasm, and the ALT activity is increased to indicate that the hepatic cells are damaged and the permeability of cell membranes is enhanced. The experimental results show (figure 3) that the administration of lactobacillus casei CCFM1052 can significantly reduce the contents of glutamic pyruvic transaminase (ALT) and gamma-glutamyl transpeptidase (gamma-GT) in the serum of mice exposed by PFOA. The lactobacillus casei 8-9 is proved to be capable of obviously relieving the damage of the structure and the function of the liver cell membrane of the mouse caused by PFOA.
Example 4 Lactobacillus casei CCFM1052 can significantly reduce the activity of superoxide dismutase SOD and the content of malondialdehyde MDA in liver homogenate of mice exposed to PFOA
The mice in example 3 were weighed on day 13, followed by euthanasia and liver harvest. Weighing liver tissue with a certain weight on ice, adding precooled normal saline according to the proportion of 1:9, homogenizing the tissue to prepare 10% liver homogenate, and centrifuging at 4000 Xg for 15 minutes to obtain supernatant. The protein concentration content of the liver homogenate is determined according to the requirements of the specification. And calculating the MDA content according to the following formula: MDA content (nmol/mgprot) ═ [ (measured OD value-control OD value)/(standard OD value-blank OD value) ] × standard concentration (nmol/mL) ÷ protein concentration of sample to be measured (mgprot/mL)
Weighing liver tissue with a certain weight on ice, adding precooled normal saline according to the proportion of 1:9, homogenizing the tissue to prepare 10% liver homogenate, and centrifuging at 4000 Xg for 15 minutes to obtain supernatant. The protein concentration content of the liver homogenate is determined according to the requirements of the specification. And calculating the SOD content according to the following formula:
SOD inhibition (%) ═ [ (a control-a control blank) - (a assay-a assay blank) ]/(a control-a control blank)
SOD activity (U/mL) ═ SOD inhibition rate ÷ 50% × reaction system dilution factor × dilution factor before sample test
The experimental result shows (figure 4) that the activity of superoxide dismutase SOD and the content of malondialdehyde MDA in liver homogenate of mice exposed by PFOA can be obviously reduced by taking lactobacillus casei CCFM 1052. Under normal conditions, the generation and elimination of Reactive Oxygen Species (ROS) in cells are effectively controlled by organisms and are in a dynamic balance state, if the cells are unbalanced in oxidative stress, the accumulation of ROS can damage DNA, protein, lipid and the like, and SOD is an important antioxidant enzyme in the bodies. Malondialdehyde MDA is the major product of lipid peroxide degradation, and its high or low content indirectly reflects the severity of free radical attack on body cells. By taking lactobacillus casei CCFM1052, free radical attack on mice exposed to PFOA can be obviously reduced, the oxidative stress level of liver tissues is obviously relieved, and liver injury of the mice exposed to PFOA is reduced.
Example 5: lactobacillus casei CCFM1052 can obviously reduce the abundance of Allobaculum in the intestinal tract of mice exposed by PFOA, increase the abundance of Clostridiaceae, Adlercreutzia and Bacteroides, improve the intestinal disorder caused by PFOA exposure and reduce the occurrence of liver diseases and metabolic diseases.
Fresh feces from the mice on day 12 in example 3 were taken and total DNA was extracted from the feces samples of the mice using the feces kit of MP. The specific operation steps are mainly carried out according to the kit instructions. Using mouse fecal genome as template, upstream primer 520F (5 ' -AYTGGGYDTAAAGNG-3 '), downstream primer 802R (5 ' -TACNV)GGGTATCTAATCC-3') is used as a primer to amplify a 16S rDNA V3-V4 region fragment, and the length of the target fragment is about 247 bp. And after the PCR reaction is finished, performing electrophoresis again on all the PCR samples with the observed target bands, preparing 2.0% agarose gel, performing electrophoresis for 40min under the condition of 120V, and after the gel is run, rapidly cutting the target bands under an ultraviolet lamp. The recovery of the target band Gel was carried out according to the QIAquick Gel Extraction Kit Gel recovery Kit instructions. The DNA concentration of the Sample is detected according to a Qubit DNA3.0 Kit, then a library is constructed according to a TurSeq DNA LT Sample Preparation Kit and the description thereof, and finally the concentration is determined on an Illumina Miseq sequencer according to a MiSeq Regent Kit and the description thereof. After the sequencing is finished, single sequences with the sequence length less than 200bp, primer sequences and non-splicing sequences are removed, and the single sequences are overlapped according to the basic groups>10bp standard splicing sequence without mismatch. Defining the sequence with similarity greater than 97% as a classification Unit (OTU), by RibosolDatabase Project (RDP)
Figure RE-GDA0002228731820000121
Bayessclasifier to determine species. Calculating the alpha-diversity and the beta-diversity of the sample to evaluate the flora diversity of the sample. Wherein α -diversity is characterized by a chao1 index, and the results in figure 5 show that the intestinal flora α diversity is elevated in the model group of mice, indicating that PFOA exposure is accompanied by some degree of intestinal disturbance. The lactobacillus casei CCFM1052 can obviously reduce the alpha diversity of intestinal flora and improve the intestinal disorder condition after being taken. Determining the key role that major intestinal microbial components play in various diseases is of great importance in preventing or reversing dysbiosis and thus preventing or controlling diseases.
Fig. 5 shows that the abundance of alobaculum is increased in the PFOA model group mice intestine, and the administration of lactobacillus casei CCFM1052 can significantly reverse this situation, and the abundance of alobaculum also increases in gulf war disease, and further induces neuroinflammation and gastrointestinal disorders in gulf war disease in conjunction with intestinal leaky gut and systemic endotoxemia-induced TLR4 activation. Previous researches on the increase of the abundance of Allobaculum genus have made it possible to serve as one of the risk indicators of female hepatocellular carcinoma and one of the key variables before host cancer occurs due to carcinogen exposure. Clostridia are the largest group of firmicutes and represent the most diverse group of bacteria in the human microbiota, and clostridia are a group of anaerobic, gram-positive, sporulating, coryneform bacteria. Plays a role in the regulation of the immune balance of the gastrointestinal tract. The decrease of the abundance of the family Clostridium clonedicarboxidae is found in the nonalcoholic fatty liver disease, and the abundance of the family Clostridium clonedicarboxidae in the intestinal tract of the healthy control group is obviously increased in the intestinal tract of the chronic functional constipation patient and the intestinal tract flora of the healthy control group. Therefore, the administration of the cheese milk rod office CCFM1052 reduces the occurrence and development of nonalcoholic fatty liver disease and constipation. Bacteroides (Bacteroides), also known as Bacteroides, is a genus of Bacteroides family, gram-negative, spore-free, obligately anaerobic, microbacterium. Bacteroides normally inhabit the intestinal, oral, upper respiratory and reproductive tracts of humans and animals. Bacteroides are normal flora abundantly present in humans and animals, accounting for approximately 1/4 or more of the intestinal flora of adult individuals. Is a nutritional source of intestinal bacteria; capable of regulating the expression of a variety of host genes, including those involved in nutrient absorption, mucosal barrier enhancement, and angiogenesis; activating a T cell-dependent immune response; affecting the expression of Pan cell protein; limiting colonization of the gastrointestinal tract by pathogens. In the clinical research of metabolic diseases, the metabolic disease parameters are in inverse relation with obesity and related metabolic disease parameters, and the abundance of bacteroides in mice in a high-sugar diet group is obviously lower than that in a normal group in an animal experiment of high-sugar diet. The Adlercreutzia genus is originally found in human feces, can produce short-chain fatty acids, is a microbe of anti-inflammatory bacteria, is reduced in intestinal microbiota of patients with primary sclerosing cholangitis and multiple sclerosis, is obviously improved in hyperglycemia, lipid metabolism dysfunction and inflammation in the process of using traditional Chinese medicines to treat type II diabetes rats, and has remarkably increased abundance, and shows that the Adlercreutzia genus is closely related to relevant indexes of type II diabetes.
Example 6: lactobacillus casei CCFM1052 can increase the content of acetic acid and butyric acid in the intestinal tract of PFOA-exposed mice
Taking fresh excrement of the 12 th day of the mouse in the example 3, and weighing 100mg of the excrement to 2mLEP tubes; adding 500uL saturated NaCl, shaking uniformly (soaking for 30min, shaking and crushing for 3 times by a tissue grinder at 70Hz/30 s); adding 40uL 10% sulfuric acid, and uniformly shaking in a vortex for 30 s; adding 1mL of diethyl ether, uniformly shaking in a vortex, centrifuging for 15min at 18000 Xg, and centrifuging at 4 ℃; after centrifugation, taking the supernatant, transferring the supernatant to a new 2mLEP tube, and adding 0.25g of anhydrous sodium sulfate; 18000 Xg for 15min, and centrifuging at 4 deg.C; 500uL of the supernatant was transferred to a gas vial and placed in the machine.
SCFA provide energy to intestinal mucosal cells, maintain the integrity of the intestinal barrier, regulate inflammatory responses, and inhibit pathogenic proliferation. Acetic acid can maintain the integrity of the epithelium of Caco-2 cells during invasion of the back hemorrhagic Escherichia coli, and can increase the secretion of host antibacterial peptide to play an antibacterial role. And butyric acid is a main energy source of the intestinal epithelial cells, and can not only perform oxidation energy supply on the intestinal epithelial cells, maintain water electrolyte balance, adjust intestinal flora balance, adjust intestinal barrier function and the like. And can play the role of inhibiting the proliferation and differentiation of tumor cells and inducing apoptosis at millimolar concentration, and can activate the expression of a sodium ion/glucose combined carrier gene (SLC5A8) to play the role of inducing apoptosis. In addition, oral administration of butyrate favors the progression of diabetes. The short-chain fatty acid can play an anti-Inflammatory role through two signal pathways of a G protein-coupled receptor (GPCRs) activation pathway and a Histone Deacetylase (HDACs) inhibition pathway, and has a remarkable improvement effect on Inflammatory Bowel Disease (IBD). FIG. 6 shows that the determination of short-chain fatty acids in feces shows that Lactobacillus casei CCFM1052 can obviously increase the content of acetic acid (P <0.05) in the intestinal tracts of mice exposed by PFOA and has a better increasing trend on the content of butyric acid.
Example 7: relieving effect of lactobacillus casei CCFM1052 on constipation of mice
SPF-grade male BALB/c mice 40 (20-25g) were randomly assigned to 5 groups: a blank control group, a constipation model control group, a lactobacillus casei CCFM1052 intervention group, a lactobacillus plantarum control group and a phenolphthalein treatment control group, wherein each group contains 10 mice.
Suspending the freeze-dried powder of Lactobacillus casei CCFM1052 in skimmed milk powder to make the concentration of 4.0 × 109CFU/mL of bacterial suspension. The concentration of the prepared feed for the mice in the intervention group is 4.0 multiplied by 10 every day 14 days before the experiment9CFU/mL lactobacillus casei CCFM1052 skim milk suspension 0.25mL, lactobacillus plantarum group gavage equal amount of L.plantarum ST-III, the other 3 groups gavage equal amount of bacteria-free skim milk. On the 15 th to 17 th days of the test, the negative control group is perfused with 0.25mL of physiological saline, the other four groups are perfused with 0.25mL of loperamide solution with the concentration of 1mg/mL, and the perfusion amount of the mouse loperamide is ensured to be 10 mg/kgBW.
1h after the completion of gavage, the concentration of the prepared mice in the negative control group and the constipation model control group which are gavage skim milk and lactobacillus casei CCFM1052 interference group is 4.0 multiplied by 109CFU/mL lactobacillus casei CCFM10520.25mL, phenolphthalein treatment control group gavage 0.25mL, 7mg/mL phenolphthalein solution, ensuring that the gavage amount of phenolphthalein in mice is 70 mg/kgBW. The plant lactobacillus group is intragastrically administered by 0.25mL, 4.0 multiplied by 109CFU/mL of L.plantarum ST-III.
Mouse feces were collected every day during the experiment and used for calculation of water content of the mouse feces, which was calculated as follows. Stool water content (%) - (stool wet weight-stool dry weight)/stool wet weight x 100. On the 17 th morning, except for a blank control group, the other groups were perfused with stomach saline, and after 1h of perfusion, all mice were perfused with 0.25mL of an activated carbon and gum arabic aqueous solution, and then each mouse was placed in a clean stainless steel cage paved with absorbent paper separately, and the time (min) from the beginning of the perfusion of the activated carbon to the discharge of the first black stool was recorded as the first black stool discharge time for evaluating the effect of lactobacillus casei CCFM1052 on the relief of constipation of the mice, during which the mice had free access to food and water. FIG. 7 is a measurement result of the water content of mouse feces, which shows that the water content of feces in the constipation model group is significantly reduced, while the phenolphthalein treatment group can significantly increase the water content of constipation mouse feces (P <0.001), and the effect of increasing the water content of constipation mouse feces by Lactobacillus casei CCFM1052 (P <0.01) is better than that of the control strain Lactobacillus plantarum ST-III (P < 0.05). The result of the black stool discharging of the first mouse grain shows (figure 8), the black stool discharging time of the first mouse grain is remarkably delayed in the constipation model group, the stool discharging time of the constipation model group is remarkably shortened in the phenolphthalein treatment group (P <0.001), the lactobacillus casei CCFM1052 has the same excellent effect of shortening the black stool discharging time of the first mouse grain as phenolphthalein (P <0.001), the lactobacillus plantarum ST-III also remarkably shortens the black stool discharging time of the first mouse grain (P <0.05), but the effect is obviously inferior to that of the lactobacillus casei CCFM 1052.
Example 8: lactobacillus casei CCFM1052 can promote the proliferation of high-sugar induced INS-1 cells and the expression of Maf A mRNA
The experiments were divided into 5 groups: normal group (common culture broth containing 11.1mmol/L glucose), high sugar
Group (high-sugar culture solution containing 22.2mmol/L glucose), rosiglitazone group (high-sugar culture solution + 80. mu. mol/L rosiglitazone), and CCFM1052 group (high-sugar culture solution + 1X 10. mu.L)9CFU/mL CCFM1052 bacteria liquid) LGG group (high sugar culture solution + containing 1 x109CFU/mL LGG strain).
INS-1 cells (accession No.: BH-AC0530) were cultured in RPMI-1640 medium (containing 11.1mmol/L glucose, 10% FBS, 50. mu. mol/L2-mercaptoethanol, 1mmol/L pyruvic acid, 10mmol/L HEPES), and placed at 37 ℃ in 5% CO2In an incubator.
The CCK-8 method is used for detecting cell proliferation: the well-conditioned cells were digested, centrifuged and plated on 96-well plates, each well at approximately 5X 103Cells, peripheral wells of the plate were not seeded with cells, and a PBS solution was added thereto at the same time to prevent edge effects. When the cells adhere to the wall, RPMI-1640 culture medium containing 0.5% fetal bovine serum is added into each hole, and the synchronous treatment is carried out for 24 h. And after synchronization, adding corresponding culture media into each hole according to groups for culturing for 48h, wherein each group is provided with three multiple holes and a zero setting hole. After the drug intervention, the old culture medium is aspirated, washed with PBS for 2 times, added with 180. mu.L of serum-free culture medium and 20. mu.L of CCK-8 solution, and incubated for 3-4 h. At the end of incubation, the absorbance of each well was measured using a microplate reader at 450 nm.
Determination of Maf a mRNA expression: extracting RNA by Trizol method, removing stock culture solution in 6-well plate, washing with precooled PBS for 2 times, adding 1.0mL Trizol into each well to lyse cells, transferring the lysate containing cells to enzyme-free EP tube, blowing with pipette until no obvious precipitate is formed, standing for 5min. 0.2mL of chloroform was added to each EP tube, shaken vigorously for 15s, and left at room temperature for 2-3 min. Centrifuging at 12000rpm for 15min at 4 deg.C, sucking supernatant about 0.4m L, transferring into another enzyme-free EP tube, adding 0.5mL isopropanol, mixing, and standing at room temperature for 10 min. Centrifuge at 12000rpm for 10min at 4 ℃, carefully discard the supernatant, add 1.0mL 75% ethanol and mix by inversion. Centrifuging at 12000rpm for 5min at 4 deg.C, discarding supernatant, and drying at room temperature for 2-5 min. Add 20. mu.L of treated water to dissolve and store at 80 ℃ until use. The concentration and mass of the RNA were determined and reverse transcription was performed according to the reverse transcription kit instructions. The cDNA obtained by reverse transcription was subjected to q RT-PCR detection with MafA specific primers: 5'-atcactctgcccaccatcac-3' for F and 5'-atgacctcctccttgctgaa-3' for R. The PCR system is as follows: f (10. mu.M), 0.50. mu.L; r (10. mu.M), 0.50. mu.L; c DNA Template, 1.00. mu.L; dd H2O, 3.00 μ L; mix, 5.00. mu.L. PCR procedure: at 95 ℃ for 2 min;
(95 ℃, 30 sec; 60 ℃, 30 sec; 72 ℃, 20 sec.) 35; 72 ℃ for 5 min; after the target gene is detected by Real-timePCR, 2 is adopted-△△CTThe method carries out relative gene expression analysis. CFX Manager software was used to analyze the expression level of the target gene in rat INS-1 cells in each group, and then the expression level in the normal group was 1, and the other groups were compared with each other to calculate the gene expression level in each group.
The CCK-8 method results are shown in FIG. 9, compared with the normal group, the growth of the cells in the hyperglycosylated group is obviously reduced (P <0.05), the cell proliferation of the rosiglitazone control group is obviously increased (P <0.05) compared with the hyperglycosylated group, and the cell proliferation of the CCFM1052 group is also obviously increased (P <0.05) compared with the hyperglycosylated group.
Maf A mRNA expression As shown in FIG. 10, the Maf A mRNA expression level of cells in the hyperglycosylated group was significantly lower than that in the normal group (P <0.05), while the Maf A mRNA expression level of cells in the rosiglitazone positive control group and CCFM1052 group was significantly higher than that in the hyperglycosylated group (P < 0.05).
Example 9: production of fermented food containing Lactobacillus casei CCFM1052
Selecting fresh vegetables, cleaning, squeezing to obtain juice, instantly sterilizing at high temperature, heat sterilizing at 140 deg.C for 2 s, immediately cooling to 37 deg.C, and further sterilizingInoculating the lactobacillus casei CCFM1052 microbial inoculum starter prepared by the invention to ensure that the concentration of the starter reaches 106More than CFU/mL, and storing at 4 ℃ in a refrigeration way, thus obtaining the fruit and vegetable beverage containing the viable bacteria of the lactobacillus casei CCFM 1052.
The invention can be used for preparing other fermented foods by fermenting the lactobacillus casei CCFM1052, wherein the fermented foods comprise solid foods, liquid foods and semi-solid foods. The fermented food comprises dairy products, bean products and fruit and vegetable products, wherein the dairy products comprise milk, sour cream and cheese; the fruit and vegetable products comprise cucumber, carrot, beet, celery and cabbage products.
The lactobacillus casei CCFM1052 can obviously relieve hepatotoxicity and intestinal flora imbalance caused by perfluorooctanoic acid (PFOA) exposure and can obviously relieve constipation, and belongs to the technical field of microorganisms. The lactobacillus casei has high adsorption effect on PFOA in vitro, does not colonize in intestinal tracts, obviously relieves liver oxidative stress injury and serum biochemical indexes caused by the PFOA, obviously improves spleen atrophy caused by PFOA exposure, and obviously improves the imbalance of intestinal microorganisms in intestinal tracts such as Clostridiaceae, Adlercutzia, Allobaculum, Bacteroides and the like caused by the PFOA exposure. Remarkably improves the metabolic disturbance of intestinal flora caused by PFOA exposure, and remarkably improves the content of acetic acid and propionic acid in intestinal tracts. And can obviously improve the water content of the excrement and the first black excrement time of the constipation mice. In addition, lactobacillus casei CCFM1052 can obviously improve the proliferation of INS-1 cells and the expression of MafA genes under the action of high sugar, and has the potential of relieving PFOA related diabetes. The lactobacillus casei CCFM1052 is used for preparing a medicinal composition and a fermented food for relieving PFOA toxicity and constipation, reducing the occurrence of liver diseases, metabolic diseases and potential carcinogenicity, and has wide application prospect.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Claims (10)

1. Lactobacillus casei CCFM1052 with a collection number GDMCC No: 60650.
2. a fermented food product characterized by: the fermented food is prepared by fermenting with Lactobacillus casei CCFM1052, and comprises solid food, liquid food, and semi-solid food.
3. The fermented food product according to claim 2, wherein: the fermented food comprises dairy products, bean products and fruit and vegetable products, wherein the dairy products comprise milk, sour cream and cheese; the fruit and vegetable products comprise cucumber, carrot, beet, celery and cabbage products.
4. Application of lactobacillus casei CCFM1052 in preparing non-colonized probiotics in vivo.
5. Application of lactobacillus casei CCFM1052 in preparing medicines and health products for relieving toxicity of PFOA, preventing and treating constipation, liver disease and antimetabolite diseases.
6. Use according to claim 5, characterized in that: the lactobacillus casei CCFM1052 can adsorb PFOA, improve spleen atrophy caused by PFOA exposure, reduce the content of glutamic pyruvic transaminase (ALT) and gamma-glutamyl transpeptidase (gamma-GT) in serum after PFOA exposure, improve intestinal flora disturbance after PFOA exposure, reduce the abundance of Alobaculum in intestinal tracts, increase the abundance of Clostridiaceae, Adlercutzia, Bacteroides and Holdmania, reduce the occurrence of liver diseases and metabolic diseases, improve the intestinal flora metabolic disturbance caused by PFOA exposure, increase the content of acetic acid and butyric acid in the intestinal tracts, increase the water content of excrement and the first-grain defecation time of constipation patients, and prevent and treat constipation.
7. The use of claim 6, wherein: the lactobacillus casei CCFM1052 can also improve the proliferation of INS-1 cells and the expression of MafA genes under the action of high sugar, and relieve PFOA related diabetes.
8. Use of the fermented food according to claim 2 or 3 for producing a functional food for alleviating PFOA toxicity, preventing and treating constipation, anti-liver diseases and antimetabolites.
9. The use of claim 8, wherein: the lactobacillus casei CCFM1052 can adsorb PFOA, improve spleen atrophy caused by PFOA exposure, reduce the content of glutamic pyruvic transaminase (ALT) and gamma-glutamyl transpeptidase (gamma-GT) in serum after PFOA exposure, improve intestinal flora disturbance after PFOA exposure, reduce the abundance of Alobaculum in intestinal tracts, increase the abundance of Clostridiaceae, Adlercutzia, Bacteroides and Holdmania, reduce the occurrence of liver diseases and metabolic diseases, improve the intestinal flora metabolic disturbance caused by PFOA exposure, increase the content of acetic acid and butyric acid in the intestinal tracts, increase the water content of excrement and the first-grain defecation time of constipation patients, and prevent and treat constipation.
10. The use of claim 9, wherein: the lactobacillus casei CCFM1052 can also improve the proliferation of INS-1 cells and the expression of MafA genes under the action of high sugar, and relieve PFOA related diabetes.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020259564A1 (en) * 2019-06-28 2020-12-30 江南大学 Multifunctional lactobacillus capable of relieving pfoa toxic effects and application thereof
CN113956993A (en) * 2021-04-27 2022-01-21 江南大学 Lactobacillus casei with function of relieving cell oxidative damage
CN113956993B (en) * 2021-04-27 2022-04-29 江南大学 Lactobacillus paracasei with effect of relieving oxidative damage of human keratinocytes

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