CN111117916B

CN111117916B - Pediococcus pentosaceus CCFM1103 for relieving PFOS toxic effects, and fermented food and application thereof

Info

Publication number: CN111117916B
Application number: CN201911386358.1A
Authority: CN
Inventors: 王刚; 陈卫; 孙姗姗; 乌翛冰; 赵建新; 张灏
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2019-12-29
Filing date: 2019-12-29
Publication date: 2021-10-26
Anticipated expiration: 2039-12-29
Also published as: CN111117916A

Abstract

The invention discloses a multifunctional pediococcus pentosaceus CCFM1103 for relieving PFOS toxic action, a fermented food and an application thereof, wherein the CCFM1103 has good adsorption capacity on PFOS in vitro so as to reduce PFOS concentration, has good capacity of removing diphenyl trinitrophenyl hydrazine free radical, and removing hydroxyl radical and reducing capacity, improves hepatomegaly caused by PFOS exposure, reduces the content of TNF-alpha in liver, reduces the content of glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase and alkaline phosphatase in serum, improves the levels of CAT, SOD and antioxidant capacity, improves intestinal flora disorder, reduces the abundance of S24-7 family, lactobacillus, bifidobacterium and parabacteroides in intestinal tract, and reduces the occurrence tendency of diseases such as liver disease, hypertension, diabetes and obesity.

Description

Pediococcus pentosaceus CCFM1103 for relieving PFOS toxic effects, and fermented food and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to pediococcus pentosaceus CCFM1103 for relieving PFOS 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. In addition, PFOS (perfluorooctane sulfonate) is one of the final transformation products of many fluorine compounds, and can directly cause harm to human body through environmental exposure, or enter into animal and plant bodies to accumulate in human body through food chain, so that it is receiving more and more attention from researchers. Although PFOS was regarded as a potential contaminant in the early 60's 20 th century, PFOS was detected in human blood and organisms until the 21 st century, and received extensive attention and research on the related aspects. PFOS toxicity and hazards are increasingly recognized, and thus a number of procedures have progressively limited the production and use of PFOS. However, PFOS is still in heavy use in some countries and residual PFOS 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 PFOS content in blood of the exposed population and possible health effects shows that the PFOS exposure may have a more obvious relationship with the increase of total cholesterol concentration in blood, the increase of blood sugar and blood fat, the increase of the concentration of liver enzymes ALT and ALP and the reduction of body weight. These signs suggest that PFOS 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 PFOS has various toxic effects such as hepatotoxicity, immunotoxicity, reproductive toxicity, developmental toxicity, neurotoxicity, and the like. PFOS may cause hepatomegaly and induce oxidative stress of liver tissues to increase abnormal free radicals, which may be the main cause of liver injury. PFOS exposure causes different degrees of damage to a plurality of immune organs of the immune system of aquatic animals and rodents, causes atrophy and aging of spleen and thymus of the immune organs, and obviously interferes with the expression of splenic interleukins of zebra fish and the apoptosis and the decline of lymphocytes. In an exposure experiment for mice, the high-dose PFOS exposure reduces the proliferation function of T lymphocytes and B lymphocytes of the mice, obviously induces the liver edema degeneration and vacuole formation of the mice, reduces the content of bile acid, and can mediate JAK2/STAT3 channels in astrocytes, cause abnormal TNF-alpha secretion to cause neurotoxicity to interfere with the steady state of estrogen and androgen so as to influence the growth and development.

At present, methods for relieving PFOS toxicity are mostly based on natural chemical substances with high antioxidant activity, such as quercetin, 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 PFOS toxicity without any other possible deleterious 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, in one aspect of the present invention, the present invention overcomes the deficiencies in the prior art and provides a pediococcus pentosaceus CCFM1103 with the deposit number GDMCC No: 60897.

as another aspect of the present invention, the present invention overcomes the disadvantages of the prior art by providing a fermented food product, wherein: the fermented food is prepared by fermenting pediococcus pentosaceus CCFM1103, and the fermented food 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, and 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 pediococcus pentosaceus CCFM1103 in preparing non-colonizing probiotics in vivo.

As another aspect of the invention, the invention overcomes the defects in the prior art and provides the application of pediococcus pentosaceus CCFM1103 in preparing medicines and health products for relieving the toxic action of PFOS, and resisting liver diseases, hypertension, diabetes, obesity and the like.

As a preferred embodiment of the application of the present invention: the pediococcus pentosaceus CCFM1103 has good adsorption capacity on PFOS (Perfluorooctane phosphate) in vitro to reduce PFOS concentration, has good capacity of removing diphenyl trinitrophenyl hydrazine free radical (DPPH), hydroxyl radical removing capacity and reducing capacity, improves liver enlargement caused by PFOS exposure, reduces the content of TNF-alpha in liver, reduces the content of alanine Aminotransferase (ALT), glutamic oxaloacetic transaminase (AST) and alkaline phosphatase (ALP) in serum, improves the levels of CAT, SOD and antioxidant capacity, improves intestinal flora disorder, reduces the abundance of S24-7 family, Lactobacillus (Lactobacillus), Bifidobacterium (Bifidobacterium) and Parabacteroides (Parabacteroides) in intestinal tract, and reduces the incidence tendency of diseases such as liver disease, hypertension, diabetes and obesity.

As another aspect of the invention, the invention overcomes the defects in the prior art and provides the application of the fermented food in functional foods for relieving PFOS toxicity, resisting liver diseases, resisting hypertension, resisting diabetes and resisting obesity.

The invention has the beneficial effects that: the pediococcus pentosaceus CCFM1103 disclosed by the invention has good adsorption capacity on PFOS (Perfluorooctane-dehydrogenase) in vitro so as to reduce the concentration of PFOS (Perfluorooctane-dehydrogenase), has good capacity of removing diphenyl trinitrophenyl hydrazine free radical (DPPH), and removing hydroxyl free radical and reducing capacity, improves hepatomegaly caused by PFOS exposure, reduces the content of TNF-alpha in liver, reduces the content of glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminase (AST) and alkaline phosphatase (ALP) in serum, improves the levels of CAT, SOD and antioxidant capacity, improves intestinal flora disorder, reduces the abundance of S24-7 family, Lactobacillus (Lactobacillus), Bifidobacterium (Bifidobacterium) and Parabacteroides (Parabacteroides) in intestinal tract, and reduces the occurrence tendency of diseases such as liver diseases, hypertension, diabetes and obesity. The pediococcus pentosaceus CCFM1103 can be used for preparing foods, health-care products and medicines for relieving PFOS toxicity, and has very wide application prospects.

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 PFOS after the bacterial strain is resuspended in PFOS with the concentration of 20mg/L in vitro, passes through a 0.22 mu m water-based filter membrane, enters an ultra-high performance liquid chromatography-mass spectrometer after being shaken at 37 ℃ and 200rpm for 12 hours, and is adsorbed.

FIG. 2 shows that the strain of the present invention has the capacity of eliminating diphenyl trinitrophenyl hydrazine free radical (DPPH) and hydroxyl radical eliminating capacity and reducing capacity.

FIG. 3 is a graph showing the change in specific gravity of the liver of mice exposed to PFOS 15 days after the intervention of the strain of the present invention. With the different letter representations having significant differences.

FIG. 4 is a graph showing the levels of ALP, ALT and AST in the serum of mice exposed to PFOS 15 days after the intervention of the strain of the present invention. With the different letter representations having significant differences.

FIG. 5 is a graph showing the changes of CAT, SOD and antioxidant ability in the liver of mice exposed to PFOS 15 days after the intervention of the strain of the present invention. With the different letter representations having significant differences.

FIG. 6 is a diagram showing the tumor necrosis factor alpha in the liver of mice exposed to PFOS 15 days after the intervention of the strain of the present invention. With the different letter representations having significant differences.

FIG. 7 is a schematic diagram of the strain of the invention, which shows that after 15 days of intervention, mice are exposed to PFOS, and the diversity of the intestinal flora alpha of the mice is changed; with the different letter representations having significant differences.

FIG. 8 is a graph showing the change in the abundance of S24-7 family, Lactobacillus (Lactobacillus), Bifidobacterium (Bifidobacterium) and Bacteroides (Parabacteroides) in the intestine of mice exposed to PFOS 15 days after the intervention with the strain of the present invention; with the different letter representations having significant differences.

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 Pediococcus pentosaceus (Pediococcus pentosaceus) CCFM1103 is preserved in Guangdong province microbial strain preservation center in 11 and 22 months in 2019, the preservation address is No. 59 building 5 of Michelia Tokyo 100 of Guangzhou city, the preservation number is GDMCC No: 60897.

the pediococcus pentosaceus has the following biological properties:

(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, round, 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 PFOS adsorption capacity in vitro;

(5) the in vitro has good capacity of removing diphenyl trinitrophenyl hydrazine free radical (DPPH), hydroxyl free radical removing capacity and reducing capacity;

(6) pediococcus pentosaceus CCFM1103 can remarkably improve PFOS-exposed hepatomegaly;

(7) pediococcus pentosaceus CCFM1103 can significantly reduce the levels of ALP, ALT and AST in serum exposed by PFOS;

(8) pediococcus pentosaceus CCFM1103 can significantly improve the levels of CAT, SOD and antioxidant ability in PFOS-exposed liver.

(9) The pediococcus pentosaceus CCFM1103 can obviously reduce the content of TNF-alpha in the liver exposed by PFOS;

(10) the pediococcus pentosaceus CCFM1103 can remarkably reduce the abundance of S24-7 family, Lactobacillus (Lactobacillus), Bifidobacterium (Bifidobacterium) and Parabacteroides (Parabacteroides) in intestinal tracts, improve the intestinal flora disorder caused by PFOS exposure and reduce the incidence tendency of diseases such as liver diseases, hypertension, diabetes, obesity and the like.

The extraction method of the pediococcus pentosaceus CCFM1103 comprises the following steps:

separating and screening lactic acid bacteria:

(l) Collecting several stool samples of healthy adults, and enriching the samples in culture medium containing sorbitol GM17 at 35 ℃ for 12 h;

(2) performing gradient dilution on the enriched sample, then coating the enriched sample on a GM17 solid plate added with 0.02% of olcresol purple, and culturing for 24-48 h;

(3) selecting single bacterial colony with obvious color changing circle and according with the basic morphology of lactobacillus, carrying out plate streaking purification, and screening and separating out lactobacillus;

(4) and culturing the single colony in a liquid GM17 culture solution for 24h, performing gram staining, and selecting gram-positive bacteria for subsequent tests.

(II) preliminary identification of lactic acid bacteria: caldolytic ring assay

(l) Culturing the lactic acid bacteria obtained by screening in the step (I) in a liquid sorbitol GM17 culture solution for 24h, and then centrifuging l mL of culture at 8000rpm for 2 min;

(2) with 0.05M KH₂PO₄Washing the solution twice;

(3) resuspending the resulting pellet and streaking on sorbitol GM 17-0.75% CaCO₃Culturing for 24 hours on the solid culture medium;

(4) selecting bacterial colonies which are obvious in calcium-dissolving ring, round in convex surface, fine, dense and white in color and sterile filaments, and preliminarily determining to be cocci when the bacteria are spherical after gram staining and observed by a microscope.

(III) molecular biological identification of lactic acid bacteria:

(l) Extracting a single-bacterium genome:

A. culturing the lactic acid bacteria obtained by screening in the step (II) overnight, taking l mL of the overnight-cultured bacterial suspension into a 1.5mL centrifuge tube, centrifuging at 10000rpm for 2min, and removing the supernatant to obtain thalli;

B. purging the thalli with l mL of sterile water, centrifuging at 10000rpm for 2min, and removing the supernatant to obtain the thalli;

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.50μLddH₂re-dissolving the precipitate with O 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，38μL。

PCR conditions:

95℃5min；95℃10s；55℃30s；72℃30s；step2-4 30×；72℃5min；12℃2min；

(3) preparing 1% agarose gel, mixing the PCR product with 10000 × 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 similarity of the obtained sequence result in GeneBank by using BLAST, selecting a newly discovered strain identified as belonging to pediococcus pentosaceus as a sequencing result, and preserving at-80 ℃ for later use.

Example 1: pediococcus pentosaceus CCFM1103 has good PFOS adsorption capacity

Thallus adsorption is used for purifying and activating the pediococcus pentosaceus CCFM1103, inoculating the pediococcus pentosaceus CCFM1103 into an MRS liquid culture medium according to the inoculation amount of 1% (v/v), and culturing for 12h 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 a 20mg/L potassium PFOS solution (potassium perfluorooctane sulfonate is used as an example in the present invention) and the final cell concentration was adjusted to 1g dry cells/L (the wet cells were resuspended in ultrapure water containing no PFOS as a blank control). The pH of the aqueous PFOS 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 PFOS 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 culture at 37 ℃ and 200rpm, and after 12 hours, sampling was performed, and 2 parallel experiments were averaged.

Measurement of PFOS adsorption amount: after the adsorption experiment, the sample was centrifuged at 8000r/min for 5min and filtered with a 0.22 μm water membrane, PFOS concentration was measured by 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

t/min	0-0.5	0.5-5.0	5.0-7.0	7.0-7.5
					Ratio of solvent A	70％	70-100％	100％	100-70％

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 PFOS adsorption amount of the lactic acid bacteria according to the concentration difference of the PFOS before and after adsorption. The results are shown in FIG. 1, and the adsorption rate of CCFM1103 to PFOS of 20mg/L is up to 72.3% (67.0% + -5.3%).

Example 2: the pediococcus pentosaceus CCFM1103 has good capacity of removing diphenyl trinitrophenyl hydrazine free radical (DPPH), hydroxyl free radical removing capacity and reducing capacity in vitro;

1mL of Pediococcus pentosaceus CCFM1103 whole cell suspension and 1mL of freshly prepared DPPH absolute ethanol solution (0.2mmol/L) are fully mixed, and then the mixture is subjected to a light-shielding reaction at 37 ℃ for 30 min. DPPH was mixed with PBS (pH7.2) as a control sample, and cultured under the same conditions. After centrifugation at 7000 Xg for 10min, the absorbance was measured at 517nm and the ability of the lactic acid bacteria to scavenge DPPH radicals was calculated according to the following formula:

DPPH radical clearance (%) [1-a517 (sample)/a 517 (control) ] × 100%.

1mL of 1, 10-phenanthroline 1mLPBS (pH7.2), 1mL of Pediococcus pentosaceus CCFM1103 whole cell suspension or 1mLFeSO4 were mixed uniformly (referred to as "mixture 1"). To "mixture 1" was added 1mLH₂O₂The absorbance was measured at 536nm in a water bath at 37 ℃ for 1.5h and expressed as A536 (sample). The whole cell suspension in "mixture 1" was replaced with the same volume of distilled water, cultured under the same conditions and tested, and denoted as A536 (blank). H to "mixture 1" is added₂O₂The cells were cultured under the same conditions and examined by changing to the same volume of distilled water, and designated as A536 (control). The ability of lactic acid bacteria to scavenge hydroxyl radicals is calculated according to the following formula:

hydroxyl radical clearance (%) - [ a536 (sample) -a536 (blank) ]/[ a536 (control) -a536 (blank) ] × 100%

0.5mL of a suspension of intact cells of Pediococcus pentosaceus CCFM1103 was mixed with the same volume of potassium ferricyanide (1%) and PBS buffer (pH6.6), and the mixture was shaken to homogenize the system. Distilled water was mixed with potassium ferricyanide and PBS as a blank control. The mixed system was incubated at 50 ℃ for 20min, rapidly cooled and 0.5mL of 10% trichloroacetic acid was added. After centrifugation at 2000 Xg for 5min, 1mL of the supernatant was mixed with 1mL of 0.1% ferric chloride and reacted for 10 min. The absorbance was then measured at a wavelength of 700nm and Cysteine (Cysteine) was used as a standard for characterizing the reducing power. The results of the experiment are shown in FIG. 2.

Example 3: the pediococcus pentosaceus CCFM1103 can obviously improve 24 male C57BL/6J mice with 6-week-old liver enlargement of PFOS exposed mice, and after being adapted to the environment for one week, the mice are randomly divided into four groups according to the weight: blank control group, model group, quercetin intervention group, pediococcus pentosaceus CCFM1103 intervention group, each group containing 6 mice, animal grouping and treatment method are shown in table 2.

TABLE 2 animal experiment grouping and processing method

The mice in example 3 were weighed on day 31, then euthanized, bled from the eye sockets, and the liver was weighed wet to calculate organ coefficients, which were calculated according to the following formula:

liver organ coefficient ═ liver wet weight (g)/weight of euthanasia mice (100g)

The experimental results are shown in fig. 3, and the results show that the liver enlargement of the mice caused by PFOS exposure can be obviously relieved by taking the pediococcus pentosaceus CCFM 1103.

Example 4: the pediococcus pentosaceus CCFM1103 can obviously reduce the levels of ALT, ALP and AST in serum of mice exposed by PFOS

Blood in example 3 is taken and stood for one hour, then 3500r/min is carried out, centrifugation is carried out for 15min, and serum is taken for testing. And detecting the contents of ALT, ALP and AST in the serum by using a full-automatic biochemical analyzer. ALT, AST, as a non-specific functional enzyme in the hepatocyte cytoplasm and mitochondria, is released into the blood when hepatocytes are disrupted. ALP is normally excreted by the liver and enters the blood stream via the lymphatic channels and the hepatic sinuses when there is damage to the liver. The experimental result shows (figure 4) that the content of ALT, ALP and AST in the blood serum of a mouse exposed by PFOS can be obviously reduced by taking the pediococcus pentosaceus CCFM 1103. The result shows that the damage of the structure and the function of the liver cell membrane of the mouse caused by PFOS can be obviously relieved by taking the pediococcus pentosaceus CCFM 1103.

Example 5: pediococcus pentosaceus CCFM1103 can obviously improve the levels of CAT, SOD and antioxidant capacity in the liver of a PFOS-exposed mouse

The mouse liver in example 3 was taken to prepare a 10% homogenate, the liver was accurately weighed according to the weight (g): volume (ml): 1:9 ratio, 9 volumes of physiological saline was added, mechanical homogenization was performed in an ice water bath, cells were sufficiently disrupted, 5000r/min was performed, centrifugation was performed for 10 minutes, and the supernatant was taken for measurement. The levels of SOD, CAT and antioxidant capacity T-AOC in the liver were determined according to the kit provided by Nanjing's built kit. CAT is mainly present in peroxisomes and is responsible for H₂O₂Reduction of (a) and oxidative protection of unsaturated fatty acids in cell membranes. SOD is important antioxidant enzyme in vivo, and has important scavenging effect on ROS. Antioxidant capacity may indicate the level of oxidative stress the body treats. Experimental results show that (figure 5), the administration of pediococcus pentosaceus CCFM1103 can significantly improve the levels of CAT, SOD and antioxidant capacity in the liver of mice exposed by PFOS. The pediococcus pentosaceus CCFM1103 has an effective improving effect on the liver oxidative stress injury caused by the PFOS.

Example 6: pediococcus pentosaceus CCFM1103 can obviously reduce the content of TNF-alpha in liver of mice exposed by PFOS

The mouse liver in example 3 was taken to prepare a 10% homogenate, the liver was accurately weighed according to the weight (g): volume (ml): 1:9 ratio, 9 times volume of physiological saline was added, mechanical homogenate was performed in ice water bath, cells were sufficiently disrupted, 5000r/min was centrifuged for 10 minutes, and the supernatant liver homogenate was taken for measurement. Cytokine TNF-. alpha.content in the liver cytokine levels were determined according to the ELISA kit (RD) instructions. The experimental result shows that the liver inflammation injury of mice caused by PFOS exposure can be obviously improved by taking the pediococcus pentosaceus CCFM1103 (figure 6).

Example 7: the pediococcus pentosaceus CCFM1103 can remarkably reduce the abundance of S24-7 family, Lactobacillus, Bifidobacterium and Parabacteroides in the intestinal tract of mice exposed by PFOS (Perfluorooctane-associated syndrome), improve the intestinal tract disorder caused by PFOS exposure, and reduce the tendency of diseases such as liver disease, hypertension, diabetes, obesity and the like

Fresh feces from the mice on day 31 of example 3 were taken and total DNA was extracted from the fecal samples of the mice using the feces kit from MP. The specific operation steps are mainly carried out according to the kit instructions. A V3-V4 region fragment of 16S rDNA is amplified by taking a mouse fecal genome as a template and taking an upstream primer 520F (5 '-AYTGGGYDTAAAGNG-3') and a downstream primer 802R (5 '-TACNVGGGTATCTAATCC-3') as primers, and the length of a 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 Ribosomal Database Project (RDP)

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 was characterized by chao1 and the observed species index, the results (figure 7) show an increased intestinal flora α diversity in the model group mice, indicating that PFOS exposure is accompanied by some degree of intestinal disturbance. The alpha diversity of intestinal flora can be obviously reduced by taking the pediococcus pentosaceus CCFM1103, and the intestinal disorder condition can be improved.

The abundances of S24-7 family, Lactobacillus (Lactobacillus), Bifidobacterium (Bifidobacterium) and Bacteroides (Parabacteroides) in feces were increased after PFOS infection, while the abundances of S24-7 family, Lactobacillus (Lactobacillus), Bifidobacterium (Bifidobacterium) and Bacteroides (Parabacteroides) after PFOS infection were significantly decreased by administration of Pediococcus pentosaceus CCFM11103 (FIG. 8); s24-7 is highly localized in the gastrointestinal tract of warm-blooded animals, gram-negative non-motile anaerobic microorganisms, capable of fermenting a wide variety of carbohydrates, and is involved in the development of nonalcoholic fatty liver disease and hypertension. Lactobacillus (Lactobacillus) is a part of normal gastrointestinal tract and genitourinary apparatus, is common probiotics, and has the condition that the abundance of PFOS model group and contamination model is increased in the lactic acid bacteria prevention experiment, and the lactic acid bacteria may have negative feedback regulation after PFOS exposure. Bifidobacteria (bifidobacteria) are widely present in the human and animal habitat, such as the digestive tract, vagina and oral cavity, and one of the important components of the human and animal intestinal flora can be used as probiotics in the fields of food, medicine and feed. Under normal conditions, intestinal microorganisms in the human body form a relatively balanced state. After PFOS contamination, intestinal microbial balance is disrupted, resulting in intestinal flora disturbance, and certain intestinal microorganisms such as clostridium perfringens proliferate excessively in the intestinal tract and produce harmful substances such as ammonia, amines, hydrogen sulfide, skatole, indole, nitrite, bacterial toxins, and the like, thereby further affecting health conditions. At this time, Bifidobacterium (Bifidobacterium) can generate feedback regulation, increase abundance to inhibit the growth of harmful bacteria, resist the infection of foreign pollutants, synthesize vitamins required by human body, promote the absorption of mineral substances by human body, generate organic acids such as acetic acid, propionic acid, butyric acid and lactic acid to stimulate intestinal peristalsis, promote defecation, prevent constipation and intestinal putrefaction, purify intestinal environment, decompose carcinogenic substances, stimulate the immune system of human body, and improve disease resistance. Bacteroides paraBacteroides (Parabacteroides) is one of human core flora, and in vivo and in vitro experiments show that the Bacteroides Parabacteroides (Parabacteroides) has cholic acid conversion function, can also produce a large amount of succinic acid, can activate an intestinal FXR signal path, improve lipid metabolism disorder, repair intestinal walls and activate intestinal gluconeogenesis, thereby regulating appetite, promoting liver glycogen synthesis and improving host glycometabolism disorder. In addition, the research shows that the content of the Parabacteroides paradise (Parabacteroides) is reduced, and the tendency of the body to suffer from diseases such as obesity, non-alcoholic fatty liver disease, diabetes mellitus and the like is increased. The results show that the pediococcus pentosaceus CCFM1103 has the functions of regulating the intestinal flora, regulating the immunity and the intestinal barrier, and reducing the occurrence of liver diseases, hypertension, diabetes and obesity on the basis of relieving the PFOS toxicity.

Example 8: fermented food of the pediococcus pentosaceus CCFM1103 prepared by the method

Selecting fresh vegetables, washing, juicing, carrying out high-temperature instant sterilization, carrying out high-temperature heat sterilization at 140 ℃ for 2 seconds, immediately cooling to 37 ℃, and inoculating the fermentation agent of the pediococcus pentosaceus CCFM1103 microbial inoculum prepared by the invention to ensure that the concentration of the fermentation agent reaches 10⁶The fruit and vegetable beverage containing the viable bacteria of the pediococcus pentosaceus CCFM1103 of the present invention can be obtained by storing the beverage at a temperature of 4 ℃ under refrigeration at a concentration of CFU/mL or more.

The method can be used for producing other fermented foods by fermenting the pediococcus pentosaceus CCFM1103, 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 pediococcus pentosaceus CCFM1103 has a good adsorption effect on PFOS in vitro; the pediococcus pentosaceus CCFM1103 has high antioxidation in the capabilities of scavenging diphenyl trinitrophenyl hydrazine free radical (DPPH), scavenging hydroxyl free radical and reducing; the pediococcus pentosaceus CCFM1103 can remarkably improve the hepatomegaly caused by PFOS exposure; the content of TNF-alpha in the liver after PFOS exposure is obviously reduced; the pediococcus pentosaceus CCFM1103 can remarkably reduce the contents of alanine Aminotransferase (ALT), aspartate Aminotransferase (AST) and alkaline phosphatase (ALP) in serum after PFOS exposure; the pediococcus pentosaceus CCFM1103 increases the CAT and GSH content in the liver and improves the anti-oxidation capacity of the liver; the pediococcus pentosaceus CCFM1103 can improve the intestinal flora disturbance after PFOS exposure, reduce the abundance of S24-7 family, Lactobacillus (Lactobacillus), Bifidobacterium (Bifidobacterium) and Parabacteroides (Parabacteroides) in the intestinal tract, and reduce the incidence tendency of liver disease, constipation, hypertension, diabetes and obesity.

In PFOS model mouse experiments, the liver enlargement of mice caused by PFOS exposure can be obviously improved by taking pediococcus pentosaceus CCFM 1103; the content of TNF-alpha in the liver of a mouse exposed by PFOS can be obviously reduced by taking pediococcus pentosaceus CCFM 1103; taking Pediococcus pentosaceus CCFM1103 to reduce the content of ALT, AST and ALP in the serum of the mice exposed by PFOS; the content of CAT and GSH in the liver is increased by taking Pediococcus pentosaceus CCFM1103, and the antioxidation capability of the liver is improved; the administration of the composition can improve the intestinal flora disorder of PFOS-exposed mice, reduce the abundance of S24-7 family, Lactobacillus (Lactobacillus), Bifidobacterium (Bifidobacterium) and Parabacteroides (Parabacteroides) in intestinal tract, normalize the intestinal flora, and reduce the occurrence of liver disease, constipation, hypertension, diabetes and obesity. In vitro experiments show that pediococcus pentosaceus CCFM1103 can well adsorb PFOS, can effectively remove diphenyl trinitrophenyl hydrazine free radical (DPPH), can remove hydroxyl free radical, and has good reducing capability.

The invention screens out the probiotics which have high adsorption capacity to the PFOS, are not colonized in a human body and have high oxidation resistance, can inhibit the oxidative stress caused by the PFOS, and can fundamentally remove the PFOS in the human body. The pediococcus pentosaceus CCFM1103 can be used for preparing foods, health-care products and medicines for relieving PFOS toxicity, and has a very 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

1. Pediococcus pentosaceus (Pediococcus pentosaceus) CCFM1103 with deposit number GDMCC No: 60897.

2. a fermented food product characterized by: the fermented food is prepared by fermentation production using the CCFM1103 strain of claim 1, and the fermented food includes 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. Use of the CCFM1103 strain of claim 1 for the preparation of non-colonizing probiotic bacteria in vivo.

5. Use of the CCFM1103 strain of claim 1 for the preparation of a medicament for alleviating liver diseases, hypertension, diabetes and obesity caused by PFOS toxicity.

6. The use of claim 5, wherein: the CCFM1103 strain has good adsorption capacity on PFOS in vitro so as to reduce PFOS concentration, has good capacity of removing diphenyl trinitrophenyl hydrazine free radical (DPPH), hydroxyl radical removing capacity and reducing capacity, improves liver swelling caused by PFOS exposure, reduces the content of TNF-alpha in PFOS-exposed liver, reduces the content of glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase and alkaline phosphatase in PFOS-exposed serum, and improves the levels of CAT, SOD and antioxidant capacity in PFOS-exposed liver.

7. The use of claim 6, wherein: the CCFM1103 strain improves the intestinal flora disorder caused by PFOS exposure, reduces the abundance of S24-7 family, Lactobacillus (Lactobacillus), Bifidobacterium (Bifidobacterium) and Parabacteroides (Parabacteroides) in the intestinal tract, and reduces the incidence tendency of liver diseases, hypertension, diabetes and obesity caused by the toxicity of PFOS.

8. Use of the fermented food according to claim 2 or 3 for the preparation of a health food for aiding in the relief of liver diseases, hypertension, diabetes, obesity caused by PFOS toxicity.

9. The use of claim 8, wherein: the CCFM1103 strain has good adsorption capacity on PFOS in vitro so as to reduce PFOS concentration, has good capacity of removing diphenyl trinitrophenyl hydrazine free radical (DPPH), hydroxyl radical removing capacity and reducing capacity, improves liver swelling caused by PFOS exposure, reduces the content of TNF-alpha in PFOS-exposed liver, reduces the content of glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase and alkaline phosphatase in PFOS-exposed serum, and improves the levels of CAT, SOD and antioxidant capacity in PFOS-exposed liver.

10. The use of claim 9, wherein: the CCFM1103 strain improves the intestinal flora disorder caused by PFOS exposure, reduces the abundance of S24-7 family, Lactobacillus (Lactobacillus), Bifidobacterium (Bifidobacterium) and Parabacteroides (Parabacteroides) in the intestinal tract, and reduces the incidence tendency of liver diseases, hypertension, diabetes and obesity caused by the toxicity of PFOS.

11. The use of the CCFM1103 strain of claim 1 for the preparation of a health product for the adjuvant relief of hypertension, diabetes, and obesity caused by PFOS toxicity.