CN108384735A - Lactobacillus plantarum CCFM1019, its fermented food and its application in medicine preparation - Google Patents
Lactobacillus plantarum CCFM1019, its fermented food and its application in medicine preparation Download PDFInfo
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- CN108384735A CN108384735A CN201810213917.8A CN201810213917A CN108384735A CN 108384735 A CN108384735 A CN 108384735A CN 201810213917 A CN201810213917 A CN 201810213917A CN 108384735 A CN108384735 A CN 108384735A
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- Prior art keywords
- dehp
- plasticiser
- metabolite
- lactobacillus plantarum
- ccfm1019
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Classifications
-
- 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
- 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
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/065—Microorganisms
-
- 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
- A61P39/02—Antidotes
-
- 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
- A23V2400/169—Plantarum
-
- 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
- A61K2035/11—Medicinal preparations comprising living procariotic cells
- A61K2035/115—Probiotics
-
- 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
- C12R2001/25—Lactobacillus plantarum
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses lactobacillus plantarum CCFM1019, its fermented food and its applications in medicine preparation.The bacterium is not only significantly better than enteron aisle in terms of the effect of promoting DEHP and MEHP to be discharged and resides flora Escherichia coli and enterococcus faecalis, and is better than commercial strain Lactobacillus rhamnosus LGG.Therefore, lactobacillus plantarum CCFM1019 of the invention can be as the effective means for preventing and alleviating the body injury caused by DEHP and MEHP, and does not have the toxic side effect of drug.Lactobacillus plantarum CCFM1019 can be used for preparing the pharmaceutical composition and fermented food for alleviating, preventing DEHP and its metabolite toxicity, have very extensive application prospect.
Description
Technical field
The invention belongs to microorganisms technical fields, and in particular to lactobacillus plantarum CCFM1019, its fermented food and its
Prepare the application in drug.
Background technology
(2- ethylhexyls) ester of phthalic acid two (hereinafter referred to as DEHP) is used as a kind of plasticiser, addition in the plastic may be used
Increase its flexibility and plasticity.Because cheap, DEHP is widely used in the plastics such as production medical devices, chemical products
Product is China's most popular plasticiser at present.In these plastic products, DEHP mainly with hydrogen bond and Van der Waals force and
The form of non-covalent bond is combined with other molecules, therefore is easily escaped during use, to move to environment even people
In body, harm is generated to animals and plants and health.
Studies have shown that DEHP lasts about 4~8h in people's body absorption and distribution phase, it largely can be interior for 24 hours complete by human body
Full metabolism.Urine is the main discharge approach of DEHP.DEHP enter in vivo for 24 hours after, less than 10% DEHP stostes directly through urine
Liquid is discharged, and about 67% DEHP is converted into five kinds of secondary metabolic products and is discharged through urine.Since DEHP has fat-soluble, minority
DEHP is then stranded in fat or milk.The DEHP being stored in adipose tissue cannot be metabolized completely for a long time, long half time
Up to 156h.
DEHP is mainly sent out the toxic effect of body by its metabolite phthalic acid list (2- ethylhexyls) ester MEHP
It waves.The DEHP and its metabolite MEHP accumulated in vivo can generate a variety of toxicity, including genotoxicity, hepatotoxicity, embryo
Toxicity, toxicity of thyroid, neurotoxicity etc..Since DEHP is a kind of agent for peroxisome proliferator, can break intracellular
Redox equilibrium makes the Free Radical Levels such as intracellular ROS increase.In addition, DEHP and its metabolite can also be anti-by influencing
The expression of oxidoreductase gene causes ROS accumulation to cause lipid peroxidation, generates the lipid peroxide such as malonaldehyde, cause oxygen
Change damage.At present mainly by taking some such as vitamin C, vitamin E, Proflavanol, flavonoids etc. are turned into antioxygen
Bioactive substance prevents and alleviates damages of the DEHP to human body.However, to be alleviated by supplement activity substance
The toxic effect of DEHP needs larger dosage, and excess ingestion active material can also cause body to generate side effect, such as
The intake of excessive Flavonoid substances can then cause human endocrine disorderly.And above method can not reduce in body intake and
Remaining plasticiser.Therefore, a kind of novel, safe prevention and treatment method of searching seems particularly necessary.
In the document having disclosed at present, has no and the patent that plasticiser is poisoned is alleviated by probiotics.According to existing text
Report is offered, probiotics is only in vitro 9.62% to DEHP highest adsorption rates, and adsorption effect is unsatisfactory.Therefore one kind is developed
Have the probiotics of stronger adsorption capacity to plasticiser, and proves that it there can be good alleviation in vivo by zoopery
The effect of plasticiser toxic damages is particularly important.
Invention content
The purpose of this part is to summarize some aspects of the embodiment of the present invention and briefly introduce some preferably to implement
Example.It may do a little simplified or be omitted to avoid our department is made in this section and the description of the application and the title of the invention
Point, the purpose of abstract of description and denomination of invention it is fuzzy, and this simplification or omit and cannot be used for limiting the scope of the invention.
In view of above-mentioned technological deficiency, it is proposed that the present invention.
Therefore, as one aspect of the present invention, the present invention overcomes the deficiencies in the prior art, provides a plant
Object lactobacillus CCFM1019, (Lactobacilus plantarum) were preserved in Guangdong Province's microbial bacteria on 2 11st, 2018
Kind collection, preservation address are 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100 Guangdong Microbes Inst, preservation
Number is GDMCC No.60333.
As another aspect of the invention, the present invention overcomes the deficiencies in the prior art, provides a kind of fermented food.
In order to solve the above technical problems, the present invention provides following technical solutions：The fermented food is to use
CCFM1019 fermenting and producings are made, and the fermented food includes solid-state food, food liquid, semisolid food.
As a kind of preferred embodiment of fermented food of the present invention, wherein：The fermented food includes dairy products, beans
Product, fruit and vegetable product, the dairy products include milk, sour cream, cheese；The fruit and vegetable product include cucumber, carrot, beet,
Celery, cabbage product.
As another aspect of the invention, the present invention overcomes the deficiencies in the prior art, provides lactobacillus plantarum
Applications of the CCFM1019 in preparing field planting probiotics in vivo.
As another aspect of the invention, the present invention overcomes the deficiencies in the prior art, provides lactobacillus plantarum
Applications of the CCFM1019 in the drug for preparing the discharge for promoting internal plasticiser and its metabolite.
The row for promoting internal plasticiser and its metabolite is being prepared as lactobacillus plantarum CCFM1019 of the present invention
A kind of preferred embodiment of application in the drug gone out, wherein：The lactobacillus plantarum CCFM1019 can stomach juice-resistant, bile tolerance,
Promote plasticiser and its metabolite from internal discharge, reduce content in serum of plasticiser and its metabolite, significantly
Mitigate the damage of plasticiser and its metabolite to testis tissue and liver organization.
The row for promoting internal plasticiser and its metabolite is being prepared as lactobacillus plantarum CCFM1019 of the present invention
A kind of preferred embodiment of application in the drug gone out, wherein：The plasticiser includes phthalic acid two (2- ethylhexyls) ester
With phthalic acid list (2- ethylhexyls) ester.
As another aspect of the invention, the present invention overcomes the deficiencies in the prior art, provides Claims 2 or 3
Application of the fermented food in the functional food for preparing the discharge for promoting internal plasticiser and its metabolite.
As fermented food of the present invention in the functionality for preparing the discharge for promoting internal plasticiser and its metabolite
A kind of preferred embodiment of application in food, wherein：The fermented food can stomach juice-resistant, bile tolerance, promote plasticiser and its
Metabolite from internal discharge, reduce content in serum of plasticiser and its metabolite, significantly mitigate plasticiser and its
Damage of the metabolite to testis tissue and liver organization.
Beneficial effects of the present invention：The lactobacillus plantarum CCFM1019 of the present invention has good stomach juice-resistant bile tolerance special
Property, plasticiser DEHP and its metabolite MEHP can be promoted to reduce its content in serum from internal discharge, significantly subtract
Its light detrimental effect to testis tissue and liver organization.
(being respectively 52.22% and 70.54%) is not only significantly better than in terms of the bacterium adsorbs DEHP and MEHP effects in vitro
Enteron aisle resides flora Escherichia coli (DEHP:5.86%, MEHP:And enterococcus faecalis (DEHP 0.28%):1.75%, MEHP：
0.17%), and it is better than commercial strain Lactobacillus rhamnosus LGG (DEHP:13.75%, MEHP:15.60%).In rat body,
DEHP and MEHP contents in serum (comparison blank control group reduces by 54.3% and 55.7% respectively) can be significantly reduced, are improved simultaneously
Contents of the DEHP and MEHP in excrement (34.18% and 93.88% has been respectively increased in comparison blank control group).Therefore, this hair
Bright lactobacillus plantarum CCFM1019 can as prevent and alleviate caused by DEHP and MEHP body injury effective means,
And the toxic side effect without drug.Lactobacillus plantarum CCFM1019, which can be used for preparing, to be alleviated, prevents DEHP and its metabolite
The pharmaceutical composition and fermented food of toxicity have very extensive application prospect.
Description of the drawings
In order to illustrate the technical solution of the embodiments of the present invention more clearly, required use in being described below to embodiment
Attached drawing be briefly described, it should be apparent that, drawings in the following description are only some embodiments of the invention, for this
For the those of ordinary skill of field, without having to pay creative labor, it can also be obtained according to these attached drawings other
Attached drawing.Wherein：
Fig. 1 is this patent bacterial strain and control strain Lactobacilus rhamnosus GG (LGG), Escherichia
Coli and Enterococcus faecalis are in vitro to the comparison schematic diagram of the adsorption capacity of DEHP and MEHP；
Fig. 2 is influence schematic diagram of this patent bacterial strain to DEHP and MEHP contents in serum and excrement；
Fig. 3 is improvement situation schematic diagram of this patent bacterial strain to testosterone levels in DEHP exposure model rat blood serums；
Fig. 4 is influence schematic diagram of this patent bacterial strain to malonaldehyde (MDA) content in DEHP exposure model rat testicles；
Fig. 5 is improvement situation schematic diagram of this patent bacterial strain to DEHP exposure model rat testicle site tissue damages；
Fig. 6 is this patent bacterial strain to ALP in DEHP exposure model rat blood serums, the influence schematic diagram of AST and ALT contents.
Specific implementation mode
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, with reference to specific embodiment pair
The specific implementation mode of the present invention is described in detail.
Many details are elaborated in the following description to facilitate a thorough understanding of the present invention, still the present invention can be with
Implemented different from other manner described here using other, those skilled in the art can be without prejudice to intension of the present invention
In the case of do similar popularization, therefore the present invention is not limited by following public specific embodiment.
Secondly, " one embodiment " or " embodiment " referred to herein refers to that may be included at least one realization side of the present invention
A particular feature, structure, or characteristic in formula." in one embodiment " that different places occur in the present specification not refers both to
The same embodiment, nor the individual or selective embodiment mutually exclusive with other embodiment.
The lactobacillus plantarum CCFM1019 (Lactobacilus plantarum) of the present invention was protected on 2 11st, 2018
It is hidden in Guangdong Province's Culture Collection, preservation address is 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100 Guangdong Province
Institute of microbiology, deposit number are GDMCC No.60333.
The lactobacillus plantarum CCFM1019 has following biological characteristics：
(1) thalline feature：In Gram-positive, there is acid resistance, grown under pH 3.0-7.2 environmental conditions good
It is good, do not form spore.Thalline about (0.9-1.2) μ m (3.0-8.0) μm, nose circle straight-bar bacterium, single, pairs of or short chain shape lead to
Often lack flagellum but can move；
(2) colony characteristics：Apparent bacterium colony is formed on MRS culture mediums, diameter is round between 0.3-3.0mm, convex surface
Or lenticular, it is fine and closely woven, color is white, it is smooth to muciform soft-surface, does not form mycelium；
(3) growth characteristics：The bacterium is facultative anaerobic bacteria, and optimum growth temperature is 36-38 DEG C, 32-38 DEG C of well-grown, 15
It DEG C can also grow.Most suitable initial pH is 6-7.The well-grown in the culture solution containing glucose；
(4) there is preferable tolerance to manual simulation's gastro-intestinal Fluid；
(5) damage of DEHP and metabolite MEHP to testis can be alleviated；
(6) it is horizontal that DEHP and MEHP in excrement can be dramatically increased；
(7) it is horizontal to can significantly reduce DEHP and MEHP in serum, reduces accumulation of the DEHP and MEHP in body；
(8) glutamic-pyruvic transaminase ALT in serum (glutamic-pyruvic transaminase), millet straw can be significantly improved
Transaminase AST (glutamic oxalacetic transaminase) and alkaline phosphatase (alkaline
Phosphatase) horizontal.
The extracting method of this bacterial strain is：
(1) separation screening of lactic acid bacteria
(l) the pickles sample for originating from different regions is collected, sample is enriched with 12h in containing sorbierite MRS culture mediums；
(2) enriched sample be coated on after gradient dilution being added to 0.02% and smelling on the MRS solid plates of cresol-purple,
Cultivate 24-48h；
(3) selection change chromosphere is apparent, and the single bacterium colony for meeting lactic acid bacteria grown form carries out plate streaking purifying, screening
Isolate lactic acid bacteria；
(4) above-mentioned single bacterium colony is incubated to cultivate in liquid MRS culture solutions and carries out Gram's staining afterwards for 24 hours, it is blue to choose leather
Family name's positive bacteria carries out follow-up test.
(2) Preliminary Identification of lactobacillus plantarum：Molten calcium circle measuring method
(l) lactic acid bacteria that step (1) is screened is cultivated for 24 hours in liquid sorbitol MRS culture solutions, then takes l
8000 × g of mL cultures centrifuges 2min；
(2) 0.05M KH are used2PO4Solution washes twice；
(3) gained bacterium mud is resuspended, is crossed in sorbierite MRS-0.75%CaCO3Solid medium on, culture for 24 hours；
(4) it is apparent to choose molten calcium circle, and in convex, fine and closely woven color it is white, without mycelial bacterium colony, shown after Gram's staining
It is lactobacillus that micro mirror observation thalline, which is rod-shaped i.e. preliminary judgement,.
(3) molecular biology identification of lactobacillus plantarum
(l) single bacterium genome extracts
A. lactic acid bacteria overnight incubation step (2) screened, take the bacteria suspension lmL of overnight incubation in 1.5mL from
Heart pipe, 10000 × g centrifuge 2min, abandon supernatant and obtain thalline；
B. after using l mL sterile waters purging thalline, 10000 × g centrifuges 2min, abandons supernatant and obtains thalline；
C. 200 μ L SDS lysates, 80 DEG C of water-bath 30min are added；
D. 200 μ L of phenol-chloroform solution are added in cellular lysate liquid, wherein the constituent and volume of phenol-chloroform solution
Than for Tris saturated phenols:Chloroform:Isoamyl alcohol=25:24:1, after overturning mixing, 12000 × g centrifuges 5-10min, takes 200 μ of supernatant
L；
E. 400 μ L ice ethyl alcohol or ice isopropanol is added in 200 μ L supernatants, ﹣ 20 DEG C of standings 1h, 12000 × g centrifuge 5-
10min abandons supernatant；
F. 500 μ L 70% (percentage by volume) ice ethyl alcohol are added, precipitation is resuspended, 12000 × g centrifuges 1-3min, abandons supernatant；
G.60 DEG C baking oven drying or naturally dry；
H.50μLddH2The molten precipitation of O weights is in case PCR；
(2)16S rDNA PCR
A. 50 μ LPCR reaction systems of bacterial 16 S rDNA：
10 × Taq buffer, 5 μ L；DNTP, 5 μ L；27F, 0.5 μ L；1492R, 0.5 μ L；Taq enzyme, 0.5 μ L；Template,
0.5μL；ddH2O, 38 μ L.
B.PCR conditions：
95℃5min；95℃10s；55℃30s；72℃30s；step2-4 30×；72℃5min；12℃2min；
(3) 1% Ago-Gel is prepared, is later mixed PCR product with 10 × loading buffer, 5 μ L of applied sample amount,
120V runs 30min, then carries out gel imaging；
(4) PCR product of 16S rDNA is subjected to sequencing analysis, obtained sequence results is existed using BLAST
It scans for comparing with similitude in GeneBank, chooses sequencing result and be accredited as a kind of new lactic acid for belonging to Lactococcus lactis
Zoopagales bacterial strain, -80 DEG C of preservations are spare.
Embodiment 1：Lactobacillus plantarum CCFM1019 has good tolerance to simulation gastro-intestinal Fluid
By the lactobacillus plantarum CCFM1019 streak inoculations of freezen protective in MRS solid mediums, have for 37 DEG C in temperature
Oxygen stationary culture for 24 hours, then after MRS culture solutions secondary culture 2~3 times, takes lactobacillus plantarum CCFM1019 culture solutions, 8000 ×
G centrifuges 5min and collects thalline, is resuspended in (1:1) artificial simulation gastric juices (MRS containing 1% pepsin, pH=2.5 of pH 2.5
Culture medium) mixing, the then aerobic culture at 37 DEG C is sampled in beginning (0h), 1h, 2h and 3h respectively, with MRS agar cultures
Base cast culture carries out plate count, measures viable count and calculates its survival rate.Survival rate is the work in the culture solution
Bacterium number and the ratio between viable count in 0h, are indicated with %.
It takes the culture solution of lactobacillus plantarum CCFM1019,8000 × g to centrifuge 5min and collect thalline, is resuspended in (1:1) artificial
In simulated intestinal fluid (the MRS culture mediums containing 0.3% bovine bile, 1% trypsase, pH=8.0), the aerobic culture at 37 DEG C, point
It is not sampled in 0h, 1h, 2h, 3h and 4h, carries out plate count with the cast culture of MRS agar mediums, measure viable count
And calculate its survival rate.Survival rate is viable count when being sampled in the culture solution and the ratio between viable count in 0h, with % tables
Show.Experimental result is as shown in table 1, table 2, it can be seen that lactobacillus plantarum CCFM1019 has artificial stomach, intestinal juice good resistance to
By property.
Tolerances of the 1 lactobacillus plantarum CCFM1019 of table in artificial simulation gastric juices
Tolerances of the 2 lactobacillus plantarum CCFM1019 of table in artificial simulation intestinal juice
Embodiment 2：Lactobacillus plantarum CCFM1019 in vitro in DEHP containing plasticiser or MEHP aqueous solutions to DEHP and
MEHP has good adsorption capacity
Thalline adsorbs：After experimental bacteria is carried out purifying and activation culture, according to the access MRS liquid training of 1% (v/v) inoculum concentration
Base is supported, 20h is cultivated in 37 DEG C, and (E.coli is based on 37 DEG C of shake cultures using LB cultures；E.faecalis uses MRS Liquid Cultures
Cultivated based on 37 DEG C), 20min then is centrifuged under the conditions of 8000 × g, outwells supernatant, is continued after being used in combination ultra-pure water to be resuspended
20min is centrifuged under the conditions of 8000 × g, outwells supernatant up to viable bacteria body cell, i.e. wet thallus.Wet thallus is resuspended in 50mg/L
In DEHP 10mg/L MEHP aqueous solutions, and final cell concentration is made to reach 1g wet thallus/L, blank control is wet thallus weight
It is suspended from the ultra-pure water without DEHP and MEHP.Every group of volume is 1mL.By mixed liquor in 37 DEG C of incubated under agitation 4h, then in
3500 × g centrifuges 10min, after collecting supernatant and crossing 0.22 μm of filter membrane, measures DEHP or MEHP in filtrate with UPLC-MS and contains
Amount, 3 parallel laboratory tests are averaged.
DEHP and MEHP adsorbances measure：It is measured using the UPLC-MS of Waters EYNAPT MS systems remaining in filtrate
DEHP or MEHP content, C18 columns (2.1 × 100mm, 1.7 μm, Waters Co.), 35 DEG C of column temperature, sample size be 1 μ L.A,
B eluents are respectively 100% methanol and 0.1% (v/v) aqueous formic acid, gradient elution, flow velocity 0.3mL/min.Gradient is washed
De- condition is as shown in table 3.
3 condition of gradient elution of table
| t/min | 0-0.5 | 0.5-7.0 | 7.0-7.5 | 7.5-10.0 |
| Eluent A ratios | 60% | 60-100% | 100-60% | 60% |
Mass Spectrometry Conditions：Ionization source is the sources ESI；MRM detects (DEHP:MS+；MEHP:MS-)；Capillary (capillary):
3.0KV；Conc (centrum):40.00V；Source Temperature (radiation source temperature)：120℃；Desolvation (is gone molten
Agent) temperature：400℃；Conc Gas Flow:50L/h；Desolvation Gas Flow:700L/h.Gas flow rate is
0.1mL/min；Mass-to-charge ratio scanning range：100-2000；Surface sweeping time 1s is spaced 0.061s.As a result MassLynxV4.1 is used
(Waters companies) analyzes；During this investigation it turned out, the minimum detection limit of DEHP and MEHP is respectively 0.05ppm and 0.1ppm.Root
The adsorption rate of lactic acid bacteria is calculated according to the front and back DEHP MEHP concentration differences of absorption, adsorption rate clicks formula calculating：
Adsorption rate (%)=[(content of plasticiser in content-ultra-pure water of plasticiser in the preceding aqueous solution of absorption)-(absorption
Afterwards in supernatant in content-blank control of plasticiser supernatant plasticiser content)]/(plasticiser in aqueous solution before absorption
The content of plasticiser in content-ultra-pure water) × 100.
These measurement results are listed in attached drawing 1, from Fig. 1 it will be evident that with business bacterium LGG (DEHP:13.75%,
MEHP:And enteron aisle resident bacteria E.coli (DEHP 15.60%):5.86%, MEHP:And E.faecalis (DEHP 0.28%):
1.75 MEHP:0.17%) it compares, adsorption rate (DEHPs of the lactobacillus plantarum CCFM1019 of the present invention to DEHP and MEHP:
52.22%, MEHP:70.54%) control bacterium, and the absorption for the DEHP 9.62% significantly larger than having been reported are all remarkably higher than
Rate.Therefore, lactobacillus plantarum CCFM1019 has good adsorption capacity to DEHP and MEHP.
Embodiment 3：Lactobacillus plantarum CCFM1019 is to SD rats without acute toxic side effect
Lactobacillus plantarum CCFM1019 is resuspended in 2% (w/v) sucrose solution, cell density is 1.0 × 109CFU/
mL.The healthy male SD rat 10 for taking weight 100g or so gives concentration suspension gavage 2mL daily, observes one week, record
Dead and body weights.
These test results are listed in Table 4 below.The result shows that feeding concentration 1.0 × 109The lactobacillus plantarum of CFU/mL
CCFM1019 does not cause to significantly affect to rat, and weight is generated without significant changes, the no phenomena of mortality.Rat appearance is without apparent pathology
Symptom.
The variation of 4 rat body weight of table and death condition
Note：-：Rat is without death
Embodiment 4：Lactobacillus plantarum CCFM1019 is to DEHP and MEHP content shadows in DEHP exposed Rats serum and excrement
It rings
The healthy male SD rat 18 for taking weight 100g or so, is randomly divided into 3 groups：Blank control group (control),
DEHP exposure models group (DEHP), lactobacillus plantarum CCFM1019 intervention groups (DEHP+CCFM1018), every group contains rat 6.Examination
It tests the 1-7 days, blank control group and daily 2% (w/v) sucrose solutions of gavage 2mL of DEHP exposure model groups, lactobacillus plantarum
CCFM1019 intervention group rats feed a concentration of 1.0 × 10 prepared by this specification embodiment 39The lactobacillus plantarum of CFU/mL
CCFM1019 suspensions 2mL.8th day, the drinking-water of blank control group is changed to containing 0.05% (m/V) sucrose-fatty aqueous solution of ester；
DEHP is dissolved in 0.05% (m/V) sucrose-fatty aqueous solution of ester, other press 3000mg/kg weight day in addition to blank group
Poisoning dosage carry out drinking-water contamination, continue during contamination probiotics and control 2% (w/v) sucrose solution gavage.Continuously
After four weeks of contaminating, collects excrement and by animal euthanasia, collect testis and serum, measure DEHP and MEHP in serum and excrement
Content, the measurement result are listed in attached drawing 2.* indicate that there is conspicuousness compared with the DEHP contents in control group；* P ＜
0.05, * * P ＜ 0.01, * * * P<0.005, * * * * P<0.001；#P<0.05, ##P<0.01, ###P<0.005, ####P<
0.001。
The experimental results showed that after lactobacillus plantarum CCFM1019 intervenes, DEHP and MEHP contents in serum can be significantly reduced
(comparison blank control group reduces by 54.3% and 55.7% respectively), while improving the content (comparison of DEHP and MEHP in excrement
Blank control group has been respectively increased 34.18% and 93.88%), it can be seen that lactobacillus plantarum CCFM1019 can be effectively facilitated
Discharges of the DEHP and metabolite MEHP from body.
Embodiment 5：Mitigations of the lactobacillus plantarum CCFM1019 to DEHP exposed Rats genotoxicities
The serum that Example 4 obtains measures testosterone concentration in serum according to method shown in ELISA kit.As a result such as
Shown in Fig. 3.In Fig. 3, * is indicated compared in control group, * P ＜ 0.05, * * P ＜ 0.01.
The testis tissue obtained in embodiment 4 is weighed, by 1：Physiological saline is added in 9 (m/m) ratios, in organizing in Syrup-homogenizing instrument
It is broken, 10% testis tissue homogenate is obtained, it is horizontal to measure malonaldehyde (malonaldehyde, MDA) in homogenate.Measure knot
Fruit is listed in attached drawing 4.* indicates there is conspicuousness compared with the control group in Fig. 4, and # indicates to have compared with DEHP model groups notable
Property；* P ＜ 0.05, #P ＜ 0.05.
It takes testis tissue to carry out paraffin section operation and carries out conventional H dyeing.Coloration result is as shown in Figure 5.
By in testosterone concentration, testis in DEHP exposure models group and lactobacillus plantarum CCFM1019 intervention group serum
The comparison of MDA levels and testis disease marker finds that lactobacillus plantarum CCFM1019 of the present invention can be alleviated and is led by DEHP intakes
Exception (the DEHP exposure model groups of testosterone concentration in the rat blood serum of cause：19.65mmol/L CCFM1019 intervention groups：
35.99mmol/L), content (the DEHP exposure model groups of MDA in testis tissue are reduced：50.28nmol/mg CCFM1019 intervenes
Group：29.32nmol/mg), alleviate testis tissue damage, Reproductive Damage caused by being taken in DEHP plays significant alleviate and makees
With.
Embodiment 6：Improvement of the lactobacillus plantarum CCFM1019 to hepar damnification index in DEHP exposed Rats serum
Serum includes glutamic-pyruvic transaminase ALT, glutamic-oxalacetic transaminease for measuring the blood parameters of rat in Example 4
AST and alkaline phosphatase.The results are shown in Figure 6, and * indicates there is conspicuousness compared in control group in Fig. 6；* P ＜ 0.05.
It is compared with lactobacillus plantarum CCFM1019 intervention group serological index by DEHP exposure models group, as can be seen from Figure 6, this hair
AST, ALT and ALP Indexes Abnormality caused by DEHP takes in can be effectively relieved in bright lactobacillus plantarum CCFM1019.AST is in sky
The content of white control group, DEHP exposure models group and lactobacillus plantarum CCFM1019 intervention groups is respectively 58.35U/L,
124.58U/L, 71.45U/L；ALP in blank control group, DEHP exposure models group and lactobacillus plantarum CCFM1019 intervention groups
Content is respectively 174.25U/L, 252.60U/L, 205.25U/L；ALT is in blank control group, DEHP exposure models group and plant
The content of lactobacillus CCFM1019 intervention groups is respectively 20.00U/L, 36.74U/L, 28.63U/L.
Embodiment 7：The beverage made of fruits or vegetables containing the bacterium is manufactured using lactobacillus plantarum CCFM1019 of the present invention
It squeezes the juice after selecting fresh vegetables to clean, then carries out flash pasteurization, the high temperature thermal sterilization 2 at 140 DEG C of temperature
After second, it is cooled to 37 DEG C immediately, then accesses lactobacillus plantarum CCFM1019 bacteria fermentation agent prepared by the present invention, its concentration is made to reach
To 106CFU/mL or more, it is stored refrigerated at 4 DEG C of temperature, it then obtains containing lactobacillus plantarum CCFM1019 viable bacterias of the present invention
Beverage made of fruits or vegetables.
Other fermented foods, the fermentation can be prepared using the present invention using lactobacillus plantarum CCFM1019 fermenting and producings
Food includes solid-state food, food liquid, semisolid food.The fermented food includes dairy products, bean product, fruit and vegetable product,
The dairy products include milk, sour cream, cheese；The fruit and vegetable product includes cucumber, carrot, beet, celery, cabbage system
Product.The lactobacillus plantarum CCFM1019 can stomach juice-resistant, bile tolerance, promote plasticiser and its metabolite from internal discharge,
Reduce content in serum of plasticiser and its metabolite, significantly mitigate plasticiser and its metabolite to testis tissue and
The damage of liver organization.
The lactobacillus plantarum CCFM1019 of the present invention has good stomach juice-resistant bile tolerance characteristic, can promote plasticiser
DEHP and its metabolite MEHP reduces its content in serum from internal discharge, significantly mitigate its to testis tissue and
The detrimental effect of liver organization.
(being respectively 52.22% and 70.54%) is not only significantly better than in terms of the bacterium adsorbs DEHP and MEHP effects in vitro
Enteron aisle resides flora Escherichia coli (DEHP:5.86%, MEHP:And enterococcus faecalis (DEHP 0.28%):1.75%, MEHP：
0.17%), and it is better than commercial strain Lactobacillus rhamnosus LGG (DEHP:13.75%, MEHP:15.60%).In rat body,
DEHP and MEHP contents in serum (comparison blank control group reduces by 54.3% and 55.7% respectively) can be significantly reduced, are improved simultaneously
Contents of the DEHP and MEHP in excrement (34.18% and 93.88% has been respectively increased in comparison blank control group).Therefore, this hair
Bright lactobacillus plantarum CCFM1019 can as prevent and alleviate caused by DEHP and MEHP body injury effective means,
And the toxic side effect without drug.Lactobacillus plantarum CCFM1019, which can be used for preparing, to be alleviated, prevents DEHP and its metabolite
The pharmaceutical composition and fermented food of toxicity have very extensive application prospect.
It should be noted that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although with reference to preferable
Embodiment describes the invention in detail, it will be understood by those of ordinary skill in the art that, it can be to the technology of the present invention
Scheme is modified or replaced equivalently, and without departing from the spirit of the technical scheme of the invention and range, should all be covered in this hair
In bright right.
Claims (9)
1. a kind of lactobacillus plantarum CCFM1019 (Lactobacilus plantarum) was preserved in wide on 2 11st, 2018
East saves Culture Collection, and preservation address is 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100 Guangdong Province microorganism
Research institute, deposit number are GDMCC No.60333.
2. a kind of fermented food, it is characterised in that：The fermented food is to be made using CCFM1019 fermenting and producings, the fermentation
Food includes solid-state food, food liquid, semisolid food.
3. fermented food as claimed in claim 2, it is characterised in that：The fermented food includes dairy products, bean product, fruits and vegetables
Product, the dairy products include milk, sour cream, cheese；The fruit and vegetable product includes cucumber, carrot, beet, celery, justifies in vain
Dish product.
4. applications of the lactobacillus plantarum CCFM1019 in the drug for preparing the discharge for promoting internal plasticiser and its metabolite.
5. application as claimed in claim 4, it is characterised in that：The lactobacillus plantarum CCFM1019 being capable of stomach juice-resistant, resistance to courage
Salt promotes plasticiser and its metabolite from internal discharge, reduces content in serum of plasticiser and its metabolite, significantly
Ground mitigates the damage of plasticiser and its metabolite to testis tissue and liver organization.
6. application as described in claim 4 or 5, it is characterised in that：The plasticiser includes (the 2- ethyl hexyls of phthalic acid two
Base) ester and phthalic acid list (2- ethylhexyls) ester.
7. fermented food according to claim 2 or 3 is in the function of preparing the discharge for promoting internal plasticiser and its metabolite
Application in property food.
8. the use as claimed in claim 7, it is characterised in that：The fermented food being capable of stomach juice-resistant, bile tolerance, promotion plasticizing
Agent and its metabolite from internal discharge, reduce content in serum of plasticiser and its metabolite, significantly mitigate plasticizing
The damage of agent and its metabolite to testis tissue and liver organization.
9. application as claimed in claim 8, it is characterised in that：The mitigation plasticiser and its metabolite to testis tissue and
The damage of liver organization, including alleviate AST, ALT and ALP Indexes Abnormality caused by the intake of the plasticiser.
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