CN101402923A - Plant lactobacillus M1-UVs29 and uses thereof - Google Patents
Plant lactobacillus M1-UVs29 and uses thereof Download PDFInfo
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Abstract
The invention discloses a Lactobacillus plantarum M1-UVs29 with an accession number of CGMCC No.2591. The Lactobacillus plantarum M1-UVs29 can be isolated from fermented meat products of various resources, such as preserved ham, sausage, salami, Xuanwei ham and the like, and is obtained after induced mutation and optimization. The Lactobacillus plantarum M1-UVs29 is a new strain of the induced mutation and isolation and has superior safety and minimal toxic and side effects, and can relieve classical clinical symptoms of coronary heart diseases, atherosclerosis, hyperlipemia and the like and treat cardiovascular diseases of various coronary heart diseases, atherosclerosis, hyperlipemia and the like incurred by hyperlipemia in a safe and effective way. The Lactobacillus plantarum M1-UVs29 is applied to the production of beverages, and health products of dairy products, fermented milk, acidsoy milk and the like and food additives. The obtained products of beverages, health products and food additives, containing the fermented Lactobacillus plantarum M1-UVs29 or metabolins, cell debris or secretions thereof, can effectively degrade cholesterol self-cumulated by human bodies and self-contained by food.
Description
One, technical field
The present invention relates to microbial technology field, particularly be the new bacterial strain of plant lactobacillus of mutagenesis screening and the application of this bacterial strain.
Two, background technology
Cardiovascular and cerebrovascular diseases (cerebrovascular disease; Cerebrovascular disorder; CVD) main pathological basis is coronary heart disease (coronary heart disease, CHD), atherosclerosis (atherosclerosis, AS), hyperlipidaemia (hyperlipoidemia,) etc., the control blood lipid level is the important step that prevents and reduce CHD, AS and HL and prevention cardiovascular and cerebrovascular diseases, and high anteserum cholesterol is the important factor of bringing out these diseases, and cholesterol in the human diet and the cholesterol in the blood also are proportionate.The cardiovascular and cerebrovascular diseases characteristics of incidence is complicated, many environment, multifactor interaction, is subjected to the influence of factors such as heredity, diet, living habit and external environment.Research to it is a lot, and this disease can betide any age, and men and women's sickness rate does not have significant difference, and with regard to hypertension, only in 6～18 years old students in middle and primary schools of China, hypertensive sickness rate just reaches 8%.
At present, for the main ingredient thing is auxilliary, internal medicine gets involved complex therapy with the diet in anti-Zhiduo County of CVD, and the control acute attack reduces recurrence, prevents complication.The ideal medicament of most reducing blood-fat treatments of present stage as lipid-lowering statins, only has curative effect in short-term, and for a long time with apparent side effect then occurring, therefore expectation can be developed safer replacement therapy scheme.
Current research shows, the characteristics that microorganism species is superior with its security, toxic side effect is little are by pay attention to day by day, milk-acid bacteria in the probiotic bacterium, bifidus bacillus and faecalis etc. studies show that through the microbial ecology expert, with the degraded of cholesterol certain relation is arranged, but the curative effect of the present probiotic bacterium treatment CVD that reports still can't reach perfect condition, and the bacterial strain that screening is best becomes the key of probiotic bacterium treatment CVD.
Three, summary of the invention
The objective of the invention is at the problems referred to above, provide a kind of plant lactobacillus (Lactobacillusplantarum) M1-UVs29, hereinafter referred to as plant lactobacillus M1-UVs29; Another object of the present invention is to apply it in the production that prevents and treats cardiovascular disorder or beverage, healthcare products and foodstuff additive, and the human body self of effectively degrading is accumulated the cholesterol with food product bodies.
For achieving the above object, the present invention has adopted following technical scheme: the invention discloses a kind of plant lactobacillus M1-UVs29, its preserving number is CGMCC No.2591.Plant lactobacillus M1-UVs29 can as separating in bacon, sausage, air-dry sausage, the Xuanwei ham etc., obtain after the mutagenesis optimization from fermented meat prods, is the isolating brand new strain of a kind of mutagenesis.
Plant lactobacillus M1-UVs29 is used to prevent and treat cardiovascular disorder; Described cardiovascular disorder is for being accumulated the coronary heart disease that caused, atherosclerosis, hyperlipidaemia etc. by cholesterol.
Plant lactobacillus M1-UVs29 is used for producing drink, contains above-mentioned plant lactobacillus M1-UVs29 in the drink product of being produced, or its meta-bolites, cell debris or secretory product.
Plant lactobacillus M1-UVs29 is used to produce healthcare products, contains above-mentioned plant lactobacillus M1-UVs29 or its meta-bolites, cell debris or secretory product in the healthcare products product of being produced.
Plant lactobacillus M1-UVs29 is used to produce foodstuff additive, contains above-mentioned plant lactobacillus M1-UVs29 or its meta-bolites, cell debris or secretory product in the foodstuff additive product of being produced.
The positively effect that the present invention has:
1, plant lactobacillus M1-UVs29 of the present invention can as separating in bacon, sausage, air-dry sausage, the Xuanwei ham etc., obtain after the mutagenesis optimization from the fermented meat prods in various sources, is the isolating brand new strain of a kind of mutagenesis, and security is superior, toxic side effect is little.
2, plant lactobacillus M1-UVs29 of the present invention, typical clinical symptoms such as coronary heart disease, atherosclerosis, hyperlipidaemia can be alleviated, cardiovascular disordeies such as the various coronary heart disease that cause by hyperlipidemia, atherosclerosis, hyperlipidaemia can be treated safely, effectively.
3, utilize plant lactobacillus M1-UVs29 of the present invention can be prepared into beverage, healthcare products or foodstuff additive; Or make and contain plant lactobacillus M1-UVs29, or the beverage of its meta-bolites, cell debris or secretory product, healthcare products, as milk-product, fermented-milk, soybean milk yoghurt etc.; Or foodstuff additive, be used for the intravital cholesterol level of reduction machine.
Four, description of drawings
Fig. 1 is form under the plant lactobacillus M1-UVs29 bacterium liquid smear mirror of the present invention (Gram ' s dyeing, * 1000).
Fig. 2 is plant lactobacillus M1-UVs29 electron-microscope scanning of the present invention figure (25KV2.50KX 10 μ mKYKY-2800B SEM SN:1341) as a result.
Fig. 3 is to the figure as a result that influences of Serum TC, TG and HDL-C content among the embodiment 2.
Fig. 4 be among the embodiment 2 in rats'liver body ratio influence figure as a result.
Fig. 5 is rats in normal control group hepatic tissue section figure among the embodiment 2.
Fig. 6 is a high dose group liver tissues of rats slice map among the embodiment 2.
Fig. 7 is a hyperlipidemia model group liver tissues of rats slice map among the embodiment 2.
Fig. 8 is a middle dosage group liver tissues of rats slice map among the embodiment 2.
Fig. 9 is a low dose group liver tissues of rats slice map among the embodiment 2.
Preservation information:
Strain name: plant lactobacillus (Lactobacillus plantarum) M1-UVs29
Deposit number: CGMCC No.2591
Preservation date: 2008 7 years 14 days
Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC)
Depositary institution address: Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica
Five, embodiment
Strain name: plant lactobacillus (Lactobacillus plantarum) M1-UVs29, carried out preservation at the Chinese typical culture collection center (CGMCC) that is positioned at the BeiJing, China city in 2008 7 years 14 days, preserving number is CGMCC No.2591.
The present invention is described in further detail below by specific embodiment.
Embodiment 1:
The isolation identification of plant lactobacillus M1-UVs29 and biological characteristics thereof
1, material
1.1 sample: the fermented meat prods in various sources, as bacon, sausage, air-dry sausage, Xuanwei ham etc.
1.2 reagent:
Lactic acid standard substance chromatographically pure U.S. Sigma company
Viola crystallina analytical pure Tianjin emerging magnificent chemical reagent factory
Sarranine analytical pure Tianjin emerging magnificent chemical reagent factory
Iodine analytical pure Tianjin emerging magnificent chemical reagent factory
Potassiumiodide analytical pure Tianjin emerging magnificent chemical reagent factory
Dihua worker company limited is won in tween 80 analytical pure Tianjin
The biological company limited in precious Tyke, halfcystine analytical pure Guangdong
Plumbic acetate analytical pure Tianjin emerging magnificent chemical reagent factory
Cholesterol chromatographically pure U.S. Sigma company
Cholate analytical pure U.S. Sigma company
Sucrose ester biochemical reagents Zhengzhou Yuan Feng foodstuff additive company limited
The biological company limited in precious Tyke, Tris analytical pure Guangdong
The biological company limited in precious Tyke, EDTA analytical pure Guangdong
The biological company limited in precious Tyke, SDS analytical pure Guangdong
The biological company limited in precious Tyke, Proteinase K biochemical reagents Guangdong
The biological company limited in precious Tyke, balance phenol analytical pure Guangdong
CTAB analytical pure Dalian precious biotechnology company limited
La Taq enzyme biochemical reagents Dalian precious biotechnology company limited
Loading Buffer analytical pure Dalian precious biotechnology company limited
Agarose analytical pure oxoid
EB surrogate analytical pure Dalian precious biotechnology company limited
Sky, biochemical identification pipe Hangzhou and microorganism reagent company limited
Other reagent commonly used is analytical pure
1.3 laboratory apparatus:
XSP-2CA type opticmicroscope Shanghai Precision Scientific Apparatus Co., Ltd
The general universal apparatus company limited of analysing in T6 new millennium type uv-spectrophotometric instrument Beijing
JBT/C-YCL400/3P (D) Jining Sinobest Electronics Co., Ltd.
Adjustable ultrasonic wave medicine handler
JY92-2D ultrasonic cell disruptor Ningbo new sesame biotechnology stock company
YXQ-SG46-280A portable pressure steam steriliazer Shanghai living silver-colored Medical Instruments instrument company
DH4000 type electro-heating standing-temperature cultivator Tianjin Tai Site Instr Ltd.
HZQ-F160 shaking culture case Harbin Dong Lian Electronic Technology Development Co.
JD100-3B electronic analytical balance Shenyang Longteng Electronic Co., Ltd.
Micropipet (10,20,200,1000 μ L) Finland thunder C Compaq
BCN-136000 Bechtop Harbin Dong Lian Electronic Technology Development Co.
PHS-2 type acidometer Shanghai thunder magnetic instrument plant
LGR10-4.2 high speed freezing centrifuge Beijing Medical Centrifugal Machine Factory
DDY-III type electrophoresis apparatus Liuyi Instruments Plant, Beijing
TC-25/H gene-amplificative instrament Hangzhou BIOER Technology Co., Ltd
Gel images analytical system Beijing monarch east electrophoresis equipment company that anticipates
Instrument plant is contained by Jintan City, SZ-1 type quick vortex mixer Jiangsu Jin Cheng state
2. culture medium preparation:
2.1MRS substratum: peptone 10g, extractum carnis 10g, yeast extract 5g, K2HPO4 2g, dibasic ammonium citrate 2g, sodium acetate 5g, glucose 20g, tween 80 1mL, MgSO47H2O 0.5g, MnSO40.25g, distilled water 1000mL, 6.2～6.4,121 ℃ of sterilizations of pH value 20min.Add 1.8% agar on the basis of solid medium liquid medium within.
2.2 salt tolerance test medium: the MRS liquid nutrient medium adds 2%, 4% and 6% NaCl respectively.
2.3 nitrate resisting test medium: the MRS liquid nutrient medium adds the NaNO of 50mg/kg, 100mg/kg, 150mg/kg respectively
2
2.4 produce the mucus substratum: MRS consubstantiality substratum, the sucrose with 5% replaces glucose.
2.5 produce H
2S substratum (plumbic acetate paper slip method): peptone 10g, NaCl 5g, extractum carnis 10g, halfcystine 0.5g, distilled water 1000mL, pH7.0～7.4,112 ℃ sterilization 20～30min.In addition common filter paper is cut into the wide paper slip of 0.5～1cm, length is decided according to test tube and substratum height.Plumbic acetate with 5～10% soaks into paper slip, uses oven for drying then, and it is standby to be put in the culture dish sterilization.
2.6H
2O
2Produce and detect substratum
Basic medium: extractum carnis 0.5g, yeast extract paste 0.5g, tween 80 0.05mL, MnSO
44H
2O0.01g, agar 1.5g, pH6.5,121 ℃ of 15min.The aseptic sugar 1% of independent sterilization, pour plate.Skim is toppled on the upper strata, and (1～2mL) same substratum adds 4%MnO
2
2.7 bacteriostatic test substratum:
LB substratum: Tryptones 10g, yeast extract 5g, NaCl 5g, distilled water 1000mL, pH value 7.0.
2.8 protein degrading activity test medium: in the MRS solid medium, add 15% skimmed milk powder.
2.9 lipolysis activity test medium: in the MRS solid medium, add 15% lard and toluylene red indicator.
2.10 hypercholesterolemia MRSO-CHOL substratum: in the liquid nutrient medium of 1000mL, contain the preparation of 1.0mg/mL cholesterol micellar solution: accurately weighing cholesterol 0.1g puts into small beaker, adding 0.2g cholate, 0.1g sucrose ester, 1mL tween 80 stir, pipette the glacial acetic acid heating for dissolving of 5mL again, behind lysate usefulness ultrasonication 15min, join fast in the liquid nutrient medium for preparing, stir while adding, make it form uniform and stable colloidal solution.
2.11 preparation X-gal solution (20m/mL)
With diameter 0.22 μ m disposable filter filtration sterilization, getting 50 μ L, to be laid on the MRS solid medium coating evenly standby.
2.12 the lactic acid ply of paper is analysed reagent
2.12.1 developping agent: water, phenylcarbinol, propyl carbinol mix with 1: 5: 5 amount, add 1% formic acid then.
2.12.2 developer: 0.04% tetrabromophenol sulfonphthalein spirituous solution, regulate pH to 6.70 with 0.1N NaOH.
3, the separation of plant lactobacillus M1-UVs29, purifying
3.1 strains separation: the fermented meat prods in various sources such as bacon, sausage, air-dry sausage, Xuanwei ham, get the 20g sample respectively in the aseptic peptone water of 180mL (peptone 1g/L, Nacl 0.85g/L, tween 80 lmL) concussion (concussion shaking table 200r/min, concussion 60min) mixing, serial dilution, get supernatant liquor and make the substratum pour plate with MRS+3%CaCO3, picking produces single bacterium colony of molten calcium circle, the line separation and purification, pure bacterium carries out gramstaining, chooses Gram-positive and catalase negative bacterium, carries out the inclined-plane and preserves.
3.2 primary dcreening operation: the milk-acid bacteria that goes out to sift out is carried out the fermentation character test (produce clingtest, salt tolerance experiment, the salt of anti-nitrous acid experiment, produce H
2O
2, produce ammonia experiment, the experiment of glucose aerogenesis, produce the H2S experiment, the enzyme experiment of betraing is taken off in litmus milk experiment, the experiment of nitrate reduction ability, amino acid, bacteriostatic experiment, acid producing ability experiment, lactic acid ply of paper analyse reagent) in fermented meat prods, dominant strain should have that anti-6%Nacl and 150mg/kgNaNO2, acid production speed are fast, glucose fermentation not aerogenesis, do not produce H
2S, hydrolysis arginine do not produce ammonia, do not have amino acid takes off the shuttle enzymic activity, does not produce mucus, does not produce H
2O
2, can suppress intestinal bacteria and streptococcus aureus, fermentable carbohydrates product are mainly lactic acid, from isolating milk-acid bacteria, screen strain excellent.
3.3 multiple sieve: the mensuration of carrying out cholesterol degradation rate in the mensuration of cholesterol degradation rate and the food substrate.
3.3.1 the mensuration of lactobacter growth curve and pH value
Lactobacillus inoculum is cultivated 24h for 30 ℃ in the MRS liquid nutrient medium, is blank with MRS, and every 2h measures the OD value with ultraviolet spectrophotometer in 650nm, measures the pH value with acidometer.
3.3.2 the cholesterol degradation shaker test method of different strain
After will activating enlarged culturing with the confession examination bacterium of kind, respectively change in 5 liquid tube substratum, after optimal temperature is cultivated 48h, adopt colony counting method survey in the selection substratum that contains identical cholesterol micellar concentration of bacterial classification in the liquid line of the identical cell count magnitude of the somatic cells number of each pipe → get → be linked into preparation simultaneously by 3% inoculum size → after cultivating the regular hour under the suitable temperature → qualitative again and measure cholesterol level quantitatively, and with do not inoculate blank group contrast, compare the cholesterol degradation rate of each bacterial classification, and then select the strong bacterial strain of cholesterol degradation ability.
3.3.3 the cholesterol degradation experimental technique of different incubation times
(cell concentration is 10 by 3% inoculum size with the seed selection bacterial strain
8CFU/mL) be inoculated in and contain in the 1mg/mL cholesterol micellar substratum, cultivate under the optimal temperature, regularly get nutrient solution, measure content of cholesterol, pH value and bacterium number, calculate the cholesterol degradation rate, and the significance of difference, the dependency of pH value and cholesterol degradation rate and the variation of bacterium number between processing are discussed in analysis.
3.3.4 the cholesterol degradation experimental technique of different vaccination amount
Tested bacterial strain is diluted to 10 with stroke-physiological saline solution
8CFU/mL, respectively with different inoculum sizes: 1%, 2%, 3%, 4%, 5%, 6%, be inoculated in and contain in the 1mg/mL cholesterol micellar substratum, cultivate 3d under the optimal temperature after, the variation of measuring and calculating cholesterol degradation rate, pH value and bacterium number respectively.
3.3.5 the degradation experiment method of various biliary sterol micellar concentration
(somatic cells concentration all is 10 by identical inoculum size with the seed selection bacterial strain
8CFU/mL) be inoculated in and contain 0.5mg/mL, 1.0mg/mL, 1.5mg/mL, 2.0mg/mL in the different concns cholesterol micellar substratum, adopt tested bacterial strain optimum growth temperature to cultivate respectively, after incubation time is 3d, measure and calculate the variation of cholesterol degradation rate, pH value and bacterium number.
3.4 bacterial strain preliminary evaluation
3.4.1 phenotypic characteristic
The solid culture feature; The liquid culture feature.
3.4.2 physiological and biochemical property
3.4.3PCR identify: the amplification of 16SrDNA, adopt PCR method that the 16SrDNA gene of strain isolated is increased.PCR adopts the Idiotype primer.
4, colony characteristics (oxytolerant test):
Plant lactobacillus M1-UVs29 presents the moistening bacterium colony of circle, white, projection, the about 2-4mm of diameter, neat in edge after MRS solid medium plate anaerobism is cultivated 48h.
5, the form under microscope and the scanning electron microscope:
Fig. 1 is a plant lactobacillus M1-UVs29 bacterium liquid smear, provides the Gram-positive bacillus in the picture, and is in the majority with the rod-short bacillus, single or paired.
Fig. 2 is that plant lactobacillus M1-UVs29 warp is fixing, and the bacterium liquid sem photograph behind gradient dehydration, gradient displacement, spray platinum plated film, the bacterium that provides in the picture, and bacterium colony smooth surface, complete, the form homogeneous is corynebacterium, the blunt circle in two, no gemma.(25KV?2.50KX?10μm?KYKY-2800B?SEM?SN：1341)
6, the main fermentation character of milk-acid bacteria is identified
6.1 product adhesive test: adopt and produce the viscosity culture medium inoculated, cultivate 24h for 37 ℃, with inoculating needle picking colony direct viewing.
6.2 salt tolerance test: isolating bacterial classification inoculation in the salt tolerance test medium, is cultivated 24h for 37 ℃, measure the OD value, make blank with the MRS substratum, relatively its growing state with spectrophotometer 650nm.
6.3 nitrate resisting test: isolating bacterial classification inoculation to anti-nitre test medium, is cultivated 24h for 37 ℃, and 650nm measures the OD value, and relatively its growing state is made blank with the MRS substratum.
6.4H
2O
2Produce: the fresh culture thing is in H
2O
2Produce and detect the substratum line, cultivate 5d, observe periphery of bacterial colonies, become defecator H
2O
2Positive.
6.5 produce H
2S: with reference to the method for eastern elegant pearl (2001).
6.6 the MRS solid medium that bacteriostatic test: 20mL does not add sodium acetate melts about postcooling to 45 ℃, with dull and stereotyped behind indicator (intestinal bacteria and streptococcus aureus) the fresh culture thing 100 μ L mixings, solidifying the back is that the aseptic punch tool of 7mm punches on flat board with diameter, the lactic acid bacteria culture solution of accurately measuring 40 μ L adds in the aperture, flat board places 4 ℃ of refrigerator diffusions, place 37 ℃ of constant temperature culture 24h then, observe aperture inhibition zone on every side.
6.7 protein degrading activity test: the protein degrading activity test medium is made aseptic flat board, get equivalent (0.1mL, 108CFU/mL) bacteria suspension of separated bacterial strain is evenly coated planar surface, cultivate 48h for 37 ℃ and observe cultivation results, when if casein is decomposed in the milk powder, transparent circle can appear in periphery of bacterial colonies, and is promptly positive, otherwise negative.
6.8 the lipolysis activity test: the lipolysis activity test medium is made aseptic flat board, (0.1mL, 108CFU/mL) bacteria suspension of separated bacterial strain in the planar surface coating evenly, cultivating 48h for 37 ℃ observes, the generation lipid acid if this bacterial strain can reduce fat, red transparent spot then appears on flat board, and promptly positive, otherwise negative.
6.9 bacterial strain fatal temperature measuring method: respectively with MRS solid medium and liquid tube substratum under differing temps (55 ℃, 60 ℃, 65 ℃ etc.), water-bath preheating 30min, be incubated 30min after inoculating the corresponding bacterial strain that is in logarithmic phase fast, putting into frozen water immediately descends temperature, 37 ℃ of constant temperature culture 24h, the growing state of observation bacterial strain.Situation in the liquid medium within is judged by the variable color of purpurum bromocresolis.
7.10 the lactic acid ply of paper is analysed test: get size to fit filter paper bar, at the standardized line in 3cm place, distance base, dip in the sample liquid that takes a morsel with clean glass capillary, every sample of 2～2.5cm point (comprising 2% lactic acid standard, blank nutrient solution and contain the bacteria culture fluid sample), every some sample dries up with hair dryer immediately; Paper is put into chromatography cylinder, make saturated for some time, add developping agent again and begin chromatography with developping agent.Behind the up 25cm, take out airing, whether the spray developer yellow spotting occurs on the observation sample, and compares with 2% lactic acid sample, and calculates the Rf value, to determine whether inoculum contains lactic acid.Concrete outcome sees Table 1 and table 2.
The milk-acid bacteria thalli morphology of table 1 screening and main fermentation character
The Rf value that table 2 lactic acid and fermented liquid ply of paper are analysed
7, MRS-X-gal substratum color reaction
Plant lactobacillus M1-UVs29 after the separation and purification evaluation is inoculated on the MRS-X-gal substratum, and anaerobism is cultivated 48h, takes out plate and is positioned in 4 ℃, observes bacterium colony colour developing situation behind the several minutes.The result shows: bacterium colony is light blue on the MRS-X-gal plate.
8, plasmid detects
Collect bacterium mud extracting plasmid according to a conventional method, 1.0% agarose gel electrophoresis detects plant lactobacillus M1-UVs29 and whether has plasmid.Detected result is not seen plasmid.
9, genetic characteristics
Collect the bacterium mud of plant lactobacillus M1-UVs29 and extracting DNA according to a conventional method, carry out the pcr amplification of milk-acid bacteria 16s rDNA with the Auele Specific Primer of lactobacillus, 1.0% agarose gel electrophoresis detects the PCR product, and visible 400bp place has a bright band identical with the target fragment size.
Plant lactobacillus M1-UVs29 irritates the observation of curative effect of stomach to the hyperlipidemia mouse.Animal model is divided into normal control group (NC), the high fat control group of inductive (HFC), low dose group (LD), middle dosage group (MD) and high dose group (HD) give plant lactobacillus M1-UVs29 respectively and irritate stomach and raise, and observe the body weight of respectively organizing mouse, pathological change etc.The result shows: plant lactobacillus M1-UVs29 can reduce the blood lipid level of laboratory animal, alleviates clinical symptom, can effectively prevent and treat cardiovascular disordeies such as the various coronary heart disease that caused by hyperlipidemia, atherosclerosis, hyperlipidaemia.
One, experiment material and method:
1, laboratory animal grouping and raising
Select 60 of the Wistar rats of male and female half and half for use, in 6.8 ages in week, body weight 180～220g purchases in Changchun biological products company, raises in a cleaning level Animal House.After adaptability raised for 1 week, be divided into 4 groups by body weight: normal control group (NC), high fat control group (HFC), low, in and high dose group (physiological saline dilution bacterium liquid is 1 * 10 for LD, MD and HD
4CFU/mL, 1 * 10
7CFU/mL, 1 * 10
10Standby behind the CFU/mL.
The experiment basis feed is: Semen Maydis powder 30%, soybean cake powder 20%, wheat bran 25%, flour 16%, fish meal 5%, bone meal 2%, yeast powder 1%, salt 1%;
The experiment high lipid food is: basal feed 93%, cholesterol 1.5%, Sodium cholic acid 0.5%, lard 5%.
Normal control group (NC) basal feed of feeding is all fed high lipid food for all the other four groups.
1.2 main agents
Plant lactobacillus M1-UVs29 bacteria suspension, cholesterol, the pig cholate, triglyceride level, TC, TG, HDL-C test kit (giving birth to biological reagent company in Beijing provides), beef powder (Beijing extensive and profound in meaning star biotechnology responsibility company limited, 20040607), tween .80 (the rich power laboratory in Xi'an, lot number 021125), phosphoric acid dioxy potassium (Henan Province's Jiaozuo City chemical industry three factories, lot number 20010404), agar (Beijing extensive and profound in meaning star biotechnology responsibility company limited), Salazosulfamide adjoins pyridine sheet (SASP, Fuda Pharmaceutical Co., Ltd., Shanghai, lot number 041207), DSS (molecular weight 5000, Sigma company).Other reagent all adopts analytical pure.
1.3 key instrument
Biochemical fully-automatic analyzer, Hitachi 7020; High speed tabletop centrifuge (TGL.16G) Shanghai peaceful whizzer factory; 721 spectrophotometers, Shanghai the 3rd analytical instrument factory; The homogenate device; Electric-heated thermostatic water bath, Shanghai Medical Apparatus and Instruments Factory; The liquid flash mixer; The JEM-1200EX electron microscope.
2, experimental technique
2.1 experiment bacterium: plant lactobacillus M1-UVs29 method as described above separates, identifies gained.Bacterium anaerobism in the MRS substratum is cultivated after 24 hours and collected bacterium colony, and is standby with spectrophotometer counting back.
2.2 animal model: artificial feeding raised high cholesterol diet to make the animal hyperlipidemia model.Probiotic bacterium (irritate stomach and all begin to begin earlier to carry out in preceding 2 days from drinking bacteria suspension, and every mouse is irritated stomach once every day, each 0.3ml/20g by plant lactobacillus M1-UVs29 bacterium liquid.
2.3 experiment grouping: experiment Wistar rat, be divided into 5 groups at random, it is as follows that each group is 12 grouping situations.
A, normal control group: basal feed, normal diet does not have special processing.
B, positive high fat control group: feed with high lipid food.
C, high dose group: feed 1 * 10 with high lipid food
10CFU/mL bacterium liquid is irritated stomach.
D, middle dosage group: feed 1 * 10 with high lipid food
7CFU/mL bacterium liquid is irritated stomach.
E, low dose group: feed 1 * 10 with high lipid food
4CFU/mL bacterium liquid is irritated stomach.
2.4 testing index and method
2.4.1 lipid determination: tested for the 8th weekend, 10h adopts a frame blood on an empty stomach, measures TC, TG and HDC-C content, and TC, TG, HDL-C all adopt and give birth to biological reagent company kit measurement in Beijing.
2.4.2 liver lipid and liver body ratio measurement: tested for the 8th weekend,, win liver immediately, weigh through femoral vein sacrificed by exsanguination animal.Get fresh liver tissue 5g and be prepared into the liver homogenate, soak 72h, filter with chloroform, methanol solution, 65 ℃ of evaporates to dryness, the petroleum ether dissolution constant volume is measured TC and TG, and TC, TG all adopt and give birth to biological reagent company kit measurement in Beijing.
2.4.3 liver histopathology inspection: after the stripped naked eyes visual inspection of liver, use 10% formalin fixed, routine paraffin wax embedded section, HE dyeing.
2.4.4 LPO measures in serum and the tissue: tested for the 8th weekend, 10h on an empty stomach gets the blood and the heart, brain, hepatic tissue is used the Riely method and surveyed LPO content.
2.4.5 whole blood SOD and GSH-Px measure: tested for the 8th weekend, 10h gets whole blood and surveys SOD, GSH-Px vigor on an empty stomach, and SOD generates the inhibition method with nitrite, and GSH-Px is with methods such as Xia Yiming.
2.5 data statistic analysis
Every data all adopt biometrics SAS (6.12 editions) software to carry out variance analysis on computers, relatively check with T between two groups.
3, result
3.1 the influence that body weight changes
Respectively organize mouse body weight difference before the experiment and do not have apparent property, experiment back model group, high fat positive controls, treatment group body weight all obviously descend, the body weight difference of body weight poor (difference before experiment back and the experiment) and normal control group relatively has apparent property difference (P＜0.05), treat six groups of body weight decline is arranged slightly, relatively there is not apparent property difference (P＞0.05) with the body weight difference of normal control group, as shown in table 3 to the influence of mouse body weight.
The influence of table 3 pair mouse body weight (x ± s)
Compare with the normal control group a:P＜0.05; Compare with low dose group b:P＜0.05
By table 3 as seen, add bacteria suspension the LD group and HD group weight of mice amount all than the NC group height that does not add wheat germ, and all have notable difference (P＜0.05); The body weight gain amount of HD group is obviously than LD group high (P＜0.05).As seen with the increase of adding bacteria suspension concentration, weight increase is more obvious.
3.2 dead mouse situation
Have 15 dead mouses in the experimentation, the wherein not clear cause of death is 4, die from thrombus totally 11, is respectively 1 of model group, 1 of negative control group, 1 of positive controls, two groups 3 of treatments, three groups 2 of treatments, four groups 3 of treatments.
3.3 influence to rat fat
To the influence of rat fat such as Fig. 3, table 4.Fig. 3 be feed raise the plant lactobacillus M1-UVs29 bacterium liquid of different concns after, Serum TC, TG and HDL-C content figure, the index result of variations figure for each experimental group mouse serum before and after the experiment that provides in the picture be provided.Associative list 4, shown in Figure 3, TC, the TG of HFC group are significantly higher than NC group (P＜0.05), and HDL-C significantly is lower than NC group (P＜0.05), shows that the high lipid food of feeding can cause serum lipids in rats; LD, MD and HD group TC, TG extremely significantly are lower than HFC group (P＜0.01), and the HDL-C utmost point is significantly higher than HFC group (P＜0.01).The prompting bacteria suspension has reducing blood lipid, and MD group lipid-lowering effect is better than LD and HD group.
The influence of table 4 pair Serum TC, TG and HDL-C content (x ± s)
Compare with the normal control group a:P＜0.01, and compare with high fat control group b:P＜0.01.
3.4 influence to rats'liver lipid and liver body ratio
To the influence of rats'liver lipid and liver body ratio shown in Fig. 4, table 5.Fig. 4 is that rats'liver body proportion influences figure after feeding the plant lactobacillus M1-UVs29 bacterium liquid of raising different concns, and that provide in the picture is the figure as a result that differs of six each experimental group mouse liver body proportions.Because high lipid diet, the rat liver lipid accumulation, TC in the hyperlipidemia model group liver tissues of rats and the TG utmost point are higher than normal control group (P＜0.01) significantly, and three groups of TC, TG that irritate the liver tissues of rats of stomaches compare with the normal control group and significant difference are also arranged (P＜0.05), and the MD group reduces the effect of TC, TG content in the liver tissues of rats and is better than LD and HD group.The result of liver body ratio has also shown the existence of same effect, shows to reduce the liver lipid accumulation significantly, has good lipotropic effect.
The influence of TC, TG content and liver body ratio in the table 5 pair rat liver (x ± s)
Compare with the normal control group a:P＜0.01, and compare with high fat control group b:P＜0.05.
3.5 liver histopathology inspection
Visual control: hyperlipidemia model rat liver volume is big, the coating anxiety, and color and luster is yellow greasy, is typical liver fat and becomes; The rats'liver fat of feeding becomes alleviating in various degree, and wherein the HD group is the lightest, and part liver outward appearance is near normal.
Microscopy: accompanying drawing 5 is rats in normal control group hepatic tissue section figure, provides the anatomical slice figure (HE * 200) of compared with control rats liver in the picture, no abnormal phenomenon; Accompanying drawing 7 is hyperlipidemia model group liver tissues of rats slice maps, provide the anatomical slice figure (HE * 200) of compared with control rats liver in the picture, hyperlipidemia model group rat liver is the diffusivity fatty degeneration of liver, the most serious with liver lobule peripheral band pathology, liver cell obviously swells, there are a large amount of fat to drip in the endochylema, and point-like and focus necrotic area occur.Accompanying drawing 6, accompanying drawing 8, accompanying drawing 9 are respectively high dose group, and the mouse hepatic tissue section figure of middle dosage group and low dose group provides the anatomical slice figure (HE * 200) of three dosage control group mouse livers in the picture.More as can be seen, the rat liver pathology of the bacterium liquid of feeding is lighter, HD group especially, liver cell is arranged, form almost with the normal group no significant difference.This result and liver lipid assay result match.
3.6 influence to LPO content in rat blood serum and the tissue
As shown in table 6 to the influence of LPO content in rat blood serum and the tissue, hyperlipidemia rats when blood fat raises, the content of LPO also significantly raise (P＜0.05) in serum and the tissue.Can significantly reduce LPO content (P＜0.01) in experimental hyperlipidemia rat blood serum and the tissue, and in the tissue LPO content there were significant differences (P＜0.05) between LD and MD group.
The influence of LPO content in table 6 pair rat blood serum and the tissue (x ± s)
A:P＜0.01 and normal control group compare, and compare with high fat control group b:P＜0.05, and compare with high fat control group c:P＜0.01, and d:P＜0.05 and low dosage control group are relatively.
3.7 to rat whole blood SOD and the active influence of GSH-Px
As shown in table 7 to rat whole blood SOD and the active influence of GSH-Px, high lipid food can significantly reduce the activity (P＜0.05) of rat whole blood SOD and GSH-Px, LD group, MD group and HD group SOD activity are significantly higher than HFC group (P＜0.05, P＜0.01), make the SOD activity improve 20.1%, 50.7% and 51.1% respectively, LD group, MD group and HD group GSH-Px activity are significantly higher than HFC group (P＜0.05), make the GSH-Px activity improve 13.9%, 18.5% and 23.9% respectively.Show the activity that can improve SOD and GSH-Px in the rat whole blood, have remarkable enhancing Antioxidation Ability of Rats, with regard to SOD, HD group effect obviously is better than the LD group.
Table 7 couple rat whole blood SOD and the active influence of GSH-Px (x ± s)
Compare with the normal control group a:P＜0.05, and compare with high fat control group b:P＜0.05, and c:P＜0.01 and high fat control group are relatively.
Above presentation of results, serum TC, its variation direct reaction of TG content as the hyperlipidaemia important indicator this bacteria suspension and can be significantly improved experimental hyperlipidemia rat blood serum HDL-C content, simultaneously, significantly reduce serum TC and TG content, generalized case and intestinal mucosa pathologic finding and normal control group do not have difference.
Claims (6)
1, a kind of plant lactobacillus (Lactobacillus plantarum) M1-UVs29, its preserving number is CGMCC No.2591.
2, fermenting plant Bacterium lacticum according to claim 1 (Lactobacillus plantarum) M1-UVs29 is characterized in that: described fermenting plant Bacterium lacticum (Lactobacillus plantarum) M1-UVs29 is used for the treatment of cardiovascular disorder.
3, ((Lactobacillus plantarum) M1-UVs29 is characterized in that: described fermenting plant Bacterium lacticum M1-UVs29 is used for the treatment of by cholesterol and accumulates coronary heart disease, atherosclerosis, the hyperlipidaemia that is caused fermenting plant Bacterium lacticum according to claim 2.
4, fermenting plant Bacterium lacticum according to claim 1 (Lactobacillus plantarum) M1-UVs29, it is characterized in that: described fermenting plant Bacterium lacticum M1-UVs29 is used for producing drink, contain fermenting plant Bacterium lacticum (Lactobacillus plantarum) M1-UVs29 in the drink product of being produced, or its meta-bolites, cell debris or secretory product.
5, fermenting plant Bacterium lacticum according to claim 1 (Lactobacillus plantarum) M1-UVs29, it is characterized in that: described fermenting plant Bacterium lacticum (Lactobacillus plantarum) M1-UVs29 is used to produce healthcare products, contain fermenting plant Bacterium lacticum (Lactobacillus plantarum) M1-UVs29 in the healthcare products product of being produced, or its meta-bolites, cell debris or secretory product.
6, fermenting plant Bacterium lacticum according to claim 1 (Lactobacillus plantarum) M1-UVs29, it is characterized in that: described fermenting plant Bacterium lacticum (Lactobacillus plantarum) M1-UVs29 is used to produce foodstuff additive, contain fermenting plant Bacterium lacticum (Lactobacillus plantarum) M1-UVs29 in the foodstuff additive product of being produced, or its meta-bolites, cell debris or secretory product.
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