CN102329762A - Lactobacillus helveticus H9 and application thereof - Google Patents
Lactobacillus helveticus H9 and application thereof Download PDFInfo
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Abstract
The invention relates to Lactobacillus helveticus H9 and application of the Lactobacillus helveticus H9 to the resistance of hypertension and the prevention of cardiovascular disease. A preparation method comprises the following steps of: isolating strains, identifying the strains, preserving the strains, culturing the strains, preparing a bacterial liquid, preparing fermented milk, and screening a strain with angiotensin-converting enzyme (ACE) inhibitory activity; and further evaluating the influence of Lactobacillus helveticus H9 fermented milk on hypertension (SHR, spontaneously hypertensive rats) and normal blood pressure (WHY rats, Wistar-Kyoto rats). The research shows that strain fermented milk has a better in-vivo antihypertensive effect, has the advantages of long onset time and the like, does not affect blood circulation, and has no adverse effects on the normal blood pressure.
Description
[technical field]
The invention belongs to the microbial technique Application Areas.More specifically; The present invention relates to a kind of lactobacterium helveticus (Lactobacillus helveticus) H9; Also relate to the purposes of said lactobacterium helveticus H9 in the medicine of preparation preventing cardiovascular disease, also relate to the purposes of said lactobacterium helveticus H9 in producing protective foods.
[background technology]
Hypertension is to cause heart trouble, cerebro-vascular diseases, nephropathy to take place and dead main Hazard Factor; Be a kind of serious threat human health and influence quality of life; And the modal chronic disease that has caused tremendous economic to lose to society; And have age of onset constantly to reduce, the trend that sickness rate rises year by year.Though clinical proof chemical synthetic drug has hypotensive effect, life-time service has spinoff in various degree.Thereby antihypertensive active material research becoming the already focus of preventing and treating the hypertension research field in food source.
Milk-acid bacteria is used for multiple leavened food processing as the mikrobe of the safety of generally acknowledging, by the human consumption more than one thousand years, and the immunomodulatory of milk-acid bacteria, antitumor, reducing blood-fat and many physiological functions such as hypotensive are generally acknowledged by medical circles.The hypotensive activity of milk-acid bacteria is roughly from four aspects: the 1. proteolyzing through milk-acid bacteria discharges active antihypertensive peptide from food proteins (like, casein); 2. the thalline composition of milk-acid bacteria is like polysaccharide-Polysaccharides, peptide complexes composition; 3. part milk-acid bacteria especially reaches the probiotic lactobacillus of enteron aisle with the viable bacteria form, in enteron aisle, promote body to part can blood pressure regulation the absorption of mineral substance; 4. the part exocellular polysaccharide that produces of milk-acid bacteria.Lactobacterium helveticus (Lactobacillushelveticus) is to prevent and treat the hypertension aspect, utilizes one of maximum mikrobe.
CN102098923A discloses the new bacterial strain of the lactobacterium helveticus that can produce a large amount of blood pressure lowering peptides, particularly IPP, VPP and LPP.This invention also relates to the mixture that contains tripeptides IPP, VPP and LPP and the cultured milk prod of lactobacterium helveticus bacterial strain.The mixture that this invention also relates to the specific peptide mixt be made up of tripeptides IPP, VPP and LPP and tunning or peptide is used to reduce in food, food supplement or pharmaceutical composition or the purposes of preventing hypertension.
CN101420969A disclose a kind of have can improve blood vessel endothelium correlation function, various diseases that prevention is relevant with function of vascular endothelium, or have the effect that suppresses vascellum tunica interna incrassation, be expected to have effect, excellent in safety such as kovakorisan, have the medicament that improves function of vascular endothelium and suppress at least a effect of vascellum tunica interna incrassation; And kovakorisan agent.The medicament of this invention contains (a) Xaa Pro Pro or (b) contains the caseic hydrolyzate of animal milk or its enriched material of Xaa Pro Pro or (c) make the fermented product that contains IlePro Pro and/or Val Pro Pro that the fermenting raw materials that contains milk protein obtains as effective constituent through the bacterial strain that belongs to the lactobacterium helveticus kind.
At present, also there are not bibliographical information lactobacterium helveticus H9 biological characteristics, screening method and application.Therefore, the inventor has accomplished the present invention finally through lot of experiments.
[summary of the invention]
[technical problem that will solve]
The purpose of this invention is to provide a kind of lactobacterium helveticus (Lactobacillus helveticus) H9.
Another object of the present invention provides the purposes of a kind of lactobacterium helveticus H9 in the medicine of preparation preventing cardiovascular disease.
Another object of the present invention provides the purposes of a kind of lactobacterium helveticus H9 in producing protective foods.
[technical scheme]
The present invention realizes through following technical proposals.
The present invention relates to a kind of lactobacterium helveticus (Lactobacillus helveticus) H9.This bacteria strain on April 28th, 2011 in the preservation of common micro-organisms DSMZ of China Committee for Culture Collection of Microorganisms, preserving number is CGMCC 4811.
The invention still further relates to the purposes of described lactobacterium helveticus H9 in the medicine of preparation preventing cardiovascular disease.
According to the present invention, described cardiovascular disorder is coronary heart disease, hypertension, exercise related sudden death, stenocardia, acute myocardial infarction, coronary heart disease or hyperlipidemia.
Described medicine by described lactobacterium helveticus H9 pulvis with form at pharmaceutically acceptable carrier.
At pharmaceutically acceptable carrier is that one or more are selected from the carrier of normally used weighting agent, tackiness agent, wetting agent, disintegrating agent, absorption enhancer, tensio-active agent, absorption carrier, lubricant or correctives pharmaceutically.
The formulation of said medicine is tablet, pill, capsule or microcapsule formulations.
The invention still further relates to the purposes of lactobacterium helveticus H9 of the present invention in producing protective foods.
According to the present invention, described protective foods is bread, dessert or the beverage that contains lactobacterium helveticus H9 according to the invention.
Lactobacterium helveticus H9 of the present invention obtains according to following screening method screening.
This screening method comprises the steps:
A, with traditional zymotic acid yak milk as the milk-acid bacteria sample separation, get the said sour yak milk of 2-3mL and put into the sterilization small test tube, get the sour yak milk of the said fermentation of 0.5-1mL again and put into sterilization CaCO is housed
3In the aseptic sampling tube of powder, refrigeration is subsequent use under temperature 2-6 ℃ condition;
B, draw the said sour yak milk sample of 0.5-1mL and be inoculated in 5-10mL reindeer moss degreasing milk medium with the sterilization suction pipe; And place 30～40 ℃ of thermostat containers of temperature to increase bacterium to be cultured to acidifying and to solidify and present pink;
C, at step B) increase bacterium and cultivate after, with the streak inoculation of disinfection inoculation ring picking in the BL nutrient agar that contains 10ppm cycloheximide and 10ppm Polymyxin E sulfate by weight; Put it in the anaerobic jar, under the condition of 30～40 ℃ of temperature, cultivated 48～72 hours, behind the formation bacterium colony, use the transfering loop picking colony, be inoculated in the MRS nutrient solution, place incubator under the condition of 30～40 ℃ of temperature, to cultivate 24～48 hours again;
D, treat step C) after strain growth is good; Streak inoculation is in the BL nutrient agar once more; Place incubator under the condition of 30～40 ℃ of temperature, to cultivate 24～48 hours, observed and recorded colonial morphology and gramstaining cell morphological characteristic, and carry out catalase test; Isolate the bacillus of in said test, being negative, said bacillus is a probiotic lactobacillus;
E, described probiotic lactobacillus then carry out purifying under the condition of 37 ℃ of temperature in substratum MRS cultivates, and under temperature-80 ℃ to-85 ℃ condition, carries out freezing then and preserves, for use;
F, get the 5mL skimming milk, be chilled to 40 ℃ of temperature at 121 ℃ of temperature sterilization 7min down; Inoculate then in its sterilization skimming milk weight 11% in step e) the freezing preservation probiotic lactobacillus that obtains, carry out the activation of the 1st generation under the condition of 37 ℃ of temperature; Then,, under the condition of 37 ℃ of temperature, cultivate 18h and carry out the activation of the 2nd generation, use the BCP nutrient agar to count then being transferred in the MRS substratum in its sterilization skimming milk weight 2% the 1st generation activated seed; Again the centrifugal back of bacterium liquid is used and contain by weight the PBS protective material of 1% Tryptones and 0.1% Sodium Glutamate and in 4 ℃ of refrigerators, preserve, be used for the preparation of fermented-milk;
G, the bacterial strain that will count are with 5 * 10
6The cfu/mL inoculum size is in 95 ℃ of temperature down in 11% skimming milk of sterilization 10min,, sample cooled off rapidly, and sample is carried out acidity, OD during to pH value 4.5 at 37 ℃ of following ferment at constant temperature of temperature
600Value, FAN and ACE suppress active mensuration, thereby filter out the probiotic lactobacillus with hypertension potential;
H, employing blood pressure measuring method and target organ are organized observation analysis method under the mirror, research and the influence of evaluation lactobacterium helveticus H9 fermented-milk to trouble hypertension rat and normal arterial pressure mouse;
I, this bacterial strain processed supply examination bacterium liquid, draw the 1.0mL suspension bacteria liquid and join in the simulated gastric fluid of 9.0mL pH value 2.5, at 37 ℃ of temperature digestion 3h down, in its process respectively at 1h, 2h, 3h sampling and measuring viable count, and calculating bacterial strain survival rate; Then, that draws pH value 2.5 behind the 1.0mL digestion 3h contains the bacterium simulated gastric fluid, joins in the artificial pancreatic juice of 9.0mL, and continuation is cultivated under 37 ℃ of temperature, respectively at 0h, 4h, 8h sampling and measuring viable count, and according to computes bacterial strain survival rate:
N
1---bacterial strain is handled the back viable count;
N
0---the initial viable count of bacterial strain;
According to fermented-milk acidity, OD
600Value, FAN and ACE suppress activity and the bacterial strain survival rate selects the lactobacterium helveticus H9 with good antihypertensive effect.
Below the present invention will be described in more detail.
The present invention relates to a kind of lactobacterium helveticus H9.This bacteria strain on April 28th, 2011 in the preservation of common micro-organisms DSMZ of China Committee for Culture Collection of Microorganisms, preserving number is CGMCC 4811.
Lactobacterium helveticus H9 of the present invention adopts following screening method to obtain.Be described below in conjunction with 1 pair of this method steps of accompanying drawing:
A, with traditional zymotic acid yak milk as the milk-acid bacteria sample separation, get the said sour yak milk of 2-3mL and put into the sterilization small test tube, get the sour yak milk of the said fermentation of 0.5-1mL again and put into sterilization CaCO is housed
3In the aseptic sampling tube of powder, be placed in the refrigerator, under 2-6 ℃ of condition, refrigerate, with subsequent use as subsequent use sample.
Traditional zymotic acid yak milk is taken from the geographic sour yak milk of Nagqu; Chemical constitution, mikrobe about this sour yak milk formed, microbiological property; See also the contriver at " International Journalof Food Sciences and Nutrition ", 2009,60 (S7): 243-250 and " International Journalof Dairy Technology "; 2010,63 (3): specify among the 437-444.
Sterilization CaCO is housed in aseptic sampling tube
3The purpose of powder is to stablize the pH environment.The aseptic sampling tube that the present invention uses is a product sold in the market, the aseptic sampling tube that for example friendly Kang Jiye biotechnology (Beijing) ltd produces.
B, draw the said sour yak milk sample of 0.5-1mL and be inoculated in 5-10mL reindeer moss degreasing milk medium with the sterilization suction pipe; And place 30～40 ℃ of thermostat containers to increase bacterium to be cultured to acidifying and to solidify and present pink.
Reindeer moss is a kind of pH indicator, when pH rises to 8.3 alkalescence, shows blue, and pH shows red when reducing to 4.5 acidity; PH is near neutral, and nonvaccinated substratum is a hyacinthine.Reindeer moss also is a kind of redox indicator, can be reduced to white, so litmus milk is a kind of differential medium that is used for measuring several kinds of metabolisming properties of seized bacterium.
The reindeer moss degreasing milk medium is to be added to reindeer moss alcoholization solution in the fresh skimming milk, is distributed into tubule again, uses steam sterilizing to obtain then.
C, at step B) increase bacterium and cultivate after, with the streak inoculation of disinfection inoculation ring picking in the BL nutrient agar that contains 10ppm cycloheximide and 10ppm Polymyxin E sulfate.Put it in the anaerobic jar, place under 30～40 ℃ of conditions and cultivated 48～72 hours, behind the formation bacterium colony, use the transfering loop picking colony, be inoculated in the MRS nutrient solution, place incubator under the condition of 30～40 ℃ of temperature, to cultivate 24～48 hours again.
Cycloheximide (Actidione) by carry in the actinomycetes fermentation liquor that produces cycloheximide a kind of microbiotic.It is a clear crystal, 119～121 ℃ of fusing points, and it is soluble in methyl alcohol, ethanol and the acetone, is slightly soluble in the water, and it is acidproof, and is also heat-resisting.It is prone to decompose under alkaline condition.It has restraining effect to pathogenic bacterias such as yeast, mould, protozoon etc., and bacterium is not had remarkable restraining effect.
Colistine sulfate is white or near-white powder, and is soluble in water, is slightly soluble in methyl alcohol, the ethanol, is dissolved in acetone, the ether equal solvent.Stable in pH 3-7.5 scope.Colistine sulfate is produced by poly-viscosity bacillus, and Gram-negative bacteria is had stronger anti-microbial effect.
The BL nutrient agar is the substratum that the technician in present technique field knows; It is the GB standard medium; It contains liver and soaks powder, peptone, beef extract powder, yeast and soak powder, pancreas casein peptone, Zulkovsky starch, L-halfcystine, glucose, potassium primary phosphate, potassium hydrogenphosphate, soya peptone, sal epsom, ferrous sulfate, sodium-chlor, agar, manganous sulfate, tween 80, pH value 7.2 ± 0.1.
The MRS substratum is the substratum that the technician in present technique field knows, and it contains beef protein powder, flesh of fish juice, Yeast diffusion juice powder, glucose, sodium-acetate, dibasic ammonium citrate, tween 80, sal epsom, manganous sulfate, and pH 6.2～6.4.
D, treat step C) after strain growth is good; Streak inoculation is in the BL nutrient agar once more; Place incubator under the condition of 30～40 ℃ of temperature, to cultivate 24～48 hours, observed and recorded colonial morphology and gramstaining cell morphological characteristic, and carry out catalase test; Isolate the bacillus of in said test, being negative, said bacillus is a probiotic lactobacillus.
Observing colonial morphology is neat in edge, smooth surface, the white colony that central authorities are flat
Observing the gramstaining cellular form is: behind gramstaining, be purple, be gram-positive microorganism.
Catalase test is bacterium colony one transfering loop on the picking solid medium, places in the clean tube, drips 3% superoxol 2mL; Observations; Catalatic bacterium is arranged, can generate water and nascent oxygen by catalyzing hydrogen peroxide, form molecular oxygen then and bubble occurs.Can the bubbling person positive, the bubbling person be not negative.In the GPC, staphylococcus and micrococci all produce katalase, so test is usually used in tentatively hiving off of GPC, and streptococcus is negative.
Confirm as milk-acid bacteria by colonial morphology, gramstaining cellular form and catalase test.
E, described probiotic lactobacillus then carry out purifying under the condition of 37 ℃ of culture temperature in substratum MRS cultivates, and under temperature-80 ℃ to-85 ℃ condition, carries out freezing then and preserves, for use.
According to the present invention, purifying cultivates to should be appreciated that it is a kind of cultural method that can isolate single culture.
F, get the 5mL skimming milk, be chilled to 40 ℃ of temperature at 121 ℃ of temperature sterilization 7min down; In the sterilization skimming milk that obtains, inoculation in its sterilization skimming milk weight 11% in step e) the freezing preservation probiotic lactobacillus that obtains, under the condition of 37 ℃ of temperature, carry out the activation of the 1st generation; Then; Being transferred in the MRS substratum in its sterilization skimming milk weight 2% the 1st generation activated seed; Under the condition of 37 ℃ of temperature, cultivate 18h and carry out the activation of the 2nd generation, said lactobacillus strains vigor is fully recovered, the BCP nutrient agar (purpurum bromocresolis solid medium) that uses Japanese Rong Yan Co., Ltd. to produce is then counted; Its method of counting is referring to Guo; Z. wait In vitro comparison of probiotic propertiesof Lactobacillus casei Zhang, a potential new probiotic, with selected probioticstrains [J] .LWT-Food Science and Technology; 2009,42 (10): 1640-1646.
The centrifugal back of bacterium liquid used contain by weight the PBS protective material of 1% Tryptones and 0.1% Sodium Glutamate and in 4 ℃ of refrigerators, preserve, be used for the preparation of fermented-milk.
The composition of BCP nutrient agar is yeast extract paste 25g, peptone 5g, and glucose 5g, purpurum bromocresolis 0.04g, agar 15g are dissolved in the 1000ml water, transfer pH to 7.0.During with BCP nutrient agar separating lactic acid bacterium, can separate, be purified into lactic-acid-bacterium with colonial morphology (bacterium colony of lactic-acid-bacterium is generally tip-like) according to periphery of bacterial colonies substratum change in color (yellow) by purple stain.
G, the bacterial strain that will count are with 5 * 10
6The cfu/mL inoculum size is (95 ℃ of sterilization 10min) in 11% skimming milk, during 37 ℃ of ferment at constant temperature to pH values 4.5, sample is cooled off rapidly, and according to the method that describes below sample is carried out acidity, OD
600Value, FAN and ACE suppress active mensuration, thereby filter out the probiotic lactobacillus with hypertension potential, and the step of going forward side by side is carried out blood pressure lowering effect research in the body.
H, organize observation analysis method under the mirror through blood pressure measuring method and target organ, research with estimate of the influence of lactobacterium helveticus H9 fermented-milk to hypertension (SHR mouse) and normal arterial pressure (WKY mouse).
Blood pressure measuring method is a tail sleeve method; Specifically referring to Antihypertensiveeffect of peptides from royal jelly in spontaneously hypertensive rats [J] .Biological & Pharmaceutical Bulletin such as document K Tokunaga; 2004,27 (2): 189-192.).Observe blood pressure through feeding mouse, can analyze of the influence of its fermented-milk the mouse blood pressure with lactobacterium helveticus H9 fermented-milk.
Target organ organizes that the observation analysis method is hematoxylin-eosin staining method (hematoxylin-eosin staining under the mirror; Be called for short the HE staining); Concrete operation can reference Kiernan JA (2008) Histological and Histochemical Methods:Theory andPractice.4th ed.Bloxham, UK:Scion.Observe the pathological change that left ventricle and kidney are cut into slices through feed mouse with lactobacterium helveticus H9 fermented-milk, can the evaluation analysis fermented-milk to the influence of rat heart muscle fiber, blood vessel, renal glomerulus and uriniferous tubules unit organization pathology.
Two kinds of methods are measured as a result binding analysis lactobacterium helveticus H9 fermented-milk to the influence of Hypertensive Rats disease severity.
I, this bacterial strain processed supply examination bacterium liquid; Drawing the 1.0mL suspension bacteria liquid joins in the simulated gastric fluid of 9.0mL pH value 2.5; Digest 3h down for 37 ℃ in temperature; In its process, measure viable count with the BCP nutrient agar of Japanese Rong Yan Co., Ltd. preparation respectively at 1h, 2h, 3h sampling, and calculating bacterial strain survival rate.Then; That draws pH value 2.5 behind the 1.0mL digestion 3h contains the bacterium simulated gastric fluid; Join in the artificial pancreatic juice of 9.0mL; Continuation is cultivated under 37 ℃ of temperature, measures viable count respectively at 0h, 4h, 8h sampling with the BCP nutrient agar of Japanese Rong Yan Co., Ltd. preparation, and calculating bacterial strain survival rate.
Simulated gastric fluid is according to " Journal of Dairy Science ", and 1999,82 (2): the 243-248. preparation, its composition is NaCl 0.2%, stomach en-(pepsin, sigma) 0.30%, pH value 2.5.
Artificial pancreatic juice is according to " Journal of Dairy Science ", 1999,82 (2): the 243-248. preparation, its simulated intestinal fluid preparation method: following a liquid and b liquid are simulated intestinal fluid with mixing in 2: 1.A. pancreas liquid: trypsin Trypsin, sigma) 0.1%, after adjustment pH was 8.0, filtration sterilization was subsequent use.B. bile: Bile Salts (Difco) 1.8%, adjustment pH is 8.0, filtration sterilization is subsequent use.
N
1---bacterial strain is handled the back viable count;
N
0---the initial viable count of bacterial strain
Various measuring methods are described respectively below.
A, acid test method
Accurately weighing 5.00g fermented-milk sample places the 100mL triangular flask, removes CO toward wherein adding 40mL
2Water adds 0.5mL 0.5% (weight) phenolphthalein ethanolic soln again, carefully shakes up, till in 1min, not disappearing to blush with the titration of 0.1mol/L sodium hydroxide standard water solution.Consume 0.1mol/L sodium hydroxide standard water solution milliliter number divided by sample g number, multiply by 100 again, obtain acidity (° T).
B, OD
600Values determination method
Get 0.5mL breast appearance; Toward wherein adding 4.5mL 0.2% (weight), pH 12 ice-cold EDTA (EDTA Disodium) aqueous solution; The vibration mixing uses the spectrophotometer of day island proper Tianjin company with trade(brand)name UV-1700 sale then, is tested liquid to add the breast appearance that does not connect bacterium among the EDTA; Be determined at the absorbance at 600nm place, obtain OD
600Value.
C, free amine group Miniature coulomb titrimeter for measuring nitrogen content
50mL 100mM borax solution, 5mL 20% (weight) lauryl sodium sulfate aqueous solution, 80mg OPA (being dissolved in the 2mL methyl alcohol) and 200 μ L beta-mercaptoethanols are mixed, dissolve to 100mL surely with deionized water then, obtain a kind of reaction reagent.Then, 50 μ L fermented-milk samples are added in the said reaction reagent of 2mL, at room temperature reacted two minutes, use UV-1700 (Tianjin, island, Japan) to detect absorbancy at the 340nm place again.With casein trypsinase peptone as standard substance according to ordinary method drawing standard curve, calculate the free amine group nitrogen content according to detecting absorbancy again.
D, Zinc metallopeptidase Zace1 (ACE) suppress determination of activity
A. the fermented-milk with above-mentioned preparation carries out centrifugal 10min under the 8000g condition; The supernatant that obtains uses the 10mol/L NaOH aqueous solution with its pH regulator to 8.3 again; Under the 8000g condition, carry out centrifugal 10min again, the supernatant that obtains suppresses active testing sample as measuring ACE.
B. hippuryl histidyl-leucine (HHL) and Zinc metallopeptidase Zace1 (ACE) are dissolved in the 0.1mol/L sodium borate buffer solution (pH 8.3) that contains 0.3mol/L NaCl.In the 1.5mL centrifuge tube, add 50 μ L0.010mol/L hippuryl-histidyl-s-leucine (HHL) solution and the above-mentioned supernatant of 50 μ L; Abundant concussion mixing is hatched 2min then under 37 ℃ of temperature, add 50 μ L0.010U/mLACE solution again; Fully shake mixing; React 30min down for 37 ℃ in temperature, in 85 ℃ of water-baths of temperature, heat 10min again, make enzyme deactivation and termination reaction.Adopt reversed-phased high performace liquid chromatographic (RP-HPLC) to be determined at urobenzoic acid (Hippruic acid, HA) content in the reaction solution.The chromatographic column of using: ZORBAX C18 (4.6 * 250mm, 5 μ m, Agilent, USA); Moving phase: by by volume 22% acetonitrile (containing 0.1%TFA (trifluoroacetic acid)) and 78% deionized water (containing 0.1%TFA); Flow velocity: 1.5mL/min; Detect wavelength: 228nm; Column temperature: 30 ℃; Sample size: 20 μ L.
The ACE inhibiting rate calculates according to the following equation:
ACE inhibiting rate (%)=([HA] c-[HA] s)/([HA] c-[HA] h) * 100%
In the formula:
[HA] c is a control sample urobenzoic acid concentration (adding deionized water);
[HA] s is a testing sample urobenzoic acid concentration;
[HA] h is a urobenzoic acid concentration in the HHL standard substance.
The index screenings such as fermentation time, acidity, free amine group nitrogen content and ACE inhibition activity of taking all factors into consideration this strain fermentation breast go out to have the probiotic lactobacillus of hypertension potential, lactobacterium helveticus H9.Organize observation analysis research under the mirror, estimated of the influence of lactobacterium helveticus H9 fermented-milk through blood pressure and target organ hypertension (SHR) and normal arterial pressure (WKY mouse).Research shows that this strain fermentation breast has the interior blood pressure lowering effect of body preferably, has onset time length simultaneously, does not influence blood circulation, to characteristics such as normal arterial pressure rat (WKY) have no adverse effect.
The biological characteristics of said lactobacterium helveticus H9 is listed in the table 1.
Table 1: the biological characteristics of lactobacterium helveticus H9
In the table: L representes that the lactic acid opticity is left-handed; W representes the weak positive; + expression is positive;-expression is negative.
Cultural method and the medium preparation of described lactobacterium helveticus H9 are following:
The preparation of substratum: glucose 20.460g/L, soy peptone 10g/L, yeast powder 5g/L, Tryptones 10g/L, NaA
C6.835g/L, K
2HPO
42.929g/L, Trisodium Citrate 0.976g/L, MgSO
47H
2O 200.0mg/L, MnSO
45H
2O 54.0mg/L, L-halfcystine 0.25g/L, Tween-80 are 1g/L, and above-mentioned each component is miscible in the 943g deionized water, promptly prepare substratum.
Culture condition and equipment: the nutrient solution that will connect bacterium places LRH-500F constant incubator (Shanghai Yiheng Scientific Instruments Co., Ltd) to cultivate 15-18 hour for 37 ℃.
The present invention relates to the purposes of described lactobacterium helveticus H9 in the medicine of preparation preventing cardiovascular disease.
According to the present invention, described cardiovascular disorder is coronary heart disease, hypertension, exercise related sudden death, stenocardia, acute myocardial infarction, coronary heart disease or hyperlipidemia.
Described medicine by described lactobacterium helveticus H9 pulvis with form at pharmaceutically acceptable carrier.At pharmaceutically acceptable carrier is that one or more are selected from the carrier of normally used weighting agent, tackiness agent, wetting agent, disintegrating agent, absorption enhancer, tensio-active agent, absorption carrier, lubricant or correctives pharmaceutically.
The formulation of said medicine is tablet, pill, capsule or microcapsule formulations.
The invention still further relates to the purposes of described lactobacterium helveticus H9 in producing protective foods.
The 3.1st (function) food that will keep healthy of GB16740-1997 " health care (function) food universal standard " is defined as; Health care (function) food is a kind of food, has the general character of normal food, can regulate the function of human body; It is edible to be suitable for specific crowd, but is not purpose with the treatment disease.
According to this standard, in the present invention, described protective foods is food such as the bread that contains lactobacterium helveticus H9 according to the invention, dessert, beverage.
Lactobacterium helveticus (Lactobacillus helveticus) H9 on May 5th, 2011 in common micro-organisms DSMZ of China Committee for Culture Collection of Microorganisms (CGMCC) preservation, preserving number is CGMCC 4811.
[beneficial effect]
The fermented-milk that contains lactobacterium helveticus H9 bacterial strain of the present invention has blood pressure lowering effect in the good body, have simultaneously onset time long, do not influence blood circulation, to normal arterial pressure beneficial effect such as have no adverse effect.
[description of drawings]
Fig. 1 is separation, the purifying of lactobacterium helveticus H9 and preserves operational flowchart;
Fig. 2 representes repeatedly to irritate the influence of clothes lactobacterium helveticus H9 fermented-milk to the rat systolic pressure;
Fig. 3 representes repeatedly to irritate the influence of clothes lactobacterium helveticus H9 fermented-milk to the rat diastolic pressure;
Fig. 4 representes repeatedly to irritate clothes lactobacterium helveticus H9 fermented-milk to be influenced rat left ventricle exponential;
Fig. 5 is a rat left ventricular tissues HE dyeing pathological section (* 200);
Fig. 6 is that rat kidney is organized HE dyeing pathological section (* 200);
Fig. 7 is the tolerance of lactobacterium helveticus H9 to artificial gastrointestinal fluid.
[embodiment]
Embodiment 1: irritate clothes and contain the influence of the fermented-milk of lactobacterium helveticus H9 to test mouse blood pressure
A, preparation contain the fermented-milk of lactobacterium helveticus H9:
Lactobacterium helveticus H9 is pressed 5 * 10
6In the skimming milk of cfu/mL access 11%, 37 ℃ of fermentations, the pH value was reduced to 4.5 o'clock, and chilling is handled.
B, test materials and method
The SHR mouse: 30, male, 7 ages in week, body weight 131 ± 6g, systolic pressure 140 ± 13mmHg is divided into 2 groups at random, 15 every group (control group and H9 group).
The WKY mouse: 15, male, 7 ages in week, body weight 150.7 ± 26.7g, systolic pressure 118 ± 15mmHg.
These mouse are after the laboratory adapted to for 1 week; 3 groups of rats are implemented to irritate stomach every day: control group (saline water, 15mL/kg BW), H9 organize the (fermented-milk that contains lactobacterium helveticus H9; 15mL/kg BW) and WKY group (lactobacterium helveticus H9 fermented-milk; 15mL/kg BW), and weekly adjust its dosage (it is long-pending to irritate body of stomach) according to changes of weight.Respectively 0 day, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, these test mouse are carried out blood pressure determination during 7 weeks.The BP-98A intelligence blood pressure instrument that adopts tail sleeve method to produce with the soft grand company in Beijing under the specified condition of its specification sheets, measures SHR the caudal artery systolic pressure (Systolic Blood Pressure, SBP) and diastolic pressure (Diastolic Blood Pressure, DBP).
C, test-results
The test-results of present embodiment is listed in Fig. 2 and Fig. 3.Fig. 2 and Fig. 3 show that the fermented-milk that contains lactobacterium helveticus H9 repeatedly irritates the influence of blood pressure of stomach to the test mouse.Can be found out that by Fig. 2 and Fig. 3 the SBP of all test SHR mouse and DBP all rise with the prolongation of irritating all numbers of stomach, to be the SHR blood pressure rise with the growth in mouse age its reason.Research is proof also, and the SHR mouse has formed left ventricular hypertrophy since 6 week elevations of blood pressure in age during 10 weeks, 22-36 week blood pressure stabilization, and 30-36 week begins to occur the left chamber function obstacle.It was 7 ages in week that the present invention selects the SHR mouse for use, did not also arrive all ages of blood pressure stabilization, so the blood pressure of all SHR experimental group all raises with the increase in mouse age.
The fermented-milk group that contains lactobacterium helveticus H9 is compared with control group, and its SBP and DBP are irritating 5 weeks of stomach and 6 weeks back generation remarkable effect (P＜0.05) respectively, and this pressure reduction effect is retained to this off-test.These results of study show that the fermented-milk that contains lactobacterium helveticus H9 has good blood pressure lowering effect in vivo, and normal arterial pressure is not produced adverse influence.
Embodiment 2: the fermented-milk that the filling clothes contain lactobacterium helveticus H9 influences the left ventricular mass exponential
A, the fermented-milk that contains lactobacterium helveticus H9 that uses embodiment 1 to prepare.
B, test materials and method
The SHR mouse: 30, male, 7 ages in week, body weight 131 ± 6g, systolic pressure 140 ± 13mmHg is divided into 2 groups at random, 15 every group (control group and H9 group).
The WKY mouse: 15, male, 7 ages in week, body weight 150.7 ± 26.7g, systolic pressure 118 ± 15mmHg.
These mouse adapted to for 1 week in the laboratory after; 3 groups of rats are implemented to irritate stomach every day: control group (saline water, 15mL/kg BW), H9 organize the (fermented-milk that contains lactobacterium helveticus H9; 15mL/kg BW) and the WKY mouse group (fermented-milk that contains lactobacterium helveticus H9; 15mL/kg BW), and weekly adjust its dosage (it is long-pending to irritate body of stomach) according to changes of weight.
Off-test recession food 20h, the weighing the weight of animals is put to death through the femoral artery blood sampling again, cuts chest, abdominal cavity rapidly open, takes out the heart and kidney.The heart that takes out with normal saline flushing, filter paper blot, weigh, separate left ventricle, the left ventricle of weighing; Be divided into three then; Wherein 1 places formalin solution to fix, and all the other 2 are sub-packed in that frozen Guan Zhongyong liquid nitrogen is anxious to be frozen, and are stored in then in-80 ℃ of refrigerators.The kidney that takes out blots, weighs with normal saline flushing, filter paper, divides three then, and wherein 1 places Superlysoform to fix, and all the other 2 are sub-packed in frozen Guan Zhongyong liquid nitrogen urgency and freeze, and are stored in then in-80 ℃ of refrigerators.The liver of taking out blots, weighs, divides then three with normal saline flushing, filter paper, and wherein 1 places Superlysoform to fix, and all the other 2 are sub-packed in frozen Guan Zhongyong liquid nitrogen urgency and freeze, and are stored in then in-80 ℃ of refrigerators.
In order to study the influence of the fermented-milk that contains lactobacterium helveticus H9 to SHR mouse left ventricular hypertrophy; Then; Above-mentioned 3 groups of laboratory animal have been carried out left ventricular mass index (Left Ventricular MassIndex; LVMI), i.e. the mensuration of the ratio of left ventricular mass (mg) and body weight (g), it is measured the result and lists in Fig. 4.
C, test-results
Fig. 4 shows that the left ventricular mass index of normal arterial pressure WKY mouse significantly low (P＜0.05) is in essential hypertension (SHR mouse).In SHR mouse test group, the left ventricular mass index of fermented-milk group (H9) that contains lactobacterium helveticus H9 is significantly less than control group, explains that irritating fermented-milk that clothes contain lactobacterium helveticus H9 has good inhibition effect to the left ventricular hypertrophy of original hypertensive rat.
Embodiment 3: irritate clothes and contain the influence of the fermented-milk of lactobacterium helveticus H9 to heart and renal tissue pathology
The SHR mouse: 30, male, 7 ages in week, body weight 131 ± 6g, systolic pressure 140 ± 13mmHg is divided into 2 groups at random, 15 every group (control group and H9 group).
The WKY mouse: 15, male, 7 ages in week, body weight 150.7 ± 26.7g, systolic pressure 118 ± 15mmHg.
After the laboratory adapted to for 1 week; 3 groups of rats are implemented to irritate stomach every day: control group (saline water, 15mL/kg BW), H9 organize the (fermented-milk that contains lactobacterium helveticus H9; 15mL/kg BW) and the WKY mouse group (fermented-milk that contains lactobacterium helveticus H9; 15mL/kg BW), and weekly adjust dosage (it is long-pending to irritate body of stomach) according to changes of weight.
Experiment finishes recession food 20h, and the weighing the weight of animals is put to death through the femoral artery blood sampling again, cuts chest, abdominal cavity rapidly open, takes out the heart and kidney.Take out that normal saline flushing behind the heart, filter paper blot, weigh, separate left ventricle, the left ventricle of weighing, divide three then, wherein 1 places Superlysoform to fix, and all the other 2 are sub-packed in that frozen Guan Zhongyong liquid nitrogen is anxious to be frozen, and are stored in then in-80 ℃ of refrigerators.The kidney that takes out blots, weighs, divides then three with normal saline flushing, filter paper, and wherein 1 places Superlysoform to fix, and all the other 2 are sub-packed in frozen Guan Zhongyong liquid nitrogen urgency and freeze, and are stored in then in-80 ℃ of refrigerators.The liver of taking out blots, weighs, divides then three with normal saline flushing, filter paper, and wherein 1 places Superlysoform to fix, and all the other 2 are sub-packed in frozen Guan Zhongyong liquid nitrogen urgency and freeze, and are stored in then in-80 ℃ of refrigerators.
The myocardium of left ventricle pathological observation:
A. the making of section
Get about 0.3cm * 0.3cm size tissue block as experiment material from the rat left ventricle.Be fixed in 24h in 4% Paraformaldehyde 96/0.1M PBS solution.
(2) repair piece and flushing: material is repaired after in stationary liquid, taking out, and puts into the copper sieve then in tap water flushing 24h.
(3) dehydration: with tissue block carry out step by step that gradient alcohol dehydration: 50%1.5h, 75% spends the night, 85%1h, 95% (I) and 95% (II) each 45min, 100% (I) and 100% (II) each 30min.
(4) transparent: YLENE (I), each 15min of YLENE (II).I, II implication are described
(5) waxdip: in 60 ℃ of incubators, will organize and immerse 30min in the low melt wax.
(6) embedding: will organize from low melt wax and take out, and be embedded in and carry out embedding in the high melting-point wax.
(7) section: tissue block is cut into the thick film of 5 μ m with slicing machine.
(8) exhibition sheet: in 30% alcohol, the tissue slice that cuts is launched.
(9) paster: will open up good tissue slice in 40 ℃ of water-baths and be affixed on the slide glass.
The HE dyeing (dyeing of Hematorylin one Yihong) of B paraffin section:
(1) roasting sheet: roasting 2h on 45 ℃ of roasting sheet machines.
(2) dewaxing: YLENE 10min * 2 time.
(3) aquation: absolute ethyl alcohol 3min * 2 time, 95% ethanol 2min, 80% ethanol 2min, 70% ethanol 2min.
(4) dye nuclear: instant haematoxylin dyeing liquid 10min.
(5) flushing: tap water is washed till that observation of cell is colourless under the mirror.
(6) differentiation: 0.5% acidic alcohol differentiation, 1～2s.
(7) oil blackeite: tap water washes that observation of cell nuclear is blue to the mirror, and endochylema is colourless.
(8) dye endochylema: 95% ethanol 1min, 0.5% Yihong ethanol staining fluid 1～2min.
(9) differentiation: 80% ethanol suitably breaks up 30s.
(10) dehydration: 95% ethanol 5min * 2 time.
(11) dehydration: 100% ethanol dehydration 3min * 2 time.
(12) transparent: YLENE 5min * 2 time.
(13) neutral gum mounting.
C. microscopic examination and taking pictures.
Adopt HE dyeing tissue slice light microscopic detection method to detect above-mentioned three groups of test samples, its result lists in Fig. 5 and 6.
Fig. 5 shows, irritates and obeys the pathology situation that the fermented-milk that contains lactobacterium helveticus H9 obviously reduces the myocardium of left ventricle of SHR mouse.The myocardium of left ventricle pathological study characteristics of each test group are following:
SHR mouse control group (contrast): the cardiac muscle fibre form is changeable, and thickness does not wait, and obviously hyperplasia is loose, and arrangement disorder has the trend (A) of fracture; And the myocardial cell is loose, and (B) deepened in nucleus increase, accidental apoptosis, necrotic myocardium cell, endochylema dyeing; Vascular wall is thicker, and a peripheral part is hyaline degeneration (C).
The fermented-milk group (H9) that contains lactobacterium helveticus H9: fibrosis obviously alleviates (A) between the slightly loose phenomenon of cardiac muscle fibre, cardiac muscle fibre; Circumvascular collegen filament reduce (B).
Normotensive group (WKY mouse): normal group myocardial cell's marshalling, nucleus size homogeneous, endochylema even dyeing.
The renal tissue pathological observation:
Fig. 6 shows, irritates and obeys the pathology situation that the fermented-milk that contains lactobacterium helveticus H9 obviously reduces the renal tissue of SHR mouse.Each test group pathological observation characteristic is following:
SHR mouse control group (contrast): tube wall obviously thickens, luminal stenosis or obturation.The tube wall generation sex change, the necrosis that have become homogeneous red translucent (A); The periphery inflammatory cell infiltration, the renal glomerulus structure deteriorate is obvious, its cyst wall and surrounding tissue adhesion, matter inflammatory cell infiltration (B) between kidney.
The fermented-milk group (H9) that contains lactobacterium helveticus H9: the quantity that lesion vessels appears in the kidney arteriole reduces (A), and lesion degree alleviates; SHR mouse treatment group glomerular volume slightly becomes greatly, structure comparatively complete (B).
Normotensive group (WKY mouse): vessel wall does not have and thickens, and does not see pathological change (A); Normal group renal glomerulus structural integrity, volume is little, the uriniferous tubules clear in structure, matter does not have inflammatory cell infiltration (B) between kidney.
Embodiment 4: the tolerance of lactobacterium helveticus H9 in Gl tract
In the pH value is respectively 2.0,2.5 sterilization PBS, adds the 3.0g/L stomach en-with the filtration sterilization method and process simulated gastric fluid; In the sterilization PBS of pH 8.0 (with 0.1mol/L NaOH adjustment), the trypsinase that adds 1.0g/L with the filtration sterilization method is processed artificial pancreatic juice.
Get 1.0ml lactobacterium helveticus H9 bacterium liquid and join in the simulated gastric fluid among the 9.0ml 1.2.5,37 ℃ of digestion 3h measure viable count respectively at 1h, 2h, 3h sampling with colony counting method.Afterwards, pH value 2.5 manual works behind the absorption 1.0ml digestion 3h contain bacterium gastric juice, join in the artificial pancreatic juice of 9.0ml, continue at 37 ℃ of cultivations, measure viable count respectively at 0h, 4h, 8h sampling with colony counting method.
The bacterial strain survival rate is used following formula and is calculated
[114]:
N
1---bacterial strain is handled the back viable count;
N
0---the initial viable count of bacterial strain
Learn that from Fig. 7 lactobacterium helveticus H9 bacterial strain 2h in pH 2.5 simulated gastric fluids decreases with interior survival rate but difference not significantly (P＞0.05).Prolong survival rate with digestion time and have downtrending, but shown that this bacterial strain has tolerance preferably to the simulated gastric fluid of pH 2.5.Lactobacterium helveticus H9 bacterial strain from pH 2.5 simulated gastric fluids 37 ℃ keep 3h after, change in the simulated intestinal fluid of pH 8.0 and keep 8h, its result is as shown in Figure 7.From figure, find out that this bacterial strain survival rate has obvious ascendant trend in the simulated intestinal fluid of pH 8.0, this bacterial strain survival rate has obtained phenomenal growth (P＜0.05) when being retained to 8h.
Claims (8)
1. a lactobacterium helveticus (Lactobacillus helveticus) H9, this bacteria strain on April 28th, 2011 in the preservation of common micro-organisms DSMZ of China Committee for Culture Collection of Microorganisms, preserving number is CGMCC 4811.
2. the purposes of lactobacterium helveticus H9 according to claim 1 in the medicine of preparation preventing cardiovascular disease.
3. purposes according to claim 2 is characterized in that described cardiovascular disorder is coronary heart disease, hypertension, exercise related sudden death, stenocardia, acute myocardial infarction, coronary heart disease or hyperlipidemia.
4. purposes according to claim 2, it is characterized in that described medicine by described lactobacterium helveticus H9 pulvis with form at pharmaceutically acceptable carrier.
5. purposes according to claim 3 is characterized in that at pharmaceutically acceptable carrier it being that one or more are selected from the carrier of normally used weighting agent, tackiness agent, wetting agent, disintegrating agent, absorption enhancer, tensio-active agent, absorption carrier, lubricant or correctives pharmaceutically.
6. purposes according to claim 2, the formulation that it is characterized in that said medicine are tablet, pill, capsule or microcapsule formulations.
7. the purposes of lactobacterium helveticus H9 according to claim 1 in producing protective foods.
8. purposes according to claim 7 is characterized in that described protective foods is bread, dessert or the beverage that contains the said lactobacterium helveticus H9 of claim 1.
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