A kind of lactobacillus plantarum CW 006 with antihypertensive function
Technical field
The present invention relates to the new bacterial strain of a kind of plant lactobacillus (Lactobacillus plantarum), and this bacterial strain is being produced and manufacturing has application aspect hypertension (and/or hypotensive) the effect product, for example purposes in food-processing and the pharmacy industry.Belong to the microbial product technical field.
Technical background
Hypertension is that a kind of the rising with arteries systolic pressure or diastolic pressure is the clinical syndrome of feature, is a kind of common cardiovascular disorder.Its sickness rate height, often be attended by disease and the functional or organic changes of heart, blood vessel, brain and kidney and other organs such as hyperlipidemia, coronary heart disease, diabetes, atherosclerosis, causing very big threat to healthy, is global great public health problem.
In organism, many systems all with the confidential relation of having kept of blood pressure, wherein important have central nervous system, suprarenin system, renin-angiotensin (RAS) system, bradykinin-kassinin kinin-prostaglandin(PG) (KKS) system and a blood vessel endothelium relaxation factor-contraction factor system etc.These systems are subjected to the influence of internal and external environment and change, thereby participate in the regulation and control of blood pressure.
(Angiotensin Converting Enzyme ACE) finds to have vital role in the blood pressure regulation process by Skeggs (1954) to Zinc metallopeptidase Zace1 first from horse blood.ACE can be hydrolyzed to activated Angiotensin II (AngII) with the angiotensin I (AngI) of non-activity in the RAS system on the one hand, promotes the contraction of blood vessel, makes elevation of blood pressure; On the other hand, in the KKS system, it can make the vasodilatory loss of activity of BK with bradykinin (BK) hydrolysis, has further increased the weight of the rising of blood pressure.When taking angiotensin converting enzyme inhibitor (Angiotensin converting enzyme inhibitor, ACEI) time, ACEI can suppress the activity of ACE enzyme effectively, make AngI can not be converted into AngII, BK also still keeps its vasodilatory activity, thereby keeps the blood pressure stabilization of organism.
At present, a line antihypertensive drug of WHO/ISH recommendation is six big classes such as diuretic(s), beta receptor blocker, angiotensin converting enzyme inhibitor (ACEI), calcium channel blocker (CaA), alpha receptor blocking agent, Angiotensin (Ang) receptor antagonist.Different medicines has different characteristics, and certain side effect is also arranged.
ACEI is effective to each phase hyperpietic, does not influence the regulating effect of sympathetic nerve to vascular smooth muscle, does not also cause the heart rate quickening, and the main adverse reaction of synthetic ACEI is for easily causing fash, proteinuria, oligoleukocythemia etc.Potential changes to the metabolism aspect in the use antihypertensive drug therapeutic process, particularly there is detrimentally affect fat, sugar and uric acid metabolism aspect; Pharmacological agent also is the reason that causes Incidence of CHD to increase; Drug-induced blood pressure drops might cause brain blood to reduce, and can cause in cerebral ischemia, the ischemic cerebral apoplexy and visual disorder etc.Therefore hypertensive non-drug therapy more and more draws attention.The stepped care scheme that WHO recommends with non-drug therapy as the hypertensive basis of treatment.
Since (1970) such as Ferreira from a kind of snake venom, found first a kind of have suppress the active little peptide of ACE since, in various food separation and purification to the antihypertensive peptide that differs of various structures, sequence and length scale.1979, reported first such as Oshima derive from the antihypertensive peptide of food protein, the researchist utilizes the Collagenase gelatin hydrolysate, obtain nine and had the inhibiting little peptide of ACE, wherein six have significant ACE enzyme inhibition, and its C-end all has Ala-Hyp (Hyp:Hydroxyproline, oxyproline) structure.Japan scholar Suetsuna etc. is used to come from the denazyme AP enzymic hydrolysis sardines a kind of by name of aspergillus oryzae, obtain two molecular weight sizes and be the little peptide of 1000-2000, this little peptide has stronger ACE enzyme inhibition activity, measure through experiment in vitro, IC50 is (under the condition determination of ACE enzyme inhibition activity, the concentration that can suppress active needed this little peptide of ACE enzyme 50%, same down) be respectively 39.7mg/100ml and 90.1mg/100ml.Yano etc. utilize thermophilic protein enzymic hydrolysis zein, obtained a large amount of little peptide fragment, the IC50 of the ACE enzyme inhibition activity of total hydrolyzate is 24.5 μ g/ml, and wherein a tripeptides (Leu-Arg-Pro) ACE enzyme inhibition activity is the strongest, also has antihypertensive activity in vivo.In addition, vinasse (Saito etc., 1994a, b), royal jelly (Matsui etc., 2002), tuna (Matsumura etc., 1993; Yokoyama etc., 1992), also found the existence of antihypertensive peptide in the milk-acid bacteria extract food proteins such as (Furushiro etc., 1990).
When microorganism that part is specific such as lactobacterium helveticus (Lactobacillus helveticus) and cereuisiae fermentum (Saccharomyces cerevisiae) and lactobacterium helveticus (Lactobacillus helveticus) LBK-16H grow in the matrix that with the breast is base-material, hydrolytic action by secreted proteolytic enzyme, be that extracellular protein enzymic digestion animals milk protein produces various peptide fragment, the peptide fragment that is produced further generates low-molecular-weight peptide through the effect of peptase, these particular peptide fragments are discharged, thereby produce hypotensive activity in vivo.Yamoto etc. disclose lactobacterium helveticus CM4 in International Patent Application WO 99/16862, FERM BP-6060 can produce tripeptides Val-Pro-Pro and/or Ile-Pro-Pro, and the tripeptides of these two kinds of sequences has stronger ACE restraining effect.The United States Patent (USP) 5,449,661 of Nakamura etc. has been described and has been adopted lactobacillus helveticus strain jcm 1004 fermented skim milks to prepare the method for Val-Pro-Pro tripeptides.Annika etc. are at United States Patent (USP) 6,890, and 529 have described lactobacterium helveticus LBK-16H, and DSM13137 can produce biologically active peptides Val-Pro-Pro and Ile-Pro-Pro and can promote NO production, thereby reduce hypertension.In addition, the part microorganism can be synthesized some material in process of growth, also has hypotensive activity in primary hypertension mouse (SHR) body.
Be separated to a kind of polysaccharide-glycopeptide complex in the aqtocytolysis thing as lactobacterium casei YIT9018, SHR takes back blood pressure drops 10-20mmHg.
The milk-acid bacteria of antihypertensive function at present research focuses mostly in lactobacterium helveticus and this two big bacterial strain of lactobacterium casei, and searching can be used as starter and probiotic bacterium and can be used as that natural product are used and to be used for the new effective microorganism of pharmacy industry significant in foodstuffs industry in this field.
Summary of the invention
Purpose of the present invention: at above problem, with traditional fermented food (fermented soya bean crust, beans sauce, sour milk, tibet koumiss) milk-acid bacteria in is the target flora, adopt specific in-vitro screening model, therefrom filter out the beneficial natural disposition milk-acid bacteria that fermented-milk produces the step-down peptide later on, and further studied its function and application aspect hypertension.
Technical scheme of the present invention: a kind of lactobacillus plantarum CW 006 is provided, and this bacterium has antihypertensive effect.
According to the present invention, the bacterial preparation that contains the bacterial strain lactobacillus plantarum CW 006 is provided for the human consumer, this bacterial preparation can also contain other microorganism, and this bacterial preparation can be freeze dried powder or capsule, uses at this point or is used for food, functional product or medicine production.
The present invention relates to contain above-mentioned bacterial strains or milk-product or beverage product, beverage product preferred whey beverage, fruit beverage or beer by using above-mentioned bacterial strains to prepare.
The invention still further relates to and utilize the application of bacterial strain lactobacillus plantarum CW 006 in preparation hypertension product.
The sepn process of plant lactobacillus among the present invention (Lactobacillus plantarum) CW006 is as follows: with traditional fermented food (fermented soya bean crust, beans sauce, sour milk, tibet koumiss) is sample, sample is after pre-treatment, PBS damping fluid with pH6.8 soaks mixing and gradient dilution, select suitable extent of dilution solution coat on MRS and LBS flat board, 37 ℃ of anaerobism were cultivated 48 hours, picking colonies typical (bacterium colony circle, smooth and the neat in edge of surface wettability etc.) be forwarded to the dull and stereotyped purifying of MRS, picking list bacterium colony is transferred and is increased bacterium into MRS liquid, obtains lactic bacterium strains and preservation.
Switching is gone in the fresh MRS substratum milk-acid bacteria cultivated 24h in the MRS substratum under 37 ℃ of conditions after, cultivate 12h with condition, thalline is centrifugal, resuspended with the PBS damping fluid, in the skimmed milk (SKM) with 1% access 10%, 37 ℃ of fermentation certain hours, fermented-milk is through 4 ℃ of 8500 * g frozen centrifugation 10min, it is 8.3 that the gained clear liquid is transferred pH with 10mol/LNaOH, preserves to be measured under-70 ℃ of conditions.Suppress the method that the active mensuration of ACE adopts Cushman and Cheung.For having obvious inhibiting bacterial strain to carry out strain identification to ACE, obtain lactobacillus plantarum CW 006, and measure the hypotensive activity of its tunning in spontaneous hypertensive rat (SHR) external.
This bacterial strain has following characteristic:
Lactobacterium plantarum strain CW006 is Gram-positive, staff cell.The temperature range of growth is 17-52 ℃, and optimum temperuture is 34-40 ℃.
In the time of 34-40 ℃, lactobacillus plantarum CW 006 produces 1.8%-2.1% lactic acid (DL) at the Ruzhong well-grown.
The optimal pH of growth is 5.8-6.5.In the cultivation of not carrying out pH regulator, the pH reduction scope of breast is 4.2-4.5.
This bacterial strain also well-grown in being generally used for the substratum of genus lactubacillus.
The carbohydrate of lactobacillus plantarum CW 006 utilizes situation as follows:
The comparison of table 1 API qualification result and classical qualification result
API groove number
1 |
Carbohydrate |
CW006
2 |
Lactobacillus plantarum
3 |
4 5 |
Pectinose ribose |
+ + |
d + |
6 10 11 12 13 15 18 19 23 25 26 27 28 29 30 31 32 34 35 47 |
Wood sugar semi-lactosi dextrose fructose seminose rhamnosyl N.F,USP MANNITOL sorbyl alcohol Vitamin B17 Vitamin C2 saligenin cellobiose maltose lactose melibiose sucrose trehalose melizitose raffinose glyconic acid |
d + + + + - + + + + + + + + + + + + + + |
d + + + + - + + + + + + + + d + + + + + |
1 represents API test kit reaction groove number.
2 represent the strain identification result of CW006 through the API system.
3Bergey ' s handbook is anti-about the carbohydrate of plant lactobacillus
Should describe: "+" positive, "-" feminine gender, " d " is not obvious.
Lactobacillus plantarum CW 006 has proteolytic activity.Measure (this method is stated and measured protein hydrolysis degree based on Phthalyldicarboxaldehyde) by the OPA method in following examples, the proteolytic activity of this bacterial strain is 0.3-0.4.Can utilize proteolytic activity as starter and the preparation that is used for biological active agents.
Lactobacillus plantarum CW 006 has fabulous tolerance to low pH value and bile.From the angle of keeping quality and physiologically active, this is an important properties.The result shows that this bacterial strain can be by surviving behind stomach and the small intestine in digestive tube, and still can survive in colon.
Lactobacillus plantarum CW 006 has the ability that produces biologically active peptides.And antihypertensive peptide still keeps 80% original activity behind the simulation gastro-intestinal digestion.
Beneficial effect of the present invention: the lactobacillus plantarum CW 006 among the present invention can be used to prepare the specialty products that contain biologically active peptides admirably, or as the composition or the additive of Edible material.Can the single culture lactobacillus plantarum CW 006 to form pure growth, also can cultivate this bacterial strain, individually or with using that routine is used with obtainable starter of commerce or probiotic bacterium with the form of mixed culture.
Description of drawings
Fig. 1 contrasts different lactic bacterial protein hydrolysis to be changed and the ACEI variation relation
Fig. 2 lactobacillus plantarum CW 006 tunning is irritated behind the stomach in 24 hours the influence to the spontaneous hypertensive rat blood pressure
The biological material specimens preservation
Plant lactobacillus (Lactobacillus plantarum) CW006 has been preserved in Chinese typical culture collection center, is called for short CCTCC, and preservation date is on April 10th, 2006, and deposit number is CCTCCM206032.
Embodiment
Below will the present invention be described in more detail by embodiment.These embodiment only have illustrative, and the present invention is not subjected to the restriction of these embodiment.
Embodiment 1 preliminary screening
With traditional fermented food (fermented soya bean crust, beans sauce, sour milk, tibet koumiss) is sample, experiment separates milk-acid bacteria with catalase through gramstaining, purifying, preserve, switching is gone in the fresh MRS substratum milk-acid bacteria that preserves cultivated 24h in the MRS substratum under 37 ℃ of conditions after, cultivate 12h with condition, thalline is centrifugal, and is resuspended with the PBS damping fluid, in the skimmed milk (SKM) with 1% access 10%, 37 ℃ of fermentation certain hours, fermented-milk is through 4 ℃ of 8500 * g frozen centrifugation 10min, and it is 8.3 that the gained clear liquid is transferred pH with 10mol/LNaOH, preserves to be measured under-70 ℃ of conditions.Suppress the method that the active mensuration of ACE adopts Cushman and Cheung (1971) in the fermented-milk.
O.D
ALight absorption value when not having inhibitor
O.D
BLight absorption value when having inhibitor and ACE
O.D
CLight absorption value when not existing for inhibitor and ACE
Obtain primary election bacterial strain such as following table
Table 2ACE suppresses the living lactic acid bacteria results of preliminary screening
Strain number |
Cell count * 10
8CFU/mL
|
Titration acidity (%) |
ACEI (%) |
Colonial morphology |
Thalli morphology |
The source |
CZ0032 CZ0043 CZ0027 CZ0023 CW006 DC25 |
2.4 8.1 9.9 9.5 3.0 2.0 |
0.498 1.026 0.917 1.604 1.769 0.411 |
61.7 81.8 79.5 80.6 83.1 29.9 |
The white mellow and full milky white grain of rice shape breast of milky white transparent milky white product slimy milk is transparent from mellow and full milky white |
Ball quarter butt quarter butt bar bar ball |
Tibet koumiss tibet koumiss tibet koumiss tibet koumiss tibet koumiss fermented soya bean crust |
DC05 DC10 S3 S4 B6 DJ201 |
3.8 4.1 3.7 4.7 6.9 4.8 |
0.517 0.615 1.635 0.742 1.109 0.917 |
47.8 35.4 68.8 51.7 72.4 38.2 |
It is mellow and full that milky white product slimy milk Virgin's milk produces the white transparent milky white of slimy milk Virgin's milk in vain |
Bar ball quarter butt bar quarter butt quarter butt |
Fermented soya bean crotons fermented soya beans, salted or other wise crust pickles pickles pickles Korea beans sauce |
The acidproof anti-bile characteristic of embodiment 2 milk-acid bacterias
Get experimental strain and suspend respectively in the MRS of pH2.0 and pH3.0 substratum, cultivated 3 hours for 37 ℃, measure the viable bacteria survival rate with the plate count method.
Experimental strain inserts respectively among MRS and the MRSO (MRS that adds 0.3% oxgall) by 1% inoculum size, and 37 ℃ of cultivations reach time difference that 0.3 unit increases judging basis as bacterial strain bile tolerance with MRS and MRSO nutrient solution at A620nm.
The survival rate of table 3 milk-acid bacteria under sour environment
Bacterium numbering |
Viable bacteria survival rate (%) |
pH3.0 |
pH2.0 |
CZ0032 CZ0043 CZ0027 CZ0023 CW006 DC25 DC05 DC10 S3 S4 B6 DJ201 |
81.54 50.77 80.00 64.44 79.32 52.78 72.60 74.55 79.20 81.35 88.17 60.94 |
32.31 20.77 31.11 24.44 29.67 19.67 27.95 10.30 17.17 29.42 26.45 26.56 |
Table 4 milk-acid bacteria increases progressively the time contrast in MRS and MRSO
Strain number |
The A620nm value reaches 0.3 unit and increases the needed time (h) |
MRS |
MRSO (0.3% oxgall) |
LT
b(h)
|
CZ0032 CZ0043 C70027 CZ0023 |
4.12 5.79 7.14 8.63 |
4.81 6.67 10.69 10 |
0.69 0.88 3.55 1.37 |
CW006 DC25 DC05 DC10 S3 S4 B6 DJ201 |
3.69 8.46 6.77 5.19 7.69 5.88 5.61 11.21 |
4.20 12.22 7.80 7.91 9.30 6.06 5.83 13.79 |
0.51 3.76 1.03 2.72 1.61 0.18 0.22 2.58 |
Embodiment 3 simulation gastro-intestinal digestions suppress active influence to ACE
Be that (removing to see through liquid) is used for measuring after the cellulose membrane uf processing of 10000Da with the above-mentioned whey for preparing through molecular weight cut-off.Method according to Sandrine Parrot, with 1M hydrochloric acid whey pH is transferred to 2.0, get 10ml sample liquid and add 1ml pepsin solution (the 0.1g stomach en-is dissolved in 1L0.01M hydrochloric acid), wherein E/S is 1: 200, in 37 ℃ of shaking baths (160 times/min) digestion reaction 90min, termination reaction behind 85 ℃ of heating in water bath 5min then, with 4% sodium hydroxide sample liquid pH is transferred to 7.5, add 0.2ml trypsin solution (0.5g trypsinase is dissolved in the 1L deionized water), wherein E/S is about 1: 200, above-mentionedly react 240min, again through 85 ℃ of heating in water bath 5min termination reactions with under the condition.Use gastric pepsin digestion 90min, tryptic digestion 30min respectively, 120min takes a sample during 240min, measures its ACE and suppresses active.
Table 5 simulation gastro-intestinal digestion suppresses active influence to ACE
The numbering of bacterium |
ACEI before the digestion |
ACEI behind the gastric pepsin digestion 90min |
ACEI behind the tryptic digestion 30min |
ACEI behind the tryptic digestion 120min |
ACEI behind the tryptic digestion 240min |
CZ0032 CZ0043 CZ0027 CZ0023 CW006 DC25 DC05 DC10 S3 S4 B6 DJ201 |
65.2 80.4 77.6 79.8 82.2 35.4 45.2 33.7 70.9 48.7 68.8 36.2 |
48.4 64.5 77.7 82.1 85.6 49.3 33.4 18.7 74.5 35.2 74.5 24.1 |
39.6 46.3 48.7 76.4 74.5 25.1 34.2 9.8 60.1 30.7 62.7 19.5 |
26.2 35.8 60.3 66.8 76.7 20.6 20.7 30.6 65.7 31.5 60.9 21.2 |
16.7 28.9 50.2 61.3 72.3 22.7 39.4 30.6 63.5 29.8 60.3 19.8 |
The mensuration of embodiment 4 lactic bacterial protein hydrolytic activities
With reference to o-phthalaldehyde(OPA) (OPA) method of Church, the activatory milk-acid bacteria is inoculated in the degreasing reconstituted milk (NFS is 10%) with 1% inoculum size, cultivated 12 hours down at 37 ℃, use not contain the corresponding newborn sample of inoculum as blank.Accurately draw 2.5 ml samples, add in the test tube, add 0.5ml double distilled water mixing again, and then add 0.5ml0.75mol/l trichoroacetic acid(TCA) (TCA), through the vortex mixing, under room temperature, left standstill 10 minutes, under 3000r/min centrifugal 5 minutes, it was standby to get supernatant liquor.Get supernatant liquor 150ul and add in the test tube, add 3ml o-phthalaldehyde(OPA) (OPA) reagent again, behind the mixing, at room temperature reacted 2 minutes, survey absorbancy in the 340nm place, as shown in Figure 1.
5 couples of SHR of embodiment irritate stomach CW006 fermented whey and measure its systolic pressure variation
After 48 hours, fermented supernatant fluid obtains powdered mixture, as laboratory sample to lactobacillus plantarum CW 006 after centrifugal ultrafiltration is handled to 37 ℃ of fermentations of 10% skimmed milk.
Laboratory animal: spontaneous hypertensive rat (SHR), male 26 ages in week, body weight 388 ± 30 gram is provided by Chinese Academy of Sciences's Shanghai Experimental Animal Center, animal conformity certification number: Shanghai is moving closes the card word No. 152.
Clear-headed rat blood pressure is measured and is adopted SHR rat electronic blood pressure instrument (production of Beijing China-Japan Friendship Hospital) to carry out the indirect manometry of caudal artery, places 38 ℃ of incubator internal heating 15-20min. to measure systolic pressure rat.
8 of spontaneous hypertensive rats are divided into two groups at random: give the control group of physiological saline, give the low dose group of 0.6g fermented-milk lyophilized powder/Kg body weight, 4 every group.Rat begins pressure measurement in administration the last week, treats to begin experiment behind the blood pressure stabilization, measures blood pressure before the rat administration earlier, gastric infusion then, and the step-down of medicine to rat measured at every interval in 2 hours respectively.
The result shows that the hypotensive effect of tunning arrived peaking in 8 hours.
Table 6 lactobacillus plantarum CW 006 tunning is irritated the influence of stomach to the spontaneous hypertensive rat blood pressure
The experiment grouping |
Blood pressure (mmHg) before the administration |
Blood pressure after the administration (mmHg) |
First day |
The 5th day |
The 16 day |
The control group low dose group |
207±14 198±15 |
204±18 174±20 |
199±13 168±16 |
203±12 185±23 |
Application Example 1 utilizes the lactobacillus plantarum CW 006 manufacturing to have the lactobacillus drink of antihypertensive function
Raw dairy (skimmed milk, fresh milk, recovery milk etc.) is handled postcooling to 5 ℃ through the high-strength hot in twice 95 ℃, 20 minutes or 140 ℃, 2 seconds, add lactobacillus plantarum CW 006 of the present invention, make its concentration reach 10
9Cfu/ml, 5 ℃ of stored refrigerated promptly obtain the viable bacteria milky-drinks.
Application Example 2 utilizes the lactobacillus plantarum CW 006 manufacturing to have the milk powder of antihypertensive function
Raw dairy (skimmed milk, fresh milk, recovery milk etc.) is handled postcooling to 5 ℃ through the high-strength hot in twice 95 ℃, 20 minutes or 140 ℃, 2 seconds, connect the bacterium amount with 4% of raw dairy volume and add lactobacillus plantarum CW 006 of the present invention, 37 ℃ fermented the centrifuging and taking supernatant liquor 48 hours.With homogeneous in the raw dairy after 1: 3 (V/V) adding sterilization, after vacuum concentration, spray drying treatment, obtain hypotensive milk powder, or make hypotensive capsule product.
Application Example 3 utilizes the lactobacillus plantarum CW 006 preparation to have the sour milk of antihypertensive function
Raw dairy (skimmed milk, fresh milk, recovery milk etc.) is handled postcooling to 5 ℃ through the high-strength hot in twice 95 ℃, 20 minutes or 140 ℃, 2 seconds, amount with the 3%-5% volume percent adds lactobacillus plantarum CW 006 of the present invention, and add can symbiotic preparation sour milk commercial starter lactobacillus bulgaricus and thermophilus streptococcus, 37 ℃ of mixed fungus fermentation to titration acidities are 0.6%-0.7% (in lactic acid), and refrigeration to 5 ℃ and stored refrigerated promptly obtains sour milk with function of lowering blood pressure.