CN104593307B - With the Lactobacillus rhamnosus and purposes for inhibiting angiotensins enzyme effect - Google Patents

With the Lactobacillus rhamnosus and purposes for inhibiting angiotensins enzyme effect Download PDF

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CN104593307B
CN104593307B CN201510088465.1A CN201510088465A CN104593307B CN 104593307 B CN104593307 B CN 104593307B CN 201510088465 A CN201510088465 A CN 201510088465A CN 104593307 B CN104593307 B CN 104593307B
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lactobacillus rhamnosus
bacterium colony
ace
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邹积宏
刘红梅
贺龙惠
袁杰利
金涛
姜岸
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Harbin jiaoyangji Biotechnology Co., Ltd
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Heilongjiang University
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Abstract

With the Lactobacillus rhamnosus and purposes for inhibiting angiotensins enzyme effect, with deepening continuously for the researchs such as associated biomolecule technology, physical-fitness medicine subject, the effects that new bioactive ingredients of hypertension, functional gene, functional protein novel targets successive discovery, to filter out efficient decompression, safety, the probiotic products of high activity provide the foundation condition.The present invention is by being collected in spontaneous fermentation bean product, and through detaching, purifying, physical mutagenesis, the high activity bacterial strain preferably obtained, mycology specific name actobacillus rhamnosus HLH 20120717, bacteria characteristic is：It is placed in the culture medium of 37 DEG C of constant incubators and is cultivated, find that diluted concentration is 10 by culture‑5When bacterium growth it is better, bacterium colony dispersion；Bacterium colony growth under the same conditions varies, and bacterium colony is rounded, and milky, neat in edge, surface is smooth, wherein smaller bacterium colony is yellowish；Preserving number is：CGMCC No.7084.The present invention provides basis for further the probiotics functional health-care food of research and development prevention and treatment hypertension and drug.

Description

With the Lactobacillus rhamnosus and purposes for inhibiting angiotensins enzyme effect
Technical field
The present invention is by preferably obtaining a kind of Lactobacillus rhamnosus HLH 20120717 (Lactobacillusrhamnosus HLH 20120717) inhibits angiotoninase (ACE) to make for fermenting to prepare to have Soya-bean milk and relevant food.
Background technology
With the continuous development of world economy, people’s lives level improves, using hypertension as the angiocardiopathy of representative Harm increasingly aggravate.All precautionary measures, treatment means are taken, generation, the development of hypertension is controlled, is depressured with reasonable application Drug, which reduces its illness rate and the death rate, has become the international common recognition for being directed to hypertension.Current clinically primary treatment hypertension Drug such as diuretics, β-receptor blocking pharmacon, calcium channel blocker, angiotensin converting enzyme inhibitor, angiotensinⅡ by Body blocking agent etc..But there are certain side effect or adverse reaction, easily draw hepatic and renal function harm, as prophylactic or long-term clothes With just more unreasonable.
At present, the pathogenic factors of corresponding hypertension, can be by improving bad life habits, rational diet, strengthening health religion The generation of modes preventing hypertension to greatest extent such as educate, it has also become the emphasis of world's publicity is reached with functional food Prevention, the dual purpose for the treatment of hypertension, the exploitation of wherein probiotics and products thereof is more popular.
To the research and application starting of probiotics than later, government and place are paid much attention to, and have been put into state at present in China The emphasis of family's research in new high-tech development plan supports project.But research and development has angiotensin converting enzyme (ACE) suppression System activity probiotic composition report or it is fewer, with the continuous depth of the researchs such as associated biomolecule technology, physical-fitness medicine subject Enter, the successive discovery of novel targets the effects that the new bioactive ingredients of hypertension, functional gene, functional protein, to filter out height Effect decompression, safety, the probiotic products of high activity provide the foundation condition.
Invention content
The object of the present invention is to provide one plant of fermentation with the rhamnose for inhibiting angiotoninase (ACE) effect soya-bean milk Lactobacillus HLH 20120717 (Lactobacillus rhamnosus HLH 20120717) prevents for further research and development Basis is provided with the probiotics functional health-care food and drug for the treatment of hypertension.
Above-mentioned purpose is achieved through the following technical solutions：
One kind has the Lactobacillus rhamnosus for inhibiting angiotoninase (ACE) effect from fermented soybean milk, by acquiring In spontaneous fermentation bean product, through detaching, purifying, physical mutagenesis, the high activity bacterial strain preferably obtained, mycology specific name Actobacillus rhamnosus HLH 20120717, bacteria characteristic are：Be placed in the culture medium of 37 DEG C of constant incubators into Row culture, bacterium grows better when finding that diluted concentration is 10-5 by culture, bacterium colony dispersion；Bacterium colony is given birth under the same conditions Length varies, and bacterium colony is rounded, and milky, neat in edge, surface is smooth, wherein smaller bacterium colony is yellowish；Preserving number is： CGMCC No.7084。
Described has the Lactobacillus rhamnosus for inhibiting angiotoninase (ACE) effect, bacterium from fermented soybean milk Characteristic is learned to further include：Gram's staining result is positive；Spore staining thalline is in green；There is no bubble production in catalase test It is raw；Molten calcium circle method culture observes that transparent circle occurs in periphery of bacterial colonies, the feature for having apparent lactic acid bacteria.
Described has the Lactobacillus rhamnosus for inhibiting angiotoninase (ACE) effect, warp from fermented soybean milk 16S rDNA partial sequences carry out BLAST comparisons online, as a result with Lactobacillus Pseudomonas rhamnosus strains Central genetic characteristic：
16S rDNA sequencings partial sequence is as follows：
The Lactobacillus rhamnosus from fermented soybean milk with inhibition angiotoninase (ACE) effect, it is described Culture medium prescription is：
The ratio is ratio of weight and number.
1 Lactobacillus rhamnosus HLH 20120717 of table is through VITEK 2Compact qualification results
A kind of soya-bean milk made using above-mentioned Lactobacillus rhamnosus, by the inoculation in through soybeans soaking, is ground It grinds, be homogenized, boil, in soya-bean milk made from filtering, high-temperature sterilization, 37 DEG C -42 DEG C ferment 24 hours or more, and tamarind slurry is made.
A kind of production method of soya-bean milk described in preceding：
It weighs to be put in 250mL tap water after 100g washing soybeans and impregnates 12h at room temperature；After peeling, clear water It cleans, by soaked soybean with beans water ratio tissue mashing machine's defibrination of weight ratio 1: 8, is heated to after boiling 10 minutes With after three layers of filtered through gauze.The 15min that sterilizes in triangular flask in 121 DEG C is poured into, it is sterile to be chosen with oese after gauze sealing cooling The 20120717 bacterium colony five rings of Lactobacillus rhamnosus HLH of MRS test tube slants culture is taken, after shaking uniformly, is placed on 37 DEG C of trainings Support 12 hours, be cooled to room temperature, after be put into that 4 DEG C of refrigerator is cold to be deposited, as spare soya-bean milk；
It ibid weighs to be put in 50000mL tap water after 2000g washing soybeans and impregnates 12h at room temperature；Through peeling Afterwards, clear water is cleaned, by soaked soybean with the beans water ratio soy bean milk making machine defibrination of 1: 8 weight ratio, after being heated to boiling, Centrifugal filtration removes bean dregs；Through Pasteurizer 135 DEG C of sterilizings, 10 seconds quick sterilizations, it is placed in the good fermentation tank of washing and sterilizing, Above-mentioned spare fermented soybean milk is added in by 5% volume ratio, with 30r/min, 37-40 DEG C of fermentation 2 hours then dispenses, is close It seals in Yoghourt plastic cup, after room temperature is fermented 12 hours, is put into that 4 DEG C of refrigerator is cold to be deposited；
Remainder is placed in the good large beaker of washing and sterilizing, and it is more than hour to ferment 24 for room temperature after gauze sealing, works as pH Reach 4.9 hereinafter, tamarind slurry is all stirred into flowable character, the enamel of tiling 2cm thickness by tamarind slurry in shape, blender is solidified In disk, -40 DEG C to 30 DEG C freeze-dryings into crisp yellow solid, are ground into 100 mesh powders, through Kjeldahl nitrogen determination Protein content 47%.Low temperature drying is sealed to preserve.
By the above-mentioned Lactobacillus rhamnosus from fermented soybean milk with inhibition angiotoninase (ACE) effect, use Inhibit angiotoninase (ACE), the purposes for making the health food of prevention and treatment hypertension and drug in making.
Advantageous effect：
Probiotics has plurality of health care functions, reduces and adjust the function of blood pressure, the comparison of reporting for work of research is more, probiotics pair Hypertension health-care effect has attracted great attention.Why probiotics can generate decompression and adjust the effect of blood pressure, mainly special Determine the extracellular proteinase of probiotics, digest food proteins, generation can release antihypertensive activity peptide matters, as angiotensins turns Change enzyme inhibition peptide etc.；Discharging in cell wall has antihypertensive effect peptide glycan and minerals, has significant；Third, part is prebiotic Bacterium can promote absorption of the body to certain minerals for adjusting blood pressure.Probiotics blood pressure lowering is prepared using modern biotechnology to produce Product have wide Development volue and market prospects.
Angiotensin-Converting (Angiotensin Converting Enzyme, ACE) is that last century mid-term finds anti- Hypertension drug target position enzyme passes through the adjusting of feritin-angiotensins-kinin system.Angiotensin I can be converted Into the Angiotensin II with vasoconstriction, boosting；ACE degrades the bradykinin with buck functionality simultaneously, It is allowed to lose antihypertensive effect, cause elevation of the blood pressure activity.Angiotensin converting enzyme inhibitor (ACEI) is clinically wide at present The novel antihypertensive medicament of general use, significant in efficacy, effect safety.It is mainly used for treating angiocardiopathy, is especially treating It just plays an increasingly important role in the prevention of the diseases such as hypertension, heart failure, myocardial ischemia, atherosclerosis.It is existing In generation, is research shows that angiotensin converting enzyme inhibitor (ACEI) while hypertension is reduced, also prevents heart failure heart failure, prolongs The generation of slow complication with diabetes.Also play the role of to incomplete brain blood supply type dementia gratifying.Therefore, ACEI activity levels can be used to As the reference evaluation index of antihypertensive health care product and drug, extensive use and food and pharmaceutical research field ACE content highests Tissue site be brain choroid plexus, nigro-striatal pathway and basal ganglia region tissue.[HONGGER1] ACEI reduces blood pressure blood pressure Mechanism, mainly can competitively be combined in vivo with the active aglucon zinc ion of ACE at [HONGGER2], inhibit the work of ACE Property, angiotensin I is blocked to be transformed into angiotensinⅡ；Renal vascular is directly influenced, further passes through sympathetic nervous system System and influence Aldosterone Secretion, and then the degradation of bradykinin is reduced, play decompression.ACEI is suitable for various hypertension, is being depressured While, without reflectivity increased heart rate；Urine proteins can be reduced and improve renal function；It is not easy to cause electrolyte disturbance and lipid Dysbolism；Can prevent with reversing hypertension patient vessel's wall thicken with myocardiocyte hypertrophy hypertrophy, the direct heart can be played Dirty protective effect etc..With the further investigation to ACE chemical constitutions and its property, the stronger ACEI of large quantities of activity is first at present After list, will be that treatment hypertension plays better clinical efficacy.
Food proteins are the important sources of the nutritional ingredients such as body essential amino acid, albumen, the generated life of degradation Object active peptide has different physiological roles (such as：Decompression, anti-allergic drug, anti-oxidant, raising immunity etc.).For other Decompression and the adverse reaction of vasodilator drug, exploitation safety, natural, efficient, particularly food source ACEI health products and medicine Product will become cardiovascular treatment and the new concept and developing direction of prevention.At present report in from soybean, small red bean, garlic, A variety of different ACE peptide for inhibiting are successfully obtained in corn, wheat germ, milk, fish, shellfish, egg, vinasse.From fermentation lactoprotein The ACEI researchs extracted in source are more deep.But it is also seldom to obtain ACEI relevant reports by the use of fermented soybean as source.
ACEI is obtained by probiotics fermention lactoprotein research shows that, contained more bioactivity peptide fragments in lactoprotein, It can be digested by probiotics metabolic enzyme and the hydrolysis of lactic acid releases.Foreign study scholar passes through Lactobacillus helveticus CP790 eggs White enzyme hydrolysis α-casein (α-CN) and β-casein (β-CN), the ACE peptide for inhibiting of acquisition, there is preferable decompression to live Property.Other scholars isolate the 5-12 peptides with ACE inhibitory activity using different methods from casein hydrolysate.Function Property investigate also indicate that, take for a long time milk-derived ACE peptide for inhibiting can not only reach prevention, control, alleviation, the high blood of auxiliary treatment The purpose of pressure, also with certain health care and nutritive value.China is the original producton location of soybean, and the people in many areas drink beans The custom of slurry and edible bean product, using soybean, exploitation soybean source has ACEI effects, prevents and treats the work(of hypertension Energy sexual health product will become the hot spot in functional food industry and market.
Pharmaceutical research
20120717 fermented soybean milk acute toxicity testing of 1, Lactobacillus rhamnosus HLH is studied
1.1 experimental animal
Kunming mouse, weight range 18-22g, half male and half female are provided by Harbin Medical University's Experimental Animal Center；
The preparation of 1.2 fermented soybean milk samples
The above-mentioned tamarind slurry being stored in Yoghourt packing plastics cup is added in into isometric distilled water (100ml), uses blender (Kjeldahl's method measures protein content 3%) is spare after mixing
1.3 experimental method
40 kunming mice half male and half females are randomly divided into control group and experimental group, continuous three days of experimental group, each gavage 0.1mL fermented soybean milk sample solutions, the physiological saline of control group gavage 0.1mL, quantitatively add feed and drinking-water every morning, 21 ± 3 DEG C of constant temperature, relative humidity 50-70%, 12h illumination, continuous observation 7 days.Record weight, sign and the death of mouse Situation.Test group and control group are respectively taken out 10 mouse to put to death, liver, spleen is taken out, weighs, calculate liver body ratio, spleen body Than.
Bacterial translocation infection experiment
Remaining mouse continuous gavage after sterile working eyeball takes blood, is put to death immediately to after 28 days, takes out liver, spleen, It weighs, calculates liver body ratio, spleen body ratio.Sterile saline is added in by the amount of 1mL/g, is smashed to pieces with sterile tissue homogenizer, takes liver 100 μ L tissue homogenates of spleen and blood are coated on MRS solid medium tablets, and 37 DEG C are inverted culture 48 hours, are seen at any time Examine bacterium colony growing state.
Experimental result
Tamarind slurry acute toxicity testing and transposition experimental result table prepared by the fermentations of Lactobacillus rhamnosus HLH 20120717 Bright, death, during the experiment experimental group and control group sign (weight, behavior, activity, hair, color do not occur for each group experiment mice Pool, diet, excrement etc.) indices Non Apparent Abnormality；There was no significant difference for the physical signs of internal organs (liver, spleen), and And have no bacterial strain transposition infection phenomenons, tamarind slurry safety prepared by preliminary proof Lee's sugar lactobacillus HLH 20120717 fermentations Property.
20120717 fermented soybean milk of 2, Lactobacillus rhamnosus HLH influences original hypertensive rat (SHR)：
2.1 experimental animals and instrument reagent
Male original hypertensive rat (SHR) is born 60 days or so, weight 180-220g, by Harbin Medical University Experimental Animal Center provides.
Male SD rats are born 60 days or so, and weight 180-220g is carried by Harbin Medical University's Experimental Animal Center For.
Experimental rat is freely eaten and drinks water, specialized management.Animal housing light 12h is illuminated, and room temperature is controlled 21 ± 3 DEG C, relative humidity 50-70%.
Full-automatic non-invasive blood pressure measuring system Chengdu TME Technology Co., Ltd.
T-CHOL (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-G) kit build up life by Nanjing Object Graduate School of Engineering provides.
Influence research of the fermented soybean milk to SHR hypotensive activities
2.2.1 experiment sample
The preparation of fermented soybean milk sample：The above-mentioned tamarind slurry being stored in Yoghourt packing plastics cup is added in into isometric distillation Water (100ml), it is spare with (Kjeldahl's method measures protein content 3%) after blender mixing
Soya-bean milk detects the preparation of sample：By the above-mentioned soya-bean milk after pasteurization prepared used in fermented soybean milk, measure 100ml adds in isometric distilled water (100ml).With tissue mashing machine's mixing.It is spare
2.2.2 experiment packet
SHR rats are randomly divided into control group, low-dosage sample group, middle dosage sample sets, three groups of high dose sample sets, Every group of 8 SHR rats
Separately 8 SD rats is taken to be set as SD control groups.
Control group：Sample gavage is detected with 10mL/Kg soya-bean milk
High dose sample sets：With 15mL/Kg fermented soybean milk sample gavages；
Middle dosage sample sets：With 10mL/Kg fermented soybean milk sample gavages
Low-dosage sample group：With 5mL/Kg fermented soybean milk sample gavages；
SD control groups：10mL/Kg acidified milks.
Short-term antihypertensive effect experiment
All experimental animals are raised one week after buying in zoopery room, and 8 points of every morning and at 3 points in afternoon use BP- The full-automatic non-invasive blood pressure measuring systems of 600A respectively survey a rat-tail blood pressure, and after stablizing three days, the gavage time is every morning 8:00. Each experimental group according to dosage gavage, continuous gavage 3d, 0h, 2h, 4h, 6h, 8h, 10h after each gavage, for 24 hours, measures SHR Caudal artery blood pressure, every repetition 3 times, is averaged, experimental data is represented with " mean+SD ".
Long-term antihypertensive effect evaluation
All experimental animals are raised one week after buying in zoopery room, and 8 points of every morning and at 3 points in afternoon use BP- The full-automatic non-invasive blood pressure measuring systems of 600A respectively survey a rat-tail blood pressure, and after stablizing three days, the gavage time is every morning 8:00. According to dosage gavage, continuous gavage 4 weeks, weekly timing measure a blood pressure and weight, repeat 3 times, be averaged each experimental group, Experimental data is represented with " mean+SD "；After four weeks, animal fasting 20h, through heart puncturing extracting blood, 37 DEG C are incubated 2h, 2500r/min centrifuges 20min, takes serum, measures T-CHOL in serum, triglycerides, high-density lipoprotein cholesterol and contains Amount.
Experimental result
Table 2：Feeding fermented sour soybean is starched in short term to SHR rat antihypertensive effect experimental results
Note：*0.01<P<0.05, compared with the control group in significant difference；**P<0.01, compared with the control group in extremely aobvious Write sex differernce.
Table 3：Feeding fermented sour soybean slurry for a long time tests SHR rats antihypertensive effect
Note：*0.01<P<0.05, compared with the control group in significant difference；**P<0.01, compared with the control group in extremely aobvious Write sex differernce.
Table 4：Influence of the fermented soybean milk to SHR rat body weights
Note：*0.01<P<0.05, compared with the control group in significant difference；**P<0.01, compared with the control group in extremely aobvious Write sex differernce.
Table 5：Fermented soybean milk influences SHR rat blood serums cholesterol, triglycerides, high-density lipoprotein testing result
Note：*0.01<P<0.05, compared with the control group in significant difference；**P<0.01, compared with the control group in extremely aobvious Write sex differernce.
Feeding is by fermented soybean milk prepared by Lactobacillus rhamnosus HLH 20120717 to original hypertensive rat (SHR) The influence research experiment of hypotensive activity shows：The short-term and high, medium and low dosage fermented soybean milk group of long-term feeding, to SHR rats Tail systolic arterial pressure changes significant influence；Control group and SD control groups change without conspicuousness；In addition feeding it is high, in, Low dosage fermented soybean milk group is in addition to weight, to T-CHOL, triglycerides, high-density lipoprotein content in serum also significant shadow It rings.It is therefore evident that the fermented soybean milk that feeding is prepared by Lactobacillus rhamnosus HLH 20120717 is to original hypertensive rat (SHR) hypotensive activity is apparent.
Specific embodiment：
Embodiment 1：
One kind has the Lactobacillus rhamnosus for inhibiting angiotoninase (ACE) effect from fermented soybean milk, by acquiring In spontaneous fermentation bean product, through detaching, purifying, physical mutagenesis, the high activity bacterial strain preferably obtained, mycology specific name Actobacillus rhamnosus HLH 20120717, bacteria characteristic are：Be placed in the culture medium of 37 DEG C of constant incubators into Row culture, bacterium grows better when finding that diluted concentration is 10-5 by culture, bacterium colony dispersion；Bacterium colony is given birth under the same conditions Length varies, and bacterium colony is rounded, and milky, neat in edge, surface is smooth, wherein smaller bacterium colony is yellowish；Preserving number is： CGMCC No.7084。
Embodiment 2：
Having from fermented soybean milk described in embodiment 1 inhibits the rhamnose breast of angiotoninase (ACE) effect Bacillus, bacteria characteristic further include：Gram's staining result is positive；Spore staining thalline is in green；Do not have in catalase test There is bubble generation；Molten calcium circle method culture observes that transparent circle occurs in periphery of bacterial colonies, the feature for having apparent lactic acid bacteria.
Embodiment 3：
There is the sandlwood for inhibiting angiotoninase (ACE) effect from fermented soybean milk described in embodiment 1 or 2 Sugared lactobacillus carries out BLAST comparisons, as a result with Lactobacillus Pseudomonas online through 16S rDNA partial sequences The central genetic characteristic of rhamnosus strains：
16S rDNA sequencings partial sequence is as follows：
Embodiment 4：
There is the sandlwood for inhibiting angiotoninase (ACE) effect from fermented soybean milk described in embodiment 1,2,3 Sugared lactobacillus, the culture medium prescription are：
The ratio is ratio of weight and number.
Embodiment 5：
The soya-bean milk made using the Lactobacillus rhamnosus described in embodiment 1 or 2 or 3 or 4, the above embodiments are obtained The inoculation arrived is ground, is homogenized, is boiled, filter, in soya-bean milk made from high-temperature sterilization, 37 DEG C -42 DEG C in through soybeans soaking Tamarind slurry is made in fermentation 24 hours or more.
Embodiment 6：
The mouse that having from fermented soybean milk described in the above embodiments is inhibited into angiotoninase (ACE) effect Lee's sugar lactobacillus, for make inhibit angiotoninase (ACE), for make prevention and treatment hypertension health food and The purposes of drug.
Embodiment 7：
One kind has the Lactobacillus rhamnosus for inhibiting angiotoninase (ACE) effect from fermented soybean milk
It is micro- to be preserved in China on January 4th, 2013 by Lactobacillus rhamnosus HLH 20120717 Biological inoculum preservation administration committee common micro-organisms center, address is：City of BeiJing, China Chaoyang District North Star West Road 1 institute 3 Number (100101) phone：64807355 preserving numbers are：CGMCC No.7084.
Lactobacillus rhamnosus Lactobacillus rhamnosus HLH 20120717, are by being collected in spontaneous fermentation beans In product, through detaching, purifying, physical mutagenesis, the high activity bacterial strain preferably obtained, it is placed in 37 DEG C of constant incubators and is trained It supports, better by cultivating bacterium growth, bacterium colony dispersion, not of uniform size, bacterium colony is rounded, milky, neat in edge, surface light It is sliding, wherein smaller bacterium colony is yellowish.
The obtained Lactobacillus rhamnosus Lactobacillus rhamnosus HLH 20120717 of optimization, condition of culture For：Culture medium is 10 parts of peptone, 10. parts of beef extract, 5 parts of yeast extract, 2 parts of diammonium hydrogen citrate [(NH4) 2HC6H5O7] 20 parts of glucose (C6H12O6H2O), 1 part of Tween 80,5 parts of sodium acetate (CH3COONa3H2O), dipotassium hydrogen phosphate (K2HPO43H2O) 2 parts, 0.58 part of magnesium sulfate (MgSO47H2O), 0.25 part of manganese sulfate (MnSO4H2O), agar 18 parts, 1000 parts of distilled water, 6.2~6.6,37 DEG C of constant temperature incubations of pH.
Gram's staining result is positive；Spore staining thalline is in green；There is no bubble generation in catalase test；Molten calcium Circle method culture is observed that transparent circle occurs in periphery of bacterial colonies, through advanced automatic bacterial identification/susceptibility system (VITEK2Compact) has the feature of apparent lactic acid bacteria.
Lactobacillus rhamnosus HLH 20120717 is through VITEK 2Compact qualification results
BLAST comparisons are carried out online through 16S rDNA partial sequences, as a result with Lactobacillus Pseudomonas The central genetic characteristic of rhamnosus strains.
Table 6：Lactobacillus rhamnosus HLH 20120717 is through VITEK 2Compact qualification results
Heilongjiang University
Embodiment 8：
Using 20120717 fermented sour soybean sizing process of Lactobacillus rhamnosus Lactobacillus rhamnosus HLH：
It weighs to be put in 250mL tap water after 100g washing soybeans and impregnates 12h at room temperature；After peeling, clear water It cleans, by soaked soybean with 1: 8 beans water ratio tissue mashing machine's defibrination, is used after being heated to boiling 10 minutes, three After layer filtered through gauze.The 15min that sterilizes in triangular flask in 121 DEG C is poured into, it is sterile to be chosen with oese after five layers of gauze sealing cooling 20120717 bacterium colonies of Lactobacillus rhamnosus HLH of MRS test tube slants culture, about five rings are taken, after shaking uniformly, is placed on 37 DEG C culture 12 hours, is cooled to room temperature, after be put into that 4 DEG C of refrigerator is cold to be deposited, be ready for use on subsequent fermentation.
It ibid weighs to be put in 50000mL tap water after 2000g washing soybeans and impregnates 12h at room temperature；Through peeling Afterwards, clear water is cleaned, by soaked soybean with 1: 8 beans water ratio soy bean milk making machine defibrination, after being heated to boiling, centrifugal filtration Remove bean dregs.Through Pasteurizer 135 DEG C of sterilizings, 10 seconds quick sterilizations, it is placed in the good fermentation tank of washing and sterilizing, by 5% body Product ratio adds in above-mentioned spare fermented soybean milk, and with 30r/min, 37-40 DEG C of fermentation 2 hours, then part is with every glass 100mL packing is sealed in 150mL Yoghourt plastic cups, and after the fermentation 12 hours of (25 DEG C or so) of room temperature, it is cold to be put into 4 DEG C of refrigerator It deposits.
Remainder is placed in the good 100mL large beakers of washing and sterilizing, and room temperature fermentation is 24 small after five layers of gauze sealing When more than, when pH reach 4.9 hereinafter, tamarind slurry in solidification shape, blender by tamarind slurry all stir into flowable character, tile In the enamel disk of 2cm thickness, -40 DEG C to 30 DEG C freeze-dryings into crisp yellow solid, are ground into 100 mesh powders, pass through Kjeldahl nitrogen determination protein content 47%.Sealing low temperature drying saves backup.
Embodiment 9：
Fermented soybean milk detects the preparation of sample
The above-mentioned tamarind slurry being stored in Yoghourt packing plastics cup is added in into isometric distilled water (100ml), uses blender After mixing, 8000r/min centrifugation 10min collect supernatant, detection PH values, with the NaOH and 0.1mol/LNaOH of 5mol/L Its pH is adjusted to 8.2-8.4, then to centrifuge 10min under 8000r/min, takes supernatant in -4 DEG C of preservations.It is spare
Soya-bean milk detects the preparation of sample
By the above-mentioned soya-bean milk after pasteurization prepared used in fermented soybean milk, measure 100ml and add in isometric distilled water (100ml), 8000r/min centrifugation 10min collect supernatant, detection PH values, with the NaOH and 0.1mol/ of 5mol/L LNaOH adjusts its pH to 8.2-8.4, then to centrifuge 10min under 8000r/min, takes supernatant in -4 DEG C of preservations.It is spare
Reference substance solution is prepared：(0.1mol/L pH8.3 borate buffer solutions)
19.068g sodium tetraborates and 8.766gNaCl are weighed respectively, 400mL distillation water dissolutions are added in, with 5M NaOH PH to 8.3 is adjusted, 500mL is settled to distilled water.It is spare
Being surveyed with visible spectrophotometry detection fermented soybean milk inhibits ACE activity to operate
50 μ L (1mmol/L) FAPGG, 40 μ L (0.1U/mL) ACE of 37 DEG C of preheating 10min in advance are measured respectively, respectively It is quickly adding into three and is assembled with 96 well culture plate of 40 μ L fermented soybean milks sample liquids, 40 μ L soya-bean milk solution and 40 μ L reference substances liquid In, mixing, in 0min, 30min, METHOD FOR CONTINUOUS DETERMINATION light absorption value is three times under microplate reader 340nm wavelength respectively at 37 DEG C.By such as Lower formula calculates.
Wherein A：Control group light absorption value decreasing value after 30min；B ：Sample sets light absorption value decreasing value after 30min.
Testing result：It detects fermented soybean milk and its reference substance inhibits ACE enzymatic activity results
Table 7：It detects fermented soybean milk and its reference substance inhibits ACE enzymatic activity results
Testing result shows：Tamarind slurry prepared by the fermentations of Lactobacillus rhamnosus HLH 20120717 has significant inhibit ACE enzymatic activitys.
16S rDNA sequencings partial sequence is as follows：
<110>Heilongjiang University
<120>Have from fermented soybean milk and inhibit angiotoninase（ACE）The Lactobacillus rhamnosus of effect
<141>Date
<160>1
<210> Lactobacillus rhamnosus HLH 20120717
<211>1562
<212>RNA
<213>Lactobacillus rhamnosus
<223>N=a or g or c or t
<400>Nucleotide sequence and/or amino acid sequence
aaggaggtgatccagccgca ggttctccta cggctacctt gttacgactt caccctaatc 60
atttgtcccaccttagacgg ctcgctccct aaaagggtta cgccaccggc ttcgggtgtt 120
acaaactctcatggtgtgac gggcggtgtg tacaaggccc gggaacgtat tcaccgcggc 180
gtgctgatccgcgattacta gcgattccga cttcgtgtag gcgagttgca gcctacagtc 240
cgaactgagaatggctttaa gagattagct tgacctcgcg gtctcgcaac tcgttgtacc 300
atccattgtagcacgtgtgt agcccaggtc ataaggggca taatgatttg acgtcatccc 360
caccttcctccggtttgtca ccggcagtct tactagagtg cccaactaaa tgctggcaac 420
tagtcataagggttgcgctc gttgcgggac ttaacccaac atctcacgac acgagctgac 480
gacaaccatgcaccacctgt cattttgccc ccgaagggga aacctgatct ctcaggtgat 540
caaaagatgtcaagacctgg taaggttctt cgcgttgctt cgaattaaac cacatgctcc 600
accgcttgtgcgggcccccg tcaattcctt tgagtttcaa ccttgcggtc gtactcccca 660
ggcggaatgcttaatgcgtt agctgcggca ctgaagggcg gaaaccctcc aacacctagc 720
attcatcgtttacggcatgg actaccaggg tatctaatcc tgttcgctac ccatgctttc 780
gagcctcagcgtcagttaca gaccagacag ccgccttcgc cactggtgtt cttccatata 840
tctacgcatttcaccgctac acatggagtt ccactgtcct cttctgcact caagtttccc 900
agtttccgatgcacttcctc ggttaagccg agggctttca catcagactt aaaaaaccgc 960
ctgcgctcgctttacgccca ataaatccgg ataacgcttg ccacctacgt attaccgcgg 1020
ctgctggcacgtagttagcc gtggctttct ggttggatac cgtcacgccg acaacagtta 1080
ctctgccgaccattcttctc caacaacaga gttttacgac ccgaaagcct tcttcactca 1140
cgcggcgttgctccatcaga cttgcgtcca ttgtggaaga ttccctactg ctgcctcccg 1200
taggagtttgggccgtgtct cagtcccaat gtggccgatc aacctctcag ttcggctacg 1260
tatcattgccttggtgagcc gttacctcac caactagcta atacgccgcg ggtccatcca 1320
aaagcgatagcttacgccat ctttcagcca agaaccatgc ggttcttgga tttatgcggt 1380
attagcatctgtttccaaat gttatccccc acttaagggc aggttaccca cgtgttactc 1440
acccgtccgccactcgttca aaattaaatc aagatgcaag cacctttcaa taatcagaac 1500
tcgttcgacttgcatgtatt aggcacgccg ccagcgttca tcctgagcca ggatcaaact 1560
ct 1562
<110>Heilongjiang University
<120>Have from fermented soybean milk and inhibit angiotoninase（ACE）The Lactobacillus rhamnosus of effect
<141>Date
<160>1
<210> Lactobacillus rhamnosus HLH 20120717
<211>1562
<212>RNA
<213>Lactobacillus rhamnosus
<223>N=a or g or c or t
<400>Nucleotide sequence and/or amino acid sequence
aaggaggtga tccagccgca ggttctccta cggctacctt gttacgactt caccctaatc 60
atttgtccca ccttagacgg ctcgctccct aaaagggtta cgccaccggc ttcgggtgtt 120
acaaactctc atggtgtgac gggcggtgtg tacaaggccc gggaacgtat tcaccgcggc 180
gtgctgatcc gcgattacta gcgattccga cttcgtgtag gcgagttgca gcctacagtc 240
cgaactgaga atggctttaa gagattagct tgacctcgcg gtctcgcaac tcgttgtacc 300
atccattgta gcacgtgtgt agcccaggtc ataaggggca taatgatttg acgtcatccc 360
caccttcctc cggtttgtca ccggcagtct tactagagtg cccaactaaa tgctggcaac 420
tagtcataag ggttgcgctc gttgcgggac ttaacccaac atctcacgac acgagctgac 480
gacaaccatg caccacctgt cattttgccc ccgaagggga aacctgatct ctcaggtgat 540
caaaagatgt caagacctgg taaggttctt cgcgttgctt cgaattaaac cacatgctcc 600
accgcttgtg cgggcccccg tcaattcctt tgagtttcaa ccttgcggtc gtactcccca 660
ggcggaatgc ttaatgcgtt agctgcggca ctgaagggcg gaaaccctcc aacacctagc 720
attcatcgtt tacggcatgg actaccaggg tatctaatcc tgttcgctac ccatgctttc 780
gagcctcagc gtcagttaca gaccagacag ccgccttcgc cactggtgtt cttccatata 840
tctacgcatt tcaccgctac acatggagtt ccactgtcct cttctgcact caagtttccc 900
agtttccgat gcacttcctc ggttaagccg agggctttca catcagactt aaaaaaccgc 960
ctgcgctcgc tttacgccca ataaatccgg ataacgcttg ccacctacgt attaccgcgg 1020
ctgctggcac gtagttagcc gtggctttct ggttggatac cgtcacgccg acaacagtta 1080
ctctgccgac cattcttctc caacaacaga gttttacgac ccgaaagcct tcttcactca 1140
cgcggcgttg ctccatcaga cttgcgtcca ttgtggaaga ttccctactg ctgcctcccg 1200
taggagtttg ggccgtgtct cagtcccaat gtggccgatc aacctctcag ttcggctacg 1260
tatcattgcc ttggtgagcc gttacctcac caactagcta atacgccgcg ggtccatcca 1320
aaagcgatag cttacgccat ctttcagcca agaaccatgc ggttcttgga tttatgcggt 1380
attagcatct gtttccaaat gttatccccc acttaagggc aggttaccca cgtgttactc 1440
acccgtccgc cactcgttca aaattaaatc aagatgcaag cacctttcaa taatcagaac 1500
tcgttcgact tgcatgtatt aggcacgccg ccagcgttca tcctgagcca ggatcaaact 1560
ct 1562

Claims (2)

1. one kind has the Lactobacillus rhamnosus for inhibiting angiotoninase (ACE) effect, which is characterized in that the sandlwood Sugared lactobacillus（Lactobacillus rhamnosus）Bacterial strain number be HLH 20120717, preserving number CGMCC No.7084。
2. a kind of tamarind made using Lactobacillus rhamnosus described in claim 1 is starched, it is characterized in that：By claim 1 institute The inoculation stated grinds, is homogenized, boiling, filtering, in soya-bean milk made from high-temperature sterilization, 37 DEG C -42 DEG C are sent out in through soybeans soaking Tamarind slurry is made in ferment 24 hours or more.
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