CN104593307A - Lactobacillus rhamnosus with angiotensinase inhibiting function and application of lactobacillus rhamnosus - Google Patents
Lactobacillus rhamnosus with angiotensinase inhibiting function and application of lactobacillus rhamnosus Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C11/00—Milk substitutes, e.g. coffee whitener compositions
- A23C11/02—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins
- A23C11/10—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins
- A23C11/103—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins containing only proteins from pulses, oilseeds or nuts, e.g. nut milk
- A23C11/106—Addition of, or treatment with, microorganisms
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
Abstract
The invention discloses lactobacillus rhamnosus with an angiotensinase inhibiting function and application of the lactobacillus rhamnosus. With continuous development of study on the correlated biotechnology and the health medicine subject, novel bioactive constituents, functional genes, functional proteins and other novel action targets of hypertension are discovered in succession, so that basic conditions for screening safe and highly active probiotics capable of efficiently lowering blood pressure are provided. The lactobacillus rhamnosus is a highly active bacterial strain collected from a naturally fermented bean product and formed by separation, purification, physical mutagenesis and optimization, wherein the bacterial systematic name is lactobacillus rhamnosus HLH 20120717; the bacterial characteristics are as follows: the bacteria are placed in a culture medium of a 37 DEG C constant temperature incubator and cultured, and through the culture, the bacteria grow well when the diluted concentration is 10<-5>, and the bacterial colonies are dispersed; under the same conditions, the growth sizes of the bacterial colonies are different; the bacterial colonies are round, milk white, ordered in edges, and smooth in surfaces; the smaller bacterial colonies are pale yellow; the collection number is CGMCC No.7084. The lactobacillus rhamnosus disclosed by the invention provides a basis for further study and development of probiotic functional health food and drugs capable of preventing and treating hypertension.
Description
Technical field
The present invention by preferably obtain a kind of lactobacillus rhamnosus HLH 20120717 (Lactobacillus rhamnosus HLH 20120717), to get everything ready the soya-bean milk and relevant food that suppression hypertensinase (ACE) acts on for fermentation.
Background technology
Along with the development of world economy, the standard of living of people improves, and is that the harm of the cardiovascular disorder of representative increases the weight of day by day with hypertension.Take all preventive measures, treatment means, control hypertensive generation, development, reduce its morbidity and mortality ratio become international for hypertensive common recognition rationally to apply antihypertensive drugs.The current hypertensive medicine of primary treatment is clinically as diuretic(s), receptor blocking agent, calcium channel blocker, angiotensin converting enzyme inhibitor, angiotensin ii receptor blocker etc.But there are certain side effect or untoward reaction, easily draw hepatic and renal function harm, just more unreasonable as prophylactic or long-term taking.
At present, corresponding hypertensive pathogenic factors, by improving the generation of the modes such as bad life habits, rational diet, reinforcement health education preventing hypertension to greatest extent, become the emphasis of world's publicity, reach prevention with functional food, treat hypertensive dual purpose, wherein the exploitation of probiotic bacterium and products thereof is more popular.
The investigation and application starting of China to probiotic bacterium is more late, and government and local great attention, the emphasis being put into national research in new high-tech evolutionary operation(EVOP) at present supports project.But research and development have angiotensin-converting enzyme (ACE) inhibit activities probiotic composition report or fewer, along with deepening continuously of the research such as associated biomolecule technology, sanipratics subject, the discovery in succession of the effect such as the bioactive ingredients that hypertension is new, functional gene, functional protein novel targets, for filtering out efficient step-down, safety, highly active probiotic products provide the foundation condition.
Summary of the invention
The object of this invention is to provide a strain fermentation and there is the lactobacillus rhamnosus HLH 20120717 (Lactobacillus rhamnosus HLH 20120717) that suppression hypertensinase (ACE) acts on soya-bean milk, providing basis for researching and developing the hypertensive probiotic bacterium functional health-care food of prevention and therapy and medicine further.
Above-mentioned purpose is achieved through the following technical solutions:
One derives from fermented soybean milk and has the lactobacillus rhamnosus that suppression hypertensinase (ACE) acts on, by being collected in spontaneous fermentation bean product, through the high reactivity bacterial strain of separation, purifying, physical mutagenesis, preferably acquisition, mycology specific name actobacillus rhamnosus HLH 20120717, bacteria characteristic is: the substratum being placed in 37 DEG C of constant incubators is cultivated, when finding that weaker concn is 10-5 by cultivating, bacteria growing is comparatively good, and bacterium colony disperses; Colony growth varies under the same conditions, and bacterium colony is rounded, oyster white, neat in edge, smooth surface, the micro-yellow of wherein less bacterium colony; Preserving number is: CGMCC No.7084.
The described fermented soybean milk that derives from has the lactobacillus rhamnosus that suppression hypertensinase (ACE) acts on, and its bacteria characteristic also comprises: gramstaining result is positive; Spore staining thalline is in green; Bubble is not had to produce in catalase test; Molten calcium circle method cultivation is observed periphery of bacterial colonies and is occurred transparent circle, possesses the feature of obvious milk-acid bacteria.
The described fermented soybean milk that derives from has the lactobacillus rhamnosus that suppression hypertensinase (ACE) acts on, carry out BLAST comparison online through 16S rDNA partial sequence, result has the central genetic characteristic of Lactobacillus Pseudomonas rhamnosus bacterial classification:
16S rDNA order-checking partial sequence is as follows:
The described fermented soybean milk that derives from has the lactobacillus rhamnosus that suppression hypertensinase (ACE) acts on, and described substratum prescription is:
Described ratio is ratio of weight and number.
Lactobacillus rhamnosus HLH 20120717 is through VITEK 2Compact qualification result
Lactobacillus rhamnosus HLH 20120717 is through VITEK 2Compact qualification result
Lactobacillus rhamnosus HLH 20120717 is through VITEK 2Compact qualification result
Utilize the soya-bean milk that above-mentioned lactobacillus rhamnosus makes, by described inoculation in through soybeans soaking, grinding, homogenate, to boil, filter, in soya-bean milk that high-temperature sterilization is obtained, 37 DEG C-42 DEG C fermentations more than 24 hours, obtained tamarind is starched.
The making method of the soya-bean milk described in a kind of preceding:
Be put in after taking 100g washing soybean in 250mL tap water and soak 12h under room temperature; After peeling, clear water is cleaned, by soaked soybean with the beans water of weight ratio 1: 8 than using tissue mashing machine's defibrination, be heated to boiling use after 10 minutes, after three layers of filtered through gauze.Pour in triangular flask in 121 DEG C of sterilizing 15min, after gauze sealing cooling, Lee's sugar Bacterium lacticum HLH 20120717 bacterium colony five rings of cultivating MRS test tube slant chosen by aseptic transfering loop, after jolting evenly, be placed on 37 DEG C to cultivate 12 hours, be cooled to room temperature, after put into that 4 DEG C, refrigerator is cold to be deposited, as soya-bean milk for subsequent use;
Be put in 50000mL tap water after the same 2000g of taking washing soybean and soak 12h under room temperature; After peeling, clear water is cleaned, and by soaked soybean with beans water ratio the soybean milk maker defibrination of 1: 8 weight ratio, after being heated to seethe with excitement, bean dregs are gone out in centrifuging; Through bus Sterilizers 135 DEG C of sterilizings 10 seconds quick sterilizations, be placed in the fermentor tank that washing and sterilizing is good, above-mentioned fermented soybean milk for subsequent use is added by 5% volume ratio, with 30r/min, the fermentation of 37-40 DEG C 2 hours, then packing, be sealed in Yoghourt plastic cup, after room temperature is fermented 12 hours, put into that 4 DEG C, refrigerator is cold to be deposited;
Remainder is placed in the good large beaker of washing and sterilizing, after gauze sealing, room temperature is fermented more than 24 hours, when pH reaches less than 4.9, tamarind slurry is in solidifying shape, and tamarind slurry is all stirred into flowable shape by agitator, in the enamel dish of tiling 2cm thickness,-40 DEG C to 30 DEG C lyophilizes, become crisp yellow solid, be ground into 100 object powder, through Kjeldahl nitrogen determination protein content 47%.Sealing cryodrying is preserved.
The above-mentioned fermented soybean milk that derives from being had the lactobacillus rhamnosus that suppression hypertensinase (ACE) acts on, suppressing hypertensinase (ACE), for making the purposes of the hypertensive protective foods of prevention and therapy and medicine for making.
Beneficial effect:
Probiotic bacterium has plurality of health care functions, reduces and adjust the function of blood pressure, and reporting for work of research is many, and probiotic bacterium attracts great attention to hypertension health-care effect.Why probiotic bacterium can produce the effect of step-down and adjustment blood pressure, the mainly extracellular proteinase of specific probiotic bacterium, enzymolysis food proteins, and generation can discharge antihypertensive activity peptide matters, as angiotensin converting enzyme-inhibiting peptide etc.; In release cells wall, there is hypotensive effect peptidoglycan and mineral substance, have significant; Three is that part probiotic bacterium can promote that body regulates the absorption of the mineral substance of blood pressure to some.Utilize modern biotechnology to prepare probiotic bacterium antihypertensive product, there are wide Development volue and market outlook.
Zinc metallopeptidase Zace1 (Angiotensin Converting Enzyme, ACE) finds antihypertensive drug target position enzyme mid-term in last century, by the adjustment of renin-angiotensin-kinin system.Angiotensin I converting one-tenth can be had the Angiotensin II of vasoconstriction, boosting; ACE will have the bradykinin degraded of buck functionality simultaneously, and make it to lose hypotensive effect, cause elevation of the blood pressure activity.Angiotensin converting enzyme inhibitor (ACEI) is the novel antihypertensive medicament extensively adopted clinically at present, evident in efficacy, effect safety.Be mainly used in Cardiovarscular, especially just play an increasingly important role in the control of the diseases such as treatment hypertension, heart failure, myocardial ischemia, atherosclerosis.Modern study shows that angiotensin converting enzyme inhibitor (ACEI) is reducing hypertensive while, also has prevention heart failure heart failure, delays the generation of complication with diabetes.Also gratifying effect is had to incomplete brain blood supply type dementia.Therefore, ACEI activity level can be used as the reference evaluation index of antihypertensive health care product and medicine, and widespread use and food and the highest tissue site of pharmaceutical research field ACE content are brain choroid plexus, nigro-striatal pathway and basal ganglia region tissue.[HONGGER1] ACEI reduces blood pressure the mechanism of blood pressure, mainly can be combined with the active aglucon zine ion of ACE competitively in [HONGGER2] body, suppress the activity of ACE, block angiotensin I and be transformed into angiotensinⅡ; Renal vascular is directly affected, further by sympathetic nervous system and affect Aldosterone Secretion, and then reduces the degraded of bradykinin, play step-down.ACEI is applicable to various hypertension, while step-down, without reflectivity increased heart rate; Urine protein can be reduced and improve renal function; Not easily cause electrolyte disturbance and lipidosis; Can prevent with thickening with myocardiocyte hypertrophy of reversing hypertension patient vessel wall loose, can direct cardioprotection etc. be played.Along with the further investigation to ACE chemical structure and character thereof, the ACEI successively listing that current large quantities of activity is stronger, will play better clinical efficacy for treatment hypertension.
Food proteins is the important sources of the nutritive ingredients such as body indispensable amino acid, albumen, its degraded generate biologically active peptides have different physiological roles (as: step-down, anti-allergic drug, anti-oxidant, improve immunizing power etc.).For the untoward reaction of other step-downs and vasodilation medicine, develop safe, natural, eat source ACEI healthcare product and medicine efficiently, particularly, new concept and the developing direction of cardiovascular treatment and prevention will be become.From soybean, little red bean, garlic, corn, wheat germ, milk, fish, shellfish, egg, vinasse, various different ace inhibitory peptide is successfully obtained in current report.The ACEI research of extracting from fermented-milk protein source is more deep.But it is also little to utilize fermented soybean to obtain ACEI relevant report as source.
Obtain ACEI research by probiotics fermention milk-protein to show, many biologically active peptidess section contained in milk-protein, the hydrolysis by probiotic bacterium metabolic enzyme enzymolysis and lactic acid discharges.Foreign study scholar is by lactobacterium helveticus CP790 protease hydrolysis alpha-casein (α-CN) and beta-casein (β-CN), and the ace inhibitory peptide of acquisition, has good antihypertensive activity.Other scholars adopt diverse ways to isolate from casein hydrolysate to have the 5-12 peptide of ACE inhibitory activity.Functional investigation also shows, long-term taking ACE-inhibitory peptides not only can reach the object of prevention, control, alleviation, assisting in treating hypertension, also has certain health care and nutritive value.China is the country of origin of soybean, the people in a lot of area have the custom of drinking soya-bean milk and edible bean product, utilize soybean, and exploitation soybean source has ACEI effect, the hypertensive functional health product of prevention and therapy, will become the focus in functional foodstuff industry and market.
Pharmaceutical research
1, Lee's sugar Bacterium lacticum HLH 20120717 fermented soybean milk acute toxicity test research
1.1 laboratory animal
Kunming mouse, weight range 18-22g, male and female half and half, are provided by Harbin Medical University's Experimental Animal Center.
The preparation of fermented soybean milk sample
The above-mentioned tamarind slurry be stored in Yoghourt packing plastics cup is added equal-volume distilled water (100ml), with after agitator mixing (Kjeldahl determination records protein content 3%). for subsequent use
1.3 experimental technique
40 kunming mice male and female half and half are divided into control group and experimental group at random, continuous three days of experimental group, each gavage 0.1mL fermented soybean milk sample solution, the physiological saline of control group gavage 0.1mL, every morning quantitatively adds feed and drinking-water, constant temperature 21 ± 3 DEG C, and relative humidity is 50-70%, 12h throws light on, continuous observation 7 days.Record the body weight of mouse, sign and death condition.Test group and control group are respectively taken out 10 sacrifice, takes out liver, spleen, weigh, calculate liver body ratio, spleen body ratio.
Bacterial translocation infection experiment
After residue mouse continuous gavage to 28 day, after aseptic technique eyeball gets blood, put to death immediately, take out liver, spleen, weigh, calculate liver body ratio, spleen body ratio.Add stroke-physiological saline solution by the amount of 1mL/g, smash to pieces with sterile tissue homogenizer, get liver spleen 100 μ L tissue homogenate and blood is coated on MRS solid medium flat board, be inverted cultivation 48 hours for 37 DEG C, observe colony growth situation at any time.
Experimental result
The tamarind slurry acute toxicity test that Lee's sugar Bacterium lacticum HLH 20120717 fermentation is standby and transposition experimental result show, there is not death in each group of experiment mice, experimental session experimental group and control group sign (body weight, behavior, activity, hair, color and luster, diet, ight soil etc.) indices Non Apparent Abnormality; The physical signs there was no significant difference of internal organs (liver, spleen), and have no bacterial strain transposition and infect phenomenon, the tamarind slurry security that preliminary proof Lee sugar Bacterium lacticum HLH 20120717 fermentation is standby.
2, Lee's sugar Bacterium lacticum HLH 20120717 fermented soybean milks affect original hypertensive rat (SHR):
2.1 laboratory animal and instrument reagent
Male original hypertensive rat (SHR), be born about 60 days, body weight 180-220g, is provided by Harbin Medical University's Experimental Animal Center.
Male SD rat, be born about 60 days, body weight 180-220g, is provided by Harbin Medical University's Experimental Animal Center.
The equal free choice feeding of experimental rat and drinking-water, specialized management.Animal housing light 12h throws light on, and room temperature controls at 21 ± 3 DEG C, and relative humidity is 50-70%.
Full-automatic non-invasive blood pressure measuring system Chengdu TME Technology Co., Ltd.
Total cholesterol (TC), triglyceride level (TG), high-density lipoprotein cholesterol (HDL-G) test kit builds up Bioengineering Research Institute by Nanjing provides.
Fermented soybean milk is to the influence research of SHR hypotensive activity
2.2.1 test sample
The preparation of fermented soybean milk sample: the above-mentioned tamarind slurry be stored in Yoghourt packing plastics cup is added equal-volume distilled water (100ml), with after agitator mixing (Kjeldahl determination records protein content 3%). for subsequent use
Soya-bean milk detects the preparation of sample: prepare the fermented soybean milk soya-bean milk after bus sterilizing used by above-mentioned, measure 100ml and add equal-volume distilled water (100ml).Mix with tissue mashing machine.For subsequent use
2.2.2 grouping is tested
SHR rat is divided into control group at random, low-dosage sample group, middle dose sample group, high dosage sample sets three groups, often organizes 8 SHR rats
Separately get 8 SD rats and be set to SD control group.
Control group: detect sample gavage with 10mL/Kg soya-bean milk
High dosage sample sets: by 15mL/Kg fermented soybean milk sample gavage;
Middle dose sample group: by 10mL/Kg fermented soybean milk sample gavage
Low-dosage sample group: by 5mL/Kg fermented soybean milk sample gavage;
SD control group: 10mL/Kg fermented-milk.
Short-term antihypertensive effect is tested
All laboratory animal buy after the indoor raising of experimentation on animals one week, every morning 8 and at 3 in afternoon respectively survey a mouse tail blood pressure by the full-automatic non-invasive blood pressure measuring system of BP-600A, 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, 24h after each gavage terminates, measure SHR caudal artery blood pressure, and every only repetition 3 times, average, experimental data represents with " mean+SD ".
Long-term antihypertensive effect evaluation
All laboratory animal buy after the indoor raising of experimentation on animals one week, every morning 8 and at 3 in afternoon respectively survey a mouse tail blood pressure by the full-automatic non-invasive blood pressure measuring system of BP-600A, and after stablizing three days, the gavage time is every morning 8:00.Each experimental group according to dosage gavage, continuous gavage 4 weeks, timing measures a blood pressure and body weight weekly, and repeat 3 times, average, experimental data represents with " mean+SD "; After surrounding, animal fasting 20h, through heart puncturing extracting blood, hatch 2h for 37 DEG C, the centrifugal 20min of 2500r/min, gets serum, measures the content of total cholesterol, triglyceride level, high density lipoprotein cholesterol in serum.
Experimental result
Table: feeding fermented sour soybean slurry short-term is to SHR rat antihypertensive effect experimental result
Note: * 0.01<P<0.05, in significant difference compared with control group; * P<0.01, in pole significant difference compared with control group.
Table: feeding fermented sour soybean slurry is tested SHR rat antihypertensive effect for a long time
Note: * 0.01<P<0.05, in significant difference compared with control group; * P<0.01, in pole significant difference compared with control group.
Table: fermented soybean milk on the impact of SHR rat body weight (
, g)
Note: * 0.01<P<0.05, in significant difference compared with control group; * P<0.01, in pole significant difference compared with control group.
Table: fermented soybean milk is on SHR rat blood serum cholesterol, triglyceride level, the impact of high-density lipoprotein detected result
Note: * 0.01<P<0.05, in significant difference compared with control group; * P<0.01, in pole significant difference compared with control group.
The fermented soybean milk that feeding is prepared by Lee's sugar Bacterium lacticum HLH 20120717 is tested the influence research of original hypertensive rat (SHR) hypotensive activity and is shown: short-term and long-term feeding high, medium and low dosage fermented soybean milk group, all have the impact of significance on the change of SHR rat tail artery systolic pressure; Control group and SD control group change without significance; Feeding high, medium and low dosage fermented soybean milk group is except body weight in addition, also has a significant impact total cholesterol, triglyceride level, high-density lipoprotein content in serum.Therefore confirm that the fermented soybean milk that feeding is prepared by Lee's sugar Bacterium lacticum HLH 20120717 is obvious to original hypertensive rat (SHR) hypotensive activity.
Embodiment:
Embodiment 1:
One derives from fermented soybean milk and has the lactobacillus rhamnosus that suppression hypertensinase (ACE) acts on, by being collected in spontaneous fermentation bean product, through the high reactivity bacterial strain of separation, purifying, physical mutagenesis, preferably acquisition, mycology specific name actobacillus rhamnosus HLH 20120717, bacteria characteristic is: the substratum being placed in 37 DEG C of constant incubators is cultivated, when finding that weaker concn is 10-5 by cultivating, bacteria growing is comparatively good, and bacterium colony disperses; Colony growth varies under the same conditions, and bacterium colony is rounded, oyster white, neat in edge, smooth surface, the micro-yellow of wherein less bacterium colony; Preserving number is: CGMCC No.7084.
Embodiment 2:
The fermented soybean milk that derives from described in embodiment 1 has the lactobacillus rhamnosus that suppression hypertensinase (ACE) acts on, and its bacteria characteristic also comprises: gramstaining result is positive; Spore staining thalline is in green; Bubble is not had to produce in catalase test; Molten calcium circle method cultivation is observed periphery of bacterial colonies and is occurred transparent circle, possesses the feature of obvious milk-acid bacteria.
Embodiment 3:
The fermented soybean milk that derives from described in embodiment 1 or 2 has the lactobacillus rhamnosus that suppression hypertensinase (ACE) acts on, carry out BLAST comparison online through 16S rDNA partial sequence, result has the central genetic characteristic of Lactobacillus Pseudomonas rhamnosus bacterial classification:
16S rDNA order-checking partial sequence is as follows:
Embodiment 4:
The fermented soybean milk that derives from described in embodiment 1,2,3 has the lactobacillus rhamnosus that suppression hypertensinase (ACE) acts on, and described substratum prescription is:
Described ratio is ratio of weight and number.
Embodiment 5:
Utilize the soya-bean milk that embodiment 1 or 2 or the lactobacillus rhamnosus described in 3 or 4 make, inoculation the above embodiments obtained in through soybeans soaking, grinding, homogenate, to be boiled, and filters, in soya-bean milk that high-temperature sterilization is obtained, 37 DEG C-42 DEG C ferment more than 24 hours, obtained tamarind slurry.
Embodiment 6:
The fermented soybean milk that derives from described in the above embodiments being had the lactobacillus rhamnosus that suppression hypertensinase (ACE) acts on, suppressing hypertensinase (ACE), for making the purposes of the hypertensive protective foods of prevention and therapy and medicine for making.
Embodiment 7:
One derives from fermented soybean milk and has the lactobacillus rhamnosus Lactobacillus rhamnosus HLH 20120717 that suppression hypertensinase (ACE) acts on, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on January 4th, 2013, address is: No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute No. 3 (100101) phones: 64807355 preserving numbers are: CGMCC No.7084.
Lactobacillus rhamnosus Lactobacillus rhamnosus HLH 20120717, being by being collected in spontaneous fermentation bean product, through the high reactivity bacterial strain of separation, purifying, physical mutagenesis, preferably acquisition, being placed in 37 DEG C of constant incubators and cultivating, better by cultivating bacteria growing, bacterium colony disperses, and not of uniform size, bacterium colony is rounded, oyster white, neat in edge, smooth surface, the micro-yellow of wherein less bacterium colony.
Optimize the lactobacillus rhamnosus Lactobacillus rhamnosus HLH 20120717 obtained, culture condition is: substratum is peptone 10 parts, extractum carnis 10. parts, yeast extract paste 5 parts, diammonium hydrogen citrate [(NH4) 2HC6H5O7] 2 parts of glucose (C6H12O6H2O) 20 parts, tween 80 1 part, sodium acetate (CH3COONa3H2O) 5 parts, dipotassium hydrogen phosphate (K2HPO43H2O) 2 parts, 0.58 part, magnesium sulfate (MgSO47H2O), manganous sulfate (MnSO4H2O) 0.25 part, 18 parts, agar, distilled water 1000 parts, pH 6.2 ~ 6.6, 37 DEG C of constant temperature culture.
Gramstaining result is positive; Spore staining thalline is in green; Bubble is not had to produce in catalase test; Molten calcium circle method cultivation can be observed periphery of bacterial colonies and occur transparent circle, possesses the feature of obvious milk-acid bacteria through senior automatic bacterial qualification/susceptibility system (VITEK 2Compact).
Table 3: lactobacillus rhamnosus HLH 20120717 is through VITEK 2Compact qualification result
Carry out BLAST comparison online through 16S rDNA partial sequence, result has the central genetic characteristic of Lactobacillus Pseudomonas rhamnosus bacterial classification.
Table: lactobacillus rhamnosus HLH 20120717 is through VITEK 2Compact qualification result
Heilongjiang University
Embodiment 8:
Adopt lactobacillus rhamnosus Lactobacillus rhamnosus HLH 20120717 fermented sour soybean sizing process:
Be put in after taking 100g washing soybean in 250mL tap water and soak 12h under room temperature; After peeling, clear water is cleaned, by soaked soybean with 1: 8 beans water than using tissue mashing machine's defibrination, be heated to boiling use after 10 minutes, after three layers of filtered through gauze.Pour in triangular flask in 121 DEG C of sterilizing 15min, after five layers of gauze sealing cooling, Lee's sugar Bacterium lacticum HLH 20120717 bacterium colony cultivated MRS test tube slant chosen by aseptic transfering loop, about five rings, after jolting evenly, be placed on 37 DEG C and cultivate 12 hours, be cooled to room temperature, after put into that 4 DEG C, refrigerator is cold to be deposited, be ready for use on subsequent fermentation.
Be put in 50000mL tap water after the same 2000g of taking washing soybean and soak 12h under room temperature; After peeling, clear water is cleaned, by soaked soybean with 1: 8 beans water ratio soybean milk maker defibrination, after being heated to seethe with excitement, bean dregs are gone out in centrifuging.Through bus Sterilizers 135 DEG C of sterilizings 10 seconds quick sterilizations, be placed in the fermentor tank that washing and sterilizing is good, above-mentioned fermented soybean milk for subsequent use is added by 5% volume ratio, with 30r/min, the fermentation of 37-40 DEG C 2 hours, and rear section with every glass of 100mL packing, be sealed in 150mL Yoghourt plastic cup, after room temperature (about 25 DEG C) is fermented 12 hours, put into that 4 DEG C, refrigerator is cold to be deposited.
Remainder is placed in the good 100mL large beaker of washing and sterilizing, after five layers of gauze sealing, room temperature is fermented more than 24 hours, when pH reaches less than 4.9, tamarind slurry is in solidifying shape, and tamarind slurry is all stirred into flowable shape by agitator, in the enamel dish of tiling 2cm thickness,-40 DEG C to 30 DEG C lyophilizes, become crisp yellow solid, be ground into 100 object powder, through Kjeldahl nitrogen determination protein content 47%.Sealing cryodrying saves backup.
Embodiment 9:
Fermented soybean milk detects the preparation of sample
The above-mentioned tamarind slurry be stored in Yoghourt packing plastics cup is added equal-volume distilled water (100ml), after agitator mixing, the centrifugal 10min of 8000r/min, collect supernatant liquor, detect pH value, its pH to 8.2-8.4 is regulated with NaOH and 0.1mol/LNaOH of 5mol/L, again with 10min centrifugal under 8000r/min, get supernatant liquor in-4 DEG C of preservations.For subsequent use
Soya-bean milk detects the preparation of sample
The fermented soybean milk soya-bean milk after bus sterilizing used is prepared by above-mentioned, measure 100ml and add equal-volume distilled water (100ml), the centrifugal 10min of 8000r/min, collect supernatant liquor, detect pH value, its pH to 8.2-8.4 is regulated with NaOH and 0.1mol/LNaOH of 5mol/L, again with 10min centrifugal under 8000r/min, get supernatant liquor in-4 DEG C of preservations.For subsequent use
Reference substance solution is prepared: (0.1mol/L pH8.3 borate buffer solution)
Take 19.068g sodium tetraborate and 8.766gNaCl respectively, add 400mL distilled water and dissolve, adjust pH to 8.3 with 5M NaOH, be settled to 500mL with distilled water.For subsequent use
Detect fermented soybean milk by visible spectrophotometry and survey the operation of suppression ACE activity
Measure 50 μ L (1mmol/L) FAPGG, 40 μ L (0.1U/mL) ACE of 37 DEG C of preheating 10min in advance respectively, joining rapidly three is respectively assembled with in 40 μ L fermented soybean milk sample liquid, 40 μ L soya-bean milk solution and 40 μ L reference substance liquid 96 well culture plates, mixing, at 37 DEG C respectively when 0min, 30min under microplate reader 340nm wavelength METHOD FOR CONTINUOUS DETERMINATION light absorption value three times.Be calculated as follows.
Wherein control group light absorption value decreasing value after A:30min; Sample sets light absorption value decreasing value after B:30min.
Detected result: detect fermented soybean milk and reference substance suppression ACE enzymic activity result thereof
Table: detect fermented soybean milk and reference substance suppression ACE enzymic activity result thereof
Detected result shows: the standby tamarind slurry of Lee's sugar Bacterium lacticum HLH 20120717 fermentation has and suppresses ACE enzymic activity significantly.
16S rDNA order-checking partial sequence is as follows:
。
<110> Heilongjiang University
<120> derives from fermented soybean milk and has the lactobacillus rhamnosus that suppression hypertensinase (ACE) acts on
the <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 aminoacid 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
Claims (7)
1. one kind derives from fermented soybean milk and has the lactobacillus rhamnosus that suppression hypertensinase (ACE) acts on, it is characterized in that: by being collected in spontaneous fermentation bean product, through the high reactivity bacterial strain of separation, purifying, physical mutagenesis, preferably acquisition, mycology specific name actobacillus rhamnosus HLH 20120717, bacteria characteristic is: the substratum being placed in 37 DEG C of constant incubators is cultivated, and finds that weaker concn is 10 by cultivating
-5time bacteria growing comparatively good, bacterium colony disperses; Colony growth varies under the same conditions, and bacterium colony is rounded, oyster white, neat in edge, smooth surface, the micro-yellow of wherein less bacterium colony; Preserving number is: CGMCC No.7084.
2. the fermented soybean milk that derives from according to claim 1 has the lactobacillus rhamnosus that suppression hypertensinase (ACE) acts on, and it is characterized in that: its bacteria characteristic also comprises: gramstaining result is positive; Spore staining thalline is in green; Bubble is not had to produce in catalase test; Molten calcium circle method cultivation is observed periphery of bacterial colonies and is occurred transparent circle, possesses the feature of obvious milk-acid bacteria.
3. the fermented soybean milk that derives from according to claim 1 and 2 has the lactobacillus rhamnosus that suppression hypertensinase (ACE) acts on, it is characterized in that: carry out BLAST comparison online through 16S rDNA partial sequence, result has the central genetic characteristic of Lactobacillus Pseudomonas rhamnosus bacterial classification:
16S rDNA order-checking partial sequence is as follows:
<110> Heilongjiang University
<120> derives from fermented soybean milk and has the lactobacillus rhamnosus that suppression hypertensinase (ACE) acts on
The <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 aminoacid sequence
4. the fermented soybean milk that derives from according to claim 1 or 2 or 3 has the lactobacillus rhamnosus that suppression hypertensinase (ACE) acts on, and it is characterized in that: described substratum prescription is:
Described ratio is ratio of weight and number.
5. the soya-bean milk utilizing claim 1 or 2 or the lactobacillus rhamnosus described in 3 or 4 to make, it is characterized in that: by the inoculation described in claim 1 or 2 or 3 or 4 in through soybeans soaking, grinding, homogenate, to boil, in the soya-bean milk that filtration, high-temperature sterilization obtain, 37 DEG C-42 DEG C ferment more than 24 hours, obtained tamarind slurry.
6. a making method for soya-bean milk according to claim 5, is characterized in that:
Be put in after taking 100g washing soybean in 250mL tap water and soak 12h under room temperature; After peeling, clear water is cleaned, by soaked soybean with the beans water of weight ratio 1: 8 than using tissue mashing machine's defibrination, be heated to boiling use after 10 minutes, after three layers of filtered through gauze.Pour in Erlenmeyer flask in 121 DEG C of sterilizing 15min, after gauze sealing cooling, choose Lee's sugar Bacterium lacticum HLH 20120717 bacterium colony five rings of cultivating MRS test tube slant with transfering loop under aseptic condition, after jolting evenly, be placed on 37 DEG C to cultivate 12 hours, be cooled to room temperature, after put into that 4 DEG C, refrigerator is cold to be deposited, as soya-bean milk for subsequent use;
Be put in 50000mL tap water after the same 2000g of taking washing soybean and soak 12h under room temperature; After peeling, clear water is cleaned, and by soaked soybean with beans water ratio the soybean milk maker defibrination of 1: 8 weight ratio, after being heated to seethe with excitement, bean dregs are gone out in centrifuging; Through bus Sterilizers 135 DEG C of sterilizings 10 seconds quick sterilizations, be placed in the fermentor tank that washing and sterilizing is good, above-mentioned fermented soybean milk for subsequent use is added by 5% volume ratio, with 30r/min, the fermentation of 37-40 DEG C 2 hours, then packing, be sealed in Yoghourt plastic cup, after room temperature is fermented 12 hours, put into that 4 DEG C, refrigerator is cold to be deposited;
Remainder is placed in the good large beaker of washing and sterilizing, after gauze sealing, room temperature is fermented more than 24 hours, when pH reaches less than 4.9, tamarind slurry is in solidifying shape, and tamarind slurry is all stirred into flowable shape by agitator, in the enamel dish of tiling 2cm thickness,-40 DEG C to 30 DEG C lyophilizes, become crisp yellow solid, be ground into 100 object powder, through Kjeldahl nitrogen determination protein content 47%.Sealing cryodrying is preserved.
7. the fermented soybean milk that derives from described in claim 1 or 2 or 3 or 4 being had the lactobacillus rhamnosus that suppression hypertensinase (ACE) acts on, suppressing hypertensinase (ACE), for making the purposes of the hypertensive protective foods of prevention and therapy and medicine for making.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105154370A (en) * | 2015-09-30 | 2015-12-16 | 山东大学 | Lactobacillus rhamnosus of high-yield cell membrane protease and application thereof |
| CN107960481A (en) * | 2017-12-19 | 2018-04-27 | 苏州金记食品有限公司 | It is a kind of that the biofermentation method that soya-bean milk trypsin inhibitor inactivates is made based on Lactobacillus rhamnosus |
| IT202100026282A1 (en) * | 2021-10-13 | 2023-04-13 | Alfasigma Spa | FERMENTED SURNATANT AND POSTBIOTIC COMPOSITIONS USEFUL IN THE PREVENTION AND TREATMENT OF CARDIOVASCULAR DISEASES |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006290835A (en) * | 2005-04-14 | 2006-10-26 | Mitsukan Group Honsha:Kk | Functional composition exhibiting ace inhibitory activity and having hypotensive activity |
| CN101053346A (en) * | 2007-05-11 | 2007-10-17 | 扬州大学 | Preparation method for probiotics fermented milk |
| CN101314763A (en) * | 2007-06-01 | 2008-12-03 | 统一企业(中国)投资有限公司 | Short bifidobacteria with functions of anti-gastrointestinal tract pathogen, oxidation resistance and blood pressure reduction |
| CN103330001A (en) * | 2013-05-25 | 2013-10-02 | 吉林农业大学 | Preparation method for soya-bean milk fermented by lactobacillus rhamnosus |
| CN103421715A (en) * | 2013-08-03 | 2013-12-04 | 青岛蔚蓝生物集团有限公司 | Lactobacillus rhamnosus and application thereof |
-
2015
- 2015-02-26 CN CN201510088465.1A patent/CN104593307B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006290835A (en) * | 2005-04-14 | 2006-10-26 | Mitsukan Group Honsha:Kk | Functional composition exhibiting ace inhibitory activity and having hypotensive activity |
| CN101053346A (en) * | 2007-05-11 | 2007-10-17 | 扬州大学 | Preparation method for probiotics fermented milk |
| CN101314763A (en) * | 2007-06-01 | 2008-12-03 | 统一企业(中国)投资有限公司 | Short bifidobacteria with functions of anti-gastrointestinal tract pathogen, oxidation resistance and blood pressure reduction |
| CN103330001A (en) * | 2013-05-25 | 2013-10-02 | 吉林农业大学 | Preparation method for soya-bean milk fermented by lactobacillus rhamnosus |
| CN103421715A (en) * | 2013-08-03 | 2013-12-04 | 青岛蔚蓝生物集团有限公司 | Lactobacillus rhamnosus and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| A PIHLANTO 等: "Angiotensin I converting enzyme (ACE) inhibitory activity and antihypertensive effect of fermented milk", 《INTERNATIONAL DAIRY JOURNAL》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105154370A (en) * | 2015-09-30 | 2015-12-16 | 山东大学 | Lactobacillus rhamnosus of high-yield cell membrane protease and application thereof |
| CN105154370B (en) * | 2015-09-30 | 2018-03-20 | 山东大学 | One plant of Lactobacillus rhamnosus for producing cell envelope protease and its application |
| CN107960481A (en) * | 2017-12-19 | 2018-04-27 | 苏州金记食品有限公司 | It is a kind of that the biofermentation method that soya-bean milk trypsin inhibitor inactivates is made based on Lactobacillus rhamnosus |
| IT202100026282A1 (en) * | 2021-10-13 | 2023-04-13 | Alfasigma Spa | FERMENTED SURNATANT AND POSTBIOTIC COMPOSITIONS USEFUL IN THE PREVENTION AND TREATMENT OF CARDIOVASCULAR DISEASES |
| WO2023061978A1 (en) | 2021-10-13 | 2023-04-20 | Alfasigma S.P.A. | Fermented supernatant and postbiotic compositions useful in the prevention and treatment of cardiovascular diseases. |
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