CN104789488A - Lactobacillus rhamnosus having cholesterol lowering effect, and uses thereof - Google Patents
Lactobacillus rhamnosus having cholesterol lowering effect, and uses thereof Download PDFInfo
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Abstract
The present invention provides Lactobacillus rhamnosus having a cholesterol lowering effect, and uses thereof. In the prior art, if people uptakes too high high-cholesterol foods, the synthesis and the metabolism of the cholesterol in the body overload, and the blood cholesterol rises so as to cause generation of cholesterol precipitation on the artery vessel, and induction of atherosclerosis and other cardiovascular diseases. According to the present invention, the high activity strain is collected from the naturally-fermented soybean product and is obtained through separation, purification, physical mutation and preferable selection, the mycology classification name is Lactobacillus rhamnosus SCF 20120718, the mycology characteristics comprise that: the bacteria growth is good and the colonies are dispersed when the dilution concentration is 10<-5> after the bacterial are culture in a culture medium in a 37 DEG C constant temperature incubator, the growing colonies have characteristics of different sizes, round shape, milk white color, neat edges and smooth surface under the same conditions, and the small colonies have the slight-yellow color, and the preservation number is CGMCC No.7085. The present invention provides the probiotic functional health product raw materials for prevention and treatment of cardiovascular and cerebrovascular diseases.
Description
Technical field:
The present invention relates to and a kind of obtain a kind of lactobacillus rhamnosus (Lactobacillus rhamnosus) SCF 20120718 by preferred institute, can be used for fermentation and to get everything ready the soya-bean milk of reduction cholesterol effect and relevant healthcare product and medicine.
Background technology:
Cholesterol in human body is normally free to be present in biological cells and tissues with cholesteryl ester two kinds of forms, distribute in brain and nervous tissue, kidney, skin, liver and bile higher, it is the important component in body internal membranous structure, participate in forming microbial film, also play an important role in the conductive process of plasma lipoprotein synthesis, lipid metabolism, nervous excitation, or bile acide, the synthesis material of vitamins D and multiple hormone, therefore, cholesterol maintains the indispensable important substance of organism physiology function.While but cholesterol plays its vital role in body, also can produce certain harm as too high in body especially blood, have influence on metabolism, bring out cancer; The too high meeting of cardio-vascular endothelial cells dysfunction, particularly low-density lipoprotein can also be caused to have influence on calcium ion regulatory abnormal, be that cardiovascular function is not normal, causes the impaired and disease of cardiovascular and cerebrovascular to occur.Also likely bring out the generation of senile dementia.
Normal circumstances lower body cholesterol 2/3rds is primarily of the endogenous synthesis of the organ such as liver, small bowel, all the other mainly contain poultry eggs, pluck and muscle tissue such as pork liver, fat meat external source by the food containing cholesterol in diet and obtain, if people's chronic dietary unreasonable structure, absorption high-cholesterol diet is too high, synthesis and the metabolic burden of body inner cholesterol are overweight, Blood Cholesterol raises, cause the precipitation comprising arterial blood tube wall cholesterol, bring out the generation of the diseases such as arteriosclerosis etc. is cardiovascular, serious threat human health.
Summary of the invention:
The object of this invention is to provide a strain fermentation and there is lactobacillus rhamnosus (Lactobacillus rhamnosus) SCF20120718 reducing cholesterol effect soya-bean milk, provide and research and develop prevention and therapy further due to the too high caused probiotic bacterium functional health-care food of cardiovascular and cerebrovascular diseases of cholesterol and the raw material of medicine.
Above-mentioned purpose is achieved through the following technical solutions:
A kind of fermented soybean milk that derives from has lactobacillus rhamnosus (Lactobacillus rhamnosus) SCF 20120718 reducing cholesterol effect soya-bean milk, by being collected in spontaneous fermentation bean product, through the high reactivity bacterial strain of separation, purifying, physical mutagenesis, preferably acquisition, mycology specific name Lactobacillusrhamnosus, bacteria characteristic: 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, being deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, registers on the books and is numbered in preservation center: CGMCC No.7085.
The described fermented bean products that derives from has lactobacillus rhamnosus (Lactobacillus rhamnosus) SCF20120718 reducing cholesterol effect soya-bean milk, and the bacteria characteristic of described high reactivity bacterial strain is: 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.
The described fermented bean products that derives from has lactobacillus rhamnosus (Lactobacillus rhamnosus) SCF20120718 reducing cholesterol effect soya-bean milk, bacterial strain carries out BLAST comparison online through 16S rDNA partial sequence, and result has the central genetic characteristic of Lactobacillus Pseudomonas rhamnosus bacterial classification;
16S rDNA order-checking partial sequence is as follows:
There is lactobacillus rhamnosus and the purposes of decreasing cholesterol effect
The described fermented bean products that derives from has lactobacillus rhamnosus (Lactobacillus rhamnosus) SCF20120718 reducing cholesterol effect soya-bean milk, and described substratum prescription is:
Described ratio is ratio of weight and number.
Table: lactobacillus rhamnosus SCF 20120718 is through VITEK 2Compact qualification result
Table: lactobacillus rhamnosus SCF 20120718 is through VITEK 2Compact qualification result
Utilize the soya-bean milk that above-mentioned lactobacillus rhamnosus makes, by above-mentioned 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.
A kind of preparation method of above-mentioned soya-bean milk:
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 parts by weight 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 SCF 20120718 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, 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, and by soaked soybean with beans water ratio the soybean milk maker defibrination of parts by weight 1: 8, 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, add above-mentioned fermented soybean milk for subsequent use by 5% volume ratio, with the fermentation 2 hours of 30r/min, 37-40 DEG C, then packing, sealing, 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 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 45.1%.Sealing cryodrying saves backup.
The described fermented bean products that derives from is had lactobacillus rhamnosus (Lactobacillus rhamnosus) SCF 20120718 reducing cholesterol effect soya-bean milk, can be used for fermentation and to get everything ready the soya-bean milk and relevant healthcare product and medicine that reduce cholesterol effect.
Beneficial effect:
Along with the raising of expanding economy and people's living standard, healthy and health care consciousness is strengthened, particularly to the raising of the too high harm awareness to body of cholesterol level, caused worldwide to the attention controlling cholesterol, China has stepped into the high-incidence season of cardiovascular disorder.Do not have effective means at current Cardiovarscular, prevention and health care just seem particularly important.
Control the rising of cholesterol, it is one of Main Means of the first vascular disease of current prevention and therapy, control the comparison effective means of cholesterol in the world, except the mode of making the life better and adjustment of diet drink, it is high that heat content is taken in restriction, fat is high, the food (as animal tallow etc.) that cholesterol is high is taken in, rational motion, have is exactly by the special class (fenofibrate of shellfish again, bezafibrate, Win-35833 etc.) and Hydroxymethylglutaryl list acyl CoA reductase inhibitor (Simvastatin, lovastatin, Pravastatin) etc. pharmacological agent, reduce the absorption of cholesterol, synthesis and promotion metabolism.But clinical drug therapy action specificity is stronger, interim reduction cholesterol index can only be reached, the function damage of Body adverse reaction and histoorgan can be caused in addition.Produce hypercholesterolemia reason more complicated (as unreasonable in diet, inherited genetic factors, pathogenic cholesterol metabolic are disorderly), at present for overall effect reason, the treatment means of system is very undesirable.By eating the channel in source, while guarantee body full nutrition and energy supply, the functional health care food being reduced cholesterol by strengthening reaches long-term care and prophylactic effect, has become the fashion of modern medicine study and consumption.
Probiotic bacterium and meta-bolites thereof play health-care effect by adjustment intestinal microflora function, attract great attention, people just find having the Massai people of diet cultured milk prod and American's serum cholesterol seldom to occur exception very early, but also cause academic sensation.People was had to demonstrate Lactobacterium acidophilum in food by experiment with animal afterwards and thermophilus streptococcus has obvious decreasing cholesterol effect.There is people from sausage, bacon, pickles, cheese, Yoghourt, filter out the higher milk-acid bacteria of multiple decreasing cholesterol ability successively.Scientific research personnel both domestic and external also confirms the certainty of different probiotic effect simultaneously in human trial.Achievement in research also shows the diversity of decreasing cholesterol probiotic strain and the otherness of function simultaneously.But about to have the probiotic bacterium reducing cholesterol effect in bean product from fermenting, and be developed to product and have not been reported.
It is definite that probiotic bacterium comprises the effect reducing cholesterol, but due to the complicacy of probiotic bacterium physiological function and the diversity of meta-bolites, current study mechanism also thorough, and this is also the one of the main reasons of restriction probiotic products exploitation.Current research shows, probiotic bacterium promotes cholate and cholesterol deposits by the lactic acid produced in metabolic process, and thalline adheres to and absorbs, and have impact on the absorption of enteron aisle to cholesterol, reaches the effect reducing blood cholesterol levels,
Abroad to probiotic bacterium research ratio deeply, China also has very large gap, constrain the development of probiotic bacterium research and industry, wherein there is the reason that fundamental research is inadequate and mechanism is unclear, it is main that what be is that probiotic bacterium study on the industrialization is inadequate, as bacteria selection, the stability of optimization and probiotic bacterium function is also pure at wretched insufficiency, some achievements in research, not through systematic research, the Ergonomy checking of human body and animal or cannot be unable to undergo and evaluate, mass-producing probiotic products (the Yoghourt of our country's production at present, bifidobacteria health-care product) bacterial classification and mostly or dependence on import, seriously constrain the development of industrialization and industry, therefore, probiotic bacterium comprise the basic researchs such as the screening of excellent species, a lot of work is also had to do, shoulder heavy responsibilities!
Along with China's Economic development, the raising of people's living standard and health care consciousness, the market demand of probiotic composition has continuous increase trend, China is soybean source region, people have the custom of long-term human consumption soybean, soybean prod product, inherently there is certain nourishing function (hypotensive, reducing blood-fat, raising immunizing power, improve blood heart and brain pipe function etc.), wherein the function of decreasing cholesterol is also obvious, therefore adopt probiotics fermention to make product, strengthen and increase its nourishing function meaning more great.Also contribute to driving China's Soybean Industry and improving added value of product, promote the development in related industries and market, for healthy and health care provide the theory of innovation and ground-breaking means to make due contribution.
Lactobacillus rhamnosus SCF 20120718 of the present invention is for the research reducing cholesterol effect
For examination bacterial classification: Lactobacillus rhamnosus SCF 20120718
For examination control strain: SJ20120511, SJ20120601, SJ20120611, SJ20120612, HHL201206, HHL20120311, HHL20120322, HHL20120405, HHL20120516, HHL20120622, totally 10 strains derive from commercially available yoghourt, pickles, sauerkraut, cheese, all preserve under 4 DEG C of conditions with the form of slant preservation.Place, Heilongjiang University's Life Science College pharmacological evaluation room
The preparation of experiment reagent
The preparation of 1.0mg/mL cholesterol solution
Accurately take cholesterol 100.00mg, use 50mL anhydrous alcohol solution, and be settled to 100mL;
The preparation of 0.1mg/mL cholesterol standardized solution
Accurate measuring (2.1.1) 1.0mg/mL cholesterol solution 10.0mL, uses 50mL anhydrous alcohol solution, and is settled to 100mL.
The preparation of ammonium ferric sulfate solution liquid
Accurately take ferric ammonium sulfate solid 4.463g, with dissolving with 50mL85% phosphoric acid rear and being settled to 100mL sealing, store in moisture eliminator.
The preparation of ferric ammonium sulfate nitrite ion
Accurate measuring ferric ammonium sulfate storing solution 10.0mL, with using 50mL concentrated sulfuric acid dissolution, and is settled to 100mL, seals and be placed in moisture eliminator to preserve.
The drafting of typical curve
Accurate measuring (2.1.2) 0.1mg/mL cholesterol standardized solution 0.4mL, 0.8mL, 1.2mL, 1.6mL, 2.0mL, 2.4mL, 2.8mL, 3.2mL are placed in 5mL volumetric flask, and use dehydrated alcohol constant volume scale respectively, accurately respectively measure 2mL instillation and add in the test tube of 2.0mL ferric ammonium sulfate nitrite ion in advance, jolting mixing 2min, adopts ultraviolet spectrophotometer to measure light absorption value in 560nm place.Take cholesterol concentration as X-coordinate, OD
560for ordinate zou, drawing standard curve.
For the mensuration of examination fermented liquid cholesterol level
Substratum is prepared
Lactic-acid-bacterium substratum (MRS) substratum is prepared
Accurately take peptone 10.0g, extractum carnis 10.0g, yeast extract paste 5.0g, diammonium hydrogen citrate [(NH4) 2HC6H5O7] 2.0g, glucose (C6H12O6H2O) 20.0g tween 80 1.0mL, sodium acetate (CH3COONa3H2O) 5.0g, dipotassium hydrogen phosphate (K2HPO43H2O) 2.0g, magnesium sulfate (MgSO47H2O) 0.58g, manganous sulfate (MnSO4H2O) 0.25g, to in the beaker being preinstalled with 500mL distilled water, heated and stirred is dissolved, and be settled to 1000mL, pH controls at 6.2-6.4, damp and hot 121 DEG C of sterilizing 15min, for subsequent use.
Cholesterol MRS (CHOL-MRS) substratum is prepared
Accurately take cholesterol 1g, be settled to 100mL with a small amount of anhydrous alcohol solution, after the filtering with microporous membrane of 0.45 μm, the amount according to 2% joins in MRS liquid nutrient medium.
The cultivation of strains tested
The activation culture of experimental strain
Strains tested and 10 strains are seeded in 10mL MRS liquid nutrient medium for examination control strain respectively, 37 DEG C of constant temperature culture 48h.
Experimental strain collecting cells
1. standard is prepared than turbid liquid
Measure respectively in 1% sulphuric acid soln 8.0mL, 1% barium chloride solution 2.0mL to 10mL volumetric flask, mix, for subsequent use.
2. sample liquid preparation is detected
Get strains tested and 10 strains respectively and carry out the centrifugal 15min of 4000r/min for examination control strain fermented liquid, bacterial sediment is washed three times by stroke-physiological saline solution, bacterial sediment is transferred to standard than in the identical volumetric flask of turbid liquid test tube, and is diluted to concentration (thalline turbidity and 10 wheat formula turbidity standard pipes suitable 3 × 10 with sterile saline
9the bacteria suspension of individual/mL).
The hot housing of experimental strain cholesterol
Strains tested and 10 strains are inoculated in 10mLCHOL-MRS substratum for examination control strain bacteria suspension by inoculum size by 2% respectively, 37 DEG C of constant temperature culture 72h.
For the mensuration of cholesterol level in examination fermented liquid
The each strains tested of accurate measuring and 10 strains, in examination control strain fermented liquid 0.2mL to 10mL centrifuge tube, add 9.8mL dehydrated alcohol, the centrifugal 15min of jolting 1min, 4000r/min.The centrifugal rear supernatant liquor 2.0mL that gets respectively is in test tube, and slowly add 2.0mL ferric ammonium sulfate nitrite ion along tube wall, jolting fully mixes, and to room temperature, compares with the substratum processed equally, measures light absorption value OD
560.Calculate removing cholesterol rate.
Result and discussion
Table: strains tested body removes the result of cholesterol ability
| Strain number | Decreasing ratio (%) | Strain number | Decreasing ratio (%) |
| SJ20120511 | 23.3 | SCF20120718 | 79.5 |
| SJ20120601 | 22.4 | HHL20120311 | 7.2 |
| SJ20120611 | 19.5 | HHL20120322 | 11.3 |
| SJ20120612 | 16.2 | HHL20120405 | 27.8 |
| HHL201206 | 10.9 | HHL20120516 | 19.7 |
| HHL20120622 | 7.4 |
Adopt ferric ammonium sulfate method to detect probiotic bacterium Cholesterol removal evaluation experimental result to show: strains tested and 10 strains all have fermenting process cholesterol ability in various degree for examination control strain, experiment obtain this patent project Lactobacillus rhamnosus strain SCF20120718, compare with the probiotic bacterium obtained from commercially available yoghourt, pickles, sauerkraut, cheese, there is the effect of cholesterol in stronger removal substratum, for further zooscopy is laid a good foundation! Experimental strain pharmaceutical research
1, lactobacillus rhamnosus SCF 20120718 fermented soybean milk acute toxicity test research
Laboratory animal
Kunming mouse, weight range 18-22g, male and female half and half, purchased from Nangang District, Harbin City Jade Hare plant.
The preparation of fermented soybean milk sample
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
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 3.4%). for subsequent use
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 lactobacillus rhamnosus SCF 20120718 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 lactobacillus rhamnosus SCF 20120718 fermentation is standby.
Table: fermented soybean milk acute toxicity test result
Note: P>0.05, there was no significant difference compared with control group.
Lactobacillus rhamnosus SCF 20120718 ferments and reduces blood cholesterol levels experimental study
Laboratory animal and reagent
Laboratory animal:
Healthy male mouse of kunming 100, purchased from Nangang District, Harbin City Jade Hare plant, body weight 20-24g.Animal general state is good, fur gloss, and feed is normal with activity.Animal rearing environment: Heilongjiang University's Life Science College Animal Lab., indoor clean, well-ventilated.
Experimental implementation
Laboratory animal divides into groups
Mouse adapts to 6d in experimental situation, and reject body weight defective, body surface has the abnormal mouse of wound, sign, equal free choice feeding and drinking-water before experiment, specialized management in experimentation.Animal housing light 12h throws light on, and room temperature controls at 21 ± 3 DEG C, and relative humidity is 50-70%.
Experiment contrast group: get healthy male mice 20 20-24g, be divided into 2 groups at random,
Contrast I group (n=10 only): normal ad lib mouse basal feed and drinking-water, put to death after 4 weeks, take liver spleen and blood.
Contrast II group (n=10 only): normal ad lib mouse basal feed and drinking-water, continue 8 weeks.5th week starts physiological saline gavage, within the 9th week, puts to death, takes liver spleen and blood.
Hyperlipidemia model group
Mouse high fat diet: take mouse basal feed 2kg, by 2% cholesterol, 10% lard, 0.5% Sodium cholic acid, heat fused mixes, and mixes briquetting while hot with basal feed, puts to room temperature.
Get healthy male mice 20,20-24g is divided into 2 groups at random, often organizes 10,
Hypercholesterolemia model group I (n=10 only): normal ad lib mouse high lipid food and drinking-water, puts to death after 4 weeks, takes liver spleen and blood.
Hypercholesterolemia modelⅱ group (n=10 only): normal ad lib mouse high fat diet and drinking-water, continue 8 weeks.5th week starts physiological saline gavage.Within 9th week, put to death, take liver spleen and blood.Drug treatment group
Test group: get body weight healthy male mice 30 20-24g, be divided into 2 groups at random,
High dose group (n=10 only): normal ad lib mouse high fat diet and drinking-water, continue 8 weeks, starts by 30mL/Kg fermented soybean milk sample gavage on the 5th week.Within 9th week, put to death, take liver spleen and blood.
Middle dosage group (n=10 only): normal ad lib mouse high fat diet and drinking-water, continue 8 weeks, starts by 20mL/Kg fermented soybean milk sample gavage on the 5th week.Within 9th week, put to death, take liver spleen and blood.
Low dose group (n=10 only): normal ad lib mouse high fat diet and drinking-water, continue 8 weeks, starts by 10mL/Kg fermented soybean milk sample gavage on the 5th week.Within 9th week, put to death, take liver spleen and blood.
Mouse sign is observed
Test each group of appetite of observing mouse at any time, behavior, ight soil, hair and animal dead situation.
Prepared by blood biochemistry checking Indexs measure mice serum
The sampling time neck that breaks established according to grouping respectively gets blood.Mouse whole blood is through the centrifugal 10min of 3000rpm, and separation of serum is for subsequent use.
The mensuration (sulphur ferrophosphorus method) of mice serum total cholesterol
The preparation of reagent
2%FeCl
3solution preparation: take the iron trichloride (FeCl after grinding
36H
2o) 2g, is dissolved in 100ml strong phosphoric acid, dark place hold over night.
Sulphur ferrophosphorus preparation of reagents: get above-mentioned 2%FeCl
38ml, puts into 100ml volumetric flask, adds the vitriol oil to scale, and mixing, opens in dark place.
Cholesterol standardized solution is prepared: accurately take recrystallization, dry cholesterol 30mg, be dissolved in methyl alcohol to 100ml.
Experimental technique and step
1. cholesterol typical curve is made: be mixed with molten for 0.3mg/ml cholesterol standard the cholesterol solution that concentration is respectively 0.03mg/ml, 0.06mg/ml, 0.09mg/ml, 0.12mg/ml, 0.15mg/ml.Get the cholesterol solution 3ml adding above-mentioned 5 kinds of concentration in 5 test tubes respectively, add methanol solution in another test tube as blank tube.Slowly add sulphur ferrophosphorus reagent 3ml along tube wall respectively, immediately rapidly jolting mixing, place cooling after 15 minutes blank tube for carry out colorimetric in comparison with 520nm, to take cholesterol concentration as X-coordinate drawing standard curve,
2. get centrifuge tube numbering, accurately add mice serum 0.1ml, then add methyl alcohol 4.9ml at the bottom of pipe.Block the mouth of pipe with glassine paper, jolting makes it mixing, and room temperature places 15min, then jolting mixing, then centrifugal 5 minutes (2000r/min), gets supernatant liquor.In mensuration pipe, add above-mentioned methanol extract liquid 3ml respectively, in blank tube, add methyl alcohol 3ml.Add sulphur ferrophosphorus reagent 3ml in each pipe respectively, slowly must add, be divided into two-layer with methanol solution along tube wall, jolting mixing rapidly, places cooling 15 minutes immediately, with blank for carry out colorimetric in comparison with 520nm, calculates
The light absorption value of serum methanol extract liquid is y, brings typical curve equation into and obtains x value.
The mensuration (colorimetry) of mice serum gpt
The preparation of reagent
PH=7.4 phosphate buffered saline buffer: precision measures potassium primary phosphate 2.69g, moves in 100ml volumetric flask after dipotassium hydrogen phosphate 13.97g is dissolved in water, adding distil water, to scale, is stored in refrigerator for subsequent use.
Substrate solution (matrix liquid): precision measures DL-Alanine 1.79g, α-one the eleventh of the twelve Earthly Branches diacid 29.2mg, is first dissolved in about 50ml phosphoric acid buffer, then with 1mol/L NaOH school to pH=7.4, be diluted to 100ml with phosphate buffered saline buffer again, add chloroform number and drip anticorrosion, be stored in refrigerator.
μ g/ml pyruvate standard liquid: precision takes the pyruvic acid 12.64mg being dried to weight, is placed in 100ml volumetric flask, is diluted to scale with the phosphoric acid buffer of pH=7.4.(the necessary prepared before use of this liquid, can not deposit)
0.02%2.4-dinitrophenylhydrazine liquid: take 2.4-dinitrophenylhydrazine 20mg, is dissolved in 10N HCl 10ml, after dissolving again adding distil water to 100ml.
Experimental technique and step
Test according to operation below:
Table: mouse gpt detects operation steps
Table: mouse gpt detects operation steps
After static 10 minutes, select the monochromatic ray of 520nm, with distilled water zeroing, measure each pipe optical density(OD).
By calculating:
Pathology of hepar checks
Make belly median incision after getting blood and enter abdominal cavity, observe each internal organs general condition, carefully differentiate each group of mouse liver appearance change.Complete resection liver also claims weight in wet base, calculates liver index=(liver weight in wet base/body weight) × 100%.And liver segmentation is become fritter, drop in 10% formaldehyde fixing, paraffin embedding, section, HE dyes, light Microscopic observation.Its experiment flow is as follows:
(1) fixing: the fritter liver split to be placed in 10% formaldehyde solution and to fix 24h;
(2) wash: be placed in flowing water and wash liver organization 12h;
(3) dewater: propyl carbinol dewaters: 1. 70% alcohol, 1.5h; 2. 80% alcohol, 1.5h; 3. propyl carbinol (I), 1.5h; 4. propyl carbinol (II), 1.5h; 5. propyl carbinol (III), 2h.
Waxdip: 1. wax I (fusing point 48 DEG C-50 DEG C), 20min; 2. wax II (fusing point 52 DEG C of-54C), 20min; 3. wax III (fusing point 56 DEG C-58 DEG C), 20min.
Embedding: adopt largest face embedding, gently press tissue block arched part with tweezers in embedding process, make it smooth in bottom;
(6) cut into slices: getting thickness is that 5 μ g cut into slices;
(7) open up sheet and drag for sheet: cut into slices in warm water (42 DEG C-45 DEG C) launch, and fish for complete and non-wrinkled section, are pasted on the centre of in slide 1/3 and lower 1/3;
(8) roasting sheet: will slide roasting sheet 1h in 60 DEG C of baking ovens of section be posted;
(9) dewax: baked slide is placed in dimethylbenzene dewaxing (2 times); Alcohol then from the alcohol of high density to lower concentration takes off dimethylbenzene;
(10) dye: brazilwood extract dyeing 8min, then washes the dye liquor combined in a slice with water, with 1% hydrochloric acid color separation, washes 10h with flowing water, cause section to be faded in case hydrochloric acid is residual, finally enter Yihong and redye 5min;
(11) dewater: the idiographic flow of section dehydration is: 70% alcohol 2min, 80% alcohol 2min, 95% alcohol 2min, 100% alcohol 5min, 100% alcohol 5min, dimethylbenzene 5min, dimethylbenzene 5min;
(12) microscopy: light Microscopic observation evaluating liver steatosis changes, and takes pictures.
Fatty degeneration of liver degree judging criterion: become (-) without liver cell fat, in liver lobule, < 30% liver cell generation fat becomes (+), 30%-50% person is mild fatty liver (++), 50%-75% person is moderate fatty liver (+++), and > is severe fatty liver (++++).
Experimental result
When carrying out, in experiment manufacture pathology high blood lipid model process, hyperlipidemia model group and control group compare: in cholesterol feed process, initial stage appetite is good, and body weight gain is rapid, and sign occurs some abnormal (mouse hair owes gloss, the soft flavescence of loose and watery stool) gradually.Started to occur appetite decline phenomenon at the 13 day, and started at the 16 day obviously to decline.Body weight, liver weight in wet base, liver index, blood cholesterol and serum glutamic pyruvic transminase compare with Normal group significant difference (P < 0.01).
Table: manufacturing hyperlipidemic mice affects sign and liver physiology and chemistry
Note: * * represents and compares P < 0.01 with normal control I group
Directly perceived and the pathological section of hepatic tissue also shows simultaneously: feeding high cholesterol diet mouse liver and control group compare, and volume increases, and edge circle is blunt, and matter is soft and crisp, tangent plane is greasy, color whiting.There is fatty pathology in tissue, occurs that fat drips in liver cell kytoplasm, and in severe patient cell, large fat drips and nucleus is pressed to side.Testing the method taked impels normal mouse sign, hepatic tissue and biochemical indicator generation pathologic to change, and manufactures high blood lipid model success.
Table: murine liver tissue sex change is observed
Feeding fermented soybean milk is on the impact of feeding high cholesterol diet mouse
Feeding lactobacillus rhamnosus SCF 20120718 fermented soybean milk each dosage group and feeding hypercholesterolemia control group compare sign (appetite increase not obvious, figure is stiff, loose and watery stool is soft) obviously to be improved (appetite, reaction, ight soil recover normal gradually), middle and high dosage group is improved the most obvious, close to Normal group mouse.Hepatic tissue is dissected and pathology section examination demonstrates result too, and body weight, liver weight in wet base, liver index, blood cholesterol and serum glutamic pyruvic transminase compare with hypercholesterolemia feeding control group significant difference (P < 0.01).Result proves that the physically different and liver tissue injury of mouse that feeding fermented soybean milk causes hypercholesterolemia feeding has reparation and therapeutic action
Table: manufacturing hyperlipidemic mice affects sign and liver physiology and chemistry
Note: * * represents that comparing P < 0.01, * represents and compare P < 0.05 with normal control II group with normal control II group
★ ★represent and compare P < 0.01 with model comparison II group,
★represent and compare P < 0.05 with model comparison II group
Table-14 murine liver tissue pathology section examination results
Take fermented soybean milk human body effect experimental of the present invention
Suffer from 10 years camber Patients with Fatty Liver for one, take 1000mL lactobacillus rhamnosus fermentation every day for tamarind slurry after two months, health check-up is reported the result and is shown, vexed before edible, feel run-down, have a sleepless night, the symptom such as dizzy is very serious, health check-up report (figure below) that on February 27th, 2012, large health examination Harbin Xiangfang branch carried out in power-section, Harbin, wherein all there is the exception of significance in triglyceride level (TG), glutamic-oxal(o)acetic transaminase (AST), gpt (ALT) and ratio (AST/ALT).Take on May 26th, 1 and carry out blood biochemical detection check (figure below) in No.211 Hospita, PLA.Find after twice detected result, ALT, AST, TG, glutamyltranspeptidase (GGT), alkaline phosphatase (ALP) content all obviously decline, except LDL-C slightly raises, millet straw/paddy third numerical value recovers normal, illustrate that the lactobacillus rhamnosus fermentation that patent adopts not only has for tamarind slurry the effect reducing ALT, TC, AST, GGT, TG multiaction can also be reduced simultaneously, can be used as the protective foods of the cardiovascular and cerebrovascular diseases that prevention and therapy raises with hyperlipidemia.There is the value of very high further research and development.
Accompanying drawing illustrates:
First group of accompanying drawing, each test group murine liver tissue internal anatomy
Fig. 1: normal control I group of murine liver tissue internal anatomy Fig. 2: model comparison I group of murine liver tissue internal anatomy
Fig. 3: normal control II group of murine liver tissue internal anatomy Fig. 4: model comparison II group of murine liver tissue internal anatomy
Fig. 5: high dose group murine liver tissue internal anatomy Fig. 6: low dose group murine liver tissue internal anatomy.
Second group of accompanying drawing, each test group murine liver tissue pathological section figure
Fig. 7: control group mice hepatic tissue pathology slice map (-) Fig. 8: high dose group murine liver tissue pathological section figure (+)
Fig. 9: low dose group murine liver tissue pathological section figure (++) Figure 10: middle dosage group murine liver tissue pathological section figure (+++)
Figure 11: model control group murine liver tissue pathological section figure (++++).
3rd group of accompanying drawing, trier takes blood parameters detected result before and after fermented soybean milk
Figure 12: take trier's blood parameters detected result (on February 27th, 2012) before fermented soybean milk
Figure 13: take trier's blood parameters detected result (on May 26th, 2012) after fermented soybean milk.
Embodiment:
Embodiment 1:
A kind of fermented bean products that derives from has the lactobacillus rhamnosus reducing cholesterol effect, by being collected in spontaneous fermentation bean product, through the high reactivity bacterial strain of separation, purifying, physical mutagenesis, preferably acquisition, mycology specific name Lactobacillus rhamnosus, by cultivating, bacteria characteristic: the substratum being placed in 37 DEG C of constant incubators is cultivated, finds that weaker concn is 10
-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, and preserving number is: CGMCC No.7085.
Embodiment 2:
The fermented soybean milk that derives from described in embodiment 1 has the lactobacillus rhamnosus reducing cholesterol effect, and the bacteria characteristic of described high reactivity bacterial strain is: 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.
Embodiment 3:
The fermented soybean milk that derives from described in embodiment 1 or 2 has the lactobacillus rhamnosus reducing cholesterol effect, described bacterial strain carries out BLAST comparison online through 16S rDNA partial sequence, and result has the central genetic characteristic of Lactobacillus Pseudomonas rhamnosus bacterial classification;
16S rDNA order-checking partial sequence is as follows:
There is lactobacillus rhamnosus and the purposes of decreasing cholesterol effect
Embodiment 4:
The fermented soybean milk that derives from described in embodiment 1 or 2 or 3 has the lactobacillus rhamnosus reducing cholesterol effect, and described substratum prescription is:
Described ratio is ratio of weight and number.
Embodiment 5:
A kind of soya-bean milk utilizing the lactobacillus rhamnosus described in embodiment 1,2,3 to make, by the inoculation of one of embodiment 1-4 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 ferment more than 24 hours, obtained tamarind slurry.
Embodiment 6:
The preparation method of the soya-bean milk described in a kind of embodiment 5:
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 parts by weight 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 SCF 20120718 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, 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, and by soaked soybean with beans water ratio the soybean milk maker defibrination of parts by weight 1: 8, 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, add above-mentioned fermented soybean milk for subsequent use by 5% volume ratio, with the fermentation 2 hours of 30r/min, 37-40 DEG C, then packing, sealing, 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 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 45.1%.Sealing cryodrying saves backup.
Embodiment 7:
The above-mentioned fermented bean products that derives from is had the lactobacillus rhamnosus reducing cholesterol effect, reduces the hyperlipemia comprising cholesterol raise for making, for the purposes of the protective foods and medicine that make prevention and therapy cardiovascular and cerebrovascular diseases.
Embodiment 8:
A kind of fermented soybean milk that derives from has lactobacillus rhamnosus (Lactobacillus rhamnosus) SCF 20120718 reducing cholesterol effect, 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 (100101) phone: 64807355 preserving numbers are: CGMCC No.7085.
Lactobacillus rhamnosus (Lactobacillus rhamnosus) SCF 20120718, being by being collected in spontaneous fermentation bean product (dry bean curd), 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 SCF 20120718 obtained, culture condition is: substratum is peptone 10 parts, extractum carnis 10. parts, yeast extract paste 5 parts, diammonium hydrogen citrate [(NH
4)
2hC
6h
5o
7] 2 parts of glucose (C
6h
12o
6h
2o) 20 parts, tween 80 1 part, sodium acetate (CH
3cOONa3H
2o) 5 parts, dipotassium hydrogen phosphate (K
2hPO
43H
2o) 2 parts, magnesium sulfate (MgSO
47H
2o) 0.58 part, manganous sulfate (MnSO
4h
2o) 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: lactobacillus rhamnosus SCF 20120718 is through VITEK 2Compact qualification result
Table: lactobacillus rhamnosus SCF 20120718 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.
Order-checking partial sequence is as follows:
There is lactobacillus rhamnosus and the purposes of decreasing cholesterol effect
Embodiment 9:
Adopt lactobacillus rhamnosus (Lactobacillus rhamnosus) SCF 20120718 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 SCF 20120718 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 45.1%.Sealing cryodrying saves backup.
Embodiment 10:
A kind of fermented soybean milk that derives from has lactobacillus rhamnosus (Lactobacillus rhamnosus) SCF20120718 reducing cholesterol, 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 (100101) phone: 64807355 preserving numbers are: CGMCC No.7085.
Lactobacillus rhamnosus (Lactobacillus rhamnosus) SCF20120718, being by being collected in spontaneous fermentation bean product (soft beancurd is commonly called as large bean curd), 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.
Embodiment 11:
Described lactobacillus rhamnosus (Lactobacillus rhamnosus) SCF20120718, carry out BLAST comparison online through 16S rDNA partial sequence, result has the central genetic characteristic of Serratia Pseudomonas marcescens bacterial classification.It is characterized in that: 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, adopts Physiology and biochemistry assessing instrument to identify to have the physiological and biochemical property of following table to lactobacillus rhamnosus (Lactobacillus rhamnosus) SCF20120718.
Gene order table
<110> Heilongjiang University
<120> has lactobacillus rhamnosus and the purposes of decreasing cholesterol effect
The <141> date
<160>1
<210> 1
<211>1561
<212>RNA
<213> lactobacillus rhamnosus
<223> n=a or g or c or t
<400> nucleotide sequence and/or aminoacid sequence
gagtttgatc ctggctcagg atgaacgctg gcggcgtgcc taatacatgc aagtcgaacg 60
agttctgatt attgaagggt gcttgcatct tgatttaatt ttgaacgagt ggcggacggg 120
tgagtaacac gtgggtaacc tgcccttaag tgggggataa catttggaaa cagatgctaa 180
taccgcataa atccaagaac cgcatggttc ttggctgaaa gatggcgtaa gctatcgctt 240
ttggatggac ccgcggcgta ttagctagtt ggtgaggtaa cggctcacca aggcaatgat 300
acgtagccga actgagaggt tgatcggcca cattgggact gagacacggc ccaaactcct 360
acgggaggca gcagtaggga atcttccaca atggacgcaa gtctgatgga gcaacgccgc 420
gtgagtgaag aaggctttcg ggtcgtaaaa ctctgttgtt ggagaagaat ggtcggcaga 480
gtaactgttg tcggcgtgac ggtatccaac cagaaagcca cggctaacta cgtgccagca 540
gccgcggtaa tacgtaggtg gcaagcgtta tccggattta ttgggcgtaa agcgagcgca 600
ggcggttttt taagtctgat gtgaaagccc tcggcttaac cgaggaagtg catcggaaac 660
tgggaaactt gagtgcagaa gaggacagtg gaactccatg tgtagcggtg aaatgcgtag 720
atatatggaa gaacaccagt ggcgaaggcg gctgtctggt ctgtaactga cgctgaggct 780
cgaaagcatg ggtagcgaac aggattagat accctggtag tccatgccgt aaacgatgaa 840
tgctaggtgt tggagggttt ccgcccttca gtgccgcagc taacgcatta agcattccgc 900
ctggggagta cgaccgcaag gttgaaactc aaaggaattg acgggggccc gcacaagcgg 960
tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accaggtctt gacatctttt 1020
gatcacctga gagatcaggt ttccccttcg ggggcaaaat gacaggtggt gcatggttgt 1080
cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttatgact 1140
agttgccagc atttagttgg gcactctagt aagactgccg gtgacaaacc ggaggaaggt 1200
ggggatgacg tcaaatcatc atgcccctta tgacctgggc tacacatgtg ctacaatgga 1260
tggtacaacg agttgcgaga ccgcgaggtc aagctaatct cttaaagcca ttctcagttc 1320
ggactgtagg ctgcaactcg cctacacgaa gtcggaatcg ctagtaatcg cggatcagca 1380
cgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca tgagagtttg 1440
taacacccga agccggtggc gtaacccttt tagggagcga gccgtctaag gtgggacaaa 1500
tgattagggt gaagtcgtaa caaggtagcc gtaggagaac ctgcggctgg atcacctcct 1560
t 1561。
Claims (7)
1. one kind derives from the lactobacillus rhamnosus that fermented soybean milk has decreasing cholesterol effect, 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 Lactobacillusrhamnosus SCF 20120718, by cultivating, bacteria characteristic: the substratum being placed in 37 DEG C of constant incubators is cultivated, finds that weaker concn is 10
-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, and preserving number is: CGMCC No.7085.
2. according to claim 1ly derive from the lactobacillus rhamnosus that fermented soybean milk has decreasing cholesterol effect, it is characterized in that: the bacteria characteristic of described high reactivity bacterial strain is: 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.
3. according to claim 1 and 2ly derive from the lactobacillus rhamnosus that fermented soybean milk has decreasing cholesterol effect, it is characterized in that: described bacterial strain carries 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:
Lactobacillus rhamnosus SCF 20120718
<110> Heilongjiang University
<120> has lactobacillus rhamnosus and the purposes of decreasing cholesterol effect
<160>1
<210>Lactobacillus rhamnosus SCF 20120718
<211>1561
<212>RNA
<213> lactobacillus rhamnosus
<223>n=a or g or c or t
<400> nucleotide sequence and/or aminoacid sequence
gagtttgatc ctggctcagg atgaacgctg gcggcgtgcc taatacatgc aagtcgaacg 60
agttctgatt attgaagggt gcttgcatct tgatttaatt ttgaacgagt ggcggacggg 120
tgagtaacac gtgggtaacc tgcccttaag tgggggataa catttggaaa cagatgctaa 180
taccgcataa atccaagaac cgcatggttc ttggctgaaa gatggcgtaa gctatcgctt 240
ttggatggac ccgcggcgta ttagctagtt ggtgaggtaa cggctcacca aggcaatgat 300
acgtagccga actgagaggt tgatcggcca cattgggact gagacacggc ccaaactcct 360
acgggaggca gcagtaggga atcttccaca atggacgcaa gtctgatgga gcaacgccgc 420
gtgagtgaag aaggctttcg ggtcgtaaaa ctctgttgtt ggagaagaat ggtcggcaga 480
gtaactgttg tcggcgtgac ggtatccaac cagaaagcca cggctaacta cgtgccagca 540
gccgcggtaa tacgtaggtg gcaagcgtta tccggattta ttgggcgtaa agcgagcgca 600
ggcggttttt taagtctgat gtgaaagccc tcggcttaac cgaggaagtg catcggaaac 660
tgggaaactt gagtgcagaa gaggacagtg gaactccatg tgtagcggtg aaatgcgtag 720
atatatggaa gaacaccagt ggcgaaggcg gctgtctggt ctgtaactga cgctgaggct 780
cgaaagcatg ggtagcgaac aggattagat accctggtag tccatgccgt aaacgatgaa 840
tgctaggtgt tggagggttt ccgcccttca gtgccgcagc taacgcatta agcattccgc 900
ctggggagta cgaccgcaag gttgaaactc aaaggaattg acgggggccc gcacaagcgg 960
tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accaggtctt gacatctttt 1020
gatcacctga gagatcaggt ttccccttcg ggggcaaaat gacaggtggt gcatggttgt 1080
cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttatgact 1140
agttgccagc atttagttgg gcactctagt aagactgccg gtgacaaacc ggaggaaggt 1200
ggggatgacg tcaaatcatc atgcccctta tgacctgggc tacacatgtg ctacaatgga 1260
tggtacaacg agttgcgaga ccgcgaggtc aagctaatct cttaaagcca ttctcagttc 1320
ggactgtagg ctgcaactcg cctacacgaa gtcggaatcg ctagtaatcg cggatcagca 1380
cgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca tgagagtttg 1440
taacacccga agccggtggc gtaacccttt tagggagcga gccgtctaag gtgggacaaa 1500
tgattagggt gaagtcgtaa caaggtagcc gtaggagaac ctgcggctgg atcacctcct 1560
t 1561。
4. according to claim 1 or 2 or 3, derive from the lactobacillus rhamnosus that fermented soybean milk has decreasing cholesterol effect, it is characterized in that: described substratum prescription is:
Described ratio is ratio of weight and number.
5. the soya-bean milk utilizing the lactobacillus rhamnosus described in claim 1,2,3 to make, it is characterized in that: by the inoculation of 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 preparation 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 parts by weight 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 SCF 20120718 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, 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, and by soaked soybean with beans water ratio the soybean milk maker defibrination of parts by weight 1: 8, 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, add above-mentioned fermented soybean milk for subsequent use by 5% volume ratio, with the fermentation 2 hours of 30r/min, 37-40 DEG C, then packing, sealing, 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 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 45.1%.Sealing cryodrying saves backup.
7. one kind will derive from the lactobacillus rhamnosus that fermented soybean milk has decreasing cholesterol effect described in claim 1 or 2 or 3 or 4, the hyperlipemia comprising cholesterol for making that comprising sees the bottom raises, for the protective foods of exploitation and composition prevention and therapy cardiovascular disorder and the purposes of medicine.
Priority Applications (1)
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113088470A (en) * | 2021-04-14 | 2021-07-09 | 广东医科大学 | Lactobacillus rhamnosus L.rB16 and application thereof |
| IT202100026282A1 (en) * | 2021-10-13 | 2023-04-13 | Alfasigma Spa | FERMENTED SURNATANT AND POSTBIOTIC COMPOSITIONS USEFUL IN THE PREVENTION AND TREATMENT OF CARDIOVASCULAR DISEASES |
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