CN104560790A - Functional lactobacillus plantarum and preparation method of compound bacterial powder of functional lactobacillus plantarum - Google Patents
Functional lactobacillus plantarum and preparation method of compound bacterial powder of functional lactobacillus plantarum Download PDFInfo
- Publication number
- CN104560790A CN104560790A CN201410763406.5A CN201410763406A CN104560790A CN 104560790 A CN104560790 A CN 104560790A CN 201410763406 A CN201410763406 A CN 201410763406A CN 104560790 A CN104560790 A CN 104560790A
- Authority
- CN
- China
- Prior art keywords
- powder
- bacillus natto
- freeze
- natto
- plant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
-
- 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
- C12N1/14—Fungi; Culture media therefor
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Botany (AREA)
- Mycology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to a functional lactobacillus plantarum plant subspecies having a holesterol lowering function and a preparation method of a compound bacterial powder of the plant subspecies, and belongs to the application fields of functional food and microecologics. According to the preparation method, the lactobacillus plantarum plant subspecies Zhang-LL CGMCC No. 6936 is used for preparing Zhang-LL strain freeze-dried powder by virtue of culture activation, fermentation, bacterial sludge collection and freeze-drying, and the viable count of the freeze-dried powder is 3.50*10<11> CFU/g. Bacillus natto BNZ3 CGMCC No. 9146 is used for preparing bacillus natto power by virtue of culture activation, enlarged cultivation, fermentation and drying, and the activity of nattokinase in the bacillus natto power is 2000IU/g; monacus purpureus Zhang-MP CGMCC No. 9221 is used for preparing monacus purpureus powder by virtue of culture activation, enlarged cultivation, solid-state fermentation, drying and grinding; as being tested, the content of the active substance Monacolin K in the monacus purpureus powder is 4.98mg/g. The prepared lactobacillus plantarum plant subspecies powder is thoroughly mixed with the bacillus natto power and the monacus purpureus powder in the ratio of 1:1:1(w/w), thereby obtaining compound bacterial powder having the serum cholesterol lowering effect.
Description
Technical field
The present invention relates to a kind of plant lactobacillus plant subspecies with decreasing cholesterol ability, and with plant lactobacillus bacterium powder for main raw material, add natto powder and Hongqu powder (red colouring agent), prepare a kind of composite bacterium powder with decreasing cholesterol function, belong to functional food and probiotics Application Areas.
Background technology
Serum cholesterol level is higher is the Major Risk Factors causing cardiovascular disorder, plant lactobacillus plant subspecies of the present invention, bacillus natto, monascus purpureus are edible probiotic bacterium, the functional composite bacterium powder be made up of these three kinds of bacterial strains, can hypercholesterolemia be prevented, to human body, there is good health-care effect.
Probiotic lactobacillus can promote the absorption of the nutritive substances such as protein, produces a large amount of benefit materials such as vitamin B complex, improves human intestinal function, recover colony balance in human intestinal, strengthen body immunity, antitumor, also there is decreasing cholesterol, reducing blood-fat, hypotensive activity; Can interior free yl be eliminated, there is antidotal effect.
Bacillus natto can change human intestinal microflora ecology, helps normal gut function, maintains body physiological environment.Secrete various enzyme and VITAMIN, promote the propagation of small intestinal mucosa cell.In addition, bacillus natto, when utilizing soybean protein to grow, produces Nattokinase and vitamin K, has treatment three height (hyperglycemia, hypertension, hyperlipidemia) and thrombolytic effect.
There is in the meta-bolites of red colouring agent for food, also used as a Chinese medicine the natural statin substance that can reduce serum cholesterol, particularly containing lovastatin (a kind of active substance and Monacolin K with remarkable blood lipid regulation ability), many compositions useful to human body can also be produced, as lipid acid etc. is closed in indispensable amino acid, insatiable hunger simultaneously.Large quantity research finds, red colouring agent for food, also used as a Chinese medicine has very powerful reduction total cholesterol, low density lipoprotein cholesterol, serum triglyceride, atherogenic index, the remarkable comprehensive therapeutic effect of high density lipoprotein increasing cholesterol.Take that red colouring agent for food, also used as a Chinese medicine security is high, side effect is little, and effectively can treat the cardiovascular and cerebrovascular diseases such as coronary heart disease, cerebral apoplexy and the disease relevant to hyperlipidemia, as diabetes, nephrotic syndrome and fatty liver, be considered to the most promising current lipopenicillinase material.
At present, have a lot of research to functional plants milk-acid bacteria both at home and abroad, " strain has plant lactobacillus and the application thereof of decreasing cholesterol function " (CN201010034128.1) of Jilin Academy of Agricultural Science application discloses the lactobacterium plantarum strain that a strain has decreasing cholesterol ability.(CN200910069088.1's " preparation method of the probiotics preparation of a kind of reducing blood-fat and regulating intestinal canal flora " of University Of Science and Technology Of Tianjin's application) ferments to Kefir lactobacillu plantarurn, freeze-drying, and describes the physiological function that it has decreasing cholesterol ability and regulating intestinal canal flora.In addition, also have some patent documentations to relate to the plant lactobacillus of decreasing cholesterol ability, but all not yet relate to natto and red colouring agent for food, also used as a Chinese medicine is composite, make functional bacterium powder.Accordingly, the present invention mainly utilizes a strain to have the plant lactobacillus plant subspecies of decreasing cholesterol ability and stronger acid resistance, bile tolerance ability, Hongqu powder (red colouring agent) prepared by the Bacillus natto powder prepared with bacillus natto and monascus purpureus, prepares a kind of composite bacterium powder with decreasing cholesterol function.
Summary of the invention
The object of the invention is to provide a kind of plant lactobacillus plant subspecies bacterium powder with decreasing cholesterol ability, and Hongqu powder (red colouring agent) prepared by the natto powder prepared with bacillus natto and monascus purpureus is composite, prepares a kind of composite bacterium powder with decreasing cholesterol function.
1, the preparation method of plant lactobacillus plant subspecies bacterium powder
Lactobacillus plantarum plant subspecies (the Lactobacillus plantarum subsp.plantarum) Zhang-LL that the present invention relates to, be preserved in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 04th, 2012, Institute of Microorganism, Academia Sinica), its preserving number is: CGMCC No.6936.It is characterized in that MRS substratum, cultivate 2d, thalli morphology: thalline rod-short, 0.5 × 1.33 ~ 2.0 μm, single, in pairs or in short catenation, spore of not sprouting, Gram-positive.
The preparation process of plant lactobacillus plant subspecies bacterium powder:
(1) actication of culture: by the plant lactobacillus plant subspecies Zhang-LL bacterial classification after activation enlarged culturing, be inoculated in 2L modified MRS culture medium, 5L automatic fermenter is utilized to carry out high density fermentation, temperature controls at 34 DEG C, fermented liquid pH 6.5, mixing speed is 150r/min, and fermentation 20 ~ 28h, obtains fermented liquid.
In aforesaid method, MRS liquid culture medium formula: Tryptones 10.0g, extractum carnis 10.0g, yeast leaching powder 5.0g, Triammonium citrate 2.0g, glucose 20.0g, tween 80 1.0mL, sodium acetate 5.0g, dipotassium hydrogen phosphate 2.0g, magnesium sulfate 0.5g, manganous sulfate 0.25g, water 1L, pH6.5,0.1Mpa sterilizing 15min.
(2) collect bacterium mud: fermented liquid is placed in whizzer, in 4 DEG C, the centrifugal 10min of 8000r/min, collect and obtain bacterium mud.
(3) pre-freeze and lyophilize: mixed with the protective material of 1/10 original fermented solution volume by plant lactobacillus plant subspecies bacterium mud, lyophilized vaccine composition is 15% skimmed milk powder, after mixing, obtains starter culture concentrates; By starter culture concentrates in-35 DEG C of refrigerators after pre-freeze 13h, obtain pre-freeze active bacteria formulation, utilize vacuum freeze drier by pre-freeze active bacteria formulation in-55 DEG C, under the condition of vacuum tightness 0.08mBar, freeze-drying 48h, to complete drying state, obtains plant lactobacillus plant subspecies freeze-dried vaccine powder (active bacteria formulation).
2, the preparation method of bacillus natto natto powder
A strain bacillus natto (Bacillus natto) BNZ3 that the present invention relates to, be preserved in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 12nd, 2014, Institute of Microorganism, Academia Sinica), preserving number is: CGMCC No.9146.It is characterized in that glucose agar medium is cultivated, cultivate 36h, thalli morphology: thalline is shaft-like, (0.7-0.8) μm × (2.0-3.0) μm, does not form filament, forms statospore, Gram-positive.
The preparation process of bacillus natto natto powder:
(1) actication of culture and enlarged culturing: bacillus natto slant strains is accessed and is equipped with in the 100mL triangular flask of 20mL seed culture medium, 37 DEG C, 200r/min shake flask cultivation 24 ~ 36h; Getting the immigration of 1mL seed liquor is again equipped with in the 250mL triangular flask of the fermention medium of 50mL, and 37 DEG C, 200r/min shaking table cultivation 24 ~ 36h, obtain bacillus natto and expand seed liquor.
In aforesaid method, seed culture based formulas: peptone 2g, extractum carnis 3g, glucose 3g, NaCl 5g, water 1L, pH 7.0,0.1Mpa sterilizing 15min.
(2) ferment: human consumption soybean is crushed to 100 orders, adds water by suitable proportion, in 0.1Mpa sterilizing 60min, after being cooled to 40 DEG C, inoculation bacillus natto expands seed liquor, after stirring, in 37 DEG C of fermentation 24h, obtains bacillus natto to ferment thing.
(3) dry: by bacillus natto to ferment thing after 55 ~ 65 DEG C of dryings, cross 60 mesh sieve, obtain bacillus natto natto powder.
3, the preparation method of monascus purpureus Hongqu powder (red colouring agent)
A strain monascus purpureus (Monascus purpureus) Zhang-MP that the present invention relates to is Laboratories Accession bacterial classification, be preserved in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 12nd, 2014, Institute of Microorganism, Academia Sinica), preserving number is: CGMCC No.9221.In MEA substratum, cultivate 7d, colony diameter 30 ~ 32mm for 25 DEG C, color swallow chin is red, and bacterium colony quality is velvet shape, and the irregular branch of mycelia, has tabula, wall has orange crystal, and width is 4 ~ 5.5 μm; The single raw or chain life of conidium, raw on mycelia top or side shoot top, falls pyriform to spherical; Thecaspore ovalize.
The preparation process of monascus purpureus Hongqu powder (red colouring agent):
(1) actication of culture and enlarged culturing: be inoculated on malt agar by monascus purpureus slant strains, cultivates 48h in 30 DEG C, 4 DEG C of preservations; The strain inoculation that picking activates, in solid Spore cultivation base, after cultivating 5d, is washed lower spore with physiological saline, is obtained monascus purpureus spore liquid in 30 DEG C.
(2) solid state fermentation: by spore liquid with the centrifugal 20min of 10 000r/min, get spore mud, join in solid-state fermentation culture medium, stir, cultivates 12 ~ 15d in 32 DEG C, obtains Red kojic rice.
In aforesaid method, solid-state fermentation culture medium formula is: substratum based on long-grained nonglutinous rice 70%+ buckwheat 30%, and adds 0.1% glucose, 0.3%501 rapeseed protein peptone and 0.04%ZnSO
4.
(3) dry and pulverizing: the Red kojic rice completely that will ferment, after 50 DEG C of dryings, is pulverized, and crosses 60 mesh sieve, obtains monascus purpureus Hongqu powder (red colouring agent).
4, plant lactobacillus plant subspecies bacterium powder prepares composite bacterium powder method with bacillus natto natto powder, monascus purpureus Hongqu powder (red colouring agent).
The above-mentioned made plant lactobacillus plant subspecies bacterium powder got ready is fully mixed in 1: 1: 1 (w/w) ratio with bacillus natto natto powder, monascus purpureus Hongqu powder (red colouring agent), become the composite bacterium powder with serum cholesterol-lowering effect, granule, tablet or capsule can be made, preserve under shady and cool, drying, ventilation condition.
Above-mentioned method, the method for bacillus natto natto powder and the method for monascus purpureus Hongqu powder (red colouring agent) preparing plant lactobacillus plant subspecies bacterium powder, method preparing composite bacterium powder and products thereof, and patent bacterial classification belongs to scope.
Embodiment
Experimental technique in following embodiment, if no special instructions, is ordinary method.
Percentage composition in following embodiment, if no special instructions, is the percentage composition of weight to weight.
The preparation method of embodiment 1, plant lactobacillus plant subspecies bacterium powder
1, the preparation of plant lactobacillus plant subspecies bacterium powder
(1) actication of culture: by the plant lactobacillus plant subspecies Zhang-LL bacterial classification after activation enlarged culturing, be inoculated in 2L modified MRS culture medium, 5L automatic fermenter is utilized to carry out high density fermentation, temperature controls at 34 DEG C, fermented liquid pH 6.5, mixing speed is 150r/min, and fermentation 20 ~ 28h, obtains fermented liquid.
MRS liquid culture medium is filled a prescription: Tryptones 10.0g, extractum carnis 10.0g, yeast leaching powder 5.0g, Triammonium citrate 2.0g, glucose 20.0g, tween 80 1.0mL, sodium acetate 5.0g, dipotassium hydrogen phosphate 2.0g, magnesium sulfate 0.5g, manganous sulfate 0.25g, water 1L, pH6.5,0.1Mpa sterilizing 15min.
(2) collect bacterium mud: fermented liquid is placed in whizzer, in 4 DEG C, the centrifugal 10min of 8000r/min, collect and obtain bacterium mud.
(3) pre-freeze and lyophilize: mixed with the protective material of 1/10 original fermented solution volume by plant lactobacillus plant subspecies Zhang-LL bacterium mud, lyophilized vaccine composition is 15% skimmed milk powder, after mixing, obtains starter culture concentrates; By starter culture concentrates in-35 DEG C of refrigerators after pre-freeze 13h, obtain pre-freeze active bacteria formulation, utilize vacuum freeze drier by pre-freeze active bacteria formulation in-55 DEG C, under the condition of vacuum tightness 0.08mBar, freeze-drying 48h, to complete drying state, obtains plant lactobacillus plant subspecies freeze-dried vaccine powder (active bacteria formulation).
2, the mensuration of plant lactobacillus plant subspecies freeze-dried vaccine powder viable count
Adopt gradient dilution colony counting method to measure number of viable before and after freeze-drying, and calculate the bacteria containing amount in survival rate and bacterium powder.As seen from the experiment, utilize 15% skimmed milk powder as the lyophilized vaccine of plant lactobacillus plant subspecies Zhang-LL, after freeze-drying, bacterium powder viable count can reach 3.50 × 10
11cFU/g.
The preparation method of embodiment 2, bacillus natto natto powder
1, the preparation of bacillus natto natto powder
(1) actication of culture and enlarged culturing: bacillus natto slant strains is accessed and is equipped with in the 100mL triangular flask of 20mL seed culture medium, 37 DEG C, 200r/min shake flask cultivation 24 ~ 36h; Getting the immigration of 1mL seed liquor is again equipped with in the 250mL triangular flask of the fermention medium of 50mL, and 37 DEG C, 200r/min shaking table cultivation 24 ~ 36h, obtain bacillus natto and expand seed liquor.
In aforesaid method, seed culture based formulas: peptone 2g, extractum carnis 3g, glucose 3g, NaCl 5g, water 1L, pH 7.0,0.1Mpa sterilizing 15min.
(2) ferment: human consumption soybean is crushed to 100 orders, adds water by suitable proportion, in 0.1Mpa sterilizing 60min, after being cooled to 40 DEG C, inoculation bacillus natto expands seed liquor, after stirring, in 37 DEG C of fermentation 24h, obtains bacillus natto to ferment thing.
(3) dry: by bacillus natto to ferment thing after 55 ~ 65 DEG C of dryings, cross 60 mesh sieve, obtain bacillus natto natto powder.
2, the mensuration of natto kinase activity in bacillus natto natto powder
Utilize fibrin plate method to measure the activity of Nattokinase, it is that reference urokinase (UK) or the measuring method for activity knitting plasminogen activator (tRA) are set up.Fibrin plate method, the agarose-fibrin plate method reported according to Astrup etc., to record fibrinolysis circle area to represent the activity of NK.Take urokinase as standard, the solution of preparation different concns, gets 10 μ L point samples in fibrin plate, in 37 DEG C of thermostat containers after 18h, measures two perpendicular diameter of solusphere, with the relative size of the molten fine vigor of its product size reflection, and drawing standard curve.Just can measure the vigor of Nattokinase in this way.
The relation of the vigor of Nattokinase and its proportional routine function of solusphere area on fibrin plate, its straight-line equation is y=0.0044x, then the Bacillus natto powder after 1.0g freeze-drying is taken, be diluted to certain multiple, the enzyme measuring Nattokinase with fibrin plate method is lived, and in bacillus natto natto powder, natto kinase activity can reach 2000IU/g.
The preparation method of embodiment 3, monascus purpureus Hongqu powder (red colouring agent)
1, the preparation of monascus purpureus Hongqu powder (red colouring agent)
(1) actication of culture and enlarged culturing: be inoculated on malt agar by monascus purpureus slant strains, cultivates 48h in 30 DEG C, 4 DEG C of preservations; The strain inoculation that picking activates, in solid Spore cultivation base, after cultivating 5d, is washed lower spore with physiological saline, is obtained monascus purpureus spore liquid in 30 DEG C.
(2) solid state fermentation: by spore liquid with the centrifugal 20min of 10 000r/min, get spore mud, join in solid-state fermentation culture medium, stir, cultivates 12 ~ 15d in 32 DEG C, obtains Red kojic rice.
In aforesaid method, solid-state fermentation culture medium formula is: substratum based on long-grained nonglutinous rice 70%+ buckwheat 30%, and adds 0.1% glucose, 0.3%501 rapeseed protein peptone and 0.04%ZnSO
4.
(3) dry and pulverizing: the Red kojic rice completely that will ferment, after 50 DEG C of dryings, is pulverized, and crosses 60 mesh sieve, obtains monascus purpureus Hongqu powder (red colouring agent).
2, the mensuration of Monacolin K content in monascus purpureus Hongqu powder (red colouring agent)
Take 1.0g monascus purpureus Hongqu powder (red colouring agent), put in 200mL volumetric flask, add ethanolic soln and be diluted to scale.Shake up, 3000r/min, 20min are centrifugal, get supernatant liquor; It is appropriate that another precision takes reference substance, adds dissolve with ethanol and be quantitatively diluted to the solution containing 10 μ g in every 1mL, product solution in contrast.Through the absorbancy of 752 type ultraviolet spectrophotometers in 237nm place working sample supernatant liquor and reference substance, the final content measuring active substance Monacolin K in monascus purpureus Hongqu powder (red colouring agent) is 4.98mg/g.
Embodiment 4, plant lactobacillus plant subspecies bacterium powder prepare composite bacterium powder method with bacillus natto natto powder, monascus purpureus Hongqu powder (red colouring agent)
The above-mentioned made plant lactobacillus plant subspecies bacterium powder got ready is fully mixed in 1: 1: 1 (w/w) ratio with bacillus natto natto powder, monascus purpureus Hongqu powder (red colouring agent), become the composite bacterium powder with serum cholesterol-lowering effect, granule, tablet or capsule can be made, preserve under shady and cool, drying, ventilation condition.
Embodiment 5, Gl tract inverse ring border plant lactobacillus plant subspecies decreasing cholesterol ability test
Hypercholesterolemia MRSO-CHOL substratum: in the liquid nutrient medium of 1000mL, prepare containing 0.1mg/mL cholesterol micellar solution: precise 0.1g cholesterol, put into small beaker, add 0.3g bovine bile, 0.1g sucrose ester, 1mL tween-80 stirs, pipette the glacial acetic acid of 5mL, 60 DEG C of heating for dissolving and then carry out break process (being set to of broken time: work 5s with ultrasonic disruption machine, interval 3s, 9 circulations altogether), lysate is used while hot the membrane filtration of 0.45 μm, join in the autoclaved MRS substratum of the 1000mL prepared fast, stir while adding, it is made to form uniform and stable colloidal solution, obtain about containing the MRS broth culture of the cholesterol of 0.1mg/mL.Adjust medium pH between 6.0 ~ 7.0 with the 6M NaOH of bacterium of having gone out, measure with pH test paper.
Gl tract inverse ring border decreasing cholesterol ability test method: after test plant Bacterium lacticum plant sp. strain was activated for 2 generations in MRS substratum, be inoculated in the PBS damping fluid of pH3.0 by 10% (v/v) inoculum size, 37 DEG C of cultivations, respectively at 0min, 30min, 60min, 90min, 120min, be inoculated into containing 0.3% bovine bile by 10% inoculum size again, in the liquid MRS substratum of 0.1% cholesterol, cultivate 8h for 37 DEG C, the centrifugal 10min/ of nutrient solution 1000r/min measures the content of supernatant liquor cholesterol according to o-phthalaldehyde method, light absorption value (the A that 8h measures cholesterol is at 550nm
550nm).Often organize experiment do three parallel, to sample respectively before cultivation and after cultivating, and to add in liquid MRS substratum that the 10% PBS damping fluid not connecing bacterium is linked into containing 0.3% bovine bile, 0.1% cholesterol as blank.Calculate the cholesterol reduced rate of plant lactobacillus plant subspecies.
O-phthalaldehyde method: by the bacterium liquid after cultivation in 4 DEG C, the centrifugal 10min of 10000r/min, get supernatant liquor 0.5mL in 18mm × 18cm test tube, add successively 3mL volume fraction be 95% ethanol and 2mL mass concentration be the potassium hydroxide solution of 0.5g/mL, vortex oscillation mixing is placed on saponification 10min in 60 DEG C of water-baths, 5mL normal hexane is added after rapid cooling, vortex oscillation 1min extracts, 3mL water is added after leaving standstill 10min, 10min is left standstill in room temperature after repeating vibration 1min, 2.5mL normal hexane layer is got in 15mm × 15cm test tube after layering, be placed in 60 DEG C of water-bath nitrogen and dry up solvent, add 4mL o-phthalaldehyde(OPA) developer, 10min is left standstill after vortex oscillation 1min, add 2mL vitriol oil vibration 1min, room temperature surveys absorbancy in wavelength 550nm place after leaving standstill 10min, according to the mass concentration of cholesterol and absorbance standard curve regression equation calculation cholesterol.Experiment repetition 3 times, result is taken the mean.
Cholesterol removal rate calculation formula is:
Cholesterol removal rate/%=(1-ρ
1/ ρ
0) × 100
In formula: ρ
1for cultivating the mass concentration of rear supernatant liquor cholesterol;
ρ
0for the mass concentration of initial medium total cholesterol.
The reduced rate of table 1 bacterial strain Zhang-LL cholesterol level
From table 1, plant lactobacillus plant subspecies Zhang-LL through the PBS damping fluid of pH3.0 against environmental treatment 0min, after 30min, 60min, 90min, 120min time, be linked in the liquid MRS substratum containing 0.3% bovine bile, 0.1% cholesterol, cultivate 8h for 37 DEG C, cholesterol reduced rate decreases along with the growth of time.Bacterial strain cholesterol reduced rate after cultivation 8h without the PBS damping fluid process of pH3.0 is 35.86%, and the bacterial strain cholesterol reduced rate after cultivation 8h through the PBS damping fluid process 120min of pH3.0 still can reach 20.96%.As can be seen here, this bacterial strain has stronger decreasing cholesterol ability.
Embodiment 6, rat test
The hypolipemic function of composite bacterium powder product is evaluated according to protective foods inspection and assessment technical specifications experimentation on animals evaluation method.Experimentation on animals shows, and this product has the function reducing blood fat.
Animal grouping and test method: get 200g healthy rat, rat is divided into 3 groups, often organize 20.Animal adaptability gets fasting blood through eye socket, separation of serum after raising 7d, detects serum TC, TG, HDL-C and LDL-C level.According to TC level, with reference to TG level and body weight, rat is divided into Basal control group, hyperlipidemia model group and test group (composite bacterium powder).Basal control group is fed basal feed, and hyperlipidemia model group and test group are fed high lipid food (high lipid food: 2% cholesterol, the lard of 10%, the Sodium cholic acid of 0.5%).Test group gavage tested material, control group and model group give distilled water, continuous 8 weeks.Detect weekly hepatic tissue TC, TG, HDL-C and LDL-C level.
Total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) biochemical reagents box, by Beijing, Zhongsheng Beikong Biological Science & Technology Co., Ltd. provides.
Table 2 composite bacterium powder is on the impact of the 8th week rat hypercholesterolemia blood fat
Have table 2 known, showing 3 groups of rat fats has different change, and TC, TG content of hyperlipidemia model group and blank group rat liver has extremely significantly (p < 0.01) difference; TC and TG content in rats in test groups serum extremely significantly (p < 0.01), lower than hyperlipidemia model group, shows that composite bacterium powder has the effect of TC and the TG content extremely significantly reducing rat blood serum; The HDL-C content of hyperlipidemia model group and Basal control group rat blood serum has extremely significantly (p < 0.01) difference, the HDL-C content extremely remarkable (p < 0.01) of rats in test groups serum, higher than hyperlipidemia model group, shows that composite bacterium powder has the effect extremely significantly raising rat liver HDL-C content; The LDL-C content of rats in test groups serum, lower than hyperlipidemia model group, shows that composite bacterium powder has the effect to a certain degree reducing rat blood serum LDL-C content.
Project 1 belonging to this patent: the sub-problem of the Department of Science and Technology " 12 " national high-tech research evolutionary operation(EVOP) (863) project " livestock product pathogenic bacteria safety control technology ".
Item number: 2012AA101606-05
The project beginning and ending time: 2012.01-2015.12
Project leader: Zhang Hongxing
Project 2 belonging to this patent: Beijing institution of higher education directly under the jurisdiction of a municipal government high-level personnel Introduction and cultivation planning item " natural lactobacillin is to the antimicrobial mechanism of important pathogenic bacteria in livestock product and Effect study ".
Item number: CIT & TCD20140315
The project beginning and ending time: 2014.01-2016.12
Project leader: Zhang Hongxing
Project 3 belonging to this patent: Department of Science and Technology's national science and technology key special subjects " disease-resistant transgenic sheep rearing new variety " sub-problem " foundation that disease-resistant transgenic sheep expansion traditional font is/disease-resistant transgenic goat-anti disease, production performance and safety evaluation " item number: 2013ZX08008-005
The project beginning and ending time: 2013.01-2013.12
Project leader: Liu Hui.
Claims (4)
1. plant lactobacillus plant subspecies (Lactobacillus plantarum subsp.plantarum) Zhang-LLCGMCC No.6936.
2. bacillus natto (Bacillus natto) BNZ3CGMCC No.9146.
3. monascus purpureus (Monascus purpureus) Zhang-MP CGMCC No.9221.
4. the preparation of a function plant lactobacillus and composite bacterium powder thereof, it is characterized in that: the preparation process of (1) plant lactobacillus plant subspecies bacterium powder: 1. actication of culture: by the plant lactobacillus plant subspecies Zhang-LL bacterial classification after activation enlarged culturing, be inoculated in 2L modified MRS culture medium, 5L automatic fermenter is utilized to carry out high density fermentation, temperature controls at 34 DEG C, fermented liquid pH 6.5, and mixing speed is 150r/min, fermentation 20 ~ 28h, obtains fermented liquid; 2. collect bacterium mud: fermented liquid is placed in whizzer, in 4 DEG C, the centrifugal 10min of 8000r/min, collect and obtain bacterium mud; 3. pre-freeze and lyophilize: mixed with the protective material of 1/10 original fermented solution volume by plant lactobacillus plant subspecies Zhang-LL bacterium mud, lyophilized vaccine composition is 15% skimmed milk powder, after mixing, obtains starter culture concentrates; By starter culture concentrates in-35 DEG C of refrigerators after pre-freeze 13h, obtain pre-freeze active bacteria formulation, utilize vacuum freeze drier by pre-freeze active bacteria formulation in-55 DEG C, under the condition of vacuum tightness 0.08mBar, freeze-drying 48h, to complete drying state, obtains plant lactobacillus plant subspecies freeze-dried vaccine powder; (2) preparation process of bacillus natto natto powder: 1. actication of culture and enlarged culturing: bacillus natto slant strains is accessed and is equipped with in the 100mL triangular flask of 20mL seed culture medium, 37 DEG C, 200r/min shake flask cultivation 24 ~ 36h; Getting the immigration of 1mL seed liquor is again equipped with in the 250mL triangular flask of the fermention medium of 50mL, and 37 DEG C, 200r/min shaking table cultivation 24 ~ 36h, obtain bacillus natto and expand seed liquor.2. ferment: human consumption soybean is crushed to 100 orders, adds water by suitable proportion, in 0.1Mpa sterilizing 60min, after being cooled to 40 DEG C, inoculation bacillus natto expands seed liquor, after stirring, in 37 DEG C of fermentation 24h, obtains bacillus natto to ferment thing; 3. dry: by bacillus natto to ferment thing after 55 ~ 65 DEG C of dryings, cross 60 mesh sieve, obtain bacillus natto natto powder; (3) preparation process of monascus purpureus Hongqu powder (red colouring agent): 1. actication of culture and enlarged culturing: be inoculated on malt agar by monascus purpureus slant strains, cultivates 48h in 30 DEG C, 4 DEG C of preservations; The strain inoculation that picking activates, in solid Spore cultivation base, after cultivating 5d, is washed lower spore with physiological saline, is obtained monascus purpureus spore liquid in 30 DEG C; 2. solid state fermentation: by spore liquid with the centrifugal 20min of 10000r/min, get spore mud, join in solid-state fermentation culture medium, stir, cultivates 12 ~ 15d in 32 DEG C, obtains Red kojic rice; 3. dry and pulverizing: the Red kojic rice completely that will ferment, after 50 DEG C of dryings, is pulverized, and crosses 60 mesh sieve, obtains monascus purpureus Hongqu powder (red colouring agent); (4) plant lactobacillus plant subspecies bacterium powder prepares composite bacterium powder method with bacillus natto natto powder, monascus purpureus Hongqu powder (red colouring agent): fully mixed in 1: 1: 1 (w/w) ratio with bacillus natto natto powder, monascus purpureus Hongqu powder (red colouring agent) by the above-mentioned made plant lactobacillus plant subspecies bacterium powder got ready, become the composite bacterium powder with serum cholesterol-lowering effect.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410763406.5A CN104560790A (en) | 2014-12-15 | 2014-12-15 | Functional lactobacillus plantarum and preparation method of compound bacterial powder of functional lactobacillus plantarum |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410763406.5A CN104560790A (en) | 2014-12-15 | 2014-12-15 | Functional lactobacillus plantarum and preparation method of compound bacterial powder of functional lactobacillus plantarum |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104560790A true CN104560790A (en) | 2015-04-29 |
Family
ID=53077871
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410763406.5A Pending CN104560790A (en) | 2014-12-15 | 2014-12-15 | Functional lactobacillus plantarum and preparation method of compound bacterial powder of functional lactobacillus plantarum |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104560790A (en) |
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104560799A (en) * | 2014-12-25 | 2015-04-29 | 北京北农红泽生物科技有限公司 | Preparation method of bacteriocin-producing Lactobacillus plantarum subsp. Plantarum Zhang-LL active bacterial preparation |
| CN106399154A (en) * | 2016-08-29 | 2017-02-15 | 中国疾病预防控制中心传染病预防控制所 | Lactobacillus probiotics CGMCC NO. 12422 and application of lactobacillus probiotics in preparing lipid-lowering drug |
| CN106434427A (en) * | 2016-08-29 | 2017-02-22 | 中国疾病预防控制中心传染病预防控制所 | Lactic acid bacillus probiotic CGMCC NO.12421 and applicaiton to preparing lipid-lowering drugs |
| CN106834186A (en) * | 2017-03-06 | 2017-06-13 | 北京农学院 | A kind of Lactobacillus plantarum probiotics and its preparation method and application |
| CN107353049A (en) * | 2017-08-10 | 2017-11-17 | 黑龙江九穗谷农业科技发展有限公司 | A kind of preparation method of multiple-effect biological bacterium enzyme |
| CN107594086A (en) * | 2017-09-28 | 2018-01-19 | 北京农学院 | A kind of composite microbial compositions and feed for nursery-age pig |
| CN109022331A (en) * | 2018-09-12 | 2018-12-18 | 北京农学院 | A kind of compound micro-ecological preparation and its preparation and application |
| CN109187394A (en) * | 2018-10-18 | 2019-01-11 | 华南理工大学 | A kind of measuring method of cholesterol concentration |
| CN109757730A (en) * | 2019-01-31 | 2019-05-17 | 山东环亿生物科技有限公司 | A kind of composition and preparation method thereof with reducing blood lipid, blood pressure and blood glucose |
| CN110734880A (en) * | 2019-11-19 | 2020-01-31 | 扬州大学 | Lactobacillus plantarum Bama06 derived from sibima and having high vitamin B yield and application thereof |
| CN110964640A (en) * | 2019-12-17 | 2020-04-07 | 北京农学院 | Freeze-drying protective agent, fermentation agent, and preparation method and application thereof |
| CN113832046A (en) * | 2021-06-15 | 2021-12-24 | 安徽农业大学 | Difunctional Bacillus belgii and application thereof in red yeast rice fermentation |
| CN113841891A (en) * | 2021-09-17 | 2021-12-28 | 江苏食品药品职业技术学院 | Food nutrient and preparation method thereof |
| CN114028524A (en) * | 2021-12-06 | 2022-02-11 | 重庆市中药研究院 | Compound for improving blood lipid metabolism and preparation method and application thereof |
Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101214263A (en) * | 2007-12-26 | 2008-07-09 | 浙江大学宁波理工学院 | Method for preparing freeze-dried powder containing bacillus natto and nattokinase |
| JP2008280311A (en) * | 2007-04-11 | 2008-11-20 | Oriza Yuka Kk | Prophylactic/curative agent for hyperlipemia |
| CN101407825A (en) * | 2008-10-31 | 2009-04-15 | 江南大学 | Method for producing water-soluble functional red koji powder |
| CN101559082A (en) * | 2009-06-01 | 2009-10-21 | 天津科技大学 | Method for preparing probiotic preparation for reducing blood lipid and adjusting intestinal flora |
| CN101864375A (en) * | 2010-01-15 | 2010-10-20 | 吉林省农业科学院 | Lactobacillu plantarurn with function of reducing cholesterol and application thereof |
| CN102038046A (en) * | 2010-11-16 | 2011-05-04 | 蒙顿茶制品(大连)有限公司 | Production method of Pu'er tea ferment liquid, ferment powder and ferment beverage |
| CN102670917A (en) * | 2012-01-20 | 2012-09-19 | 华子昂 | Composite enzyme and its preparation method |
| CN103404757A (en) * | 2013-05-27 | 2013-11-27 | 胡安然 | Special diet for hyperglycemia patients |
| CN104109700A (en) * | 2014-07-09 | 2014-10-22 | 江南大学 | Method of producing monacolin K functional red yeast rice by solid-liquid co-fermentation |
| CN104388322A (en) * | 2014-12-09 | 2015-03-04 | 北京农学院 | Solid state fermentation method of lipid-lowering Monascus purpureus Zhang-MP |
| CN104531562A (en) * | 2014-12-09 | 2015-04-22 | 北京农学院 | Preparation method of (Lactobacillus planetarium subsp. plantarum)Zhang-LL and its listeria monocytogene-resistant bacteriocin |
| CN104531561A (en) * | 2014-12-09 | 2015-04-22 | 北京农学院 | (Lactobacillus planetarium subsp. plantarum)Zhang-LL bacteriocin and Nisin composite bacteriostatic agent and its use in chilled meat |
| CN104726360A (en) * | 2014-11-14 | 2015-06-24 | 北京农学院 | Construction and application of shuttle vector between Escherichia coli and Lactobacilli |
-
2014
- 2014-12-15 CN CN201410763406.5A patent/CN104560790A/en active Pending
Patent Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008280311A (en) * | 2007-04-11 | 2008-11-20 | Oriza Yuka Kk | Prophylactic/curative agent for hyperlipemia |
| CN101214263A (en) * | 2007-12-26 | 2008-07-09 | 浙江大学宁波理工学院 | Method for preparing freeze-dried powder containing bacillus natto and nattokinase |
| CN101407825A (en) * | 2008-10-31 | 2009-04-15 | 江南大学 | Method for producing water-soluble functional red koji powder |
| CN101559082A (en) * | 2009-06-01 | 2009-10-21 | 天津科技大学 | Method for preparing probiotic preparation for reducing blood lipid and adjusting intestinal flora |
| CN101864375A (en) * | 2010-01-15 | 2010-10-20 | 吉林省农业科学院 | Lactobacillu plantarurn with function of reducing cholesterol and application thereof |
| CN102038046A (en) * | 2010-11-16 | 2011-05-04 | 蒙顿茶制品(大连)有限公司 | Production method of Pu'er tea ferment liquid, ferment powder and ferment beverage |
| CN102670917A (en) * | 2012-01-20 | 2012-09-19 | 华子昂 | Composite enzyme and its preparation method |
| CN103404757A (en) * | 2013-05-27 | 2013-11-27 | 胡安然 | Special diet for hyperglycemia patients |
| CN104109700A (en) * | 2014-07-09 | 2014-10-22 | 江南大学 | Method of producing monacolin K functional red yeast rice by solid-liquid co-fermentation |
| CN104726360A (en) * | 2014-11-14 | 2015-06-24 | 北京农学院 | Construction and application of shuttle vector between Escherichia coli and Lactobacilli |
| CN104388322A (en) * | 2014-12-09 | 2015-03-04 | 北京农学院 | Solid state fermentation method of lipid-lowering Monascus purpureus Zhang-MP |
| CN104531562A (en) * | 2014-12-09 | 2015-04-22 | 北京农学院 | Preparation method of (Lactobacillus planetarium subsp. plantarum)Zhang-LL and its listeria monocytogene-resistant bacteriocin |
| CN104531561A (en) * | 2014-12-09 | 2015-04-22 | 北京农学院 | (Lactobacillus planetarium subsp. plantarum)Zhang-LL bacteriocin and Nisin composite bacteriostatic agent and its use in chilled meat |
Non-Patent Citations (4)
| Title |
|---|
| 孙明 等: "红曲改善高血脂大鼠血液流变学作用的研究", 《卫生研究》 * |
| 赵爽 等: "泡菜直投式乳酸菌发酵剂的制备", 《食品工业科技》 * |
| 靳志强，王延样: "植物乳杆菌在人体肠道的益生特性", 《中国乳品工业》 * |
| 韩彦彬 等: "纳豆胶囊对高脂血症模型大鼠血脂调节作用的研究", 《中国卫生检验杂志》 * |
Cited By (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104560799A (en) * | 2014-12-25 | 2015-04-29 | 北京北农红泽生物科技有限公司 | Preparation method of bacteriocin-producing Lactobacillus plantarum subsp. Plantarum Zhang-LL active bacterial preparation |
| CN106399154B (en) * | 2016-08-29 | 2019-10-18 | 中国疾病预防控制中心传染病预防控制所 | Bacillus acidi lactici probiotics CGMCC NO.12422 and preparing the application in fat-reducing medicament |
| CN106399154A (en) * | 2016-08-29 | 2017-02-15 | 中国疾病预防控制中心传染病预防控制所 | Lactobacillus probiotics CGMCC NO. 12422 and application of lactobacillus probiotics in preparing lipid-lowering drug |
| CN106434427A (en) * | 2016-08-29 | 2017-02-22 | 中国疾病预防控制中心传染病预防控制所 | Lactic acid bacillus probiotic CGMCC NO.12421 and applicaiton to preparing lipid-lowering drugs |
| CN106834186A (en) * | 2017-03-06 | 2017-06-13 | 北京农学院 | A kind of Lactobacillus plantarum probiotics and its preparation method and application |
| CN106834186B (en) * | 2017-03-06 | 2020-07-28 | 北京农学院 | Lactobacillus plantarum microecological preparation and preparation method and application thereof |
| CN107353049A (en) * | 2017-08-10 | 2017-11-17 | 黑龙江九穗谷农业科技发展有限公司 | A kind of preparation method of multiple-effect biological bacterium enzyme |
| CN107594086A (en) * | 2017-09-28 | 2018-01-19 | 北京农学院 | A kind of composite microbial compositions and feed for nursery-age pig |
| CN109022331A (en) * | 2018-09-12 | 2018-12-18 | 北京农学院 | A kind of compound micro-ecological preparation and its preparation and application |
| CN109187394A (en) * | 2018-10-18 | 2019-01-11 | 华南理工大学 | A kind of measuring method of cholesterol concentration |
| CN109757730A (en) * | 2019-01-31 | 2019-05-17 | 山东环亿生物科技有限公司 | A kind of composition and preparation method thereof with reducing blood lipid, blood pressure and blood glucose |
| CN109757730B (en) * | 2019-01-31 | 2021-06-04 | 山东环亿生物科技有限公司 | Composition with functions of reducing blood fat, blood pressure and blood sugar and preparation method thereof |
| CN110734880A (en) * | 2019-11-19 | 2020-01-31 | 扬州大学 | Lactobacillus plantarum Bama06 derived from sibima and having high vitamin B yield and application thereof |
| CN110734880B (en) * | 2019-11-19 | 2021-05-18 | 扬州大学 | Lactobacillus plantarum Bama06 derived from Guangxi Bama and having high vitamin B yield and application thereof |
| CN110964640A (en) * | 2019-12-17 | 2020-04-07 | 北京农学院 | Freeze-drying protective agent, fermentation agent, and preparation method and application thereof |
| CN110964640B (en) * | 2019-12-17 | 2021-11-30 | 北京农学院 | Freeze-drying protective agent, fermentation agent, and preparation method and application thereof |
| CN113832046A (en) * | 2021-06-15 | 2021-12-24 | 安徽农业大学 | Difunctional Bacillus belgii and application thereof in red yeast rice fermentation |
| CN113841891A (en) * | 2021-09-17 | 2021-12-28 | 江苏食品药品职业技术学院 | Food nutrient and preparation method thereof |
| CN114028524A (en) * | 2021-12-06 | 2022-02-11 | 重庆市中药研究院 | Compound for improving blood lipid metabolism and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104560790A (en) | Functional lactobacillus plantarum and preparation method of compound bacterial powder of functional lactobacillus plantarum | |
| CN102618456B (en) | Lactobacillus rhamnosus capable of relieving chronic alcohol liver injury and application thereof | |
| CN102356912B (en) | Preparation method of probiotic fermented rice milk | |
| CN104430851A (en) | Fermented milk capable of reducing cholesterol and preparation method of fermented milk | |
| CN105132318A (en) | Lactobacillus plantarum grx16 and application thereof | |
| CN105018379A (en) | Lactobacillus plantarum with high antioxidant activity and application of lactobacillus plantarum | |
| CN109810912A (en) | One lactobacillus plantarum LH-511 and its application | |
| CN102559560B (en) | Bacillus licheniformis for producing lactic acid at high yield after fermentation, and preparation and application of Bacillus licheniformis | |
| CN104388322A (en) | Solid state fermentation method of lipid-lowering Monascus purpureus Zhang-MP | |
| CN101356950A (en) | Preparation method of composite bacteria fermentation bed | |
| CN107156638B (en) | Preparation method of lipid-lowering red yeast powder | |
| CN108165512A (en) | A kind of extracellular polysaccharide spatial plant lactobacillus SS18-119 and its application in biological antioxidant activity is improved | |
| CN101463335A (en) | Piggery fermentation bed composite microbial preparation and preparation thereof | |
| CN103468600B (en) | Lactic acid bacterium used for fermenting cereal and applications thereof | |
| CN104560799A (en) | Preparation method of bacteriocin-producing Lactobacillus plantarum subsp. Plantarum Zhang-LL active bacterial preparation | |
| CN104531550A (en) | High-temperature kitchen waste degradation microbial agent and preparing and using method thereof | |
| CN102899276A (en) | Streptococcus thermophilus capable of lowering cholesterol levels and application thereof | |
| CN109810913A (en) | One plant of Lactobacillus rhamnosus ASD-9 and its application | |
| CN112126599B (en) | High-density culture method of lactobacillus helveticus, preparation of high-activity bacterium powder and application of high-density culture method | |
| CN108330086A (en) | A kind of extracellular polysaccharide spatial plant lactobacillus SS18-33 and its application in improving biological antioxidant activity | |
| CN110777097A (en) | Lactobacillus strain with strong acid resistance and screening and fermenting processes thereof | |
| CN103343107A (en) | Human lactobacillus casei gr x 12 with antioxidant function and application thereof | |
| CN111714572B (en) | Lactobacillus plantarum-based probiotic tablet and preparation method thereof | |
| CN102524794B (en) | Preparation method of serum cholesterol reduction Kluyveromyces marxianus freeze-dried powder | |
| CN104305168A (en) | A preparing method of lactobacillus rhamnosus particle food relieving alcoholic liver injury |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150429 |