CN105779337A - Method for comprehensively utilizing radix isatidis - Google Patents
Method for comprehensively utilizing radix isatidis Download PDFInfo
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- CN105779337A CN105779337A CN201610159197.2A CN201610159197A CN105779337A CN 105779337 A CN105779337 A CN 105779337A CN 201610159197 A CN201610159197 A CN 201610159197A CN 105779337 A CN105779337 A CN 105779337A
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- radix isatidis
- described step
- comprehensive utilization
- lactic acid
- acid bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
Abstract
The invention discloses a method for comprehensively utilizing radix isatidis. The method includes steps of (1), preparing and sterilizing MRS media; (2), amplifying and cultivating lactic acid bacteria; (3), preparing radix isatidis coarse powder; (4), preparing fermentation media; (5), carrying out inoculation and fermentation; (6), carrying out centrifugal separation; (7), carrying out detection; (8), carrying out ethanol precipitation. The method has the advantages that the method is simple, effective and environmentally friendly, is high in yield and low in cost and has good benefits; the radix isatidis is fermented by the aid of the lactic acid bacteria, accordingly, the radix isatidis polysaccharide extraction rate can be increased, purified radix isatidis polysaccharides can be directly used as immunologic adjuvants, medicine residues obtained after the radix isatidis polysaccharides are extracted contain abundant probiotics-lactic acid bacteria, are excellent microecological preparations and can be directly reasonably utilized as feed additives, waste is prevented in integral procedures, the radix isatidis can be sufficiently utilized, and industrial automatic production can be completely implemented.
Description
Technical field
The invention belongs to processing technique of Chinese herbs field, the method being specifically related to the comprehensive utilization of a kind of Radix Isatidis.
Background technology
Radix Isatidis is the dry root of crucifer tetraploid isatis, begins to be loaded in Shennong's Herbal, its property
Cold, taste is the most micro-sweet rear pained, the thoughts of returning home, stomach warp.Have clearing heat and detoxicating, anti-bacteria and anti-virus, preventing cold,
Effect of profit pharynx.Be mainly used in treating febrile virulent maculae, tongue dark reddish purple dark, scarlet fever, epidemic meningitis,
The diseases such as pneumonia.Modern study shows, Radix Isatidis not only has antibacterial, antiviral effect, the most again
Find its platelet aggregation-against, strengthen body's immunity and anti-endotoxin effect.
Banlangen Polysaccharide is one of main effective active composition of Radix Isatidis, has multiple biologically active, to special
Property immunity and nospecific immunity all have certain facilitation, be preferable immunopotentiator.It can improve
The function of body immune system, can not only promote the immunocytes such as T cell, B cell, NK cell, M cell
Function, moreover it is possible to promote white between the generation of the cell factor such as element, interferon.Patent No. CN201080068430
Patent disclose Banlangen Polysaccharide preparation for treatment and/or the disease that causes of flu-prevention virus and
Purposes in the medicine of complication, it was demonstrated that the mechanism of Banlangen Polysaccharide antivirus action is to suppress influenza virus
It is adsorbed onto on host cell.
Traditional method extracting Banlangen Polysaccharide is mainly water extraction and alcohol precipitation method, and this method is also by " Chinese Pharmacopoeia "
Recording, mainly use thermophilic digestion, ethanol precipitates.This method is not required to special installation, but has the operating time
The longest, energy consumption is big, and active ingredient high temperature easily decomposes, and recovery rate is the lowest, and the dregs of a decoction after extraction are the most disposable
Etc. shortcoming, it is clear that be not suitable with industrialized production.Probiotics fermention Chinese herbal medicine is utilized to become research in recent years
Hot fields, the most lactic acid bacteria, Bacillus, the report of saccharomycetes to make fermentation Chinese herbal medicine.Research shows,
Microorganism has very abundant enzyme system, has the ability of powerful decomposition and inversion material, and Chinese herbal medicine is sent out
After ferment, its active ingredient and active material are discharged to greatest extent, can also produce new work simultaneously
Property material.Number of patent application CN201210449873 (CN102894185A) discloses a kind of utilization addicted to yogurt
Bacillus and the method for streptococcus fecalis fermentation Radix Isatidis, the method fermentation time is longer, and process is the most extensive, carries
Take rate the highest, more cannot be carried out industrialized production.
Summary of the invention
In order to solve the problems referred to above, the present invention provides a kind of method that Radix Isatidis comprehensively utilizes.
The technical solution used in the present invention is:
The method of a kind of Radix Isatidis comprehensive utilization, comprises the following steps:
(1) preparation of MRS culture medium and sterilizing: preparation MRS fluid nutrient medium, is placed in sterilizing in autoclave；
(2) expansion of lactic acid bacteria is cultivated: expanded in sterilized MRS fluid nutrient medium by lactobacillus inoculum
Big cultivation；
(3) preparation of Radix Isatidis meal: chromatogram of Radix Isatidis is placed in baking oven drying, then pulverizes, sieve；
(4) preparation fermentation medium: preparation fermentation medium, is placed in sterilizing in autoclave；
(5) inoculate and ferment: will be enlarged by the lactobacillus inoculum after cultivating to the fermentation medium containing Radix Isatidis meal
Middle fermentation；
(6) centrifugal: zymotic fluid fermentation obtained is centrifuged, and washes precipitation with water, is again centrifuged, and merges twice
Centrifugal supernatant；
(7) detection: will centrifugal after the precipitation i.e. dregs of a decoction be dried, lactic acid bacteria living bacteria count in the detection dregs of a decoction and upper
The content of Banlangen Polysaccharide in clear liquid；
(8) alcohol precipitation: the supernatant concentration after merging, adds ethanol and makes it precipitate, and then transfer is precipitated to vacuum
Low temperature drying in drying box, obtains Banlangen Polysaccharide.
In described step (1), the temperature of autoclave is 121 DEG C, and sterilization time is 15 minutes.
Expanding the temperature cultivated in described step (2) is 32-38 DEG C, and incubation time is 20-36 hour.
In described step (3), the temperature of baking oven is 55 DEG C, and drying time is 4 hours, crosses No. two medicines after pulverizing
Sieve.
In described step (4), the component of fermentation medium is tryptone 3-6 part, dusty yeast 4-6 part, corn
Slurry 0.5-2 part, potassium dihydrogen phosphate 1-3 part, magnesium sulfate 0.2-1 part, 6 parts of Radix Isatidis powder, remaining is distillation
Water, 150ml is in 500ml triangular flask in preparation, and adjusting initial pH was 6.8, in 121 DEG C of sterilizings 10 minutes.
In described step (5), the inoculum concentration of lactic acid bacteria is 1%-3%.
Rotating speed centrifugal in described step (6) is 3500-5000 rev/min, and centrifugation time is 5-15 minute.
Described step (7) precipitation is dried 5 hours in an oven, and the temperature of baking oven is 45 DEG C.
The concentration of described step (8) ethanol is 80%.
The temperature of described step (8) vacuum drying chamber is 40 DEG C, and vacuum is 0.085MPa.
The invention has the beneficial effects as follows: the present invention provide a kind of simple, effectively, environmental protection, yield height, low cost,
Profitable method, utilizes lactobacillus-fermented Radix Isatidis, has both improve the recovery rate of Banlangen Polysaccharide, purifies
After Banlangen Polysaccharide can directly as immunologic adjuvant, simultaneously extract after the dregs of a decoction rich in there being abundant probio-breast
Acid bacterium, is good probiotics, can be directly as feed addictive, after efficiently solving traditional Chinese medicine extraction
The reluctant difficult problem of the dregs of a decoction, whole process no waste produce, Radix Isatidis is fully used, should during
Lactic acid bacteria expands cultivation, sweat, extraction process etc. and has now the most been capable of industrial automation production.
Detailed description of the invention
The method of a kind of plate Radix Isatidis comprehensive utilization, comprises the following steps:
(1) preparation of MRS culture medium and sterilizing: preparation MRS fluid nutrient medium, is placed in sterilizing in autoclave；
(2) expansion of lactic acid bacteria is cultivated: expanded in sterilized MRS fluid nutrient medium by lactobacillus inoculum
Big cultivation；
(3) preparation of Radix Isatidis meal: chromatogram of Radix Isatidis is placed in baking oven drying, then pulverizes, sieve；
(4) preparation fermentation medium: preparation fermentation medium, is placed in sterilizing in autoclave；
(5) inoculate and ferment: will be enlarged by the lactobacillus inoculum after cultivating to the fermentation medium containing Radix Isatidis meal
Middle fermentation；
(6) centrifugal: zymotic fluid fermentation obtained is centrifuged, and washes precipitation with water, is again centrifuged, and merges twice
Centrifugal supernatant；
(7) detection: the precipitation i.e. dregs of a decoction after centrifugal are dried, lactic acid bacteria living bacteria count and supernatant in the detection dregs of a decoction
The content of Banlangen Polysaccharide in liquid；
(8) alcohol precipitation: the supernatant concentration after merging, adds ethanol and makes it precipitate, and then transfer is precipitated to vacuum
Low temperature drying in drying box, obtains Banlangen Polysaccharide.
In described step (1), the formula of MRS fluid nutrient medium is peptone 1 part, beef extract 1 part, yeast
Cream 0.5 part, glucose 2 parts, dipotassium hydrogen phosphate 0.2 part, diammonium hydrogen citrate 0.2 part, sodium acetate 0.1
Part, 0.06 part of magnesium sulfate, manganese sulfate 0.025 part, Tween 80 0.1 part, remaining is distilled water.
In described step (1), the temperature of autoclave is 121 DEG C, and sterilization time is 15 minutes.
Expanding the temperature cultivated in described step (2) is 32-38 DEG C, and incubation time is 20-36 hour.
In described step (3), the temperature of baking oven is 55 DEG C, and drying time is 4 hours, crosses No. two medicines after pulverizing
Sieve.
In described step (4), the component of fermentation medium is tryptone 3-6 part, dusty yeast 4-6 part, corn
Slurry 0.5-2 part, potassium dihydrogen phosphate 1-3 part, magnesium sulfate 0.2-1 part, 6 parts of Radix Isatidis powder, remaining is distillation
Water, 150ml is in 500ml triangular flask in preparation, and adjusting initial pH was 6.5, in 121 DEG C of sterilizings 10 minutes.
In described step (5), the inoculum concentration of lactic acid bacteria is 1%-3%.
Rotating speed centrifugal in described step (6) is 3500-5000 rev/min, and centrifugation time is 5-15 minute.
Described step (7) precipitation is dried 5 hours in an oven, and the temperature of baking oven is 45 DEG C.
The concentration of described step (8) ethanol is 80%.
The temperature of described step (8) vacuum drying chamber is 40 DEG C, and vacuum is 0.085MPa, and drying time is
1-3 hour.
Embodiment 1
The method of a kind of Radix Isatidis comprehensive utilization, comprises the following steps:
(1) preparation MRS fluid nutrient medium, MRS Liquid Culture based formulas: peptone 1 part, beef extract 1 part,
Yeast extract 0.5 part, glucose 2 parts, dipotassium hydrogen phosphate 0.2 part, diammonium hydrogen citrate 0.2 part, sodium acetate
0.1 part, 0.06 part of magnesium sulfate, manganese sulfate 0.025 part, Tween 80 0.1 part, remaining is distilled water, pH:
6.5 ± 0.2,121 DEG C of sterilizings 15 minutes；
(2) the lactic acid bacteria 3-5 ring separated from commercially available yoghourt with oese picking is inoculated into MRS fluid nutrient medium
In, cultivation temperature 35 DEG C, incubation time 24h；
(3) take 10ml lactobacillus solution, with former blank cultures for comparison, at 600nm, detect absorbance
It is 0.504；
(4) by chromatogram of Radix Isatidis in baking oven 55 DEG C dry 4 hours, took out after pulverizing No. two medicines sieve；
(5) fermentation medium is prepared according to the following formulation: tryptone 4 parts, dusty yeast 6 parts, corn steep liquor 1 part,
Potassium dihydrogen phosphate 2 parts, 1 part of magnesium sulfate, 6 parts of Radix Isatidis powder, remaining is distilled water；Preparation 150ml in
In 500ml triangular flask, adjust initial medium PH6.5,121 DEG C of sterilizings 10 minutes；
(6) by seed liquor 1% inoculation fermentation shaking flask by volume, shaking flask is placed in shaking table in 33 DEG C of cultivations, every
Shaking table is started 5 minutes with 150 revs/min, fermentation time 60 hours every 3 hours；
(7) zymotic fluid that fermentation obtains is transferred in centrifuge, 4000 revs/min, centrifugation time 10 minutes,
The precipitation i.e. dregs of a decoction are 45 DEG C dry 5 hours in baking oven；
(8) the dried dregs of a decoction are according to the method for plate culture count, detect lactic acid bacteria living bacteria count；
(9) supernatant after centrifugal takes 10ml in 100ml volumetric flask, adds 4ml 30% zinc sulfate, and water-bath adds
Heat 5 minutes, adds the potassium ferrocyanide test solution of 4ml under shaking, constant volume, filters, collects filtrate, filter
Liquid, with dextrose standard sample for comparison, measures polyoses content according to phend-sulphuric acid；
(10) different glucose standard items absorbance
The absorbance of table 1-1 dextrose standard sample solution
(11) supernatant after being centrifuged, is concentrated into and crude drug equivalent, adds the absolute ethyl alcohol of 4 times amount, ethanol
Concentration be 80%, 2-4 DEG C stand 10-14 hour, supernatant discarded, precipitation add appropriate petroleum ether 2
Secondary；Transfer is precipitated to low temperature drying in vacuum drying chamber, obtains Banlangen Polysaccharide dry product, vacuum drying chamber
Temperature is 40 DEG C, and vacuum is 0.085MPa, and drying time is 1-3 hour；
(12) according to embodiment 1 method, obtaining lactic acid bacteria Radix Isatidis mixed powder 5.80g, living bacteria count is
9.6×1010Cfu/g, Banlangen Polysaccharide 2.28g, Banlangen Polysaccharide recovery rate 25.3%.
Embodiment 2
The method of a kind of Radix Isatidis comprehensive utilization, comprises the following steps:
(1) preparation MRS fluid nutrient medium, MRS Liquid Culture based formulas: peptone 1 part, beef extract 1 part,
Yeast extract 0.5 part, glucose 2 parts, dipotassium hydrogen phosphate 0.2 part, diammonium hydrogen citrate 0.2 part, sodium acetate
0.1 part, 0.06 part of magnesium sulfate, manganese sulfate 0.025 part, Tween 80 0.1 part, remaining is distilled water, pH:
6.5 ± 0.2,121 DEG C of sterilizings 15 minutes；
(2) the lactic acid bacteria 3-5 ring separated from commercially available yoghourt with oese picking is inoculated into MRS fluid nutrient medium
In, cultivation temperature 37 DEG C, incubation time 30h；
(3) take 10ml lactobacillus solution, with former blank cultures for comparison, at 600nm, detect absorbance
It is 0.686；
(4) by chromatogram of Radix Isatidis in baking oven 55 DEG C dry 4 hours, take out and after pulverizing, cross No. two medicines sieve；
(5) fermentation medium is prepared according to the following formulation: tryptone 6 parts, dusty yeast 4 parts, corn steep liquor 1.5 parts,
Potassium dihydrogen phosphate 3 parts, 0.5 part of magnesium sulfate, 6 parts of Radix Isatidis powder, remaining is distilled water；Preparation 150ml
In 500ml triangular flask, adjust initial medium PH6.5,121 DEG C of sterilizings 10 minutes；
(6) by seed liquor 1% inoculation fermentation shaking flask by volume, shaking flask is placed in 35 DEG C of cultivations in shaking table, at interval of
Within 4 hours, start shaking table 5 minutes with 150 revs/min, fermentation time 58 hours；
(7) zymotic fluid that fermentation obtains is transferred in centrifuge, 5000 revs/min, centrifugation time 5 minutes,
The precipitation i.e. dregs of a decoction are 45 DEG C dry 5 hours in baking oven；
(8) the dried dregs of a decoction are according to the method for plate culture count, detect lactic acid bacteria living bacteria count；
(9) supernatant after centrifugal takes 10ml in 100ml volumetric flask, adds 4ml 30% zinc sulfate, and water-bath adds
Heat 5 minutes, adds the potassium ferrocyanide test solution of 4ml under shaking, constant volume, filters, collects filtrate, filter
Liquid, with dextrose standard sample for comparison, measures polyoses content according to phend-sulphuric acid；
(10) supernatant after centrifugal, is concentrated into and crude drug equivalent, adds the absolute ethyl alcohol of 4 times amount, 2-4 DEG C
Standing 10-14 hour, supernatant discarded, precipitation adds appropriate petroleum ether 2 times.Transfer is precipitated to vacuum
Low temperature drying in drying box, obtains Banlangen Polysaccharide dry product, and the temperature of vacuum drying chamber is 40 DEG C, vacuum
For 0.085MPa, drying time is 1-3 hour；
(11) according to embodiment 2 method, obtaining lactic acid bacteria Radix Isatidis mixed powder 5.76g, living bacteria count is
1.4×1011Cfu/g, Banlangen Polysaccharide 2.53g, Banlangen Polysaccharide recovery rate 28.1%.
Embodiment 3
The method of a kind of Radix Isatidis comprehensive utilization, comprises the following steps:
(1) preparation MRS fluid nutrient medium, MRS Liquid Culture based formulas: peptone 1 part, beef extract 1 part,
Yeast extract 0.5 part, glucose 2 parts, dipotassium hydrogen phosphate 0.2 part, diammonium hydrogen citrate 0.2 part, sodium acetate
0.1 part, 0.06 part of magnesium sulfate, manganese sulfate 0.025 part, Tween 80 0.1 part, remaining is distilled water, pH:
6.5 ± 0.2,121 DEG C of sterilizings 15 minutes；
(2) the lactic acid bacteria 3-5 ring separated from commercially available yoghourt with oese picking is inoculated into MRS fluid nutrient medium
In, cultivation temperature 37 DEG C, incubation time 34h；
(3) take 10ml lactobacillus solution, with former blank cultures for comparison, at 600nm, detect absorbance
It is 0.915；
(4) by chromatogram of Radix Isatidis in baking oven 55 DEG C dry 4 hours, take out and after pulverizing, cross No. two medicines sieve；
(5) fermentation medium is prepared according to the following formulation: tryptone 3 parts, dusty yeast 4 parts, corn steep liquor 1 part,
Potassium dihydrogen phosphate 2 parts, 1 part of magnesium sulfate, 6 parts of Radix Isatidis powder, remaining is distilled water；Preparation 150ml in
In 500ml triangular flask, adjust initial medium PH6.5,121 DEG C of sterilizings 10 minutes；
(6) by seed liquor 1% inoculation fermentation shaking flask by volume, shaking flask is placed in 37 DEG C of cultivations in shaking table, at interval of
Within 4 hours, start shaking table 5 minutes with 150 revs/min, fermentation time 54 hours；
(7) zymotic fluid that fermentation obtains is transferred in centrifuge, 5000 revs/min, centrifugation time 8 minutes,
The precipitation i.e. dregs of a decoction are 45 DEG C dry 5 hours in baking oven；
(8) the dried dregs of a decoction are according to the method for plate culture count, detect lactic acid bacteria living bacteria count；
(9) supernatant after centrifugal takes 10ml in 100ml volumetric flask, adds 4ml 30% zinc sulfate, and water-bath adds
Heat 5 minutes, adds the potassium ferrocyanide test solution of 4ml under shaking, constant volume, filters, collects filtrate, filter
Liquid, with dextrose standard sample for comparison, measures polyoses content according to phend-sulphuric acid；
(10) supernatant after centrifugal, is concentrated into and crude drug equivalent, adds the absolute ethyl alcohol of 4 times amount, 2-4 DEG C
Standing 10-14 hour, supernatant discarded, precipitation adds appropriate petroleum ether 2 times；Transfer is precipitated to vacuum
Low temperature drying in drying box, obtains Banlangen Polysaccharide dry product, and the temperature of vacuum drying chamber is 40 DEG C, vacuum
For 0.085MPa, drying time is 1-3 hour；
(11) according to embodiment 3 method, obtaining lactic acid bacteria Radix Isatidis mixed powder 5.69g, living bacteria count is
1.2×1011Cfu/g, Banlangen Polysaccharide 2.47g, Banlangen Polysaccharide recovery rate 27.4%.
Embodiment 4
The method of a kind of Radix Isatidis comprehensive utilization, comprises the following steps:
(1) preparation MRS fluid nutrient medium, MRS Liquid Culture based formulas: peptone 1 part, beef extract 1 part,
Yeast extract 0.5 part, glucose 2 parts, dipotassium hydrogen phosphate 0.2 part, diammonium hydrogen citrate 0.2 part, sodium acetate
0.1 part, 0.06 part of magnesium sulfate, manganese sulfate 0.025 part, Tween 80 0.1 part, remaining is distilled water, pH:
6.5 ± 0.2,121 DEG C of sterilizings 15 minutes；
(2) the lactic acid bacteria 3-5 ring separated from commercially available yoghourt with oese picking is inoculated into MRS fluid nutrient medium
In, cultivation temperature 33 DEG C, incubation time 35h；
(3) take 10ml lactobacillus solution, with former blank cultures for comparison, at 600nm, detect absorbance
It is 0.785；
(4) by chromatogram of Radix Isatidis in baking oven 55 DEG C dry 4 hours, took out after pulverizing No. two medicines sieve；
(5) fermentation medium is prepared according to the following formulation: tryptone 5 parts, dusty yeast 5 parts, corn steep liquor 2 parts,
Potassium dihydrogen phosphate 1 part, 0.2 part of magnesium sulfate, 6 parts of Radix Isatidis powder, remaining is distilled water；Preparation 150ml
In 500ml triangular flask, adjust initial medium PH6.8,121 DEG C of sterilizings 10 minutes；
(6) by seed liquor 2% inoculation fermentation shaking flask by volume, shaking flask is placed in 40 DEG C of cultivations in shaking table, at interval of
Within 3 hours, start shaking table 5 minutes with 180 revs/min, fermentation time 55 hours；
(7) zymotic fluid that fermentation obtains is transferred in centrifuge, 3500 revs/min, centrifugation time 15 minutes,
The precipitation i.e. dregs of a decoction are 45 DEG C dry 5 hours in baking oven；
(8) the dried dregs of a decoction are according to the method for plate culture count, detect lactic acid bacteria living bacteria count；
(9) supernatant after centrifugal takes 10ml in 100ml volumetric flask, adds 4ml 30% zinc sulfate, and water-bath adds
Heat 5 minutes, adds the potassium ferrocyanide test solution of 4ml under shaking, constant volume, filters, collects filtrate, filter
Liquid, with dextrose standard sample for comparison, measures polyoses content according to phend-sulphuric acid；
(10) supernatant after centrifugal, is concentrated into and crude drug equivalent, adds the absolute ethyl alcohol of 4 times amount, 2-4 DEG C
Standing 10-14 hour, supernatant discarded, precipitation adds appropriate petroleum ether 2 times；Transfer is precipitated to vacuum
Low temperature drying in drying box, obtains Banlangen Polysaccharide dry product, and the temperature of vacuum drying chamber is 40 DEG C, vacuum
For 0.085MPa, drying time is 1-3 hour；
(11) according to embodiment 3 method, obtaining lactic acid bacteria Radix Isatidis mixed powder 5.72g, living bacteria count is
1.2×1011Cfu/g, Banlangen Polysaccharide 2.49g, Banlangen Polysaccharide recovery rate 27.6%；
Embodiment 5
The method of a kind of Radix Isatidis comprehensive utilization, comprises the following steps:
(1) preparation MRS fluid nutrient medium, MRS Liquid Culture based formulas: peptone 1 part, beef extract 1 part,
Yeast extract 0.5 part, glucose 2 parts, dipotassium hydrogen phosphate 0.2 part, diammonium hydrogen citrate 0.2 part, sodium acetate
0.1 part, 0.06 part of magnesium sulfate, manganese sulfate 0.025 part, Tween 80 0.1 part, remaining is distilled water, pH:
6.5 ± 0.2,121 DEG C of sterilizings 15 minutes；
(2) the lactic acid bacteria 3-5 ring separated from commercially available yoghourt with oese picking is inoculated into MRS fluid nutrient medium
In, cultivation temperature 38 DEG C, incubation time 28h；
(3) take 10ml lactobacillus solution, with former blank cultures for comparison, at 600nm, detect absorbance
It is 0.653；
(4) by chromatogram of Radix Isatidis in baking oven 55 DEG C dry 4 hours, took out after pulverizing No. two medicines sieve；
(5) fermentation medium is prepared according to the following formulation: tryptone 3 parts, dusty yeast 4 parts, corn steep liquor 0.5 part,
Potassium dihydrogen phosphate 2 parts, 1 part of magnesium sulfate, 6 parts of Radix Isatidis powder, remaining is distilled water；Preparation 150ml in
In 500ml triangular flask, adjust initial medium PH6.8,121 DEG C of sterilizings 10 minutes；
(6) by seed liquor 3% inoculation fermentation shaking flask by volume, shaking flask is placed in 30 DEG C of cultivations in shaking table, at interval of
Within 6 hours, start shaking table 5 minutes with 100 revs/min, fermentation time 62 hours；
(7) zymotic fluid that fermentation obtains is transferred in centrifuge, 5000 revs/min, centrifugation time 8 minutes,
The precipitation i.e. dregs of a decoction are 45 DEG C dry 5 hours in baking oven；
(8) the dried dregs of a decoction are according to the method for plate culture count, detect lactic acid bacteria living bacteria count；
(9) supernatant after centrifugal takes 10ml in 100ml volumetric flask, adds 4ml 30% zinc sulfate, and water-bath adds
Heat 5 minutes, adds the potassium ferrocyanide test solution of 4ml under shaking, constant volume, filters, collects filtrate, filter
Liquid, with dextrose standard sample for comparison, measures polyoses content according to phend-sulphuric acid；
(10) supernatant after centrifugal, is concentrated into and crude drug equivalent, adds the absolute ethyl alcohol of 4 times amount, 2-4 DEG C
Standing 10-14 hour, supernatant discarded, precipitation adds appropriate petroleum ether 2 times；Transfer is precipitated to vacuum
Low temperature drying in drying box, obtains Banlangen Polysaccharide dry product, and the temperature of vacuum drying chamber is 40 DEG C, vacuum
For 0.085MPa, drying time is 1-3 hour；
(11) according to embodiment 3 method, obtaining lactic acid bacteria Radix Isatidis mixed powder 5.78g, living bacteria count is
1.0×1011Cfu/g, Banlangen Polysaccharide 2.36g, Banlangen Polysaccharide recovery rate 26.2%.
Conclusion: the above is only preferred embodiment, and every technical scheme carried out based on the present invention is repaiied
Change and should be included within the scope of the present invention.
Claims (10)
1. the method for a Radix Isatidis comprehensive utilization, it is characterised in that: comprise the following steps:
(1) preparation of MRS culture medium and sterilizing: preparation MRS fluid nutrient medium, is placed in sterilizing in autoclave；
(2) expansion of lactic acid bacteria is cultivated: cultivate being enlarged in lactobacillus inoculum to sterilized MRS fluid nutrient medium；
(3) preparation of Radix Isatidis meal: chromatogram of Radix Isatidis is placed in baking oven drying, then pulverizes, sieve；
(4) preparation fermentation medium: preparation fermentation medium, is placed in sterilizing in autoclave；
(5) inoculate and ferment: will be enlarged by the lactobacillus inoculum after cultivating and ferment in the fermentation medium containing Radix Isatidis meal；
(6) centrifugal: zymotic fluid fermentation obtained is centrifuged, and washes precipitation with water, is again centrifuged, merge twice centrifugal supernatant；
(7) detection: the precipitation i.e. dregs of a decoction after centrifugal are dried, the lactic acid bacteria living bacteria count in the detection dregs of a decoction and the content of the Banlangen Polysaccharide in supernatant；
(8) alcohol precipitation: the supernatant concentration after merging, adds ethanol and makes it precipitate, and then transfer is precipitated to low temperature drying in vacuum drying chamber, obtains Banlangen Polysaccharide dry product.
The method of a kind of Radix Isatidis the most according to claim 1 comprehensive utilization, it is characterised in that: in described step (1), the temperature of autoclave is 121 DEG C, and sterilization time is 15 minutes.
The method of a kind of Radix Isatidis the most according to claim 1 comprehensive utilization, it is characterised in that: expanding the temperature cultivated in described step (2) is 32-38 DEG C, and incubation time is 20-36 hour.
The method of a kind of Radix Isatidis the most according to claim 1 comprehensive utilization, it is characterised in that: in described step (3), the temperature of baking oven is 55 DEG C, and drying time is 4 hours, crosses No. two medicine sieves after pulverizing.
The method of a kind of Radix Isatidis the most according to claim 1 comprehensive utilization, it is characterized in that: in described step (4), the component of fermentation medium is tryptone 3-6 part, dusty yeast 4-6 part, corn steep liquor 0.5-2 part, potassium dihydrogen phosphate 1-3 part, magnesium sulfate 0.2-1 part, 6 parts of Radix Isatidis powder, remaining is distilled water, and 150ml is in 500ml triangular flask in preparation, adjusting initial pH was 6.8, in 121 DEG C of sterilizings 10 minutes.
The method of a kind of Radix Isatidis the most according to claim 1 comprehensive utilization, it is characterised in that: in described step (5), the inoculum concentration of lactic acid bacteria is 1%-3%.
The method of a kind of Radix Isatidis the most according to claim 1 comprehensive utilization, it is characterised in that: rotating speed centrifugal in described step (6) is 3500-5000 rev/min, and centrifugation time is 5-15 minute.
The method of a kind of Radix Isatidis the most according to claim 1 comprehensive utilization, it is characterised in that: described step (7) precipitation is dried 5 hours in an oven, and the temperature of baking oven is 45 DEG C.
The method of a kind of Radix Isatidis the most according to claim 1 comprehensive utilization, it is characterised in that: during described step (8) alcohol precipitation, the concentration of ethanol is 80%.
The method of a kind of Radix Isatidis the most according to claim 1 comprehensive utilization, it is characterised in that: in described step (8), the temperature of vacuum drying chamber is 40 DEG C, and vacuum is 0.085Mp.
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| CN107569530A (en) * | 2017-10-31 | 2018-01-12 | 河南黑马动物药业有限公司 | A kind of preparation method of compound isatis-root injection for animals |
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