CN105420143A - Acetobacter orientalis and method for producing astragalus polysaccharide through same - Google Patents
Acetobacter orientalis and method for producing astragalus polysaccharide through same Download PDFInfo
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Abstract
The invention provides acetobacter orientalis. The preservation name of the acetobacter orientalis is Acetobacter orientalis, the acetobacter orientalis is preserved in China Center for Type Culture Collection on July 16, 2015, and the preservation serial number is CCTCC NO: M 2015455. The acetobacter orientalis has good stability, is high in metabolic capability and safety and has good medicinal value. The invention further provides a method for producing astragalus polysaccharide through the acetobacter orientalis. The content of the astragalus polysaccharide obtained through fermented culture is not lower than 30%.
Description
Technical field
The present invention relates to microorganism field, particularly relate to the method for a kind of east acetobacter and production astragalus polysaccharides thereof.
Background technology
East acetobacter comes from the distinctive a kind of Yoghourt in Qinghai-Tibet Platean, and colony colour is creamy white, and late stage of culture colony colour turns yellow, bacterium colony is round point shape, intermediate projections neat in edge, and smooth surface is moistening, have special tart flavour, Gram-reaction is positive, and optical microphotograph Microscopic observation is rod-short.
Astragalus polysaccharides has enhancing animal immunizing power, the ability that induced animal Interferon, rabbit produces, there is obvious low temperature resistant effect, the anti-survival time of normal mouse and deficient mouse can be extended, there is the effect of two-ways regulation blood sugar, the glucose level of mouse after glucose load can be reduced, there is similar functions of hormones, extend the rutting sedson of mouse, have desirable influence to the growth of mouse.
Based on pharmacological action and the clinical value of astragalus polysaccharides, the existing demand commercially for astragalus polysaccharides is increasing, however from the Radix Astragali method of extracting directly astragalus polysaccharides, there is extraction yield low, poor stability, purifying difficulty is large, the problems such as operation steps is complicated, and the cycle is long.
By the fermentable Radix Astragali, not only can improve the yield of astragalus polysaccharides, the decomposition of some simultaneously adjoint during the fermentation composition or transform the new effective constituent of the Radix Astragali pharmacological action that may have an impact, reach the effect of efficacy enhancing and toxicity reducing; Utilize the biological characteristics of east acetobacter and the feature of growth and breeding, separation and purification is carried out to it and selects excellent bacterial classification, and its growth conditions is optimized, use it for fermentation astragalus polysaccharides and there is important economic implications and social effect.
Summary of the invention
In order to solve the problem, the invention provides the method for a kind of east acetobacter and production astragalus polysaccharides thereof.
The technical solution used in the present invention is:
A kind of east acetobacter, the preservation name of described east acetobacter is called Acetobacterorientalis, and be preserved in China typical culture collection center on July 16th, 2015, deposit number is CCTCCNO:M2015455.
East as claimed in claim 1 acetobacter produces a method for astragalus polysaccharides, comprises the following steps:
(1) get 1ml Qinghai-Tibet Platean Yoghourt and carry out 10 times of serial dilutions to 10
-6, then get the bacterium liquid 50ul after dilution in containing in the culture dish of MRS nutrient agar, and even with spreading rod coating, be placed in incubator and cultivate 48h ~ 72h at 37 DEG C ± 1 DEG C, obtain single bacterium colony;
(2) by single colony inoculation in slant medium, the test tube containing slant medium is placed in incubator 37 DEG C ± 1 DEG C cultivate 48h ~ 72h, obtain slant strains;
(3) slant strains is inoculated in Shake flask medium, is positioned in shaking table by Shake flask medium and cultivates, shaking table temperature 37 DEG C ± 1 DEG C, rotating speed 100r/min ~ 120r/min, obtains shaking flask bacterium liquid after 24h ~ 28h;
(4) the bacterium liquid obtained is inoculated in liquid nutrient medium, fermentor tank containing liquid nutrient medium is placed in cannery retort after 121 DEG C of autoclave sterilization 30min, be put in shaking table and cultivate, shaking table temperature is 37 DEG C ± 1 DEG C, rotating speed is 100r/min ~ 120r/min, put tank after cultivating 48h ~ 52h, obtain fermented liquid;
(5) gained fermented liquid is through the centrifugal 10min of 1500r/min, gets its supernatant, and supernatant obtains astragalus polyose solution through concentrated.
Slant medium in described step (2) is MRS improved culture medium, comprise the component of following massfraction: the fish protein peptone of 0.5%-1%, the yeast extract of 0.5%-1%, the Tryptones of 0.5%-1%, the citric acid hydrogen diamine of 0.2%-0.4%, the dipotassium hydrogen phosphate of 0.2%-0.4%, the sodium acetate of 0.5%-1%, four Magnesium sulfate heptahydrates of 0.02%-0.05%, the calcium carbonate of 0.3%-0.5%, the tween-80 of 0.1%-0.2%, the bitter salt of 0.02%-0.05%, the glucose of 0.8%-1.5%, all the other are distilled water.
In described step (3), Shake flask medium comprises the component of following massfraction: the fish protein peptone of 0.5%-1%, the lactose of 1%-2%, the yeast extract paste of 1%-2%, the dipotassium hydrogen phosphate of 0.25%-0.4%, the potassium primary phosphate of 0.25%-0.4%, all the other are distilled water.
In described step (3), the pH of Shake flask medium is 6.5.
In described step (4), liquid nutrient medium comprises the component of following massfraction: the nitrogenous source of 0.5%-1%, the carbon source of 1%-2%, the dipotassium hydrogen phosphate of 0.25%-0.4%, the potassium primary phosphate of 0.25%-0.4%, the astragalus membranaceus powder of 8%-10%, all the other are distilled water.
Described carbon source is lactose or glucose, and nitrogenous source is at least one in fish protein peptone, Tryptones or yeast extract paste.
In described step (4), the volume ratio of bacterium liquid and liquid nutrient medium is 1:50.
The invention has the beneficial effects as follows: east provided by the invention acetobacter has satisfactory stability, and metabolic capacity is high, and security is high, has good pharmaceutical use, the content through the astragalus polysaccharides of fermentation culture acquisition is not less than 30%.
preservation illustrates:
Strain name: east acetobacter;
Latin name: Acetobacterorientalis;
Strain number: T1-1;
Deposit number: CCTCCNO:M2015455;
Preservation mechanism: China typical culture collection center;
Preservation mechanism is called for short: CCTCC;
Address: China, Wuhan University;
Preservation date: on July 16th, 2015.
Embodiment
A kind of east acetobacter, the preservation name of described east acetobacter is called Acetobacterorientalis, and be preserved in China typical culture collection center on July 16th, 2015, deposit number is CCTCCNO:M2015455.
East as claimed in claim 1 acetobacter produces a method for astragalus polysaccharides, comprises the following steps:
(1) get 1ml Qinghai-Tibet Platean Yoghourt and carry out 10 times of serial dilutions to 10
-6, then get the bacterium liquid 50ul after dilution in containing in the culture dish of MRS nutrient agar, and even with spreading rod coating, be placed in incubator and cultivate 48h ~ 72h at 37 DEG C ± 1 DEG C, obtain single bacterium colony;
(2) by single colony inoculation in slant medium, the test tube containing slant medium is placed in incubator 37 DEG C ± 1 DEG C cultivate 48h ~ 72h, obtain slant strains;
(3) slant strains is inoculated in Shake flask medium, is positioned in shaking table by Shake flask medium and cultivates, shaking table temperature 37 DEG C ± 1 DEG C, rotating speed 100r/min ~ 120r/min, obtains shaking flask bacterium liquid after 24h ~ 28h;
(4) the bacterium liquid obtained is inoculated in liquid nutrient medium, fermentor tank containing liquid nutrient medium is placed in cannery retort after 121 DEG C of autoclave sterilization 30min, be put in shaking table and cultivate, shaking table temperature is 37 DEG C ± 1 DEG C, rotating speed is 100r/min ~ 120r/min, put tank after cultivating 48h ~ 52h, obtain fermented liquid;
(5) gained fermented liquid is through the centrifugal 10min of 1500r/min, gets its supernatant, and supernatant obtains astragalus polyose solution through concentrated.
Slant medium in described step (2) is MRS improved culture medium, comprise the component of following massfraction: the fish protein peptone of 0.5%-1%, the yeast extract of 0.5%-1%, the Tryptones of 0.5%-1%, the citric acid hydrogen diamine of 0.2%-0.4%, the dipotassium hydrogen phosphate of 0.2%-0.4%, the sodium acetate of 0.5%-1%, four Magnesium sulfate heptahydrates of 0.02%-0.05%, the calcium carbonate of 0.3%-0.5%, the tween-80 of 0.1%-0.2%, the bitter salt of 0.02%-0.05%, the glucose of 0.8%-1.5%, all the other are distilled water.
In described step (3), Shake flask medium comprises the component of following massfraction: the fish protein peptone of 0.5%-1%, the lactose of 1%-2%, the yeast extract paste of 1%-2%, the dipotassium hydrogen phosphate of 0.25%-0.4%, the potassium primary phosphate of 0.25%-0.4%, all the other are distilled water.
In described step (3), the pH of Shake flask medium is 6.5.
In described step (4), liquid nutrient medium comprises the component of following massfraction: the nitrogenous source of 0.5%-1%, the carbon source of 1%-2%, the dipotassium hydrogen phosphate of 0.25%-0.4%, the potassium primary phosphate of 0.25%-0.4%, the astragalus membranaceus powder of 8%-10%, all the other are distilled water.
Described carbon source is lactose or glucose, and nitrogenous source is at least one in fish protein peptone, Tryptones or yeast extract paste.
In described step (4), the volume ratio of bacterium liquid and liquid nutrient medium is 1:50.
Embodiment 1
East as claimed in claim 1 acetobacter produces a method for astragalus polysaccharides, comprises the following steps:
(1) get 1ml Qinghai-Tibet Platean Yoghourt and carry out 10 times of serial dilutions to 10
-6, then get the bacterium liquid 50ul after dilution in containing in the culture dish of MRS nutrient agar, and even with spreading rod coating, be placed in incubator and cultivate 48h ~ 72h at 37 DEG C ± 1 DEG C, obtain single bacterium colony;
(2) get a complete single bacterium colony to be inoculated in 18*18 by the method for line and to be equipped with on the inclined-plane of 10ml substratum, then the test tube containing slant medium is placed in incubator 37 DEG C ± 1 DEG C and cultivates 48h ~ 72h, obtain slant strains, be stored in 4 DEG C of refrigerators for subsequent use;
(3) get cell age short, the slant strains that growing way is good is inoculated in Shake flask medium, is positioned in shaking table by Shake flask medium and cultivates, shaking table temperature 37 DEG C ± 1 DEG C, and rotating speed 100r/min ~ 120r/min, obtains shaking flask bacterium liquid after 48h;
(4) the bacterium liquid obtained is inoculated in liquid nutrient medium, fermentor tank containing liquid nutrient medium is placed in cannery retort after 121 DEG C of autoclave sterilization 30min, be put in shaking table and cultivate, shaking table temperature is 37 DEG C ± 1 DEG C, rotating speed is 100r/min ~ 120r/min, put tank after cultivating 48h, obtain fermented liquid;
(5) Centrifuge Cup poured into by gained fermented liquid, then whizzer is put into centrifugal, the rotating speed of whizzer is 1500r/min, after centrifugal 10min terminates, getting supernatant liquor, to be placed in 4 DEG C of Refrigerator stores for subsequent use, centrifugal again after sediment fraction poach, the supernatant liquor of two times centrifugal is merged, concentrates the rear content measuring astragalus polysaccharides; The method measuring the content of astragalus polysaccharides adopts zinc sulfate and yellow prussiate of potash process tunning, measures under 490nm wavelength with phend-sulphuric acid.
The preparation method of MRS nutrient agar in step (1): first configure MRS broth culture: casease digest 10 parts, 10 parts, extractum carnis powder, yeast powder 4 parts, Triammonium citrate 2 parts, sodium acetate 5 parts, bitter salt 0.2 part, glucose 20 parts, four anhydrous manganese 0.05 part, tween-80 1.08 parts; Take MRS broth culture 54g again, add 20g agar and dissolve in 1000ml water.
Slant medium in described step (2) is MRS improved culture medium, comprises the component of following massfraction: the fish protein peptone of 0.5%, the yeast extract of 0.5%, the Tryptones of 0.5%, the citric acid hydrogen diamine of 0.2%, the dipotassium hydrogen phosphate of 0.2%, the sodium acetate of 0.5%, four Magnesium sulfate heptahydrates of 0.02%, the calcium carbonate of 0.3%, the tween-80 of 0.1%, the bitter salt of 0.02%, the glucose of 0.8%, all the other are distilled water.
In described step (3), Shake flask medium comprises the component of following massfraction: the fish protein peptone of 0.5%, the lactose of 1%, the yeast extract paste of 1%, the dipotassium hydrogen phosphate of 0.25%, the potassium primary phosphate of 0.25%, all the other are distilled water, and pH is 6.5.
In described step (4), liquid nutrient medium comprises the component of following massfraction: lactose 1%, fish protein peptone 0.5%, yeast extract paste 1%, dipotassium hydrogen phosphate 0.25%, potassium primary phosphate 0.25%, astragalus membranaceus powder 8%, all the other are distilled water.
In described step (4), the volume ratio of bacterium liquid and liquid nutrient medium is 1:50.
Experiment reagent needed for this experiment and experimental installation, be the biological fermentation equipment of conventional analytical pure, chemical pure, biological pure reagent and routine, easily commercially obtain.
East of the present invention acetobacter is stored in 4 DEG C of refrigerators, and long-term preservation needs whiteruss to seal up for safekeeping.
East provided by the invention acetobacter is after liquid fermenting, and the pH of fermented liquid is 3.5-4.5, and the quality of astragalus polysaccharides is not less than 30% of the added Radix Astragali, higher than common water extraction.
Radix Astragali residue after namely treated fermented liquid extracts astragalus polysaccharides and east acetobacter can be used for fodder additives, medicine and microbial preparation.
Claims (8)
1. an east acetobacter, is characterized in that: the preservation name of described east acetobacter is called Acetobacterorientalis, and be preserved in China typical culture collection center on July 16th, 2015, deposit number is CCTCCNO:M2015455.
2. east as claimed in claim 1 acetobacter produces a method for astragalus polysaccharides, it is characterized in that: comprise the following steps:
(1) get 1ml Qinghai-Tibet Platean Yoghourt and carry out 10 times of serial dilutions to 10
-6, then get the bacterium liquid 50ul after dilution in containing in the culture dish of MRS nutrient agar, and even with spreading rod coating, be placed in incubator and cultivate 48h ~ 72h at 37 DEG C ± 1 DEG C, obtain single bacterium colony;
(2) by single colony inoculation in slant medium, the test tube containing slant medium is placed in incubator 37 DEG C ± 1 DEG C cultivate 48h ~ 72h, obtain slant strains;
(3) slant strains is inoculated in Shake flask medium, is positioned in shaking table by Shake flask medium and cultivates, shaking table temperature 37 DEG C ± 1 DEG C, rotating speed 100r/min ~ 120r/min, obtains shaking flask bacterium liquid after 24h ~ 28h;
(4)the bacterium liquid obtained is inoculated in liquid nutrient medium, fermentor tank containing liquid nutrient medium is placed in cannery retort after 121 DEG C of autoclave sterilization 30min, be put in shaking table and cultivate, shaking table temperature is 37 DEG C ± 1 DEG C, rotating speed is 100r/min ~ 120r/min, put tank after cultivating 48h ~ 52h, obtain fermented liquid;
(5)gained fermented liquid, through the centrifugal 10min of 1500r/min, gets its supernatant, and supernatant obtains astragalus polyose solution through concentrated.
3. a kind of east according to claim 2 acetobacter produces the method for astragalus polysaccharides, it is characterized in that: the slant medium in described step (2) is MRS improved culture medium, comprise the component of following massfraction: the fish protein peptone of 0.5%-1%, the yeast extract of 0.5%-1%, the Tryptones of 0.5%-1%, the citric acid hydrogen diamine of 0.2%-0.4%, the dipotassium hydrogen phosphate of 0.2%-0.4%, the sodium acetate of 0.5%-1%, four Magnesium sulfate heptahydrates of 0.02%-0.05%, the calcium carbonate of 0.3%-0.5%, the tween-80 of 0.1%-0.2%, the bitter salt of 0.02%-0.05%, the glucose of 0.8%-1.5%, all the other are distilled water.
4. a kind of east according to claim 2 acetobacter produces the method for astragalus polysaccharides, it is characterized in that: in described step (3), Shake flask medium comprises the component of following massfraction: the fish protein peptone of 0.5%-1%, the lactose of 1%-2%, the yeast extract paste of 1%-2%, the dipotassium hydrogen phosphate of 0.25%-0.4%, the potassium primary phosphate of 0.25%-0.4%, all the other are distilled water.
5. a kind of east according to claim 2 acetobacter produces the method for astragalus polysaccharides, it is characterized in that: in described step (3), the pH of Shake flask medium is 6.5.
6. a kind of east according to claim 2 acetobacter produces the method for astragalus polysaccharides, it is characterized in that: in described step (4), liquid nutrient medium comprises the component of following massfraction: the nitrogenous source of 0.5%-1%, the carbon source of 1%-2%, the dipotassium hydrogen phosphate of 0.25%-0.4%, the potassium primary phosphate of 0.25%-0.4%, the astragalus membranaceus powder of 8%-10%, all the other are distilled water.
7. a kind of east according to claim 6 acetobacter produces the method for astragalus polysaccharides, and it is characterized in that: described carbon source is lactose or glucose, nitrogenous source is at least one in fish protein peptone, Tryptones or yeast extract paste.
8. a kind of east according to claim 2 acetobacter produces the method for astragalus polysaccharides, it is characterized in that: in described step (4), the volume ratio of bacterium liquid and liquid nutrient medium is 1:50.
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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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| CN115216421A (en) * | 2022-04-21 | 2022-10-21 | 广东海洋大学 | Bacillus cereus strain No.1 specially presenting island and application thereof in improving yield of traditional Chinese medicine extract |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110548075A (en) * | 2019-10-18 | 2019-12-10 | 河南黑马动物药业有限公司 | Preparation method of veterinary ephedra, apricot kernel, gypsum and licorice oral liquid |
| CN115216421A (en) * | 2022-04-21 | 2022-10-21 | 广东海洋大学 | Bacillus cereus strain No.1 specially presenting island and application thereof in improving yield of traditional Chinese medicine extract |
| CN115216421B (en) * | 2022-04-21 | 2023-03-14 | 广东海洋大学 | Bacillus cereus strain No.1 specially presenting island and application thereof in improving yield of traditional Chinese medicine extract |
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