CN109136145A - One kind separating Bifidobacterium and its purification process from rabbit excrement - Google Patents
One kind separating Bifidobacterium and its purification process from rabbit excrement Download PDFInfo
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- CN109136145A CN109136145A CN201811056559.0A CN201811056559A CN109136145A CN 109136145 A CN109136145 A CN 109136145A CN 201811056559 A CN201811056559 A CN 201811056559A CN 109136145 A CN109136145 A CN 109136145A
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- 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
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Abstract
The invention discloses one kind to separate Bifidobacterium and its purification process from rabbit excrement, is related to microorganisms technical field.The present invention includes step 1: preparation sample liquid；Step 2: dilute sample liquid；Step 3: it is separately cultured；Step 4: pure medium is prepared；Step 5: purifying culture.The present invention solves the problems, such as that the existing bifidobacteria viable bacteria amount isolated is lower by being separately cultured the purifying for improving strain；By optimizing to pure medium, incubation time is effectively shortened, solves that existing incubation time is longer at high cost, the lower problem of efficiency.
Description
Technical field
The invention belongs to microorganisms technical fields, and Bifidobacterium and its purifying side are separated from rabbit excrement more particularly to one kind
Method.
Background technique
The bacterium of Bifidobacterium is one of important composition member of humans and animals intestinal flora, is widely present in people and moves
In the habitats such as the alimentary canal of object and oral cavity；The bacterial strain of some Bifidobacteriums can be used as probiotics and be used in food, medicine and feeding
Material aspect；In order to which for Different Individual, using suitable Bifidobacterium, common method is to isolate Bifidobacterium by excrement
Strain cultivated；But the bifidobacteria viable bacteria amount developed in such method is lower, survival not easy to maintain, while existing
Required incubation time is longer when culture medium culture, higher cost, and efficiency is lower.
Summary of the invention
The purpose of the present invention is to provide one kind to separate Bifidobacterium and its purification process from rabbit excrement, by being separately cultured
The purifying for improving strain solves the problems, such as that the existing bifidobacteria viable bacteria amount isolated is lower；By to pure medium
It optimizes, effectively shortens incubation time, solve that existing incubation time is longer at high cost, the lower problem of efficiency.
In order to solve the above technical problems, the present invention is achieved by the following technical solutions:
The present invention is that one kind separates Bifidobacterium and its purification process from rabbit excrement, comprising the following steps:
Step 1: preparation sample liquid；It takes new fresh rabbit excrement to be placed in peptone buffer agent, stirs, it is primary to be centrifuged
Upper layer turbid is collected after processing；Appropriate peptone buffer agent is added in upward turbid, collects upper layer turbid after centrifugal treating again, i.e.,
For sample liquid；
Step 2: dilute sample liquid；A certain amount of sample liquid is taken to be diluted with decimal dilution method using peptone buffer agent,
Dilution gradient is 10-1To 10-9；
Step 3: it is separately cultured；Taking a certain amount of dilution gradient respectively is 10-4To 10-9Dilute sample liquid be placed in culture
On base plate, the dilute sample liquid of each dilution gradient does 2 to 3 Duplicate Samples；It is being moved after being cultivated 45-50 hours under anaerobic condition
To cultivating 5-7 hours under the conditions of aerobic；
Step 4: pure medium is prepared；Nitrogen source and carbon source optimizing are carried out to modified MRS culture medium；
Step 5: it purifying culture: is selected, will be selected according to the bacterium colony of the culture in Bifidobacterium character pair step 3
Strain be seeded to step 4 preparation purifying culture solution in carry out purifying culture.
Further, rabbit excrement quality and peptone buffer agent volume ratio are 1:10 in the step 1.
Further, cultivation temperature is 36-39 degrees Celsius in the step 3.
Further, nitrogen source in the step 4 are as follows: soy peptone, tryptone, casein hydrolysate and beef leaching
Cream；Carbon source are as follows: glucose, isomaltose and lactose.
Further, the soy peptone, tryptone, casein hydrolysate and beef extract mass ratio are followed successively by 2:
1:1:1；The glucose, isomaltose and lactose mass ratio are followed successively by 2:1:1.
Further, condition of culture is to cultivate 10-16 in the case where temperature is 37-39 degrees Celsius of anaerobic condition in the step 5
Hour.
The invention has the following advantages:
1, the present invention is by centrifugal treating twice, and aerobic culture processing is carried out in being separately cultured, and effectively reduces
The separation and purifying difficulty of each strain morphologically, improve the number of viable and purifying rate of Bifidobacterium.
2, the present invention is effectively shortened the incubation time of double gas bacillus, is improved by the optimization to pure medium
Culture efficiency is effective to reduce production cost.
Certainly, it implements any of the products of the present invention and does not necessarily require achieving all the advantages described above at the same time.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's all other embodiment obtained without creative efforts belongs to the model that the present invention protects
It encloses.
The present invention is that one kind separates Bifidobacterium and its purification process from rabbit excrement, comprising the following steps:
Step 1: preparation sample liquid；New fresh rabbit excrement is taken to be placed in peptone buffer agent, rabbit excrement quality and peptone buffer agent
Volume ratio is 1:10, is stirred, and sets centrifugal force 600-1500g, collects upper layer turbid after centrifugation five minutes；It is turbid upwards
Appropriate peptone buffer agent is added in liquid, sets centrifugal force 2000-4000g, collects upper layer turbid after being centrifuged five minutes again, as
Sample liquid；
Step 2: dilute sample liquid；A certain amount of sample liquid is taken to be diluted with decimal dilution method using peptone buffer agent,
Dilution gradient is 10-1To 10-9；
Step 3: it is separately cultured；Taking a certain amount of dilution gradient respectively is 10-4To 10-9Dilute sample liquid be placed in culture
On base plate, the dilute sample liquid of each dilution gradient does 2 to 3 Duplicate Samples；Under anaerobic, temperature is that 36-39 is Celsius
Degree, culture are cultivated 5-7 hours in the case where moving to aerobic condition again after 45-50 hours；Intersection training is carried out by anaerobic and aerobic environment
Supporting can sufficiently be such that various strains are grown, and distinguish conducive to from colonial morphology, improve the bacterium colony purity isolated；
Step 4: pure medium is prepared；Nitrogen source and carbon source optimizing are carried out to modified MRS culture medium；By optimizing nitrogen source
With the ratio of in carbon source composition, the culture speed of Bifidobacterium can be effectively improved, improves purifying rate and production efficiency, is reduced
Production cost；
Step 5: it purifying culture: is selected, will be selected according to the bacterium colony of the culture in Bifidobacterium character pair step 3
Strain to be seeded in the purifying culture solution of step 4 preparation the culture 10-16 in the case where temperature is 37-39 degrees Celsius of anaerobic condition small
When.
Wherein, nitrogen source in step 4 are as follows: soy peptone, tryptone, casein hydrolysate and beef extract；Carbon source
Are as follows: glucose, isomaltose and lactose.
Wherein, soy peptone, tryptone, casein hydrolysate and beef extract mass ratio are followed successively by 2:1:1:1；Portugal
Grape sugar, isomaltose and lactose mass ratio are followed successively by 2:1:1.
Embodiment one:
Step 1: preparation sample liquid；It takes new fresh rabbit excrement 1g to be placed in 10ml peptone buffer agent, stirs, if
Determine centrifugal force 600-1500g, collects upper layer turbid after centrifugation five minutes；Appropriate peptone buffer agent, setting is added in upward turbid
Centrifugal force 2000-4000g collects upper layer turbid, as sample liquid after being centrifuged five minutes again；
Step 2: dilute sample liquid；Take 1ml sample liquid to be added in the peptone buffer agent of 9ml with decimal dilution method according to
It is secondary to be diluted, dilution gradient 10-1To 10-9；
Step 3: it is separately cultured；The dilution gradient for taking 0.1ml respectively is 10-4To 10-9Dilute sample liquid be placed in culture
On base plate, the dilute sample liquid of each dilution gradient does 3 Duplicate Samples；Under anaerobic, temperature is 36-39 degrees Celsius,
Culture is cultivated 5 hours in the case where moving to aerobic condition again after 45-50 hours；
Step 4: pure medium is prepared；Nitrogen source and carbon source optimizing are carried out to modified MRS culture medium；Optimization of C/C composites are as follows: ferment
Mother leaches powder 5g, ferric citrate 2g, sodium acetate 5g, disodium-hydrogen 2g, manganese sulfate 0.05g, half Guang ammonia of manganese chloride 0.5g, L-
Sour 0.5g, X-Gal0.06g, Tween-80 1ml, agar 25g, distilled water 1000ml；Nitrogen source and carbon source is added, adjusting pH value is 7,
It sterilizes 10-20 minutes under 120-130 degrees Celsius；
Wherein nitrogen source: soy peptone 10g, tryptone 5g, casein hydrolysate 5g and beef extract 5g；Carbon source: Portugal
Grape sugar 10g, isomaltose 5g and lactose 5g；
Step 5: purifying culture: according to the characteristic of Bifidobacterium character such as thallus shape, color and anaerobism in step 3
Culture bacterium colony carry out scribing line partition method select, by the single colonie strain selected be seeded to step 4 preparation purifying culture solution
In temperature be 37-39 degrees Celsius of anaerobic condition under cultivate 20 hours；And from starting every 4 hours to bacterium colony progress light absorption value survey
Fixed and record.
Comparative example one:
Step 1: preparation sample liquid；It takes new fresh rabbit excrement 1g to be placed in 10ml peptone buffer agent, stirs, if
Determine centrifugal force 600-1500g, collects upper layer turbid after centrifugation five minutes；Appropriate peptone buffer agent, setting is added in upward turbid
Centrifugal force 2000-4000g collects upper layer turbid, as sample liquid after being centrifuged five minutes again；
Step 2: dilute sample liquid；Take 1ml sample liquid to be added in the peptone buffer agent of 9ml with decimal dilution method according to
It is secondary to be diluted, dilution gradient 10-1To 10-9；
Step 3: it is separately cultured；The dilution gradient for taking 0.1ml respectively is 10-4To 10-9Dilute sample liquid be placed in culture
On base plate, the dilute sample liquid of each dilution gradient does 3 Duplicate Samples；Under anaerobic, temperature is 36-39 degrees Celsius,
Culture is cultivated 5 hours in the case where moving to aerobic condition again after 45-50 hours；
Step 4: pure medium is prepared；Nitrogen source and carbon source optimizing are carried out to modified MRS culture medium；Optimization of C/C composites are as follows: ferment
Mother leaches powder 5g, ferric citrate 2g, sodium acetate 5g, disodium-hydrogen 2g, manganese sulfate 0.05g, half Guang ammonia of manganese chloride 0.5g, L-
Sour 0.5g, X-Gal0.06g, Tween-80 1ml, agar 25g, distilled water 1000ml；Nitrogen source and carbon source is added, adjusting pH value is 7,
It sterilizes 10-20 minutes under 120-130 degrees Celsius；
Wherein nitrogen source: soy peptone 5g, tryptone 5g, casein hydrolysate 5g and beef extract 10g；Carbon source: Portugal
Grape sugar 5g, isomaltose 5g and lactose 5g；
Step 5: purifying culture: according to the characteristic of Bifidobacterium character such as thallus shape, color and anaerobism in step 3
Culture bacterium colony carry out scribing line partition method select, by the single colonie strain selected be seeded to step 4 preparation purifying culture solution
In temperature be 37-39 degrees Celsius of anaerobic condition under cultivate 20 hours；And from starting every 4 hours to bacterium colony progress light absorption value survey
Fixed and record.
Comparative example two:
Step 1: preparation sample liquid；It takes new fresh rabbit excrement 1g to be placed in 10ml peptone buffer agent, stirs, if
Determine centrifugal force 600-1500g, collects upper layer turbid after centrifugation five minutes；Appropriate peptone buffer agent, setting is added in upward turbid
Centrifugal force 2000-4000g collects upper layer turbid, as sample liquid after being centrifuged five minutes again；
Step 2: dilute sample liquid；Take 1ml sample liquid to be added in the peptone buffer agent of 9ml with decimal dilution method according to
It is secondary to be diluted, dilution gradient 10-1To 10-9；
Step 3: it is separately cultured；The dilution gradient for taking 0.1ml respectively is 10-4To 10-9Dilute sample liquid be placed in culture
On base plate, the dilute sample liquid of each dilution gradient does 3 Duplicate Samples；Under anaerobic, temperature is 36-39 degrees Celsius,
Culture is cultivated 5 hours in the case where moving to aerobic condition again after 45-50 hours；
Step 4: pure medium is prepared；Nitrogen source and carbon source optimizing are carried out to modified MRS culture medium；Optimization of C/C composites are as follows: ferment
Mother leaches powder 5g, ferric citrate 2g, sodium acetate 5g, disodium-hydrogen 2g, manganese sulfate 0.05g, half Guang ammonia of manganese chloride 0.5g, L-
Sour 0.5g, X-Gal0.06g, Tween-80 1ml, agar 25g, distilled water 1000ml；Nitrogen source and carbon source is added, adjusting pH value is 7,
It sterilizes 10-20 minutes under 120-130 degrees Celsius；
Wherein nitrogen source: soy peptone 5g, tryptone 10g, casein hydrolysate 5g and beef extract 5g；Carbon source: Portugal
Grape sugar 5g, isomaltose 5g and lactose 10g；
Step 5: purifying culture: according to the characteristic of Bifidobacterium character such as thallus shape, color and anaerobism in step 3
Culture bacterium colony carry out scribing line partition method select, by the single colonie strain selected be seeded to step 4 preparation purifying culture solution
In temperature be 37-39 degrees Celsius of anaerobic condition under cultivate 20 hours；And from starting every 4 hours to bacterium colony progress light absorption value survey
Fixed and record.
Comparative example three:
Step 1: preparation sample liquid；It takes new fresh rabbit excrement 1g to be placed in 10ml peptone buffer agent, stirs, if
Determine centrifugal force 600-1500g, collects upper layer turbid after centrifugation five minutes；Appropriate peptone buffer agent, setting is added in upward turbid
Centrifugal force 2000-4000g collects upper layer turbid, as sample liquid after being centrifuged five minutes again；
Step 2: dilute sample liquid；Take 1ml sample liquid to be added in the peptone buffer agent of 9ml with decimal dilution method according to
It is secondary to be diluted, dilution gradient 10-1To 10-9；
Step 3: it is separately cultured；The dilution gradient for taking 0.1ml respectively is 10-4To 10-9Dilute sample liquid be placed in culture
On base plate, the dilute sample liquid of each dilution gradient does 3 Duplicate Samples；Under anaerobic, temperature is 36-39 degrees Celsius,
Culture is cultivated 5 hours in the case where moving to aerobic condition again after 45-50 hours；
Step 4: pure medium is prepared；Nitrogen source and carbon source optimizing are carried out to modified MRS culture medium；Optimization of C/C composites are as follows: ferment
Mother leaches powder 5g, ferric citrate 2g, sodium acetate 5g, disodium-hydrogen 2g, manganese sulfate 0.05g, half Guang ammonia of manganese chloride 0.5g, L-
Sour 0.5g, X-Gal0.06g, Tween-80 1ml, agar 25g, distilled water 1000ml；Nitrogen source and carbon source is added, adjusting pH value is 7,
It sterilizes 10-20 minutes under 120-130 degrees Celsius；
Wherein nitrogen source: soy peptone 5g, tryptone 5g, casein hydrolysate 10g and beef extract 5g；Carbon source: Portugal
Grape sugar 5g, isomaltose 10g and lactose 5g；
Step 5: purifying culture: according to the characteristic of Bifidobacterium character such as thallus shape, color and anaerobism in step 3
Culture bacterium colony carry out scribing line partition method select, by the single colonie strain selected be seeded to step 4 preparation purifying culture solution
In temperature be 37-39 degrees Celsius of anaerobic condition under cultivate 20 hours；And from starting every 4 hours to bacterium colony progress light absorption value survey
Fixed and record.
By carrying out different proportions to composition in nitrogen source and carbon source, the light absorption value surveyed is compared, statistics such as table
The comparison of one, bacterium colony light absorption value；
The comparison of one, bacterium colony light absorption value of table
Wherein nitrogen source component mass ratio is followed successively by soy peptone, tryptone, casein hydrolysate and beef extract；Carbon
Source component mass ratio is followed successively by glucose, isomaltose and lactose；
It can be seen that bacterium colony starts to increase rapidly before 12h by upper table, growth rate region is steady after 12h；Illustrate bacterium
Reach increased logarithmic phase when falling 12h, compare existing conventional medium culture, the pure medium after present invention optimization is effective
The incubation time for shortening Bifidobacterium improves the quantity of viable bacteria；As preferably the incubation time of purifying culture can be into
One step is preferably 12-13 hours, improves the efficiency of culture；
By being compared with comparative example one, comparative example two and comparative example three as can be seen that using the nitrogen in embodiment one
Source and carbon source component mass-energy density effectively improve the number of viable of bacterium colony, and growth rate is accelerated；Therefore in embodiment one
Nitrogen source and carbon source component mass ratio as most preferred selection.
In the description of this specification, the description of reference term " one embodiment ", " example ", " specific example " etc. means
Particular features, structures, materials, or characteristics described in conjunction with this embodiment or example are contained at least one implementation of the invention
In example or example.In the present specification, schematic expression of the above terms may not refer to the same embodiment or example.
Moreover, particular features, structures, materials, or characteristics described can be in any one or more of the embodiments or examples to close
Suitable mode combines.
Present invention disclosed above preferred embodiment is only intended to help to illustrate the present invention.There is no detailed for preferred embodiment
All details are described, are not limited the invention to the specific embodiments described.Obviously, according to the content of this specification,
It can make many modifications and variations.These embodiments are chosen and specifically described to this specification, is in order to better explain the present invention
Principle and practical application, so that skilled artisan be enable to better understand and utilize the present invention.The present invention is only
It is limited by claims and its full scope and equivalent.
Claims (6)
1. one kind separates Bifidobacterium and its purification process from rabbit excrement, which comprises the following steps:
Step 1: preparation sample liquid；It takes new fresh rabbit excrement to be placed in peptone buffer agent, stirs, a centrifugal treating
Upper layer turbid is collected afterwards；Appropriate peptone buffer agent is added in upward turbid, collects upper layer turbid, as sample after centrifugal treating again
Product liquid；
Step 2: dilute sample liquid；It takes a certain amount of sample liquid to be diluted with decimal dilution method using peptone buffer agent, dilutes
Gradient is 10-1To 10-9；
Step 3: it is separately cultured；Taking a certain amount of dilution gradient respectively is 10-4To 10-9Dilute sample liquid to be placed in culture medium flat
On plate, the dilute sample liquid of each dilution gradient does 2 to 3 Duplicate Samples；It is being moved to after being cultivated 45-50 hours under anaerobic condition
It is cultivated 5-7 hours under the conditions of oxygen；
Step 4: pure medium is prepared；Nitrogen source and carbon source optimizing are carried out to modified MRS culture medium；
Step 5: purifying culture；It is selected according to the bacterium colony of the culture in Bifidobacterium character pair step 3, the bacterium that will be selected
Kind is seeded in the purifying culture solution of step 4 preparation and carries out purifying culture.
2. one kind according to claim 1 separates Bifidobacterium and its purification process from rabbit excrement, which is characterized in that described
Rabbit excrement quality and peptone buffer agent volume ratio are 1:10 in step 1.
3. one kind according to claim 1 separates Bifidobacterium and its purification process from rabbit excrement, which is characterized in that described
Cultivation temperature is 36-39 degrees Celsius in step 3.
4. one kind according to claim 1 separates Bifidobacterium and its purification process from rabbit excrement, which is characterized in that described
Nitrogen source in step 4 are as follows: soy peptone, tryptone, casein hydrolysate and beef extract；Carbon source are as follows: glucose, different wheat
Bud sugar and lactose.
5. one kind according to claim 4 separates Bifidobacterium and its purification process from rabbit excrement, which is characterized in that described
Soy peptone, tryptone, casein hydrolysate and beef extract mass ratio are followed successively by 2:1:1:1；The glucose, different wheat
Bud sugar and lactose mass ratio are followed successively by 2:1:1.
6. one kind according to claim 1 separates Bifidobacterium and its purification process from rabbit excrement, which is characterized in that described
Condition of culture is to cultivate 10-16 hours in the case where temperature is 37-39 degrees Celsius of anaerobic condition in step 5.
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| CN201811056559.0A CN109136145A (en) | 2018-09-11 | 2018-09-11 | One kind separating Bifidobacterium and its purification process from rabbit excrement |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114774324A (en) * | 2022-05-10 | 2022-07-22 | 安徽农业大学 | Method for optimizing and separating probiotics |
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2018
- 2018-09-11 CN CN201811056559.0A patent/CN109136145A/en not_active Withdrawn
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114774324A (en) * | 2022-05-10 | 2022-07-22 | 安徽农业大学 | Method for optimizing and separating probiotics |
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