CN101715908A - Method for improving efficiency of removing microcystin by probiotics - Google Patents
Method for improving efficiency of removing microcystin by probiotics Download PDFInfo
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- CN101715908A CN101715908A CN200910250614A CN200910250614A CN101715908A CN 101715908 A CN101715908 A CN 101715908A CN 200910250614 A CN200910250614 A CN 200910250614A CN 200910250614 A CN200910250614 A CN 200910250614A CN 101715908 A CN101715908 A CN 101715908A
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- probiotics
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Abstract
The invention relates to a method for improving efficiency of removing microcystin by probiotics, which belongs to the technical field of food organisms. The fermentation liquor which is abundant in probiotics is prepared by adopting the probiotics as a biological catalyst through liquid deep fermentation or the fermentation liquor is refined to obtain bacterial mud; the obtained probiotic fermentation liquor or the bacterial mud is charged into water; meanwhile, glucose as an external additive is placed to carry out vibration culture at 37 DEG C for 24-30 hours by 150rpm, and the removing efficiency of the probiotics on the microcystin is obviously improved. Because the glucose and the probiotics have no harm to the safety of humans and livestock and have the health effect, the invention can be widely applied to the field of foods, and a healthy and safe product with rich nutrient components is obtained.
Description
Technical field
A kind of method that improves efficiency of removing microcystin by probiotics belongs to technical field of food biotechnology.
Background technology
(Microcystins MCs) is a kind of ring-type seven peptide materials that produced by blue-green algaes such as the microcystic aeruginosa in the eutrophication water, wawter bloom anabena, beads algaes to Microcystin.Microcystin is the effect target organ with the animal's liver, thereby the CKIs phosphatase activity brings out a series of pathologies such as cancer specifically.Epidemiology survey shows that the incidence of disease of primary carcinoma of liver has very big correlation among the pollution of the Microcystin in the drinking water and the crowd.
The sweep-out method of Microcystin mainly contains chemical oxidation removing, physisorphtion and bioanalysis at present, the microorganism that bioanalysis is removed the utilization of algae toxin mainly concentrates on Sphingol single-cell, pseudomonas aeruginosa and acide eating Darfot bacteria, but above-mentioned bacterial classification all is not suitable for being applied to remove the Microcystin in the food system.
Probio is an important physical bacterium among the human intestine, have antitumor, alleviate lactose intolerance, strengthen immunity, reduce cholesterol, adjust physiological actions such as gut flora, this research department has obtained good effect with the algae toxin that probio is used for removing food system, but compare with other bacterial classification, probio is also lower to the elimination efficiency of Microcystin, presses for the efficient that improves removing microcystin by probiotics.
Summary of the invention
Technical problem to be solved by this invention provides a kind of method that improves efficiency of removing microcystin by probiotics.
In order to solve the problems of the technologies described above, the present invention proposes following technical scheme:
The present invention adopts probio as biocatalyst, be rich in the zymotic fluid of probio or with the refining bacterium mud that obtains of zymotic fluid by liquid deep layer fermenting preparation, gained probiotics fermention liquid or bacterium mud are dropped in the water body, drop into glucose as adjuvant, 37 ℃, 150rpm shaken cultivation 24-30h.
In the described method, probio is Lactobacillus salivarius (Lactobacillus salivarius ATCC 29602) or lactobacillus fermenti (Lactobacillus fermentum ATCC 9338).
In the described method, the cultural method of probio is:
Cultivate and adopt the MRS culture medium, the MRS culture medium consists of: peptone 10g, beef extract 10g, dusty yeast 5g, K
2HPO
42g, dibasic ammonium citrate 2g, sodium acetate 5g, glucose 20g, Tween 80 1mL, MgSO
47H
2O 0.58g, MnSO
44H
2O 0.25g, distilled water 1000mL;
Condition of culture is as follows: get the immigration of 2-3 ring probio from well-grown flat board and fill the little centrifuge tube of the aseptic glycerine of 0.5ml (glycerol concentration is 15%-20%), the back that closes the lid is put-80 ℃ of refrigerators and is preserved with sealing the film sealing orifice.Glycerine is guaranteed the probio access MRS fluid nutrient medium of Tibetan, leave standstill at 37 ℃ and cultivate 20h to the logarithm middle and later periods.
In the described method, the process for purification of bacterium mud is as follows: the probiotics fermention liquid of the middle and later periods in growth period of taking the logarithm, and 4 ℃, the centrifugal 10min collection of 3200rpm bacterium mud, bacterium mud is collected thalline through centrifugal 2 times of phosphate buffer (pH 7.0) washing.
In the described method, the making time of glucose is: glucose and probio are dropped in the water body synchronously.
In the described method, the probio counting adopts viable bacteria counting method, and bacteria suspension is diluted certain multiple, is coated with on flat board, calculates clump count.
In the described method, the method for efficient liquid phase is adopted in the detection of Microcystin:
Chromatographic column: Agilent ZORBAX Eclipse XDB-C18 (4.6 * 150mm, 5.0 μ m)
Phase flows: acetonitrile: H
2O (0.05%TFA)=40: 60 (v/v)
Sample size: 20 μ l; Flow velocity: 1mL/min; Column temperature: 40 ℃
Detector: DAD 238nm; MC-LR retention time: 3.48min
The present invention adopts the method for adding glucose to improve the efficient of removing microcystin by probiotics, has improved the effect of probio in field of food, has solved in the food residual Microcystin to the problem of health hazard.Adopt the present invention to handle the product that obtains, have advantages such as nutritional labeling is abundant, Microcystin content is low, be particularly suitable for removing the Microcystin in the aquatic products.
The specific embodiment
Embodiment 1
The fermenting and producing of Lactobacillus salivarius:
Lactobacillus salivarius (Lactobacillus salivarius ATCC 29602) adopts the MRS culture medium: peptone 10g, beef extract 10g, dusty yeast 5g, K
2HPO
42g, dibasic ammonium citrate 2g, sodium acetate 5g, glucose 20g, Tween 80 1mL, MgSO
47H
2O 0.58g, MnSO
44H
2O 0.25g, distilled water 1000mL.
The cultural method of Lactobacillus salivarius is as follows: get the immigration of 2-3 ring Lactobacillus salivarius from well-grown flat board and fill the little centrifuge tube of the aseptic glycerine of 0.5ml (glycerol concentration is 15%-20%), the back that closes the lid is put-20 ℃ of refrigerators and is preserved with sealing the film sealing orifice.The Lactobacillus salivarius of glycerine being guaranteed the Tibetan inserts the MRS fluid nutrient medium, leaves standstill at 37 ℃ and cultivates 20h to the logarithm middle and later periods.
Making with extra care of Lactobacillus salivarius bacterium mud:
The take the logarithm Lactobacillus salivarius zymotic fluid of middle and later periods in growth period, 4 ℃, the centrifugal 10min of 3200rpm are collected bacterium mud, and bacterium mud obtains Lactobacillus salivarius bacterium mud through centrifugal 2 times of phosphate buffer (pH 7.0) washing.
Glucose improves the method that Lactobacillus salivarius is removed algae toxin efficient:
The counting of Lactobacillus salivarius adopts viable bacteria counting method, and bacteria suspension is diluted certain multiple, is coated with on flat board, calculates clump count.
With 10
9The lactobacillus fermenti input of CFU/mL contains in the water body of Microcystin of 150 μ g/L (pH 7.0), adds the glucose of 10% (w/v), and 37 ℃, 150rpm vibration are placed 30h, Microcystin clearance rate 100%.
Embodiment 2
The fermenting and producing of Lactobacillus salivarius:
With embodiment 1
Making with extra care of Lactobacillus salivarius bacterium mud:
With embodiment 1
Glucose improves the method that Lactobacillus salivarius is removed algae toxin efficient:
The counting of Lactobacillus salivarius adopts viable bacteria counting method, and bacteria suspension is diluted certain multiple, is coated with on flat board, calculates clump count.
With 10
10The lactobacillus fermenti input of CFU/mL contains in the water body of Microcystin of 1800 μ g/L (pH 7.0), adds the glucose of 10% (w/v), 37 ℃, 150rpm shaken cultivation 24h, Microcystin clearance rate 84.8%.
Embodiment 3
The fermenting and producing of lactobacillus fermenti:
Lactobacillus fermenti (Lactobacillus fermentum ATCC 9338) adopts the MRS culture medium: peptone 10g, beef extract 10g, dusty yeast 5g, K
2HPO
42g, dibasic ammonium citrate 2g, sodium acetate 5g, glucose 20g, Tween 80 1mL, MgSO
47H
2O 0.58g, MnSO
44H
2O 0.25g, distilled water 1000mL.
The cultural method of lactobacillus fermenti is as follows: get the immigration of 2-3 environment-development kefir milk bacillus from well-grown flat board and fill the little centrifuge tube of the aseptic glycerine of 0.5ml (glycerol concentration is 15%-20%), the back that closes the lid is put-20 ℃ of refrigerators and is preserved with sealing the film sealing orifice.The lactobacillus fermenti of glycerine being guaranteed the Tibetan inserts the MRS fluid nutrient medium, leaves standstill at 37 ℃ and cultivates 20h to the logarithm middle and later periods.
Making with extra care of lactobacillus fermenti bacterium mud:
The take the logarithm lactobacillus fermenti zymotic fluid of middle and later periods in growth period, 4 ℃, the centrifugal 10min of 3200rpm are collected bacterium mud, and bacterium mud obtains lactobacillus fermenti bacterium mud through centrifugal 2 times of phosphate buffer (pH 7.0) washing.
Glucose improves the method that lactobacillus fermenti is removed algae toxin efficient:
The counting of lactobacillus fermenti adopts viable bacteria counting method, and bacteria suspension is diluted certain multiple, is coated with on flat board, calculates clump count.
With 10
9The lactobacillus fermenti input of CFU/mL contains in the water body of Microcystin of 150 μ g/L (pH 7.0), adds the glucose of 10% (w/v), and 37 ℃, 150rpm vibration are placed 30h, Microcystin clearance rate 100%.
Embodiment 4
The fermenting and producing of lactobacillus fermenti:
With embodiment 3
Making with extra care of lactobacillus fermenti bacterium mud:
With embodiment 3
Glucose improves the method that lactobacillus fermenti is removed algae toxin efficient:
The counting of lactobacillus fermenti adopts viable bacteria counting method, and bacteria suspension is diluted certain multiple, is coated with on flat board, calculates clump count.
With 10
10The lactobacillus fermenti input of CFU/mL contains in the water body of Microcystin of 1800 μ g/L (pH 7.0), adds the glucose of 10% (w/v), and 37 ℃, 150rpm vibration are placed 24h, Microcystin clearance rate 79.9%.
The comparative example 1
The fermenting and producing of Lactobacillus salivarius:
With embodiment 1
Making with extra care of Lactobacillus salivarius bacterium mud:
With embodiment 1
The application of Lactobacillus salivarius bacterium mud:
The counting of Lactobacillus salivarius adopts viable bacteria counting method, and bacteria suspension is diluted certain multiple, is coated with on flat board, calculates clump count.
With 10
9The Lactobacillus salivarius input of CFU/mL contains in the water body of Microcystin of 150 μ g/L (pH 7.0), and 37 ℃, 150rpm vibration are placed 30h, Microcystin clearance rate 41%.
The comparative example 2
The fermenting and producing of lactobacillus fermenti:
With embodiment 3
Making with extra care of lactobacillus fermenti bacterium mud:
With embodiment 3
The application of lactobacillus fermenti bacterium mud:
The counting of lactobacillus fermenti adopts viable bacteria counting method, and bacteria suspension is diluted certain multiple, is coated with on flat board, calculates clump count.
With 10
9The lactobacillus fermenti input of CFU/mL contains in the water of Microcystin of 150 μ g/L (pH 7.0), and 37 ℃, 150rpm vibration are placed 30h, Microcystin clearance rate 41%.
Be understandable that, for those of ordinary skills, can be equal to replacement or change according to technical scheme of the present invention and inventive concept thereof, and all these changes or replacement all should belong to the protection domain of the appended claim of the present invention.
Claims (5)
1. method that improves the Microcystin elimination efficiency, it is characterized in that, adopt probio as biocatalyst, be rich in the zymotic fluid of probio or with the refining bacterium mud that obtains of zymotic fluid by liquid deep layer fermenting preparation, gained probiotics fermention liquid or bacterium mud are dropped in the water body, drop into glucose as adjuvant, 37 ℃, 150rpm shaken cultivation 24-30h.
2. probio according to claim 1, it is characterized in that, adopt commercial Lactobacillus salivarius (Lactobacillussalivarius ATCC 29602) or lactobacillus fermenti (Lactobacillus fermentum ATCC 9338) as starting strain.
3. bacterium mud according to claim 1, it is characterized in that process for purification is: the probiotics fermention liquid of the middle and later periods in growth period of taking the logarithm, the centrifugal 10min of 3200rpm collects bacterium mud under the room temperature, bacterium mud is collected thalline through centrifugal 2 times of phosphate buffer (pH 7.0) washing.
4. probiotics fermention method according to claim 1 and 2 is characterized in that, the probiotic's culture method is as follows:
1) the used culture medium of probiotics fermention consists of: peptone 10g, beef extract 10g, dusty yeast 5g, K
2HPO
42g, dibasic ammonium citrate 2g, sodium acetate 5g, glucose 20g, Tween 80 1mL, MgSO
4.7H
2O 0.58g, MnSO
44H
2O 0.25g, distilled water 1000mL;
2) the probiotic's culture condition is as follows: get the immigration of 2-3 ring probio from well-grown flat board and fill the little centrifuge tube of the aseptic glycerine of 0.5ml (glycerol concentration is 15%-20%), the back that closes the lid is put-20 ℃ of refrigerators and is preserved with sealing the film sealing orifice.Glycerine is guaranteed the probio access MRS fluid nutrient medium of Tibetan, leave standstill at 37 ℃ and cultivate 20h to the logarithm middle and later periods.
5. glucose input method according to claim 1 is characterized in that glucose and probio are dropped in the water body synchronously.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102021127A (en) * | 2010-07-28 | 2011-04-20 | 江南大学 | Lactobacillus paracasei and application thereof |
| CN103146619A (en) * | 2013-03-21 | 2013-06-12 | 青岛蔚蓝天成生物科技有限公司 | Halomonas sulfidaeris and application thereof |
| CN104056595A (en) * | 2014-06-12 | 2014-09-24 | 众合发(北京)生物科技发展有限公司 | Degrader for toxins of alga in water body |
| CN105753170A (en) * | 2016-01-29 | 2016-07-13 | 江苏绿科生物技术有限公司 | Application of lactobacillus plantarum in inhibiting microcystic toxins |
| CN106176821A (en) * | 2016-08-31 | 2016-12-07 | 汪金小 | A kind of Tiny ecosystem vagina microbial inoculum and its preparation method and application |
| EP3351259A1 (en) * | 2017-01-18 | 2018-07-25 | Symrise AG | Probiotics for aggregation with disease-associated species in the oral cavity |
| US11020441B2 (en) | 2016-01-19 | 2021-06-01 | Symrise Ag | Probiotics for use as anti-inflammatory agents in the oral cavity |
| US11198848B2 (en) | 2016-01-19 | 2021-12-14 | Symrise Ag | Probiotics for altering the composition of oral biofilms |
-
2009
- 2009-12-11 CN CN200910250614A patent/CN101715908A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102021127A (en) * | 2010-07-28 | 2011-04-20 | 江南大学 | Lactobacillus paracasei and application thereof |
| CN103146619A (en) * | 2013-03-21 | 2013-06-12 | 青岛蔚蓝天成生物科技有限公司 | Halomonas sulfidaeris and application thereof |
| CN104056595A (en) * | 2014-06-12 | 2014-09-24 | 众合发(北京)生物科技发展有限公司 | Degrader for toxins of alga in water body |
| US11020441B2 (en) | 2016-01-19 | 2021-06-01 | Symrise Ag | Probiotics for use as anti-inflammatory agents in the oral cavity |
| US11198848B2 (en) | 2016-01-19 | 2021-12-14 | Symrise Ag | Probiotics for altering the composition of oral biofilms |
| CN105753170A (en) * | 2016-01-29 | 2016-07-13 | 江苏绿科生物技术有限公司 | Application of lactobacillus plantarum in inhibiting microcystic toxins |
| CN105753170B (en) * | 2016-01-29 | 2018-08-28 | 江苏绿科生物技术有限公司 | Application of the lactobacillus plantarum on inhibiting Microcystin |
| CN106176821A (en) * | 2016-08-31 | 2016-12-07 | 汪金小 | A kind of Tiny ecosystem vagina microbial inoculum and its preparation method and application |
| EP3351259A1 (en) * | 2017-01-18 | 2018-07-25 | Symrise AG | Probiotics for aggregation with disease-associated species in the oral cavity |
| WO2018134256A1 (en) * | 2017-01-18 | 2018-07-26 | Symrise Ag | Probiotics for aggregation with disease-associated species in the oral cavity |
| US11338001B2 (en) | 2017-01-18 | 2022-05-24 | Symrise Ag | Probiotics for aggregation with disease-associated species in the oral cavity |
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Application publication date: 20100602 |