CN101701034B - Method for preparing high-purity nisin - Google Patents
Method for preparing high-purity nisin Download PDFInfo
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- CN101701034B CN101701034B CN2009101529308A CN200910152930A CN101701034B CN 101701034 B CN101701034 B CN 101701034B CN 2009101529308 A CN2009101529308 A CN 2009101529308A CN 200910152930 A CN200910152930 A CN 200910152930A CN 101701034 B CN101701034 B CN 101701034B
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Abstract
The invention provides a method for preparing high-purity nisin, which comprises the steps of taking coarse powder of nisin as a raw material, dissolving, stirring, then standing, carrying out centrifugal phase separation, removing impurities, sampling, dissolving, carrying out the centrifugal phase separation again, removing the impurities, sampling, and drying, thereby preparing a product with the purity of more than 98%. The method has safe and simple technological method and high product purity, thereby being a good nisin purifying technology and being extensively used in the large-scale industrial production process.
Description
Technical field
The present invention relates to a kind of high purity biological preservative, specifically, relate to a kind of preparation method of high-purity nisin.
Background technology
Nisin is claimed nisin again; English name Nisin; Some streptococcus acidi lactici that is N type serum synthesizes the little peptide that has very strong germicidal action with excretory in metabolic process; Can effectively suppress many growth, breedings that cause the gram-positive microorganism of food spoilage, especially heat-resistant bacillus, Clostridium botulinum and listeria spp had had strong inhibitory effects, be a kind of efficient, nontoxic, safely, the antiseptics for natural food that has no side effect.
At present, nisin has been widely used in food, but purity is generally lower, and the ubiquity process step is many again to the purification of nisin, and purge process is complicated, the big and not high shortcoming of purity of equipment input.
Summary of the invention
Of the present invention being solves the problem that purge process is complicated, purity is not high that prior art exists, and a kind of preparation method of high-purity nisin is provided.
In order to realize the object of the invention, the preparation method of a kind of high-purity nisin of the present invention comprises the steps:
1) earlier the nisin meal is dissolved in the mixed solution of acid solution, organic solvent or acid and organic solvent;
2) stir then leave standstill, centrifugal phase-splitting, extracting lactic acid streptostacin layer;
3) again the nisin layer is used organic solvent dissolution, the recentrifuge phase-splitting, impurity elimination, drying forms.
Wherein, the content of nisin is 5-10% in the said nisin meal.
The pH of said acid solution is 2-6, is generally hydrochloric acid, acetic acid, sulfuric acid etc.
Said organic solvent can be: alcohols, ethers or carboxylic-acid, and such as ethanol, ether, acetate.
The ratio of said nisin meal and its lysate is 1: 10-20.
Can add salt when step 2) stirring and make solution saturated, fully stirring and dissolving leaves standstill.Churning time is 1-3 hour, and time of repose is 6-16 hour.The ratio of salt and nisin meal is 0.5-2: 1.
It is 2000-10000 rev/min that centrifugal rotational speed is adopted in said centrifugal phase-splitting, and the time is 5-40 minute.The whole mixed solution in centrifugal back is divided into four layers, is respectively light phase, nisin layer, heavy phase and impurity layer, removes residue (a large amount of impurity) and weight phase, extracting lactic acid streptostacin layer.
Dissolve nisin, centrifugal phase-splitting, impurity elimination, drying with the used solution of step 1) in the step 3).
Said drying means can be:
Spraying drying: get the deposition after centrifugal, with the solution dissolving, stir, spraying drying should constantly stir in the spray process and guarantees to mix, and promptly obtains highly purified nisin dry powder after the spraying drying.
Vacuum-drying: get the deposition after centrifugal,, drain the final vacuum drying with solution dissolving back suction filtration, dry accomplish to pulverize promptly obtain highly purified nisin dry powder.
Described solution is alcohols, ethers, carboxylic-acid organic solvent when dry.
The preparation method of high-purity nisin of the present invention, its process step is simple, and required equipment is less, and products obtained therefrom purity reaches more than 98%.
Safe in process of the present invention, easy, product purity is high, is a good nisin purification technique, can be widely used in the large-scale industrial production process.
Embodiment
Further describe the present invention with embodiment below, help understanding, but said embodiment only is used to explain the present invention rather than restriction the present invention the present invention and advantage thereof, better effects if.
Embodiment 1
Get Nisin bullion (saltout after the wet-milling of little filter press gained, the nisin weight percentage is about 7%) 300 grams, pulverize the back and add 4 liters of hydrochloric acid (pH is 2) and 2 liters of ethanol, fully stirred 1 hour.Slowly add 400 gram refined salt stirring and dissolving and make solution saturated, left standstill 6 hours.Centrifugal phase-splitting (rotating speed is 2000rpm) 40 minutes, the whole mixed solution in centrifugal back is divided into four layers, is respectively light phase (mainly being ethanol), nisin layer, heavy phase and impurity layer; Remove residue (a large amount of impurity) and weight phase, extracting lactic acid streptostacin layer, the Nisin layer is pearl; After adding 2 liters of ethanol stirring and evenly mixings; Liquid is removed in recentrifuge phase-splitting (rotating speed is 2000rpm, 40 minutes), gets the beds of precipitation; This moment, the Nisin layer was creamy white, and was not that oyster white needs centrifugal repeatedly phase-splitting to be creamy white until color like color.Carry out spraying drying after 4 liters of ethanol of taking precipitate adding stir, the stirring that in spray-drying process, should not stop mixes it, promptly obtains highly purified nisin product after the spraying drying.
Be 39394IU/mg through tiring of biological assay the finished product, be 40000IU/mg, but to invent the nisin product purity that used method makes be 98.49% to knowledge capital as the tiring of pure article of mark article.
Embodiment 2
Get Nisin bullion (saltout after the wet-milling of little filter press gained, the streptococcus acidi lactici cellulose content is about 10%) 300 grams, pulverize the back and add 2 liters of hydrochloric acid (pH is 6) and 1 liter of acetic acid, fully stirred 3 hours.Slowly add 600 gram refined salt stirring and dissolving and make solution saturated, left standstill 10 hours.Centrifugal phase-splitting (rotating speed is 5000rpm) 10 minutes, the whole mixed solution in centrifugal back is divided into four layers, is respectively light phase, nisin layer, heavy phase and impurity layer; Remove residue (a large amount of impurity) and weight phase; Extracting lactic acid streptostacin layer adds behind 2 liters of acetic acid stirring and evenly mixings the recentrifuge phase-splitting and removes liquid, and the beds of precipitation are the Nisin layer and get the beds of precipitation; This moment, the Nisin layer was creamy white, and was not that oyster white needs centrifugal repeatedly phase-splitting to be creamy white until color like color.Suction filtration behind 4 liters of acetate dissolutions of taking precipitate adding is drained the final vacuum drying, and dry completion is pulverized and promptly obtained highly purified nisin dry powder.
Through the biological assay the finished product tire for: 39276IU/mg, the nisin product purity that the used method of the present invention makes is 98.19%.
Embodiment 3
Get Nisin bullion (saltout after the wet-milling of little filter press gained, the streptococcus acidi lactici cellulose content is about 9%) 300 grams, pulverize the back and add 1 liter of hydrochloric acid (pH is 4), soaks 2.5 liters of hydrochloric acid of adding and 2.5 liters of ethanol after 6 hours, fully stirred 2 hours.Slowly add 150 gram refined salt and stirred 2 hours, left standstill 16 hours.Whole mixed solution is divided into four layers, is respectively light phase (mainly being ethanol), Nisin layer, heavy phase and impurity layer.Extract out gently and the Nisin layer, residue (a large amount of impurity) and weight phase are removed in centrifugal phase-splitting (rotating speed is 10000rpm) 5 minutes, and centrifugal Nisin layer afterwards is pearl.Liquid is removed in the recentrifuge removal of impurities after adding 2 liters of ethanol stirring and evenly mixings, and the beds of precipitation are the Nisin layer and get the beds of precipitation, and this moment, the Nisin layer was creamy white, and is not that oyster white needs centrifugal repeatedly phase-splitting to be creamy white until color like color.Add 4 liters of ethanol and stir, get 2 liters and carry out spraying drying, the stirring that in spray-drying process, should not stop makes its mixing.Other 2 liters are carried out suction filtration, drain back 60 ℃ of vacuum-dryings 12 hours, to be measured after pulverizing.
Spray-dired condition: inlet temperature 190-200 ℃
Temperature out 90-100 ℃
Shower nozzle rotating speed 5000rpm
Inlet amount 2L/h
Final spraying drying gets 8.5g, and biological assay is tired and is that 39294IU/mg, nisin product purity are 98.24%.
Vacuum drying 8.3g, biological assay is tired and is that 39376IU/mg, nisin product purity are 98.44%.
Although the present invention has been done detailed explanation and quoted some specific exampless as proof, for a person skilled in the art, only otherwise leave the spirit and scope of the present invention, it is obvious doing various variations or correction.
Claims (4)
1. the preparation method of a high-purity nisin is characterized in that, it comprises the steps:
1) earlier the nisin meal is dissolved in the mixed solution of acid solution, organic solvent or acid and organic solvent; The ratio of said nisin meal and its lysate is 1: 10-20; Said acid solution is hydrochloric acid, acetic acid or sulfuric acid, and said organic solvent is an ethanol;
2) add salt then and make solution saturated, churning time is 1-3 hour, and time of repose is 6-16 hour, and the ratio of salt and nisin meal is 0.5-2: 1, and centrifugal phase-splitting, extracting lactic acid streptostacin layer;
3) again the nisin layer is used dissolve with ethanol, the recentrifuge phase-splitting, impurity elimination, drying forms.
2. the described method of claim 1 is characterized in that, the pH of acid solution described in the step 1) is 2-6.
3. method according to claim 1 and 2 is characterized in that, it is 2000-10000 rev/min that centrifugal rotational speed is adopted in said centrifugal phase-splitting, and the time is 5-40 minute.
4. method according to claim 1 is characterized in that, said drying is spraying drying or vacuum-drying.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101529308A CN101701034B (en) | 2009-09-22 | 2009-09-22 | Method for preparing high-purity nisin |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101529308A CN101701034B (en) | 2009-09-22 | 2009-09-22 | Method for preparing high-purity nisin |
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| Publication Number | Publication Date |
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| CN101701034A CN101701034A (en) | 2010-05-05 |
| CN101701034B true CN101701034B (en) | 2012-06-06 |
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| CN2009101529308A Expired - Fee Related CN101701034B (en) | 2009-09-22 | 2009-09-22 | Method for preparing high-purity nisin |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102304172B (en) * | 2011-08-12 | 2013-06-05 | 郑州奇泓生物科技有限公司 | Nisin extraction method |
| CN113527447A (en) * | 2021-07-21 | 2021-10-22 | 山东元泰生物工程有限公司 | Extraction method of nisin |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1293201A (en) * | 2000-10-18 | 2001-05-02 | 中国科学院微生物研究所 | Process for separating and extracting nisin from fermented liquid |
| CN1743339A (en) * | 2004-08-31 | 2006-03-08 | 天津康益生物工程有限公司 | Nisin separating and preparing method |
| CN1876674A (en) * | 2005-06-07 | 2006-12-13 | 上海高科联合生物技术研发有限公司 | Natural preservative industrialized purification process |
-
2009
- 2009-09-22 CN CN2009101529308A patent/CN101701034B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1293201A (en) * | 2000-10-18 | 2001-05-02 | 中国科学院微生物研究所 | Process for separating and extracting nisin from fermented liquid |
| CN1743339A (en) * | 2004-08-31 | 2006-03-08 | 天津康益生物工程有限公司 | Nisin separating and preparing method |
| CN1876674A (en) * | 2005-06-07 | 2006-12-13 | 上海高科联合生物技术研发有限公司 | Natural preservative industrialized purification process |
Non-Patent Citations (2)
| Title |
|---|
| Cheeseman G C et.al..An improved Method of Preparing Nisin.《Biochem J》.1957,第65卷(第3期), * |
| 李安林 等.水- 乙醇- 硫酸铵体系及在分离提取天然产物中的应用.《安徽农业科学》.2007,第35卷(第12期), * |
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