CN102304172B - Nisin extraction method - Google Patents
Nisin extraction method Download PDFInfo
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- CN102304172B CN102304172B CN 201110230940 CN201110230940A CN102304172B CN 102304172 B CN102304172 B CN 102304172B CN 201110230940 CN201110230940 CN 201110230940 CN 201110230940 A CN201110230940 A CN 201110230940A CN 102304172 B CN102304172 B CN 102304172B
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Abstract
The invention relates to a nisin extraction method which is characterized by comprising the following steps: (1) adjusting the pH value of a nisin fermentation concentrated solution to 1.7-3.0, and mixing the nisin fermentation concentrated solution with a water-microsoluble organic solvent or water-insoluble organic solvent, wherein the organic solvent is any one of dichloromethane, n-butyl alcohol, ethyl acetate and carbon tetrachloride, or a combination of any two; and (2) stirring, standing, carrying out solid-phase separation to obtain a nisin wet cake, and drying while depressurizing in vacuum to obtain the nisin product. The invention has the following main advantage: the nisin is precipitated by means of the organic solvent, thereby overcoming the defects of the existing process such as high salt content and low product purity, and ensuring that the product purity of nisin is higher than 50%. The method is safe, convenient, practical and low in cost.
Description
Technical field
The present invention relates to a kind of preparation method of antiseptics for natural food, specifically a kind of from streptococcus acidi lactici or Lactococcus lactis Fermented Condensed liquid the method for separation and Extraction nisin.
Background technology
Nisin claims again Nisin or nisin, English name Nisin, it is the polypeptide with very strong germicidal action that is produced in metabolic process by streptococcus acidi lactici (Streptococcus lactis) or Lactococcus lactis (Lactococcus lactis), primary structure is comprised of 34 amino acid, and molecular weight is about 3510Da.Nisin is a kind of antiseptics for natural food, can suppress most of G such as Staphylococcus, streptococcus and bacillus
+The Growth and reproduction of bacterium and gemma thereof has the advantages such as germ resistance is strong, nontoxic to human body, good water solubility, Heat stability is good, has been widely used in the anti-corrosive fresh-keeping of milk-product, tinned pre-, meat product and beverage.
Since nineteen sixties, to extract the ways and means of nisin and set up successively from fermented liquid, its removal process mainly comprises the steps: fermented liquid acidifying, heating, (or spraying drying), vacuum-drying concentrate, saltout.Concentration method mainly contains: cryogenic vacuum method of enrichment, ultrafiltration and concentration method, foam concentrate, Adsorption Concentration.Absorption method adds solid particulate sorbent material absorbing lacto streptococus element in fermented liquid, after desorb, stripping liquid is saltoutd or spraying drying, makes Powdered nisin product; At first membrane filter method adopts Plate Filtration to carry out pre-treatment to fermented liquid, removes the solid substances such as thalline, and after filtrate was concentrated with ultra-filtration membrane, concentrated solution was saltoutd or spraying drying, makes the nisin powder.
Patent of invention " method of separating and extracting nisin from fermented liquid " (application number: 00129928, publication number: 1293201) adopt the sorbent material absorbing lacto streptococus elements such as diatomite, macroporous resin, with spraying drying after the acidic aqueous solution desorption of polyoxyethylene glycol, although the cost of sorbent material is cheap, but not also the most desirable, and desorption agent polyoxyethylene glycol is difficult to remove, and can enter the nisin finished product, affects product purity.
Patent of invention " method of separating lactic acid streptostacin from the Lactococcus lactis fermented liquid " (application number: 200510066481.7, publication number: 1687110) adopt membrane filtering method, remove thalline and the solid equimolecular quantity material larger than nisin molecular weight with mineral membrane or tubular membrane from fermented liquid; Remove the little material of molecular weight ratio nisin molecular weight from fermented liquid with the rolled film ultrafiltration, the concentrate of fermentation liquid that obtains adds solid salt, and after spraying drying, salt content is 10-50%.
Patent of invention " a kind of industrialized purification process of natural antiseptic agent " (Granted publication CN 100554276C) adopts the ion exchange layer analysis method to obtain high-purity nisin, and the method does not also possess industrial utility value because cost of investment, running cost are higher.
Patent of invention " a kind of preparation method of nisin " (Granted publication number: 101113463B) adopt ultrafiltration and concentration, the concentrated two kinds of concentration techniques of foam to complete the concentrated of fermented liquid, adopt at last spray drying technology to obtain the nisin product, still do not solve the low problem of product purity.
At present, the nisin purifying technique mainly adopts saltouts and spray-dired mode, and in product, salts contg is high, and nisin purity is 20%, and extraction step is loaded down with trivial details, and purge process is complicated, has the defectives such as extraction cost height.
Summary of the invention
Purpose of the present invention is just in above-mentioned prior art, existing salts contg is high, extraction process is complicated and the extracting method of a kind of new nisin of the defective of product purity the and special development.
The objective of the invention is to be achieved through the following technical solutions:
The extracting method of nisin of the present invention comprises the following steps:
(1) with the nisin fermentation concentrated solution, transferring pH with hydrochloric acid is 1.7-3.0, mix with water microsolubility organic solvent or water-insoluble organic solvents, the volume ratio of organic solvent used and nisin fermentation concentrated solution is 1: 5-1: 1, described organic solvent is any one in methylene dichloride, propyl carbinol, ethyl acetate, tetracol phenixin, or any two kinds of combinations; When organic solvent was two kinds of combinations, two kinds of solvent volume ratios were: 1: 1-1: 10.
(2) stirring is standing, and solid phase is separated, and gets the wet cake of nisin, and vacuum decompression is dry in the baking oven of 40 ℃-60 ℃, namely gets the nisin product.
In the present invention, the concentrated solution that the nisin fermentation concentrated solution is that lactic bacteria streptococcus element fermented liquid that fermentation is completed adopts that cryogenic vacuum is concentrated, ultrafiltration and concentration, foam are concentrated, method Adsorption Concentration is removed thalline and obtain.
Service temperature in whole leaching process is 4-50 ℃.
Major advantage of the present invention is to adopt the organic solvent deposit nisin, has overcome the defective that existing technique salts contg is high, product purity is low, and the nisin product purity is higher than 50%.Method safety is easy, practical, cost is low.
Embodiment
Embodiment one:
In the present embodiment, nisin extracts according to the following steps:
(1) transfer pH: (desorb after thalline absorption makes after the filtering thalline extracting lactic acid streptostacin concentrated broth, and tiring is 51.8 ten thousand/ml) 1L, transfers pH to 2.0 with hydrochloric acid.
(2) precipitation: add the 1L methylene dichloride in above-mentioned nisin concentrated solution, 20 ℃ are fully stirred, and isolate the upper strata water after standing, and water is mixed with lower batch fermentation liquid, again concentrate; Lower floor's solid-liquid separation reclaims methylene dichloride, gets the wet cake of nisin;
(3) drying: with the wet cake of nisin vacuum decompression drying in 50 ℃ of baking ovens, namely get the nisin product of food grade after dried cake is pulverized.
The biological assay of the present embodiment nisin product is tired and is that 20321IU/mg, product purity are 50.80%.
Embodiment two:
(1) transfer pH: extracting lactic acid streptostacin concentrated broth (cryogenic vacuum is concentrated to be made, and tires 58.4 ten thousand) 1L, transfer pH to 1.7 with hydrochloric acid.
(2) precipitation: add the 0.5L propyl carbinol in above-mentioned nisin concentrated solution, 50 ℃ are fully stirred, and isolate lower floor's water after standing, and water is mixed with lower batch fermentation liquid, again concentrate; The upper strata solid-liquid separation reclaims propyl carbinol, gets the wet cake of nisin;
(3) drying: with the wet cake of nisin vacuum decompression drying in 60 ℃ of baking ovens, namely get the nisin product of food grade after dried cake is pulverized.
The biological assay of the present embodiment nisin product is tired and is that 26432IU/mg, product purity are 66.08%.
Embodiment three:
(1) transfer pH: extracting lactic acid streptostacin concentrated broth (after Plate Filtration, ultrafiltration and concentration makes, and tires 55.3 ten thousand) 1L, transfer pH to 3.0 with hydrochloric acid.
(2) precipitation: add the 0.2L tetracol phenixin in above-mentioned nisin concentrated solution, 30 ℃ are fully stirred, and standing rear separation upper strata water mixes water with lower batch fermentation liquid, again concentrate; Lower floor's solid-liquid separation reclaims tetracol phenixin, gets the wet cake of nisin;
(3) drying: with the wet cake of nisin vacuum decompression drying in 60 ℃ of baking ovens, namely get the nisin product of food grade after dried cake is pulverized.
The biological assay of the present embodiment nisin product is tired and is that 23081IU/mg, product purity are 57.70%.
Embodiment four:
(1) transfer pH: extracting lactic acid streptostacin concentrated broth (make after foam is concentrated, tire 31.7 ten thousand) 1L, transfer pH to 2.0 with hydrochloric acid.
(2) precipitation: add 0.5L methylene dichloride and 0.5L ethyl acetate in above-mentioned nisin concentrated solution, 10 ℃ are fully stirred, and standing rear water phase separated is mixed water with lower batch fermentation liquid, again concentrated; Organic solvent phase solid-liquid separation gets the wet cake of nisin;
(3) drying: with the wet cake of nisin vacuum decompression drying in 40 ℃ of baking ovens, namely get the nisin product of food grade after dried cake is pulverized.
The biological assay of the present embodiment nisin product is tired and is that 22932IU/mg, product purity are 57.33%.
Embodiment five:
(1) transfer pH: extracting lactic acid streptostacin concentrated broth (desorb after thalline absorption makes after the filtering thalline, tires 51.8 ten thousand) 1L, transfer pH to 2.0 with hydrochloric acid.
(2) precipitation: add 0.025L methylene dichloride and 0.25L propyl carbinol in above-mentioned nisin concentrated solution, 10 ℃ are fully stirred, and standing rear water phase separated is mixed water with lower batch fermentation liquid, again concentrated; Organic solvent phase solid-liquid separation gets the wet cake of nisin;
(3) drying: with the wet cake of nisin vacuum decompression drying in 40 ℃ of baking ovens, namely get the nisin product of food grade after dried cake is pulverized.
The biological assay of the present embodiment nisin product is tired and is that 26354IU/mg, product purity are 65.88%.
Claims (6)
1. the extracting method of a nisin, it is characterized in that: this extracting method comprises the following steps:
(1) with the nisin fermentation concentrated solution, accent pH is 1.7-3.0, mix with water microsolubility organic solvent or water-insoluble organic solvents, described organic solvent is any one in methylene dichloride, propyl carbinol, ethyl acetate, tetracol phenixin, or the combination of the combination of methylene dichloride and propyl carbinol, methylene dichloride and ethyl acetate; The concentrated solution that described nisin fermentation concentrated solution is that lactic bacteria streptococcus element fermented liquid that fermentation is completed adopts that cryogenic vacuum is concentrated, ultrafiltration and concentration, foam are concentrated, method Adsorption Concentration is removed thalline and obtain;
(2) stirring is standing, and solid phase is separated, and gets the wet cake of nisin, and vacuum decompression is dry, namely gets the nisin product.
2. the extracting method of nisin according to claim 1, it is characterized in that: the volume ratio of organic solvent used and nisin fermentation concentrated solution is 1: 5-1: 1.
3. the extracting method of nisin according to claim 1 is characterized in that: when organic solvent was two kinds of combinations, two kinds of solvent volume ratios were: 1: 1-1: 10.
4. the extracting method of nisin according to claim 1, it is characterized in that: the vacuum decompression drying process is to carry out in the baking oven of 40 ℃-60 ℃.
5. the extracting method of nisin according to claim 1, it is characterized in that: the acid of adjust pH is hydrochloric acid.
6. the extracting method of nisin according to claim 1 is characterized in that: tiring when the concentrated solution that adopts adsorption concentration method is 51.8 ten thousand/ml; Tiring when the concentrated solution that adopts the cryogenic vacuum concentration method is 58.4 ten thousand/ml; Tiring when the concentrated solution that adopts the ultrafiltration and concentration method is 55.3 ten thousand/ml; Tiring when the concentrated solution that adopts the foam concentration method is 31.7 ten thousand/ml.
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| CN 201110230940 CN102304172B (en) | 2011-08-12 | 2011-08-12 | Nisin extraction method |
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| CN 201110230940 CN102304172B (en) | 2011-08-12 | 2011-08-12 | Nisin extraction method |
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| CN104212858B (en) * | 2013-11-06 | 2017-03-22 | 西藏天虹科技股份有限责任公司 | Adsorption separation coupling fermentation process with high nisin yield, |
| CN104839855B (en) * | 2015-04-21 | 2018-07-27 | 山东福瑞达生物科技有限公司 | A kind of preparation method of liquid Nisin preservatives |
| CN106047966B (en) * | 2016-07-12 | 2019-06-28 | 汕头大学 | A kind of antibacterial peptide Nisin and its extracting method and application |
| CN107286226A (en) * | 2017-06-28 | 2017-10-24 | 浙江新银象生物工程有限公司 | The preparation method of high clarity and the high-titer nisin aqueous solution |
| CN107494718A (en) * | 2017-08-14 | 2017-12-22 | 佛山推启农业研究院（普通合伙） | A kind of method using the fresh-keeping hickory chick of nisin |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1587277A (en) * | 2004-07-08 | 2005-03-02 | 浙江大学 | Process for preparing collagen active polypeptide from avian animal tissue |
| CN101701034A (en) * | 2009-09-22 | 2010-05-05 | 浙江银象生物工程有限公司 | Method for preparing high-purity nisin |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1587277A (en) * | 2004-07-08 | 2005-03-02 | 浙江大学 | Process for preparing collagen active polypeptide from avian animal tissue |
| CN101701034A (en) * | 2009-09-22 | 2010-05-05 | 浙江银象生物工程有限公司 | Method for preparing high-purity nisin |
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