CN101776665A - HPLC detection method of nisin - Google Patents
HPLC detection method of nisin Download PDFInfo
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- CN101776665A CN101776665A CN200910045204A CN200910045204A CN101776665A CN 101776665 A CN101776665 A CN 101776665A CN 200910045204 A CN200910045204 A CN 200910045204A CN 200910045204 A CN200910045204 A CN 200910045204A CN 101776665 A CN101776665 A CN 101776665A
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- 108010053775 Nisin Proteins 0.000 title claims abstract description 32
- 238000001514 detection method Methods 0.000 title claims abstract description 32
- NVNLLIYOARQCIX-MSHCCFNRSA-N Nisin Chemical compound N1C(=O)[C@@H](CC(C)C)NC(=O)C(=C)NC(=O)[C@@H]([C@H](C)CC)NC(=O)[C@@H](NC(=O)C(=C/C)/NC(=O)[C@H](N)[C@H](C)CC)CSC[C@@H]1C(=O)N[C@@H]1C(=O)N2CCC[C@@H]2C(=O)NCC(=O)N[C@@H](C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(NCC(=O)N[C@H](C)C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCSC)C(=O)NCC(=O)N[C@H](CS[C@@H]2C)C(=O)N[C@H](CC(N)=O)C(=O)N[C@H](CCSC)C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(N[C@H](C)C(=O)N[C@@H]3C(=O)N[C@@H](C(N[C@H](CC=4NC=NC=4)C(=O)N[C@H](CS[C@@H]3C)C(=O)N[C@H](CO)C(=O)N[C@H]([C@H](C)CC)C(=O)N[C@H](CC=3NC=NC=3)C(=O)N[C@H](C(C)C)C(=O)NC(=C)C(=O)N[C@H](CCCCN)C(O)=O)=O)CS[C@@H]2C)=O)=O)CS[C@@H]1C NVNLLIYOARQCIX-MSHCCFNRSA-N 0.000 title claims abstract description 31
- 239000004309 nisin Substances 0.000 title claims abstract description 31
- 235000010297 nisin Nutrition 0.000 title claims abstract description 31
- 238000004128 high performance liquid chromatography Methods 0.000 title claims description 23
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 111
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 8
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 22
- 150000007524 organic acids Chemical class 0.000 claims description 9
- 235000014897 Streptococcus lactis Nutrition 0.000 claims description 8
- 238000000855 fermentation Methods 0.000 claims description 7
- 230000004151 fermentation Effects 0.000 claims description 7
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 4
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 3
- 235000019253 formic acid Nutrition 0.000 claims description 3
- 241000194035 Lactococcus lactis Species 0.000 claims 1
- 238000004811 liquid chromatography Methods 0.000 abstract 1
- 238000010829 isocratic elution Methods 0.000 description 10
- 229920001817 Agar Polymers 0.000 description 8
- 239000008272 agar Substances 0.000 description 8
- 238000000926 separation method Methods 0.000 description 8
- 244000057717 Streptococcus lactis Species 0.000 description 7
- 238000009792 diffusion process Methods 0.000 description 6
- 235000013305 food Nutrition 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000010828 elution Methods 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000013558 reference substance Substances 0.000 description 4
- 230000002421 anti-septic effect Effects 0.000 description 3
- 229940064004 antiseptic throat preparations Drugs 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 239000003643 water by type Substances 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 241000282461 Canis lupus Species 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 108010062877 Bacteriocins Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 241000194036 Lactococcus Species 0.000 description 1
- 101710115767 Lantibiotic nisin-Z Proteins 0.000 description 1
- 206010034133 Pathogen resistance Diseases 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 108010073771 Soybean Proteins Proteins 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- PQLVXDKIJBQVDF-UHFFFAOYSA-N acetic acid;hydrate Chemical compound O.CC(O)=O PQLVXDKIJBQVDF-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 108010027597 alpha-chymotrypsin Proteins 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000000680 avirulence Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
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- 239000006172 buffering agent Substances 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 235000020992 canned meat Nutrition 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
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- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 235000019985 fermented beverage Nutrition 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N ferric oxide Chemical compound O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 235000013622 meat product Nutrition 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 238000004848 nephelometry Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 235000013324 preserved food Nutrition 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 235000019710 soybean protein Nutrition 0.000 description 1
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- 239000005720 sucrose Substances 0.000 description 1
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Abstract
The invention discloses a High Perfomance Liquid Chromatography (HPLC) detection method of nisin, the flowing phase contains acetonitrile and water, the initial concentration of the acetonitrile is 5-25 vol.% while the final concentration thereof is 35-100 vol.%.
Description
Technical field
The present invention relates to the detection of target product in the fermentation liquor, relate in particular to a kind of high performance liquid chromatography (High Performance Liquid Chromatography, HPLC) detection method of nisin.
Background technology
Nisin (Nisin, claim galactococcus peptide or streptococcus lactis peptide again) be small-molecular peptides with strong bacteriostasis by streptococcus lactis (Lactococcus lactis ssp.lactis) synthetic and secretion in metabolic process, also be research at present at most, use one of wider bacteriocin.Nisin allows one of minority antiseptics for natural food of commercially producing at present in the world, the anti-corrosive fresh-keeping that is widely used in dairy products, fermented beverage, canned food, meat and meat products, it can effectively suppress to cause the gram-positive bacterium (G of food apoilage
+) and the breeding of pathogen.Compare with other antiseptics, Nisin is produced and responsive especially to proteinase by naturally occurring Lactococcus bacterium, is decomposed by alpha-chymotrypsin very soon in alimentary canal.Therefore it does not produce cross-resistance with medical microbiotic to the basic avirulence of human body yet, can degraded harmlessly in enteron aisle.Its structure is seen Fig. 1.
Nisin was at first found by Rogerst Whittier in nineteen twenty-eight.That realize suitability for industrialized production the earliest is Britain Aplin﹠amp; Berrett company, also to ratify Nisin in succession be antiseptics for natural food in more than 50 countries and regions such as the U.S., Britain subsequently.China starts from 1989 to the research of Nisin.That the bacterial classification L.laticsAL2 of China's realization suitability for industrialized production produces is NisinZ.Nineteen ninety, Nisin is put into the nineteen ninety of China GB GB2780-86 and augments in the product.
In early days, before proposition agar diffusion methods such as Tramer, what people generally adopted is test tube dilution technology and nephelometry (Turbidity Assay).Tramer in 1964 and Fowler have set up the basic skills of agar diffusion method, but because the molecular weight of nisin is bigger, and dissolubility is better under the acid condition, and solubleness is very little under neutral environment, spreads so be difficult in the agar.Wolf etc. further optimized agar diffusion method afterwards.They have optimum parameters: added buffering agent etc. in the concentration of the degree of depth of indicator bacteria, agar and concentration, Tween20, the nutrient culture media.Improving one's methods of Wolf etc. compared with the initial method of Tramer etc., degree of accuracy has improved 57%, accuracy has improved 12%, therefore the most frequently used method that is used for detecting food streptococcus lactis peptide content at present is an agar diffusion method (analytical control Food Science 2005, Vol.26, No.7175, cylinder-plate method is measured the improvement that nisin tires; Food Science 2007, Vol.28, No.03175, optimization of Agar Diffusion Bioassay for Nisin; External medical microbiotic fascicle the 25th the 5th phase of volume of September in 2004, the detection method of bacteriocin Nisin).
HPLC and agar diffusion method relatively have fast, accurately, and advantages such as good reproducibility.Existing HPLC technology is mentioned the post with RP-HPLC Hi-Pore RP-318 (BioRad), 250x10mm, using mobile phase A at 30 ℃ is that 10% (V/V) acetonitrile adds 0.1% trifluoroacetic acid, and Mobile phase B is that 90% acetonitrile adds 0.07% trifluoroacetic acid gradient elution.The initial concentration of Mobile phase B is that 25% termination concentration is 28%, and concentration is changed to (the Eur.J.Biochem.201 that increases progressively gradually in gradient elution, 581-584,1991, Identification and characterization of the lantibiotic nisin Z, anatural nisin variant).Time-histories with the method analytic sample is long, and nearly 1 hour, the gradient elution baseline wander caused the integration difficulty, sample determination result's reappearance, accuracy equal difference.The inventor does not have target peak by this method in the HPLC spectrum.
Therefore, this area presses for the HPLC method that a kind of working time weak point, baseline stability, degree of separation good detection nisin is provided.
Summary of the invention
The present invention aims to provide a kind of high performance liquid chromatogram detection method of nisin.
In the present invention, a kind of detection method of nisin is provided, described detection method is high performance liquid chromatography (High Performance Liquid Chromatography, HPLC), contain acetonitrile and water in its moving phase, the initial concentration of acetonitrile is 5-25vol.%, and ultimate density is 35-100vol.%.
In another preference, the acetonitrile in the moving phase was incremented to 35-100vol.% at 15-40 minute from 5-25vol.%.
In another preference, the acetonitrile in the moving phase was incremented to 35-80vol.% at 20-35 minute from 10-22vol.%.
In another preference, the acetonitrile in the moving phase was incremented to 50-70vol.% at 15-30 minute from 10-22vol.%, was incremented to 90-100vol.% at 5-10 minute from 50-70vol.% then.
In another preference, the acetonitrile in the moving phase was incremented to 50-70vol.% at 15-30 minute from 10-22vol.%, kept 50-70vol.% at 1-3 minute then, was incremented to 90-100vol.% at 5-10 minute from 50-70vol.% again.
In another preference, described method comprises step:
(1) acetonitrile in the described moving phase remained 5-25vol.% in 2-5 minute;
(2) acetonitrile in the described moving phase was incremented to 35-100vol.% at 15-40 minute from 5-25vol.%; With
(3) acetonitrile in the described moving phase was decremented to 5-25vol.% at 1-5 minute from 35-100vol.%.
In another preference, the acetonitrile described in the step (2) in the moving phase was incremented to 35-80vol.% at 20-35 minute from 10-22vol.%.
In another preference, the acetonitrile described in the step (2) in the moving phase was incremented to 50-70vol.% at 15-30 minute from 10-22vol.%, was incremented to 90-100vol.% at 5-10 minute from 50-70vol.% then.
In another preference, the acetonitrile described in the step (2) in the moving phase was incremented to 50-70vol.% at 15-30 minute from 10-22vol.%, kept 50-70vol.% at 1-3 minute then, was incremented to 90-100vol.% at 5-10 minute from 50-70vol.% again.
In another preference, the acetonitrile described in the step (1) in the moving phase remained 5-25vol.% in 2-5 minute.
In another preference, the acetonitrile described in the step (1) in the moving phase remained 10-22vol.% in 2-5 minute.
In another preference, in the preceding step that also comprises of step (1):
With streptococcus lactis fermentation liquor and mixed in hydrochloric acid, the centrifugal supernatant that obtains containing nisin.
In another preference, also contain the organic acid of 0.05-0.2vol.% in the moving phase of the HPLC that the present invention uses; More preferably, the organic acid that contains 0.08-0.12vol.%; Described organic acid is selected from formic acid, acetate, trifluoroacetic acid or butyric acid; Be preferably trifluoroacetic acid.
In another preference, described HPLC uses anti-phase C4 chromatographic column, and the detection wavelength is 200-220nm.
In view of the above, the invention provides the HPLC method of a kind of working time of weak point, baseline stability, degree of separation good detection nisin.
Description of drawings
Fig. 1 is the nisin structural drawing.
Fig. 2 is that the HPLC of Nisin reference substance (standard items) detects collection of illustrative plates.
Fig. 3 is that the HPLC of Nisin sample detects collection of illustrative plates.
Embodiment
The inventor is through extensive and deep research, by a large amount of tests, grope to have obtained a kind of nisin HPLC detection method.The inventor has found effective separation, the acetonitrile that detects nisin and the volume ratio of water, further, the inventor finds that also the volume ratio that makes acetonitrile and water in the regular hour progressively changes (promptly adopting certain gradient elution mode), can obtain better separation, detect effect.
The inventor uses anti-phase C4 chromatographic column, contains acetonitrile and water in the moving phase, and wherein the initial concentration of acetonitrile is 5-25vol.%, and it stops concentration is 35-100vol.%.The detection wavelength is 200-220nm.Can also contain a spot of organic acid in the moving phase, described organic acid is selected from formic acid, acetate, trifluoroacetic acid or butyric acid; Preferred trifluoroacetic acid.
In a preferred embodiment of the invention, contain mobile phase A and/or Mobile phase B in the moving phase; The volume ratio of water and trifluoroacetic acid is 100: 0.1 in the described mobile phase A, and the volume ratio of acetonitrile and trifluoroacetic acid is 100: 0.1 in the described Mobile phase B.Wherein the initial concentration of Mobile phase B is 5-25vol.%, and it stops concentration is 35-100vol.%.
In another preference, the percent by volume of the Mobile phase B in the described moving phase was incremented to 35-100vol.% at 15-40 minute from 5-25vol.%.
In another preference, the percent by volume of the Mobile phase B in the described moving phase was incremented to 35-80vol.% at 20-35 minute from 10-22vol.%.
In another preference, the percent by volume of the Mobile phase B in the described moving phase was incremented to 50-70vol.% at 15-30 minute from 10-22vol.%, was incremented to 90-100vol.% at 5-10 minute from 50-70vol.% then.
In another preference, the percent by volume of the Mobile phase B in the described moving phase was incremented to 50-70vol.% at 15-30 minute from 10-22vol.%, keep 50-70vol.% at 1-3 minute then, be incremented to 90-100vol.% at 5-10 minute from 50-70vol.% again.
In a preference of the present invention, described method comprises step:
(a) percent by volume of the Mobile phase B in the described moving phase remained 5-25vol.% in 2-5 minute;
(b) percent by volume of the Mobile phase B in the described moving phase was incremented to 35-100vol.% at 15-40 minute from 5-25vol.%; With
(c) percent by volume of the Mobile phase B in the described moving phase was decremented to 5-25vol.% at 1-5 minute from 35-100vol.%.
In another preference, the percent by volume of the Mobile phase B described in the step (b) in the moving phase was incremented to 35-80vol.% at 20-35 minute from 10-22vol.%.
In another preference, the percent by volume of the Mobile phase B described in the step (b) in the moving phase was incremented to 50-70vol.% at 15-30 minute from 10-22vol.%, was incremented to 90-100vol.% at 5-10 minute from 50-70vol.% then.
In another preference, the percent by volume of the Mobile phase B described in the step (b) in the moving phase was incremented to 50-70vol.% at 15-30 minute from 10-22vol.%, keep 50-70vol.% at 1-3 minute then, be incremented to 90-100vol.% at 5-10 minute from 50-70vol.% again.
In another preference, the percent by volume of the Mobile phase B described in the step (a) in the moving phase remained 10-22vol.% in 2-5 minute.
In another preference, the percent by volume of the Mobile phase B described in the step (a) in the moving phase remained 10-22vol.% in 2-5 minute.
In another preference, in the preceding step that also comprises of step (a):
With streptococcus lactis fermentation liquor and mixed in hydrochloric acid, the centrifugal supernatant that obtains containing nisin.
In a preferred embodiment of the invention, initial Mobile phase B is 5-22vol.%, more preferably is 20vol.%, isocratic elution 3 minutes; The Mobile phase B continuity is incremented to 40-70vol.% subsequently, more preferably is 40vol.%, and wash-out 12-30 minute more preferably is 27 minutes; Mobile phase B was got back to 20vol.%, balance in last 2 minutes.
The above-mentioned feature that the present invention mentions, or the feature that embodiment mentions can combination in any.All features that this case instructions is disclosed can with any composition forms and usefulness, each feature that is disclosed in the instructions can anyly provide the alternative characteristics of identical, impartial or similar purpose to replace.Therefore removing has special instruction, and the feature that is disclosed only is the general example of equalization or similar features.
Major advantage of the present invention is:
1, optimizes detection method, the theoretical cam curve at product peak is improved greatly, and appearance time shortens nearly one times, reagent dosage and manpower have obtained saving greatly, reach well with impurity peaks and to separate, really reach purpose fast and accurately thereby allow HPLC detect;
2, lacking working time, only is 30 minutes; Go out the peak early, just could in 16 minutes.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is usually according to the normal condition or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise all percentage, ratio, ratio or umber by weight.
Unit in the percent weight in volume among the present invention is well-known to those skilled in the art, for example is meant the weight of solute in 100 milliliters solution.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
Test condition in the embodiment of the invention is as follows:
Instrument and chromatographic condition:
Instrument: Waters company high performance liquid chromatograph (Waters 2996 diode array detector, Waters1525 binary HPLC pump)
Chromatographic column: Waters symmetryC4 (5 μ m, 3.9 * 250mm) posts numbering: 000286
Mobile phase A: double steaming solution adds 0.1% trifluoroacetic acid
Mobile phase B: second eyeball solution adds 0.1% trifluoroacetic acid
Flow velocity: 1m/min
Column temperature: 30 ℃
Detect wavelength: select for use 215nm as detecting wavelength.
The preparation of solution
Reference substance solution: precision takes by weighing the about 10mg of Nisin reference substance, puts in the 10ml volumetric flask, with 0.02NHCl dissolving and be diluted to scale, product solution in contrast.
Sample solution: scrape extracting lactic acid streptococcus (Lactococcus lactis ssp.lactis) on the inclined-plane, it is inserted seed liquor, and (prescription is soybean protein isolate 0.5%, peptone 0.5%, yeast extract 0.25%, beef extract 0.5%, glucose 0.5%, ascorbic acid 0.05%, Na
2HPO
412H
2O 1.0%, MgSO
47H
2O 0.012%, pH6.8) cultivates 20h for 37 ℃.Seed liquor inserted (culture medium prescription is: sucrose 2.0%, yeast extract 3.0%, peptone 2.0% in the fermentation liquor, magnesium sulphate 0.02%, potassium dihydrogen phosphate 1.0%, NaCl0.2%, Tween-80 0.5%, pH6.8) 30 ℃ fermented 12-14 hour, stopped fermentation, get a certain amount of Nisin fermentation liquor, transferring pH with 0.02NHCL is 2.0, high speed centrifugation 12000-14000rpm, 10 minutes, get supernatant with water film filtering after, as sample solution.
Initial Mobile phase B: mobile phase A=20: 80 isocratic elution 3 minutes, the Mobile phase B continuity increases progressively from 20% and is raised to 40% wash-out 27 minutes gradually subsequently, and Mobile phase B got back to 20%, balance in last 2 minutes.The reference substance retention time is 16.38min under the method, and degree of separation 1.08, and theoretical cam curve reaches 16738.See Fig. 2 and Fig. 3.
Embodiment 2
Initial Mobile phase B: mobile phase A=20: 80 isocratic elution 3 minutes, the Mobile phase B continuity increases progressively from 20% and is raised to 40% wash-out 12 minutes gradually subsequently, and Mobile phase B got back to 20%, balance in last 2 minutes.Appearance time is about 10min under the method, baseline wander, and theoretical cam curve is 12800.
Embodiment 3
Initial Mobile phase B: mobile phase A=20: 80 isocratic elution 3 minutes, the Mobile phase B continuity increases progressively from 20% and is raised to 60% wash-out 20 minutes gradually subsequently, Mobile phase B continues to increase progressively from 60% and rises to 80% wash-out 5 minutes subsequently, Mobile phase B kept 80% wash-out 2 minutes, Mobile phase B got back to 20%, balance in last 2 minutes.Appearance time is about 17min under the method, and degree of separation is better, and theoretical cam curve is 11600.
Embodiment 4
Initial Mobile phase B: mobile phase A=20: 80 isocratic elution 3 minutes, the Mobile phase B continuity increases progressively from 20% and is raised to 60% wash-out 12 minutes gradually subsequently, Mobile phase B continues to increase progressively from 60% and rises to 80% wash-out 2 minutes subsequently, Mobile phase B kept 80% wash-out 2 minutes, Mobile phase B got back to 20%, balance in last 2 minutes.Appearance time is about 9min under the method, baseline wander, and theoretical cam curve is 11000.
Embodiment 5
Initial Mobile phase B: mobile phase A=20: 80 isocratic elution 3 minutes, the Mobile phase B continuity increases progressively from 20% and is raised to 60% wash-out 23 minutes gradually subsequently, Mobile phase B continues to increase progressively from 60% and rises to 80% wash-out 5 minutes subsequently, Mobile phase B kept 80% wash-out 2 minutes, Mobile phase B got back to 20%, balance in last 2 minutes.Appearance time is about 18min under the method, and theoretical cam curve is 12000.
Embodiment 6
Initial Mobile phase B: mobile phase A=5: 95 isocratic elution 3 minutes, the Mobile phase B continuity increases progressively from 5% and is raised to 60% wash-out 27 minutes gradually subsequently, Mobile phase B: mobile phase A=60: 40 isocratic elution 5 minutes, Mobile phase B continues to increase progressively from 60% and rises to 100% wash-out 5 minutes subsequently, Mobile phase B kept 100% wash-out 5 minutes, Mobile phase B got back to 5% in last 2 minutes, and appearance time is about 25min under the method, and theoretical cam curve is 14100.
Embodiment 7
Initial Mobile phase B: mobile phase A=10: 90 isocratic elution 3 minutes, the Mobile phase B continuity increases progressively from 10% and is raised to 60% wash-out 27 minutes gradually subsequently, Mobile phase B continues to increase progressively from 60% and rises to 100% wash-out 5 minutes subsequently, Mobile phase B kept 100% wash-out 2 minutes, this moment, appearance time was about 20min, and degree of separation is better, and theoretical cam curve is 14800.
Embodiment 8
Initial Mobile phase B: mobile phase A=22: 78 isocratic elution 3 minutes, the Mobile phase B continuity increases progressively from 22% and is raised to 70% wash-out 27 minutes gradually subsequently, and Mobile phase B got back to 22%, balance in last 2 minutes.Appearance time is about 19min under the method, and baseline is steady and degree of separation is better, and theoretical cam curve is 15000.
The above only is preferred embodiment of the present invention, be not in order to limit essence technology contents scope of the present invention, essence technology contents of the present invention is broadly to be defined in the claim scope of application, any technology entity or method that other people finish, if it is defined identical with the claim scope of application, also or a kind of change of equivalence, all will be regarded as being covered by among this claim scope.
Claims (16)
1. the detection method of a nisin, it is characterized in that (HighPerformance Liquid Chromatography contains acetonitrile and water to high performance liquid chromatography in moving phase HPLC), the initial concentration of acetonitrile is 5-25vol.%, and ultimate density is 35-100vol.%.
2. detection method as claimed in claim 1 is characterized in that the acetonitrile in the moving phase was incremented to 35-100vol.% at 15-40 minute from 5-25vol.%.
3. detection method as claimed in claim 1 is characterized in that the acetonitrile in the moving phase was incremented to 35-80vol.% at 20-35 minute from 10-22vol.%.
4. detection method as claimed in claim 1 is characterized in that, the acetonitrile in the moving phase was incremented to 50-70vol.% at 15-30 minute from 10-22vol.%, was incremented to 90-100vol.% at 5-10 minute from 50-70vol.% then.
5. detection method as claimed in claim 1, it is characterized in that, acetonitrile in the moving phase was incremented to 50-70vol.% at 15-30 minute from 10-22vol.%, kept 50-70vol.% at 1-3 minute then, was incremented to 90-100vol.% at 5-10 minute from 50-70vol.% again.
6. detection method as claimed in claim 1 is characterized in that, described method comprises step:
(1) acetonitrile in the described moving phase remained 5-25vol.% in 2-5 minute;
(2) acetonitrile in the described moving phase was incremented to 35-100vol.% at 15-40 minute from 5-25vo1.%; With
(3) acetonitrile in the described moving phase was decremented to 5-25vol.% at 1-5 minute from 35-100vol.%.
7. detection method as claimed in claim 6 is characterized in that, the acetonitrile described in the step (2) in the moving phase was incremented to 35-80vol.% at 20-35 minute from 10-22vol.%.
8. detection method as claimed in claim 6 is characterized in that, the acetonitrile described in the step (2) in the moving phase was incremented to 50-70vol.% at 15-30 minute from 10-22vol.%, was incremented to 90-100vol.% at 5-10 minute from 50-70vol.% then.
9. detection method as claimed in claim 6, it is characterized in that, acetonitrile described in the step (2) in the moving phase was incremented to 50-70vol.% at 15-30 minute from 10-22vol.%, keep 50-70vol.% at 1-3 minute then, be incremented to 90-100vol.% at 5-10 minute from 50-70vol.% again.
10. detection method as claimed in claim 6 is characterized in that, the acetonitrile described in the step (1) in the moving phase remained 5-25vol.% in 2-5 minute.
11. detection method as claimed in claim 9 is characterized in that, the acetonitrile described in the step (1) in the moving phase remained 10-22vol.% in 2-5 minute.
12. detection method as claimed in claim 1 is characterized in that, also contains the organic acid of 0.05-0.2vol.% in the moving phase; Described organic acid is selected from formic acid, acetate, trifluoroacetic acid or butyric acid.
13. detection method as claimed in claim 12 is characterized in that, also contains the organic acid of 0.08-0.12vol.% in the moving phase.
14. detection method as claimed in claim 12 is characterized in that, described organic acid is a trifluoroacetic acid.
15. detection method as claimed in claim 1 is characterized in that, uses anti-phase C4 chromatographic column, the detection wavelength is 200-220nm.
16. detection method as claimed in claim 6 is characterized in that, in the preceding step that also comprises of step (1):
With streptococcus lactis fermentation liquor and mixed in hydrochloric acid, the centrifugal supernatant that obtains containing nisin.
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| CN112114062A (en) * | 2020-08-03 | 2020-12-22 | 河北圣雪大成制药有限责任公司 | Method for detecting nisin by high performance liquid chromatography |
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