Background technology
Nisin (Nisin, claim galactococcus peptide or streptococcus lactis peptide again) be the 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 present the most frequently used method for detection of streptococcus lactis peptide content in the food is 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), 250 * 10mm, 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 efficient liquid phase 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), it contains acetonitrile and water in flowing mutually, the initial concentration of acetonitrile is 5-25vol.%, and ultimate density is 35-100vol.%.
In another preference, the acetonitrile in flowing mutually was incremented to 35-100vol.% at 15-40 minute from 5-25vol.%.
In another preference, the acetonitrile in flowing mutually was incremented to 35-80vol.% at 20-35 minute from 10-22vol.%.
In another preference, the acetonitrile in flowing mutually 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 flowing mutually 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) described acetonitrile in flowing mutually remained 5-25vol.% in 2-5 minute;
(2) described acetonitrile in flowing mutually was incremented to 35-100vol.% at 15-40 minute from 5-25vol.%; With
(3) described acetonitrile in flowing mutually was decremented to 5-25vol.% at 1-5 minute from 35-100vol.%.
In another preference, the acetonitrile in flowing mutually described in the step (2) was incremented to 35-80vol.% at 20-35 minute from 10-22vol.%.
In another preference, the acetonitrile in flowing mutually described in the step (2) 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 flowing mutually described in the step (2) 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 in flowing mutually described in the step (1) remained 5-25vol.% in 2-5 minute.
In another preference, the acetonitrile in flowing mutually described in the step (1) 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, the HPLC that the present invention uses flow mutually in also contain the organic acid of 0.05-0.2vol.%; More preferably, the organic acid that contains 0.08-0.12vol.%; Described organic acid is selected from formic acid, acetic acid, 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.
Accordingly, the invention provides the HPLC method of a kind of working time of weak point, baseline stability, degree of separation good detection nisin.
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 (namely adopting certain gradient elution mode), can obtain better separation, detect effect.
The inventor uses anti-phase C4 chromatographic column, contains acetonitrile and water in flowing mutually, 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 flowing mutually, described organic acid is selected from formic acid, acetic acid, 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 flowing mutually; 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 described Mobile phase B in flowing mutually was incremented to 35-100vol.% at 15-40 minute from 5-25vol.%.
In another preference, the percent by volume of described Mobile phase B in flowing mutually was incremented to 35-80vol.% at 20-35 minute from 10-22vol.%.
In another preference, the percent by volume of described Mobile phase B in flowing mutually 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 described Mobile phase B in flowing mutually 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 described Mobile phase B in flowing mutually remained 5-25vol.% in 2-5 minute;
(b) percent by volume of described Mobile phase B in flowing mutually was incremented to 35-100vol.% at 15-40 minute from 5-25vol.%; With
(c) percent by volume of described Mobile phase B in flowing mutually was decremented to 5-25vol.% at 1-5 minute from 35-100vol.%.
In another preference, the percent by volume of the Mobile phase B in flowing mutually described in the step (b) 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 flowing mutually described in the step (b) 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 flowing mutually described in the step (b) 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 in flowing mutually described in the step (a) remained 10-22vol.% in 2-5 minute.
In another preference, the percent by volume of the Mobile phase B in flowing mutually described in the step (a) 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 discloses can with any composition forms and usefulness, each feature that discloses in the instructions can anyly provide the alternative characteristics of identical, impartial or similar purpose to replace.Therefore except special instruction is arranged, the feature that discloses 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 for explanation the present invention 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 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 refers to the weight of solute in 100 milliliters solution.
Unless otherwise defined, employed all specialties are identical with the meaning that 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.
Embodiment 1
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.