CN104212857A - Method for separation purification of antibacterial peptide from lactobacillus paracasei fermented milk as raw material - Google Patents
Method for separation purification of antibacterial peptide from lactobacillus paracasei fermented milk as raw material Download PDFInfo
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- 238000000926 separation method Methods 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 27
- 238000000746 purification Methods 0.000 title claims abstract description 19
- 235000015140 cultured milk Nutrition 0.000 title claims abstract description 17
- 239000002994 raw material Substances 0.000 title claims abstract description 14
- 241000186605 Lactobacillus paracasei Species 0.000 title abstract 3
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- PLXBWHJQWKZRKG-UHFFFAOYSA-N Resazurin Chemical compound C1=CC(=O)C=C2OC3=CC(O)=CC=C3[N+]([O-])=C21 PLXBWHJQWKZRKG-UHFFFAOYSA-N 0.000 description 2
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- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
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- Peptides Or Proteins (AREA)
Abstract
The invention discloses a method for separation purification of an antibacterial peptide from lactobacillus paracasei fermented milk as a raw material. The method utilizes lactobacillus paracasei subsp. tolerans FX-6-1 as a fermentation strain, and the fermentation strain is preserved by the China center for type culture collection on February 23, 2014 and has a preservation number of CCTCC NO: M2014043. The method comprises the following steps of preparing a lactobacillus paracasei fermentation product, adding distilled water into the fermentation product, mixing the materials to obtain a uniform mixture, carrying standing and centrifugation, carrying out filtration by a filtration membrane, carrying out rotary evaporation concentration on the supernatant, carrying out freeze drying to obtain an antibacterial crude extract, carrying out cellulose anion DEAE-52 exchange chromatographic separation, concentrating the active ingredient, and carrying out high performance liquid chromatography (HPLC)-based separation and condensation to obtain an antibacterial peptide active single-peak ingredient. The antibacterial peptide active single-peak ingredient comprises 12 amino acids and has effects of substantially inhibiting bacterial pathogens such as escherichia coli and staphylococcus aureus, and mould such as aspergillus flavus and rhizopus nigricans. The method can be used for separation purification of the antibacterial peptide.
Description
Technical field
The present invention relates to a kind of separation purification method of antibacterial peptide, be specifically related to a kind of with the method for lactobacillus paraceasi fermented-milk for raw material separation and purification antibacterial peptide.
Technical background
In pharmaceutical industries and livestock-raising industry, a large amount of uses of conventional antibiotic, have caused appearance and the drug residue of a large amount of endurance strain, have caused serious threat to human health and livestock and poultry breeding industry.In food service industry, along with the lifting of healthy consumption consciousness, people more and more repel the people of traditional chemical sanitas for adding.Therefore, exploitation not easily produces resistance and noresidue, has important practical significance without the new type natural antimicrobial substance of potential safety hazard, and becomes the focus of Recent study as the protein-based antimicrobial substance-antibacterial peptide of natural origin.
Although at present can by chemosynthesis and preparation antibacterial peptide, chemosynthesis and genetic engineering technique be often with high costs, and part antibacterial peptide need carry out safety evaluation.Therefore, find from natural food source and be separated antibacterial peptide and have important practical significance.
Chinese patent disclosed a kind of " separation purification method of the antibacterial peptide that lactobacillus paraceasi produces " (publication number is: CN101451158A), employing soybean protein freezes, extractum carnis, yeast extract, Na
2sO
3, NaAc, MgSO4, MnSO4 and ammonium citrate etc. are fermention medium, through the method for the means separation and purification antibacterial peptides such as refrigeration separation, cation-exchange chromatography, ultrafiltration desalination, high performance liquid chromatography separation.The method relative cost is higher, technique relative complex.
Summary of the invention
The object of the present invention is to provide a kind of with the method for lactobacillus paraceasi fermented-milk for raw material separation and purification antibacterial peptide, it adopts natural food source, and process costs is lower.Be separated the antibacterial peptide obtained by the method, to the bacteria pathogeny such as intestinal bacteria, streptococcus aureus microorganism and the mould such as flavus, bread mould, there is remarkable restraining effect.
For achieving the above object, embodiment of the present invention are: a kind of with the method for lactobacillus paraceasi fermented-milk for raw material separation and purification antibacterial peptide, comprise the following steps:
(1) to be separated from the tough and tensile subspecies FX-6-1 of the lactobacillus paraceasi of Tibet Kefir fermented milk as fermentation strain, this bacterial strain was preserved in China typical culture collection center on 02 23rd, 2014, deposit number is CCTCC M 2014043, is substratum, prepares fermented product with pure milk;
(2) get fermented product and add distilled water, material liquid volume ratio is 1 ︰ 0.5, mixes and leaves standstill, centrifugal, crosses film, and supernatant liquor, through choosing dress evaporation concentration, obtains antibacterial crude extract after lyophilize;
(3) by antibacterial crude extract through cellulose anion DEAE-52 displacement chromatography, with the NH of the 0.005-0.1 of pH8.0
4aC solution gradient wash-out, collects corresponding elutriant according to chromatographic peak, rotary evaporation removing organic solvent, lyophilize, obtained active ingredient;
(4) active ingredient step (3) obtained is through high performance preparative liquid chromatography flash liberation, chromatographic column is C18 post, moving phase is that water and acetonitrile carry out gradient elution, the elutriant corresponding to each peak is collected according to chromatographic peak, rotary evaporation removing organic solvent, lyophilize, obtains antibiont active ingredient;
(5) active ingredient step (4) obtained is through high performance preparative liquid chromatography secondary separation: chromatographic column is Shimadzu PRC-ODS (K) post, moving phase is that water and methyl alcohol carry out gradient elution, the elutriant corresponding to each peak is collected according to chromatographic peak, rotary evaporation concentrates, lyophilize, obtains antibiont active ingredient;
(6) active ingredient obtained for step (5) is analyzed liquid phase chromatographic separation through efficient preparation, chromatographic column is ECOSIL C18 post, moving phase is that water and methyl alcohol carry out gradient elution, the elutriant corresponding to each peak is collected according to chromatographic peak, rotary evaporation concentrates, lyophilize, obtains the active unimodal component of antibacterial peptide.
In step (2), fermented product and distilled water volume ratio are 1 ︰ 0.5, rotating speed 4000 r/min, time 20min during centrifugation), crossing filter membrane that film uses is 0.45 μm of filter membrane.
In step (3), antibacterial crude extract is carried out just being separated through cellulose anion DEAE-52 displacement chromatography: cellulose anion DEAE-52 post specification is 2.6 cm × 40 cm, and applied sample amount is 1% ~ 4%V
post, flow velocity is 1.5 mL/mln, and determined wavelength is 280nm, uses the 0.005M of pH8.0 respectively, the NH of 0.075M, 0.01M, 0.0125M, 0.02M, 0.1M
4aC solution gradient wash-out, every gradient 3 times of column volume elutriants.
In step (4), described C18 post specification is 480mm × 30mm, and applied sample amount is 5mL, and flow velocity is 10mL/min, and determined wavelength is 214nm and 280nm.
In step (5), during liquid chromatography secondary separation, applied sample amount is 1mL, and flow velocity is 10mL/min, and determined wavelength is 214nm and 280nm.
In step (6), when analyzing liquid phase chromatographic separation, applied sample amount is 20 μ L, and flow velocity is 1mL/min, and determined wavelength is 214nm and 280nm.
In step (4), (5), (6), containing 0.1% trifluoroacetic acid (TFA) of 0.1% in the water in moving phase.
Through confirming, the active unimodal component of antibacterial peptide that the present invention obtains is through amino acidanalyser analysis after the hydrochloric acid hydrolysis of 6mol/L, and it contains 12 seed amino acid such as Asp, Thr, Ser, Glu, Gly, Ala, Val, Ile, Leu, Orn, Lys, Pro.
The present invention confirms that the active unimodal component of the antibacterial peptide that obtains detects through 96 orifice plate micro-dilution method antibacterial experiments and analyzes, find its to intestinal bacteria (
escherlchia coli), streptococcus aureus (
staphylococcus aureus) etc. bacteria pathogeny microorganism and flavus (
aspergillus flavus), bread mould (
rhizopus nigricans) etc. mould there is remarkable restraining effect.
The invention has the advantages that: the tough and tensile subspecies FX-6-1 of (1) fermentation strain used lactobacillus paraceasi is separated from Tibet Kefir fermented milk, this bacterial strain was preserved in China typical culture collection center on 02 23rd, 2014, and deposit number is CCTCC M 2014043; (2) the present invention is with the relatively inexpensive pure milk of price for fermentation training base, freezes, extractum carnis, yeast extract, Na without the need to soybean protein
2sO
3, the reagent type substratum such as NaAc, this will be greatly cost-saving in production application; (3) separating and purifying antibacterial peptide process of the present invention is simple, without the need to desalting treatment; (4) the antibacterial spectrum width of gained antibacterial peptide of the present invention, has remarkable restraining effect to the bacteria pathogeny microorganisms such as intestinal bacteria (Escherlchia coli), streptococcus aureus (Staphylococcus aureus) and the mould such as flavus (Aspergillus flavus), bread mould (Rhizopus nigricans).
Accompanying drawing explanation
Fig. 1 is that the antibacterial crude extract of lactobacillus paraceasi tough and tensile subspecies FX-6-1 strain fermentation generation is through DEAE-52 thin layer chromatography figure;
Fig. 2 is high performance preparative liquid chromatography (RP-HPLC) isolating active component W-I;
Fig. 3 is high performance preparative liquid chromatography (RP-HPLC) secondary separation active ingredient " peak 1 ";
Fig. 4 is that efficient analysis liquid chromatography (HPLC) is separated antibacterial active constituents " peak 1-2 ".
Embodiment
(1)the preparation of the tough and tensile subspecies FX-6-1 bacterial strain of lactobacillus paraceasi of the present invention
Be inoculated in pure milk by Tibet Kefir granule, 30 DEG C of static fermentations cultivate 24 h, and the curdled milk after mixing fermentation, gets liquid fermented milk 0.9% stroke-physiological saline solution and be diluted to 10
-6~ 10
-8, draw 0.1 mL diluted sample after vortex mixing and be also coated with evenly to MC solid medium and MRS solid state rheology planar surface.30 DEG C of lower seals are cultivated 24-48h and are grown to single bacterium colony.Picking produces single bacterium colony of clear calcium circle, and again by MRS agar plate purifying to obtain single bacterium colony bacterial strain, collect 49 strains of lactic acid bacteria altogether.
By be separated to product acid single bacterium colony inoculation in MRS liquid culture medium, static sealing incubated overnight at 30 DEG C.Lactic acid bacterial liquid will be activated subsequently by inoculum size 5%(v/v) add in pure milk, at mixing latter 30 DEG C, static sealing cultivates 24 h, centrifugal (4,000 r/min of fermented liquid, after 20min) crossing film (0.45 μm of filter membrane), obtain freeze-drying sample through lyophilize.
Take intestinal bacteria as indicator, adopt Oxford cup agar diffusion method to screen the tough and tensile subspecies FX-6-1 bacterial strain of lactobacillus paraceasi of anti-microbial activity the best.This strain culturing feature is as follows: the rounded white projection of bacterium colony, and bacterium colony is moistening, bright, and neat in edge, has typical milk-acid bacteria colony characteristics.
The tough and tensile subspecies FX-6-1 of described lactobacillus paraceasi (Lactobacillus paracasei subsp.tolerans FX-6-1) bacterial strain has been preserved in and has been preserved on 02 23rd, 2014 the China typical culture collection center being positioned at Wuhan University of Wuhan City, and deposit number is CCTCC M 2014043.
(2) with the method for lactobacillus paraceasi fermented-milk for raw material separation and purification antibacterial peptide
Concrete steps and method are:
(1)the antibacterial crude extract preparation that lactobacillus paraceasi tough and tensile subspecies FX-6-1 strain fermentation produces
By the activation bacterium liquid of tough and tensile for lactobacillus paraceasi subspecies FX-6 bacterial strain by inoculum size 5%(v/v) add in pure milk, at mixing latter 35 DEG C, static sealing cultivates 72 h, get fermented product and add distilled water (fermented product and distilled water volume ratio are 1 ︰ 0.5), mixing leaves standstill, fermented liquid centrifugal (4,000 r/min, 20min) cross film (0.45 μm of filter membrane) after, obtain the antibacterial crude extract of freeze-drying through lyophilize.
(2) separation and purification of bacterial strain antibacterial substance antibacterial peptide
A. cellulose anion DEAE-52 displacement chromatography is just separated antibacterial crude extract: post specification: 2.6 cm × 40 cm, flow velocity: 1.5mL/min, applied sample amount: 1% ~ 4%V
post, moving phase: distilled water, the NH of different ions concentration
4aC solution, determined wavelength: 280 nm
The filling of b, cellulose anion DEAE-52 exchange column:
Swelling: DEAE-52 is put into distilled water respectively, soaking at room temperature 12 h ~ 24 h..
Rinsing: with distilled water rinsing removing impurity and superfine small-particle repeatedly.
Activation: first soak a few hours with 0.5moL/L NaOH, repeatedly rinse to neutrality with deionized water, then soak a few hours with 0.5moL/L HCl, deionized water rinses repeatedly to neutrality, soak a few hours with 0.5moL/L NaOH again, finally repeatedly rinse to neutrality for subsequent use with deionized water.
Dress post: by the DEAE-52 distilled water suspendible activated, pour in the cleaned glass chromatography column that is vertically fixed on iron clamp platform from upper end, while adding DEAE-52 suspension, allow water flow out from chromatography column lower end, make DEAE-52 certainly but decline equably, the chromatographic column installed can not have segmentation.
Column equilibration: after cellulose anion DEAE-52 exchange column is loaded, rinses 2 h ~ 3 h with distilled water with the flow velocity of 1.5mL/min.Use pH8 damping fluid 0.005 moL/L NH again
4aC, with same flow velocity balance 1 ~ 2 h, under 280 nm determined wavelength, record color atlas, until baseline balance.The envrionment temperature of chromatography column is room temperature.
Loading and wash-out: get antibacterial crude extract freeze-drying sample, consumption is 1% ~ 4% of column volume, and add 6mL distilled water and fully dissolve, room temperature, 4500r/min are centrifugal, and 10min is for subsequent use.After cellulose anion DEAE-52 exchange column balance, add sample solution 4.0mL, with distilled water, the 0.005M of pH8.0,0.075M, 0.01M, 0.0125M, 0.02M, 0.1M NH4AC solution, every gradient 3 times of column volume wash-outs, under 280 nm determined wavelength, the light absorption value of record elutriant, manual collection sample, collect complete, concentrated freeze-dried rear employing Oxford cup agar diffusion method analytic sample anti-microbial activity, wherein peak W-I has strong activity (Fig. 1).
(3). high performance preparative liquid chromatography (RP-HPLC) flash liberation antibacterial active constituents
The freeze drying activity component W-I distilled water be separated to by cellulose anion DEAE-52 chromatography column dissolves, and is configured to the solution of 100 mg/mL, upper efficient preparation liquid phase initial gross separation antibacterial active constituents after 0.45 μm of membrane filtration.
Chromatographic condition is: chromatographic column: 480 mm × 30 mm, C18 post; Moving phase: A: water (containing 0.1%TFA), B: acetonitrile; Flow velocity: 10 mL/min; Sample size: 5 mL; Determined wavelength: 280 nm, 214 nm; Elution requirement: acetonitrile 4%-8%(50 min).
Collect each component according to the peak that goes out in chromatograms, 55 DEG C concentrate, and the chromatographic peak sample lyophilized products anti-microbial activity adopting Oxford cup agar diffusion method analysis to collect after lyophilize freeze-drying, wherein peak 1 has remarkable anti-microbial activity (Fig. 2).
(4) high performance preparative liquid chromatography (RP-HPLC) secondary separation antibacterial active constituents
Peak 1 active ingredient distilled water high performance preparative liquid chromatography (RP-HPLC) flash liberation obtained dissolves, and is configured to the solution of 50 mg/mL, and after 0.45 μm of membrane filtration, upper high performance preparative liquid chromatography is to active ingredient secondary separation.
Chromatographic condition is: chromatographic column: Shimadzu PRC-ODS (K) post; Moving phase: A: water (containing 0.1%TFA), B: methyl alcohol; Sample size: 0.5 mL; Flow velocity: 10 mL/min; Determined wavelength: 280 nm, 214 nm; Elution requirement: methyl alcohol 5.5%(50 min).
Collect each component according to the peak that goes out in chromatograms, 55 DEG C concentrate, and adopt Oxford cup agar diffusion method to analyze each component anti-microbial activity, wherein peak 1-2 has remarkable anti-microbial activity (Fig. 3).
(5). efficient analysis liquid chromatography (HPLC) is separated antibacterial active constituents
Peak 1-2 active ingredient distilled water high performance preparative liquid chromatography (RP-HPLC) secondary separation obtained dissolves, be configured to the solution of 20 mg/mL, through 0.45 μm of membrane filtration, upper efficient analysis liquid chromatography compartment analysis.
Chromatographic condition is: chromatographic column: ECOSIL C18 (4.6 mm × 250 mm, 5 μm, Lubex, Japan); Moving phase: A: water (containing 0.1%TFA), B: methyl alcohol; Sample size: 20 μ L; Flow velocity: 1 mL/min; Determined wavelength: 280 nm, 214 nm; Elution requirement: water 100%(30 min).Collect and eachly unimodally carry out Analysis of Antimicrobial Activity, wherein peak 1-2-3 has remarkable anti-microbial activity (Fig. 4).
(3) amino acid of the present invention to bacterial strain antibacterial substance antibacterial peptide is analyzed
Adopt GB/T 5009.124-2003 to measure the amino acid composition of active single " peak 1-2-3 ", mainly it contains 12 seed amino acid such as Asp, Thr, Ser, Glu, Gly, Ala, Val, Ile, Leu, Orn, Lys, Pro.
(4) bacterial strain antibacterial substance antibacterial peptide antimicrobial spectrum of the present invention and minimum inhibitory concentration analysis
Adopt 96 orifice plate micro-dilution methods (resazurin makes indicator) to antibacterial substance antibacterial peptide antimicrobial spectrum and minimum inhibitory concentration analysis, concrete grammar is:
First 100 μ L indicator solutions (resazurin solution of 750 μ g/mL) are placed on the 11st row (blank control wells) of 96 orifice plates.The bacterium liquid (1 × 10 to be measured of about 7.5 mL indicator solutions and 5 mL
8cFU/mL) mix, then shift 100 μ L mixed solutions in 1 to 10 row (sample test hole) and the 12nd row (growth control hole) plate hole.Add 100 μ L testing sample solutions in the 1st row hole, transfer to the 2nd row after mixing from the 1st row taking-up 100 μ L, use the same method until the 10th row, the 100 μ L that the 10th row take out lose.Finally, 96 orifice plates having added sample are put into constant incubator, cultivate 5-6 h, until the dyeing of orifice plate becomes growth look for 37 DEG C: pink.If treat that examination bacterium is fungi, it is that color is from pink to blueness that incubation time is approximately the activated mark of 12-16 h..The minimum weaker concn of the colour-change occurred is considered to the minimum inhibitory concentration (MIC) of testing compound, and the OD value change of the available microplate reader of change under 600 nm of color simultaneously judges.
What utilize 96 orifice plate micro-dilution methods to measure the antimicrobial spectrum of antibacterial substance antibacterial peptides and corresponding minimum inhibitory concentration the results are shown in Table 1:
Table 1 antibacterial substance antibacterial peptide antimicrobial spectrum and minimum inhibitory concentration (X ± SD, n=3)
| Indicator | Minimum inhibitory concentration (μ g/mL) |
| Intestinal bacteria | 62.50±2.03 |
| Streptococcus aureus | 125.00±3.76 |
| Bacillus thuringiensis | 62.50±1.43 |
| Salmonellas | 250.00±3.34 |
| Shigella dysenteriae | 250.00±3.26 |
| Flavus | 250.00±4.06 |
| Aspergillus niger | 250.00±3.98 |
| Bread mould | 125.00±4.01 |
| Penicillum glaucum | 250.00±3.71 |
Can know from table 1, the antimicrobial spectrum of antibacterial substance antibacterial peptide is relatively extensive, and the Gram-negative bacteria common to part and gram-positive microorganism and fungi all have bacteriostatic action in various degree.Wherein, the strongest to the inhibition of intestinal bacteria and bacillus thuringiensis in bacterium, MIC value all reaches 62.5 μ g/mL.Except having inhibition to bacterium, be also play restraining effect to some fungi, wherein best to bread mould fungistatic effect, MIC value is 125 μ g/mL, and the MIC value of other several fungies is all at about 250 μ g/mL.Generally speaking, the antimicrobial spectrum of antibacterial substance antibacterial peptide is extensive, have effect, and antibacterial effect is strong to some bacteriums and fungi.
Claims (7)
1., with the method for lactobacillus paraceasi fermented-milk for raw material separation and purification antibacterial peptide, it is characterized in that, comprise the following steps:
(1) with the tough and tensile subspecies FX-6-1 of lactobacillus paraceasi for fermentation strain, this bacterial strain was preserved in China typical culture collection center on 02 23rd, 2014, and deposit number is CCTCC M 2014043, is substratum, prepares fermented product with pure milk;
(2) get fermented product and add distilled water, material liquid volume ratio is 1 ︰ 0.5, mixes and leaves standstill, centrifugal, crosses film, and supernatant liquor, through choosing dress evaporation concentration, obtains antibacterial crude extract after lyophilize;
(3) by antibacterial crude extract through cellulose anion DEAE-52 displacement chromatography, with the NH of the 0.005-0.1 of pH8.0
4aC solution gradient wash-out, collects corresponding elutriant according to chromatographic peak, rotary evaporation removing organic solvent, lyophilize, obtained active ingredient;
(4) active ingredient step (3) obtained is through high performance preparative liquid chromatography flash liberation, chromatographic column is C18 post, moving phase is that water and acetonitrile carry out gradient elution, the elutriant corresponding to each peak is collected according to chromatographic peak, rotary evaporation removing organic solvent, lyophilize, obtains antibiont active ingredient;
(5) active ingredient step (4) obtained is through high performance preparative liquid chromatography secondary separation: chromatographic column is Shimadzu PRC-ODS (K) post, moving phase is that water and methyl alcohol carry out gradient elution, the elutriant corresponding to each peak is collected according to chromatographic peak, rotary evaporation concentrates, lyophilize, obtains antibiont active ingredient;
(6) active ingredient obtained for step (5) is analyzed liquid phase chromatographic separation through efficient preparation, chromatographic column is ECOSIL C18 post, moving phase is that water and methyl alcohol carry out gradient elution, the elutriant corresponding to each peak is collected according to chromatographic peak, rotary evaporation concentrates, lyophilize, obtains the active unimodal component of antibacterial peptide.
2. according to claim 1 with the method for lactobacillus paraceasi fermented-milk for raw material separation and purification antibacterial peptide, it is characterized in that, in step (2), fermented product and distilled water volume ratio are 1 ︰ 0.5, rotating speed 4000 r/min, time 20min during centrifugation), crossing filter membrane that film uses is 0.45 μm of filter membrane.
3. according to claim 1 with the method for lactobacillus paraceasi fermented-milk for raw material separation and purification antibacterial peptide, it is characterized in that, in step (3), antibacterial crude extract is carried out just being separated through cellulose anion DEAE-52 displacement chromatography: cellulose anion DEAE-52 post specification is 2.6 cm × 40 cm, and applied sample amount is 1% ~ 4%V
post, flow velocity is 1.5 mL/mln, and determined wavelength is 280nm, uses the 0.005M of pH8.0 respectively, the NH of 0.075M, 0.01M, 0.0125M, 0.02M, 0.1M
4aC solution gradient wash-out, every gradient 3 times of column volume elutriants.
4. according to claim 1ly to it is characterized in that with the method for lactobacillus paraceasi fermented-milk for raw material separation and purification antibacterial peptide, in step (4), described C18 post specification is 480 mm × 30 mm, applied sample amount is 5mL, and flow velocity is 10mL/min, and determined wavelength is 214nm and 280nm.
5. according to claim 1ly to it is characterized in that with the method for lactobacillus paraceasi fermented-milk for raw material separation and purification antibacterial peptide, in step (5), during liquid chromatography secondary separation, applied sample amount is 1mL, and flow velocity is 10mL/min, and determined wavelength is 214nm and 280nm.
6. according to claim 1ly to it is characterized in that with the method for lactobacillus paraceasi fermented-milk for raw material separation and purification antibacterial peptide, in step (6), when analyzing liquid phase chromatographic separation, applied sample amount is 20 μ L, and flow velocity is 1mL/min, and determined wavelength is 214nm and 280nm.
7. according to claim 1ly to it is characterized in that with the method for lactobacillus paraceasi fermented-milk for raw material separation and purification antibacterial peptide, in step (4), (5), (6), containing 0.1% trifluoroacetic acid of 0.1% in the water in moving phase.
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| CN104855492A (en) * | 2015-04-02 | 2015-08-26 | 曹庸 | A natural preservative for meat products and a preparation and use method thereof |
| CN106018823A (en) * | 2016-05-19 | 2016-10-12 | 四川大学 | Method for rapidly detecting antibacterial peptides in lactobacillus casei fermentation process and final products |
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| CN106018823A (en) * | 2016-05-19 | 2016-10-12 | 四川大学 | Method for rapidly detecting antibacterial peptides in lactobacillus casei fermentation process and final products |
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| CN109652482A (en) * | 2018-11-06 | 2019-04-19 | 华南农业大学 | A kind of antibacterial peptide and the preparation method and application thereof |
| CN109652482B (en) * | 2018-11-06 | 2021-12-17 | 华南农业大学 | Antibacterial peptide and preparation method and application thereof |
| CN109679355A (en) * | 2018-12-14 | 2019-04-26 | 华南农业大学 | A kind of antibacterial film and its application |
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| CN110229768B (en) * | 2019-06-20 | 2022-02-18 | 内蒙古农业大学 | Lactobacillus paracasei ALAC-4 and bacteriostatic application thereof |
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