CN1570096A - Use of gene recombinant lysozyme #-[134] and gene recombinant human lysozyme and its pure products in antistaling and antiseptic agent - Google Patents
Use of gene recombinant lysozyme #-[134] and gene recombinant human lysozyme and its pure products in antistaling and antiseptic agent Download PDFInfo
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- CN1570096A CN1570096A CN 200410010842 CN200410010842A CN1570096A CN 1570096 A CN1570096 A CN 1570096A CN 200410010842 CN200410010842 CN 200410010842 CN 200410010842 A CN200410010842 A CN 200410010842A CN 1570096 A CN1570096 A CN 1570096A
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Abstract
The invention relates to a gene recombinant lysozyme#-[134] and gene recombinant human lysozyme and its uses of pure product in antistaling preservatives, especially relates to uses of pure product in fields of food, beverage and cosmetics antistaling and preservative. The material is rich and cheap, innocuous. The process is simple.
Description
Technical field:
The present invention relates to a kind of gene recombination N,O-Diacetylmuramidase
134The application in antistaling and antiseptic agent with gene recombinant human lysozyme and pure product thereof particularly relates to a kind of gene recombination N,O-Diacetylmuramidase
134With gene recombinant human lysozyme and pure product thereof at food, beverage freshness remaining is anticorrosion and the application of makeup in anticorrosion.
Background technology:
Food, especially seal-packed modern food, in long time stored or long-distance transport, taking place to go rotten rotten is the problem that people worry most.After food decay is rotten, people common to be aerogenesis, souring, fouling, stickness, wire drawing, become mildewed or the like, these phenomenons all are that food produces under microbial infection.Main component sugar, protein and fat from food: the rotten of sugar is called fermentation, causes aerogenesis, souring; The proteinic rotten corruption that is called owing to produced malodorous sulfide, nitride, causes food smelly, produces peculiar smell; Greasy rotten be called to breathe out become or become sour produced rudimentary aldehyde, ketone because lipid acid decomposes, cause to distribute and breathe out flavor.From causing the rotten microorganism of food decay, yeast, bacterium and fungi are arranged, they self are able to a large amount of breedings when causing food decay rotten, and a large amount of bacteriums is present in the water or autolyze, can make that solution becomes is glutinous, wire drawing; A large amount of fungus breedings is owing to mycelia or different " hairs " that grow different colours such as black, green, redness of spore color.In order to prevent rotting of food, people once used many methods, and such as salted, sugaring, drying, heating, vacuum-packed, irradiation or the like, and the most simple and effective method just is to use food preservatives, oxidation inhibitor.Food is subjected to effects such as microorganism, oxygen corruption can take place, go mouldy in production and storage, cause very big loss for people's life and production, and the synthetic Antisepticize and mildew preventive often brings certain detrimentally affect to human health.Therefore, natural, environmental preservation agent, and be used for the fresh-keeping research of food, be subject to people's attention day by day.In the last few years, because antibacterials is widely-used, bacterial drug resistance was constantly strengthened, and a lot of bacterium is developed into multidrug resistant by single medicine resistance.Add antibacterials in the feed, as many as lasting low dosage medication.Animal body contacts with medicine for a long time, causes resistant organism to be on the increase, and resistance also constantly strengthens.Antibacterial medicine residue makes the people also contact with medicine for a long time in animal food equally, causes the increase of resistant organism in the human body.Utilize lysozyme of chicken as the existing a lot of reports of food preservative, and the shortcoming of lysozyme of chicken is to infect avian influenza virus easily, but does not see gene recombinant human lysozyme, reorganization N,O-Diacetylmuramidase
134Pure product are anticorrosion as food fresh keeping, the report and the application of the application of makeup in anticorrosion.
Summary of the invention:
The object of the present invention is to provide a kind of gene recombination N,O-Diacetylmuramidase
134The application in antistaling and antiseptic agent with gene recombinant human lysozyme and pure product thereof.
In fact, purpose of the present invention relates to a kind of gene recombination N,O-Diacetylmuramidase
134The application in various wrap food preservation and antisepsis with gene recombinant human lysozyme and pure product thereof.
Another object of the present invention relates to a kind of gene recombination N,O-Diacetylmuramidase
134The application in various packing makeup are anticorrosion with gene recombinant human lysozyme and pure product thereof.
Relate to a kind of gene recombination N,O-Diacetylmuramidase
134The application in various packing grape wine, dry red winew, Dry white wine and various fruit wine preservation and antisepsis with gene recombinant human lysozyme and pure product thereof.
Relate to a kind of gene recombination N,O-Diacetylmuramidase
134The application in various packing milk foods preservation and antisepsis with gene recombinant human lysozyme and pure product thereof.
Relate to a kind of gene recombination N,O-Diacetylmuramidase
134The application in various packed drinks and fruit juice food preservation and antisepsis with gene recombinant human lysozyme and pure product thereof.
Relate to a kind of gene recombination N,O-Diacetylmuramidase
134With the application in various packing fresh meats and cold cuts food fresh keeping are anticorrosion of gene recombinant human lysozyme and pure product thereof.
Relate to a kind of gene recombination N,O-Diacetylmuramidase
134With the application in various packing fresh fish meat and ripen fish food fresh keeping are anticorrosion of gene recombinant human lysozyme and pure product thereof.
Relate to a kind of gene recombination N,O-Diacetylmuramidase
134The application in various packing fruit food fresh keepings are anticorrosion with gene recombinant human lysozyme and pure product thereof.
Relate to a kind of gene recombination N,O-Diacetylmuramidase
134The application in various packing fresh-keeping of vegetables are anticorrosion with gene recombinant human lysozyme and pure product thereof.
Relate to a kind of gene recombination N,O-Diacetylmuramidase
134With the application in various packaging of meat cans and sauce can and fruit can food fresh keeping are anticorrosion of gene recombinant human lysozyme and pure product thereof.
Relate to a kind of gene recombination N,O-Diacetylmuramidase
134The application in various packing cheese food preservation and antisepsis with gene recombinant human lysozyme and pure product thereof.
In order to understand essence of the present invention better, below with gene recombinant human lysozyme and reorganization N,O-Diacetylmuramidase
134The experiment of pure product and result illustrate that it is anticorrosion at food, beverage freshness remaining, the application of makeup in anticorrosion.
Therefore the toxicity of natural fresh-keeping sanitas composition, seek the research of natural fresh-keeping sanitas composition to cause various countries scientist's attention from nature in recent years well below the toxicity of synthetic antistaling and antiseptic agent composition.N,O-Diacetylmuramidase is a kind of natural antimicrobial substance that people, zooblast produce, because of N,O-Diacetylmuramidase itself promptly has in animal body, so use gene recombinant human lysozyme, reorganization N,O-Diacetylmuramidase
134Pure product replace synthetic antistaling and antiseptic agent composition commonly used can avoid bringing certain detrimentally affect to human health.
The gene recombination N,O-Diacetylmuramidase
134Material: the gene recombination N,O-Diacetylmuramidase
134The expression system feature be to adopt methyl alcohol nutritional type yeast as host bacterium " bacterial strain of pichia spp Pseudomonas ".On the phenotype of methyl alcohol utilization, both adopt the quick type of methyl alcohol utilization (mut+), and also used methyl alcohol to utilize type (muts or must-) at a slow speed.The main smd1165 (his4.prb1) that adopts pichia spp, smd1163 (his.pep4.prb1), smd1168 (his4.prb1) Gs115, km71 bacterial strain.Gene recombination N,O-Diacetylmuramidase of the present invention
134Plasmid mainly be on the plasmid integration above-mentioned bacterial strains karyomit(e) with pPIC9k series.
1, gene source:
With the synthetic reorganization of chemical synthesis N,O-Diacetylmuramidase
134Gene.(rich inferior biological company limited is synthetic by Shanghai) again by the DNA recombinant technology, in the pUC19 plasmid vector, clone strain turns out to be the reorganization N,O-Diacetylmuramidase by the nucleotide DNA sequential analysis with gene clone
134Gene.
2, reorganization N,O-Diacetylmuramidase
134The preparation of gene:
The reorganization N,O-Diacetylmuramidase
134Gene design and with the synthetic oligonucleotide primer of chemical synthesis:
(p1-5 '-CCTCGAGAAAAGAGAGGCTGAAGCTAAGGTCTTTGAAAGGTG-3 ' P2-5 '-GGAATTCTTACACTCCACAACCTTG-3 ', rich inferior biological company limited is synthetic by Shanghai) employing PCR method (reaction system: RT product 10 μ l, 10 * PCR damping fluid, 5 μ l, 10mmol/L dNTP 1 μ l, 15Pmol/L P1 primer 1 μ l, 15Pmol/L P2 primer 1 μ l, Taq enzyme 1 μ l (5u), redistilled water 31 μ l, cumulative volume 50 μ l; Reaction conditions: 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 2 minutes, 35 circulations).Reclaim the dna fragmentation of test kit (Shanghai China Shun Bioisystech Co., Ltd product) the about 420bp of purified pcr product with DNA glue.With the PCR product D NA of EcoRI and SalI double digestion purifying, and be cloned into that (method for transformation is CaCl in the pUC19 plasmid
2Method, host bacterium are e. coli jm109), obtain recombinant plasmid pUC19-LZ/JM109 transformant, transformant is cut with nucleotide sequence analysis through enzyme and is turned out to be human lysozyme gene and many four amino acid A E A E of N end again.
3, the structure of expression vector:
With resulting reorganization N,O-Diacetylmuramidase in 1
134Gene recombination plasmid pUC19-LZ is a template, by PCR and DNA recombinant technology, and the N,O-Diacetylmuramidase of will recombinating
134Gene is inserted among pichia (Pichia pastoris) the secreted expression carrier pPIC9K, obtains the N,O-Diacetylmuramidase of recombinating
134Expression vector pPIC9K-LZ as shown in Figure 1.
4, human lysozyme gene expression vector establishment step:
Four amino acid A E A E more than also N holds again with 1 income earner's lysozyme gene.Recombinant plasmid pUC19-hLZ is a template, uses primer
(p1-5 '-CCTCGAGAAAAGAGAGGCTGAAGCTAAGGTCTTTGAAAGGTG-3 ' P2-5 '-GGAATTCTTACACTCCACAACCTTG-3 ', rich inferior biological company limited is synthetic by Shanghai), adopt PCR method to amplify the reorganization N,O-Diacetylmuramidase
134Gene and alpha factor signal peptide are read the corresponding XhoI of frame and EcoRI restriction enzyme site and TAA termination codon.(reaction system is: pUC19-hLZ DNA 1 μ l (50ng), 10XPCR damping fluid 5 μ l, 10mmol/L dNTP 1 μ l, 15Pmol/L P3 primer 1 μ l, 15Pmol/L P4 primer 1 μ l, pfu taq enzyme 1 μ l (the biological company limited of 5u Shanghai lottery industry product), redistilled water 40 μ l, cumulative volume 50 μ l); 94 ℃ 30 seconds, 58 ℃ 30 seconds, after 72 ℃ of 30 circulations in 1 minute, 72 ℃ 10 minutes, 4 ℃ 2 minutes.Cut the PCR target DNA fragment of purifying with XhoI and EcoRI enzyme again and be cloned on the secretion type Pichi expression vector pPIC9, obtain recon plasmid pPIC9-hLZ.With 2910 transformants of gained, dibbling is in 2mg/ml G418 YEPD culture dish respectively, cultivate after 2 days for 30 ℃, screen 82 of the transformants of anti-2mg/ml G418, by last method, take out corresponding 82 transformant dibbling 3mg/ml G418 YEPD substratum plates again, cultivate after 2 days for 30 ℃ from the MMD bacterial classification, be sieved to 26 anti-3mg/ml transformants, 13 of anti-4mg/ml G418 transformants screen an anti-4mg/ml G41867 transformant at last.
5, reorganization N,O-Diacetylmuramidase
134Preparation process
Step; 1, shake-flask seed preparation: to prepare 200 milliliters of substratum is benchmark, uses H
3PO
4(phosphoric acid) 4-8 milliliter, MgSO
4(sal epsom) 1-5 gram, K
2SO
4(vitriolate of tartar) 2-6 gram, KOH (potassium hydroxide) 1-3 gram, CaSO
42H
2O (calcium sulfate) 1-3.5 gram, adding distil water to 200 milliliter, inoculation glycerine pipe seed behind the autoclaving, the shaking table revolution is that per minute 250 changes, culture temperature is 20-35 ℃, cultivates 36-48 hour on the constant temperature bed.2, seed tank culture: be upgraded to benchmark with seeding tank maximum volume 1, preparation substratum H
3PO
4(phosphoric acid) 25-19 milliliter, MgSO
4(sal epsom) 13-17 gram, CaSO
42H
2O (calcium sulfate) 0.4-0.8 gram, K
2SO
4(vitriolate of tartar) 16-20 gram, KOH (potassium hydroxide) 2.12-6.12 gram, adding distil water to 500 milliliter, inoculation shaking table seed behind the autoclaving; Fermentation reactor operating parameters temperature is 20-32 ℃, and pH value is 2.0-8.0; Dissolved oxygen concentration is 10-60%, and when the cell density in the nutrient solution is increased to about OD200, seed culture finishes.3, produce a jar cultivation: be upgraded to 10 and produce a jar preparation substratum, use H
3PO
4(phosphoric acid) 93-97 milliliter, MgSO
4(sal epsom) 51-55 gram, CaSO
42H
2O (calcium sulfate) 19-23 gram, K
2SO
4(vitriolate of tartar) 61-65 gram, KOH (potassium hydroxide) 13-17 gram adding distil water to 7 liter, inoculation seeding tank seed behind the autoclaving, fermentation reactor operating parameters temperature is controlled at 25-32 ℃, and pH value is 4.0-8.0, and dissolved oxygen concentration is 10-60%; After cell in the nutrient solution reached certain density, stream added methanol induction in nutrient solution, the time length 50-60 in this stage hour, put a jar cultivation then and finished.
Purifying process: the pichia spp bacteria culture fluid of human lysozyme that will contain gene engineering expression is collected filtrate with the 10-50 ten thousand molecular weight membrane ultrafiltration that dams; With the 5000 molecular weight ultrafiltration of damming, collect dope; Carboxymethyl cellulose on the dope, the elution buffer wash-out is collected elutriant, crosses G-50 post desalination, and frost drying is measured protein content, purity and lysozyme activity.
Human lysozyme of the present invention, reorganization N,O-Diacetylmuramidase
134Pure product generally add 0.0001-0.05% by following portion rate and add in the food-drink; With 3%NaCl, 12.5ppmNaNO
2, 30～1000ppm human lysozyme or the reorganization N,O-Diacetylmuramidase
134Pure product blending water dilution is made liquid and is added in the food-drink; Human lysozyme, reorganization N,O-Diacetylmuramidase
134Pure product 300U～150000U% adds in the food-drink.Butchering fowl immerses in the ice-water bath of this liquid after cleaning, and soaks more than 1 hour.Can pack after the taking-up, fresh keeping time is more than 20 days.
One, reorganization N,O-Diacetylmuramidase
134Body in sterilization experiment
(1), material
1. bacterial strain: streptococcus aureus, 2. animal: BALB/c mouse
(2), method
Streptococcus aureus, the experiment of infection BALB/c mouse are with 10
5～10
9The streptococcus aureus of the logarithmic phase of CFU (colony-forming unit), be inoculated in the BALB/c mouse in 6 ages in week through the abdominal cavity respectively, observations is calculated mortality ratio.
The reorganization N,O-Diacetylmuramidase
134Protect active evaluation with 1 * 10
9The streptococcus aureus of logarithmic phase, abdominal cavity are inoculated in the BALB/c mouse in 6 ages in week as infecting contrast, and N,O-Diacetylmuramidase protection group is injected the reorganization N,O-Diacetylmuramidase of various dose at inoculated bacteria simultaneously
134, observe 7 days results, calculate protection, protection ratio %=[(experimental group surviving rate-control group surviving rate)/the control group mortality ratio] * 100%.
(3), result
The deadly experiment of streptococcus aureus, infection BALB/c mouse is with 10
5～10
9CFU streptococcus aureus, infection BALB/c mouse, the result is from 10
6～10
9CFU infects BALB/c and all can cause death, and death mostly occurs within 3 days, and is basicly stable after 4 days, no longer dead.Wherein 10
8CFU streptococcus aureus, the death of infection BALB/c mouse is rule comparatively, and 10
9The CFU streptococcus aureus, infect in the BALB/c mouse 3 days all dead.We adopt 10
9The CFU streptococcus aureus, carry out the anti-streptococcus aureus of N,O-Diacetylmuramidase, infect the experiment of BALB/c mouse passive protection.
With four various dose reorganization N,O-Diacetylmuramidases
134BALB/c mouse is carried out the protection of streptococcus aureus, infection and identify, observed continuously 7 days, all dead in the control group BALB/c mouse 2 days as a result, the protection group death time slightly postponed, at 2-3 days.Not dead BALB/c mouse was no longer dead in 7 days in 3 days.Four various dose reorganization N,O-Diacetylmuramidases
134The activity that all has passive protection opposing streptococcus aureus, infection, wherein protection ratio reaches 80% during 300,000 units/kilogram.
The anti-streptococcus aureus monoclonal antibody immunity protection of table 2. experimental result
Tab2.protertion?of?lysozyme?against Staphylococcus
Dose(u/kg) NO.test No.death Protective?rate(％)
30×10
4 20 4 80
15×10
4 20 5 75
7.5×10
4 20 7 65
3.75×10
4 20 11 45
0 15 15 0
Two, sterilization experiment in the body of human lysozyme
(1), material
1. bacterial strain: streptococcus aureus, 2. animal: BALB/c mouse
(2), method
Streptococcus aureus, the experiment of infection BALB/c mouse are with 10
5～10
9The streptococcus aureus of the logarithmic phase of CFU (colony-forming unit), be inoculated in the BALB/c mouse in 6 ages in week through the abdominal cavity respectively, observations is calculated mortality ratio.
Human lysozyme protects active evaluation with 1 * 10
9The streptococcus aureus of logarithmic phase, abdominal cavity are inoculated in the BALB/c mouse in 6 ages in week as infecting contrast, and N,O-Diacetylmuramidase protection group is injected the reorganization N,O-Diacetylmuramidase of various dose at inoculated bacteria simultaneously
134, observe 7 days results, calculate protection, protection ratio %=[(experimental group surviving rate-control group surviving rate)/the control group mortality ratio] * 100%.
(3), result
The deadly experiment of streptococcus aureus, infection BALB/c mouse is with 10
5～10
9CFU streptococcus aureus, infection BALB/c mouse, the result is from 10
6～10
9CFU infects BALB/c and all can cause death, and death mostly occurs within 3 days, and is basicly stable after 4 days, no longer dead.Wherein 10
8CFU streptococcus aureus, the death of infection BALB/c mouse is rule comparatively, and 10
9The CFU streptococcus aureus, infect in the BALB/c mouse 3 days all dead.We adopt 10
9The CFU streptococcus aureus, carry out the anti-streptococcus aureus of N,O-Diacetylmuramidase, infect the experiment of BALB/c mouse passive protection.
Identify with the protection that four various dose human lysozymes carry out streptococcus aureus, infection to BALB/c mouse, observed continuously 7 days that all dead in the control group BALB/c mouse 2 days as a result, the protection group death time slightly postponed, at 2-3 days.Not dead BALB/c mouse was no longer dead in 7 days in 3 days.Four various dose human lysozymes all have the activity of passive protection opposing streptococcus aureus, infection, and wherein protection ratio reaches 80% during 300,000 units/kilogram.
The anti-streptococcus aureus monoclonal antibody immunity protection of table 2. experimental result
Tab2.protertion?of?lysozyme?against?Staphylococcus
Dose(u/kg) NO.test No.death Protective?rate(％)
30×10
4 20 4 80
15×10
4 20 5 75
7.5×10
4 20 7 65
3.75×10
4 20 11 45
0 15 15 0
Three, reorganization N,O-Diacetylmuramidase
134And the anti-infective experiment of the protection duroc of pure product
(1), material
1. bacterial strain: Salmonellas, 2. animal: duroc
(2), method
With the reorganization N,O-Diacetylmuramidase
134And pure product filling stomach nursing duroc, normal forage feed compares group.With 1 * 10
9The Salmonellas of logarithmic phase is irritated stomach and is inoculated in protection group and control group duroc respectively.Observations is calculated protection.Immune protective rate %=[(experimental group surviving rate-control group surviving rate)/the control group mortality ratio] * 100%
(3), result
The reorganization N,O-Diacetylmuramidase
134And the anti-infective protection experimental result of pure product protection duroc shows: behind the Salmonella infection duroc, with the reorganization N,O-Diacetylmuramidase
134And pure product filling stomach nursing duroc group protection ratio can reach 65%.The control group of normal forage feed is all dead.
Four, the anti-infective experiment of the protection duroc of human lysozyme
(1), material
1. bacterial strain: Salmonellas, 2. animal: duroc
(2), method
Irritate stomach with human lysozyme and feed duroc, normal forage feed compares group.With 1 * 10
9The Salmonellas of logarithmic phase is irritated stomach and is inoculated in protection group and control group duroc respectively.Observations is calculated protection.Immune protective rate %=[(experimental group surviving rate-control group surviving rate)/the control group mortality ratio] * 100%
(3), result
The anti-infective protection experimental result of human lysozyme protection duroc shows: behind the Salmonella infection duroc, irritate stomach nursing duroc group protection ratio with human lysozyme and can reach 65%.The control group of normal forage feed is all dead.
Five, reorganization N,O-Diacetylmuramidase
134With antibiotic synergetic antibacterial effect
(1), material
1. bacterial strain: strain surplus the clinical separation strain 400 is provided by Chengdu microbiotic industrial research.
2. pH of buffer 7.2 10mMTris-Cl damping fluids
3.LB substratum
4. clarithromycin, Roxithromycin
(2), method
The reorganization N,O-Diacetylmuramidase
134Unite use with clarithromycin, Roxithromycin difference (1: 1),
Method one (test tube method)
With pH7.2 10mM Tris-Cl damping fluid streptococcus aureus (or intestinal bacteria) is made into 10
7-10
8The bacterium liquid of CFU/mL left and right sides concentration joins respectively in the different magnificent test tubes, every pipe 2mL.The human lysozyme (100 μ g-1mg) that adds various dose in the different magnificent test tubes respectively spends the night for 37 ℃, at first gets 20 μ L at every pipe and dilute with 10 times of dilution methods, gets 100 μ L and is applied to the LB substratum, visual inspection turbidity variation or use spectrophotometer, 37 ℃ of overnight incubation; Again every pipe is added 100 μ L 10%SDS (final concentration 0.5%) and shake up, carry out than turbid (640), observations, and calculate total plate count in the original pipe.N,O-Diacetylmuramidase
134Total plate count * 100% in bacteriolyze effect=(in the control tube in total plate count-experiment tube total plate count)/control tube.
Method two (flat band method)
Behind the agarose autoclaving with pH7.2 10mM Tris-Cl damping fluid preparation 1%, be cooled to 40 ℃, add bacterium to 10
6Pour plate, punch on flat board with punch tool, the lysozyme soln (0-100mg/ml) that in the hole, adds different concns, 37 ℃ of incubators were hatched 4-6 hour, behind the LB nutrient agar medium autoclaving, be cooled to 40 ℃, be covered on the above-mentioned agarose plate, should shine 20 minutes down as for ultraviolet lamp by flat board.(the LB nutrient agar medium is covered in the agarose plate process that contains bacterium, the free bacteria on agarose surface can be sneaked in the nutrient agar medium, wherein be flushed to the bacterium on nutrient agar medium surface, because the effect of oxygen, the speed of growth is very fast, form mycoderm, influence the observation of bacterial growth situation between agarose and the nutrient agar medium layer) so kill the bacterium on nutrient agar medium surface with ultra violet lamp.37 ℃ of incubator overnight incubation are observed bacterial growth situation between agarose and the nutrient agar medium layer, measure the diameter of the inhibition zone of different concns N,O-Diacetylmuramidase formation, the diameter of inhibition zone " well diameter 4mm person is minimum effective bacteriocidal concentration.(for amphitrichous, the bacterium that motion is active can influence the formation of inhibition zone, can consider to add 0.01% phenylic acid and suppress its motion.)
(3), result
The reorganization N,O-Diacetylmuramidase
134Unite use with clarithromycin, Roxithromycin difference (1: 1), can make clarithromycin, Roxithromycin to more than its anti-microbial activity synergy 2-16 times, indivedual bacterial strain synergy multiples are at 1000 times.
Clinical separation persister totally 30 strains that this institute uses are respectively streptococcus aureus 8 strains, pseudomonas aeruginosa 3 strains, intestinal bacteria 7 strains, 1 strain of Bao eel bacillus, serratia marcescens 1 strain, Bacillus proteus 3 strains, alpha streptococcus 1 strain.The bacteriostatic experiment result shows the animal 30 strain animal resistant organisms that this institute uses, all to the reorganization N,O-Diacetylmuramidase of gene engineering expression
134Sensitivity, the reorganization N,O-Diacetylmuramidase of gene engineering expression
134Minimum inhibitory concentration to experimental strain there are differences at different bacterium, and effectively the Mlc scope is between 50 μ g-1mg/ml.Find the reorganization N,O-Diacetylmuramidase of gene engineering expression simultaneously
134Difference between the minimum inhibitory concentration of different bacterium mainly is present between the different strains of identical bacterium, and between the different bacterium and between gram-positive microorganism and the Gram-negative bacteria, because the experimental strain comparatively small amt does not find to exist notable difference.
Six, human lysozyme and pure product and antibiotic synergetic antibacterial effect
(1), material
1. bacterial strain: strain surplus the clinical separation strain 400 is provided by Chengdu microbiotic industrial research.
2. pH of buffer 7.2 10mMTris-Cl damping fluids
3.LB substratum
4. clarithromycin, Roxithromycin
(2), method
Human lysozyme and clarithromycin, Roxithromycin (1: 1) are respectively united use,
Method one (test tube method)
With pH7.2 10mM Tris-Cl damping fluid streptococcus aureus (or intestinal bacteria) is made into 10
7-10
8The bacterium liquid of CFU/mL left and right sides concentration joins respectively in the different magnificent test tubes, every pipe 2mL.The human lysozyme (100 μ g-1mg) that adds various dose in the different magnificent test tubes respectively spends the night for 37 ℃, at first gets 20 μ L at every pipe and dilute with 10 times of dilution methods, gets 100 μ L and is applied to the LB substratum, visual inspection turbidity variation or use spectrophotometer, 37 ℃ of overnight incubation; Again every pipe is added 100 μ L 10%SDS (final concentration 0.5%) and shake up, carry out than turbid (640), observations, and calculate total plate count in the original pipe.Total plate count * 100% in human lysozyme bacteriolyze effect=(in the control tube in total plate count-experiment tube total plate count)/control tube.
Method two (flat band method)
Behind the agarose autoclaving with pH7.2 10mM Tris-Cl damping fluid preparation 1%, be cooled to 40 ℃, add bacterium to 10
6Pour plate, punch on flat board with punch tool, the lysozyme soln (0-100mg/ml) that in the hole, adds different concns, 37 ℃ of incubators were hatched 4-6 hour, behind the LB nutrient agar medium autoclaving, be cooled to 40 ℃, be covered on the above-mentioned agarose plate, should shine 20 minutes down as for ultraviolet lamp by flat board.(the LB nutrient agar medium is covered in the agarose plate process that contains bacterium, the free bacteria on agarose surface can be sneaked in the nutrient agar medium, wherein be flushed to the bacterium on nutrient agar medium surface, because the effect of oxygen, the speed of growth is very fast, form mycoderm, influence the observation of bacterial growth situation between agarose and the nutrient agar medium layer) so kill the bacterium on nutrient agar medium surface with ultra violet lamp.37 ℃ of incubator overnight incubation are observed bacterial growth situation between agarose and the nutrient agar medium layer, measure the diameter of the inhibition zone of different concns N,O-Diacetylmuramidase formation, the diameter of inhibition zone " well diameter 4mm person is minimum effective bacteriocidal concentration.(for amphitrichous, the bacterium that motion is active can influence the formation of inhibition zone, can consider to add 0.01% phenylic acid and suppress its motion.)
(3), result
Human lysozyme and clarithromycin, Roxithromycin (1: 1) are respectively united use, can make clarithromycin, Roxithromycin to more than its anti-microbial activity synergy 2-16 times, and indivedual bacterial strain synergy multiples are at 1000 times.Clinical separation persister totally 30 strains that this institute uses are respectively streptococcus aureus 8 strains, pseudomonas aeruginosa 3 strains, intestinal bacteria 7 strains, 1 strain of Bao eel bacillus, serratia marcescens 1 strain, Bacillus proteus 3 strains, alpha streptococcus 1 strain.The bacteriostatic experiment result shows the animal 30 strain animal resistant organisms that this institute uses, all to the human lysozyme sensitivity of gene engineering expression, the human lysozyme of gene engineering expression there are differences at different bacterium the minimum inhibitory concentration of experimental strain, and effectively the Mlc scope is between 50 μ g-1mg/ml.The human lysozyme of finding gene engineering expression simultaneously mainly is present between the different strains of identical bacterium to the difference between the minimum inhibitory concentration of different bacterium, and between the different bacterium and between gram-positive microorganism and the Gram-negative bacteria, because the experimental strain comparatively small amt does not find to exist notable difference.
Seven, reorganization N,O-Diacetylmuramidase
134Body in sterilization experiment ()
(1), material
1, bacterial strain: intestinal bacteria
2, animal: BALB/c mouse
(2), method
The experiment of coli-infection BALB/c mouse is with 10
5～10
9The intestinal bacteria of the logarithmic phase of CFU (colony-forming unit) are inoculated in the BALB/c mouse in 6 ages in week respectively through the abdominal cavity, observations is calculated mortality ratio.
The reorganization N,O-Diacetylmuramidase
134Protect active evaluation with 1 * 10
9The intestinal bacteria abdominal cavity of logarithmic phase is inoculated in the BALB/c mouse in 6 ages in week as infecting contrast, the reorganization N,O-Diacetylmuramidase
134The protection group is injected the reorganization N,O-Diacetylmuramidase of various dose at inoculated bacteria simultaneously
134, observe 7 days results, calculate protection, protection ratio %=[(experimental group surviving rate-control group surviving rate)/the control group mortality ratio] * 100%.
(3), result
The deadly experiment of coli-infection BALB/c mouse is with 10
5～10
9CFU coli-infection BALB/c mouse, the result is from 10
6～10
9CFU infects BALB/c and all can cause death, and death mostly occurs within 3 days, and is basicly stable after 4 days, no longer dead.Wherein 10
8The death of CFU coli-infection BALB/c mouse is rule comparatively, and 10
9All dead in the CFU coli-infection BALB/c mouse 3 days.We adopt 10
9The CFU intestinal bacteria N,O-Diacetylmuramidase of recombinating
134Chinese People's Anti-Japanese Military and Political College enterobacteria infects the experiment of BALB/c mouse passive protection.
With four various dose reorganization N,O-Diacetylmuramidases
134BALB/c mouse is carried out the protection of coli-infection and identify, observed continuously 7 days, all dead in the control group BALB/c mouse 2 days as a result, the protection group death time slightly postponed, at 2-3 days.Not dead BALB/c mouse was no longer dead in 7 days in 3 days.Four various dose reorganization N,O-Diacetylmuramidases
134The activity that all has passive protection opposing coli-infection, wherein protection ratio reaches 80% during 300,000 units/kilogram.
Table 1. Chinese People's Anti-Japanese Military and Political College enterobacteria monoclonal antibody immunity protection experimental result
Tab2.protertion?of?lysozyme?against?V.E.coli
Dose(u/kg) NO.test No.death Protective?rate(％)
30×10
4 20 4 80
15×10
4 20 5 75
7.5×10
4 20 7 65
3.75×10
4 20 11 45
0 15 15 0
Eight, sterilization experiment () in the body of human lysozyme
(1), material
1, bacterial strain: intestinal bacteria
2, animal: BALB/c mouse
(2), method
The experiment of coli-infection BALB/c mouse is with 10
5～10
9The intestinal bacteria of the logarithmic phase of CFU (colony-forming unit) are inoculated in the BALB/c mouse in 6 ages in week respectively through the abdominal cavity, observations is calculated mortality ratio.
Human lysozyme protects active evaluation with 1 * 10
9The intestinal bacteria abdominal cavity of logarithmic phase is inoculated in the BALB/c mouse in 6 ages in week as infecting contrast; human lysozyme protection group at inoculated bacteria simultaneously; inject the human lysozyme of various dose; observe 7 days results; calculate protection, protection ratio %=[(experimental group surviving rate-control group surviving rate)/the control group mortality ratio] * 100%.
(3), result
The deadly experiment of coli-infection BALB/c mouse is with 10
5～10
9CFU coli-infection BALB/c mouse, the result is from 10
6～10
9CFU infects BALB/c and all can cause death, and death mostly occurs within 3 days, and is basicly stable after 4 days, no longer dead.Wherein 10
8The death of CFU coli-infection BALB/c mouse is rule comparatively, and 10
9All dead in the CFU coli-infection BALB/c mouse 3 days.We adopt 10
9The CFU intestinal bacteria carry out human lysozyme Chinese People's Anti-Japanese Military and Political College enterobacteria and infect the experiment of BALB/c mouse passive protection.
Identify with the protection that four various dose human lysozymes carry out coli-infection to BALB/c mouse, observed continuously 7 days that all dead in the control group BALB/c mouse 2 days as a result, the protection group death time slightly postponed, at 2-3 days.Not dead BALB/c mouse was no longer dead in 7 days in 3 days.Four various dose human lysozymes all have the activity of passive protection opposing coli-infection, and wherein protection ratio reaches 80% during 300,000 units/kilogram.
Table 1. Chinese People's Anti-Japanese Military and Political College enterobacteria monoclonal antibody immunity protection experimental result
Tab2.protertion?of?lysozyme?against?V.E.coli
Dose(u/kg) NO.test No.death Protective?rate(％)
30×10
4 20 4 80
15×10
4 20 5 75
7.5×10
4 20 7 65
3.75×10
4 20 11 45
0 15 15 0
Nine, reorganization N,O-Diacetylmuramidase
134Body in sterilization experiment (two)
(1), material
1. bacterial strain: suis
2. animal: BALB/c mouse
(2), method
The experiment of streptococcal infection BALB/c mouse is with 10
5～10
9The vibrio alginolyticus of the logarithmic phase of CFU (colony-forming unit) is inoculated in the BALB/c mouse in 6 ages in week respectively through the abdominal cavity, observations is calculated mortality ratio.
The reorganization N,O-Diacetylmuramidase
134Protect active evaluation with 2 * 10
8The suis abdominal cavity of logarithmic phase is inoculated in the BALB/c mouse in 6 ages in week as infecting contrast, the reorganization N,O-Diacetylmuramidase
134The protection group is injected the reorganization N,O-Diacetylmuramidase of various dose in the inoculation suis simultaneously
134, observe 7 days results, calculate protection, protection ratio %=[(experimental group surviving rate-control group surviving rate)/the control group mortality ratio] * 100%.
(3), result
The deadly experiment of streptococcal infection BALB/c mouse is with 10
5～10
9CFU streptococcal infection BALB/c mouse, the result is from 10
6～10
9CFU infects BALB/c and all can cause death, and death mostly occurs within 3 days, and is basicly stable after 4 days, no longer dead.Wherein 5 * 10
7～5 * 10
8The death of CFU streptococcal infection BALB/c mouse is rule comparatively, and 10
9All dead in the CFU streptococcal infection BALB/c mouse 3 days.We adopt 2 * 10
8The CFU suis N,O-Diacetylmuramidase of recombinating
134Streptococcus infects the experiment of BALB/c mouse passive protection.
With four various dose reorganization N,O-Diacetylmuramidases
134BALB/c mouse is carried out the protection of streptococcal infection and identify, observed continuously 7 days, all dead in the control group BALB/c mouse 3 days as a result, the protection group death time slightly postponed, at 3-5 days.Not dead BALB/c mouse was no longer dead in 7 days in 5 days.Four various dose reorganization N,O-Diacetylmuramidases
134The activity that all has passive protection opposing streptococcal infection, wherein protection ratio reaches 85% during 300,000 units/kilogram.
Table 2. streptococcus monoclonal antibody immunity protection experimental result
Tab2.protertion?of?lysozyme?against?Streptococcus
Dose(u/kg) NO.test No.death Protective?rate(％)
30×10
4 20 3 85
15×10
4 20 5 75
7.5×10
4 20 10 50
3.75×10
4 20 11 45
0 15 15 0
Ten, sterilization experiment (two) in the body of human lysozyme and pure product thereof
(1), material
1. bacterial strain: suis
2. animal: BALB/c mouse
(2), method
The experiment of streptococcal infection BALB/c mouse is with 10
5～10
9The vibrio alginolyticus of the logarithmic phase of CFU (colony-forming unit) is inoculated in the BALB/c mouse in 6 ages in week respectively through the abdominal cavity, observations is calculated mortality ratio.
Human lysozyme protects active evaluation with 2 * 10
8The suis abdominal cavity of logarithmic phase is inoculated in the BALB/c mouse in 6 ages in week as infecting contrast, and human lysozyme protection group is injected the reorganization N,O-Diacetylmuramidase of various dose in the inoculation suis simultaneously
134, observe 7 days results, calculate protection, protection ratio %=[(experimental group surviving rate-control group surviving rate)/the control group mortality ratio] * 100%.
(3), result
The deadly experiment of streptococcal infection BALB/c mouse is with 10
5～10
9CFU streptococcal infection BALB/c mouse, the result is from 10
6～10
9CFU infects BALB/c and all can cause death, and death mostly occurs within 3 days, and is basicly stable after 4 days, no longer dead.Wherein 5 * 10
7～5 * 10
8The death of CFU streptococcal infection BALB/c mouse is rule comparatively, and 10
9All dead in the CFU streptococcal infection BALB/c mouse 3 days.We adopt 2 * 10
8The CFU suis carries out the human lysozyme streptococcus and infects the experiment of BALB/c mouse passive protection.
Identify with the protection that four various dose human lysozymes and pure product thereof carry out streptococcal infection to BALB/c mouse, observed continuously 7 days that all dead in the control group BALB/c mouse 3 days as a result, the protection group death time slightly postponed, at 3-5 days.Not dead BALB/c mouse was no longer dead in 7 days in 5 days.Four various dose reorganization N,O-Diacetylmuramidases
134The activity that all has passive protection opposing streptococcal infection, wherein protection ratio reaches 85% during 300,000 units/kilogram.
Table 2. streptococcus monoclonal antibody immunity protection experimental result
Tab2.protertion?of?lysozyme?against?Streptococcus
Dose(u/kg) NO.test No.death Protective?rate(％)
30×10
4 20 3 85
15×10
4 20 5 75
7.5×10
4 20 10 50
3.75×10
4 20 11 45
0 15 15 0
11, reorganization N,O-Diacetylmuramidase
134The fresh fowl of protection, poultry, fish
(1), material
1, bacterial strain: streptococcus aureus
2, fresh fowl, poultry, the flesh of fish
(2), method
The reorganization N,O-Diacetylmuramidase
134Protect fresh fowl, poultry, the anti-infective experiment of the flesh of fish with 3%NaCl, 12.5ppmNaNO2,30～1000ppm human lysozyme or reorganization N,O-Diacetylmuramidase
134Liquid is made in pure product blending water dilution, in fresh fowl, poultry, flesh of fish fowl dipping.With 1 * 10
9The fresh fowl of the infection of staphylococcus aureus of logarithmic phase, poultry, the flesh of fish.Observations is calculated protection.Protection ratio %=[(experimental group-control group)/the control group infection rate] * 100%
(3), result
The reorganization N,O-Diacetylmuramidase
134Protect fresh fowl, poultry, the anti-infective experimental result of the flesh of fish to show: with the fresh fowl of infection of staphylococcus aureus, poultry, the flesh of fish, with human lysozyme or reorganization N,O-Diacetylmuramidase
134Liquid is made in pure product blending water dilution, and 20 days protection ratios in fresh fowl, poultry, the flesh of fish fowl dipping can be reached more than 95%.Control group all infects.
12, the fresh fowl of the protection of human lysozyme, poultry, fish
(1), material
1, bacterial strain: streptococcus aureus
2, fresh fowl, poultry, the flesh of fish
(2), method
With 3%NaCl, 12.5ppmNaNO
2, 30～1000ppm human lysozyme or the reorganization N,O-Diacetylmuramidase
134Liquid is made in pure product blending water dilution, in fresh fowl, poultry, flesh of fish fowl dipping.With 1 * 10
9The fresh fowl of the infection of staphylococcus aureus of logarithmic phase, poultry, the flesh of fish.Observations is calculated protection.Protection ratio %=[(experimental group-control group)/the control group infection rate] * 100%
(3), result
Human lysozyme protects fresh fowl, poultry, the anti-infective experimental result of the flesh of fish to show: with the fresh fowl of infection of staphylococcus aureus, poultry, the flesh of fish, with human lysozyme or reorganization N,O-Diacetylmuramidase
134Liquid is made in pure product blending water dilution, and 20 days protection ratios in fresh fowl, poultry, the flesh of fish fowl dipping can be reached more than 95%.Control group all infects.
13, human lysozyme and reorganization N,O-Diacetylmuramidase
134The anti-infective experiment of protection milk
(1), material
1. bacterial strain: intestinal bacteria
2. fresh milk
(2), method
The reorganization N,O-Diacetylmuramidase
134The anti-infective experiment human lysozyme of protection fresh milk, reorganization N,O-Diacetylmuramidase
134Pure product generally add 0.0001-0.05% by following ratio of weight and number and add fresh milk, establish the normal control group.With 5 * 10
8The intestinal bacteria of logarithmic phase are inoculated in protection group and control group fresh milk respectively.Observations is calculated protection.Immune protective rate %=[(experimental group-control group)/the control group infection rate] * 100%
(3), result
The reorganization N,O-Diacetylmuramidase
134The anti-infective experimental result of protection fresh milk shows: use the coli-infection fresh milk, human lysozyme, reorganization N,O-Diacetylmuramidase
13440 days protection ratios of pure product protection fresh milk can reach 95%, and the normal control group all infects.
14, human lysozyme and reorganization N,O-Diacetylmuramidase
134The anti-infective experiment of protection fruit wine
(1), material
1, bacterial strain: intestinal bacteria
2, grape wine, dry red winew, Dry white wine
(2), method
Human lysozyme and reorganization N,O-Diacetylmuramidase
134The anti-infective experiment human lysozyme of protection fruit wine, reorganization N,O-Diacetylmuramidase
134Pure product generally add 0.0001-0.05% by following ratio of weight and number and add grape wine, dry red winew, Dry white wine, establish the normal control group.With 5 * 10
8The intestinal bacteria of logarithmic phase are inoculated in protection group and control group grape wine, dry red winew, Dry white wine respectively.Observations is calculated protection.Immune protective rate %=[(experimental group-control group)/the control group infection rate] * 100%
(3), result
The reorganization N,O-Diacetylmuramidase
134The anti-infective experimental result of protection grape wine, dry red winew, Dry white wine shows: with coli-infection grape wine, dry red winew, Dry white wine, and human lysozyme, reorganization N,O-Diacetylmuramidase
134Pure product protection grape wine, dry red winew, 40 days protection ratios of Dry white wine can reach 95%, and the normal control group all infects.
15, human lysozyme and reorganization N,O-Diacetylmuramidase
134The anti-infective experiment of protection fresh vegetables fruit
(1), material
1. bacterial strain: streptococcus aureus, suis, intestinal bacteria,
2. fresh vegetables fruit
(2), method
Human lysozyme and reorganization N,O-Diacetylmuramidase
134The anti-infective experiment human lysozyme of protection fresh vegetables fruit, reorganization N,O-Diacetylmuramidase
134Reach pure product 300U～150000U% and protect fresh vegetables fruit, establish the normal control group with the mode of spraying.With 1 * 10
9The mode of the streptococcus aureus of logarithmic phase, suis, intestinal bacteria spraying is inoculated in protection group and control group fresh vegetables fruit respectively.Observations is calculated protection.Immune protective rate %=[(experimental group-control group)/control group] * 100%
(3), result
Human lysozyme and reorganization N,O-Diacetylmuramidase
134The anti-infective experimental result of protection fresh vegetables fruit shows: after infecting fresh vegetables fruit respectively with the mode of streptococcus aureus, suis, intestinal bacteria spraying, with the human lysozyme and the N,O-Diacetylmuramidase of recombinating
134Spraying protection fresh vegetables fruit 20 days, protection ratio can reach more than 95%.Normal control group all rots.
16, human lysozyme and reorganization N,O-Diacetylmuramidase
134The anticorrosion experiment of protection makeup
(1), material
1, bacterial strain: mould
2, makeup
(2), method
Human lysozyme and reorganization N,O-Diacetylmuramidase
134The anticorrosion experiment of protection makeup, human lysozyme, reorganization N,O-Diacetylmuramidase
134Pure product generally add in the 0.0001-0.05% adding makeup by following ratio of weight and number protects makeup, establishes the normal control group.With 1 * 10
9The mould of logarithmic phase is inoculated in protection group and control group makeup respectively.Observations is calculated protection.Immune protective rate %=[(experimental group-control group)/control group] * 100%
(3), result
Human lysozyme and reorganization N,O-Diacetylmuramidase
134The anticorrosion experimental result of protection makeup shows: after infecting makeup respectively with mould, with human lysozyme and reorganization N,O-Diacetylmuramidase
134The anticorrosion experimental result of protection makeup 240 days, protection ratio can reach more than 95%.Normal control group is all mouldy.
Positively effect of the present invention is that its raw material sources are abundant, inexpensive, has no side effect; Preparation technology is simple.
Embodiment:
The present invention will be further described below in conjunction with embodiment:
Embodiment 1:
Human lysozyme and pure product thereof are joined in the wrap food as antistaling and antiseptic agent in 0.0001% ratio.
Embodiment 2:
Human lysozyme and pure product 0.05% thereof are joined in the packing fruit as antistaling and antiseptic agent.
Embodiment 3:
The N,O-Diacetylmuramidase of will recombinating
134And pure product join in the vegetable based food as antistaling and antiseptic agent in 0.03% ratio.
Embodiment 4:
The N,O-Diacetylmuramidase of will recombinating
134And pure product join grape wine, dry red winew, Dry white wine and various fruit wine as antistaling and antiseptic agent in 0.03% ratio.
Embodiment 5:
With 3%NaCl, 12.5ppmNaNO
2, the dilution of 30ppm human lysozyme and pure product blending water thereof makes liquid and adds in the packing cheese food as antistaling and antiseptic agent.
Embodiment 6:
With 3%NaCl, 12.5ppmNaNO
2, the dilution of 1000ppm human lysozyme and pure product blending water thereof makes liquid and adds in the beverage as antistaling and antiseptic agent.
Embodiment 7:
With 3%NaCl, 12.5ppmNaNO
2, the 80ppm N,O-Diacetylmuramidase of recombinating
134And pure product blending water dilution is made in the liquid adding milk as antistaling and antiseptic agent.
Embodiment 8:
With 3%NaCl, 12.5ppmNaNO
2, the 900ppm N,O-Diacetylmuramidase of recombinating
134And pure product blending water dilution is made liquid adding nectar as in the antistaling and antiseptic agent.
Embodiment 9:
Human lysozyme and pure product 300U dilute with water thereof are made liquid, fresh fowl, poultry, fish are butchered cleaning after, immerse in the ice-water bath of this liquid; soak more than 1 hour; can pack after the taking-up, fresh keeping time is more than 20 days, and protection ratio can reach more than 95%.
Embodiment 10:
Human lysozyme and pure product 150000U dilute with water thereof are made liquid, fresh fowl, poultry, fish are butchered cleaning after, immerse in the ice-water bath of this liquid; soak more than 1 hour; can pack after the taking-up, fresh keeping time is more than 20 days, and protection ratio can reach more than 95%.
Embodiment 11:
The N,O-Diacetylmuramidase of will recombinating
134And pure product join in the makeup as antistaling and antiseptic agent by 1000U.
Embodiment 12:
The N,O-Diacetylmuramidase of will recombinating
134And pure product join packaging of meat can and sauce can and fruit can food preservative by 120000U.
Claims (10)
1, gene recombination N,O-Diacetylmuramidase
134The application in various wrap food preservation and antisepsis with gene recombinant human lysozyme and pure product thereof.
2, gene recombination N,O-Diacetylmuramidase
134The application in various packing makeup are anticorrosion with gene recombinant human lysozyme and pure product thereof.
3, gene recombination N,O-Diacetylmuramidase
134The application in grape wine, dry red winew, Dry white wine and various fruit wine preservation and antisepsis with gene recombinant human lysozyme and pure product thereof.
4, gene recombination N,O-Diacetylmuramidase
134The application in various packing milk, beverage and fruit juice food preservation and antisepsis with gene recombinant human lysozyme and pure product thereof.
5, gene recombination N,O-Diacetylmuramidase
134With the application in various packing fresh meats and cold cuts food fresh keeping are anticorrosion of gene recombinant human lysozyme and pure product thereof.
6, gene recombination N,O-Diacetylmuramidase
134With the application in various packing fresh fish meat and ripen fish food fresh keeping are anticorrosion of gene recombinant human lysozyme and pure product thereof.
7, gene recombination N,O-Diacetylmuramidase
134The application in various packing fruits, vegetable based food preservation and antisepsis with gene recombinant human lysozyme and pure product thereof.
8, gene recombination N,O-Diacetylmuramidase
134With the application in various packaging of meat cans and sauce can and fruit can food fresh keeping are anticorrosion of gene recombinant human lysozyme and pure product thereof.
9, gene recombination N,O-Diacetylmuramidase
134The application in various packing cheese food preservation and antisepsis with gene recombinant human lysozyme and pure product thereof.
10, comprise that according to claim 1,2,3,4,5,6,7,8,9, described N,O-Diacetylmuramidase the human lysozyme of gene engineering expression, the aminoterminal of gene engineering expression human lysozyme have (L-glutamic acid-L-Ala)
2Or (L-glutamic acid-L-Ala)
3Human lysozyme, gene engineering expression or the chemosynthesis mutant human lysozyme modified.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410010842 CN1570096A (en) | 2004-05-12 | 2004-05-12 | Use of gene recombinant lysozyme #-[134] and gene recombinant human lysozyme and its pure products in antistaling and antiseptic agent |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410010842 CN1570096A (en) | 2004-05-12 | 2004-05-12 | Use of gene recombinant lysozyme #-[134] and gene recombinant human lysozyme and its pure products in antistaling and antiseptic agent |
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| Publication Number | Publication Date |
|---|---|
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
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Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1570096A (en) |
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2004
- 2004-05-12 CN CN 200410010842 patent/CN1570096A/en active Pending
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