CN101235408A - Method for preparing Lactoferrins antibiotic peptide by enzyme method - Google Patents
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Abstract
The invention relates to a method for preparing lactoferrin antibacterial peptide through an enzyme method which belongs to the technical field for preparing the lactoferrin antibacterial peptide. The method of the invention comprises: taking lactoferrin as raw materials, adopting pig pepsin to hydrolyze the lactoferrin under certain condition, and finally obtaining lactoferrin antibacterial peptide mixture with stronger antibacterial activity. The method has simple process and strong practicability, the lactoferrin antibacterial peptide which is obtained has stronger antibacterial property and is broad-spectrum antibacterial peptide, gram-negative bacteria and gram-positive bacteria which contain pathogenic escherichia coli can be inhibited, eukaryotic cells are not affected, and the lactoferrin antibacterial peptide is functional antibacterial peptide and has better application prospect. The method for preparing the lactoferrin antibacterial peptide has important significance for promoting dairy products deep processing and colostrum resources comprehensive utilization, increasing health level of our people especially infants, and researching and developing functional food additives.
Description
Technical field
The present invention relates to a kind of method of preparing Lactoferrins antibiotic peptide by enzyme method, being specifically related to the lactoferrin is raw material, adopts the porcine pepsin lactoferrin hydrolysate, and is the test bacterial classification with intestinal bacteria, detects the activity of antibacterial peptide.Belong to the lactoferrin antimicrobial peptide preparing technical field.
Background technology
Modern biological metabolism is discovered: the protein of human picked-up only absorbs with amino acid whose form unlike thinking in the past through after the gastral plurality of enzymes hydrolysis, more is that the form with little peptide directly absorbs.Wherein some little peptide can not only provide the human body required nutritive substance that grows, and have the important physical function simultaneously, as promote mineral substance absorb, prevent the hepatopathy encephalopathic, antibiotic, suppress the angiotensin-converting enzyme vigor, reduce the blood vessel cholesterol level, improve the function of body immunity etc.These little peptides are called bioactive peptide by people.
Bioactive peptide can be divided into four classes by the acquisition methods difference: (one) extracts (as peptide antibiotics, hormone etc.) from the natural biological body; (2) chemical synthesis; (3) DNA recombination method; (4) external enzymolysis protein produces.Diverse ways is fit to different purposes, depends on length, quantity and the purposes of purpose peptide.Every kind of method has its relative merits.The content of natural biologically active peptides seldom, the biologically active peptides that can extract is wherein studied, but can not large-scale development uses.Chemical synthesis is widely used in producing the pharmacology level peptide of high price, but its cost height, and also byproduct is harmful.Recombinant DNA technology is applicable to long-chain polypeptide and proteinic production, also is widely used in laboratory and the industrial production now, develops prematurity still but compare with other method.The Production by Enzymes functional peptides has many good qualities, and high and can locate and produce specific peptide as working condition gentleness, Product Safety, cost is low, has become the topmost production method of biologically active peptides.
Antibacterial peptide is the little peptide of the heat stable alkalescence of a class, and molecular weight is about 4 KD, iso-electric point 8.9-10.7.Anti-microbial activity (general pressing down killed concentration all in micromole's level) with broad-spectrum high efficacy is to multiple Gram-negative (G
-) bacterium and Gram-positive (G
+) bacterium, particularly endurance strain have stronger inhibitory or killing effect, also can press down and kill some fungi, virus and protozoon, and most cancer cells and animal solid tumor are had tangible lethal effect.Antibiotic mechanism uniqueness does not have splitting action to normal mammalian cell and yeast, and bacterium is developed immunity to drugs and intercrossing.Promise to be antiseptic-germicide of new generation.Antibacterial peptide is also different with traditional antitumor drug, can not cause damage to hemopoietic system, thereby avoid infringement that the human body antineoplastic immune is caused.Most of antibacterial peptides nearly all are cationic in essence, also are cationic peptide.
Owing to have broad-spectrum antibacterial action, can kill the antibiotics resistance bacterial strain, and bactericidal mechanism makes pathogenic bacteria be difficult for producing the resistance sudden change, and can press down some fungies, virus, parasite and the characteristics such as tumour cell and solid tumor of killing, antibacterial peptide is expected to exploitation becomes antibacterium of new generation, antimycotic, antiviral and cancer therapy drug.Because it has a extensive future, now become a research focus of life science.At present, existing abroad more than 10 kind of antibacterial peptide enters human clinical's experiment as the new drug of treatment bacterium, viral infection and tumour.
Raising along with people's living standard, people are also more and more higher to the requirement of food, picked-up food is not only in order to solve the basic living problems, and also is not only in order to obtain essential nutrition surviving, but also requires it to have the function of certain adjusting physiological activity.At present, functional foodstuff has become a research focus of food service industry, is described as the food of 21 century, and representative is when the trend of food substitutes development.From the research of physiological activity material, or from specific human consumer's particular requirement, carrying out the exploitation of new type functional food, is the developing direction of present functional foodstuff.
Biological activity protein natural for deriving from, have potential application foreground in the food and medicine field, lactoferrin is worth people's more concern.Since 1992, the international symposium of relevant lactoferrin view has been held repeatedly, and all previous meetings have been carried out extensive discussion with regard to structure, character, physiologically active and the application clinically of lactoferrin.This shows that the distinctive biological significance of lactoferrin has been subjected to worldwide extensive attention.Recent studies proves, though lactoferrin has anti-microbial activity, its thermotolerance is relatively poor, and not only solvability is good, digestibility is high for newborn peptide, acidproof, heat-resisting, and has multiple physiological regulation function.
Summary of the invention
The purpose of this invention is to provide a kind of is the method for the stronger antibacterial peptide of feedstock production germ resistance with the lactoferrin.
Technical scheme of the present invention: a kind of method of preparing Lactoferrins antibiotic peptide by enzyme method is a raw material with the lactoferrin, adopts the porcine pepsin lactoferrin hydrolysate, and is the test bacterial classification with intestinal bacteria, detects the bacteriostatic activity of antibacterial peptide.
The lactoferrin solution of mass concentration 2%-3.5% is added constantly stirring in the isothermal reaction container, after reaching 40-50 ℃, insulation 5min, HCl with 1.0mol/L regulates pH2.0-2.5, add porcine pepsin, enzyme concentration is the 1%-2% of substrate lactoferrin quality, in reaction process, constantly stir, and dropping 1.0mol/LHCl is invariable to keep pH, and at 40-50 ℃ of following enzymolysis 2-2.5h, control DH value 10%-12% is a reaction end, stop to stir, and temperature is raised to 80 ℃ rapidly, and keep 15min with deactivating enzyme, add 1.0mol/LNaOH and adjust pH to 7.0,15000 * g frozen centrifugation 30min, the supernatant liquor lyophilize, the lyophilized powder that obtains is lactoferrin antimicrobial peptide, and its molecular weight is distributed in 200～6000Da mostly.
Optimized process conditions is: the lactoferrin solution of mass concentration 3% is added in the isothermal reaction container constantly stirs, reach 45 ℃ after, insulation 5min regulates pH2.5 with the HCl of 1.0mol/L.Add porcine pepsin, enzyme concentration is 1% of a substrate lactoferrin quality.In reaction process, constantly stir, and dropping 1.0mol/L HCl is invariable to keep pH.At 45 ℃ of following enzymolysis 2h, control DH value 10%-12% is a reaction end, stops to stir, and temperature is raised to 80 ℃ rapidly, keeps 15min with deactivating enzyme.Add 1.0mol/LNaOH and adjust pH to 7.0,15000 * g frozen centrifugation 30min.The supernatant liquor lyophilize, the lyophilized powder that obtains is lactoferrin antimicrobial peptide, and its molecular weight is distributed in 200～6000Da mostly.
Beneficial effect of the present invention: flow process of the present invention is simple, practical, resulting lactoferrin antimicrobial peptide germ resistance is stronger, be a kind of broad-spectrum antimicrobial peptide, can suppress to comprise the Gram-negative bacteria and the gram-positive microorganism of pathogenic colon bacillus, to not influence of eukaryotic cell, it is a kind of functional antibiosis peptide, its solvability is good, digestibility is high, acidproof, heat-resisting, and have multiple physiological regulation function, have application promise in clinical practice.The present invention improves particularly infant's health level of China people for the deep processing that promotes milk-product and the comprehensive utilization of colostrum resource, and the research and development functional food ingredient all has great importance.
Analytical procedure
1, the mensuration of degree of hydrolysis
Get the 0.25mL enzymolysis solution, joining 10mL contains in the test tube of 1% sodium laurylsulfonate (SDS) solution, in 75 ℃ of water-baths, be incubated 15min, and constantly shake test tube, be diluted to required concentration with SDS then, get this diluent 0.25mL, add the phosphate buffer soln of 2mL 0.21mol/L, pH8.20 and trinitro-benzene-sulfonic acid (TNBS) solution of 1.0mL 0.1%, put into 50 ℃ of thermostat water baths, behind the lucifuge reaction 1h, the HCl stopped reaction that adds 4mL 0.1%, room temperature is placed 30min, 340nm colorimetric.The degree of hydrolysis formula:
In the formula: h
TotPeptide bond sum (mmol/g) is about 8.3 in the-substrate.
2, the mensuration of anti-microbial activity
The mensuration of bacteriostasis rate adopts colony counting method.With the bacterial classification inoculation of preserving (some) on nutrient agar slant medium,, activate continuously 2 times in 37 ℃ of cultivation 24h.Through 2 activatory experimental strains, add stroke-physiological saline solution washing after, be transferred to sterile test tube and vibration is evenly distributed thalline.According to absorbance (A
560nm) concern typical curve with bacterial concentration, bacterium liquid is transferred to 10
6Cfu/mL.Get the test tube that 4mL meat soup protein culture medium (peptone 1%, beef extract 0.3% and NaCl0.5%) is housed, number respectively, it is 10 that each test tube adds bacterium dense
6The bacterium liquid 0.5mL of cfu/mL adds lactoferrin range of hydrolysed peptides solution 0.5mL under the different enzymatic hydrolysis conditions then respectively in the experimental group test tube, place 37 ± 1 ℃ of shaking tables to cultivate then.After cultivating 6h, plate count.The biocidal property size represents that with Y calculation formula is:
3, aminoacid component analysis
Automatic analyzer for amino acids method: lactoferrin range of hydrolysed peptides solution example under the different enzymatic hydrolysis conditions is placed the hydrolysis pipe, the HCl solution that adds 6mol/L, vacuum seal, at 110 ℃ of following hydrolysis 24h, cooling back constant volume, filtration, evaporate to dryness, the HCl solution that adds 0.02mol/L is again placed 30min in air, last machine is measured aminoacids content.The results are shown in Table 1.
The amino acid of table 1 lactoferrin and antibacterial peptide thereof is formed
| Amino acid | Lactoferrin (%) | Lactoferrin antimicrobial peptide (%) |
| ASP GLU SER HIS GLY THR ARG ALA TYR CYS VAL MET PHE ILE LEU LYS PRO | 9.76 13.82 5.70 2.44 4.51 5.27 7.25 6.26 4.32 2.65 5.26 0.12 5.29 2.77 10.30 9.96 4.32 | 10.80 13.95 5.64 2.43 4.13 4.91 6.76 6.47 4.02 1.65 5.33 1.00 5.09 2.66 10.50 9.96 4.69 |
4, relative molecular mass Determination of distribution
(high performance size exclusion chromatography, HPSEC) relative molecular mass of detection lactoferrin polypeptide distributes with the high performance liquid phase exclusion chromatography.
Instrument: Waters 600 high performance liquid chromatographs (joining 2487 UV-detector (wavelength 220nm) and M 32 workstations);
Chromatographic column: TSKgel 2000 SWXL 300nm * 7.8mm
Mobile phase volume ratio: ethanol: water: Mono Chloro Acetic Acid=45: 54: 1;
Detect wavelength: 220nm;
Flow: 0.5mL/min;
Column temperature: 30 ℃;
Specimen preparation: draw sample 2mL in the 10mL volumetric flask, be diluted to scale, with the laggard sample of millipore filtration membrane filtration with moving phase.Institute's test sample product molecular weight is distributed in 200-6000Da mostly.
Determining of processing condition
1, single factor experiment
The screening of enzyme: select 537 aspartic proteases, stomach en-, AS1398 neutral protease, Alcalase Sumizyme MP, 5 kinds of protease hydrolysis lactoferrins of trypsinase respectively for use, and with analytical procedure 1,2
Survey the different time degree of hydrolysis, and the bacteriostasis rate of last hydrolysate.
Determining of enzymolysis time: DH is along with the prolongation of hydrolysis time constantly raises, but differential responses time response trend is obviously different.DH significantly raises in 15 min of beginning, and along with the carrying out of hydrolysis, the increase trend of DH is slowed down, but still in rising trend.It is milder that 2～4h increases trend, changes very little.Therefore, the hydrolysis time with lactoferrin is decided to be 2-2.5h.
Determining of hydrolysis temperature: temperature influences the efficient of enzyme digestion reaction from two aspects of stability of proteolytic enzyme catalyzed reaction speed and proteolytic enzyme.Pepsic optimum temperuture is generally between 35 ℃～55 ℃, and is different with the difference of substrate.At pH2.0, enzyme concentration ([E]/[S]) 3%, under the condition of [S] 50mg/mL, enzymolysis 2h measures the DH under the differing temps.The result shows that temperature has considerable influence to the hydrolysis of lactoferrin, and along with temperature raises, DH increases afterwards earlier and reduces.DH rises very slowly in the time of 35 ℃～40 ℃, but has stronger activity at 40 ℃～45 ℃ enzymes, and DH rises the fastest.In the time of 45 ℃～55 ℃, DH descends rapidly.Therefore, temperature is comparatively suitable at 40-50 ℃.
Determining of concentration of substrate: at pH2.0, [E]/[S] is 3%, and temperature is enzymolysis 2h under 45 ℃ the condition, measures different [S] influence to DH.The result shows that under the enzyme concn controlled condition, (10mg/mL, 20mg/mL), along with the increase of [S] concentration, DH also increases gradually when [S] is very little.When [S] was 30mg/mL, DH reached maximum value.Increase along with [S] continues, DH descends rapidly.Therefore, determine that [S] is 20-35mg/mL, preferred [S] is 30mg/mL.
The enzyme-to-substrate concentration ratio: at pH2.0, [S] 30mg/mL, enzymolysis 2h under the condition that temperature is 45 ℃ measures the DH under different [E]/[S] conditions, and the result shows, is 0.5% o'clock at [E]/[S], and DH is less, is 1%～4% o'clock at [E]/[S], and DH increases not obvious.Therefore, selecting [E]/[S] is that 1%-2% is comparatively suitable.Preferably [E]/[S] is 1%.
2, response surface regression analysis
Adler-Nissen thinks that influencing the enzyme digestion reaction primary variables is temperature, pH, [E]/[S].So select pepsin hydrolysis LF for use, determine that [S] is that 30mg/mL, hydrolysis time are 2h, to hydrolysis temperature (X
1), enzyme-to-substrate concentration ratio (X
2) and enzymolysis pH (X
3) carry out quadratic regression design experiment and analysis, be response value with DH, bacteriostasis rate (Y/%).Adopt the Box-Behnken design that enzymatic hydrolysis condition is further optimized, see Table 2, table 3.
The level of factor that table 2 enzymolysis process is optimized
| Level | X 1 | X 2 | X 3 |
| -1 0 1 | 40 45 50 | 0.5％ 1％ 1.5％ | 2.0 2.5 3.0 |
Table 3 Box-Behnken test design and result
| Experimental point | X 1 | X 2 | X 3 | DH/％ | Y/％ |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 | -1 -1 1 1 0 0 0 0 -1 1 -1 1 0 0 0 | -1 1 -1 1 -1 -1 1 1 0 0 0 0 0 0 0 | 0 0 0 0 -1 1 -1 1 -1 -1 1 1 0 0 0 | 9.81 12.77 9.70 11.16 9.35 8.45 12.45 11.97 10.93 10.47 9.78 10.57 13.01 12.99 12.93 | 95.73 96.97 97.37 95.07 96.73 70.33 96.67 80.67 98.83 96.60 62.67 67.33 96.50 96.30 96.37 |
45 ℃ of hydrolysis temperatures, enzyme-to-substrate concentration ratio 1% and enzymolysis pH2.5 are preferred state of the art.Revision test under these processing condition (test sequence number 13-15) shows that average DH value is 13%, and bacteriostasis rate is 96%.
Description of drawings
Hydrolysis process curve and corresponding germ resistance under the optimum hydrolysising condition of Fig. 1.
Embodiment
Embodiment 1
Above-mentioned to specifications operational path adds the lactoferrin solution of mass concentration 3% in the isothermal reaction container and constantly to stir, reach 45 ℃ after, insulation 5min regulates pH2.5 with the HCl of 1.0mol/L.Add porcine pepsin, enzyme concentration is 1% of a substrate lactoferrin quality.In reaction process, constantly stir, and dropping 1.0mol/L HCl is invariable to keep pH.At 45 ℃ of following enzymolysis 2h, be reacted to terminal point, the DH value that records is 13%, stops to stir, and temperature is raised to 80 ℃ rapidly, keeps 15min with deactivating enzyme.Add 1.0mol/L NaOH and adjust pH to 7.0,15000 * g frozen centrifugation 30min.The supernatant liquor lyophilize, the lyophilized powder that obtains is lactoferrin antimicrobial peptide, and bacteriostasis rate is 96%.
Claims (2)
1, a kind of method of preparing Lactoferrins antibiotic peptide by enzyme method is characterized in that with the lactoferrin being raw material, adopts the porcine pepsin lactoferrin hydrolysate; The lactoferrin solution of mass concentration 2%-3.5% is added constantly stirring in the isothermal reaction container, after reaching 40-50 ℃, insulation 5min, HCl with 1.0mol/L regulates pH2.0-2.5, add porcine pepsin, enzyme concentration is the 1%-2% of substrate lactoferrin quality, in reaction process, constantly stir, and dropping 1.0mol/L HCl is invariable to keep pH, and at 40-50 ℃ of following enzymolysis 2-2.5h, control DH value 10%-12% is a reaction end, stop to stir, and temperature is raised to 80 ℃ rapidly, and keep 15min with deactivating enzyme, add 1.0mol/L NaOH and adjust pH to 7.0,15000 * g frozen centrifugation 30min, the supernatant liquor lyophilize, the lyophilized powder that obtains is lactoferrin antimicrobial peptide, and its molecular weight is distributed in 200～6000Da mostly.
2, method according to claim 1 is characterized in that preferred processing condition are; The lactoferrin solution of mass concentration 3% is added constantly stirring in the isothermal reaction container, after reaching 45 ℃, insulation 5min, HCl with 1.0mol/L regulates pH2.5, add porcine pepsin, enzyme concentration is 1% of a substrate lactoferrin quality, in reaction process, constantly stir, and dropping 1.0mol/L HCl is invariable to keep pH, and at 45 ℃ of following enzymolysis 2h, control DH value 10%-12% is a reaction end, stop to stir, and temperature is raised to 80 ℃ rapidly, and keep 15min with deactivating enzyme, add 1.0mol/L NaOH and adjust pH to 7.0,15000 * g frozen centrifugation 30min, the supernatant liquor lyophilize, the lyophilized powder that obtains is lactoferrin antimicrobial peptide, and its molecular weight is distributed in 200～6000Da mostly.
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| CN102051398B (en) * | 2009-10-29 | 2012-10-17 | 浙江海洋学院 | Method for preparing half-fin anchovy antibacterial peptides |
| CN101744124B (en) * | 2010-01-29 | 2012-09-19 | 浙江大学 | Method for preparing milk-derived antimicrobial peptide |
| CN102477457A (en) * | 2010-11-23 | 2012-05-30 | 深圳市圣西马生物技术有限公司 | Sensitive, accurate and visual method for detecting antibacterial peptide product suitable for industrial production |
| CN104757125A (en) * | 2015-04-24 | 2015-07-08 | 光明乳业股份有限公司 | Brine gel for storing fresh Mozzarella cheese and preparation method of brine gel |
| CN104770476A (en) * | 2015-04-24 | 2015-07-15 | 光明乳业股份有限公司 | Storage method for fresh mazerella cheese |
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| CN104770476B (en) * | 2015-04-24 | 2018-01-16 | 光明乳业股份有限公司 | A kind of storage method of fresh horse Soviet Union lira cheese |
| CN106377762A (en) * | 2016-08-24 | 2017-02-08 | 方雅悯 | Application of bovine lactoferrin and zymolytes thereof in products for protecting stomach and liver |
| CN110507814A (en) * | 2019-10-12 | 2019-11-29 | 延边大学 | A kind of suspension and application method for treating young deer yellowish-white dysentery |
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Assignee: Wuxi Super Food Technology Coffee Co., Ltd. Assignor: Jiangnan University Contract record no.: 2012990000274 Denomination of invention: Method for preparing Lactoferrins antibiotic peptide by enzyme method Granted publication date: 20110105 License type: Exclusive License Open date: 20080806 Record date: 20120426 |
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