CN102051398A - Method for preparing half-fin anchovy antibacterial peptides - Google Patents
Method for preparing half-fin anchovy antibacterial peptides Download PDFInfo
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Abstract
The invention relates to a method for preparing half-fin anchovy antibacterial peptides, which comprises the following steps of: homogenating fish into fish paste, adding pepsin into the fish paste according to a ratio of 1,000-1,300U/g under the condition that the pH value is 1.5-2.5, raising the temperature to 35-45DEG C, performing enzymolysis for 2 to 3 hours, heating enzymolysis liquid at the temperature of between 95 and 100DEG C for 5 to 15 minutes for killing enzyme, cooling to the temperature of between 3 and 5DEG C, centrifuging at a rate of 6,000 to 8,000r/min for 5 to 15 minutes, filtering, taking intermediate filtrate, and performing frozen storage or freeze-dried storage at -20DEG C at least. The antibacterial peptides is prepared from a pure natural low-value marine product, namely half-fin anchovy; the half-fin anchovy antibacterial peptides are rich in amino acid necessary for human bodies, and have stable physical and chemical properties; the antibacterial peptides prepared from food-derived protein through enzymolysis have high safety, good absorptivity and good antibacterial effect, and can serve as a nutritional and functional additive in foods or is developed into drug resistant bacteria antibiotics. The preparation method is simple, economic and easy to operate, can be carried out in a common laboratory, and is suitable for industrial expanded production.
Description
Technical field
The present invention relates to the preparation method of albumen antibacterial peptide, especially a kind of preparation method of yellow crucian carp albumen antibacterial peptide.
Background technology
At the microbiotic abuse, today that Resistant strain constantly produces, the research of antibacterial peptide is attracting the sight of Chinese scholars, and people explore the new mechanism of action of antibacterial peptide constantly seeking new antibacterial peptide.(antibacterialpeptide, (antimicrobial peptide AMP) is distributed widely in bacterium, virus and the various animal and plant body antibacterial peptide, is the important component part of biological innate immune defence system ABP) to claim antimicrobial peptide again.The AMP majority is that the molecule amount is less than the 10kDa small-molecular peptides, chain length, sequence and structure have multifarious cationic amphiphic peptide class (Boman H G.Antibactericidal peptides:key components needed in immunity[J] .Cell, 1991,65:205-207; Hultmark D.Drosophila immunity:paths and pattems[J] .Current Opinion in Immunology, 2003,15:12-19.).First antibacterial peptide Cecropin be Sweden scientist Steiner etc. induced from cherish guppy giant silkworm (Hyatophora cecropia) pupa in 1981 separate obtain a kind of cecropin (Steiner H, Hultmark D,
., et al.Sequence and specificity of two antibacterial proteins involved in insect immunity[J] .Nature, 1981,292:246-248.).The preparation method of antibacterial peptide has at present: direct extraction method, chemical synthesis, gene engineering research, enzymolysis process from animal vegetable tissue.Directly extract antibacterial peptide from the natural animal-plant tissue and be confined to several main animals and plants at present, and need a large amount of solvents, the separation purification difficult is bigger; Chemical synthesis is along with the increase of amino acid no, and error rate and side reaction also increase, and the cost height; Gene engineering research since the antibacterial peptide molecule little, easily by proteasome degradation, and expression product may be harmful to the host, thereby (cloth is preced with to have influenced the high level expression of gene, Li Hongji, Yang Guoyu, Deng. the effect characteristics of antibacterial peptide and application prospect [J]. animal medicine progress, 2005,26 (3): 26-28.).Bolscher etc. think that adopting enzymolysis protein matter method to produce antibacterial peptide may be to produce the most promising method of antibacterial peptide (Bolscher J GM in enormous quantities, Van der Kraan M I A, Nazmi K., et al.A one-enzyme strategy to release an antimicrobialpeptide from the LFampin-domain of bovine lactoferrin[J] .peptides, 2006,27:1-9.).Enzymolysis process mild condition, the reaction process advantage such as control easily that responds.The protein of no antibiotic effect own can be by changing protein configuration or obtaining anti-microbial activity by the enzymolysis mode, and the albumen with anti-microbial effect can be used as precursor substance (the L ó pez Exp ó sito I with new function and stronger anti-microbial effect, Recio I.Antibacterial activity ofpeptides and folding variants from milk proteins[J] .International Dairy Journal, 2006, (16): 1294-1305.).
With food source albumen is feedstock production antibacterial peptide research, mainly concentrates on the milk protein aspect both at home and abroad, and relevant other food source albumen to be matrix directly report seldom by the research that enzyme butt formula obtains to have antibacterial active peptide.This research is raw material with the yellow crucian carp of large marine products low value fish, determine the top condition of antibacterial peptide preparation by response surface optimized method (RSM), and the nutritive ingredient and the physical and chemical stability of antimicrobial fluid measured, the result shows that essential amino acids content is abundant in the yellow crucian carp albumen antibacterial peptide, physico-chemical property is stable.Because the antibacterial peptide of food source proteolysis gained is safe, absorptivity good, so yellow crucian carp albumen antibacterial peptide has in food as nutrition and functional type Application of Additives prospect.
Summary of the invention
Technical problem to be solved by this invention provides a kind of preparation method of yellow crucian carp albumen antibacterial peptide, and prepared antibacterial peptide is safe, absorptivity good, and the preparation method is scientific and reasonable.
The present invention solves the problems of the technologies described above the technical scheme that is adopted: a kind of preparation method of yellow crucian carp albumen antibacterial peptide is characterized in that may further comprise the steps:
1) will oppress to clean and cut fritter, blend evenly, make fish meat emulsion, according to 3～5 multiple proportions example adding deionized water of fish meat emulsion quality with tissue mashing machine;
2) regulate pH value to 1.5～2.5 of fish meat emulsion and water mixed liquid, and keep constant;
3) according to the quality of fish meat emulsion, stomach en-joins in the mixed solution with the ratio of 1000～1300U/g, under the condition that keeps 35～45 ℃ of hydrolysis temperatures, and enzymolysis 2～3 hours;
4) the passivation enzyme that goes out ℃ is carried out in heat temperature raising to 95～100, goes out 5～15 minutes enzyme time;
5) be cooled to below 3～5 ℃, carried out centrifugal treating 5～15 minutes, centrifugal speed 6000-8000r/min;
6) filter, filtrate is at least in cold storage below-20 ℃ or freeze-drying preservation in the middle of getting.
Described water and fish meat emulsion quality ratio are preferably 4: 1.
The pH value is to regulate with hydrochloric acid and the 6mol/L sodium hydroxide of 6mol/L among the described preparation method, and the pH value is preferably 2.0.
Described stomach en-usage quantity is preferably 1100U/g.
Preferred 37 ℃ of described hydrolysis temperature, 2.4 hours time.
Compared with prior art, the invention has the advantages that: be to be that raw material prepares antibacterial peptide with the yellow crucian carp of the marine products low value fish of pure natural, yellow crucian carp albumen antibacterial peptide is rich in needed by human leucine, Isoleucine, Methionin, phenylalanine, methionine(Met), Threonine, Xie Ansuan, stable to heat, soda acid and digestive tube proteolytic enzyme physicochemical property, to Gram-negative bacteria (G
-) intestinal bacteria (Escherichia coli), fluorescent pseudomonas (Pseudomonas fluorescens), common distortion (Proteusvulgaris), Pseudomonas aeruginosa (Pseudomonas aeruginosa); Gram-positive microorganism (G
+) golden yellow grape ball (Staphylococcus aureus), bacillus megaterium (Bacillus megaterium), sarcina (Sarcina), Sarcina lutea (Sarcina lutea) all have bacteriostatic action.With the antibacterial peptide of food source proteolysis gained have safe, absorptivity good, the advantage of good antimicrobial effect, can be in food as nutrition and functional type additive or be developed as the resistant organism microbiotic, have broad application prospects.This preparation method is simple, economy, easy handling, can carry out in common lab, is applicable to also that industrialization enlarges to produce.
Description of drawings
Fig. 1 is a process flow sheet.
Embodiment
Below in conjunction with embodiment the present invention is described in further detail.
Embodiment 1
1) yellow crucian carp is gone internal organ clean and cut fritter, blend evenly, make fish meat emulsion, get fish meat emulsion 50 grams, add 200 gram deionized waters with tissue mashing machine;
2) the pH value to 2.0 of regulating fish meat emulsion and water mixed liquid with hydrochloric acid and the 6mol/L sodium hydroxide of 6mol/L, and keep constant;
3) stomach en-with 55000U joins in the mixed solution, under the condition that keeps 37 ℃ of hydrolysis temperatures, and enzymolysis 2.4 hours;
4) be warming up to 95～100 ℃ of heating and carry out the passivation enzyme that goes out, go out 10 minutes enzyme time;
5) be cooled to below 4 ℃, carried out centrifugal treating 10 minutes, speed 7000r/min;
6) filter, filtrate is in-20 ℃ of cold storage or freeze-drying preservation in the middle of getting.
Trichloroacetic acid precipitation (TCA) is measured in the proteic stomach en-antimicrobial fluid of yellow crucian carp the soluble peptide extraction rate reached to (81.78 ± 0.04) % in conjunction with Kjeldahl determination, and the degree of hydrolysis that triketohydrindene hydrate is measured antibiotic enzymolysis solution in conjunction with Kjeldahl determination is (18.12 ± 0.39) %.The biuret reaction of enzymolysis solution, ninhydrin reaction are all positive, and explanation is a peptide mixture, and wherein mixed peptide content is 25mg/ml.
Under response surface analysis (RSM) optimum condition, the yellow crucian carp albumen of 30 μ l antibacterial peptide is (20.3 ± 1.9) mm and theoretical value 20.2mm there was no significant difference (P＞0.05) to colibacillary antibacterial circle diameter, parameter definite under the optimum condition is described accurately and reliably, has practical value.
Below the nutritive ingredient and the performance of yellow crucian carp stomach en-enzymolysis solution under the optimum condition are done further test analysis.
One, amino acid analysis result
The yellow crucian carp stomach en-of table 1 enzymolysis solution amino acid analysis
Table?1Amino?acid?composition?of?half-fin?anchovy?pepsin?hydrolysates
Annotate: tryptophane is destroyed under the strong acid hydrolysising condition, so detected result does not comprise tryptophane,
*The expression indispensable amino acid.
Amino acid analysis shows that yellow crucian carp stomach en-enzymolysis solution contains 16 seed amino acids (destroyed failing measured in the tryptophane hydrolytic process), and wherein 7 kinds of indispensable amino acid relative contents are 37.91%.Albumen with anti-microbial effect is more similar with peptide class primary structure, the N end is rich in cationic amino acid such as Methionin and arginine, the C end is rich in nonpolar amino acids such as L-Ala, Xie Ansuan, leucine, middle portion is proline rich (Boman H G then, Hultmark D.Cell-freeimmunity in insects[J] .Annual Review ofMicrobiology, 1987,41 (2): 103-126; Jaynes J M, Burton C A, Barr S B., et al.In vitro cytocidal effect of novel lytic peptides on plasmodiumfalciparum and trypanosom a cruzfi[J] .The FASEB Journal, 1989,2 (12): 2878-2883.), yellow crucian carp albumen antimicrobial fluid contains with anti-microbial effect related amino acid and relative content higher.
Two, the molecular weight distribution of antimicrobial fluid
With bovine serum albumin (68000Da), Trypsin inhibitor,Trasylol (6512Da), vitamins B
12(1355Da), Sleep-promoting factor B (612.63Da), L-carnosine (226.23Da) be reference material, through Superdex
TM-75HR 10/300 separation (λ=280mm), according to elution volume (ml) and reference material relative molecular weight logarithm (lgM
t) the definite molecular weight standard curve of relation.Yellow crucian carp stomach en-enzymolysis solution separates under identical elution requirement, and it is the highest to draw in its antibacterial protein liquid relative molecular weight 1000～3000Da component proportion according to the molecular weight standard curve, near 70%.Three, external antimicrobial spectrum and inhibitory effect
Antimicrobial spectrum (n=3) the Table 2Antibacterial-spectrum of half-fin anchovy pepsin hydrolysates (n=3) of the yellow crucian carp stomach en-of table 2 enzymolysis solution
Annotate: the sterilization punch tool (diameter=4mm) punching, every hole adds antimicrobial fluid 25 μ l; Data are the antibacterial circle diameter mean+SD in the table ,+-expression do not form obvious inhibition zone.
Inhibition zone method is measured yellow crucian carp stomach en-enzymolysis solution to Gram-negative bacteria (G
-) and gram-positive microorganism (G
+) bacteriostatic action is all arranged, have broad-spectrum antibacterial, wherein to G
-The bacterium fungistatic effect totally is better than G
+Bacterium because most of antibacterial peptides belong to neutrality or meta-alkalescence albumen, can by with G
-Electronegative species such as lipopolysaccharides on the bacterium adventitia interact, thereby destroy membrane structure with the performance anti-microbial effect, and G
+Bacterium does not have the lipopolysaccharides film, can only be with a small amount of negative charge and antibacterial peptide (the Hancock R E W that has an effect by the teichoic acid in the polypeptide surface glycan, uronic acid etc., Chapple D S.Peptideantibiotics[J] .Antimicrobial Agents and Chemotherapy, 1999,43 (6): 1317-1323.).Neutrality and basic aminoacids relative content in the yellow crucian carp stomach en-enzymolysis solution account for 50.75%, help forming cationic antibacterial peptide class and suppress the G-bacterium.
Yellow crucian carp stomach en-enzymolysis solution relatively sees Table 3 to colibacillary restraining effect with the penbritin of different extension rates and the fungistatic effect of Vetstrep.
The inhibitory effect of the yellow crucian carp stomach en-of table 3 enzymolysis solution and control drug be (n=3) Table 3Comparing the antibacterial effectiveness of half-fin anchovy pepsin hydrolysates (HAH) andcontrols (n=3) relatively
Annotate: the sterilization punch tool (diameter=4mm) punching, every hole adds sample liquid 25 μ l; Data are the antibacterial circle diameter mean+SD in the table, and △ represents to compare there was no significant difference (P＞0.05) with penbritin inhibition zone size, ▲ expression and the big or small relatively there was no significant difference (P＞0.05) of Vetstrep inhibition zone.
Yellow as shown in Table 3 crucian carp stomach en-enzymolysis solution is to the penbritin of colibacillary antibacterial result and 128 times of dilutions (13.21～15.44mcg/ml) and 4 times Vetstrep (325.00～340.00U/ml) inhibitory effects are suitable,＞0.05) of dilution.
Physico-chemical property studies show that yellow crucian carp albumen antibacterial peptide to thermally-stabilised, heats 15 respectively at 80,90,100 and 121 ℃, behind the 30min, to colibacillary bacteriostatic action and contrast (heating) there was no significant difference (P＞0.05) relatively.At pH 2.4,3.4, under 4.4,5.4,6.4,7.4,8.4 and 9.4 conditions intestinal bacteria all there is bacteriostatic action, wherein (pH 5.4) biocidal property is the strongest under mild acid conditions.Yellow crucian carp albumen antibacterial peptide tolerance trypsin treatment, do not change (P＞0.05) through behind 0.25% trypsin acting, 1～5h colibacillary restraining effect being had significance, through β---still keep bacteriostatic action after the lactamase effect, the effective antipathogenic composition that shows yellow crucian carp albumen antibacterial peptide does not contain β---the lactam nucleus structure, can play a role to some resistant organisms, have further to be developed as the antibiotic value of resistant organism.
Embodiment 2
1) yellow crucian carp is gone internal organ clean and cut fritter, blend evenly, make fish meat emulsion, get fish meat emulsion 50 grams, add 150 gram deionized waters with tissue mashing machine;
2) the pH value to 2.0 of regulating fish meat emulsion and water mixed liquid with hydrochloric acid and the 6mol/L sodium hydroxide of 6mol/L, and keep constant;
3) stomach en-with 65000U joins in the mixed solution, under the condition that keeps 35 ℃ of hydrolysis temperatures, and enzymolysis 3 hours;
4) be warming up to 100 ℃ of heating and carry out the passivation enzyme that goes out, go out 5 minutes enzyme time;
5) be cooled to below 5 ℃, carried out centrifugal treating 15 minutes, speed 6000r/min;
6) filter, filtrate is in-22 ℃ of cold storage or freeze-drying preservation in the middle of getting.
Embodiment 3
1) yellow crucian carp is gone internal organ clean and cut fritter, blend evenly, make fish meat emulsion, get fish meat emulsion 50 grams, add 250 gram deionized waters with tissue mashing machine;
2) the pH value to 2.0 of regulating fish meat emulsion and water mixed liquid with hydrochloric acid and the 6mol/L sodium hydroxide of 6mol/L, and keep constant;
3) stomach en-with 50000U joins in the mixed solution, under the condition that keeps 45 ℃ of hydrolysis temperatures, and enzymolysis 2 hours;
4) be warming up to 95 ℃ of heating and carry out the passivation enzyme that goes out, go out 15 minutes enzyme time;
5) be cooled to below 4 ℃, carried out centrifugal treating 5 minutes, speed 8000r/min;
6) filter, filtrate is in-25 ℃ of cold storage or freeze-drying preservation in the middle of getting.
Claims (5)
1. the preparation method of a yellow crucian carp albumen antibacterial peptide is characterized in that may further comprise the steps:
1) yellow crucian meat is cleaned cut fritter, blend evenly, make fish meat emulsion, according to 3～5 multiple proportions example adding deionized water of fish meat emulsion quality with tissue mashing machine;
2) regulate pH value to 1.5～2.5 of fish meat emulsion and water mixed liquid, and keep constant;
3) according to the quality of fish meat emulsion, stomach en-joins in the mixed solution with the ratio of 1000～1300U/g, under the condition that keeps 35～45 ℃ of hydrolysis temperatures, and enzymolysis 2～3 hours;
4) heating is warming up to 95～100 ℃ and carries out the passivation enzyme that goes out, and goes out 5～15 minutes enzyme time;
5) be cooled to below 3～5 ℃, carried out centrifugal treating 5～15 minutes, centrifugal speed 6000-8000r/min;
6) filter, filtrate is at least in cold storage below-20 ℃ or freeze-drying preservation in the middle of getting.
2. preparation method according to claim 1 is characterized in that described water and fish meat emulsion quality ratio are preferably 4: 1.
3. preparation method according to claim 1 is characterized in that described pH value is to regulate with hydrochloric acid and the 6mol/L sodium hydroxide of 6mol/L, and the pH value is preferably 2.0.
4. preparation method according to claim 1 is characterized in that described stomach en-usage quantity is preferably 1100U/g.
5. preparation method according to claim 1 is characterized in that preferred 37 ℃ of described hydrolysis temperature, 2.4 hours time.
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