CN101744124B - Method for preparing milk-derived antimicrobial peptide - Google Patents
Method for preparing milk-derived antimicrobial peptide Download PDFInfo
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- CN101744124B CN101744124B CN2010101046606A CN201010104660A CN101744124B CN 101744124 B CN101744124 B CN 101744124B CN 2010101046606 A CN2010101046606 A CN 2010101046606A CN 201010104660 A CN201010104660 A CN 201010104660A CN 101744124 B CN101744124 B CN 101744124B
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- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to a method for preparing milk-derived antimicrobial peptide. In the method, casein is first dissolved in deionized water, so that casein suspension is produced, the pH value of the casein suspension is regulated as 2 to 4 by acid, and pepsin is added in the casein suspension, so that enzymatic hydrolysate is obtained; the bath temperature of the enzymatic hydrolysate is increased and then quickly reduced, and is centrifuged, so that raw supernate is obtained; trichloroacetic acid solution is added in the raw supernate, the raw supernate is kept still and then centrifuged, the supernate is dialyzed, so that pure milk-derived antimicrobial peptide solution is obtained, and after the milk-derived antimicrobial peptide solution is frozen and dried, dried milk-derived antimicrobial peptide product is obtained. The method has the advantages of simple technique, low cost, high extraction rate, safety, effectiveness and no toxic effect, the purity of the produced product is high, and the method is suitable for mass industrialized production.
Description
Technical field
This belongs to additive agent field, is specifically related to a kind of preparation method who in the animal technical field, makes the milk-derived antimicrobial peptide of nutriment.
Background technology
Milk-derived antimicrobial peptide (Casein Glyco Macro Pept ide; CGMP) be Ruzhong κ one a caseic polypeptide fragment; A common part as casein micelles; Usually the polypeptide of this solubility contains more sugar chain, then be called as " PROVON 190 ", and this type of peptide in casein source all completely is called " milk-derived antimicrobial peptide " (CGMP).The amino acid sequence of the CGMP in κ one casein source is clear, and it is to be made up of 64 residues that finish from κ one terminal 106 amino acids of caseic N (Met) beginning to 169 C ends (Val).The CGMP of ox has A and B2 kind hereditary variation type, and its difference is that 136 amino acid with 148 of this polypeptide chain are different, and 136 of the A type and 148 residue amino acid are respectively Thr and Asp, and Type B is Ile and Ala.131,133,135,136 and 142 of this induced abortion threonine residues is the position of glycocideization in addition, and 149 hungry serines are the position of phosphorylation.
In general, the molecular mass of CGMP polypeptide chain is about 9ku, but changes with pH, in acid range, like pH3.5, is approximately 10～30ku, and neutrallty condition such as pH7.0 are about 20～50ku.The preparation method of many CGMP utilizes ultrafiltration to carry out separation and purification just based on this characteristic of CGMP.
In the CGMP that pepsin κ-casein produces, contain a large amount of active polysaccharides, comprising:
(1) monose GalNAc-O-R;
(2) disaccharide Gal β 1 → 3GalNAc-O-R;
(3) trisaccharide NeuAc α 2 → 3Gal β 1 → 3GalNAc-O-R;
(4) trisaccharide Gal β 1 → 3 (NeuAc α 2 → 6) GalNAc-O-R;
(5) tetrose NeuAc α 2 → 3Gal β 1 → 3 (NeuAc α 2 → 6) GalNAc-O-R
Wherein: Gal is a galactolipin; GalNAc is the N-acetylgalactosamine; NeuAc is a sialic acid.
Sialic acid is the higher active monose of content in the milk-derived antimicrobial peptide.It is the general name of the saliva base inductor of neuraminic acid, and the pH value of sialic acid is about 2.6, mainly extensively is present in the organism outside the plant with N-n acetylneuraminic acid n (Neu5Ac) form at occurring in nature.Research shows that sialic acid has many physiological functions, and topmost physiological function is to rise than important function in the defense mechanism in vivo.The sialic acid that contains high concentration in people's the breast milk has bigger relation through discovering its growth and infection defense mechanism with nursling in a large number; In addition, current research shows that sialic acid also has the virus infections effect and the effect that improves learning ability that suppresses to cause the diarrhea reason, and simultaneously, the sialic acid of a part can be moved in the brain, thereby has bigger relation (WARREN, 1959) with the growth of brain.And the about sialic acid more than 75% in Ruzhong is present among the CGMP, so its physiological function is self-evident.
Experiment shows that this polypeptide has multiple physiological active functions in a large amount of external and body relevant with CGMP.The physiological function of milk-derived antimicrobial peptide mainly contains:
It can combine cholera and the colibacillary enterotoxin of dust Xi Shi.Research shows that the receiver of (KAWASAKI, 1992) cholera toxin industry A of unit and B is a kind of gangliosides, and the oligosaccharide structure of this gangliosides and milk-derived antimicrobial peptide is similar, and therefore, CGMP has the characteristic that combines cholera toxin and dust Xi Shi colitoxin.
Milk-derived antimicrobial peptide can suppress pathogen and comprise that virus and bacterium etc. are adhered to cell, and the protection body is avoided the infection of pathogen.Research shows; The mechanism that the carious tooth that can weaken CGMP takes place is that CGMP can make the microorganism composition that produces the tooth plaque change; Its micropopulation is become by the defence line bacterium of very weak carcinogenic by staphylococcus form, research finds that simultaneously milk-derived antimicrobial peptide not only suppresses carious tooth but also can make tooth inorganicization again.
Milk-derived antimicrobial peptide can suppress gastric secretions.The material that shrinks relation about milk-derived antimicrobial peptide and intestines and stomach has caused people's extensive interest.Stan (Stan Eya.; 1983) etc. the people thinks that weakening gastric secretions will reduce animal stomach cardia hydrolysis; Can protect the intact stomaches that pass of protein such as immunoglobulin (Ig), lactoferrin and lysozyme in source, Ruzhong like this, this can be of value to people's enhance immunity power of baby and hypoimmunity.
Can promote the growth of Bifidobacterium.In a research differentiating Bifidobacterium, people such as Gyorzy find that the oligosaccharides with N-acetylneuraminic amine acid ending can promote the growth of Bifidobacterium, and they to propose this be because terminal N-acetylneuraminic amine acid comes off, exposed the cause of a GalNAc.
Has the function (AZUMA, 1984) of regulating immune reaction.The bone-marrow-derived lymphocyte propagation that milk-derived antimicrobial peptide can suppress to be excited by LPS and by PHA (the T lymphopoiesis of plant agglutinin of blood-P) excite.Its further influence be the downward modulation immune system and weaken the tolerance in oral cavity, this will give that the animal of a birth just is a kind of can protected mechanism, makes the antigen in the wide range in the animal capable opposing environment.
The part commodity that contain CGMP have been applied in the middle of people's the life, and for example, in the U.S., the toothpaste that adds CGMP is succeeded in developing, and it can suppress the generation (Berrocal., 1993) of carious tooth and canker sore; There is article to think, joins milk-derived antimicrobial peptide in the food such as biscuit, jelly and can promote health, make body enhance immunity power; In pharmaceutical products, add the treatment that CGMP is used for DVT; CGMP can combine the characteristic of enterotoxin to make it be widely used in infant food central (Marshall S.C., 1991).
In addition, CGMP has unique amino acid and forms, and particularly contains aromatic amino acid hardly, can be used as composition first-selected in phenylketonuria patient's (PKU, a kind of hereditary disease of not energy metabolism aromatic amino acid) meals.CGMP contains more branched-chain amino acid (like Val and Ile), can be used for the treatment of multiple hepatopathy, and relevant researcher thinks; The coefficient of ratio of branched-chain amino acid and aromatic amino acid is big more; Its result of treatment to hepatopathy is good, verified more, when this coefficient is about 20 left and right sides; CGMP can be used as hepatopath's meals, and effect is fine.
Summary of the invention
The object of the invention just provides a kind of preparation method of milk-derived antimicrobial peptide.
For realizing this purpose, the inventive method is taked following concrete steps:
(1) at first casein is dissolved in and processes the casein suspension in the deionized water, the weight ratio of casein and deionized water is 1: 5～10 in the casein suspension; The pH value of using acid to regulate the casein suspension then is 2～4, places 30～50 ℃ of water-baths to be incubated 10～20 minutes, in suspension, adds pepsin, and enzymolysis 1～2 hour obtains enzymolysis liquid.Pepsic addition and casein substrate mass ratio are 1: 25～50; Used acid is HCl or H
2SO4.
(2) the enzymolysis liquid bath temperature is risen to 70～90 ℃, kept 15～25 minutes, enzyme is lost activity, reduce temperature to 3～5 ℃ rapidly, centrifugally obtain thick supernatant; Centrifugal speed is 3000～4000r/min.
(3) in thick supernatant, adding temperature is that 4～8 ℃, mass concentration are 50～60% (trichloroacetic acid) TCA solution, leaves standstill after 5～10 minutes centrifugally, gets supernatant; Centrifugal speed is that 4000～4500r/min is centrifugal.The TCA solution that adds is 1: 8～12 with the volume ratio of thick supernatant.
(4) the dialysis supernatant obtains pure milk-derived antimicrobial peptide solution.The molecular cut off of used bag filter is 7000～140000.
(5) with the milk-derived antimicrobial peptide solution that obtains through freeze drying, obtain the desciccate of milk-derived antimicrobial peptide.
The inventive method technology is easy, with low cost, recovery rate is high, safe and effective and do not have a toxic side effect, prepared product purity is high, is suitable for large-scale industrial production.
The specific embodiment
Embodiment 1
(1) at first 100 gram caseins is dissolved in the deionized water of 1L and processes the casein suspension; The pH value of using HCl to regulate the casein suspension then is 4, places 30 ℃ of water-bath insulations 20 minutes, in suspension, adds 2 gram pepsins again; Enzymolysis 1 hour obtains enzymolysis liquid.
(2) the enzymolysis liquid bath temperature is risen to 70 ℃, kept 25 minutes, reduce temperature to 4 ℃ rapidly, centrifugal under the speed of 3000r/m, get supernatant, thick supernatant.
(3) in thick supernatant, adding temperature is that 4 ℃, mass concentration are 50% TCA solution, leaves standstill after 5 minutes centrifugally, gets supernatant.The volume of used TCA is 1: 8 with the volume ratio of thick supernatant, and centrifugal rotational speed is 4000r/min.
(4) the use molecular cut off is 7000 bag filter dialysis supernatant, obtains pure milk-derived antimicrobial peptide solution.
(5) with the milk-derived antimicrobial peptide solution that obtains through freeze drying, obtain the desciccate of 24.3g milk-derived antimicrobial peptide, measuring in the product total protein content, sialic acid content through Kjeldahl, to obtain product purity be 87.6%, productive rate is 24.3%.
Embodiment 2
(1) at first 150 gram caseins is dissolved in the deionized water of 1L and processes the casein suspension, use H then
2SO
4The pH value of regulating the casein suspension is 3, places 50 ℃ of water-bath insulations 10 minutes, in suspension, adds 5 gram pepsins again, and enzymolysis 1.5 hours obtains enzymolysis liquid.
(2) the enzymolysis liquid bath temperature is risen to 80 ℃, kept 20 minutes, reduce temperature to 3 ℃ rapidly, centrifugal under the speed of 3500r/m, get supernatant, thick supernatant.
(3) in thick supernatant, adding temperature is that 6 ℃, mass concentration are 55% TCA solution, leaves standstill after 8 minutes centrifugally, gets supernatant.The volume of used TCA is 1: 10 with the volume ratio of thick supernatant, and centrifugal rotational speed is 4200r/min.
(4) the use molecular cut off is 10000 bag filter dialysis supernatant, obtains pure milk-derived antimicrobial peptide solution.
(5) with the milk-derived antimicrobial peptide solution that obtains through freeze drying, obtain the desciccate of 43.8g milk-derived antimicrobial peptide, measuring in the product total protein content, sialic acid content through Kjeldahl, to obtain product purity be 89.1%, productive rate is 29.2%.
Embodiment 3
(1) at first 200 gram caseins is dissolved in the deionized water of 1L and processes the casein suspension; The pH value of using HCl to regulate the casein suspension then is 2, places 40 ℃ of water-bath insulations 15 minutes, in suspension, adds 8 gram pepsins again; Enzymolysis 2 hours obtains enzymolysis liquid.
(2) the enzymolysis liquid bath temperature is risen to 90 ℃, kept 15 minutes, reduce temperature to 5 ℃ rapidly, centrifugal under the speed of 4000r/m, get supernatant, thick supernatant.
(3) in thick supernatant, adding temperature is that 8 ℃, mass concentration are 60% TCA solution, leaves standstill after 10 minutes centrifugally, gets supernatant.The volume of used TCA is 1: 12 with the volume ratio of thick supernatant, and centrifugal rotational speed is 4500r/min.
(4) the use molecular cut off is 14000 bag filter dialysis supernatant, obtains pure milk-derived antimicrobial peptide solution.
(5) with the milk-derived antimicrobial peptide solution that obtains through freeze drying, obtain the desciccate of 61g milk-derived antimicrobial peptide, measuring in the product total protein content, sialic acid content through Kjeldahl, to obtain product purity be 93.2%, productive rate is 30.5%.
Claims (1)
1. the preparation method of a milk-derived antimicrobial peptide is characterized in that the concrete steps of this method are:
(1) at first casein is dissolved in and processes the casein suspension in the deionized water, the weight ratio of casein and deionized water is 1: 5～10 in the casein suspension; The pH value of using acid to regulate the casein suspension then is 2～4, places 30～50 ℃ of water-baths to be incubated 10～20 minutes, in suspension, adds pepsin, and enzymolysis 1～2 hour obtains enzymolysis liquid; Pepsic addition and casein substrate mass ratio are 1: 25～50;
(2) the enzymolysis liquid bath temperature is risen to 70～90 ℃, kept 15～25 minutes, reduce temperature to 3～5 ℃ rapidly, centrifugally obtain thick supernatant; Centrifugal speed is 3000～4000r/min;
(3) in thick supernatant, adding temperature is that 4～8 ℃, mass concentration are 50～60% trichloroacetic acid solution, leaves standstill after 5～10 minutes centrifugally, gets supernatant; Centrifugal speed is that 4000～4500r/min is centrifugal; The trichloroacetic acid solution that adds is 1: 8～12 with the volume ratio of thick supernatant;
(4) the dialysis supernatant obtains pure milk-derived antimicrobial peptide solution;
(5) with the milk-derived antimicrobial peptide solution that obtains through freeze drying, obtain the desciccate of milk-derived antimicrobial peptide;
Acid described in the step (1) is HCl or H
2SO4;
The molecular cut off of the bag filter that the middle dialysis of step (4) is used is 7000～140000.
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| 王立晖.胃蛋白酶酶解酪蛋白制备酪啡肽的研究.《中国优秀硕士学位论文全文数据库》.2006, * |
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