CN102220402A - Method for extracting antibacterial peptide from pearl oyster meat - Google Patents
Method for extracting antibacterial peptide from pearl oyster meat Download PDFInfo
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- CN102220402A CN102220402A CN2011101399316A CN201110139931A CN102220402A CN 102220402 A CN102220402 A CN 102220402A CN 2011101399316 A CN2011101399316 A CN 2011101399316A CN 201110139931 A CN201110139931 A CN 201110139931A CN 102220402 A CN102220402 A CN 102220402A
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Abstract
The invention discloses a method for extracting an antibacterial peptide from pearl oyster meat, which comprises the following steps: taking the pearl oyster meat after pretreatment, adding ice water for cold homogenation so as to get homogenate; adding flavourzyme into the homogenate, uniformly mixing, then regulating the working frequency of ultrasonic waves to 35kHz, regulating the strength of the ultrasonic waves to 280-360W and regulating the temperature to 50-65 DEG C for performing ultrasonic treatment for 90-100 minutes; continuing to performing enzymolysis on the homogenate for 1-2 hours after the ultrasonic treatment still in a constant-temperature water bath at the temperature of 50-65 DEG C and then performing enzyme inactivation and cooling treatment for getting enzymatic hydrolysate; and performing centrifugal treatment on the enzymatic hydrolysate, taking supernatant liquid, regulating the operation pressure of membrane separation to 2-3bar, regulating the temperature to 3-10 DEG C, utilizing the membrane separation technology for performing ultrafiltration treatment, collecting separation liquid with the intercepting molecular weight of 5000-10000Da, freeze-drying and then getting an antibacterial peptide product of the pearl oyster meat. The method uses the ultrasonic wave technology to assist the hydrolysis of the flavourzyme for carrying out the extraction of the antibacterial peptide in the pearl oyster meat, so that the operation is simple, the hydrolysis rate and the antibacterial activity can be improved, the hydrolysis time is shortened, the production cost is low, the product is safe, waste pearl oyster meat after opening pearls can be utilized with high value and further the economic benefits of a manufacturer are improved.
Description
Technical field
The present invention relates to a kind of method of extracting antibacterial peptide, specifically relate to a kind of method of from Carnis Margarita, extracting antibacterial peptide, belong to fishery products deep process technology field.
Background technology
(Antimicrobial peptides AMPs) is the general name that the small molecule polypeptide with anti-microbial activity of genes encoding is arranged to antibacterial peptide.Antibacterial peptide all has lethal effect to bacterium, part fungi, virus, protozoon, tumour cell, and the advantage of its sterilization and the effect of safety non-toxic evil makes it have the potential using value of doing antiseptics for natural food.
The shellfish that can produce pearl in the world has kind more than 30, generally is distributed in the torrid zone, subtropics, tens of meters the seabed in subtidal zone to the depth of water.Be used to produce the topmost shellfish of marine cultured pearl at present and be Pinctadamaxima (Jameson) (claim not only white dish shellfish), pinctada fucata (but also claiming Pinctada martensii), penguin Margarita etc.Wherein the most common with pinctada fucata, be the main shellfish kind of producing cultured pearl in the world.The pearl shellfish produces a large amount of shellfish meat after opening pearl, and these Carnis Margaritas remove and are used to eat raw on a small quantity, and major part is used to be processed into feed or discarded, and added value is lower.How rationally higher value application Carnis Margarita resource is a pearl shellfish aquaculture industry urgent problem.
Because pearl shellfish itself lacks the such specific immune system of high vertebrates that is similar to, its immunity mainly depends on all kinds of immune factors in the abundant humoral defense system, its non-specific immunity system is very flourishing, and antibacterial peptide is the important component part of its non-specific immunity system, and its process of growth is produced the specificity of pearl, and antibacterial peptide therefore is richly stored with.Because the complicacy and the diversity of ocean environment, that the marine invertebrate antibacterial peptide has is stronger, more heat-resisting than terrestrial organism antibacterial peptide anti-microbial activity, characteristic such as salt tolerant more, thereby its application in food fresh keeping, storage and processing has more wide potential value.
Summary of the invention
The object of the present invention is to provide a kind of method of from Carnis Margarita, extracting antibacterial peptide, can improve the degree of hydrolysis and the high bacteriostatic activity of Carnis Margarita, thereby with depleted Carnis Margarita resource higher value application.
Above-mentioned technical purpose of the present invention realizes by following technique means: a kind of method of from Carnis Margarita, extracting antibacterial peptide, and the pretreated Carnis Margarita that comprises the steps: to learn from else's experience adds frozen water and carries out getting homogenate behind the cooled homogenate; Add in homogenate that to regulate hyperacoustic operating frequency behind the flavor protease mixing be that 35kHz, ultrasonic intensity are that 280-360W, temperature are 50-65 ℃ and carry out supersound process 90-110min; Homogenate after the supersound process continues enzymolysis 1-2h in 50-65 ℃ of water bath with thermostatic control, and go out then enzyme and cooling process get enzymolysis solution; Enzymolysis solution is carried out centrifugal treating, the working pressure of getting supernatant liquor adjusting membrane sepn is that 2-3bar, temperature are 3-10 ℃, utilize membrane separation technique to carry out uf processing, collecting molecular weight cut-off is the parting liquid of 5000-10000Da, promptly obtains Carnis Margarita antibacterial peptide product after the lyophilize.
In aforesaid method:
Carnis Margarita preprocessing process of the present invention: get freezing Carnis Margarita or bright Carnis Margarita, remove its internal organ, clean 2-3 time draining 1-10min with clear water.
The consumption of frozen water of the present invention is the 0.4-0.8 times of weight of Carnis Margarita.
The enzyme work of flavor protease of the present invention is 2.0 * 10
5U adds enzyme in Carnis Margarita weight, and its addition is 1500-2500 U/g.
The present invention continues at 50-65 ℃ of enzymolysis 1-2h after ultrasonication, enzyme 5-10 min that go out under 100 ℃ then, cooling rapidly behind the enzyme that goes out.
The detailed process that enzymolysis solution of the present invention carries out centrifugal treating is: enzymolysis solution is placed refrigerated centrifuge, and the rotating speed of regulating whizzer is 10000-15000r/min, centrifugal 10-15 min under 0-4 ℃.
The detailed process that the present invention utilizes membrane separation technique to carry out uf processing is: earlier supernatant liquor is separated with the 10000Da ultra-filtration membrane, collect filtrate, separate with the 5000Da ultra-filtration membrane again, collect the parting liquid of 5000-10000Da, promptly obtain Carnis Margarita antibacterial peptide liquid.
The present invention has following advantage:
(1) the present invention passes through enzymolysis, the integrated technology that ultrasonic technology and membrane separation technique combine, Carnis Margarita is carried out enzymolysis by flavor protease, utilize ultrasonic technology to have the double attribute of fluctuation and energy, the one, cavitation phenomenon, the energy release that promptly in liquid medium, shows as the formation of bubble and break and follow, the 2nd, medium and container also can produce the second-order effect of resonance character by the absorption of sound, so promote that under hyperacoustic booster action flavor protease is hydrolyzed to Carnis Margarita, by the time of control enzymolysis, temperature, enzyme concentration, the material-water ratio example is obtained the antibacterial peptide with high biocidal property.
(2) the present invention carries out enzymolysis to Carnis Margarita by the every processing condition that adopt flavor protease and control enzymolysis, and degree of hydrolysis is 48～55%, and the Carnis Margarita antibacterial peptide liquid that obtains is faint yellow, and solid is a buff powder; Product has stronger bacteriostatic activity, and particularly the inhibition ability to Salmonellas, Shigellae, Listeria monocytogenes is stronger, and bacteriostasis reaches more than 85%.
(3) preparation method of the present invention is simple to operate, and ultrasound-assisted enzymolysis can improve percent hydrolysis greatly, shortens hydrolysis time, and production cost is low, and product safety can improve the economic benefit of manufacturing enterprise.
(4) a large amount of shellfish meat carried out higher value application after the present invention opened pearl to the main cultured pearl shellfish in the South Sea, develop and have antibacterial peptide antibacterial, the preservation and antisepsis effect, can be widely used in light industries such as food, makeup as natural antisepsis fresh-keeping agent, the Sustainable development that promotes the marine cultured pearl industry is had important economic implications and social effect.
Embodiment
The present invention is further illustrated below in conjunction with specific embodiment, wherein following raw material or the reagent that adopts, as do not have special indicating and be commercially available getting.
Embodiment 1
The preparation method of the Carnis Margarita antibacterial peptide that present embodiment provides may further comprise the steps:
(1) shellfish meat pre-treatment: freezing pinctada fucata meat is thawed, remove internal organ, clean 2-3 time, be placed in the stainless steel framework draining 5 minutes with clear water;
(2) cooled homogenate: take by weighing shellfish meat after the pre-treatment, add the frozen water of 0.4 times of shellfish meat weight, place tissue mashing machine's homogenate 2-5 min, obtain shellfish meat homogenate;
(3) enzyme-added, ultrasonication: shellfish meat homogenate is placed stainless steel vessel, and (Denmark NOVO company product, enzyme work is 2.0 * 10 to add flavor protease
5U), add enzyme by shellfish meat weight, the addition of flavor protease is 1600 U/g, after stirring evenly, is placed in the ultrasonic instrument, and regulating the ultrasonic wave operating frequency is that 35kHz, hyperacoustic intensity are that 360W, control treatment temp are 60 ℃, and the time is 90min;
(4) further enzymolysis: after ultrasonication, the shellfish meat homogenate in the step (3) is continued at 65 ℃ of water bath with thermostatic control enzymolysis 2.0 h the enzyme 5min that under 100 ℃, goes out, cooling rapidly behind the enzyme that goes out;
(5) centrifugal: the enzymolysis solution in the step (4) is centrifugal in refrigerated centrifuge, and the rotating speed of whizzer is 10000r/min, and 0 ℃ of following centrifugal 10 min gets supernatant liquor;
(6) ultrafiltration: utilize membrane separation technique that the supernatant liquor that obtains in the step (5) is carried out membrane sepn, the working pressure of membrane sepn is 2 bar, temperature is 10 ℃, separate with the 10000Da ultra-filtration membrane earlier, collect filtrate, separate with the 5000Da ultra-filtration membrane again, collect the parting liquid of 5000-10000Da, promptly obtain pinctada fucata meat antibacterial peptide liquid, be weak yellow liquid;
(7) freeze-drying: with the pinctada fucata meat antibacterial peptide liquid Freeze Drying Equipment that obtains in the step (6), freeze-drying obtains light yellow antibacterial peptide powder.
Embodiment 2
The preparation method of the Carnis Margarita antibacterial peptide that present embodiment provides may further comprise the steps:
(1) shellfish meat pre-treatment: freezing Pinctadamaxima (Jameson) shellfish meat is thawed, remove internal organ, clean 2-3 time, be placed in the frame of plastic draining 5 minutes with clear water;
(2) cooled homogenate: take by weighing shellfish meat after the pre-treatment, add the frozen water of 0.8 times of shellfish meat weight, place tissue mashing machine's homogenate 2-5 min, obtain shellfish meat homogenate;
(3) enzyme-added, ultrasonication: shellfish meat homogenate is placed Glass Containers, and (Denmark NOVO company product, enzyme work is 2.0 * 10 to add flavor protease
5U), add enzyme by shellfish meat weight, the addition of flavor protease is 2500 U/g, after stirring evenly, is placed in the ultrasonic instrument, and regulating the ultrasonic wave operating frequency is that 35kHz, hyperacoustic intensity are that 320W, control treatment temp are 50 ℃, and the time is 100min;
(4) further enzymolysis: after ultrasonication, the shellfish meat homogenate in the step (3) is continued at 50 ℃ of water bath with thermostatic control enzymolysis 1.5h the enzyme 10min that under 100 ℃, goes out, cooling rapidly behind the enzyme that goes out;
(5) centrifugal: the enzymolysis solution in the step (4) is centrifugal in refrigerated centrifuge, and the rotating speed of whizzer is 15000r/min, and 4 ℃ of following centrifugal 15 min get supernatant liquor;
(6) ultrafiltration: utilize membrane separation technique that the supernatant liquor that obtains in the step (5) is carried out membrane sepn, the working pressure of membrane sepn is 3 bar, temperature is 3 ℃, separate with the 10000Da ultra-filtration membrane earlier, collect filtrate, separate with the 5000Da ultra-filtration membrane again, collect the parting liquid of 5000-10000Da, promptly obtain Pinctadamaxima (Jameson) shellfish meat antibacterial peptide liquid, be weak yellow liquid;
(7) freeze-drying: with the Pinctadamaxima (Jameson) shellfish meat antibacterial peptide liquid Freeze Drying Equipment that obtains in the step (6), freeze-drying obtains light yellow antibacterial peptide powder.
Embodiment 3
The preparation method of the penguin Margarita shellfish meat antibacterial peptide that present embodiment provides may further comprise the steps:
(1) shellfish meat pre-treatment: fresh penguin Margarita shellfish meat is thawed, remove internal organ, clean 2-3 time, be placed in the stainless steel framework draining 5 minutes with clear water;
(2) cooled homogenate: take by weighing shellfish meat after the pre-treatment, add the frozen water of 0.6 times of shellfish meat weight, place tissue mashing machine's homogenate 2-5 min, obtain shellfish meat homogenate;
(3) enzyme-added, ultrasonication: shellfish meat homogenate is placed stainless steel cup, and (Denmark NOVO company product, enzyme work is 2.0 * 10 to add flavor protease
5U), add enzyme by shellfish meat weight, the addition of flavor protease is 2000 U/g, after stirring evenly, is placed in the ultrasonic instrument, and regulating the ultrasonic wave operating frequency is that 35kHz, hyperacoustic intensity are that 280W, control treatment temp are 65 ℃, and the time is 110min;
(4) further enzymolysis: after ultrasonication, the shellfish meat homogenate in the step (3) is continued at 60 ℃ of water bath with thermostatic control enzymolysis 1.0h the enzyme 8min that under 100 ℃, goes out, cooling rapidly behind the enzyme that goes out;
(5) centrifugal: the enzymolysis solution in the step (4) is centrifugal in refrigerated centrifuge, and the rotating speed of whizzer is 12000r/min, and 2 ℃ of following centrifugal 12 min get supernatant liquor;
(6) ultrafiltration: utilize membrane separation technique that the supernatant liquor that obtains in the step (5) is carried out membrane sepn, the working pressure of membrane sepn is 2.5 bar, temperature is 6 ℃, separate with the 10000Da ultra-filtration membrane earlier, collect filtrate, separate with the 5000Da ultra-filtration membrane again, collect the parting liquid of 5000-10000Da, promptly obtain penguin Margarita shellfish meat antibacterial peptide liquid, be weak yellow liquid;
(7) freeze-drying: with the penguin Margarita shellfish meat antibacterial peptide liquid Freeze Drying Equipment that obtains in the step (6), freeze-drying obtains light yellow antibacterial peptide powder.
Above embodiment has carried out detailed elaboration to the different implementation process of the present invention, but embodiments of the present invention are not limited in this.The those of ordinary skill of described technical field all can be realized purpose of the present invention according to disclosed content among the present invention.
Claims (7)
1. a method of extracting antibacterial peptide from Carnis Margarita is characterized in that comprising the steps: to learn from else's experience the pre-treatment Carnis Margarita, adds frozen water and carries out getting homogenate behind the cooled homogenate; Add in homogenate that to regulate hyperacoustic operating frequency behind the flavor protease mixing be that 35kHz, ultrasonic intensity are that 280-360W, temperature are 50-65 ℃ and carry out supersound process 90-110min; Homogenate after the supersound process continues enzymolysis 1-2h in 50-65 ℃ of water bath with thermostatic control, and go out then enzyme and cooling process get enzymolysis solution; Enzymolysis solution is carried out centrifugal treating, the working pressure of getting supernatant liquor adjusting membrane sepn is that 2-3bar, temperature are 3-10 ℃, utilize membrane separation technique to carry out uf processing, collecting molecular weight cut-off is the parting liquid of 5000-10000Da, promptly obtains Carnis Margarita antibacterial peptide product after the lyophilize.
2. the method for extracting antibacterial peptide from Carnis Margarita according to claim 1, it is characterized in that: described Carnis Margarita pre-treatment is: get freezing Carnis Margarita or bright Carnis Margarita, remove its internal organ, clean 2-3 time draining 1-10min with clear water.
3. the method for extracting antibacterial peptide from Carnis Margarita according to claim 1 and 2, it is characterized in that: the consumption of frozen water is the 0.4-0.8 times of weight of shellfish meat.
4. the method for extracting antibacterial peptide from Carnis Margarita according to claim 3, it is characterized in that: the enzyme work of described flavor protease is 2.0 * 10
5U adds enzyme in Carnis Margarita weight, and its addition is 1500-2500 U/g.
5. the method for extracting antibacterial peptide from Carnis Margarita according to claim 1 is characterized in that: after ultrasonic-enzymolysis processing, and enzyme 5-10 min that under 100 ℃, go out, cooling rapidly behind the enzyme that goes out.
6. the method for from Carnis Margarita, extracting antibacterial peptide according to claim 1, it is characterized in that: the detailed process that enzymolysis solution carries out centrifugal treating is: enzymolysis solution is placed refrigerated centrifuge, the rotating speed of regulating whizzer is 10000-15000r/min, centrifugal 10-15 min under 0-4 ℃.
7. the method for from Carnis Margarita, extracting antibacterial peptide according to claim 1, it is characterized in that: the detailed process of utilizing membrane separation technique to carry out uf processing is: earlier supernatant liquor is separated with the 10000Da ultra-filtration membrane, collect filtrate, separate with the 5000Da ultra-filtration membrane again, collect the parting liquid of 5000-10000Da, promptly obtain Carnis Margarita antibacterial peptide liquid.
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| CN102835541A (en) * | 2012-09-14 | 2012-12-26 | 大连格兰清水环境工程有限公司 | Method for preparing scallop hydrolyzed protein powder by using scallop scrap wastes |
| CN103073628A (en) * | 2013-02-01 | 2013-05-01 | 通化康元生物科技有限公司 | Preparation method for extracting antibacterial peptides from wood frog bodies by using ultrasonic waves |
| CN104366416A (en) * | 2014-10-16 | 2015-02-25 | 淮阴工学院 | Method for preparing corbicula fluminea juice having sobering-up function through ultrasonic assisted extraction and enzymolysis |
| CN106636270A (en) * | 2016-11-21 | 2017-05-10 | 浙江海洋大学 | Preparation method of antibacterial peptide of cyclina sinensis |
| CN109182427A (en) * | 2018-08-13 | 2019-01-11 | 中国热带农业科学院农产品加工研究所 | The preparation method of antibacterial peptide in a kind of Sipunculus nudus coelomic fluid |
| CN109892661A (en) * | 2019-03-27 | 2019-06-18 | 集美大学 | A kind of pearl shell peptide and its production method |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102835541A (en) * | 2012-09-14 | 2012-12-26 | 大连格兰清水环境工程有限公司 | Method for preparing scallop hydrolyzed protein powder by using scallop scrap wastes |
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| CN104366416A (en) * | 2014-10-16 | 2015-02-25 | 淮阴工学院 | Method for preparing corbicula fluminea juice having sobering-up function through ultrasonic assisted extraction and enzymolysis |
| CN106636270A (en) * | 2016-11-21 | 2017-05-10 | 浙江海洋大学 | Preparation method of antibacterial peptide of cyclina sinensis |
| CN109182427A (en) * | 2018-08-13 | 2019-01-11 | 中国热带农业科学院农产品加工研究所 | The preparation method of antibacterial peptide in a kind of Sipunculus nudus coelomic fluid |
| CN109892661A (en) * | 2019-03-27 | 2019-06-18 | 集美大学 | A kind of pearl shell peptide and its production method |
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