CN114515610B - Centrifuge tube and application thereof in aspect of extracting protein peptide and polyglutamic acid - Google Patents
Centrifuge tube and application thereof in aspect of extracting protein peptide and polyglutamic acid Download PDFInfo
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- CN114515610B CN114515610B CN202210258849.3A CN202210258849A CN114515610B CN 114515610 B CN114515610 B CN 114515610B CN 202210258849 A CN202210258849 A CN 202210258849A CN 114515610 B CN114515610 B CN 114515610B
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- 229920002643 polyglutamic acid Polymers 0.000 title claims abstract description 23
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 22
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 22
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 19
- 108010020346 Polyglutamic Acid Proteins 0.000 title abstract description 6
- 238000007599 discharging Methods 0.000 claims description 7
- 238000000605 extraction Methods 0.000 claims description 4
- 230000013011 mating Effects 0.000 claims 1
- 102000004196 processed proteins & peptides Human genes 0.000 claims 1
- 238000000926 separation method Methods 0.000 abstract description 10
- 238000005265 energy consumption Methods 0.000 abstract description 4
- 238000004891 communication Methods 0.000 abstract description 2
- 239000012847 fine chemical Substances 0.000 abstract description 2
- 238000007670 refining Methods 0.000 abstract description 2
- 238000000108 ultra-filtration Methods 0.000 description 17
- 239000000047 product Substances 0.000 description 13
- 239000006228 supernatant Substances 0.000 description 12
- 239000007788 liquid Substances 0.000 description 11
- 238000000855 fermentation Methods 0.000 description 8
- 230000004151 fermentation Effects 0.000 description 8
- 238000004108 freeze drying Methods 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- 230000006978 adaptation Effects 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 235000002566 Capsicum Nutrition 0.000 description 4
- 239000006002 Pepper Substances 0.000 description 4
- 241000722363 Piper Species 0.000 description 4
- 235000016761 Piper aduncum Nutrition 0.000 description 4
- 235000017804 Piper guineense Nutrition 0.000 description 4
- 235000008184 Piper nigrum Nutrition 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 235000007650 Aralia spinosa Nutrition 0.000 description 3
- 241000949456 Zanthoxylum Species 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 238000003825 pressing Methods 0.000 description 3
- 230000017854 proteolysis Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 241000193744 Bacillus amyloliquefaciens Species 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 239000004743 Polypropylene Substances 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- -1 polypropylene Polymers 0.000 description 2
- 229920001155 polypropylene Polymers 0.000 description 2
- 238000010992 reflux Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 101800000068 Antioxidant peptide Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 235000019750 Crude protein Nutrition 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000013103 analytical ultracentrifugation Methods 0.000 description 1
- 230000000151 anti-reflux effect Effects 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 238000001085 differential centrifugation Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- ZYBWTEQKHIADDQ-UHFFFAOYSA-N ethanol;methanol Chemical compound OC.CCO ZYBWTEQKHIADDQ-UHFFFAOYSA-N 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000004091 panning Methods 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
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- 238000011218 seed culture Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D17/00—Separation of liquids, not provided for elsewhere, e.g. by thermal diffusion
- B01D17/02—Separation of non-miscible liquids
- B01D17/0217—Separation of non-miscible liquids by centrifugal force
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
Abstract
The invention relates to the technical field of separation and refining of fine chemical products, in particular to a centrifuge tube and application thereof in the aspect of extracting protein peptide and polyglutamic acid. The centrifuge tube provided by the invention is convenient to operate, and has no pollution and blockage problems; the centrifugal tube realizes the communication and the separation of the upper part and the lower part of the centrifugal tube according to the magnitude of the centrifugal force generated when the piston rotates at a high speed, the product has a simple structure, and the mass production is easy to realize; the centrifuge tube can improve the concentration of the solution, and greatly reduce the subsequent treatment cost and energy consumption.
Description
Technical Field
The invention relates to the technical field of separation and refining of fine chemical products, in particular to a centrifuge tube and application thereof in the aspect of extracting protein peptide and polyglutamic acid.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Centrifugal separation is a method for separating and extracting different particles mixed in suspension or liquids which are different in density and are mutually insoluble in the suspension according to the differences in density, shape, size and the like of different substances by utilizing high-speed rotation of a motor, and is widely applied to the fields of biology, medicine, chemical industry and the like. Common centrifugation techniques include: sedimentation centrifugation, differential centrifugation, density gradient centrifugation, analytical ultracentrifugation, centrifugal panning, zonal centrifugation, continuous flow centrifugation, and the like.
Conventional centrifuge tubes are simple in structure and single in function, and cannot meet the use demands of people more and more, so that many researchers improve the centrifuge tubes, such as placing an inner tube with a filter membrane in a centrifuge tube to form a centrifugal filter tube, and the centrifugal filter tube can be used for removing bacteria and particles from a sample, preparing an HPLC sample, separating cells from a culture medium, and separating DNA from agarose gel or acrylamide gel. ZL202021300317.4 discloses an ultrafiltration centrifuge tube device, includes: the tube cover, the filter cavity, the ultrafiltration membrane, the support sleeve and the collecting tube; the filter cavity comprises an upper cavity and a lower cavity; an opening for adding the stock solution is formed in the upper end face of the upper cavity; the upper cavity is communicated with the lower cavity; the left side and the right side of the lower cavity are symmetrically provided with filter holes; the ultrafiltration membranes are attached to the left side and the right side of the lower cavity and cover the filtration holes; the support sleeve is provided with a through hole matched with the shape of the lower cavity; a liquid guide groove for enabling liquid to flow out is arranged on the hole wall of the through hole; the supporting sleeve is sleeved on the lower cavity and is connected with the upper cavity; the collecting pipe is connected with the supporting sleeve. The scheme effectively improves the recovery rate of the target product during ultrafiltration and centrifugation and reduces the loss of the target product. ZL201610042368.3 provides a take density gradient centrifuging tube of adjustable porous diaphragm in position, including centrifuging tube body, centrifuging tube body upper end is equipped with the centrifuging tube lid, this internal diaphragm layer that is equipped with of centrifuging tube is equipped with the internal thread on the centrifuging tube body inner wall, is equipped with the external screw thread on the diaphragm layer outer wall, and the internal thread of centrifuging tube body cooperatees with the external screw thread on diaphragm layer, is equipped with a plurality of apertures on the diaphragm layer. The centrifuge tube is divided into a part for containing separation liquid and a part for adding samples to be detected through the diaphragm layer, so that premixing of the samples and the separation liquid is effectively avoided, and sample pollution caused by centrifugation and sampling is reduced; meanwhile, the diaphragm layer can be adjusted up and down by a diaphragm layer knob according to different types and volumes of samples to be processed, so that the lower end of the diaphragm layer has an optimal separation liquid volume, and the application range is enlarged. The utility model discloses an integral type ultrafiltration centrifuging tube, including body, inlet, milipore filter, bottom plate, adaptation stopper, the body top is fixed with the inlet, and the inlet is the funnel form, and the inlet top is fixed with the top cap, the inside milipore filter and the bottom plate that are fixed with down in proper order of body, the drain pipe has been seted up to the bottom plate bottom, and the exit end of drain pipe is equipped with the bottom, there is the internal thread on the inner wall of body lower part, and the body bottom is fixed with an adaptation stopper, the adaptation stopper comprises interior cock body, baffle, adaptation piece, is equipped with the external screw thread with internal screw thread matched with on the interior cock body, and interior cock body is fixed in the bottom of baffle, the baffle external diameter is the same with the body external diameter, and baffle bottom is fixed with adaptation piece. According to the ultrafiltration centrifuge tube, the tube body, the bottom plate and the liquid inlet are made into an integrated structure, so that the structure of the ultrafiltration centrifuge tube is simple, mutual collision among components is not easy to occur, and the ultrafiltration centrifuge tube is not easy to damage. The ZL202022277914.6 discloses an integral type ultrafiltration centrifuging tube, which comprises a main body, the lower extreme surface of main part is provided with places protection machanism, place protection machanism's upper end surface and be provided with the outer tube, the internal surface of outer tube is provided with prevents reverse flow mechanism, prevent reverse flow mechanism's upper end surface and be provided with the ultrafilter tube. This scheme is equipped with and places protection machanism and prevent the reverse flow mechanism, can insert the visor with the ultrafiltration centrifuging tube behind the ultrafiltration centrifuging tube use, prevents that the ultrafiltration centrifuging tube from rolling to the ground and causing the damage of ultrafiltration centrifuging tube, and the fastening ring can improve connection stability, and the shock attenuation gasket can also reduce the collision of ultrafiltration centrifuging tube and visor, and the reverse flow mechanism that prevents of setting can be when the ultrafiltration centrifuging tube uses, keeps off the flow board and can block in the liquid after the filtration is reverse flow to the ultrafiltration tube, prevents to influence the filter effect of ultrafiltration centrifuging tube.
The centrifuge tube adopting the scheme can effectively achieve the purpose of centrifugal separation and can play a role in preventing reflux to a certain extent, but the porous diaphragm in the centrifuge tube is easy to block when the centrifuge tube is reused, especially for a complex sample of fermentation liquor; the anti-reflux effect is still not ideal, and especially after the device is enlarged, a new technology is sought to solve this problem.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a centrifuge tube and application thereof in the aspect of extracting protein peptide and polyglutamic acid, and the centrifuge tube realizes the connection and separation of the upper part and the lower part of the centrifuge tube according to the centrifugal force generated by a piston in high-speed rotation so as to solve the problems of easy blockage of a diaphragm of the centrifuge tube, easy reflux of materials and the like, and has good practical application value in the aspects of extracting protein peptide and polyglutamic acid and the like.
In order to achieve the above object, the technical scheme of the present invention is as follows:
in a first aspect of the invention, a centrifuge tube is provided, comprising a tube body and a blanking tube positioned in the tube body, wherein the bottom of the blanking tube is closed, the top end of the blanking tube is open, a piston and a spring are sequentially arranged in the blanking tube from top to bottom, and a discharge hole is formed in the middle upper part of the tube wall of the blanking tube.
In one or more embodiments, a bearing member is arranged in the middle of the inner wall of the pipe body;
in one or more embodiments, a supporting body is arranged at the top end of the pipe wall of the blanking pipe, the diameter of the supporting body is equal to the inner diameter of the pipe, and the supporting body is clamped on the bearing piece;
in one or more embodiments, the top outer wall of the tube body is provided with top external threads;
in one or more embodiments, the bottom of the tube is closed and the top end is open;
in one or more embodiments, the bottom of the tube body is open, and the outer wall of the bottom of the tube body is provided with bottom external threads; the outside of the tube body 1 is provided with a bottom cover, and the tube body is matched with the bottom cover for use;
preferably, the bottom cover has a height not less than 0.5 times the height of the tube body;
preferably, the bottom inner thread is arranged at the bottom end position inside the bottom cover and is matched with the bottom outer thread arranged on the outer wall of the bottom of the pipe body.
In a second aspect of the invention, there is provided the use of the centrifuge tube described above for extracting a protein peptide, the method comprising the steps of:
adding the clear liquid containing the protein peptide into the centrifuge tube, and centrifuging for a certain time under the state of being higher than the rotating speed RS; when the bottom of the tube body is closed and the top end is open, pouring out the upper layer centrifugate, continuously using the upper layer centrifugate for proteolysis to prepare protein peptide, pressing a piston, and pouring out the lower layer centrifugate through a discharge hole; or when the bottom of the tube body is opened and is matched with the bottom cover for use, pouring out the upper layer centrifugate, continuously using the upper layer centrifugate for proteolysis to prepare protein peptide, and taking down the bottom cover and obtaining the lower layer centrifugate; freeze-drying the lower layer centrifugate to obtain a protein peptide product;
the specification of the spring is determined by experiments, and the discharge hole is opened in a centrifugal state above the rotating speed RS and is closed in a centrifugal state below the rotating speed RS due to the movement of the piston.
In a third aspect of the present invention, there is provided an application of the centrifuge tube in extracting gamma-polyglutamic acid, the method of the application comprising the steps of:
adding the clear liquid containing gamma-polyglutamic acid into the centrifuge tube, and centrifuging for a certain time at a state of being higher than the rotating speed RS; when the bottom of the tube body is closed and the top end is open, pouring out the upper layer centrifugate, continuously using the upper layer centrifugate for fermenting gamma-polyglutamic acid, pressing a piston and pouring out the lower layer centrifugate through a discharge hole; or when the bottom of the tube body is opened and is matched with the bottom cover for use, pouring out the upper layer centrifugate, continuously using the upper layer centrifugate for fermenting gamma-polyglutamic acid, and taking down the bottom cover and obtaining the lower layer centrifugate; freeze-drying the lower layer centrifugate to obtain a gamma-polyglutamic acid product;
the specification of the spring is determined by experiments, and the discharge hole is opened in a centrifugal state above the rotating speed RS and is closed in a centrifugal state below the rotating speed RS due to the movement of the piston.
The specific embodiment of the invention has the following beneficial effects:
(1) The centrifuge tube provided by the invention is convenient to operate, and has no pollution and blockage problems;
(2) The centrifugal tube provided by the invention realizes the communication and separation of the upper part and the lower part of the centrifugal tube according to the centrifugal force generated by the piston during high-speed rotation, has a simple product structure, and is easy to realize batch production;
(3) The centrifuge tube provided by the invention can improve the concentration of the solution and greatly reduce the subsequent treatment cost and energy consumption.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a schematic view of the structure of a centrifuge tube of the present invention;
FIG. 2 is a schematic view of the centrifuge tube of the present invention in a centrifuged condition.
FIG. 3 is a schematic view of an open-bottom centrifuge tube according to the present invention.
FIG. 4 is a schematic view of the bottom cover of the centrifuge tube of the present invention.
In the figure: 1-a tube body; 2-a support; 3-blanking pipe; 4-top external threads; 5-a piston; 6-a load bearing member; 7-a discharge hole; 8-a spring; 9-bottom external threads; 10-a bottom cover; 11-bottom cap internal threads.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
The invention is further illustrated and described below in connection with specific examples.
Example 1
As shown in fig. 1 and 2, a centrifugal tube comprises a tube body 1 and a blanking tube 3 positioned in the tube body, wherein the bottom of the blanking tube 3 is closed, the top end of the blanking tube is open, a piston 5 and a spring 8 are sequentially arranged in the blanking tube 3 from top to bottom, and a discharge hole 7 is formed in the middle upper part of the tube wall of the blanking tube 3;
the middle part of the inner wall of the pipe body 1 is provided with a bearing piece 6; the top end of the pipe wall of the blanking pipe 3 is provided with a supporting body 2, the diameter of the supporting body 2 is equal to the inner diameter of the pipe body 1, and the supporting body is clamped on a bearing piece 6;
the bottom of the tube body 1 is closed, and the top end is open; the outer wall of the top end of the pipe body 1 is provided with a top end external thread 4;
the pipe body 1 is made of polypropylene, the inner diameter is 50mm, the height is 200mm, and the distance between the top end of the bearing piece 6 and the top end of the pipe body 1 is 180mm; the blanking pipe 3 is made of polypropylene, the inner diameter is 15mm, and the height is 50mm; the piston 5 is made of stainless steel 316L material, the outer diameter is 15mm, and the height is 15mm; the diameter of the discharging hole 7 is 4mm;
the specifications of the spring 8 were selected and experimentally determined, and the discharge hole 7 was opened in a centrifugal state of 50000rpm (revolutions per minute) or more and the discharge hole 7 was closed in a centrifugal state of 50000rpm or less due to the movement of the piston 5.
The centrifuge tube is applied to the extraction of the pricklyash seed protein peptide, adopts the pricklyash seed protein (crude protein content is 88.05 percent, based on dry basis) which is self-made in a laboratory as a raw material, and adopts papain (enzyme activity is 2.0 multiplied by 10) purchased from Beijing Hua Maike biotechnology Limited responsible company 5 U/g) carrying out enzymolysis on pepper seed kernel protein, firstly, pretreating the pepper seed protein for 10min at 90-100 ℃, and then carrying out enzymolysis under the following conditions: substrate concentration is 30mg/mL, protease addition amount is 0.08mg/mL, pH=10, enzymolysis temperature is 60 ℃, and enzymolysis time is 3.0h. Cold waterAdding into a common centrifuge tube, centrifuging at 6000rpm at 5deg.C for 10min, and collecting supernatant. The protein peptide content in the supernatant was determined to be 29.61mg/mL according to the method used in literature (Wang Hui, tian Chengrui, ma Shoulei, et al, screening studies of enzymes for antioxidant peptide hydrolysis of pricklyash seed kernel, food and fermentation industry, 2009, 35 (3): 119-123).
200mL of supernatant is added into the centrifuge tube, and is centrifuged for 30min at 60000rpm at 5 ℃, the supernatant is poured out, and the supernatant is continuously used for proteolysis; pressing the piston 5 and pouring out the lower layer centrifugate through the discharge hole 7; and (3) placing the obtained lower layer centrifugate in a refrigerator at the temperature of 4 ℃ for standby after multiple times of operation, and determining the content of the protein peptide in the lower layer centrifugate to be 38.20mg/mL.
And taking 200mL of lower layer centrifugate, and freeze-drying to obtain 9.16g of pepper seed protein peptide product.
Example 2
The points of the present embodiment that are the same as those of embodiment 1 are not described in detail, and the difference is that:
as shown in fig. 3 and 4, the bottom of the pipe body 1 is open, and the outer wall of the bottom of the pipe body is provided with bottom external threads 9; a bottom cover 10 is arranged outside the pipe body 1, and the pipe body 1 is matched with the bottom cover 10 for use;
the height of the bottom cover 10 is not less than 0.5 times of the height of the tube body 1; the bottom internal thread 11 is arranged at the bottom end position inside the bottom cover 10 and is matched with the bottom external thread 9 arranged on the outer wall of the bottom of the pipe body.
The centrifuge tube described above was applied to the extraction of gamma-polyglutamic acid and fermentation of gamma-polyglutamic acid was performed using laboratory deposit bacillus amyloliquefaciens strain Bacillus amyloliquefaciens L536, wherein the slant medium, seed medium, fermentation medium were prepared according to the material ratios described in literature (Zeng Qingdong, li Wenjie, wang Zhiwei, et al, methanol-ethanol stepwise precipitation method for the extraction of gamma-polyglutamic acid food and fermentation industries, 2014, 40 (5): 222-228). In a specific culture, the strain is inoculated in a slant and a flat culture medium, and the strain is placed at 35 ℃ for 24 hours for culture. Inoculating 1-2 kinds of fungus to seed culture medium at 180rpm, and culturing at 35 deg.c for 24 hr. The seed inoculum was placed in a 3L fermentation tank at an inoculum size of 5% (volume percent) and cultured at 37℃and 200rpm for 48 hours to obtain a fermentation broth.
Regulating pH of the fermentation broth to 3.0, adding into a common centrifuge tube, centrifuging at 6000rpm at 5deg.C for 10min to remove thallus, and collecting supernatant, and measuring gamma-polyglutamic acid concentration to 25.18g/L. 200mL of supernatant is added into the centrifuge tube, and is centrifuged for 30min at 60000rpm at 5 ℃, the upper layer centrifugate is poured out, and the upper layer centrifugate is continuously used for fermenting gamma-polyglutamic acid; taking down the bottom cover 10 and obtaining a lower layer centrifugate, and measuring the concentration of gamma-polyglutamic acid in the lower layer centrifugate to be 30.55g/L; and (3) placing the obtained lower layer centrifugate in a refrigerator at the temperature of 4 ℃ for standby.
Taking 200mL of lower layer centrifugate, regulating the pH to 7.0, adding 4 times of ethanol, standing at low temperature for precipitation for 6 hours, filtering, and freeze-drying to obtain 5.20g of gamma-polyglutamic acid product.
Compared with the embodiment 1, the bottom of the pipe body 1 is open and is matched with the bottom cover 10, so that the centrifuged lower-layer feed liquid is easier to take out and the operation is more convenient.
Comparative example 1
The points of the present embodiment that are the same as those of embodiment 1 are not described in detail, and the difference is that:
directly taking 200mL of supernatant, and obtaining 7.03g of gamma-polyglutamic acid product after freeze drying.
Comparative example 2
The points of the present embodiment that are the same as those of embodiment 1 are not described in detail, and the difference is that:
adding 200mL of supernatant into a common centrifuge tube, centrifuging at 60000rpm for 30min at 5 ℃, sucking half of the supernatant out by a suction tube, and collecting the rest lower layer supernatant; and (3) placing the obtained lower layer centrifugate in a refrigerator at the temperature of 4 ℃ for standby after multiple times of operation, and determining the content of the protein peptide in the lower layer centrifugate to be 30.15mg/mL.
And taking 200mL of lower layer centrifugate, and freeze-drying to obtain 7.22g of pepper seed protein peptide product.
As can be seen from comparative examples 1, 2 and 3, the centrifuge tube provided in example 1 of the present invention can greatly reduce the energy consumption of the freeze-drying step by improving the yield of the protein peptide product.
Comparative example 3
The points of the present embodiment that are the same as those of embodiment 2 are not described in detail, and the difference is that:
directly taking 200mL of supernatant, regulating the pH to 7.0, adding 4 times of ethanol, standing at low temperature for precipitation for 6h, filtering, and freeze-drying to obtain 4.01g of gamma-polyglutamic acid product.
As can be seen from comparative examples 2 and 3, the centrifuge tube provided in example 2 of the present invention increases the concentration of the gamma-polyglutamic acid solution, and the yield of gamma-polyglutamic acid product is increased by 30% when the same ethanol (precipitant) is used, so that the amount of precipitant used and the energy consumption for recovering the precipitant can be greatly reduced.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. The centrifugal tube is characterized by comprising a tube body and a discharging tube positioned in the tube body, wherein the bottom of the discharging tube is closed, the top end of the discharging tube is open, a piston and a spring are sequentially arranged in the discharging tube from top to bottom, and a discharging hole is formed in the middle upper part of the tube wall of the discharging tube;
the middle part of the inner wall of the pipe body is provided with a bearing piece; the top end of the pipe wall of the blanking pipe is provided with a supporting body, the diameter of the supporting body is equal to the inner diameter of the pipe body, and the supporting body is clamped on the bearing piece;
the bottom of the pipe body is open, and the outer wall of the bottom of the pipe body is provided with bottom external threads; the outside of the tube body is provided with a bottom cover, and the tube body is matched with the bottom cover for use;
the discharge hole is communicated with the pipe body in a centrifugal state above a certain rotating speed due to the movement of the piston, and is separated from the pipe body in a centrifugal state below the certain rotating speed.
2. The centrifuge tube of claim 1, wherein the top outer wall of the tube body is provided with top external threads.
3. The centrifuge tube of claim 1, wherein the bottom cover has a height no less than 0.5 times the height of the tube body.
4. The centrifuge tube of claim 3, wherein the bottom cap has internal bottom threads at a location at the inner bottom end thereof for mating with external bottom threads provided on the outer wall of the bottom of the tube body.
5. The use of the centrifuge tube according to any one of claims 1 to 4 for extracting protein peptides.
6. The application of the centrifuge tube in the extraction of gamma-polyglutamic acid according to any one of claims 1-4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210258849.3A CN114515610B (en) | 2022-03-16 | 2022-03-16 | Centrifuge tube and application thereof in aspect of extracting protein peptide and polyglutamic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| CN103079577A (en) * | 2010-03-11 | 2013-05-01 | 安托万·图瑞兹 | Process,tube and device for the preparation of wound healant composition |
| CN106179552A (en) * | 2016-07-06 | 2016-12-07 | 江苏科华医疗器械科技有限公司 | A kind of integral type ultra-filtration centrifuge tube |
| CN106861786A (en) * | 2017-04-11 | 2017-06-20 | 中国农业科学院麻类研究所 | A kind of centrifuge tube for dividing liquid |
| CN108587884A (en) * | 2018-07-16 | 2018-09-28 | 苏州呼呼健康科技有限公司 | A kind of centrifuge tube |
| CN212441265U (en) * | 2020-07-06 | 2021-02-02 | 广州洁特生物过滤股份有限公司 | Ultrafiltration centrifuge tube device |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102051207B1 (en) * | 2018-01-30 | 2019-12-03 | 이준석 | Piston for centriguation |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103079577A (en) * | 2010-03-11 | 2013-05-01 | 安托万·图瑞兹 | Process,tube and device for the preparation of wound healant composition |
| CN106179552A (en) * | 2016-07-06 | 2016-12-07 | 江苏科华医疗器械科技有限公司 | A kind of integral type ultra-filtration centrifuge tube |
| CN106861786A (en) * | 2017-04-11 | 2017-06-20 | 中国农业科学院麻类研究所 | A kind of centrifuge tube for dividing liquid |
| CN108587884A (en) * | 2018-07-16 | 2018-09-28 | 苏州呼呼健康科技有限公司 | A kind of centrifuge tube |
| CN212441265U (en) * | 2020-07-06 | 2021-02-02 | 广州洁特生物过滤股份有限公司 | Ultrafiltration centrifuge tube device |
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