CN109280081B - Method for preparing sialoglycopeptide from tuna eggs - Google Patents
Method for preparing sialoglycopeptide from tuna eggs Download PDFInfo
- Publication number
- CN109280081B CN109280081B CN201811260150.0A CN201811260150A CN109280081B CN 109280081 B CN109280081 B CN 109280081B CN 201811260150 A CN201811260150 A CN 201811260150A CN 109280081 B CN109280081 B CN 109280081B
- Authority
- CN
- China
- Prior art keywords
- sialoglycopeptide
- tuna
- roe
- drying
- powder
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108010032838 Sialoglycoproteins Proteins 0.000 title claims abstract description 95
- 102000007365 Sialoglycoproteins Human genes 0.000 title claims abstract description 95
- 235000013601 eggs Nutrition 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 22
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 48
- 239000012043 crude product Substances 0.000 claims abstract description 19
- 238000000605 extraction Methods 0.000 claims abstract description 18
- 229920005654 Sephadex Polymers 0.000 claims abstract description 15
- 239000012507 Sephadex™ Substances 0.000 claims abstract description 14
- 239000000047 product Substances 0.000 claims abstract description 14
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims abstract description 8
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 6
- 239000000843 powder Substances 0.000 claims description 36
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 34
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Substances OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- 238000004108 freeze drying Methods 0.000 claims description 17
- 238000005238 degreasing Methods 0.000 claims description 11
- 238000001914 filtration Methods 0.000 claims description 11
- 239000008367 deionised water Substances 0.000 claims description 10
- 229910021641 deionized water Inorganic materials 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 238000001035 drying Methods 0.000 claims description 9
- 238000001179 sorption measurement Methods 0.000 claims description 9
- 238000009210 therapy by ultrasound Methods 0.000 claims description 7
- 238000000502 dialysis Methods 0.000 claims description 6
- 239000003480 eluent Substances 0.000 claims description 6
- 235000019441 ethanol Nutrition 0.000 claims description 6
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 238000010257 thawing Methods 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 abstract description 2
- 238000003809 water extraction Methods 0.000 abstract description 2
- 241000251468 Actinopterygii Species 0.000 description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 238000007710 freezing Methods 0.000 description 4
- 230000008014 freezing Effects 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000004519 grease Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- DQJCDTNMLBYVAY-ZXXIYAEKSA-N (2S,5R,10R,13R)-16-{[(2R,3S,4R,5R)-3-{[(2S,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-5-(ethylamino)-6-hydroxy-2-(hydroxymethyl)oxan-4-yl]oxy}-5-(4-aminobutyl)-10-carbamoyl-2,13-dimethyl-4,7,12,15-tetraoxo-3,6,11,14-tetraazaheptadecan-1-oic acid Chemical compound NCCCC[C@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)CC[C@H](C(N)=O)NC(=O)[C@@H](C)NC(=O)C(C)O[C@@H]1[C@@H](NCC)C(O)O[C@H](CO)[C@H]1O[C@H]1[C@H](NC(C)=O)[C@@H](O)[C@H](O)[C@@H](CO)O1 DQJCDTNMLBYVAY-ZXXIYAEKSA-N 0.000 description 1
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 102000002068 Glycopeptides Human genes 0.000 description 1
- 108010015899 Glycopeptides Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000013345 egg yolk Nutrition 0.000 description 1
- 210000002969 egg yolk Anatomy 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 210000002997 osteoclast Anatomy 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Meat, Egg Or Seafood Products (AREA)
Abstract
The invention discloses a method for preparing sialoglycopeptide from tuna eggs, which fills the blank of the tuna processing industry in the aspect of sialoglycopeptide. The method comprises the steps of obtaining crude product of the tuna egg sialoglycopeptide by ultrasonic-assisted water extraction of defatted roe, separating by a sephadex G-50 column and adsorbing by active carbon, and eluting by a methanol-water system to obtain pure product of the tuna egg sialoglycopeptide. The preparation method is simple and convenient, has high extraction rate, and improves the safety of the product.
Description
Technical Field
The invention relates to the technical field of food processing, in particular to a method for preparing sialoglycopeptide from tuna eggs.
Background
Sialoglycopeptide is a complex N-glyco-peptide and can only be isolated from natural products at present. Sialoglycopeptide is a humanized glycopeptide, and has effects of regulating permeability of glomerulus and inhibiting growth of osteoclast. The highly active sialoglycopeptide is mainly present in sexually mature animal egg cells. The main international methods for isolating and purifying sialoglycopeptide are reported in 1997 by Akira Seko, Japan. The method needs two times of sephadex separation, one time of DEAE ion exchange column and one time of CM-Sephadex separation, and has very complex process and longer period. The method is characterized in that primary dextran gel is used for separation, and acetonitrile elution is used for obtaining the egg yolk sialoglycopeptide after activated carbon adsorption, so that the process is simplified, the yield is high, but the serious potential safety hazard exists in the obtained sialoglycopeptide due to the introduction of phenol in the extraction process and the introduction of acetonitrile in the separation and purification process.
Tuna is a deep-sea economic fish with high fishing amount and is one of three kinds of nutritional fishes recommended by the international society for nutrition. Through determination, the content of the sialoglycopeptide in the mature tuna eggs accounts for about 4 percent by weight in dry weight. At present, no literature is available for reporting the peptide about the sialic acid of tuna eggs. In view of the high biological activity of sialoglycopeptide, the development of a method for producing sialoglycopeptide from tuna eggs has a positive influence on increasing the added value of tuna and promoting the development of the human health industry.
Disclosure of Invention
The invention aims to: to solve the above problems, a method for producing sialoglycopeptide from tuna eggs is provided.
The technical scheme of the invention is as follows:
a method for preparing sialoglycopeptide from tuna eggs, comprising the steps of:
(1) taking out the frozen tuna roes, thawing, homogenizing, and freeze-drying to obtain tuna roe powder;
(2) degreasing the tuna egg powder by absolute ethyl alcohol, filtering and drying to obtain degreased tuna egg powder;
(3) extracting, centrifuging and concentrating the degreased tuna egg powder under the assistance of ultrasonic waves by using deionized water to obtain a concentrated solution;
(4) separating the concentrated solution by a sephadex G-50 column, and concentrating under reduced pressure to obtain crude product of the tuna roe sialoglycopeptide;
(5) and adsorbing the crude product of the sialoglycopeptide of the tuna roe by using activated carbon, and eluting the crude product of the sialoglycopeptide of the tuna roe by using a methanol-water system to obtain a pure product of the sialoglycopeptide of the tuna roe.
Further, the thawing temperature in the step (1) is 4 ℃, the freezing temperature is-20 ℃, and the time is 12 hours.
Further, the step (2) of degreasing the tuna egg powder with absolute ethyl alcohol, filtering and drying to obtain the degreased tuna egg powder comprises the following steps: adding the tuna egg powder into 10-15 times of volume of absolute ethyl alcohol, stirring for 10h for degreasing, replacing the absolute ethyl alcohol once every 3h, filtering to remove the ethanol through double-layer filtering after degreasing, taking filter residue, and drying in a cold air dryer for 6-10h to obtain the degreased tuna egg powder.
Further, in the step (3), the defatted tuna egg powder is extracted, centrifuged and concentrated by deionized water under the assistance of ultrasonic waves, and a concentrated solution is obtained by the following steps: adding the degreased tuna roe powder into deionized water according to the weight-volume ratio of 20-30 times of the degreased tuna roe powder to extract sialoglycopeptide, performing ultrasonic treatment every hour during the extraction period to assist in obtaining the sialoglycopeptide, centrifuging after the obtaining is finished, and performing reduced pressure concentration on supernatant at the temperature of 40-45 ℃ to obtain concentrated solution.
Further, the extraction temperature is 4-10 ℃, the extraction time is 12-16h, the ultrasonic treatment time is 10-30min, and the intensity of an ultrasonic sound field is 200-300W/cm2The ultrasonic frequency is 30kHz, the centrifugal speed is 8000r/min, and the centrifugal time is 10-15 min.
Further, the concentrated solution obtained in the step (4) is separated by a sephadex G-50 column and then is concentrated under reduced pressure to obtain a crude product of the tuna roe sialoglycopeptide, which comprises: separating the concentrated solution with Sephadex G-50 column at 20-30 deg.C and flow rate of 1.0-1.5ml/min, collecting eluate every 7min, determining whether sialoglycopeptide in the eluate flows off by high performance liquid chromatography, collecting eluate containing sialoglycopeptide, concentrating under reduced pressure at 40-45 deg.C, and freeze drying to obtain crude product of sialoglycopeptide from tuna roe.
Further, the activated carbon adsorption in the step (5) is as follows: preparing the crude product of the sialoglycopeptide from the tuna roe into a solution with the concentration of 100-150mg/ml, and adding a proper amount of activated carbon to adsorb the sialoglycopeptide.
Furthermore, the dosage of the active carbon is 10mg/ml, and the adsorption time is 60-120 min.
Further, the methanol-water system in the step (5) is eluted as follows: eluting sialoglycopeptide in the activated carbon by NaCl-methanol solution with the mass fraction of 4-6% for three times, collecting eluent, dialyzing by a dialysis bag with the molecular cut-off of 200Da, concentrating under reduced pressure, and freeze-drying to obtain the pure product of the sialoglycopeptide of the tuna roe.
Further, the volume fraction of the methanol is 5-8%.
The invention provides a method for preparing sialoglycopeptide from tuna eggs, which fills the blank of the tuna processing industry in the aspect of sialoglycopeptide. The method comprises the steps of obtaining crude product of the tuna egg sialoglycopeptide by ultrasonic-assisted water extraction of defatted roe, separating by a sephadex G-50 column and adsorbing by active carbon, and eluting by a methanol-water system to obtain pure product of the tuna egg sialoglycopeptide. The preparation method is simple and convenient, has high extraction rate, and improves the safety of the product.
Detailed Description
The invention provides a method for preparing sialoglycopeptide from tuna eggs, which comprises the following steps:
removing egg membrane from tuna roe, homogenizing, and freeze-drying;
degreasing the freeze-dried roe by using absolute ethyl alcohol and then drying the roe;
extracting sialoglycopeptide with deionized water under the assistance of ultrasonic waves;
separating with Sephadex G-50 column, and concentrating under reduced pressure;
adsorbing with active carbon, and eluting with methanol-water system to obtain pure product of the tuna egg sialoglycopeptide.
The present invention will be described in further detail with reference to specific embodiments in order to make the above objects, features and advantages more apparent and understandable.
A method for preparing sialoglycopeptide from tuna eggs, comprising:
the method comprises the following steps: removing egg membrane from tuna roe, homogenizing, and freeze-drying;
this step may be specifically performed as follows: taking out frozen tuna roe, thawing at 4 deg.C, homogenizing, and freeze drying at-20 deg.C for 12 hr to obtain tuna roe powder.
Step two: degreasing the freeze-dried roe by using absolute ethyl alcohol and then drying the roe;
this step may be specifically performed as follows: adding the tuna egg powder into 10-15 times of volume of absolute ethyl alcohol, stirring for 10h for degreasing, replacing the absolute ethyl alcohol once every 3h, filtering to remove the ethanol through double-layer filtering after degreasing, taking filter residue, and drying in a cold air dryer for 6-10h to obtain the degreased tuna egg powder.
Step three: extracting sialoglycopeptide with deionized water under the assistance of ultrasonic waves;
this step may be specifically performed as follows: adding the degreased tuna roe powder into deionized water according to the weight-volume ratio of 20-30 times of the degreased tuna roe powder to perform sialoglycopeptide synthesisExtracting at 4-10 deg.C for 12-16h, performing ultrasonic treatment for 10-30min per hour during extraction period to obtain sialoglycopeptide, centrifuging for 10-15min after the acquisition is finished, collecting supernatant, and concentrating under reduced pressure at 40-45 deg.C to obtain concentrated solution, wherein the intensity of ultrasonic field is 200-300W/cm2The ultrasonic frequency is 30kHz, and the centrifugal speed is 8000 r/min.
Step four: separating with Sephadex G-50 column, and concentrating under reduced pressure;
this step may be specifically performed as follows: separating the concentrated solution with Sephadex G-50 column at 20-30 deg.C and flow rate of 1.0-1.5ml/min, collecting eluate every 7min, determining whether sialoglycopeptide in the eluate flows off by high performance liquid chromatography, collecting eluate containing sialoglycopeptide, concentrating under reduced pressure at 40-45 deg.C, and freeze drying to obtain crude product of sialoglycopeptide from tuna roe.
Step five: adsorbing with active carbon, and eluting with methanol-water system to obtain pure product of the tuna egg sialoglycopeptide.
This step may be specifically performed as follows: preparing the crude product of the sialoglycopeptide from the tuna roe into a solution with the concentration of 100-150mg/ml, adding a proper amount of activated carbon to adsorb the sialoglycopeptide, wherein the amount of the activated carbon is 10mg/ml, the adsorption time is 60-120min, eluting the sialoglycopeptide in the activated carbon by using a NaCl-methanol solution with the mass fraction of 4-6% for three times, collecting eluent, dialyzing by using a dialysis bag with the molecular cut-off of 200Da, concentrating under reduced pressure, and freeze-drying to obtain the pure product of the sialoglycopeptide from the tuna roe.
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, embodiments accompanying the present invention are further described below. The invention is not limited to the embodiments listed but also comprises any other known variations within the scope of the invention as claimed.
Reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic may be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Example 1
The embodiment shows a method for preparing sialoglycopeptide from tuna eggs according to the following steps:
taking out frozen tuna roe, thawing at 4 deg.C, homogenizing with a homogenizer, freezing at-20 deg.C for 12 hr, and lyophilizing with a lyophilizer to obtain tuna roe powder. Adding 10 times volume of anhydrous ethanol (1/10, w/v) into lyophilized roe powder, stirring for 10h, and defatting while replacing the anhydrous ethanol every 3 h. And filtering the degreased double-layer filter grease to remove ethanol, and drying the filter residue in a cold air dryer for 6 hours to obtain the degreased tuna roe powder. Adding deionized water into defatted fish egg powder at a weight volume ratio of 20 times of the defatted fish egg powder for extracting sialoglycopeptide (1/20, w/v), wherein the extraction temperature is 4 deg.C, and the extraction time is 12 h. During the period, ultrasonic treatment is carried out every hour to assist in extracting sialoglycopeptide, the treatment time is 10min, and the intensity of an ultrasonic sound field is 200W/cm2The ultrasonic frequency was 30 kHz. And centrifuging at 8000r/min for 10min after extraction. The supernatant was concentrated under reduced pressure at 40 ℃. Separating the concentrated solution with Sephadex G-50 column at 20 deg.C and flow rate of 1.0ml/min, collecting eluate every 7min, and determining whether sialoglycopeptide in the eluate flows off by high performance liquid chromatography. Collecting eluate containing sialoglycopeptide, concentrating under reduced pressure at 40 deg.C, and freeze drying to obtain crude product of sialoglycopeptide from tuna roe. Preparing the freeze-dried crude product of the tuna roe sialoglycopeptide into a solution with the concentration of 100mg/ml, adding a proper amount of activated carbon to adsorb the sialoglycopeptide, wherein the using amount of the activated carbon is 10mg/ml, the adsorption time is 60min, eluting the sialoglycopeptide in the activated carbon by using NaCl-methanol solution with the mass fraction of 4% (the volume fraction of methanol is 5%) for three times, collecting eluent, dialyzing by using a dialysis bag with the molecular cut-off of 200Da, concentrating under reduced pressure, and freeze-drying to obtain the pure product of the tuna roe sialoglycopeptide.
Example 2
The embodiment shows a method for preparing sialoglycopeptide from tuna eggs according to the following steps: frozen tunaThawing the roe at 4 deg.C, homogenizing with a homogenizer, freezing at-20 deg.C for 12h, and lyophilizing with a lyophilizer to obtain powder of tuna roe. Adding 13 times volume of anhydrous ethanol (1/12, w/v) into lyophilized roe powder, stirring for 10h, and defatting while replacing the anhydrous ethanol every 3 h. And filtering the degreased double-layer filter grease to remove ethanol, and drying the filter residue in a cold air dryer for 8 hours to obtain the degreased tuna roe powder. Adding deionized water into defatted fish egg powder 25 times of the defatted fish egg powder by weight volume ratio to extract sialoglycopeptide (1/25, w/v), wherein the extraction temperature is 8 ℃, and the extraction time is 14 h. During the period, ultrasonic treatment is carried out every hour to assist in extracting sialoglycopeptide, the treatment time is 20min, and the intensity of an ultrasonic field is 250W/cm2The ultrasonic frequency was 30 kHz. And centrifuging at 8000r/min for 13min after extraction. The supernatant was concentrated under reduced pressure at 43 ℃. Separating the concentrated solution with Sephadex G-50 column at 25 deg.C and flow rate of 1.2ml/min, collecting eluate every 7min, and determining whether sialoglycopeptide in the eluate flows off by high performance liquid chromatography. Collecting eluate containing sialoglycopeptide, concentrating under reduced pressure at 43 deg.C, and freeze drying to obtain crude product of sialoglycopeptide from tuna roe. Preparing the freeze-dried crude product of the tuna roe sialoglycopeptide into a solution with the concentration of 125mg/ml, adding a proper amount of activated carbon to adsorb the sialoglycopeptide, wherein the using amount of the activated carbon is 10mg/ml, the adsorption time is 95min, eluting the sialoglycopeptide in the activated carbon by using NaCl-methanol solution with the mass fraction of 5% (the volume fraction of methanol is 7%) for three times, collecting eluent, dialyzing by using a dialysis bag with the molecular cut-off of 200Da, concentrating under reduced pressure, and freeze-drying to obtain the pure product of the tuna roe sialoglycopeptide.
Example 3
The embodiment shows a method for preparing sialoglycopeptide from tuna eggs according to the following steps:
taking out frozen tuna roe, thawing at 4 deg.C, homogenizing with a homogenizer, freezing at-20 deg.C for 12 hr, and lyophilizing with a lyophilizer to obtain tuna roe powder. Adding 15 times volume of absolute ethyl alcohol (1/15, w/v) into the freeze-dried roe powder, stirring for 10h for degreasing, and replacing the absolute ethyl alcohol every 3 h. Double-layer filtering after degreasingFiltering to remove ethanol, and air drying the filter residue in a cold air dryer for 10h to obtain defatted tuna egg powder. Adding deionized water into defatted fish egg powder 30 times of the defatted fish egg powder by weight volume ratio to extract sialoglycopeptide (1/30, w/v), wherein the extraction temperature is 10 ℃, and the extraction time is 16 h. During the period, ultrasonic treatment is carried out every hour to assist in extracting sialoglycopeptide, the treatment time is 30min, and the intensity of an ultrasonic field is 300W/cm2The ultrasonic frequency was 30 kHz. And centrifuging at 8000r/min for 15min after extraction. The supernatant was concentrated under reduced pressure at 45 ℃. Separating the concentrated solution with Sephadex G-50 column at 30 deg.C and flow rate of 1.5ml/min, collecting eluate every 7min, and determining whether sialoglycopeptide in the eluate flows off by high performance liquid chromatography. Collecting eluate containing sialoglycopeptide, concentrating under reduced pressure at 45 deg.C, and freeze drying to obtain crude product of sialoglycopeptide from tuna roe. Preparing the freeze-dried crude product of the tuna roe sialoglycopeptide into a solution with the concentration of 150mg/ml, adding a proper amount of activated carbon to adsorb the sialoglycopeptide, wherein the using amount of the activated carbon is 10mg/ml, the adsorption time is 120min, eluting the sialoglycopeptide in the activated carbon by NaCl-methanol solution with the mass fraction of 6% (the volume fraction of the methanol is 8%) for three times, collecting eluent, dialyzing by a dialysis bag with the molecular cut-off of 200Da, concentrating under reduced pressure, and freeze-drying to obtain the pure product of the tuna roe sialoglycopeptide.
Table 1 below shows the comparison of three examples with the prior art, in particular as follows:
TABLE 1
Compared with the prior art, the invention has the beneficial effects that: simple production process, short period, simplicity, convenience, higher extraction rate, high purity and high product safety.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (3)
1. A method for preparing sialoglycopeptide from tuna eggs, comprising the steps of:
(1) taking out frozen tuna roes, thawing at 4 deg.C, homogenizing, and freeze-drying at-20 deg.C for 12 hr to obtain tuna roe powder;
(2) adding the tuna egg powder into 10-15 times of volume of absolute ethyl alcohol, stirring for 10h, degreasing, replacing the absolute ethyl alcohol once every 3h, filtering to remove the ethanol by double-layer filtering after degreasing, taking filter residue, and drying in a cold air dryer for 6-10h to obtain degreased tuna egg powder;
(3) adding the degreased tuna roe powder into deionized water according to the weight-volume ratio of 20-30 times of the degreased tuna roe powder to extract sialoglycopeptide, extracting for 12-16h at the temperature of 4-10 ℃, performing ultrasonic treatment for 10-30min per hour during extraction, wherein the intensity of an ultrasonic field is 200-300W/cm2Ultrasonic frequency of 30kHz to assist in obtaining sialoglycopeptide, centrifuging at 8000r/min for 10-15min after obtaining sialoglycopeptide, and concentrating supernatant under reduced pressure at 40-45 deg.C to obtain concentrated solution;
(4) separating the concentrated solution with Sephadex G-50 column at 20-30 deg.C and flow rate of 1.0-1.5ml/min, collecting eluate every 7min, determining whether sialoglycopeptide in the eluate flows off by high performance liquid chromatography, collecting eluate containing sialoglycopeptide, concentrating under reduced pressure at 40-45 deg.C, and freeze drying to obtain crude product of sialoglycopeptide from tuna roe;
(5) adsorbing the crude product of the sialoglycopeptide of the tuna roe by using activated carbon, and eluting by using a methanol-water system, wherein the methanol-water system comprises the following steps: eluting sialoglycopeptide in the activated carbon by NaCl-methanol solution with the mass fraction of 4-6% for three times, collecting eluent, dialyzing by a dialysis bag with the molecular cut-off of 200Da, concentrating under reduced pressure, and freeze-drying to obtain a pure product of the sialoglycopeptide of the tuna roe, wherein the volume fraction of the methanol is 5-8%.
2. The method for preparing sialoglycopeptide from tuna eggs according to claim 1, wherein the activated carbon adsorption in step (5) is: preparing the crude product of the sialoglycopeptide from the tuna roe into a solution with the concentration of 100-150mg/ml, and adding a proper amount of activated carbon to adsorb the sialoglycopeptide.
3. The method of claim 2, wherein the sialoglycopeptide is prepared from tuna eggs by: the dosage of the active carbon is 10mg/ml, and the adsorption time is 60-120 min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811260150.0A CN109280081B (en) | 2018-10-26 | 2018-10-26 | Method for preparing sialoglycopeptide from tuna eggs |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811260150.0A CN109280081B (en) | 2018-10-26 | 2018-10-26 | Method for preparing sialoglycopeptide from tuna eggs |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109280081A CN109280081A (en) | 2019-01-29 |
| CN109280081B true CN109280081B (en) | 2021-06-25 |
Family
ID=65177949
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811260150.0A Active CN109280081B (en) | 2018-10-26 | 2018-10-26 | Method for preparing sialoglycopeptide from tuna eggs |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109280081B (en) |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102050866B (en) * | 2009-11-04 | 2015-02-18 | 上海来益生物药物研究开发中心有限责任公司 | Method for purifying glycopeptide compound |
| CN102382178B (en) * | 2011-10-21 | 2013-05-29 | 山东大学 | Separation method of sialoglycopeptide |
| CN103114117B (en) * | 2013-01-24 | 2015-08-05 | 中国海洋大学 | A kind of preparation method with the fish-egg glycoprotein of osteoporosis disease effect |
| CN107868808B (en) * | 2017-11-01 | 2021-03-02 | 金腰燕（广州）食品有限公司 | Method for improving free acid in cubilose saliva |
-
2018
- 2018-10-26 CN CN201811260150.0A patent/CN109280081B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN109280081A (en) | 2019-01-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104710525B (en) | Tuna bone collagen source zinc chelated collagen peptide and preparation method and application thereof | |
| EP2823714B1 (en) | Method for producing low-ash poultry plasma protein powder by utilizing poultry blood | |
| CN104710511B (en) | Iron chelating peptide derived from hairtail fish protein and preparation method and application thereof | |
| CN103255186A (en) | Combined production method for abalone polysaccharide, lipid and protein peptide | |
| CN113151387B (en) | Cod skin collagen peptide with oxidation resistance and immunity enhancement functions and preparation method thereof | |
| CN107602665A (en) | A kind of brown croaker flesh of fish anti-oxidation peptide | |
| CN109943612B (en) | Method for producing high-activity minced fillet and product antifreeze agent by enzymolysis of silver carp scales | |
| CN114621336B (en) | Casein source pancreatic lipase inhibitory peptide and preparation method thereof | |
| CN109280081B (en) | Method for preparing sialoglycopeptide from tuna eggs | |
| CN107177650B (en) | Preparation method of antioxidant enzymolysis oligopeptide from peripherical glands of northern pacific squid | |
| CN104758925A (en) | Iron chelation application of Trichiutus haumela bone iron-chelated collagen peptide | |
| CN111393537A (en) | Method for extracting bioactive substances from sea cucumber | |
| CN111887341A (en) | Method for extracting, separating and purifying miraculin | |
| CN111803531A (en) | Preparation method and application of dandelion leaf water-soluble crude extract | |
| CN116731108A (en) | Straw mushroom antioxidant peptide and application thereof | |
| CN117820437A (en) | Jerusalem artichoke peptide, preparation method thereof and application thereof in kidney-protecting auxiliary diabetes treatment antioxidant product | |
| CN107173815B (en) | Application of antioxidant enzymolysis oligopeptide from peripherical glands of northern pacific squid | |
| CN110183550A (en) | A kind of preparation process of refined heparin sodium | |
| CN110699410B (en) | Preparation method of euphausia superba small-molecule peptide | |
| CN105200104B (en) | Preparation method of crab shell leftover antioxidant peptide chain | |
| CN114262297A (en) | Preparation method of ergothioneine | |
| CN107176973B (en) | Antioxidative enzymolysis oligopeptide from peri-pacific squid oothecal gland | |
| CN109400743B (en) | Method for extracting sulfated galactan from sea grapes | |
| CN118048425B (en) | Antioxidant collagen peptide and preparation method and application thereof | |
| CN117683150B (en) | Sea buckthorn fruit peel polysaccharide and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |